In addition, complete suppression of PEP activity by the specific inhi-bitor,Fmoc-Ala-Pyrr-CN 5 lM induced in U343 and other cell lines an enhanced, but delayed, increase in Ins1,4,5P3 co
Trang 1Modulation of inositol 1,4,5-triphosphate concentration
by prolyl endopeptidase inhibition
Ingo Schulz1, Bernd Gerhartz1, Antje Neubauer1, Andreas Holloschi2, Ulrich Heiser1, Mathias Hafner2 and Hans-Ulrich Demuth1
1
Probiodrug AG, Halle, Germany;2Department of Molecular Biology and Cell Culture Technology, Mannheim University of Applied Sciences, Germany
Prolyl endopeptidase (PEP) is a proline-specific
oligopepti-dase with a reported effect on learning and memory in
dif-ferent rat model systems Using the astroglioma cell line
U343,PEP expression was reduced by an antisense
technique Measuring different second-messenger
concen-trations revealed an inverse correlation between inositol
1,4,5-triphosphate [Ins(1,4,5)P3] concentration and PEP
expression in the generated antisense cell lines However,no
effect on cAMP generation was observed In addition,
complete suppression of PEP activity by the specific
inhi-bitor,Fmoc-Ala-Pyrr-CN (5 lM) induced in U343 and other
cell lines an enhanced, but delayed, increase in Ins(1,4,5)P3
concentration This indicates that the proteolytic activity of PEP is responsible for the observed effect Furthermore,the reduced PEP activity was found to amplify Substance P-mediated stimulation of Ins(1,4,5)P3 The effect of reduced PEP activity on second-messenger concentration indicates a novel intracellular function of this peptidase,which may have an impact on the reported cognitive enhancements due
to PEP inhibition
Keywords: antisense; inositol 1,4,5-triphosphate; prolyl endopeptidase; protease; second messenger; Substance P
Prolyl endopeptidase (PEP; also called prolyl
oligopepti-dase) is a serine peptidase characterized by oligopeptidase
activity It is grouped in family S9A in clan SC [1] Enzymes
belonging to clan SC are distinct from trypsin-type and
subtilisin-type serine peptidases in their structure and the
order of the catalytic triad residues in the primary sequence
[2,3] The recently reported three-dimensional structure of
PEP revealed a two-domain organization [4] The catalytic
domain displays an a/b hydrolase fold in which the catalytic
triad (Ser554,His680,Asp641) is covered by a so-called
b-propeller domain The propeller domain probably
con-trols the access of potential substrates to the active site of the
enzyme and excludes peptides containing more than 30
amino acids
Although the enzymatic and structural properties of
PEP are well known,its biological function is far from
being fully understood [5,6] Highly conserved in
mam-mals,it is ubiquitously distributed,with high
concentra-tions in the brain [7] Recently,the enzyme became of
pharmaceutical interest because of a reported cognitive
enhancement induced by treatment with specific PEP
inhibitors [8,9] In rats displaying scopolamine-induced
amnesia,PEP inhibition caused acetylcholine release in
the frontal cortex and hippocampus [10] Furthermore, administration of a PEP inhibitor to rats with middle cerebral artery occlusion prolonged passive avoidance latency and reduced the prolonged escape latency in the Morris water maze task [11] The potential of PEP inhibitors as antidementia drugs was further supported by reports of neuroprotective effects Inducing neurodege-neration in cerebellar granule cells led to increased neuronal survival and enhanced neurite outgrowth in the presence of a PEP inhibitor [12] Moreover,the level of
m3-muscarinic acetylcholine receptor mRNA was found
to be increased after PEP inhibition,resulting in stimu-lated phosphoinositide turnover
It has been hypothesized that these effects are due to modulation of neuropeptide bioactivity by PEP [13] In vitro, PEP is able to rapidly degrade several neuropeptides, including Substance P and arginine-vasopressin,by limited proteolysis [14,15] Such neuropeptides are known to influence learning and memory [16,17] Administration of Substance P can induce long-term potentiation,a well established parameter for learning and memory [18] Binding of Substance P to neurokinin 1 receptor stimulates
a G-protein-mediated increase in Ins(1,4,5)P3concentration and release of Ca2+ from intracellular stores within the endoplasmic reticulum [19,20] It is well established, but unconfirmed for Substance P,that Ca2+release from these stores is implicated in the induction of long-term potenti-ation and in learning and memory [21] In postsynaptic cells, long-term potentiation is prevented by the inhibition of Ins(1,4,5)P3 receptors,demonstrating the crucial role of Ins(1,4,5)P3formation and Ca2+release in this learning and memory model [22] It should be noted,however,that PEP
is primarily located in the cytosol [23],whereas the interaction between the neuropeptides and their receptors takes place on the cell surface
Correspondence to H.-U Demuth,Probiodrug AG,
Weinbergweg 22,Biocenter,D-06120 Halle (Saale),Germany.
