Protein expression of Ca-SR in isolated rat cardiac myocytes To confirm that Ca-SR was expressed in cardiac myocytes, rather than neuronal or other types of cell in heart tissue, ventricu
Trang 1Calcium and polyamine regulated calcium-sensing receptors
in cardiac tissues
Rui Wang1, Changqing Xu2, Weimin Zhao1, Jing Zhang1, Kun Cao1, Baofeng Yang2and Lingyun Wu3 1
Department of Physiology, University of Saskatchewan, Saskatoon, SK, Canada;2Department of Pathophysiology, Harbin Medical University, Harbin, P.R China;3Department of Pharmacology, University of Saskatchewan, Saskatoon, SK, Canada
Activation of a calcium-sensing receptor (Ca-SR) leads to
increased intracellular calcium concentration and altered
cellular activities The expression of Ca-SR has been
iden-tified in both nonexcitable and excitable cells, including
neurons and smooth muscle cells Whether Ca-SR was
expressed and functioning in cardiac myocytes remained
unclear In the present study, the transcripts of Ca-SR were
identified in rat heart tissues using RT-PCR that was further
confirmed by sequence analysis Ca-SR proteins were
detected in rat ventricular and atrial tissues as well as in
isolated cardiac myocytes Anti-(Ca-SR) Ig did not detect
any specific bands after preadsorption with standard Ca-SR
antigens An immunohistochemistry study revealed the
presence of Ca-SR in rat cardiac as well as other tissues An
increase in extracellular calcium or gadolinium induced a
concentration-dependent sustained increase in [Ca2+]i in
isolated ventricular myocytes from adult rats Spermine (1–10 mM) also increased [Ca2+]i Pre-treatment of cardiac myocytes with thapsigargin or U73122 abolished the extra-cellular calcium, gadolinium or spermine-induced increase in [Ca2+]i The blockade of Na+/Ca2+exchanger or voltage-dependent calcium channels did not alter the extracellular calcium-induced increase in [Ca2+]i Finally, extracellular calcium, gadolinium and spermine all increased intracellular inositol 1,4,5-triphosphate (IP3) levels Our results demon-strated that Ca-SR was expressed in cardiac tissue and car-diomyocytes and its function was regulated by extracellular calcium and spermine
Keywords: calcium-sensing receptor; heart; IP3; RT-PCR; spermine
Calcium ions were the first identified endogenous substance
to function as both a first and second messenger via the
stimulation of an extracellular calcium sensing receptor
(Ca-SR) The binding of extracellular calcium (first messenger) to
Ca-SR in plasma membrane activates Gqproteins, stimulates
phospholipase C (PLC)-b activity, and increases intracellular
IP3 levels, leading to intracellular calcium release (second
messenger) [1,2] The expression of Ca-SR has been identified
in parathyroid [2], thyroid [3], kidney [4], bone [5] and GI
tract [6], the organs involved in systemic calcium
home-ostasis Defective Ca-SRs are involved in genetic diseases
linked to calcium homeostasis Ca-SR and its isoforms or
homologous receptors may represent novel clinical targets
for treatment of these diseases and others like osteoporosis
Calcium handling is essential for the homeostatic control
of cardiovascular functions, which may not couple directly
to systemic calcium homeostasis Whether Ca-SR has a
functional role to play in the cardiovascular system is
unclear Ca-SR proteins were detected, but not the
corres-ponding transcripts, in mesenteric resistant artery tissues [7]
This observation led to the conclusion that Ca-SR was actually expressed in perivascular nerves with the corres-ponding mRNA residing in the neuronal soma away from the isolated blood vessel wall Interestingly, a recent study claimed that Ca-SR was present in smooth muscle cells
of spiral modiolar artery of gerbils [8] The functions of Ca-SR in these smooth muscle cells were not studied Cardiac tissue is very sensitive to calcium homeostasis
An increased intracellular calcium concentration, either due
to the increased extracellular calcium entry through voltage-gated calcium channels or the increased intracellular calcium release, would trigger the contraction of myocytes Overloading of cellular calcium, on the other hand, leads to cell death and heart injury To date, the expression of Ca-SR
in cardiomyocytes had not been reported, less alone the function of these receptors Several lines of evidence are presented in this communication that demonstrate the existence of Ca-SR in rat heart by identifying the mRNA and proteins of Ca-SR in cardiac tissues and by delineating the functional regulation of Ca-SR in cardiac myocytes Ca-SR may present itself as a novel target by which the cardiac functions can be modulated
Materials and methods
RT-PCR analysis of the Ca-SR Male Sprague–Dawley rats (10–12 weeks old) were used with an approved protocol (University Committee on Animal Care and Supply of University of Saskatchewan)
Correspondence to: R Wang, Department of Physiology,
University of Saskatchewan, Saskatoon, SK, Canada S7N 5E5.
