These data indicate that the re-duced insulin levels were due to increased hepatic insulin clear-ance rather than reduced pancreatic insulin secretion Figure 4.The increase in insulin cl
Trang 2Reviews Editor
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Trang 3RESEARCH PAPER
Mechanisms by which the
thiazolidinedione troglitazone
protects against
sucrose-induced hepatic fat
accumulation and
hyperinsulinaemia
Fátima O Martins1,2,3†, Teresa C Delgado1,2†, Joana Viegas2,
Joana M Gaspar2, Donald K Scott4, Robert M O’Doherty4
,
M Paula Macedo2,5‡and John G Jones1,5‡
1Metabolic Control Group, Center for Neurosciences and Cell Biology of Coimbra, Cantanhede,
Portugal,2CEDOC, Chronic Diseases Research Center, NOVA Medical School/Faculdade de
Ciências Médicas, Universidade Nova de Lisboa, Lisboa, Portugal,3Institute for Interdisciplinary
Research (IIIUC), University of Coimbra, Coimbra, Portugal,4Division of Endocrinology and
Metabolism, University of Pittsburgh, Pittsburgh, PA, USA, and5APDP-Diabetes
Portugal-Education and Research Center (APDP-ERC), Lisboa, Portugal
Correspondence
John G Jones PhD and M PaulaMacedo, Metabolic Control Group,Center for Neurosciences and CellBiology of Coimbra, UC-Biotech,Biocant Park, Nucleo 4, Lote 8,Cantanhede 3060-197, Portugal.E-mail: john.griffith.jones@gmail.com;jones@cnc.uc.pt; paula.macedo@nms.unl.pt
†These authors contributed equally to thiswork
‡These senior authors also contributedequally to this work
Received
BACKGROUND AND PURPOSE
Thiazolidinediones (TZD) are known to ameliorate fatty liver in type 2 diabetes To date, the underlying mechanisms of theirhepatic actions remain unclear
EXPERIMENTAL APPROACH
Hepatic triglyceride content and export rates were assessed in 2 week high-sucrose-fed Wistar rats treated with troglitazone andcompared with untreated high-sucrose rodent controls Fractional de novo lipogenesis (DNL) contributions to hepatic triglyceridewere quantified by analysis of triglyceride enrichment from deuterated water Hepatic insulin clearance and NO status during ameal tolerance test were also evaluated
KEY RESULTS
TZD significantly reduced hepatic triglyceride (P < 0.01) by 48%, decreased DNL contribution to hepatic triglyceride (P < 0.01)and increased postprandial non-esterified fatty acids clearance rates (P < 0.01) in comparison with the high-sucrose rodentcontrol group During a meal tolerance test, plasma insulin AUC was significantly lower (P < 0.01), while blood glucose andplasma C-peptide levels were not different Insulin clearance was increased (P < 0.001) by 24% and was associated with a 22%augmentation of hepatic insulin-degrading enzyme activity (P < 0.05) Finally, hepatic NO was decreased by 24% (P < 0.05).CONCLUSIONS
Overall, TZD show direct actions on liver by reducing hepatic DNL and increasing hepatic insulin clearance The alterations inhepatic insulin clearance were associated with changes in insulin-degrading enzyme activity, with possible modulation of NOlevels
Abbreviations
DNL, de novo lipogenesis; HOMA-IR, homeostatic model assessment-insulin resistance; IDE, insulin-degrading enzyme;MTBE, methyl tertiary butyl ether; NEFA, non-esterified fatty acids; PDI, protein disulfide isomerase; T2D, type 2 diabetes;TZD, thiazolidinediones; VLDL, very low-density lipoproteins
Pharmacology
Trang 4Thiazolidinediones (TZD) are widely used for improving
glycaemic control in type 2 diabetes (T2D) patients TZD have
been also shown to decrease hepatic triglyceride levels in
those patients that present non-alcoholic fatty liver disease
– a frequent complication of T2D (Belfort et al., 2006; Ratziu
and Poynard, 2006) TZD activate the γ isoform of the
peroxisome proliferator -activated receptor gamma (PPARγ)
(Lehmann et al., 1995), a nuclear transcription factor that is
highly expressed in adipose tissue but is poorly expressed in
other insulin sensitive tissues such as skeletal muscle, liver,
pancreas, heart and spleen (Ferre, 2004) Thus, adipose tissue
is considered to be the main site of action for TZD, and much
of their systemic beneficial effects have hitherto been
explained via their effects on adipocyte physiology and
metabolism These include accelerated adipocyte uptake,
oxidation and esterification of circulating non-esterified fatty
acids (NEFA), thereby reducing the ectopic lipid burden and its
interference of insulin signalling in other tissues such as the
skeletal muscle and liver This is accompanied by alterations in
adipokine secretion profile that can further improve control of
glucose and lipid metabolism in these tissues (Guan et al.,
2002; Boden et al., 2005) However, adipose tissue may not be
the exclusive site of action for TZD Lipoatrophic patients have
negligible adipose tissue mass, and the same is true for a mouse
model of late onset lipoatrophy Yet in both settings, TZD
ther-apy was shown to improve diabetes and hyperlipidaemia
(Burant et al., 1997; Arioglu et al., 2000)
In the liver, TZD have been shown to reduce the expression
of gluconeogenic enzymes in animal models (Way et al., 2001)
and attenuate gluconeogenicfluxes in T2D patients (Gastaldelli
et al., 2006) In addition to being a principal control site for
car-bohydrate metabolism, the liver is also highly involved in
regu-lating systemic lipidfluxes via re-esterification, lipogenesis and
very low-density lipoproteins (VLDL) export Furthermore, it
plays an active role in controlling the levels of circulating insulin
viafirst-pass clearance of secreted insulin mediated by
insulin-degrading enzyme (IDE) and protein disulfide isomerase (PDI)
(Osei et al., 2007; Lamontagne et al., 2013) Recently, it has been
suggested that NO attenuates insulin clearance (Natali et al.,
2013) via inhibition of IDE (Cordes et al., 2009) Decreased
insu-lin clearance has been associated with hyperinsuinsu-linaemia and
decreased insulin sensitivity (Ader et al., 2014; Bril et al., 2014)
Thus, the development of steatosis and hyperinsulinaemia,which in addition to glucose intolerance are defining features
of the insulin resistant state, may reflect dysfunctionalhepatic lipid metabolism as well as impaired hepatic insulinclearance We hypothesized that TZD ameliorate steatosisand hyperinsulinaemia through changes in hepatic lipidfluxes and insulin clearance To test this hypothesis, we chosetroglitazone, thefirst TZD to be used as an antidiabetic drug.Troglitazone was subsequently withdrawn from clinical usedue to severe hepatotoxicity, confirmed in subsequent studieswith human primary hepatocyte cultures and cell lines(Yamamoto et al., 2002) Troglitazone is better tolerated byrat hepatocytes (Lauer et al., 2009) For both Gunn and Wistarrats, there were no indications of liver injury following long-term administration of troglitazone at 400 mg·kg1·day1 Thisdosage is well above those previously used to study its antidia-betic effects (Lee et al., 1994; Khoursheed et al., 1995; Okuno
et al., 1998) as well as that used in our present study
We tested the hepatic effects of troglitazone in rodentmodels of short-term (14 day) high-sucrose feeding Thismodel is useful for specifically probing splanchnic complica-tions of diet-induced insulin resistance because steatosis andhyperinsulinaemia are established before significant in-creases in whole-body adiposity Moreover, troglitazone hasbeen shown to be more effective in reversing insulin resis-tance and glucose tolerance in models of high sugar and fruc-tose feeding (Lee et al., 1994; Santure et al., 2003) comparedwith high-fat feeding (Khoursheed et al., 1995) It is wellknown that high sucrose feeding induces a substantial in-crease in de novo lipogenesis (DNL) such that this pathway be-comes a significant contributor to hepatic triglyceridesynthesis (Chong et al., 2007; Richelsen, 2013) Therefore,
we further hypothesized that the reversal of steatosis in thissetting by TZD involves the attenuation of hepatic DNL
Methods Animals
All animals were handled according to the European Unionguidelines for the use of experimental animals (2010/63/EU) The experiments were approved by the Ethics Commit-tee of the Faculty of Medical Sciences at the New University
Trang 5of Lisbon All animal care and experimental procedures
com-plied with the ARRIVE guidelines (Kilkenny et al., 2010;
McGrath et al., 2010) Thirty-six 12-week-old male Wistar rats
were maintained in a 12 h light/12 h dark cycle (lights on
from 07 h to 19 h) with ad libitum access to food and water
Animals were randomly separated in standard chow (SC),
HS-fed rodents (HS-C) (35% w v-1 in drinking water) and
troglitazone-treated HS-fed rodents (0.2%, included in the
diet) (HS-T) The troglitazone dose was selected from previous
studies like the one from Okuno and colleagues (Okuno et al.,
1998) The animals were maintained on these diets for 14 days
with water and food consumption being recorded Caloric
in-take was calculated taking into account these data, the
calo-ries from the diet used in the animal facility and by the
following equations:
Calories from food: average daily weight food gð ð ÞÞ*437; 1=199
Calories from beverage HS-C and HS-T groupsð Þ :
average daily water with sucrose mlð Þ
Two parallel studies were conducted with 18 animals per
study, six per diet regime In study 1, hepatic DNL and VLDL
export were measured At 19 h of day 13, all animals of study
1 received a loading dose of 99%2H2O (3 g 100 g-1body weight),
and the drinking water was also supplemented with2H2O to a
3%final enrichment Following overnight ad libitum feeding,
animals were sacrificed the next morning after cervical
disloca-tion following ketamine i.p injecdisloca-tion (100 mg·kg1 body
weight) The liver and epididymal adipose tissue were then
immediately excised, weighed and freeze-clamped in liquid
nitrogen until further analysis
In study 2, food was withdrawn on the last evening (day
13), and animals were fasted overnight On the morning of
day 14, rats were allowed ad libitum access to their respective
diets for 120 min At predetermined intervals, plasma NEFA,
glucose, insulin and C-peptide levels were quantified Rate
constants for the decrease in plasma NEFA concentrations
were derived from the logarithm-transformed curves of the
relative reduction in plasma NEFA concentrations from 0 to
120 min (Daly et al., 1998) Livers were excised and
immedi-ately freeze-clamped in liquid nitrogen until further analysis
for enzyme activities, NO levels and protein expression
Quantification of hepatic DNL
Hepatic triglycerides can be derived from plasma NEFA,
which are taken up via lipoprotein transport and esterified
to triglycerides after hepatic uptake They can also be formed
in situ by DNL of fatty acids from acetyl-CoA Hepatic DNL
was quantified using2
H2O as previously reported (Delgado
et al., 2009; Soares et al., 2012) From the1H and2H NMR
data, triglyceride methyl 2H-enrichment levels were
esti-mated, and by relating these enrichments values to that of
plasma water, the contribution of DNL to total hepatic
triglycerides was calculated
After the livers had been dried by lyophilization, about
half of each liver was powdered and a Folch extraction, with
20 mL chloroform : methanol (2:1) g-1 dried tissue,
per-formed The mixture was continuously agitated for 20 min
at room temperature and then centrifuged for 5 min at 4°C
and 1500 g The supernatant was vigorously mixed with
100 mL 0.9% (w v-1) NaCl and then centrifuged for 5 min at4°C and 1500 g The upper phase was discarded, and thelipid-containing lower phase was recovered and evaporated
to dryness Afterwards, dried lipids were dissolved in 2 mLhexane/methyl tertiary butyl ether (MTBE) (200:3) solutionfor purification by solid-phase extraction
For triglyceride purification, reverse phase solid-phase traction columns (Discovery DSC-18, Sigma-Aldrich, Steinheim,Germany) (2 g) were initially washed with 12 mL ofhexane/MTBE (96:4) followed by 12 mL hexane The lipid frac-tion was added to the column and further washed with 10 mLhexane/MTBE (200:3) To recover triglycerides, 12 mLhexane/MTBE (96:4) was eluted in the column, and 1 mL frac-tions were collected For identification of the fractions contain-ing triglycerides, thin layer chromatography was performedusing a mixture of petroleum ether, diethyl ether and acetic acid
ex-in the proportions of 8.0:2.0:0.1 as elutant and visualization byiodine Finally, triglyceride fractions were combined and evapo-rated to dryness for NMR analysis
For the acquisition of1H and2H NMR spectra, the purifiedtriglyceride extract was dissolved in 300μL of chloroform,and deuterated pyrazine was used as an internal2H-enrich-ment standard.1H and2H NMR spectra were acquired at 25°Cwith a 14.1 T Varian Spectrometer (Varian, Palo Alto, CA,USA) equipped with a 3 mm broadband probe Proton-decoupled2H NMR spectra were acquired without lock Ac-quisition parameters included a free induction decay acquisi-tion time of 5 s, a delay of 2 s and a 90° pulse width Between
500 and 1000 transients were acquired to achieve adequatesignal to noise for signal area analysis Spectra were referencedfor tetramethylsilane using the peaks of chloroform and pyrazineresonances, at 7.27 and 8.60 p.p.m respectively Before Fouriertransformations,1H and 2H NMR spectra were multiplied by0.5 and 1.0 Hz Lorentzian functions, respectively, and signalareas determined using the signal deconvolution routine of thePC-based NMR processing software NUTS proTM (Acorn, Fre-mont, CA, USA) Plasma water2H-enrichments were determinedfrom plasma by2H NMR spectroscopy analysis, as described pre-viously (Jones et al., 2001) Briefly, a 10 μL volume of plasma wasmixed with a known amount of acetone, and the2H-enrich-ments were determined using a standard curve constructed pre-viously using2H2O-enrichment standards against the constantnatural abundance2H signal of the acetone
Quantification of hepatic VLDL-triglyceride export rates
Hepatic VLDL-triglycerides export rates were determined cording to Millar et al (Millar et al., 2005) On the morning
ac-of day 14 following overnight ad libitum feeding, rats weregiven an i.p injection of poloxomer 407 (1000 mg·kg1bodyweight) Plasma triglycerides were evaluated immediately be-fore and at pre-established time intervals after poloxomer 407injection Hepatic VLDL-triglycerides export rates were de-rived from the slope of the curves of plasma triglycerides con-centrations at 0–90 min
Biochemical assays
Plasma glucose was assessed using a standard glucometer,whereas the quantitative determination of plasma insulinand C-peptide levels was achieved by means of ELISA
Trang 6(Mercodia AB, Uppsala, Sweden) Plasma NEFA levels were
assessed using an in vitro enzymatic colorimetric method assay
(Wako Chemicals GmbH, Neuss, Germany) Plasma triglycerides
and hepatic and epididymal adipose tissue triglycerides were
de-termined, following a Folch extraction of the tissue samples, by
an automated clinical chemistry analyser (Olympus AU400
Chemistry Analyzer, Beckman Coulter Inc., CA, USA)
Assessment of insulin clearance, HOMA-IR
After quantification of plasma insulin and C-peptide levels,
insulin clearance was calculated by the ratio between
C-peptide, a surrogate of insulin secretion, and plasma
insulin levels for each point analysed Homeostatic model
assessment (HOMA) indices were assessed from basal (fasting)
glucose and insulin [homeostatic model assessment-insulin
resistance (HOMA-IR)] or fasting glucose and C-peptide
con-centrations (HOMA-β) according to the recommendations of
Wallace et al (Wallace et al., 2004) HOMA-β gives an
indica-tion of β-cell secretion, while HOMA-IR gives an indication
of insulin resistance under basal fasting conditions
Assessment of hepatic NO levels
Liver was degraded by mechanical disruption using a piston
in a buffer containing Tris-HCl 25 mM (pH 7.4), EDTA 1 mM
and EGTA 1 mM Extracts were centrifuged and supernatant
denatured with ethanol Hepatic NO levels were measured
by a chemiluminescent-based technique using a Sievers 280
NO Analyzer (Sievers Instruments, Boulder, CO, USA) as
pre-viously described (Afonso et al., 2006)
Evaluation of hepatic NOS, IDE and PDI
activities
For nitric oxide synthase (NOS) activity, a buffer composed of
25 mM Tris-HCl, pH 7.4, 1 mM EDTA and 1 mM EGTA was
used to degrade the tissue in combination with mechanical
homogenization The resulting extracts were centrifuged,
and supernatants were measured for NOS activity using the
Ultra-sensitive Assay for NOS kit (Oxford Biomedical
Re-search, Oxford, MI, USA) For quantification of IDE activity,
liver tissue was also degraded by mechanical homogenization
in a buffer containing 170 mM NaCl and 2 mM EDTA The
FRET substrate Mca-GGFLRKHGQ-EDDnp was added to the
homogenate, and a fluorometric assay was performed as
previously described (Miners et al., 2008) PDI activity was
measured after incubation of homogenized liver tissue for
1 h at 37° with Krebs-Henseleit rinsing buffer supplemented
with CaCl2 and collagenase (50 mg) Samples were then
centrifuged at 4° and 16000 g for 5 min two times, with
recov-ery of the pellet containing the hepatocytes between
centri-fugations and resuspension in Krebs-Henseleit rinsing
buffer Thefinal pellet was resuspended in lysis buffer
con-taining 50 mM Tris-HCl, 300 mM NaCl, 1:200 dilution of
tab-let protease cocktail inhibitor and 1% Triton X-100 The
mixture was submitted tofive bursts of low-level sonication
and then centrifuged at 16000 g for 30 min at 4° The
super-natant was kept, and PDI activity was measured at 630 nm
