Báo cáo Y học: Modulation of inositol 1,4,5-triphosphate concentration by prolyl endopeptidase inhibition ppt

Modulation of inositol 1,4,5-triphosphate concentration by prolyl endopeptidase inhibition Ingo Schulz 1 , Bernd Gerhartz 1 , Antje Neubauer 1 , Andreas Holloschi 2 , Ulrich Heiser 1 , Mathias Hafner 2 and Hans-Ulrich Demuth 1 1 Probiodrug AG, Halle, Germany; 2 Department 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)P 3 ] 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 l M ) induced in U343 and other cell lines an enhanced, but delayed, increase in Ins(1,4,5)P 3 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)P 3 . 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 m 3 -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)P 3 concentration and release of Ca 2+ from intracellular stores within the endoplasmic reticulum [19,20]. It is well established, but unconfirmed for Substance P, that Ca 2+ 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)P 3 receptors, demonstrating the crucial role of Ins(1,4,5)P 3 formation and Ca 2+ 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) Eur. J. Biochem. 269, 5813–5820 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03297.x Here 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)P 3 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. EXPERIMENTAL PROCEDURES Construction of antisense vector To obtain the coding sequence for the catalytic domain of PEP, total RNA from 1 · 10 7 cells of the human glioma cell line U343 was isolated with TRIzolÒ 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 Expand TM PCR System (Roche) using a pair of PEP-specific primers (5¢-CATATGCTGTCCTTC CAGTACC-3¢;5¢-GATTCCGCTGTCAGGAGGAAG 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 37 °Cina 5% CO 2 and 10% CO 2 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% CO 2 atmosphere. 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 · 10 7 ) were harvested by washing twice in NaCl/P i (Gibco BRL) and resuspended in 200 lL assay buffer (50 m M Hepes, pH 7.5, 200 m M NaCl, 1 m M EDTA, 1 m M 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 l M ) (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 software FLUCOL [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 (SuperSignal TM , West Pico; Pierce). Semiquantitative analysis of Western-blot results was performed using densitometry software ( GELSCAN 3D; BioSciTec, Marburg, Germany). Assay of Ins(1,4,5) P 3 Cells were grown in 25 cm 2 culture flasks to nearly 100% confluence. Ins(1,4,5)P 3 concentration was determined by an isotope dilution method (Amersham Phamacia Biotech) using 0.5 · 10 6 cells per measurement. To inhibit intracel- lular PEP, the cells were washed twice with NaCl/P i and incubated for up to 24 h in Optimem 1 medium (Gibco BRL) supplemented with 5 l M PEP inhibitor Fmoc-Ala- Pyrr-CN. All measurements were carried out in quadrupli- cate. The calculation of Ins(1,4,5)P 3 concentration and the statistical analysis (t test) were performed using PRISM 3.0 (Graph Pad Software). Stimulation assay Wild-type and PEP antisense U343 cell lines were cultured in duplicate in 21-cm 2 culture dishes (Greiner, Frickenhau- sen, Germany) until confluence. Before stimulation, the cells were washed twice in NaCl/P i andpreincubatedfor10hin Optimem 1 medium containing 1.6 lgÆmL )1 leupeptin (Sigma), 0.86 lgÆmL )1 chymostatin (Sigma), and 40 lgÆmL )1 bacitracin (Sigma) at 37 °Cand5%CO 2 . Substance P (Bachem) was added to a final concentration of 1 l M , 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)P 3 concentration were performed as described above. 5814 I. Schulz et al.(Eur. J. Biochem. 269) Ó FEBS 2002 cAMP 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 · 10 4 cells 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). RESULTS 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 K i of 70 p M against 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 l M 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) P 3 concentration dependent on PEP To characterize the intracellular function of PEP, Ins(1,4,5)P 3 concentration in the antisense cell lines was measured. In U343 wild-type cells, it was  0.26 ± 0.02 pmol per 10 6 cells (n ¼ 4). It was increased in all generated antisense cell lines (Fig. 2). The increase in Ins(1,4,5)P 3 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 l M ). In confirmation of the results obtained with the antisense cell lines, basal Ins(1,4,5)P 3 concentration was increased in cells treated with PEP inhibitor (Fig. 2). However, the observed change in Ins(1,4,5)P 3 concentration was only 0.16 pmol per 10 6 cells. This is much smaller than thechangeinIns(1,4,5)P 3 concentration (0.66 pmol per 10 6 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 (%) U343-wt 5.00 ± 0.14 100.0 100.0 U343-as2 2.64 ± 0.08 53.0 57.0 U343-as11 a 1.52 ± 0.04 30.0 20.0 U343-as40 2.70 ± 0.06 54.0 65.0 U343-as60 2.12 ± 0.04 42.0 33.0 U343-as70 2.18 ± 0.08 43.0 61.0 U343-as110 4.20 ± 0.10 84.0 71.0 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. Ó FEBS 2002 Ins(1,4,5)P 3 increase by prolyl endopeptidase (Eur. J. Biochem. 