Fax: + 49 345 5559901,Tel.: + 49 345 5559900,
E-mail: hans-ulrich.demuth@probiodrug.de
Abbreviations: Fmoc-Ala-Pyrr-CN,9H-fluorenyl-9-ylmethyl
N-[2-(2-cyano-1-pyrrolidinyl)-1-methyl-2-oxoethyl]carbamate;
NHMec,7-(4-methyl)coumarylamide; PEP,prolyl endopeptidase.
Enzyme: prolyl endopeptidase (EC 3.4.21.26).
(Received 16 July 2002,revised 20 September 2002,
accepted 7 October 2002)
Trang 2Here we show a novel effect of PEP inhibition that may
be related to long-term potentiation and learning and
memory Using antisense cell lines with reduced PEP
expression as well as specific inhibitors,we were able to
show an inverse correlation between Ins(1,4,5)P3
concen-tration and PEP activity The data presented strongly
suggest an indirect involvement of PEP in second-messenger
pathways with potential cross-talk to signal transduction
mediated by neuropeptides
E X P E R I M E N T A L P R O C E D U R E S
Construction of antisense vector
To obtain the coding sequence for the catalytic domain of
PEP,total RNA from 1· 107cells of the human glioma cell
line U343 was isolated with TRIzol reagent (Gibco BRL)
Then 4 lg total RNA was converted into cDNA by
RT-PCR using hexanucleotide primers and Moloney
murine leukaemia virus (M-MLV) reverse transcriptase
(Promega) The resulting cDNA pool (4 lL) was then
amplified with the ExpandTMPCR System (Roche) using a
pair of PEP-specific primers (5¢-CATATGCTGTCCTTC
CACG-3¢) The resulting PCR fragment contained the
entire ORF By PCR,using two nested primers (5¢-CAT
ATGGGAATTGATGCTTCTGATTAC-3¢; 5¢-GAATTC
TGGAATCCAGTCGACATTCAG-3¢),a 0.9-kb fragment
was generated containing the catalytic domain of the
enzyme (amino acids 442–731 of human PEP) This
fragment was cloned into pPCR-Script Cam (Stratagene)
The EcoRI restriction sites of the subcloned vector and of
the nested reverse primer were used to ligate the fragment
into the mammalian expression vector pIRESneo
(Clon-tech) The resulting transformants were analysed by PCR to
determine if the insert was present in antisense orientation,
and the correct nucleotide sequence was verified by DNA
sequencing (GATC Biotech AG)
Cell culture, transfection and stable cell lines
The human glioma cell lines U343 and LN405 were
maintained in Dulbecco’s modified Eagle’s medium
con-taining 10% fetal bovine serum (Gibco BRL) at 37C in a
5% CO2 and 10% CO2 atmosphere,respectively The
neuroblastoma cell line SH-SY5Y was grown in Dulbecco’s
modified Eagle’s medium containing 5% fetal bovine serum
in a 10% CO2atmosphere All media contained 60 lgÆmL)1
gentamicin (Gibco BRL) The mammalian expression
vectors were transfected into U343 cells using Polyfectin
reagent (Biontex,Munich,Germany) according to the
manufacturer’s protocol Stable transfectants were selected
in medium containing 400 lgÆmL)1 G418 (Duchefa,
Haarlem,the Netherlands) Single clones were isolated with
cloning rings (Clontech)
Prolyl oligopeptidase assays
Cells (1· 107) were harvested by washing twice in NaCl/Pi
(Gibco BRL) and resuspended in 200 lL assay buffer
(50 mMHepes,pH 7.5,200 mMNaCl,1 mMEDTA,1 mM
dithiothreitol) Cell lysis was achieved by three cycles of
thawing and freezing,and then the cells were removed from
the incubation flask with a cell scraper The lysate obtained was centrifuged at 18 000 g for 1 min,and the supernatant transferred to a fresh tube All steps were performed on ice The protein concentration in the supernatant was determined by the method of Bradford [24] PEP activity was measured in the assay buffer using the fluorogenic substrate Z-Gly-Pro-NHMec (10 lM) (Bachem,Heidelberg, Germany) on a Kontron spectrofluorimeter SFM 25 (excitation wavelength 380 nm,emission wavelength