by an OD assay over a 60 min period The reaction mixture
was composed of distilled water, 10 mM potassium
phos-phate buffer (pH 7.4), 8 mM GSH and 1.16 mg·mL1insulin
with afinal volume of 186 μL To this, 10 μL of the tant was added to each well in a multi-well plate In this assay,the PDI promotes the cleavage of insulin present in the reac-tion mixture, and the insulin degradation products cause anincrease in OD that is proportional to the units of PDIpresent
superna-Assessment of protein expression
Protein extracts for Western blot analysis were obtained using
a lysis buffer (1 M Tris-HCl, pH 7.5, 0.2 M EGTA, 0.2 M EDTA,1% Triton-X 100, 0.1 M sodium orthovanadate, 2 g·L1sodium fluoride, 2.2 g·L1 sodium pyrophosphate and0.27 M sucrose) to homogenize liver tissue Samples werecentrifuged, and total protein lysates were kept at20° Totalprotein lysates from liver were subjected to SDS–PAGE,electrotransferred on a PVDF membrane and probed withthe respective antibodies: IDE (sc-27265) and PDI (sc-20132)(Santa Cruz Biotechnology, Santa Cruz, CA, USA) Then,membranes were revealed in a ChemiDoc apparatus (Bio-Rad Laboratories, Inc., Hercules, CA, USA) Protein levelswere normalized toβ-actin (A5316, Sigma) for each sample
Data analysis and statistical procedures
Data are expressed as mean ± SEM of at leastfive animals pergroup Statistical significance was calculated using one-way
ANOVA(Bonferroni post hoc test)
Materials
Troglitazone was procured from Sangyo, Japan.2H2O (99%enriched) was acquired from CortecNet (Voisins-Le-Bretonneux, France), sucrose for drinking water preparationfrom Panreac (Castellar del Vallès, Barcelona, Spain) andother reagents from Sigma Aldrich (Steinheim, Germany)
Results Baseline glycaemic and lipidaemic parameters for the group fed with SC and the group fed with high sucrose (HS-C)
Plasma NEFA and triglycerides following an overnight fast orafter normal overnight feeding were similar for SC and HS-T(Table 1) Weight gain over the 2 week feeding period wasnot different between SC and HS-C, although daily caloric in-take was significantly increased for HS-C Total epididymaladipose tissue triglyceride content was not different between
SC and HS-C (Table 1) However, in agreement with earlierstudies (Huang et al., 2010), hepatic triglyceride levels werethreefold higher in HS-C compared with SC (Figure 1) More-over, fractional DNL rates (Figure 1) were increased approxi-mately twofold in HS-C-fed compared with SC-fed rats.Expression of SREBP1c, a transcription factor promoting theexpression of lipogenic enzymes and activated by insulinwas also significantly elevated in HS-C-fed compared withSC-fed rats (Figure 1) As shown in Figure 2, postprandial ex-port of hepatic triglyceride via VLDL showed a tendency to
be increased in HS-C compared with SC (P = 0.16) Also,the clearance of fasting plasma NEFA levels following a
Trang 7120 min feeding period was significantly slower for HS-C
compared with SC, as shown in Figure 3
Plasma glucose levels either after overnight fasting or after
normal overnight feeding were not significantly different
be-tween SC and HS-C groups indicating that glycaemic control
was maintained with HS feeding However, fasting plasma
in-sulin levels were significantly higher for HS-C versus SC This
translates to a significantly higher HOMA-IR index (Table 1)
and indicates a compensated insulin-resistant state for HS-C
relative to SC Meal-induced blood glucose, plasma insulin,
C-peptide excursions and insulin clearance were not different
between HS-C and SC (Figure 4) Hepatic IDE activity was not
different between HS-C and SC animals, but PDI activity was
significantly decreased in HS-C compared with SC animals
(Figure 5) Regarding IDE and PDI expression, there were no
alterations between SC-fed and HS-C-fed animals (Figure 5)
Finally, there were no alterations in NO levels and NOS activity
in HS-C-fed animals at 2 weeks of sucrose feeding (Figure 6)
Effects of troglitazone administration in rats
fed with a high sucrose diet
For animals that were placed on an HS diet and also
adminis-tered with troglitazone (HS-T), neither weight gain nor
calo-ric intake over the 2 week feeding period was different
compared with HS-C or SC Epididymal adipose tissue triglyceridecontent was not different compared with either SC or HS-C.However, hepatic lipid levels were significantly reduced com-pared with HS-C and were indistinguishable from SC Thesereductions in hepatic triglyceride were associated with signifi-cant reductions in fractional DNL rates to levels that were sim-ilar to SC (Figure 1) With troglitazone, SREBP1c expressionshowed a tendency for reverting to the lower levels measured
in SC (P = 0.095 for HS versus HS-T, Figure 1) Differences inrates of hepatic VLDL-triglyceride export were not significantbut showed a tendency to be reduced in HS-T compared withHS-C (Figure 2) HS-T had similar fasting plasma NEFA andtriglycerides levels to both HS-C and SC (Table 1) suggestingthat there was no significant effect of troglitazone supplementa-tion on fasting whole-body triglyceride dynamics However,under fed conditions, HS-T had significantly lower plasma NEFAlevels compared with both HS-C and SC (Table 1) Moreover,troglitazone potentiated the drop in plasma NEFA levels follow-ing the transition from fasting to feeding, with the fractionaldecrease in NEFA concentration exceeding both HS-C and SCgroups, as shown in Figure 3
Plasma glucose levels either after overnight fasting or afternormal overnight feeding were not significantly different be-tween HS-T and either HS-C or SC groups However, HS-T hadsignificantly reduced fasting plasma insulin and HOMA-IR
Table 1
Weight increase, adiposity and plasma metabolite and hormone levels for rats fed on the three dietary regimes
Standard chow High sucrose High sucrose + troglitazone
Initial weight (g) 293 ± 18 300 ± 16 285 ± 8
2-week weight increase (%) 29 ± 3 23 ± 3 23 ± 2
Chow intake (g day-1) 28 ± 1 22 ± 2** 20 ± 1
Troglitazone intake (mg Kg-1day-1) 176 ± 6 136 ± 13** 133 ± 9**
Caloric intake (kcal day-1) 129 ± 5 174 ± 15* 156 ± 8
Plasma NEFA (mmol l-1)a 1.66 ± 0.25 1.81 ± 0.21 0.25 ± 0.04**, ##
Plasma NEFA (mmol l-1)b 2.19 ± 0.32 1.73 ± 0.42 2.02 ± 0.39
Plasma triglycerides (mmol l-1)a 1.11 ± 0.06 1.28 ± 0.18 1.13 ± 0.20
Plasma c-peptide (nmol l-1)b 0.45 ± 0.18 0.74 ± 0.30 0.48 ± 0.21
Plasma insulin (μg l-1
)b 0.4 ± 0.1# 0.9 ± 0.1* 0.2 ± 0.1##
Plasma glucose (mmol l-1)a 6.6 ± 0.3 6.3 ± 0.3 6.9 ± 0.3
Plasma glucose (mmol l-1)b 4.4 ± 0.2 4.6 ± 0.2 4.7 ± 0.2
HOMA-IR 1.84 ± 0.62# 4.40 ± 0.71* 1.10 ± 0.22##
Epididymal fat pads weight (g) 5.1 ± 0.5 6.1 ± 0.7 6.6 ± 0.8
Total epididymal adipose tissue triglycerides
Trang 8values compared with HS-C and were similar to SC regarding sulin sensitivity (Table 1) The HS-T group had a pattern ofplasma glucose excursion following a meal that was identical
in-to HS-C and SC groups While meal-induced C-peptide profileswere not significantly different between the three groups,plasma insulin levels were significantly lower in HS-Tcomparedwith either HS-C or SC (Figure 4) These data indicate that the re-duced insulin levels were due to increased hepatic insulin clear-ance rather than reduced pancreatic insulin secretion (Figure 4).The increase in insulin clearance correlates with an increase
in hepatic IDE activity for HS-T (Figure 5) Regarding PDI ity, troglitazone effected no alteration in the decrease promoted
activ-by sucrose feeding (Figure 5) IDE and PDI expression were notaltered in HS-T animals (Figure 5) Levels of NO, an inhibitor ofIDE activity, were significantly diminished in HS-T group, andthis was associated with a decrease in NOS activity Therefore,the observed increase in insulin clearance can be linked to de-creased hepatic NO levels, which in turn were associated withincreased IDE activity (Cordes et al., 2009) (Figure 6)
Discussion
Thiazolidinediones are widely used pharmacological agentsthat improve several aspects of insulin resistance includingfatty liver and hyperinsulinaemia (Belfort et al., 2006; Ratziuand Poynard, 2006) Because troglitazone was withdrawnfrom clinical use due to hepatotoxicity, a potential concern
is that the observed hepatic metabolic changes could reflecthepatic injury rather than a true pharmacological effect on
Figure 1
Hepatic triglyceride levels (A), fractional contribution of de novo
lipogen-esis to hepatic triglyceride levels (B) and SREBP-1c expression levels
rela-tive toβ-actin (C), in standard-chow (SC), high-sucrose (HS-C) and in
2 weeks troglitazone-treated high-sucrose-fed (HS-T) rodents
Trang 10metabolicflux regulation Amelioration of sucrose or
fructose-induced insulin resistance was demonstrated in animals whose
feed was supplemented with 0.2g troglitazone per 100g feed
(Lee et al., 1994; Santure et al., 2003) In our study, the estimated
average daily intake of troglitazone was 133 mg·kg1·day1, well
below the level of 400 mg·kg1·day1that was shown not to
cause hepatic injury in either Wistar or Gunn rat strains after
3 months of administration (Watanabe et al., 2000)
The purpose of the current study was to better understand
the underlying hepatic mechanisms of these actions in the
setting of sucrose-induced hepatic insulin resistance.Our data show that troglitazone prevented the accumu-lation of hepatic lipids following 2 weeks of HS feeding
in rat models Kawaguchi et al previously showed thatpioglitazone protected against steatosis in a 2 week cho-line-deficient animal model (Kawaguchi et al., 2004).Wei et al reported that another TZD member, pioglita-zone, reduced both blood glucose levels and insulin inmice that had been fed a high-fat diet for 6 months(Wei et al., 2014)
Figure 5
Quantification of hepatic enzymes activity and expression (A) Hepatic insulin-degrading enzyme (IDE) activity; (B) hepatic IDE expression; (C)hepatic protein disulfide isomerase (PDI) activity; and (D) hepatic PDI expression after 120 min refeeding in standard-chow (SC), high-sucrose(HS-C) and in 2 weeks troglitazone-treated high-sucrose-fed (HS-T) rodents
Figure 6
Evaluation of hepatic NO production (A) Hepatic NO levels and (B) hepatic NOS activity after 120 min refeeding in standard-chow (SC), sucrose (HS-C) and in 2 weeks troglitazone-treated high-sucrose-fed (HS-T) rodents
Trang 11high-Our results demonstrate that troglitazone prevents the
ac-cumulation of hepatic lipids in 2 week HS-fed rodents not
only by reducing plasma NEFA (presumably by improved
ad-ipocyte NEFA uptake) but also by decreasing hepatic DNL
The reduction in DNL appears to be mediated at least in part
by a suppression of SREBP1c expression Given that hepatic
SREBP1c expression is known to be stimulated by insulin
via Insulin Receptor substrate 1 and 2 phosphorylation
(Kohjima et al., 2008), these differences in SREBP1c
expres-sion seem inconsistent with the fact that meal-induced
stim-ulation of insulin secretion was similar between the groups,
as measured by plasma C-peptide levels (Figure 4) However,
troglitazone significantly increased hepatic insulin
degrada-tion, which would result in a steeper drop in insulin levels
across the hepatic lobule Given that DNL activity is highest
in pericentral and perivenous regions (Schleicher et al.,
2015), it is possible that these hepatocytes experienced lower
levels of insulin due to the enhancement of hepatic insulin
degradation, thereby attenuating hepatic lipogenic activity
We demonstrated that the increased insulin clearance was
associated with elevated hepatic IDE activity, which in turn
was correlated with a fall in the levels of two endogenous
in-hibitors: NO and NEFA
If the observed decrease in plasma NEFA levels was due to
a more efficient uptake and storage in adipocytes, this would
be expected to result in an increased adipose tissue mass
While the 2 week troglitazone treatment of our study did
not significantly augment either epididymal fat pad weight
or adipose tissue triglycerides mass (Table 1), a 4 week
rosiglitazone-based therapy in Zucker lean and fatty rats
significantly increased the total weight of epididymal fat
pads (Sotiropoulos et al., 2006) Therefore, it is possible that
for over a longer period of HS feeding with troglitazone
administration, epididymal fat mass would have also shown
an increase over untreated HS-fed animals Therefore, with
our short-term HS feeding, the observed plasma NEFA
decrease can be mainly attributed to changes in hepatic fat
metabolism
The fatty acyl sources of hepatic triglycerides include
plasma NEFA released from adipocytes during fasting, dietary
fat intake during feeding and hepatic DNL Although TZD are
well known to stimulate DNL in adipocytes, their effects on
hepatic lipogenesis are more controversial Here, we showed
that troglitazone therapy was associated with a decrease in
fractional DNL for HS-fed rodents Likewise, pioglitazone
re-duced steatosis in a diet-inre-duced steatohepatitis animal model
through suppression of liver lipogenic gene expression,
includ-ing sterol regulatory element-bindinclud-ing protein-1c and fatty acid
synthase (Ota et al., 2007) On the other hand, prolonged
rosiglitazone-based and troglitazone-based therapy in obese
KKAy mice, expressing elevated hepatic PPARγ, was associated
with a worsening of steatosis and activation of lipogenic genes
and DNL (Bedoucha et al., 2001) This suggests that
administra-tion of TZD when hepatic expression of PPARγ is constitutively
high may promote rather than curtail hepatic steatosis
Recently, Recently, Beysen et al demonstrated that
rosiglitazone and pioglitazone had different effects on hepatic
DNL rates in T2D subjects (Beysen et al., 2008) A possible
expla-nation is that the actions of a given TZD may be further
poten-tiated by cross-reactivity with PPARα, whose agonists are known
to increase hepatic fatty acid oxidation activity and reduce
hepatic lipogenicfluxes (Tenenbaum and Fisman, 2012) over, in addition to functioning as PPAR agonists, TZD may alsodifferentially modify PPARα and PPARγ expression Thus,troglitazone, an agonist for both PPARα and PPARγ, was alsofound to increase PPARα expression while reducing PPARγ ex-pression in mononuclear cells of obese subjects (Aljada et al.,2001) In the liver, where constitutive PPARα levels are muchhigher than those of PPARγ, the anti-lipogenic effects oftroglitazone are best explained through its promotion and stim-ulation of PPARα
More-Thiazolidinedione therapy is also associated with a tion in peripheral serum insulin levels that accompanies animprovement in insulin sensitivity (Osei et al., 2004; Kim
reduc-et al., 2005) The insulin-sensitizing effects of PPARγ agonists,such as TZD, were recently attributed to an inhibition of aninsulin-signalling cascade, the MEK/ERK pathway, by specifi-cally blocking PPARγ phosphorylation at S273 This cascadehas also been linked with increased insulin resistance, whichled the authors to open a window for resurrecting PPARγ-targeted therapeutics to improved insulin sensitivity (Banks
et al., 2015) In this study, the authors also showed an provement in glucose tolerance and a decrease in plasma in-sulin levels in animals treated with MEK inhibitors.However, the mechanism that accounts for the reduced circu-lating insulin levels remains uncertain
im-After insulin is secreted into the portal vein, a significantand variable fraction (40–70%) is immediately cleared bythe liver, this fraction being sensitive to physiological andnutritional parameters that also inform insulin sensitivity(Radziuk and Morishima, 1985) Hence, plasma insulin levelsreflect both β-cell secretion and hepatic insulin clearance In-sulin clearance is initiated by the binding of insulin to its he-patic receptor Internalization of the bound insulin complex
is critical not only for insulin clearance but also for hepaticinsulin actions It has been suggested that 80% of secreted in-sulin binds to liver receptors After binding, insulin can beeither fully degraded or returned to the circulation eitherintact or partially degraded Insulin clearance is primarilymediated by IDE and PDI (Duckworth et al., 1998) Introglitazone-treated HS-fed rodents,β-cell insulin secretion,quantified using plasma C-peptide as a surrogate (Polonskyand Rubenstein, 1984; Polonsky et al., 1984a), was notaltered Hence, the observed decrease in plasma insulin levels
in troglitazone-treated rodents is explained by increasedhepatic insulin clearance, given by the ratio between C-peptidelevels and plasma insulin levels In agreement with our study,rosiglitazone therapy increased hepatic insulin clearance innondiabetic insulin-resistant, impaired glucose tolerant andT2D subjects (Kim et al., 2005; Osei et al., 2007) The observedincrease of hepatic insulin clearance is supported, at least inpart, by the observed IDE activity increase Previously, it hasbeen shown that increased insulin sensitivity in both animalsand human studies was associated with a stimulation of insulinclearance and decreased hyperinsulinaemia (Ahren andThorsson, 2003; Ader et al., 2014) However, Maianti et al.demonstrated that IDE inhibitors improved glucose toleranceover the short term (Maianti et al., 2014)
Both glucose and NEFA are potential regulators of IDEactivity (Hamel, 2009; Pivovarova et al., 2009) Because post-prandial glucose excursions were very similar between thethree groups, the differential effects of glucose on insulin
Trang 12clearance between the groups were likely to be insignificant.