269) 5815 PEP activity, and calls into question the correlation between PEP activity and Ins(1,4,5)P 3 concentration. Therefore, Ins(1,4,5)P 3 concentration was investigated over an exten- ded period of total inhibition. As shown in Fig. 3, Ins(1,4,5)P 3 concentration in U343 cells increased during incubation. After 12 h, the total amount of Ins(1,4,5)P 3 (1.24 ± 0.24 pmol per 10 6 cells; n ¼ 4) was higher than the concentration measured in cell line as11 (0.95 ± 0.05 pmol per 10 6 cells; n ¼ 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)P 3 concentration with a similar dependence on the incubation time to that in U343 cells. However, the increase in Ins(1,4,5)P 3 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)P 3 concentration. Influence of PEP inhibition on the cAMP pathway In addition to Ins(1,4,5)P 3 , 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) P 3 accumulation after stimulation with Substance P To investigate whether the observed effect on Ins(1,4,5)P 3 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)P 3 [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. Thecelllinetransfectedwiththevectornotharbouringaninsert (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). 5816 I. Schulz et al.(Eur. J. Biochem. 269) Ó FEBS 2002 Substance 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 l M Substance P led to a rise in Ins(1,4,5)P 3 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)P 3 after Substance P stimulation. Comparing the total values after Substance P stimulation, the Ins(1,4,5)P 3 concentration again correlated with the impaired PEP activity (Fig. 4). The change in Ins(1,4,5)P 3 concentration during Substance P stimulation is illustrated in Fig. 5. To compare the stimulation-dependent increase in Ins(1,4,5)P 3 concentration, the amount of Ins(1,4,5)P 3 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)P 3 concentra- tion always occurred after 5 s stimulation. The stimulation 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)P 3 concentra- tion (Fig. 5B), the inhibitor-treated wild-type cells showed increased stimulation of Ins(1,4,5)P 3 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. DISCUSSION 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. Ó FEBS 2002 Ins(1,4,5)P 3 increase by prolyl endopeptidase (Eur. J. Biochem. 269) 5817 PEP inhibitors are in general very specific because of the proline residue in the P 1 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)P 3 concentration. Ins(1,4,5)P 3 , 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)P 3 and 1,2-diacylglycerol [19,20]. It is known that Ins(1,4,5)P 3 binds to its receptor located in the membrane of the endoplasmic reticulum and induces release of Ca 2+ , 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)P 3 concentration 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)P 3 stimulation 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)P 3 observed 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)P 3 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)P 3 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)P 3 signal after Substance P stimula- tion (Fig. 4), but the kinetic profile of the stimulation 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)P 3 signal 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)P 3 formation or is independent of this pathway. Such an alternative pathway includes the dephosphorylation of InsP 5 to Ins(1,4,5)P 3 by 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)P 3 , 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)P 3 concentration 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 P 1 proline 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. 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Modulation of inositol 1,4,5-triphosphate concentration by prolyl endopeptidase inhibition Ingo Schulz 1 , Bernd Gerhartz 1 , Antje Neubauer 1 ,