460 nm) equipped with a four-cell changer and controlled
by an IBM-compatible personal computer The data obtained were analysed with the softwareFLUCOL[25] SDS/PAGE and immunoblotting
To generate a polyclonal antibody against human PEP, rabbits were immunized with a peptide containing the N-terminal PEP sequence of amino acids 10–25 Specific antibodies (S449) were purified from rabbit serum using
an affinity chromatography column with the immobilized peptide Analytical electrophoresis in SDS/polyacrylamide gels was performed as described by Laemmli with separation gels containing 12% acrylamide [26] The separated cell extracts were transferred to a nitrocellulose membrane (Schleicher & Schuell) following a standard procedure [27] PEP and actin were detected by the polyclonal antibody S449 (1 : 400 dilution) and monoclo-nal antibody anti-actin (1 : 2500 dilution,Sigma,A2066), respectively,and visualized by chemiluminescence accord-ing to the manufacturer’s protocol (SuperSignalTM,West Pico; Pierce) Semiquantitative analysis of Western-blot results was performed using densitometry software (GELSCAN3D; BioSciTec,Marburg,Germany)
Assay of Ins(1,4,5)P3 Cells were grown in 25 cm2culture flasks to nearly 100% confluence Ins(1,4,5)P3 concentration was determined by
an isotope dilution method (Amersham Phamacia Biotech) using 0.5· 106cells per measurement To inhibit intracel-lular PEP,the cells were washed twice with NaCl/Piand incubated for up to 24 h in Optimem 1 medium (Gibco BRL) supplemented with 5 lMPEP inhibitor Fmoc-Ala-Pyrr-CN All measurements were carried out in quadrupli-cate The calculation of Ins(1,4,5)P3concentration and the statistical analysis (t test) were performed usingPRISM 3.0 (Graph Pad Software)
Stimulation assay Wild-type and PEP antisense U343 cell lines were cultured
in duplicate in 21-cm2culture dishes (Greiner,Frickenhau-sen,Germany) until confluence Before stimulation,the cells were washed twice in NaCl/Piand preincubated for 10 h in Optimem 1 medium containing 1.6 lgÆmL)1 leupeptin (Sigma),0.86 lgÆmL)1 chymostatin (Sigma),and
40 lgÆmL)1 bacitracin (Sigma) at 37C and 5% CO2 Substance P (Bachem) was added to a final concentration of
1 lM,and the incubation was stopped at the indicated time
by rapidly aspirating the medium and adding 0.4 mL ice-cold trichloric acid Preparation of samples and measure-ment of Ins(1,4,5)P3 concentration were performed as described above
Trang 3cAMP bioactivity assay
U343 cells were transfected with a reporter
plasmid,pCRE-EGFP containing a cassette of a minimal promoter and
three cAMP-responsive elements [28] Media containing
400 lg G418 were used to select stable transfectants Cells
were seeded at a density of 1· 104cells per well in a 96-well
plate (Greiner) After 24 h,the medium was replaced by
dilutions of forskolin or Fmoc-Ala-Pyrr-CN in serum-free
medium The fluorescence was measured by a Bio Assay
fluorescence microplate reader (Perkin-Elmer,U¨berlingen,
Germany) at 485 nm excitation wavelength and 538 nm
emission wavelength Data were calculated using the Prism
3.0 (Graph Pad)
R E S U L T S
Suppression of PEP expression in U343 cells
A cell line with sufficiently high concentrations of PEP was
required to investigate the cellular role of PEP The
astroglioma cell line U343 showed the highest amount of
PEP (active and protein concentration) out of six cell lines
tested (U-138 MG,LN 2308,T 98p31,U343,SY5Y,
LN405)
Two different approaches were used to influence the
intracellular activity of PEP in U343 cells