Studies on the effects of NEFA on IDE-mediated insulin
clear-ance have yielded some contradictory conclusions On the
one hand, the studies of Wei et al indicate that NEFA,
specif-ically palmitate, induces IDE protein and activity (Wei et al.,
2014) Meanwhile, other studies indicate that NEFA inhibit
insulin degradation via IDE (Hamel, 2009) In vivo, the net
outcome of these opposing effects of NEFA on insulin
clear-ance ultimately rests on the relative potency of NEFA in
in-ducing IDE protein versus their inhibition of IDE activity
An acute twofold increase of NEFA in lean subjects promoted
a pronounced reduction in insulin clearance independently
of glucose levels (Hennes et al., 1997; Xiao et al., 2006) This
suggests that at least under these short-term conditions, the
dominant effect of NEFA was the inhibition of IDE activity,
with no major alterations on IDE expression With long-term
high-fat feeding, it is possible that there is a compensatory
in-duction of IDE to counter the effects of chronically excessive
NEFA levels, thereby explaining the observations of Wei et al
(Wei et al., 2014) Most significantly however, the study of
Wei et al and our work taken together indicate that promotion
of IDE activity and improvement of hyperinsulinaemia by TZD
is a robust outcome regardless of whether insulin resistance
was established via long-term high-fat diet or by shorter-term
HS feeding
NO has also been shown to be a regulator of IDE activity
Cordes and colleagues showed that NO directly binds to IDE
leading to its inhibition (Cordes et al., 2009) We showed
that TZD treatment resulted in a significant decrease of
he-patic NO levels and NOS activity Previous studies
demon-strated that pioglitazone administration led to a decrease
in inducible NOS (iNOS) activation in obese T2D subjects
(Esterson et al., 2013) These specific effects were only
observed in T2D patients, where iNOS activity is elevated
compared with healthy subjects
While IDE is considered to be the primary enzyme for
hepatic insulin clearance, PDI, which is also expressed by
the liver, can also alter internalized insulin The disulfide
bounds between the two chains of insulin can be cleaved
by PDI action, allowing some reactions between the
sepa-rated chains and reactive molecules surrounding them
Namely, it is known that these chains can undergo nitrosylation
by PDI-derived activity (Zai et al., 1999; Root et al., 2004)
More-over, it is known that PDI activity increase has been associated
to states of higher insulin sensitivity, namely, in the
postpran-dial state (Mikami et al., 1998) Therefore, our data indicate that
hepatic PDI activity and expression were not correlated with
either IDE activity or insulin clearance, but it is possible that
PDI may have a role in insulin degradation that is downstream
or complementary to that of IDE in specific conditions
In conclusion, our data demonstrate that troglitazone
protects against hepatic fat accumulation induced by a
short-term period of high sucrose feeding via several
mecha-nisms, acting in concert, involving both adipose tissue and
liver In addition to the canonical mechanism of increased
adipocyte NEFA uptake and esterification, thereby reducing
plasma NEFA, we also demonstrated an inhibition of hepatic
DNL and also reduced hepatic NO levels and NOS
expres-sion The reduction in hepatic lipid and NO, both of which
are inhibitors of IDE, was correlated with an increase in IDE
activity and hepatic insulin clearance This increased insulin
clearance explained the maintenance of normal plasmainsulin levels concomitant with the preservation of insulinsensitivity These novel hepatic actions of TZD offer moreprecise therapeutic strategies for reversing the onset ofnon-alcoholic fatty liver disease and countering hyperinsulinaemia in T2D subjects
Acknowledgements
The authors acknowledge financial support from thePortuguese Foundation for Science and Technology (researchgrants PTDC/EEB-BIO/99810/2008, PTDC-SAU-MET-111398-
2009, PTDC/DTP-EPI/0207/2012 and EXCL/DTP-PIC/0069/2012) The NMR spectrometers are part of the National NMRNetwork and were purchased in the framework of the NationalProgramme for Scientific re-equipment, contract REDE/1517/RMN/2005, with funds from POCI 2010 (FEDER) and thePortuguese Foundation for Science and Technology T C D.and F O M held a fellowship from the Fundação para aCiência e Tecnologia, Portugal (SFRH/BPD/46197/2008 andSFRH/BD/51194/2010 respectively)
Author contributions
F O M., T C D., J V and J M G performed the research
F O M., T C D., M P M and J G J designed the researchstudy D K S and R O contributed essential reagents ortools F O M., T C D., M P M and J G J analysed the data
F O M., T C D., M P M and J G J wrote the paper
Con flict of interest
Authors declare that they have no conflicts of interest
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Trang 15RESEARCH PAPER
Activity of botulinum toxin
type A in cranial dura:
implications for treatment of
migraine and other headaches
Zdravko Lackovi ć1, Boris Filipovi ć1,2, Ivica Matak1and Zsuzsanna Helyes3,4
1
Laboratory of Molecular Neuropharmacology, Department of Pharmacology, University of Zagreb
School of Medicine,Šalata 11, 10000 Zagreb, Croatia,2
Department of Otorhinolaryngology-Headand Neck Surgery, University Hospital Sveti Duh, Sveti Duh 64, 10000 Zagreb, Croatia,
3
Department of Pharmacology and Pharmacotherapy, University of Pécs School of Medicine, Szigeti
u 12, H-7624 Pécs, Hungary, and4János Szentágothai Research Center, 3MTA-PTE NAP B Pain
Research Group, University of Pécs School of Medicine, Ifjúság útja 20, H-7624 Pécs, Hungary
Correspondence
Zdravko Lacković, Laboratory ofMolecular Neuropharmacology,Department of Pharmacology,University of Zagreb School ofMedicine,Šalata 11, 10000 Zagreb,Croatia
E-mail: lac@mef.hr;
lackoviczdravko@gmail.com
Received
BACKGROUND AND PURPOSE
Although botulinum toxin type A (BoNT/A) is approved for chronic migraine treatment, its mechanism of action is still unknown.Dural neurogenic inflammation (DNI) commonly used to investigate migraine pathophysiology can be evoked by trigeminal pain.Here, we investigated the reactivity of cranial dura to trigeminal pain and the mechanism of BoNT/A action on DNI
EXPERIMENTAL APPROACH
Because temporomandibular disorders are highly comorbid with migraine, we employed a rat model of inflammation induced bycomplete Freund’s adjuvant, followed by treatment with BoNT/A injections or sumatriptan p.o DNI was assessed by Evans blue-plasma protein extravasation, cell histology and RIA for CGRP BoNT/A enzymatic activity in dura was assessed by immunohis-tochemistry for cleaved synaptosomal-associated protein 25 (SNAP-25)
KEY RESULTS
BoNT/A and sumatriptan reduced the mechanical allodynia and DNI, evoked by complete Freund’s adjuvant BoNT/A prevented
inflammatory cell infiltration and inhibited the increase of CGRP levels in dura After peripheral application, BoNT/A-cleavedSNAP-25 colocalized with CGRP in intracranial dural nerve endings Injection of the axonal transport blocker colchicine into thetrigeminal ganglion prevented the formation of cleaved SNAP-25 in dura
CONCLUSIONS AND IMPLICATIONS
Pericranially injected BoNT/A was taken up by local sensory nerve endings, axonally transported to the trigeminal ganglion andtranscytosed to dural afferents Colocalization of cleaved SNAP-25 and the migraine mediator CGRP in dura suggests that BoNT/Amay prevent DNI by suppressing transmission by CGRP This might explain the effects of BoNT/A in temporomandibular joint
inflammation and in migraine and some other headaches
Abbreviations
BoNT/A, botulinum toxin type A; CFA, complete Freund’s adjuvant; DNI, dural neurogenic inflammation; i.a., intra-articular;i.g., intraganglionic; SNAP-25, synaptosomal-associated protein 25; TMJ, temporomandibular joint
Pharmacology
Trang 16Botulinum toxin type A (BoNT/A) blocks the vesicular release
of neurotransmitters by proteolytic cleavage of a synaptic
protein, synaptosomal-associated protein 25 (SNAP-25)
SNAP-25 is a part of the synaptic protein complex which is
in-volved in Ca2+-dependent exocytosis (Kalandakanond and
Coffield, 2001; Blasi et al., 1993) This effect of BoNT/A at
peripheral nerve endings is the basis of its therapeutic use in
a range of neuromuscular (blepharospasm, focal dystonia
and spasticity) and autonomic disorders (hyperhidrosis and
bladder dysfunction) associated with neuronal over-activity
(Dressler, 2013) Based on large clinical studies, pericranially
injected BoNT/A has also been approved for the treatment
of chronic migraine (Diener et al., 2010) It is widely accepted
that migraine headaches involve activation of trigeminal
af-ferents innervating the meningeal blood vessels and dural
neurogenic inflammation (DNI) (Moskowitz, 1990; Geppetti
et al., 2012; Ramachandran and Yaksh, 2014) We have
re-cently found that the activation of dural afferents, measured
as plasma protein extravasation, can be evoked by
extracra-nial pain in the trigeminal region (orofacial formalin-evoked
pain and infraorbital nerve constriction-induced trigeminal
neuropathy) (Filipović et al., 2012, 2014) The plasma protein
extravasation induced by different types of pain was
prevented by peripherally injected BoNT/A The effect of
BoNT/A in the cranial dura was associated with axonal
trans-port of the toxin, because its effects were prevented by
injec-tion of colchicine directly into the trigeminal ganglion
(Filipović et al., 2012)
In the present study, we investigated the effects of BoNT/A
in a model of trigeminal pain induced by complete Freund’s
adjuvant (CFA) injection into the temporomandibular joint
(TMJ), a common model of temporomandibular disorders
(Harper et al., 2001; Villa et al., 2010) Temporomandibular
disorders involve dysfunction of both the TMJ and
mastica-tory muscles, leading to chronic pain (De Rossi et al.,
2014) BoNT/A injections into masticatory muscles have
been reported to reduce the tenderness and pain in patients
suffering from temporomandibular disorders (Sunil Dutt
et al., 2015) Severe forms of temporomandibular disorders
are highly comorbid with primary headaches– up to 86%
of patients suffer from migraine or other primary headaches
(Bevilaqua Grossi et al., 2009; Franco et al., 2010) The
un-derlying mechanism of the comorbidity is proposed to be
related to extensive innervation of cranial dura by
mandibular branch of trigeminal nerve (Schueler et al.,2013) So far, inflammation of the TMJ has been used pre-clinically to study the trigeminal sensitization associatedwith migraine (Villa et al., 2010; Thalakoti et al., 2007).CFA injection into the TMJ induces pain and inflammationleading to peripheral and central sensitization of trigeminalsystem (Villa et al., 2010) Similarly, by stimulating the TMJwith capsaicin, Thalakoti et al (2007 found widespreadperipheral sensitization in trigeminal ganglion cells Ac-cordingly, we hypothesized that TMJ pain might provide asuitable model to study trigeminal activation leading toDNI, as well as the mechanism of BoNT/A action in thetrigeminovascular system, assumed to be involved inmigraine and other headaches In the TMJ inflammationmodel, apart from neurogenic plasma protein extravasation,
we studied the effect of BoNT/A on CGRP, a neuropeptideconsidered the main mediator of trigeminal sensitization
in migraine (Bigal et al., 2013)
Here, we have found that CFA-evoked TMJ inflammationwas accompanied by inflammatory changes in the cranialdura (plasma protein extravasation and inflammatory cellinfiltration) and increased levels of CGRP Additionally,following peripheral toxin injection, cleaved SNAP-25, theproduct of BoNT/A enzymic activity, was colocalized withCGRP-expressing dural afferents BoNT/A prevented theCFA-evoked dural inflammation and CGRP peptide increase
in cranial dura
Methods Animal welfare and ethical statement
All animal care and experimental procedures were in dance with the 2010/63/EU Directive on the protection ofanimals used for scientific purposes and the recommenda-tions of International Association for the Study of Pain(Zimmerman, 1983) and were approved by the Ethical Com-mittee of University of Zagreb School of Medicine (permit
accor-no 07–76/2005–43) The experimental procedures used inthe work described in this article were as humane as possible.All animal studies are described in compliance with the AR-RIVE guidelines for reporting experiments involving animals(Kilkenny et al., 2010)
One hundred andfive male Wistar rats (average weight300–350 g; 3–3.5 months old; University of Zagreb School
These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawsonet al., 2014) and are permanently archived in the ConciseGuide to PHARMACOLOGY 2013/14 (Alexanderet al., 2013)
BJP
Trang 17of Medicine, Croatia) were used in these experiments Rats
were kept under a constant 12 h/12 h light/dark cycle with
free access to food and water The animals were randomly
al-located to different experimental treatments The
experi-menter conducting the behavioural testing was unaware of
the tretaments given to the animals
CFA-induced inflammatory pain in the TMJ
Animals were anesthetized with chloral hydrate (300 mg kg 1
i.p.) Injection into the TMJ was performed by inserting a 27
gauge needle medially through the skin below the inferior
border of the zygomatic arch and superior to the mandibular
condyl until it entered the joint capsule (Villa et al., 2010)
In-flammation of the TMJ was elicited by injection of 50 μL of
CFA into the left joint capsule Control rats were injected
intra-articularly (i.a.) with saline (0.9% NaCl) Methylene
blue was injected into a few animals, and the site of injection
was examined in a preliminary experiment to confirm
suc-cessful targeting of the TMJ
Behavioural testing
The next day (24 h) following CFA injection, behavioural
assess-ment of mechanical allodynia was performed using the von Frey
monofilaments (Stoelting Co., Wood Dale, IL, USA) as
previ-ously described in detail (Filipović et al., 2012) Filaments
pro-duced a calibrated bending force of 0.16, 0.4, 0.6, 1, 2, 4, 6, 8
and 10 The rats were placed in a transparent plastic cage for
10 min to accommodate to the experimental environment until
they assumed their normal sniffing/no locomotion position