Fax: + 1 306 966 6532, Tel.: + 1 306 966 6592,
E-mail: wangrui@duke.usask.ca
Abbreviations: Ca-SR, calcium-sensing receptor; IP 3 , inositol
1,4,5-triphosphate; TG, Thapsigargin.
(Received 9 March 2003, revised 10 April 2003,
accepted 30 April 2003)
Trang 2Total RNA was extracted from isolated tissue with an
RNeasy Total RNA Kit (Qiagen) and treated with
RNase-free DNase I (Ambion) First-strand cDNA was made by
reverse transcribing 2 lg of DNase I-treated total RNA
with MMuLV reverse transcriptase (Perkin-Elmer) using
random hexamers in a total volume of 20 lL The RT
reaction was carried out at room temperature for 15 min
followed by incubation at 42C for 1 h Five microliters of
RT reaction mixture were used for PCR amplification in a
volume of 50 lL using Advanced PCR II mixer (Clontech)
with gene specific primers designed on reported sequences of
rat Ca-SR (GenBank accession number U20289) Portions
of the Ca-SR cDNA were amplified using the primer pairs
that were Ca-SR (forward), 5¢-ttcggcatcagctttgtg-3¢; and
Ca-SR (reverse), 5¢-tgaagatgatttcgtcttcc-3¢ PCR
amplifica-tion consisted of 35 cycles of denaturaamplifica-tion at 94C for 20 s,
annealing at 60C for 20 s, and polymerization at 68 C for
30 s Aliquots (5 lL) of PCR reactions were
electropho-resed through ethidium bromide-stained 1.2% agarose gels
Nucleotide sequence analysis
Gel purified PCR-amplified Ca-SR products were cloned
into the pCR2.1 TA cloning vector (Invitrogen) The
automated sequence analysis was performed on three
independent clones using an ABI-373A (Applied
Biosys-tems Inc.) sequencer
Western blot analysis
Membrane proteins were prepared as described previously
[9] Briefly, tissues were homogenized with a Polytron
homogenizer in 1 mL Tris-buffered saline (10 mM Tris,
0.3M sucrose and 1 mM EDTA) containing protease
inhibitor mixture [9] The homogenate was centrifuged at
6000 g for 15 min at 4C to remove nuclei and undisrupted
cells The supernatant was further centrifuged at 40 000 g
for 1 h at 4C Resulting pellets were then washed and
resuspended with the same Tris-buffered saline without
sucrose Protein concentration was determined using a
Bio-Rad protein assay solution with BSA as standard
Membrane proteins (20 lg) were electrophoresed through
standard 10% SDS-PAGE in Tris-glycine electrophoresis
buffer [125 mM Tris, 959 mMglycine (pH 8.3), and 0.5%
SDS] and blotted onto nitrocellulose membrane in
transfer-ring buffer [39 mMglycine, 48 mMTris (pH 8.3) and 20%
methanol] at 80 mA for 1.5 h in a water-cooled transfer
apparatus The membrane was blocked in a blocking buffer
NaCl/Picontaining 3% skimmed milk at room temperature
for 2 h The membrane was then incubated overnight at 4C
with 1 : 500 diluted affinity-purified polyclonal antibody
against Ca-SR in blocking buffer Unless otherwise specified,
anti-(Ca-SR) Igs were from Alpha Diagnostic International
Inc (San Antonio, TX, USA)
After the membrane was washed five times in NaCl/Pi, it
was incubated with goat anti-(rabbit IgG) Ig conjugated
with horseradish peroxidase diluted to 1 : 5000 in the
blocking buffer for 2 h at room temperature Antibody–
antigen complexes were detected by chemiluminescence
using chemiluminescent substrate kit (NEN Life Sciences)
As a control, immunoblotting was carried out as described
above without anti-(Ca-SR) Ig Anti-actin Ig (Chemicon
International) was used at a dilution of 1 : 400 to detect the expression level of a-actin in the isolated tissues as the house-keeping internal control
Immunohistochemistry study Sprague–Dawley rats were anasthetized by intraperitoneal injection of sodium pentobarbital (60 mgÆkg)1 body weight) The rats were perfused through the left ventricle with ice cold NaCl/Pi(pH 7.4) for 1 min and ice cold 4% paraformaldehyde in NaCl/Pifor 2 min The tissues were removed and fixed in 4% paraformaldehyde in NaCl/Piat