Fmoc-Ala-Pyrr-CN is a potent and specific inhibitor of PEP [29] with a Kiof
70 pMagainst recombinant human PEP (data not shown)
This inhibitor is able to penetrate the cell membrane and
inhibit PEP intracellularly [30] In U343 cells,total
inhibi-tion was achieved within 1 min by adding 5 lM
Fmoc-Ala-Pyrr-CN to the medium,and inhibition persisted for up
to 24 h without the addition of fresh inhibitor A completely
different approach to reducing PEP activity was also used,
namely generation of antisense cell lines with reduced
expression of the target enzyme U343 cells were transfected
with the antisense vector,and 120 clones were isolated using
cloning rings From these clones,eight stable cell lines were
established,and all had reduced PEP activity (Table 1)
However,antisense cell lines 1,13 and 110 lost their
antisense effect during the prolonged cultivation Most of
the established cell lines displayed reduced PEP activity of
50% Cell line as11 showed the greatest reduction in PEP
activity,30% compared with wild-type U343 cells
Western-blot analysis confirmed the results obtained by activity
measurements (Fig 1,Table 1) In all antisense cell lines,
the reduced proteolytic activity resulted from decreased expression of PEP The generated antisense cell lines did not show a common change in phenotype,but individual changes were observed U343 as11 cell line showed increased trypsin sensitivity,increased cell volume (three-fold),and was no longer able to grow to 100% confluence
Modulation of Ins(1,4,5)P3concentration dependent
on PEP
To characterize the intracellular function of PEP, Ins(1,4,5)P3 concentration in the antisense cell lines was measured In U343 wild-type cells,it was 0.26 ± 0.02 pmol per 106 cells (n¼ 4) It was increased in all generated antisense cell lines (Fig 2) The increase in Ins(1,4,5)P3 concentration correlated with reduced PEP activity in the antisense cell lines tested (Fig 2C,correlation coefficient 0.997)
An alternative approach to suppressing PEP activity in U343 cells was utilized The cells were incubated for 3 h in the presence of the specific inhibitor Fmoc-Ala-Pyrr-CN (5 lM) In confirmation of the results obtained with the antisense cell lines, basal Ins(1,4,5)P3 concentration was increased in cells treated with PEP inhibitor (Fig 2) However, the observed change in Ins(1,4,5)P3concentration was only 0.16 pmol per 106cells This is much smaller than the change in Ins(1, 4, 5)P3concentration (0.66 pmol per 106 cells) observed in cell line as11,which still contained 30%
Table 1 Remaining activities and expression patterns of PEP in human glioma U343 antisense cell lines Specific activity is expressed as mean ± SD All antisense cell lines show reduced remaining activity and expression intensity compared with wild-type cell line U343 Remaining acti-vity ¼ percentage of the activity found in wild-type U343 cells; remaining expression ¼ densitometric analysis of Western blot, n ¼ 2.
Cell line
Specific activity (mUÆmg)1)
Remaining activity (%)
Remaining expression (%)
a
Changes in phenotype.
Fig 1 Western-blot analysis of PEP expression in established antisense cell lines The PEP activity remaining in each antisense cell line cor-responds to the signal intensity in the Western-blot analysis First,
1 · 10 7
cells from each cell line were extracted and analysed as des-cribed in Experimental procedures Then 20 lg total protein was loaded per lane Purified recombinant human PEP was used as a positive control (75 ng) Western blots were probed with PEP-specific antibody S449 (1 : 400) and anti-actin (1 : 2500) and detected by chemiluminescence.