For each session, a series of von Freyfilaments were applied on
the tested side of the face in ascending order, starting at 0.16 g,
with three attempts until a defined behavioural response was
elicited Each time, the measurement started on the side
contra-lateral to the CFA injection A positive reaction was interpreted
as defensive forepaw movement and/or escape/attack reaction
after stimulation of whisker pad area withfilaments In total,
the measurements were performed in three sessions in 10 min
intervals If no response was observed, we assigned 10 g as the
withdrawal threshold, because the force exerted by thicker
fila-ments (>10 g) was large enough to push the head of animals
Pharmacological treatments
BoNT/A injections
i.p.) (first anaesthetic) for different BoNT/A treatments,
(performed under the second anaesthetic) For
anesthetized rats, as described earlier For intraganglionic
injections,: rats were injected with BoNT/A in a dose of
anesthetized rats, as described previously (Matak et al., 2011;
Filipović et al., 2012) In brief, a Hamilton syringe needle
(Hamilton Microliter #701; Hamilton, Bonaduz, Switzerland)
was inserted through the skin into the infraorbital foramen
and advanced through the infraorbital canal and foramen
rotundum into the trigeminal ganglion
The multiple facial injections we made as follows: thetized rats were injected with BoNT/A at four sites: (i) bilat-erally into the rat forehead above the orbital arch and (ii)bilaterally into the whisker pad Five microlitre Injections(5μLper site) were administered using a Hamilton syringe Atotal dose of 5 U kg 1was employed and divided in four equaldoses (1.25 U kg 1per site)
anes-Sumatriptan
A group of animals were given sumatriptan, p.o., 24 h after
was calculated on the basis of previously used i.v dose
(Dallas et al., 1989; Schuh-Hofer et al., 2003) Mechanicalallodynia was measured, as described above, 2 h after theadministration of sumatriptan
Dural neurogenic plasma protein extravasation
Plasma protein extravasation, as an indicator of neurogenicinflammation, was measured 24 h after CFA injection Thiswas measured by injecting Evans blue dye which complexes
to plasma proteins Anaesthetized animals were perfusedtranscardially with 500 mL of saline 30 min after injection
of 1 mL Evans blue solution (40 mg kg 1) into the tail vein.Supratentorial dura was dissected into the left (ipsilateral toCFA treatment) and right sides (contralateral to CFA) andweighed Evans blue was extracted in formamide, and the absor-bance of Evans blue was measured spectrophotometrically Theamounts of extravasated Evans blue were calculated using thestandard concentration curve, as previously described in detail(Filipović et al., 2012)
RIA for CGRP
For the measurement of CGRP immunoreactivity withRIA, animals were injected with BoNT/A into the TMJ, asdescribed above One day after the induction of TMJinflammation, animals were deeply anesthetized withchloral hydrate (300 mg kg 1 i.p.) Approximately
100 μL of CSF was withdrawn from cisterna magna using27½ gauge syringe needle inserted percutaneously be-tween the occipital bone and atlas Only transparent CSFsamples were taken for further analysis The sample wasrapidly frozen by immersing the sealed Eppendorf tubecontaining the CSF in liquid nitrogen and kept at 80°C.Immediately following the CSF sampling, anesthetized animalswere killed by decapitation Supratentorial dura, brainstem andtrigeminal ganglion were quickly dissected, frozen in liquidnitrogen and kept at 80°C until further use The frozenbrainstem was placed in cryostat-cooled environment ( 25°C)for dissection of ipsilateral trigeminal nucleus caudalis withoutthawing The nucleus was excised manually using a pre-cooledmicrotome blade, scalpel and forceps Dissected tissue wasfurther kept at 80°C until homogenization
Tissue samples were weighed and immediately homogenizedwith 1 mL distilled water and 20 μL of aprotinin solution(Trasylol, Bayer, Germany) Trigeminal ganglia and caudal nu-cleus samples were manually homogenized in a glasshomogeniser, while dura was homogenized using a Polytron me-chanical homogenizer The samples were then centrifuged for
10 min at 8944 g, and the procedure was repeated with the
BJP
Trang 18resulting supernatant Final supernatants were kept at 30°C
until further analysis CSF was directly used as a RIA sample
without further preparation
Radioimmunoassay was performed similarly as previously
described (Németh et al., 1998; Pozsgai et al., 2012) In brief,
samples or CGRP standards (Sigma) were diluted in buffer
for RIA containing 1:120 000 anti-CGRP polyclonal antibody
(Sigma) and tracer containing radio-iodinated CGRP
stan-dard Diluted samples were incubated at 4°C for 48 h
Antigen-bound and free CGRP peptides were then separated
by adding 100μL of distilled water with 10% activated
char-coal, 2% dextran and 0.2% fat-free milk powder The samples
were vortexed and centrifuged at 2010 g for 20 min Levels of
radioactivity of the pellets containing the free peptide and
su-pernatant containing the antibody-bound peptide were
deter-mined with aγ counter Concentrations of CGRP (fmol mg 1
or fmol mL 1) in samples were calculated based on a standard
concentration curve
Histology and immunohistochemistry of the
dura mater
In order to assess inflammatory cell infiltration in the dura
mater by histology, animals were injected with BoNT/A
(5 U kg 1) and CFA into the TMJ as described above One
day after CFA, the anaesthetized animals were perfused
with saline and 250 mL of 4% paraformaldehyde in PBS
Ipsilateral and contralateral supratentorial dura were
care-fully dissected and placed in paraformaldehyde fixative
containing 15% sucrose, followed by 30% sucrose in PBS
on the next day After 48 h, the samples were stored at80°C until further use
Histological study of the cranial dural tissue was formed using standard Giemsa staining Brightfield micro-photographs were taken with Olympus BX-51 microscopecoupled with DP-70 digital camera (Olympus, Tokyo, Japan)under constant condenser light intensity and camera exposi-tion The number of Giemsa-stained cell profiles was auto-matically quantified in four to five non-overlapping visualfields (obtained at 20× magnification) per single animal,using cellSens Dimension programme (Olympus) as previ-ously described in detail (Filipović et al., 2014) Five animalsper group were examined
per-To investigate the possible spread of peripherally injectedBoNT/A to dural afferents, animals were injected in the TMJunilaterally with 5 or 15 U kg 1BoNT/A, as described above.One group of animals was injected with 15 U kg 1BoNT/Ainto the whisker pad An additional group of animals wasinjected unilaterally with a total dose of 20 U kg 1BoNT/A(7 U per 350 g rat) divided in four injection sites (1.75 U/
20μL per site) – (i) TMJ, (ii) whisker pad, (iii) medial head) and (iv) lateral (temporal) cranial region Six days afterperipheral injection of BoNT/A, animals were anesthetizedand perfused for immunohistochemistry with saline andparaformaldehydefixative
(fore-Dural samples were stained for cleaved SNAP-25 using thefree-floating procedure as previously described (Matak et al.,2014) In brief, dissected dura was washed in PBS, blockedwith 10% normal goat serum and incubated overnight at
Figure 1
BoNT/A and sumatriptan effects on bilateral allodynia induced by
unilateral TMJ inflammation BoNT/A (5 U kg 1
) was injected intothe TMJ (5 U kg 1i.a.) or trigeminal ganglion (2 U kg 1i.g.) 3 days
before CFA Facial allodynia was measured with von Freyfilaments
24 h after CFA injection into the TMJ Sumatriptan (175 mg kg 1)
was administered p.o 24 h after CFA, and allodynia was measured
2 h after sumatriptan Scatter plot represents data of individual
ani-mals, and horizontal lines and bars indicate mean ± SEM.n (animals
per group) = 5–9 *P < 0.05, ** P < 0.01, ***P < 0.001, significantly
different from saline control;+++P < 0.001, significantly different
from saline + CFA; one-way ANOVA followed by Newman–Keuls
post hoc test
Figure 2
The effect of BoNT/A and sumatriptan on Evans blue/plasma proteinextravasation in dura mater after TMJ inflammation BoNT/A wasinjected into the TMJ (5 U kg 1 i.a.) or trigeminal ganglion(2 U kg 1 i.g.) 3 days before CFA Sumatriptan (175 mg kg 1)was administered p.o 24 h after CFA Four days following BoNT/A
or 2 h after sumatriptan rats were injected with Evans blue(i.v., 40 mg kg 1) and perfused with saline Dura was collected forformamide extraction and spectrophotometric measurement ofEvans blue dye which extravasates in complex with plasma proteins.Scatter plot represents data from individual animals, and horizontallines and bars indicate mean ± SEM.n (animals per group) = 5–9
*P < 0.05, ***P < 0.001, significantly different from saline control;
Trang 19room temperature with 1:1600 anti-BoNT/A-cleaved
SNAP-25 antibody (provided by Ornella Rossetto, University of
Padua, Italy) in PBS containing 1% goat serum The antibody
binds specifically to BoNT/A-cleaved SNAP-25 and not the
in-tact SNAP-25 (Matak et al., 2011) Next day, the samples were
incubated with Alexa Fluor 555 rabbit secondary
anti-body Stained dura was carefully spread on the glass slides
and cover-slipped with an anti-fading agent In animals
injected at four different sites or only into the TMJ
(5 U kg 1), additional labelling with rabbit CGRP
anti-body (1:5000, Sigma) was performed In order to prevent a
possible cross-reactivity of cleaved SNAP-25 with CGRP, a
modified primary antibody elution procedure with
pre-heated acidic buffer (50°C, pH = 2, 25 mM glycine and 1%
SDS) was performed, as described previously in detail (Matak
et al., 2014) After the elution, the dural samples were stained
with anti-CGRP and Alexa Fluor 488 secondary antibody The
appearance of cleaved SNAP-25 Alexa Fluor 555 stainedfibre
profiles, observed before and after antibody elution, was
un-changed Cross-reactivity controls (omitted CGRP antibody)
showed no Alexa Fluor 488 signal in association with cleaved
SNAP-25fibers, as reported previously (Matak et al., 2014)
Investigation of the effect of the axonal
transport inhibitor, colchicine, on
antinociceptive activity and appearance of
cleaved SNAP-25 in dura mater following
BoNT/A injection
By blocking the axonal transport within the trigeminal
ganglion, we examined the involvement of the axonal traffic
via the trigeminal nerve of BoNT/A for its antinociceptive
activity and for the presence of cleaved SNAP-25 in thedura mater Anesthetized animals were injected in theTMJ with saline or BoNT/A (5 U kg 1) Immediately afterTMJ injection, the animals were injected with 2 μL ofsaline or an equal volume of the axonal transport blockercolchicine (5 mM) into the trigeminal ganglion, percutane-ously via the infraorbital canal as previously described(Filipović et al., 2012) Seven days after i.a and i.g treatments,the animals were treated with CFA, and the mechanicalallodynia was measured after 24 h, as described above Then,the animals were anaesthetized and perfused with saline andfixative, and the dural tissue was processed and stainedfor immunohistochemistry of BoNT/A-cleaved SNAP-25 asdescribed above
St Louis, MO, USA); sumatriptan (Glaxo Wellcome, Taplow,UK) reconstituted in drinking water; and BoNT/A diluted in0.9% saline (Botox®; Allergan Inc., Irvine, CA, USA) Oneunit (1 U) of BoNT/A preparation contains 48 pg of purifiedClostridium botulinum neurotoxin type A complex
Figure 3
Neurogenic plasma protein extravasation in dura is reduced by i.a./i.g BoNT/A and p.o sumatriptan– photographs of open cranial cavities Leftside: TMJ was injected with CFA 1 day before animal perfusion with saline BoNT/A was injected into the TMJ (5 U kg 1i.a.) or trigeminal ganglion(2 U kg 1i.g.) 3 days before CFA Sumatriptan (175 mg kg 1) was administered p.o 24 h after CFA Four days following BoNT/A or 2 h aftersumatriptan rats were intravenously injected with Evans blue (40 mg kg 1) and perfused with saline Photographs were taken upon the perfusionwith saline and the removal of brain tissue
BJP
Trang 20CFA-evoked bilateral allodynia is reduced by i.a.
and i.g BoNT/A, and oral sumatriptan
Animals treated with CFA injected into the TMJ developed
mechanical allodynia 24 h after the injection Allodynia
ap-peared bilaterally Pre-treatment with BoNT/A injected
ipsi-laterally to the CFA injection [both i.a (5 U kg 1) and i.g
(2 U kg 1)] 3 days before CFA, reduced the mechanical
allodynia bilaterally (P< 0.001) Similarly, 2 h after
BoNT/A and sumatriptan reduce plasma
protein extravasation in dura mater
Dural plasma protein extravasation was significantly
in-creased bilaterally in CFA-injected animals compared with
control values (Figures 2–4) Plasma protein extravasation in
the ipsilateral dura was double that on the contralateral side
(P< 0.001, t-test for dependent samples) BoNT/A injected
both i.a (5 U kg 1) and i.g (2 U kg 1), as well as sumatriptan
(175μg kg 1
p.o.), reduced the ipsilateral dural plasma
pro-tein extravasation (Figure 2) In the contralateral side, none
of the treatments affected the DNI
In a separate experiment, we employed four BoNT/A
low-dose bilateral injections into the face of the rats (Figure 4) As
observed with the single BoNT/A injection into the TMJ, four
injections outside of TMJ prevented both bilateral allodynia
and the CFA-evoked plasma protein extravasation (Figure 4)
TMJ inflammation induces dural tissue
infiltration with inflammatory cells, which is
prevented by BoNT/A
Histological staining of the dural tissue of CFA-treated rats
demonstrated an elevated number of automatically counted,
Giemsa-positive, cell nuclei, compared with those in
saline-treated animals (P< 0,001), indicating an inflammatory cell
infiltration Inflammatory cells present in CFA-injected
ani-mals (not present in saline control) were identified by an
ex-perienced pathologist, as lymphocytes, monocytes and
plasma cells, as previously found in a model of trigeminal
neuropathy (Filipović et al., 2014) The lack of
polymorpho-nuclear neutrophils in dura suggests the presence of a sterile
inflammation BoNT/A prevented the increased number of
Giemsa positive profiles evoked by i.a CFA (Figure 5)
TMJ inflammation induces up-regulation of
CGRP in dura and TNC, which is reduced by i.a.