4C overnight Specimens were dehydrated with 20% sucrose in NaCl/Pifor 24 h Cryostat sections (5 lm) were cut on a Micron cryostat at)20 C and thaw-mounted onto ethanol-cleaned slides coated with 1% gelatin Sections were postfixed in 4% paraformaldehyde for 20 min, followed by
15 min incubation in 5 lgÆmL)1proteinase K (Ambion) for antigen retrieval at 37C After washing with NaCl/Pi, the sections were blocked with 5% normal horse serum in NaCl/Pifor 1 h at room temperature and then incubated with 1 : 500 polyclonal Ig against Ca-SR (Alpha Diagnostic International) in NaCl/Pi containing 2.5% normal horse serum and 0.1% Triton X-100 overnight at 4C After rinsing with NaCl/Pi, staining was performed with the Vectastain Universal Elite ABC Kit (Vector Laboratories, Burlington) according to manufacturer’s instructions Briefly, after washing three times in NaCl/Pi, sections were incubated for 30 min with diluted biotinylated universal secondary IgG After washing with NaCl/Pi, the sections were exposed to Vector ABC reagent (avidin coupled to biotinylated horseradish peroxidase) for 30 min Sections were washed again in NaCl/Piand visualized by incubating with horseradish peroxidase substrate containing 0.02% diaminobenzidine, 0.3% nickel ammonium sulfate and 0.002% hydrogen peroxide (Vector Laboratories) The appearance of reaction product was monitored and photo-graphed under bright-field illumination As a control, some sections were not incubated with primary antibody Adult rat myocyte isolation
Adult (6–8 weeks old) male Sprague–Dawley rats were anesthetized with pentobarbital sodium (50 mgÆkg)1, i.p.) The heart was removed and firstly perfused via the aorta at
37C with standard Tyrode’s solution for about 5 min until the effluent was clear Standard Tyrode’s solution was composed of (in mM): NaCl, 136; KCl, 5.4; NaH2PO4, 0.33; MgCl2, 1.0; CaCl2, 2.0; dextrose, 10 and Hepes, 10 (pH adjusted to 7.4 with NaOH), and was maintained at room temperature and equilibrated with 95% O2and 5% CO2 Then the heart was perfused with Ca2+-free Tyrode’s solution for 5 min and Ca2+-free Tyrode’s solution con-taining 120 UÆmL)1collagenase for 70 min Ventricular tissues (2–3 mm in diameter) were excised and placed in a high [K+] solution composed of (in mM): glutamic acid, 70; taurin, 15; KCl, 30; KH2PO4, 10; Hepes, 10; MgCl2, 0.5; EDTA, 0.5 and glucose, 10 (pH adjusted to 7.3–7.4 with KOH) Myocytes were isolated by trituration with a Pasture pipette and collected by centrifuging at 600 r.p.m for 1 min
at room temperature Cells were re-suspended in the high [K+] solution and kept at room temperature [10]
Trang 3Fura-2 measurements of [Ca2+]i
Single ventricular myocytes attached to the glass bottom of
Petri dishes coated with laminin (10 lgÆmL)1, 500 lL per
dish, with blow-drying) Cells were loaded with 2 lMfura
2-AM (Sigma) for 60 min at room temperature in a Hepes
buffer composed of (in mM): NaCl, 125; KCl, 3.0; MgSO4,
1.2; Na2PO4, 2.0; CaCl2, 1.8; dextrose, 10.5; Hepes, 32 and
0.1% BSA (pH 7.4) Thereafter, myocytes were rinsed with
normal Hepes buffer twice to remove the remaining dye and
then equilibrated for 30 min at room temperature The
tested compounds were added directly to petri dishes to
reach the desired final concentrations The fura-2 loaded
myocytes were alternatively excited at 340 and 380 nm from
a monochromator (SpectraMASTER, Olympus America,
Melville, NY, USA) Fluorescent images of ventricular
myocytes were observed through an inverted phase-contrast
microscope (Olympus IX70, Tokyo) and video images of
fluorescence at 510 nm emission were collected at 2 Hz
using an intensified CCD camera system (AstroCam,
Olympus Life Science resources, Cambridge, UK) with