Trang 4PEP activity,and calls into question the correlation between
PEP activity and Ins(1,4,5)P3 concentration Therefore,
Ins(1,4,5)P3concentration was investigated over an
exten-ded period of total inhibition As shown in Fig 3,
Ins(1,4,5)P3 concentration in U343 cells increased during
incubation After 12 h, the total amount of Ins(1,4,5)P3
(1.24 ± 0.24 pmol per 106cells; n¼ 4) was higher than the
concentration measured in cell line as11 (0.95 ± 0.05 pmol
per 106 cells; n¼ 4),which has 30% remaining enzyme
activity To confirm the observed effect,two other cell lines, SY5Y and LN405,were incubated in the presence of Fmoc-Ala-Pyrr-CN for 24 h (Fig 3) Both cell lines showed an increase in Ins(1,4,5)P3 concentration with a similar dependence on the incubation time to that in U343 cells However, the increase in Ins(1,4,5)P3 concentration was smaller The PEP activity of SY5Y cells (1.39 ± 0.03 mUÆmg)1) and LN405 cells (1.65 ± 0.04 mUÆmg)1)
is 1.7-fold and 1.4-fold lower,respectively This confirms the observed activity dependence of PEP inhibition on Ins(1,4,5)P3concentration
Influence of PEP inhibition on the cAMP pathway
In addition to Ins(1,4,5)P3,the effect of PEP inhibition on another second messenger,cAMP,was investigated Using
a reporter plasmid (pCRE-EGFP) containing three cAMP-responsive elements,the increase in cAMP concentration was measured from the expression of enhanced green fluorescent protein (EGFP) via activation of the cAMP-responsive element Incubation with Fmoc-Ala-Pyrr-CN had no positive effect on the cAMP pathway,whereas control experiments stimulating transfected U343 cells with forskolin resulted in an increase in EGFP expression (not shown)
PEP-dependent Ins(1,4,5)P3accumulation after stimulation with Substance P
To investigate whether the observed effect on Ins(1,4,5)P3 concentration represents a novel interaction between the biological activity of neuropeptides and PEP,Substance P was chosen to stimulate U343 cells Substance P,a neuro-peptide known to be degraded by PEP in vitro [15,31], is reported to influence learning and memory via a receptor-mediated signalling cascade including the second messenger, Ins(1,4,5)P3 [32,33] Using RT-PCR, the occurrence of Substance P-specific neurokinin receptor 1 in U343 cells was confirmed (data not shown) Acknowledging that
Fig 3 Time course of Ins(1,4,5)P 3 concentration in different cell lines treated with the PEP inhibitor Fmoc-Ala-Pyrr-CN Whereas PEP activity was completely inhibited after 1 min of a single treatment with
5 l M Fmoc-Ala-Pyrr-CN,the Ins(1, 4, 5)P 3 concentration required
12 h to reach maximum concentration Results are presented as mean ± SEM from experiments carried out in quadruplicate (j) U343; (d) SY5Y; (.) LN405.
Fig 2 Analysis of Ins (1,4,5)P 3 concentration in various U343 cell lines.
(A) Reduced PEP activity induces increased Ins(1,4,5)P 3 concentration
in stable transfected cell lines Human glioma cell line U343 was
transfected with a vector (pIRES) containing the coding sequence of
the PEP catalytic domain (amino acids 442–731) in antisense direction.
The cell line transfected with the vector not harbouring an insert
(pIRES) was used as a negative control (B) Wild-type U343 cells
treated with the specific PEP inhibitor,Fmoc-Ala-Pyrr-CN (5 l M )
show an increased Ins(1,4,5)P 3 concentration; Data were obtained in
quadruplicate (mean ± SD) and analysed using the unpaired t test
(***P < 0.001; **P < 0.01; *P < 0.05; n.s.,not significant) (C) The
increase in Ins(1,4,5)P 3 concentration correlates with the remaining
PEP activity in the established antisense cell lines Correlation factor
was estimated by linear regression (***P < 0.0005).