BoNT/A
Following CFA-induced TMJ inflammation, CGRP expression
was significantly increased in dura mater and ipsilateral
cau-dal trigeminal nuclei BoNT/A injected into the TMJ
pre-vented the CGRP increase in dura mater The effect of
BoNT/A on CGRP expression in trigeminal nuclei was not
sig-nificant CGRP concentration was not significantly altered in
trigeminal ganglion and CSF (Figure 6)
Figure 4
The effect of BoNT/A injection outside the TMJ on mechanicalallodynia and dural Evans blue/plasma protein extravasation.BoNT/A (total dose 5 U kg 1) was injected at four sites (bilateralforehead and bilateral whisker pad injections) (A) Three daysafter BoNT/A rats were injected with CFA into the TMJ Facialallodynia was measured with von Frey filaments 24 h after CFAinjection After behavioural measurement, rats were injected withEvans blue (i.v., 40 mg kg 1) and perfused with saline Dura washarvested for formamide extraction and spectrophotometricmeasurement of Evans blue dye extravasated in complex withplasma proteins (A) Sites of BoNT/A bilateral injections andposition of TMJ to be injected with CFA (B) The effect of BoNT/
A on mechanical thresholds measured by von Frey filaments(mechanical allodynia) (C) The effect of bilateral Evans blue/plasma protein extravasation in the cranial dura Scatter plotrepresents individual animal values, and horizontal lines and barsindicate mean ± SEM.n (animals per group) = 5–8 *P < 0.05,
**P < 0.01, ***P < 0.001, significantly different from saline control;
+P < 0.05, significantly different from saline + CFA;+++P < 0.001,significantly different from saline + CFA; one-way ANOVA followed
by Newman–Keuls post hoc test
BJP
Trang 21Cleaved SNAP-25 colocalizes with
CGRP-expressing afferents of the dura mater
after BoNT/A peripheral treatment
In animals injected peripherally with BoNT/A, we observed
the presence of cleaved SNAP-25 in the injected-side lateral
and parietal dura near the dural blood vessels after BoNT/A
multiple (Figure 7) and single injections into TMJ and
whisker pad (not shown) Cleaved SNAP-25 was also visible
in non-vascular areas of dura All examinedfibers containing
SNAP-25 co-expressed bright granular immunoreactivity for
CGRP (Figure 7) Contralateral dura was devoid of cleaved
SNAP-25, ruling out possible systemic BoNT/A diffusion
(Figure 7) Complete colocalization of CGRP and cleaved
SNAP-25 was also visible after the single injection of
BoNT/A (5 U kg 1) into the TMJ (not shown)
Anti-nociceptive activity and enzymatic
activity of BoNT/A in dura mater are axonal
transport-dependent
The anti-nociceptive actions of BoNT/A on CFA-induced pain
was prevented by the axonal transport blocker colchicine
injected into the trigeminal ganglion (Figure 8) This is in line
with previousfindings that BoNT/A antinociceptive activity is
dependent on axonal transport (Filipović et al., 2012) Similarly,
cleaved SNAP-25 was no longer found in dura after treatment
with colchicine (Figure 8) Thisfinding suggests that, after local
peripheral injection of 5 U kg 1 in the facial area, BoNT/A isaxonally transported to the ipsilateral dural primary afferents
by microtubule-dependent mechanism through the ganglion
by DNI (Filipović et al., 2012, 2014) These observations onstrate the occurrence of DNI in experimental trigeminalpain To study migraine, other authors induced DNI more‘arti-ficially’ by different chemical or electrical stimuli (Markowitz
dem-et al., 1987; Buzzi and Moskowitz, 1990; O’Shaughnessy andConnor, 1994; Arulmani et al., 2006; Nelson et al., 2010;Akerman et al., 2013) Current opinion suggests that themigraine headache involves CNS dysfunction, accompanied
by activation of the trigeminovascular system (Williamsonand Hargreaves, 2001), and release of vasoactive peptides whichinduce DNI (Markowitz et al., 1987) This is not limited to
Figure 5
The effect of BoNT/A injection into the TMJ on inflammatory cell infiltration in dura mater in CFA-treated rats The 5 U kg 1
BoNT/A orsaline were injected into the TMJ 3 days before the induction of TMJ inflammation by CFA Histological staining of ipsilateral cranial duraltissue was performed using Giemsa staining Number of Giemsa-stained cell profiles was automatically quantified by CellSens Dimensionvisualizing programme (Olympus) Each data value represents mean of 4–5 visual fields per single animal L, lymphocyte; Mo, monocyte;
P, plasma cell, cMC, constitutive mast cell; F, fibrocyte Scale bars = 100 μm Scatter plot represents individual animal values, andhorizontal lines and bars indicate mean ± SEM.n (animals per group) = 5 *P < 0.05, significantly different from saline control; ***P < 0.001,significantly different from saline control;+++P < 0.001, significantly different from saline + CFA; one-way ANOVA followed by Newman–Keuls posthoc test
BJP
Trang 22experimental animals because it was clinically observed that
DNI accompanies migraine and cluster headache attacks (Göbel
et al., 2000; Knotkova and Pappagallo 2007) Thus, ongoing
pain in the TMJ area, as well as other orofacial pain models,
can be employed to study the sensitization of trigeminal dural
afferents, assumed to be present in migraine and other
head-aches In the present experiments, peripherally injected
BoNT/A reduced mechanical allodynia and inflammatory
changes in the cranial dura [plasma protein extravasation and
cellular inflammatory response (Figures 1, 2)] The similar
ef-fects of BoNT/A injections given directly into the ganglion
sug-gests that BoNT/A action is primarily associated with the
trigeminal system (Figures 1, 2)
The recommended protocol for BoNT/A application in
chronic migraine consists of multiple injections to 31 head
and neck sites (Diener et al., 2010) A similar protocol is
diffi-cult to replicate in rats because of the smaller cranial
dimen-sions Thus, we injected BoNT/A bilaterally to the rat
forehead region overlying the frontal bone (innervated by
V1 ophthalmic trigeminal branch) and whisker pad (V2
max-illary branch) Such BoNT/A injections at four sites were
effec-tive in preventing CFA-evoked pain and DNI similarly to the
single BoNT/A injection into the TMJ (Figure 4) This
demonstrates that the effects of BoNT/A on allodynia andDNI are not primarily mediated by its direct peripheral effect
on CFA-stimulated neurons
Plasma protein extravasation in cranial dura is a usefulmarker of trigeminal activation, often employed in preclinicalscreening of antimigraine drugs (Markowitz et al., 1987; Buzziand Moskowitz, 1990; O’Shaughnessy and Connor, 1994;Arulmani et al., 2006; Nelson et al., 2010; Akerman et al.,2013) DNI consists of two main components: vasodilation,which is mediated by CGRP, and plasma protein extravasation,which is mediated by substance P Blocking only the substance
P transmission by NK1 receptor antagonists did not reducemigraine symptoms, suggesting that CGRP transmission mightplay a more important role in the pathophysiology of migraine(Williamson and Hargreaves 2001; Peroutka, 2005) Thus, weinvestigated the possibility that the antimigraine actions ofBoNT/Awere associated with prevention of CGRP transmission.Here, we have found that peripherally injected BoNT/Aprevented the CFA-induced increase in CGRP levels in thecranial dura (Figure 6) Interestingly, in chronic migrainepatients responsive to BoNT/A, the pretreatment CGRP plasmalevels were increased in comparison with those in BoNT/A non-responsive patients (Cernuda-Morollón et al., 2014) After the
Figure 6
BoNT/A effect on concentration of CGRP protein in dura, trigeminal nucleus caudalis, trigeminal ganglion and CSF in CFA-treated rats The
5 U kg 1BoNT/A was injected into the TMJ 3 days before the CFA treatment Tissues were collected 1 day post-CFA, and the CGRP tration was analysed by RIA (A) Dura mater; (B) ipsilateral trigeminal nucleus caudalis; (C) ipsilateral trigeminal ganglion; and (D) CSF Scatterplot represents individual animal values, and horizontal lines and bars indicate mean ± SEM.n (animals per group) = 6 *P < 0.05, significantlydifferent from saline control; **P < 0.01, significantly different from saline control; ++P < 0.01, significantly different from saline + CFA;one-way ANOVA followed by Newman–Keuls post hoc test
concen-BJP
Trang 23treatment, BoNT/A normalized the elevated CGRP plasma levels
(Cernuda-Morollón et al., 2015) The authors posited that
BoNT/A inhibits the release of CGRP from peripheral trigeminal
neurons and, consequently, reduces the CGRP-mediated
trigemi-nal sensitization in migraine (Cernuda-Morollón et al., 2015)
Because the anti-migraine effect of BoNT/A is difficult to
explain by its local action on peripheral, extracranial sensory
nerves endings, it was suggested that BoNT/A exhibits its
ac-tions in pain and migraine by reaching dural trigeminal
affer-ents (Matak and Lacković, 2014; Ramachandran and Yaksh,
2014) Previously, we reported that the effects of BoNT/A on
trigeminal neuropathic pain and resulting DNI was
prevented by colchicine injected into the ganglion,
indica-tive of axonal transport of this toxin (Filipović et al., 2012)
After BoNT/A peripheral injection, we detected cleaved
SNAP-25 in the cranial dura mater (Figures 7, 8) Moreover,
cleaved SNAP-25 and CGRP were colocalized in the ipsilateral
dura (Figure 7) Peripherally administered BoNT/A may
pre-vent the SNAP-25-mediated release of CGRP in cranial
me-ninges and consequent CGRP effects presumably involved
in migraine pathophysiology (Williamson and Hargreaves,2001; Durham, 2008; Geppetti et al., 2012; Karsan andGoadsby, 2015)
It was recently found that BoNT/A reduces the cal sensitivity of extracranially projecting collaterals of duralafferents which exit the cranium through the skull bonesutures in rats (Burstein et al., 2014) However, in our experi-ments, BoNT/A effects on dura mater were present even ifthe toxin was administered away from cranial sutures (TMJand whisker pad) Additionally, blockade of the axonal trans-port of the toxin by direct i.g colchicine prevented theformation of cleaved SNAP-25 in the dura (Figure 8) and othereffects of BoNT/A on DNI (Filipović et al., 2012) Colchicineaction is limited to the injection site (Kreutzberg, 1969;Cangiano and Fried, 1977) and, therefore any possibleBoNT/A axonal traffic to the dura via extracranial collaterals
mechani-of dural afferents should not be prevented by administration
of colchicine into the ganglion These observations do notsupport an important contribution of BoNT/A local activity
on extracranially projecting dural afferent collaterals
Figure 7
Colocalization of truncated SNAP-25 and CGRP in ipsilateral cranial dura after BoNT/A injection in the periphery BoNT/A 20 U kg 1 totaldose was injected into four different sites (TMJ, whisker pad, and frontal and temporal regions; 1.75 U/20μL per site) on the left side ofthe head Animals were perfused for immunohistochemistry 6 days later (A) Upper panel: lower magnification fluorescent microphotographshows the course of a single-cleaved SNAP-25 [SNAP-25(c)]-immunoreactivefibre (arrows, red immunofluorescence) in the vicinity of duralblood vessels, which colocalizes with CGRP (green fibers) Lower panel: higher magnification image of the middle part of cleaved SNAP-25-immunoreactive fibre, which colocalizes with granular CGRP immunofluorescence (B) Microphotograph of contralateral side dura ofthe same animal without detectable cleaved SNAP-25 in CGRP-expressing afferents The images are representative of the data obtained fromfour animals Scale bars = 100μm
BJP
Trang 24The question arising from the present experiments is how
BoNT/A crosses from the trigeminal extracranial nerves to
tri-geminal nerve endings in dura Dura and extracranial
trigem-inal regions are innervated by separate sensory neurons
(Larrier and Lee, 2003; Shimizu et al., 2012) Therefore, the
transcytosis of BoNT/A from the extracranial sensory
neurons to neurons that innervate dura seems the most logical
explanation for the occurrence of cleaved SNAP-25 in dura
mater after facial injection of the toxin Up to now, transcytosis
of BoNT/A between different neurons has been demonstrated
directly inside the retina and brain (Restani et al., 2011, 2012)
In the trigeminal region, BoNT/A transcytosis within the
tri-geminal ganglion after its peripheral injection has been
sug-gested by Kitamura et al (2009 The authors investigated the
effect of BoNT/A on vesicular neurotransmitter release in
tri-geminal neurons acutely isolated from neuropathic rats
sub-jected to infraorbital nerve constriction injury BoNT/A
injected into the rat face induced a profound reduction of
vesic-ular neurotransmitter release in all neurons isolated from the
ganglion They assumed that, in order to induce a widespread
effect, BoNT/Awas transcytosed within the ganglion (Kitamura
et al., 2009) In the trigeminal ganglion, facially injected
BoNT/A reduced the expression of TRPV1 channels in neurons
projecting to the dura mater (Shimizu et al., 2012) These
authors proposed that the effects of BoNT/A were mediated bytranscytosis of the toxin, within trigeminal gangliam fromextracranially projecting neurons to neurons that innervatethe dura (Shimizu et al., 2012) The exact place and mechanism
of such putative transcytosis remain to be elucidated It is likely
to occur within the trigeminal ganglion itself (Shimizu et al.,2012), although transcytosis in the trigeminal sensory nucleicannot be excluded (Ramachandran and Yaksh, 2014) (Figure 9).The conventional antimigraine drug sumatriptan, an ago-nist of 5-HT1B/Dreceptors, reduced the pain supersensitivityand dural plasma protein extravasation in CFA-induced TMJinflammation, as well (Figures 1, 2) Sumatriptan preventsthe evoked release of CGRP and substance P in vitro and
ex vivo (Buzzi and Moskowitz, 1990; Durham and Russo,1999) Furthermore, sumatriptan reduces elevated CGRP con-centrations in blood and saliva during migraine attacks(Goadsby et al., 1990; Bellamy et al., 2006) CGRP antagonistsare reported to reduce the symptoms of acute migraine at-tacks (Edvinsson and Warfvinge, 2013) Antibodies againstCGRP and CGRP receptors might also be effective as a pro-phylactic chronic migraine treatment (Edvinsson, 2015)
In conclusion, as demonstrated here, BoNT/A might havebeneficial effect on experimental TMJ pain and the accompa-nying dural inflammation The effects of BoNT/A in the cranial
Figure 8
BoNT/A antinociceptive activity and occurrence of cleaved SNAP-25 in dura mater is dependent on axonal transport (A) Preventive effect ofi.a BoNT/A (5 U kg 1) on mechanical allodynia evoked by CFA injection into the TMJ is prevented by colchicine (5 mM) injection into thetrigeminal ganglion Scatter plot represents individual animal values, and horizontal lines and bars indicate mean ± SEM n (animals pergroup) = 5–6 ***P < 0.001, significantly different from saline i.a + CFA;+++P < 0.001, significantly different from BoNT/A i.a + CFA;one-way ANOVA followed by Newman–Keuls post hoc test (B) Colchicine prevented the occurrence of cleaved SNAP-25 immunofluorescence
in dura mater The image is representative of the data obtained from four animals per group Scale bar = 100μm
BJP
Trang 25dura could be reconstructed as follows: after peripheral
injec-tion, BoNT/A is taken up by sensory nerve endings and axonally
transported to trigeminal ganglion After transcytosis, the toxin
reaches dural nerve endings containing CGRP and suppresses
the CGRP-mediated sensitization of the trigeminovascular