the output digitized at 768· 512 pixels The ratio of the
fluorescence intensities at 340 : 380 nm excitations was
monitored and processed with computer software (
ULTRA-VIEW, PerkinElmer Life Sciences Inc., Boston, MA, USA)
Measurement of IP3formation
Isolated rat ventricular myocytes were incubated for 4 h in
serum-free and inositol-free DMEM, to which 5 lCiÆmL)1
myo-2-[3H]inositol (Du Pont Canada Inc.) were added The
cells were subjected to different stimuli for 60 mins, and the
reaction was terminated by adding 0.9 mL methanol/
chloroform/HCl (40 : 20 : 1, v/v/v) The initial
inositol-phosphate (IP) pool of the aqueous phase composed of
inositol 4-phosphate, inositol 4,5-biphosphate and IP3was
eluted consecutively by ion-exchange chromatography
(AG1-X8 resin, Bio-Rad Laboratories) The lipid phase
was counted to measure the phosphatidylinositol phosphate
(PIP) lipid pool IP3 was expressed as a relative value of
(IP3/PIP)· 103(arbitrary units) to correct for the variation
in the labeling of the lipid pool
Chemicals and data analysis
Thapsigargin (TG) was purchased from Calbiochem
U73122, U73343, spermine, nifedipine, CdCl2 and other
chemicals were from Sigma Data were expressed as
means ± SEM Differences between treatments in the
same cells were evaluated by paired Student’s t-test or in
conjunction with Newman–Keuls test A significant level
of difference was determined when P < 0.05
Results
Transcriptional expression ofCa-SR in rat cardiac tissues
Expression of Ca-SR mRNA was examined using
RT-PCR A cDNA fragment of 234 bp corresponding to the
selected Ca-SR mRNA sequence was detected in both rat
atrium and ventricle (Fig 1A) In the absence of reverse
transcriptase, no PCR-amplified fragment could be
detec-ted, indicating the tested RNA samples were free of genomic DNA contamination This 234 bp PCR fragment was gel-purified, subcloned into plasmid vectors, and sequenced The derived sequences from three independent clones were identical to the Ca-SR cDNAs from rat parathyroid, kidney [4] and brain [11] The expression level of Ca-SR mRNA in thyroid appears to be much greater than that in cardiac tissues However, these results were derived from RT-PCR, which is a qualitative rather than quantitative mRNA assay Therefore, it would be inappropriate to predict the protein levels based on RT-PCR results shown in Fig 1A
Protein expression of Ca-SR in rat cardiac tissues The expression of Ca-SR protein was examined using Western blotting on whole-tissue extract Ca-SR proteins with a relative molecular mass between 120 and 140 kDa were detected in rat atrium and ventricle (Fig 1B) or in whole heart tissues (Fig 1C) The same 120–140 kDa band was also detected in thyroid, liver, parathyroid and kidney tissues, which serve as positive control While the band of PCR product for atrium was faint (Fig 1A), the expression levels of Ca-SR proteins were similar between atrium and ventricular tissues (Fig 1B), which may indicate a relative instability of Ca-SR mRNA in rat atrium In the absence of antibody, no positive band was identified (Fig 1B) Fur-thermore, preadsorption of anti-Ca-SR antibody with standard Ca-SR antigen eliminated the 140 kDa band (Fig 1C) Together, these results indicate the specificity of the anti-(Ca-SR) Ig
Immunohistochemistry study on the expression
of Ca-SR protein in different tissues Strong immunostaining was observed in liver cells (Fig 2A and B) as reported by Canaff et al [12] In heart, deep brown immunostaining was present throughout all cardio-myocytes (Fig 2D,E), indicating the expression of Ca-SR at protein level in rat heart Lack of specific staining was demonstrated in control sections in the absence of anti-Ca-SR antibody (Fig 2C,F)
Protein expression of Ca-SR in isolated rat cardiac myocytes