Trang 5Substance P is an excellent in vitro substrate for PEP,we
investigated potential degradation of Substance P during
the incubation in the serum-free Optimem 1 medium of
U343 cells by MALDI-TOF MS analysis However,during
the incubation time of 10 min used,no PEP-specific
degradation was observed (data not shown) This is in
agreement with the fact that no PEP activity is measurable
in the medium (detection limit 0.1 lUÆmg)1)
Stimulation of the wild-type U343 cells for 5 s with 1 lM
Substance P led to a rise in Ins(1,4,5)P3 concentration
(Fig 4) Intriguingly,U343 cells treated with
Fmoc-Ala-Pyrr-CN and U343 cell line as2 had a higher concentration
of Ins(1,4,5)P3 after Substance P stimulation Comparing
the total values after Substance P stimulation,the
Ins(1,4,5)P3 concentration again correlated with the
impaired PEP activity (Fig 4) The change in Ins(1,4,5)P3
concentration during Substance P stimulation is illustrated
in Fig 5 To compare the stimulation-dependent increase in
Ins(1,4,5)P3 concentration, the amount of Ins(1,4,5)P3 in
the nonstimulated state was subtracted as a baseline All
three cell lines,U343 wild-type untreated or inhibitor
treated and as2 cells,showed a similar stimulation pattern
over the time measured Maximum Ins(1,4,5)P3
concentra-tion always occurred after 5 s stimulaconcentra-tion The stimulaconcentra-tion
produced a rapid increase in the second-messenger
concen-tration followed by a slow decline,not reaching baseline
levels until 40 s Whereas U343 wild-type and as2 cells
showed no consistent difference in Ins(1,4,5)P3
concentra-tion (Fig 5B),the inhibitor-treated wild-type cells showed
increased stimulation of Ins(1,4,5)P3 by Substance P over the whole incubation (Fig 5A) Estimation of cAMP stimulation with forskolin did not reveal any difference between wild-type U343 cells and antisense cell lines or Fmoc-Ala-Pyrr-CN-treated cells
D I S C U S S I O N
First described in 1970 as an oxytocin-inactivating enzyme [34],PEP is well understood with respect to its enzymatic and structural properties,but its physiological function remains unclear However,over the past few years,it has become of pharmaceutical interest because of reports of improved learning and memory after application of specific PEP inhibitors [10,35–37]
Fig 4 Ins(1,4,5)P 3 concentrations in various U343 cell lines stimulated
by Substance P Ins(1,4,5)P 3 concentrations were measured in U343
wild-type cells with or without incubation in the presence of 5 l M
Fmoc-Ala-Pyrr-CN for 12 h and in antisense cell line U343–as2 Each
cell line was stimulated with 1 l M Substance P for 5 s after which
Ins(1,4,5)P 3 was extracted and measured Data (mean ± SD) were
obtained in quadruplicate and statistical analysis was performed using
the paired t test.
Fig 5 Kinetic profile of Ins(1,4,5)P 3 stimulation by Substance P in U343 cells The kinetic profiles of Ins(1,4,5)P 3 stimulation by Sub-stance P show a significant increase in inhibitor treated U343 cells (A, s),antisense cell line 2 (B,m) and untreated control cells (A,B,d) Cells were stimulated with 1 l M Substance P and harvested at different time points to extract Ins(1,4,5)P 3 U343 wild-type cells were treated with 5 l M Fmoc-Ala-Pyrr-CN for 12 h ahead of the experiment All data points,presented as mean ± SD,are from experiments carried out in quadruplicate.
Trang 6PEP inhibitors are in general very specific because of
the proline residue in the P1 position (Berger and
Schlechter nomenclature [38]) However,we used two
different methods of inhibition Antisense cell lines
expressing reduced PEP enable investigation of the
biological function of nonenzymatic properties of this
two-domain protein In addition,this technique avoids
possible unspecific effects of the reactive group of the
inhibitor Eight stable antisense cell lines were developed
with PEP expression reduced by various amounts In all
cell lines a strong correlation was observed between
reduced PEP expression and remaining enzyme activity
(Table 1) Although differences in cultivation and
mor-phology of these cell lines could be observed,no common
change in the phenotype was present The observed
changes seem to be related to the method used to generate
antisense cell lines,in which the antisense encoding DNA
has to be inserted into the genome in a random manner
Phenotypic changes in U343 cells were not seen when cells
were cultivated in the presence of PEP inhibitors
A relationship between the physiological function of
neuropeptides and PEP has been suggested [13,14]
Inacti-vation of the biological activity of the neuropeptides via
limited proteolysis by PEP has been hypothesized However,
this hypothesis does not explain how an intracellular enzyme
such as PEP can interfere with the extracellular interaction
between the neuropeptide and its receptor During
cultiva-tion of U343 cells,we were unable to detect any extracellular
activity of PEP,all activity being found in the cytoplasmic