system and DNI At present, this seems as the most convincing
hypothesis of the action of BoNT/A in migraine and other
headaches
Acknowledgements
The work was supported by grants from Croatian Ministry of
Science, Education and Sport (no 108-1080003-0001 awarded
to Z L.), Croatian National Science Foundation (no
O-1259-2015 awarded to Z L.), Hungarian National Brain Research
Pro-gram (SROP4.2.2.A-11/1/KONV-2012-0024 and the KTIA
NAP_13-1-2013-0001 awarded to Z H.) and National Brain
Re-search Program B (Chronic Pain Research Group;
KTIA_NAP_13–2014-0022; awarded to Z L., 888819) We thank
Mrs Teréz Bagoly for her excellent technical contribution to the
CGRP RIA and Prof Vladimir Trkulja for the suggestions
regard-ing the presentation of results Antibody to cleaved SNAP-25
was kindly provided by Assistant Prof Ornella Rossetto
(Univer-sity of Padua, Italy) The histological analysis was performed by
pathologist Prof Mara Dominis
Author contributions
Z L., B F and I M conceived and designed the study Z L.,
B F., I M and Z H analysed and interpreted the data Z L.,
B F., I M and Z H drafted the manuscript All authors have
approved thefinal version of the manuscript All authors are
accountable for all aspects of the work in ensuring that
ques-tions related to the accuracy or integrity of any part of the
work are appropriately investigated and resolved
Con flicts of interest
The authors declare no conflict of interest
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BJP
Trang 28S Saponara1, M Durante1, O Spiga2, P Mugnai1, G Sgaragli1, TT Huong3,
PN Khanh3, NT Son3, NM Cuong3and F Fusi1
1Dipartimento di Scienze della Vita, Università degli Studi di Siena, Siena, Italy,2Dipartimento di
Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, Italy, and3Institute of
Natural Products Chemistry, Vietnam Academy of Science and Technology, Hanoi, Vietnam
Correspondence
Dr Fabio Fusi, Dipartimento di Scienzedella Vita, Università degli Studi diSiena, via Aldo Moro 2, 53100 Siena,Italy
E-mail: fabio.fusi@unisi.it
Received
BACKGROUND AND PURPOSE
The carbazole alkaloid murrayafoline A (MuA) enhances contractility and the Ca2+currents carried by the Cav1.2 channels [ICa1.2]
of rat cardiomyocytes As only few drugs stimulate ICa1.2, this study was designed to analyse the effects of MuA on vascular Cav1.2channels
EXPERIMENTAL APPROACH
Vascular activity was assessed on rat aorta rings mounted in organ baths Cav1.2 Ba2+current [IBa1.2] was recorded in single rataorta and tail artery myocytes by the patch-clamp technique Docking at a 3D model of the rat,α1ccentral pore subunit of theCav1.2 channel was simulatedin silico
KEY RESULTS
In rat aorta rings MuA, at concentrations≤14.2 μM, increased 30 mM K+-induced tone and shifted the concentration-responsecurve to K+to the left Conversely, at concentrations>14.2 μM, it relaxed high K+depolarized rings and antagonized Bay K 8644-induced contraction In single myocytes, MuA stimulated IBa1.2in a concentration-dependent, bell-shaped manner; stimulationwas stable, incompletely reversible upon drug washout and accompanied by a leftward shift of the voltage-dependent activationcurve MuA docked at theα1Csubunit central pore differently from nifedipine and Bay K 8644, although apparently interactingwith the same amino acids of the pocket Neither Bay K 8644-induced stimulation nor nifedipine-induced block of IBa1.2wasmodified by MuA
CONCLUSIONS AND IMPLICATIONS
Murrayafoline A is a naturally occurring vasoactive agent able to modulate Cav1.2 channels and dock at theα1Csubunit centralpore in a manner that differed from that of dihydropyridines © 2015 The British Pharmacological Society
Abbreviations
IBa1.2, Cav1.2 channel Ba2+current; ICa1.2, Cav1.2 channel Ca2+current; MuA, murrayafoline A; PSS, modified
Krebs–Henseleit saline solution
Pharmacology
Trang 29Murrayafoline A (1-methoxy-3-methyl-9H-carbazole; MuA;
Figure 1) was isolated for the first time from Murraya
euchrestifolia Hayata (Rutaceae) collected in Taiwan and
identified as a carbazole alkaloid by Furukawa et al (1985)
Thereafter, MuA was isolated from the root of several species
of the genus Murraya (Itoigawa et al., 2000), Glycosmis
(Glycosmis pentaphylla (Retz.) DC and Glycosmis stenocarpa
(Drake) Guilt.; Bhattacharyya and Chowdhury, 1985; Cuong
et al., 2004) and Clausena (Clausena dunniana Levl.; Cui
et al., 2002) MuA exhibits strong fungicidal activity against
Cladosporium cucumerinum and possesses growth inhibitory
activity on humanfibrosarcoma HT-1080 cells as well as cell
cycle M-phase inhibitory and apoptosis-inducing activity on
mouse tsFT210 cells (Cui et al., 2002) Furthermore, this
compound provided thefirst example of a carbazole alkaloid
able to suppress growth of the human leukemia HL-60 cell
line by inducing apoptosis through the activation of the
caspase-9/caspase-3 pathway (Ito et al., 2012) MuA
attenu-ated the Wnt/β-catenin pathway by promoting the
degrada-tion of intracellular β-catenin proteins (Choi et al., 2010)
Because molecular lesions in Wnt/β-catenin signalling and
subsequent up-regulation ofβ-catenin response transcription
occur frequently during the development of colon cancer,
MuA has been proposed as a potential chemotherapeutic
agent in this type of cancer
In addition to being an interesting and promising drug
per se, MuA represents also a useful scaffold for the design and
development of novel drugs In fact, recent results indicate that
MuA derivatives containing a 1,2,3-triazole nucleus inhibit
the LPS-stimulated production of pro-inflammatory cytokines
(IL-6, IL-12 p40 and TNF-α) in bone marrow-derived dendritic
cells, thus representing potential anti-inflammatory drugs
(Thuy et al., 2013) Finally, this carbazole alkaloid can be totally
synthesized either from 5-methyl-2-nitrophenol, through a
four-step process using the organic palladium catalysts Pd
(OAc)2, Pd2(dba)3and Dave-phos (Toan et al., 2013), or directly
from 1,2,3,4-tetrahydrocarbazol-1-one (Chakraborty et al.,
2013)
MuA has been recently shown to enhance contractility
and increase Ca2+ influx in single rat ventricular myocytes
(Son et al., 2014), behaving like a stimulator of Cav1.2
channels Therefore, in view of its possible therapeutic use,
it would be interesting to know its effects on vascular
function To this end, an in-depth analysis of MuA effects
on rat vascular Cav1.2 channel was performed in vitro both
on intact vessels and single myocytes and in silico on α1csubunit pore model of the channel MuA was shown to exert
a bimodal effect on both aorta ring contractility and ICa1.2and docked at theα1C subunit central pore in a differentway from that of the dihydropyridines
Methods Aorta ring preparation
All animal care and experimental protocols conformed to theEuropean Union Guidelines for the Care and the Use ofLaboratory Animals (European Union Directive 2010/63/EU)and were approved by the Italian Department of Health (666/2015-PR) All studies involving animals are reported in accor-dance with the ARRIVE guidelines for reporting experimentsinvolving animals (Kilkenny et al., 2010; McGrath et al., 2010)
A total of 74 animals were used in the experiments describedhere Aorta rings (2 mm wide) were prepared from male Wistarrats (300–400 g; Charles River Italia, Calco, Italy), anaesthetized(i.p.) with a mixture of Ketavet® (30 mg·kg 1 ketamine;Intervet, Aprilia, Italy) and Xilor® (8 mg·kg 1xylazine; Bio 98,San Lazzaro, Italy), decapitated and exsanguinated The endo-thelium was removed by gently rubbing the lumen of the ringwith the curved tips of a forceps Contractile isometric tensionwas recorded as described elsewhere (Cuong et al., 2014).Control preparations were challenged with the drug vehicleonly
Effect of MuA and Bay K 8644 on aorta rings depolarized with high K+concentrations
The effects of MuA and Bay K 8644 on the contraction induced
by high K+ concentrations were assessed to determine theinvolvement of Cav1.2 channels in their vascular activity.Steady tension was evoked in rings by either 30 mM or
60 mM K+; thereafter, the drug under investigation was addedcumulatively At the end of each experiment, 10μM nifedipinefollowed by 100μM sodium nitroprusside were added to testmuscle functional integrity Muscle tension was evaluated as apercentage of the initial response to K+, taken as 100%
Effect of MuA on the concentration-response curve for K+of aorta rings
To study MuA-induced sensitization to K+, a cumulativeconcentration-response curve to K+was constructed in rings
These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawsonet al., 2014) and are permanently archived in theConcise Guide to PHARMACOLOGY 2013/14 (Alexanderet al., 2013)
Trang 30preincubated for 15 min with vehicle or drug Responses were
evaluated as percentage of the contraction induced by 0.3μM
phenylephrine in modified Krebs–Henseleit saline solution
(PSS; see below for composition), taken as 100%
Functional interaction between MuA and Bay K
8644
Any potential interaction between MuA and Bay K 8644 at
the Cav1.2 channel was assessed on depolarized rings Rings
were stimulated with 60 mM K+for 15 min and then washed
for 90 min with a Ca2+-free PSS containing 1 mM EGTA The
preparations were then challenged with 0.3μM phenylephrine
to deplete the intracellular Ca2+ stores The spasmogenicresponse to 3 mM Ca2+was assessed on rings depolarized with
Ca2+-free 60 mM K+PSS and preincubated for 30 min with thedrug or vehicle At the plateau of the Ca2+-induced contrac-tion, 10 nM Bay K 8644 followed by 100μM sodium nitro-prusside were added to test Cav1.2 channels as well assmooth muscle functional integrity The response wasevaluated as a percentage of the initial response to 60 mM K+,taken as 100%
Smooth muscle cell isolation procedure and whole-cell patch clamp recordings
Smooth muscle cells were freshly isolated from either the aorta,according to Zhao et al (2001), or the main tail artery (Mugnai
et al., 2014) Briefly, a 3 mm long thoracic section of aortawas incubated at 37°C in 2 mL of Ca2+-free external solution(see below) containing 20 mM taurine (prepared by replacingNaCl with equimolar taurine), 1 mg·mL 1bovine serum albu-min, 0.75 mg·mL 1papain and 1 mg·mL 1DL-dithiothreitol,and gently bubbled with a 95% O2–5% CO2 gas mixture for20–30 min After removing the adventitia, the aorta was cut intosmall pieces and transferred into a Ca2+-free external solutioncontaining 20 mM taurine, 1 mg·mL 1 collagenase (type XI)and 1 mg·mL 1hyaluronidase for 10 min at 37°C Single cellswere released by gentle trituration of minced, proteolysed tissue,through a Pasteur pipette, stored at 4°C in the Ca2+-free externalsolution containing 20 mM taurine and used on the same day ofthe preparation
Smooth muscle cells were freshly isolated from a 5 mmlong piece of main tail artery incubated at 37°C in 2 mL of
20 mM taurine and 0.1 mM Ca2+external solution ing 1 mg·mL 1 collagenase (type XI), 1 mg·mL 1 soybeantrypsin inhibitor and 1 mg·mL 1BSA, gently bubbled with a95% O2–5% CO2gas mixture, as previously described (Fusi
contain-et al., 2001) Cells, stored in 0.05 mM Ca2+external solutioncontaining 20 mM taurine and 0.5 mg·mL 1BSA at 4°C undernormal atmosphere, were used for experiments within 2 daysafter isolation (Mugnai et al., 2014)
Whole-cell patch-clamp recordings
Cells were continuously superfused with external solutioncontaining 0.1 mM Ca2+and 30 mM tetraethylammoniumusing a peristaltic pump (LKB 2132, Bromma, Sweden), at aflow rate of 400 μL·min 1
The conventional whole-cellpatch-clamp method (Hamill et al., 1981) was employed tovoltage-clamp smooth muscle cells, as previously described(Cuong et al., 2014; see also Supporting Information).Electrophysiological responses were assessed at room temper-ature (20–22°C)
IBa1.2 and ICa1.2 recordings
The current carried by the Cav1.2 channels, IBa1.2or ICa1.2,was always recorded in external solution containing 30 mMtetraethylammonium and 5 mM Ca2+or Ba2+(tail artery) or
10 mM Ba2+(aorta) Current was elicited with 250 ms clamppulses (0.067 Hz) to 0 mV from a Vhof 50 mV (tail artery) or
to 10 mV from a Vhof 80 mV (aorta) Data were collected oncethe current amplitude had been stabilized (usually 7–10 minafter the whole-cell configuration had been obtained) by
Figure 1
Effect of MuA on high K+-induced contraction of rat aorta rings (A)
Effect of the drug on rings depolarized with either 30 mM or
60 mM K+ Responses are shown as a percentage of the initial tension
induced by 30 mM or 60 mM K+, taken as 100% Data points are
mean ± SEM (n = 6) * P< 0.05, maximum effect at 60 mM K+
versusthat at 30 mM K+, Student’s t test for unpaired samples Inset: trace
(representative of six experiments) of responses to cumulative
concentrations of MuA added to a ring precontracted with 30 mM K+
The effect of 100μM sodium nitroprusside (SNP) is also shown (B)
Concentration-response curves for K+ in the absence (2.1 mM
DMSO) or presence of various concentrations of MuA Data points
are mean ± SEM (n = 12–14) and represent the percentage of the
response to 0.3 μM phenylephrine (phe), taken as 100% The
maximal responses to 80 mM K+, recorded under the four
experi-mental conditions, were not significantly different; one-wayANOVA
and Dunnett’s post hoc test Inset: chemical structure of MuA
Trang 31using pClamp 8.2.0.232 (Molecular Devices Corporation,
Sunnyvale, CA, USA) At this point, the various protocols
were performed as detailed below IBa1.2 and ICa1.2 did not
run down during the following 40 min under these
condi-tions (Fusi et al., 2012)
Steady-state inactivation and activation curves were
obtained as previously described (Mugnai et al., 2014; see also
Supporting Information)
K+ currents were blocked with 30 mM
tetraethyl-ammonium in the external solution and Cs+in the internal
solution Current values were corrected for leakage using
10μM nifedipine, which completely blocked IBa1.2and ICa1.2
Data analysis
Data are reported as mean ± SEM; n is the number of cells or
rings analysed (indicated in parentheses), isolated from at
least three animals Analysis of data was accomplished by
using pClamp 9.2.1.8 software (Molecular Devices
Corpora-tion) and GraphPad Prism version 5.04 (GraphPad Software
Inc., San Diego, CA, USA) The AUC, used as a cumulative
measurement of drug effect, was calculated to compare the
concentration-response curves recorded at 30 mM and
60 mM K+ The area was computed, using the trapezoid rule,
in units of the X axis multiplied by the units of the Yaxis
Statistical analyses and significance as measured by
one-way or repeated measures ANOVA (followed by either
Dunnett’s or Bonferroni’s post hoc test), one sample t test or
Student’s t test for paired or unpaired samples (two-tailed)
were obtained using GraphPad InStat version 3.06 (GraphPad
Software, USA) Post hoc tests were performed only when
ANOVAfound a significant value of F and no variance in
homo-geneity In all comparisons, P< 0.05 was considered
signifi-cant The pharmacological response to each substance was
described in terms of either pEC50or pIC50
Solutions and materials
The PSS contained (in mM): NaCl 118; KCl 4.75; KH2PO4
1.19; MgSO4.1.19; NaHCO325; glucose 11.5; CaCl2.2.5; gassed
with a 95% O2–5% CO2 gas mixture to a pH of 7.4 PSS
containing KCl at a concentration greater than 4.75 mM
was prepared by replacing NaCl with equimolar KCl
External solution contained (in mM): 130 NaCl, 5.6 KCl,
10 HEPES, 20 glucose, 1.2 MgCl2and 5 Na-pyruvate; pH 7.4