To confirm that Ca-SR was expressed in cardiac myocytes, rather than neuronal or other types of cell in heart tissue, ventricular and atrial myocytes were isolated separately and the expression of Ca-SR proteins in these cells was examined Similar to the observations on whole heart tissue, Ca-SR proteins were identified in the isolated myocytes (Fig 3) Compared to ventricular and atrial membrane preparations, membrane preparation from liver cells had a low protein content as evidenced by low actin level (Figs 1 and 3) Preadsorption of the anti-Ca-SR Igs with standard Ca-SR antigen completely eliminated the
140 kDa band (Fig 3B) In these experiments, the anti-(Ca-SR) Ig was from Affinity BioRegents, Inc (Golden,
CO, USA) at dilution of 1 : 400 In all other Western blot and immnunostaining studies, anti-(Ca-SR) Ig from Alpha Diagnostic International were used The same results using
Trang 4the anti-(Ca-SR) Igs from different suppliers further validate
the specificity of Ca-SR proteins detected in rat
cardio-myocytes
Extracellular calcium, gadolinium and spermine induced
changes in intracellular calcium concentration
Elevating [Ca2+]o from 0 mM to > 1.5 mM evoked an
increase in intracellular calcium concentration in more than
90% of isolated ventricular myocytes in a given observation
field (Fig 4A) The maximal increase in intracellular
calcium concentrations was obtained with 5–10 mM
extra-cellular calcium (Fig 4B) After changing extraextra-cellular
calcium concentration back to 0 mM, the increased
intra-cellular calcium concentration declined gradually (Fig 4C)
Consecutive exposure of freshly isolated rat ventricular
myocytes to extracellular gadolinium also induced a
concentration-dependent increase in intracellular calcium
(Fig 4D)
With 1 mM Ca2+in the bath solution, spermine from
1–10 mM induced a time- and concentration-dependent
increase in intracellular calcium (Fig 5) At 10 mM,
sper-mine produced a calcium burst in a total of 27 cells from
five dishes (P < 0.05) In less than 1 min after spermine
application, all cells in the observation field contracted and quickly exploded (Fig 5A and B) This calcium burst, however, was not observed after calcium was removed completely from the bath solution As shown in Fig 5C,D, spermine still increased intracellular calcium but in a less dramatic way and all cells survived from this spermine treatment
The role of intracellular calcium release and the phospholipase C (PLC) pathway in the extracellular calcium-induced increase in [Ca2+]i
Isolated myocytes were pretreated for 10 min with 10 lM
TG that inhibits the refilling of the IP3-sensitive calcium release pools [12,13] Subsequently, extracellular calcium was changed from 0–1.5 mM, which failed to elicit any increase in [Ca2+]i This effect was observed in a total of 25 cells from six Petri dishes (n¼ 6, P < 0.05) (Fig 6A) Preincubation of myocytes with TG also abolished 0.3 mM
Gd3+-induced (n¼ 8) or 5 mMspermine-induced (n¼ 6) increase in the [Ca2+]i level (not shown) U73122 is a phosphatidylinositol-specific PLC blocker [3,14] Pretreat-ment with U73122 for 10 min eliminated the effect of extracellular calcium-induced intracellular calcium release
Fig 1 Expression of Ca-SR in rat cardiac
tissues (A) Detection of Ca-SR mRNA by
RT-PCR in rat heart in the presence or
absence of reverse transcriptase (RT) M,
DNA marker; bp, base pairs Similar results
were obtained in four other experiments (B)
Detection of Ca-SR proteins by Western blot
in various rat tissues using anti-(Ca-SR) Ig
(left) or in the absence of anti-(Ca-SR) Ig
(right) (C) Detection of Ca-SR proteins by
Western blot in various rat tissues using
anti-(Ca-SR) Ig without preadsorption (left) or
after incubation with excess Ca-SR antigens
overnight at 4 C (right).