fraction Another possible relationship may be an
intracel-lular involvement of PEP in the receptor-mediated signalling
cascade of neuropeptides The first hint of this unexpected
function came from a PEP knock-out mutant in the slime
mold Dictyostelium [39] While trying to generate a Li+
-resistant mutant of Dictyostelium,the authors found that the
PEP knock-out mutant prevented typical effects of Li+by
increasing the Ins(1,4,5)P3concentration Ins(1,4,5)P3,as a
central molecule in the signalling cascade of neuropeptides,
offers an intriguing starting point to investigate such an
unexpected relationship Neuropeptides such as Substance P
are able to activate phospholipase C via their specific
receptors and do so by inducing the second messengers
Ins(1,4,5)P3and 1,2-diacylglycerol [19,20] It is known that
Ins(1,4,5)P3binds to its receptor located in the membrane of
the endoplasmic reticulum and induces release of Ca2+,
which is believed to play a crucial role in learning and
memory [22]
Interestingly,in the mammalian cell lines U343,SY5Y,
and LN405, Ins(1,4,5)P3concentration increased according
to reduced expression of PEP and was dependent on the
proteolytic activity being suppressed by the inhibitor (Figs 2
and 3) The effects of the antisense approach and inhibitor
treatment on Ins(1,4,5)P3stimulation differ with respect to
concentration,probably because of the longer period of
reduced PEP expression in the antisense approach However,
the increased Ins(1,4,5)P3observed in the antisense cell lines
leaves open the question of which domain of PEP is
responsible for this effect The results obtained with the
specific inhibitor indicate an involvement of the catalytic
domain within the enzyme The inhibitor
used,Fmoc-Ala-Pyrr-CN,interacts with the enzyme in a substrate-like
manner and restricts changes to the active site of the enzyme
[29,40] This strongly suggests that the impaired proteolytic
activity of PEP is responsible for the elevated Ins(1,4,5)P3 concentration No effect of PEP inhibition on the alternative signal-transduction pathway of neuropeptides such as arginine-vasopressin with cAMP as second messenger was observed
The astroglioma cell line U343 expresses neurokinin 1 receptor,the specific receptor for the neuropeptide Sub-stance P, and displays typical Ins(1,4,5)P3 kinetics after Substance P stimulation (Fig 5) [41] Both U343 antisense cell lines and cells incubated with the PEP inhibitor showed
an amplified Ins(1,4,5)P3signal after Substance P stimula-tion (Fig 4),but the kinetic profile of the stimulastimula-tion was left unchanged (Fig 5) This amplification supports the hypo-thesis that PEP somehow influences the signalling cascade of neuropeptides such as Substance P However,the amplifi-cation of the Ins(1,4,5)P3signal appeared to be partially due
to the increased baseline level of the second messenger and partially due to enhanced efficacy of Substance P This raises the question of whether PEP influences the neuropeptide signalling cascade at or before phospholipase C-catalysed Ins(1,4,5)P3 formation or is independent of this pathway Such an alternative pathway includes the dephosphorylation
of InsP5to Ins(1,4,5)P3by multiple inositol polyphosphatase [42] This enzyme was reported to have increased activity in the PEP knock-out mutants of Dictyostelium [39] Neither Ins(1,4,5)P3,its precursor,nor enzymes such as phosphol-ipase C or multiple inositol polyphosphatase are substrates
of PEP,therefore,the observed effect must be indirect The extremely delayed response of Ins(1,4,5)P3concentration to total inhibition of PEP supports this suggestion (Fig 3) In addition,it is intriguing that the enzymatic activity of PEP can be suppressed by a phosphorylated residue adjacent to the P1proline residue [43]
In conclusion,the results presented strongly indicate a novel type of interaction between the signal-transduction cascades of neuropeptides such as Substance P and the serine peptidase PEP,in addition to the well reported in vitro direct inactivation Because of its intracellular localization, the effect of PEP on the signalling cascade offers a new way
in which PEP inhibitors may enhance learning and memory After submitting this manuscript,Williams and co-workers [44] have published an article where they establish
a link between the mood-stabilizing drugs lithium,car-bamazepine and valproic acid,and inositol depletion Inhibitors of prolyl endopeptidase reverse the effects of all three drugs on sensory neuron growth cone area and collapse, suggesting an influence on Ins(1,4,5)P3metabolism
by PEP which is demonstrated in the present investigation
A C K N O W L E D G E M E N T S
This study was supported by grants from the BMBF,project no
beo-312302 The MALDI-TOF MS analysis of Dr Fred Rosche is gratefully acknowledged We are indebted to Dr S Buckley and Dr S Hinke for critical reading of the manuscript and the stimulating discussion.
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