The internal solution contained (in mM) 100 CsCl, 10
HEPES, 11 EGTA, 1 CaCl2. (pCa 8.4), 2 MgCl2, 5 Na-pyruvate,
5 succinic acid, 5 oxalacetic acid, 3 Na2-ATP and 5
phospho-creatine; pH was adjusted to 7.4 with CsOH
The osmolarity of the tetraethylammonium- and Ca2+or
Ba2+-containing external solution (320 mosmol) and that of
the internal solution (290 mosmol; Stansfeld and Mathie,
1993) was measured with an osmometer (Osmostat OM
6020, Menarini Diagnostics, Florence, Italy)
Phenylephrine, ACh, collagenase (type XI), trypsin
inhib-itor, bovine serum albumin, papain, DL-dithiothreitol,
hyal-uronidase, tetraethylammonium chloride, EGTA, HEPES,
taurine, Bay K 8644 (methyl
(4S)-2,6-dimethyl-5-nitro-4-[2-
(trifluoromethyl)phenyl]-1,4-dihydropyridine-3-carboxyl-ate) and nifedipine were from Sigma Chimica (Milan, Italy);
sodium nitroprusside was from Riedel-De Hặn AG
(Seelze-Hannover, Germany) MuA was isolated from the
dried, powdered roots of Glycosmis stenocarpa (Drake) Guilt.,
as previously described (Cuong et al., 2004) MuA (473 mMstock solution), dissolved directly in DMSO, Bay K 8644and nifedipine, dissolved in ethanol, were diluted at least
1000 times prior to use All these solutions were stored at20°C and protected from light by wrapping containerswith aluminium foil The resulting concentrations of DMSOand ethanol (below 0.1%, v v 1) did not affect responses ofthe preparations Phenylephrine was dissolved in 0.1 MHCl Sodium nitroprusside was dissolved in distilled water.All other substances used were of analytical grade and usedwithout further purification
Docking experiments
Construction of the model The rat Cav1.2 channelα1Csubunitsequence (NP_036649.2) was retrieved from the NCBI ProteinDatabase (http://www.ncbi.nlm.nih.gov/protein/) This hasfour repeats, each containing six transmembrane helices(S1–S6) and a P-loop between S5 and S6 (Cheng et al., 2009).The quality of a homology model is given by the accuracy ofthe sequence alignment and the resolution of the templatestructures used A PSI-BLAST search (Altschul et al., 1997) forratα1C subunit sequences was firstly performed in order toobtain the best template of the unit and the tetrameric portion
of the model Subsequently, the sequences were aligned aspreviously reported (Zhorov and Tikhonov, 2004; Cheng et al.,2010) Here, the disposition of both P-loops and inner heliceswas derived from earlier structure templates Therefore,complete, suitable templates were the KvAP (1ORQ pdb) (Jiang
et al., 2003) and the KvAP (2R9R pdb) for the reconstruction ofthe unit and the tetramer respectively
When viewed from the extracellular side, the repeats I–IVwere arranged clockwise around the central pore (Cheng
et al., 2009) This channel model was built using theSwissPdbViewer-DeepView version 4.1 (Guex and Peitsch,1997), which allowed us also to define the consistency ofbond distances, bond angles and torsion angles with thevalues of standard proteins The structure of the channelmodel was energetically minimized using the Gromacs pack-age (Berendsen et al., 2012) equipped with the AMBER forcefield (Sorin and Pande, 2005) till a final convergence of0.01 kcal mol 1Å 1was achieved The stereochemical quality
of thefinal structure (i.e the distribution of ϕ and ψ angles)was assessed by means of PROCHECK program (Laskowski
et al., 1993) With this test, no severely disallowed atomiccontacts were detected, suggesting essentially good stereo-chemistry, with 86.1% and 11.0% of the amino acid residues
in the most favoured and additional allowed regions, tively, and with 2.1% and 0.8% residues in generouslyallowed and disallowed regions of the Ramachandran plot
respec-Docking simulations Docking of ligands (nifedipine, Bay K
8644 and MuA) was simulated by usingflexible side chainsprotocol with AutoDock/Vina version 1.1 (Trott and Olson,2010) This program used an iterated local search globaloptimizer algorithm based on a succession of steps, whichconsisted of mutation and local optimization Ligandstructures were retrieved from the PubChem database(http://www.ncbi.nlm.nih.gov/pcsubstance/), and pdbqtfiles were generated by using scripts included in the
Trang 32Molecular Graphics Laboratory (MGL) tools (Morris et al.,
2009) The generation and affinity grid maps, view
of docking poses and analysis of virtual screening
results were carried out by using AutoDock plug-in of
PyMOL The dimensions of the box for docking calculation
(60 Å × 60 Å × 60 Å) were sufficiently great to include not
only the active docking site, as previously suggested
(Cosconati et al., 2007), but also significant portions of the
surrounding surface
In silico alanine scanning mutagenesis was performed by
using the ABS-Scan tool 2 (Anand et al., 2014) Each amino
acid residue present at the binding site was computationally
mutated to alanine and the ligand interaction energy
recalculated for each mutant The correspondingΔΔG values
were computed by comparing them with the wild type
protein, thus allowing the evaluation of individual residue
contribution towards ligand interaction
Results
Effect of MuA on aorta rings contracted by high
K+concentrations
To determine the involvement of Cav1.2 channels in the
vascular activity of MuA, its effect was evaluated on the
contraction induced by both 30 mM and 60 mM K+in aorta
rings As shown in Figure 1A, MuA caused a
concentration-dependent relaxation of the preparations Rings contracted
by 60 mM K+ relaxed fully in the presence of 473μM MuA
with a pIC50value of 4.22 ± 0.13 (n = 6) and an AUC value
of 109.6 ± 13.0 Furthermore, maximal relaxation was
significantly greater than that recorded in preparations
contracted by phenylephrine (see Supporting Information
Fig S1; P< 0.05, Student’s t test for unpaired samples) When
rings were depolarized with 30 mM K+, the
concentration-response curve was shifted upward (Figure 1A), showing an
AUC value of 180.5 ± 25.3 (n = 6; P< 0.05 Student’s t test
for unpaired samples) MuA, at concentrations ≤47.3 μM,
caused an increase in K+-induced vascular tone while at
higher concentrations, partly reversed the contraction,
sho-wing a relative pIC50value of 4.07 ± 0.10μM (n = 6)
Murrayafoline A potentiated, in a concentration-dependent
manner, the contractile response to K+(Figure 1B) causing a
left-ward shift of the K+concentration-response curve pEC50values
for K+changed from 1.61 ± 0.04 (2.1 mM DMSO; n = 12) to 1.67
± 0.04 (1.4μM MuA, n = 13; P > 0.05, Dunnett’s post hoc test),
1.78 ± 0.04 (4.7μM MuA, n = 14; P < 0.05) and 1.80 ± 0.05
(14.2 μM MuA, n = 14; P < 0.05) Potentiation of responses
to K+by 14.2μM MuAwas greater at 15 mM K+
, being 409.6%
of control, as compared with that observed at higher K+
con-centrations (157.0% and 124.3% at 30 mM and 60 mM K+
respectively) MuA, however, did not modify the maximal
response to K+
Effect of Bay K 8644 on aorta rings contracted
by high K+concentrations and its interaction
with MuA
To define the effects of MuA on aorta rings, the same
exper-iments described earlier were repeated, using the Cav1.2
channel agonist Bay K 8644 instead of MuA When the effect
of Bay K 8644 on the contraction induced by high K+concentrations was evaluated in aorta rings, a concentration-dependent and marked increase of muscle tone in preparationscontracted by 30 mM K+ was observed, whilst in ringscontracted by 60 mM K+, this was considerably smaller(Figure 2A) The AUC values were 214.3 ± 36.7 (n = 7) and91.1 ± 20.6 (n = 6; P < 0.05 Student’s t test for unpairedsamples) respectively
Any potential pharmacological interactions betweenMuA and Bay K 8644 were assessed in rings contracted
by the addition of 3 mM Ca2+ to the Ca2+-free, 60 mM
K+-containing PSS In rings pre-treated with DMSO, 10 nMBay K 8644 increased Ca2+-induced contraction by about40% (Figure 2B) MuA, at concentrations of 47.3μM and
Figure 2
Effect of Bay K 8644 on high K+-induced contraction of rat aorta ringsand its functional interaction with MuA (A) Concentration-responsecurves of Bay K 8644 in rings depolarized with 30 mM or 60 mM K+.Responses are shown as percentage of the initial tension induced by
30 mM or 60 mM K+, taken as 100% Data points are mean ± SEM(n = 6–7) * P < 0.05 maximum effect at 30 mM K+
versus that at
60 mM K+; Student’s t test for unpaired samples (B) Effect of
10 nM Bay K 8644 on Ca2+-induced vascular tone of depolarized ringstreated with either vehicle (DMSO) or MuA Columns are mean ±SEM (n = 7–10) and represent the percentage of the response to
60 mM K+, taken as 100% * P< 0.05 versus control; Student’s t testfor paired samples;#P< 0.05 versus DMSO + Bay K 8644; one-way
ANOVAand Dunnett’s post hoc test
Trang 33142μM, significantly antagonized the Bay K 8644-induced
increase
Ca2+influx stimulated by Cav1.2 channel agonists may be
completely buffered by the superficial sarcoplasmic reticulum
or even prevented if channels are not pre-activated with low
K+concentrations However, addition of MuA to rings either
bathed in normal PSS or pre-treated with 1μM thapsigargin
or 15 mM K+ failed to induce mechanical responses (data
not shown) When this assay was performed with Bay K
8644, in normal PSS, a concentration-dependent contraction
was recorded in seven out of 17 preparations (pEC50value of
7.53 ± 0.24, n = 7) In rings pre-incubated with 15 mM K+or
1 μM thapsigargin, the concentration-response curves to
Bay K 8644 shifted to the left (pEC50value of 8.33 ± 0.19,
n = 11, P> 0.05 and 9.13 ± 0.41, n = 6, P < 0.05, Dunnett’s
post hoc test) In the former case (15 mM K+), an increase in
drug efficacy was also observed (data not shown)
Effect of MuA on IBa1.2and ICa1.2
The contribution of Cav1.2 channel modulation to the
effects of MuA on vascular rings was assessed on IBa1.2
recorded in isolated aorta myocytes At Vh of 80 mV,
MuA stimulated the current in a concentration-dependent
manner with a pEC50value of 5.33 ± 0.08 (n = 7) (Figure 3A)
At 47.3μM this stimulatory effect was less evident, whilst at
473.4 μM, MuA clearly inhibited IBa1.2 Similar results
(pEC50value of 5.26 ± 0.10, n = 7; P> 0.05) were obtained
in tail artery myocytes Therefore, an in-depth analysis of
MuA effects on IBa1.2 was performed on the latter cells,
whose biophysical and pharmacological properties are well
characterized (Mugnai et al., 2014 and references therein)
MuA modulation of the current through Cav1.2 channels
was not affected by changes of either the charge carrier or
Vh In fact, when equimolar Ca2+replaced Ba2+in the
exter-nal solution or when Vh was changed to 50 mV, the
stimulatory potency was not modified (pEC505.60 ± 0.12,
n = 5 and 5.44 ± 0.03, n = 9, respectively; P> 0.05)
then declined with time courses that could be fitted by a
mono-exponential function MuA did not affect significantly
either the τ for inactivation or that for activation at all
concentrations tested (data not shown)
Figure 3B shows the time course of the effects of MuA on
0 mV After the current had reached steady values, the addition
to bath solution of 14.2μM MuA produced a gradual increase
of the current that reached a plateau in about 4 min Current
amplitude stimulation was only partially reversible upon
drug washout but still blocked by the Ca2+antagonist nifedipine
Furthermore, MuA-induced stimulation of IBa1.2was stable for
about 30 min
The current–voltage relationships recorded at Vhof 50 mV
(Figure 4A) show that 14.2 μM MuA significantly increased
the peak IBa1.2without altering either the maximum at 10 mV
or the threshold at approximately 30 mV The relative
value of IBa1.2 stimulation by MuA (Figure 4, inset) was
constant in the range of membrane potential values of 20
to 30 mV In addition, drug washout partially reversed
MuA-induced stimulation of IBa1.2 at most of the tested
(see schematic diagram), measured in the absence (control) orpresence of various concentrations (μM) of MuA (B) Time course
(14.2μM) was applied at the time indicated by the arrow, andcurrent was recorded during a typical depolarization from 50 to
0 mV, applied every 15 s (0.067 Hz) and subsequently normalized
to the current recorded just prior to MuA addition Drug washoutallowed for partial recovery from stimulation IBa1.2 suppression
by 10μM nifedipine is also shown Data points are mean ± SEM(n = 7–9) Inset: average traces (recorded from seven cells) ofconventional whole-cell IBa1.2elicited with 250 ms clamp pulses
to 0 mV from a Vhof 50 mV, recorded in the absence (control)
or presence of 14.2μM MuA as well as after drug washout
Trang 34Student’s t test for paired samples) and the slope factor ( 7.57
± 0.16 mV and 7.62 ± 0.64 mV; P> 0.05) of the steady-state
inactivation curve (Figure 4B)
The activation curves, calculated from the current–
voltage relationships showed in Figure 4A, werefitted to the
Boltzmann equation MuA significantly decreased the 50%
activation potential ( 6.65 ± 1.24 mV, control and 9.47 ±
1.18 mV, MuA, n = 6; P< 0.05, repeated measuresANOVAand
Dunnett’s post hoc test) without changing the slope factor
(6.65 ± 0.51 mVand 6.32 ± 0.32 mV; Figure 4B) Drug washoutfully reversed this effect (50% activation potential 7.07 ±1.08 mVand slope factor 6.14 ± 0.28 mV; P> 0.05)
Modelling and docking
To determine in silico the interaction of MuA with the Cav1.2channel protein, the homology model of the central pore ofthe ratα1csubunit was reconstructed in accordance with Cheng
et al (2009) Docking calculations performed with the referencedihydropyridines, nifedipine and Bay K 8644, showed similar
ΔG values (Table 1) Moreover, the two molecules positionedinside the pocket in a superimposable manner (Figure 5A) Byinteracting with specific parts of the channel pore, nifedipineand Bay K 8644 formed H-bonds with the same keydihydropyridine-sensing amino acid residues of three differenthelices (Figure 5B): Tyr1178 (IIIS6) with the NH group andGln1069 (IIIS5) and Tyr1489(IVS5) with the COOCH3 groups
On the contrary, MuA, which showed a less favourableΔG value(Table 1), had a different pose (Figure 5A) characterized by theabsence of H-bonds (Figure 5B)
In silico alanine scanning mutagenesis gave rise to ably similarΔΔG values for both nifedipine and Bay K 8644(Figure 5C), whereas MuA exhibited a rather different profile,some residues even appearing unfavourable to its binding
remark-Functional interaction between MuA and Bay K
8644 or nifedipine on IBa1.2
Because docking analysis suggested that MuA binds the Cav1.2channel binding pocket at a site that can bind also nifedipineand Bay K 8644, the functional interaction between thisalkaloid and the two dihydropyridines was investigated Bay K
8644 (100 nM) stimulated IBa1.2in the range from 30 to 50 mVand shifted the maximum of the current–voltage relationships
by 10 mV in the hyperpolarizing direction (Figure 6A) Theactivation curves, calculated from the current–voltagerelationships shown in Figure 6A, werefitted to the Boltzmannequation Bay K 8644 significantly decreased the 50% activa-tion potential ( 5.72 ± 0.72 mV, control and 13.95 ± 0.82 mV,Bay K 8644, n = 6; P< 0.05, repeated measuresANOVA andDunnett’s post hoc test) and changed the slope factor (from6.22 ± 0.30 mV to 4.67 ± 0.26 mV; P< 0.05) (Figure 6A inset).The subsequent addition of 14.2μM MuA did not modifyBay K 8644-induced effects on both IBa1.2 amplitude andactivation curve (50% activation potential 13.71 ± 0.57 mVand slope factor 5.20 ± 0.20 mV; P> 0.05)