Trang 5(n¼ 4) (Fig 6B) This treatment also abolished 0.3 mM
Gd3+-induced (n¼ 4) or 5 mMspermine-induced (n¼ 4)
increase in intracellular calcium (not shown) Under the
same condition but without TG or U73122 pretreatment,
extracellular calcium induced significant increase in [Ca2+]i
(Fig 4C) On the other hand, pretreatment of cells with
U73334 at 10 lM, an inactivated analogue of U73122 [8],
for 10 min did not prevent the increase in [Ca2+]iinduced
by extracellular calcium (n¼ 5, P < 0.05) (Fig 6C) These
results suggest that activation of Ca-SR resulted in
stimu-lation of PLC pathway, and the subsequent production of
IP3 stimulated the TG-sensitive IP3 receptors, leading to
intracellular calcium increase
Involvement of extracellular calcium entry in the
extracellular calcium-induced increase in [Ca2+]i
To examine whether the increased [Ca2+]iwas related to the
activity of Na+/Ca2+ exchanger, NiCl2 (10 mM) was
applied to the isolated myocytes [15] Under this condition,
increasing [Ca2+]o from 0–1.5 mM significantly increased
[Ca2+]i(data not shown) Thus, the activity of Na+/Ca2+
exchanger in plasma membrane could not explain the
increase in [Ca2+] upon the stimulation of Ca-SR In
another series of experiments, myocytes were pretreated with 200 lMCdCl2for 10 min CdCl2treatment alone did not alter [Ca2+]i With CdCl2pretreatment, an increase in [Ca2+]iinduced by extracellular calcium was again observed (Fig 7A) Furthermore, increasing [Ca2+]ofrom 0–1.5 mM still significantly increased intracellular calcium in 30 cells from five Petri dishes in the presence of nifedipine (10 lM) (not shown)
Changes in intracellular IP3levels in response
to different Ca-SR stimuli
An increased IP3formation in rat ventricular myocytes was observed after incubation with 3 mM calcium, 0.3 mM gadolinium, or 1 mM spermine (Fig 7B) The largest IP3 response was induced by extracellular calcium when com-pared with the effects of gadolinium and spermine
Discussion
Expression of Ca-SR in cells with functions unrelated to systemic calcium homeostasis has been demonstrated in many cases For instance, expression of Ca-SR in neurons suggests the coupling of [Ca2+] to neuronal activities [11]
Fig 2 Immunohistochemical detection of Ca-SR in rat cardiac tissues Tissue sections of rat liver (A–C) and rat heart (D–F) were processed in the presence (A,B,D,E) or absence of anti-Ca-SR Ig (C and F) Magnification was · 95 (A,D,F); · 190 (B,C); · 380 (E) Representative results were shown from three different experiments.
Trang 6Identification of Ca-SR in spiral modiolar artery, located
between the eighth cranial nerve and the bond of the
cochlear modiolus, also indicates that the changes in
[Ca2+]o may somehow affect smooth muscle functions
The involvement of Ca-SR in diverse cellular functions
implies broad physiological functions beyond the regulation
of systemic calcium homeostasis Our present study for the
first time demonstrated the existence of Ca-SR in cardiac
myocytes This conclusion is based on several lines of
evidence: (a) transcripts of Ca-SR were clearly detected in
cardiac tissue and the sequences of these transcripts were
confirmed as identical to the known sequence of Ca-SR; (b)
Ca-SR proteins were identified in cardiac tissue as well as in
isolated atrial and ventricular myocytes; (c)
Immunohisto-logical staining clearly located Ca-SR proteins in cardiac
tissues; (d) increase in [Ca2+]o increased intracellular free
calcium levels, which was not mediated by extracellular
calcium entry through either voltage-gated Ca2+channels
or a Na+/Ca2+exchanger Release of intracellular calcium from thapsigargin-sensitive calcium pools after activation of PLC pathway was responsible for the extracellular calcium-induced [Ca2+]i; (e) [Ca2+]iincrease in isolated ventricular myocytes was induced by spermine at concentrations between 1–10 mM, which was the concentration range used
in many other studies to elucidate the presence of Ca-SR in different preparations [6,12,16]
Ca-SR in cardiac cells senses the changes
in extracellular calcium concentrations
An increase from 0–1.5 mMin [Ca2+]otriggered an increase
in intracellular calcium and this effect was maximal at
Fig 4 Extracellular calcium-induced intracellular calcium increase in freshly isolated rat ventricular myocytes (A) The same groups of ven-tricular myocytes were exposed consecutively to different [Ca2+] o Changes in the density of pseudo-greyscale indicate different levels of intracellular calcium concentrations with the black representing lower [Ca2+] (B) Concentration dependent effects of extracellular calcium
on [Ca2+] i in ventricular myocytes Changes in 4–6 cells in each culture dish were analysed and a total of four culture dishes were used at each calcium concentration *P < 0.05 vs data obtained at 0 m M of extracellular calcium (C) Reversibility of the extracellular calcium-induced [Ca 2+ ] o change (D) The same groups of ventricular myocytes were exposed consecutively to different [Gd 3+ ] o
Fig 3 Detection of Ca-SR receptor in isolated rat atrial and ventricular
myocytes using the anti-(SR) Igs (Affinity BioRegents, Inc.) (A)
Ca-SR proteins were detected in ventricular and atrial myocytes as well as
in liver (B) Anti-(Ca-SR) Igs were incubated with excess Ca-SR
antigens overnight at 4 C before being used in Western blot
experi-ments.
Trang 75 mMextracellular calcium The physiological relevance
of this narrow range of [Ca2+]oin cardiac myocytes should
be commented on Under what circumstances would the
extracellular calcium be in the range of 0–1.5 mMin heart?
Intracellular calcium changes have been observed in
para-thyroid hormone-releasing and calcitonin cells in response
to [Ca2+]o changes from 0.75– 3 mM [17] Brown et al
described a steep dose–response relationship of the
activa-tion of Ca-SR by extracellular calcium in parathyroid cells
[2] The plasma levels of ionized Ca2+ are between 1.0–
1.3 mM[6] The [Ca2+]ocan be significantly lowered within
the interstitial fluid of the beating heart [18], especially
within the T-tubular system of heart This system is a
restricted plasma membrane invagination and the calcium
content therein is limited The sustained membrane
depo-larization of heart membrane has been reported to lead to
calcium depletion in T-tubular system [19] The lowering of
[Ca2+]o would reduce the activity of Ca-SR in cardio-myocytes, lowering [Ca2+]iand protecting cardiac muscles from sustained contraction Upon repolarization, [Ca2+]o can be restored to a physiological level around 1.5 mM The consequent re-activation of Ca-SR would then restore normal contractility of cardiac muscles by normalizing [Ca2+]i Can [Ca2+]obe further elevated from 1.5–5 mMin cardiac muscle? Similar to our results in cardiac myocytes, Ca-SR in human antral gastrin cells has been reported to
be sensitive to extracellular calcium concentrations ranged from 1.8–5.4 mM[6] Under certain in vivo conditions, the luminal surface of the gastrin cells can be exposed to 15 mM extracellular calcium [20] As high as 40 mMof extracellular calcium in the direct vicinity of bone-osteoclasts has been observed [8] There are several scenarios for which [Ca2+]o
in the vicinity of cardiac myocytes, especially in T-tubule system, may temporarily increase, such as the extrusion of intracellular calcium from the excited myocytes and the
Fig 5 Extracellular spermine-induced [Ca2+] i in freshlyisolated rat
ventricular myocytes Changes in the density of pseudo-greyscale
indicate different levels of [Ca2+] i with black representing lower
cal-cium levels (A) A sudden exposure of ventricular myocytes to 10 m M
spermine triggered an intracellular calcium burst and cell death with
1 m M calcium in the bath solution (B) Time course of the increase in
[Ca2+] i induced by a sudden exposure to 10 m M spermine with 1 m M
calcium in the bath solution (C) Spermine induced a gradual increase
in [Ca 2+ ] i with 0 m M calcium in the bath solution All cells survived
under this gradual spermine exposure condition (D) Time course of
the increase in [Ca 2+ ] i induced by various concentrations of spermine
with 0 m M calcium in the bath solution (total 10 cells from three
dif-ferent Petri dishes).
Fig 6 Signal transduction pathways involved in the extracellular cal-cium-induced increase in [Ca 2+ ] i in isolated rat ventricular myocytes (A) Thapsigargin blocked the effect of extracellular calcium-induced increase in [Ca 2+ ] i (B) Pretreatment of cells with 10 l M U73122 eliminated the effect of extracellular calcium-induced intracellular calcium release (C) Pretreatment of cells with 10 l M U73343 did not prevent the increase in [Ca 2+ ] i induced by extracellular calcium.