Under control conditions, the current evoked at 0 mVfrom a Vhof 50 mV activated and then declined with timecourses that could befitted by mono-exponential functions(Figure 6B) Bay K 8644 (100 nM) significantly prolongedtheτ for activation and reduced that for inactivation: the sub-sequent addition of MuA brought only theτ for activationalmost to control values, without affecting that forinactivation
The antagonistic effect of nifedipine was determined undercontrol conditions as well as in myocytes pre-treated withMuA or Bay K 8644 Nifedipine inhibited IBa1.2 in aconcentration-dependent manner with a pIC50value of 7.67 ±0.06 (n = 6; Figure 7A,D) Similar results were obtained inthe presence of 14.2μM MuA (pIC50 value of 7.60 ± 0.10,
n = 6; P> 0.05, one-wayANOVAand Dunnett’s post hoc test;
Figure 4
Effect of MuA on IBa1.2–voltage relationship as well as on IBa1.2
activa-tion and inactivaactiva-tion curves (A) Effect of MuA on the current–
voltage relationship Current–voltage relationships, recorded from a
Vhof 50 mV, constructed prior to the addition of drug (control), in
presence of 14.2μM MuA and after drug wash out Data points are
mean ± SEM (n = 7) * P< 0.05 versus control.#
P< 0.05 versusMuA; repeated measuresANOVAand Bonferroni’s post hoc test Inset:
relationship between membrane potential and relative value of IBa1.2
stimulation by 14.2μM MuA Current stimulation was expressed as a
fold increase over the peak amplitude of IBa1.2evoked, in the absence
of MuA, by varying the amplitude of depolarizing pulse Data points
are mean ± SEM (n = 7) P> 0.05; one-wayANOVAand Bonferroni’s
post hoc test (B) Steady-state inactivation curves recorded from Vhof
50 mV, obtained in the absence (control) and presence of 14.2μM MuA,
werefitted to the Boltzmann equation The current measured during
the test pulse was plotted against membrane potential and expressed
as relative amplitude Activation curves obtained from the current–
voltage relationships of panel A andfitted to the Boltzmann equation
Data points are mean ± SEM (n = 6–7)
Trang 35Figure 5
Docking of murrayafoline A, nifedipine and Bay K 8644 at the Cav1.2 channelα1Csubunit model and effect of alanine scanning mutagenesis (A)Docked structures of nifedipine (yellow), MuA (blue) and Bay K 8644 (red), displayed as bold sticks, in the pore channel, displayed as molecularsurface coloured grey (B) Best docking poses of the three ligands; the pore amino acid residues [Gln1069 (IIIS5), Tyr1178 (IIIS6) and Tyr1489(IVS5)] forming H-bonds with nifedipine and Bay K 8644 are displayed in magenta (C) ABS-scan energy ploy.ΔΔG values recorded after alaninemutation of the single residues involved in the binding of nifedipine, Bay K 8644 and MuA Amino acid residues are listed in rank order according
to their contribution in the complex with nifedipine and Bay K 8644 (ΔΔG values)
Table 1
Murrayafoline A, nifedipine and Bay K 8644 docking at the channel pore of rat Cav1.2α1Csubunit
Molecule ΔGbind Surrounding amino acid residues
Murrayafoline A
(C14H13NO)
6.9 Ser1141 (IIIP), Gln1069 (IIIS5), Tyr1489 (IVS6), Ile1486 (IVS6), Met1490 (IVS6), Met1186 (IIIS6),Phe1138 (IIIP), Ile1182 (IIIS6), Thr1066 (IIIS5), Leu1137 (IIIP), Thr1142 (IIIP), Tyr1178 (IIIS6),Met1134 (IIIP), Ile1189 (IIIS6), Phe1070 (IIIS5), Phe1485 (IVS6)
Trang 36Figure 7B,D) On the contrary, when IBa1.2 was stimulated
with 100 nM Bay K 8644, the concentration-response curve
to nifedipine was shifted to the right (pIC50value of 6.42 ±
0.08, n = 7; P< 0.05; Figure 7C,D)
Discussion
Murrayafoline A has been shown recently to enhance
contractility and increase Ca2+ influx in single rat
ventricular myocytes (Son et al., 2014), behaving like astimulator of Cav1.2 channels However, its effects onvascular function are unknown The present investigationdemonstrated that MuA was able either to stimulate or toinhibit contraction of vascular smooth muscle by directlyactivating or blocking Cav1.2 channels, respectively, de-pending on the concentration used This conclusion issupported not only by indirect, functional observationsbut also by direct electrophysiological data and dockinganalysis
Figure 6
Effects of Bay K 8644 and murrayafoline A on IBa1.2–voltage relationship and kinetics in rat tail artery myocytes (A) Current–voltage relationshipsconstructed prior to the addition of drugs (control), in the presence of 100 nM Bay K 8644 and in the presence of Bay K 8644 plus 14.2μM MuA.Data points are mean ± SEM (n = 6) * P< 0.05 versus control, P > 0.05 Bay K 8644 versus +murrayafoline A, repeated measuresANOVAandBonferroni’s post hoc test Inset: steady-state activation curves obtained from the current–voltage relationships of panel A and fitted to theBoltzmann equation (see Methods section) (B) Average traces (recorded from six cells) of conventional whole-cell IBa1.2elicited with 250 msclamp pulses to 0 mV from a Vhof 50 mV, recorded in the absence (control) or presence of 100 nM Bay K 8644 (Bay) and Bay K 8644 plus14.2μM MuA (Bay + mur) Control and Bay K 8644 plus MuA traces are magnified so that the peak amplitude matched that of Bay K 8644 Inset:time constant for activation (τact) and for inactivation (τinact) measured in the absence (control) or presence of Bay K 8644 (Bay) and Bay K 8644plus MuA Columns represent mean ± SEM (n = 6) * P< 0.05 versus control, repeated measuresANOVAand Bonferroni’s post hoc test
Trang 37The mechanical and electrophysiological effects of MuA
were compared with those of the synthetic Cav1.2 channel
activator Bay K 8644 (Hess et al., 1984) Both vascular
smooth muscle active tone and IBa1.2 stimulation induced
by MuA shared some basic features with those sustained
by Bay K 8644 Thus, MuA, like Bay K 8644, stimulated the
active tone of aorta rings depolarized with 30 mM K+, this
effect disappearing when K+ concentration raised up to
60 mM (i.e in fully activated preparations) Furthermore,
it shifted the K+ concentration-response curve to the left
without changing its maximum (for Bay K 8644, see
Fusi et al., 2003) Finally, at low concentrations, both drugs
stimulated IBa1.2 in a nifedipine-sensitive manner
Collectively, thesefindings suggest that MuA, like Bay K 8644,
affected the vascular Cav1.2 channel protein
Murrayafoline A, added either before or after K+, enhanced
tissue responses to low, but not to high depolarizing stimuli
‘Sensitization’ to K+
is generally observed with drugs, like Bay
K 8644, that facilitate the voltage-dependent activation of
Cav1.2 channels (this paper), thus shifting the curve relating
tension development and depolarizing stimulus (i.e membrane
potential) to lower K+concentrations (i.e more negative values;
see Fusi et al., 2003) This hypothesis was confirmed by the
Boltzmann analysis (activation curve) of the current–voltage
relationship, showing that MuA, similarly to Bay K 8644,
signif-icantly decreased the 50% activation potential of IBa1.2
Murrayafoline A caused a parallel leftward shift of the K+
concentration-response curve as well as a relatively constant
stimulation of current amplitude over a broad range of
membrane potentials These data suggested that the drug
most likely increased the open probability of the channel
and that its action on the channel was voltage-independent
However, only single-channel recordings comparing theeffect of Bay K 8644 and MuA may provide direct evidencefor this possibility
Ca2+channel activators, such as Bay K 8644, are able toevoke full contractile, tonic responses in vascular smooth mus-cle preparations, mainly when they are depolarized with low
K+concentrations (this paper; Usowicz et al., 1995; Fusi et al.,2003) or when the Ca2+ buffering activity of the superficialsarcoplasmic reticulum is impaired (this paper; Asano andNomura, 1999) This means that, on one hand, Cav1.2 channelactivation is voltage-dependent, and therefore, channels have
to be activated in order to respond to Ca2+-agonist drugs Onthe other hand, Ca2+influx triggered by the Ca2+
-agonist drugcan induce a maximum muscle contraction only in the absence
of a functional sarcoplasmic reticulum MuA, however, did notelicit significant mechanical responses in rat aorta rings eitherunder control conditions or in the presence of thapsigargin ormoderate concentrations of K+, thus suggesting that its potencyand efficacy were much lower than those of Bay K 8644 Finally,Bay K 8644, at variance with MuA, affected IBa1.2kinetics andstimulated IBa1.2 maximally at weak depolarization values,causing a leftward shift in the maximum of the current–voltagerelationship (Wang et al., 1989; McDonald et al., 1994; Saponara
et al., 2008; this paper)
When used at high concentrations, MuA acted mostly as a
Ca2+channel blocker Several pieces of evidence concur to thisconclusion First, MuA reversed the contraction induced byhigh K+, in agreement with data already published by Wu et al.(1998), this relaxant effect becoming more pronounced at higherdepolarization levels (i.e depending on membrane potential;Bean, 1984; Kuriyama et al., 1995) Vasorelaxation induced by
Ca2+channel blockers is directly correlated to the extracellular
Figure 7
Effects of nifedipine on Bay K 8644- or murrayafoline A-induced stimulation of IBa1.2in rat tail artery myocytes (A–C) Average traces (recordedfrom six to seven cells) of conventional whole-cell IBa1.2elicited with 250 ms clamp pulses to 0 mV from a Vhof 50 mV and recorded afterthe addition of cumulative concentrations (10 nM-1μM) of nifedipine (A) in the absence (control) or presence of (B) 14.2 μM MuA and(C) 100 nM Bay K 8644 (D) Amplitude of the current normalized upon that recorded under control conditions and in the presence of eitherMuA or Bay K 8644, taken as 100% The curves show the bestfit of the points Data points are mean ± SEM (n = 6–7)
Trang 38concentration of K+, as it is the case with nifedipine, whose
po-tency increases as the membrane voltage (i.e the concentration
of extracellular K+) increases (McDonald et al., 1994) Second,
MuA-induced relaxation was lower when phenylephrine,
instead of a high concentration of K+, was employed to contract
the vessel, in line with the observation that Cav1.2 channels
play only a secondary role in this type of contraction
(McFadzean and Gibson, 2002), as observed with nifedipine
(Gurney, 1994) Third, it inhibited IBa1.2 in single myocytes
isolated from both aorta and tail artery Because this
effect was observed independently of the charge carrier used,
a Ca2+-dependent inactivation of the channel subsequent to
current stimulation can be ruled out
In the computational study, where a model of the α1C
subunit central pore region was reconstructed to perform
in silico molecular docking analysis, Bay K 8644, nifedipine
and MuA showed favourable free-energy binding values In
par-ticular, those related to dihydropyridines are in agreement with
already published data (Cosconati et al., 2007; Tikhonov and
Zhorov, 2009; Senatore et al., 2011), thus confirming the validity
of the model constructed The two dihydropyridines used in
this study showed similarΔG values and fitted inside the pocket
stabilizing the channel conformation by forming H-bonds with
key sensing amino acid residues, as previously established by
Zhorov (2013) Conversely, MuA lacked the H-bonds formed
by the dihydropyridines and showed a less favourableΔG value
In agreement with these data, the in silico alanine scanning
mutagenesis showed that theΔΔG profile shared by nifedipine
and Bay K 8644 was not reproduced for MuA, supporting the
hypothesis that MuA and the dihydropyridines shared only
some amino acid residues when they docked in the pocket of
the channel central pore unit These results corroborate those
obtained in vitro On one hand they clearly point to MuA as a
novel ligand of Cav1.2 channels, able to (i) bind as the reference,
dihydropyridine ligands to the pore-formingα1Csubunit; (ii)
prevent Bay K 8644-induced facilitation of extracellular Ca2+
influx; and (iii) partly reverse the effects of Bay K 8644 on
current kinetics On the other hand, the less favourablefit of
MuA within the pocket might explain why the drug (i) failed
to antagonize nifedipine blockade of the current, unlike Bay K
8644; (ii) displayed a low potency and efficacy; (iii) did not affect
two orders of magnitude higher; and (iv) was not characterized
by a voltage-dependence as well as a sigmoidal
concentration-dependence
When the effects of MuA on vascular and cardiac (Son et al.,
2014) Cav1.2 channels are compared, interesting similarities
emerge In both tissues, in fact, MuA induced a
concentration-dependent, nifedipine-sensitive stimulation of IBa1.2, without
altering the current kinetics Additionally, current stimulation
was bell-shaped, although the highest concentration assessed
in cardiomyocytes was only 200μM, that is, 2.5-fold lower than
that tested in vascular myocytes On the contrary, vascular
prep-arations seemed to be more sensitive to MuA as the pEC50value
was one order of magnitude lower than that recorded in
cardiomyocytes Finally, in cardiomyocytes, MuA stimulation
of Ca2+sparks and Ca2+transients depends on PKC activation
(Kim et al., 2015), while its modulatory activity on rat tail artery
Cav1.2 channel, where PKC plays a stimulatory role (Navedo
et al., 2005), was not significantly affected by the PKC inhibitors
GF109203X and Gö6976 (Supporting Information Fig S2) This
finding once more suggests that MuA might directly activate the
Cav1.2 channel in vascular myocytes
In conclusion, the presentfindings show that the zole alkaloid MuA can be included among the molecules ofnatural origin capable of modulating the voltage-dependent
carba-Cav1.2 channel in vascular as well as in cardiac (Son et al.,2014) myocytes, by docking at theα1Csubunit central pore
in a different way from that of the dihydropyridines.Vietnamese medicinal plants represent a valuable source forthe discovery of novel pharmacological agents that can beuseful in the analysis of the basic structure and function of
Cav1.2 channels
Acknowledgements
This work was supported by the National Foundation forScience and Technology Development of Vietnam (NAFOSTED;grant No 104.01-2010.25) and by the Ministero degli AffariEsteri (Rome, Italy), as stipulated by Law 212 (26-2-1992), tothe project ‘Discovery of novel cardiovascular active agentsfrom selected Vietnamese medicinal plants’ Miriam Duranteand Paolo Mugnai received a personal PhD scholarship fromthe University of Siena We wish to thank Dr M Lenoci for theassistance in some preliminary experiments
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Supporting Information
Additional Supporting Information may be found in the line version of this article at the publisher’s web-site:http://dx.doi.org/10.1111/bph.13369
on-Figure S1 Effect of murrayafoline A on induced contraction of rat aorta rings Concentration-responsecurves of MuA in endothelium-denuded rings precontracted
phenylephrine-by 0.3 M phenylephrine In the ordinate scale, response isreported as percentage of the initial tension induced by phen-ylephrine (phe), taken as 100% Data points are mean ± SEM(n = 6)
Figure S2Murrayafoline A modulation of Ba1.2 of single rattail artery myocytes Effect of GF109203X and Gö6976 onMuA-induced modulation of Ba1.2 Concentration-dependenteffect of MuA measured at Vh of 50 mV in the absence(control) or presence of either 5μM GF109203X or 100 nMGö6976 On the ordinate scale, response is given as a percentage
of control Data points are mean ± SEM (n = 5–9) * P < 0.05 vs.control (100%), one sample t test