Trang 8release of calcium from necrotic myocytes The healthy
myocyte in the neighborhood of necrotic myocytes would
face relatively high [Ca2+]o and increase their Ca-SR
activity Thus, the contractility of these healthy myocytes
would be increased to maintain the pump function
by increasing their intracellular calcium levels Contrary
to the conventional thought of a static extracellular calcium
level, [Ca2+]o in heart tissues may undergo fluctuations
depending on the activity of the heart The presence of
Ca-SR in cardiac myocytes may co-ordinate cellular
activities with the dynamic changes in [Ca2+]o in the
vicinity of cardiomyocytes [1]
The naturally occurring polyamines, including spermine,
spermidine and putrescine, are involved in the synthesis of
nucleic acids and proteins in eukaryotic and prokaryotic
cells They play an important role in the regulation of
cellular proliferation and differentiation [21] For the
regulation of cardiac function, polyamines are also
import-ant Previous studies have provided evidence that
polyam-ines promote cardiac hypertrophy [22,23] In spontaneously
hypertensive rats, an increased left ventricular mass [24] or
cardiac hypertrophy [25] was associated with increased
spermine and spermidine contents The molecular
mecha-nisms underlying the cellular actions of polyamines include
the activation of different plasmalemmal ion channels [26–28] as well as the stimulation of Ca-SR [29] In our study, spermine elicited an extracellular calcium-dependent intracellular calcium response in isolated cardiomyocytes In the absence of extracellular calcium, the spermine-induced [Ca2+]i increase was less dramatic than that in the presence
of 1 mM extracellular calcium and no calcium burst and cellular destruction were observed Similar extracellular calcium dependency of the effects of spermine on Ca-SR has been noticed in other previous studies [6,29]
The physiological concentration of plasma spermine is in the low micromolar range [29,30] In the study by Quinn
et al [29], spermine was used at concentrations from 0.1–1 mMto test the Ca-SR-mediated intracellular calcium response in Ca-SR-expressing HEK cells Ray et al [6] reported the effect of spermine on Ca-SR at concentrations between 0.1–1 mM In hepatocytes, spermine-induced [Ca2+]iincrease was manifested at spermine concentrations from 1.25–10 mM[12] Similarly, in our study, a spermine response was observed at concentrations between 1–10 mM
No effect was observed when spermine concentration was lower than 1 mM Nevertheless, the physiological signifi-cance of this spermine effect at these concentrations can still
be appreciated Polyamine secretion from some neurons has been indicated [31], presenting the possibility that local concentration of spermine can be much higher than the circulating concentration Moreover, the tissue spermine content of ventricular tissues was increased from
68 pmolÆmg)1 in normotensive Wistar–Kyoto rats to
376 pmolÆmg)1 in spontaneously hypertensive rats [24] This observation may also shed light on the pathophysio-logical significance of the effect of spermine at relatively high concentrations on Ca-SR in hearts The spermin-induced increase in [Ca2+]i alone may not suffice to demonstrate conclusively the involvement of Ca-SR but when taken in conjunction with the effects of extracellular calcium and gadolinium on [Ca2+]i, as well as the detection
of Ca-SR at mRNA and protein levels, does provide a line
of evidence for the presence and function of Ca-SR in cardiac myocytes The physiological importance of Ca-SR
in cardiomyocytes can be better understood by comparing the structure and function of hearts with or without Ca-SR deficiency Ca-SR knock-out mice provide an avenue for this kind of study However, cardiovascular functions of Ca-SR knock-out mice have not been reported to our knowledge Loss of Ca-SR in parathyroid gland in knock-out mice results in hyperparathyroidism, hypercalcemia, and growth retardation [32] These alterations may also significantly and indirectly affect cardiac function, mingled with any potential direct cardiac outcome due to the lack of cardiac Ca-SR Therefore, organ-selective or heart-selective inactivation or activation of Ca-SR in living animals should
be engineered, which may help to better determine the direct cardiac outcome of cardiac Ca-SR deficiency
In summary, Ca-SR may play an important physiological role in the modulation of cardiac functions under both physiological and pathophysiological conditions Increased local calcium concentration is sensed by myocytes via Ca-SR and lead to increased cardiac activity Increased extracellular polyamine concentration in heart, on the other hand, may stimulate Ca-SR on cardiomyocytes to promote cardiac hypertrophy Due to the limited access to specific
Fig 7 Changes in intracellular calcium and IP 3 levels (A) Effect of
CdCl 2 on the extracellular calcium-induced increase in [Ca2+] i in
isolated rat ventricular myocytes (total of 32 cells from five Petri
dishes) [Ca 2+ ] i was determined when the changes reached the
maxi-mum levels (B) IP 3 formation in isolated rat ventricular myocytes.
n ¼ 5 for each group *P < 0.05 compared with control group.
Trang 9blockers of Ca-SR, whether polyamine-induced cardiac
hypertrophy is mediated by Ca-SR cannot be readily tested
at the moment Nevertheless, delineation of the interaction
among extracellular calcium levels, polyamine
concentra-tions, functional status of Ca-SR, and myocyte apoptosis
and proliferation would help better understand the
mech-anisms of cardiac hypertrophy as well as its management
Acknowledgements
This study was supported by an operating grant from Canadian
Institutes of Health Research (CIHR) R Wang is an Investigator of
CIHR L Wu is a New Investigator of CIHR.
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