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(The FASEB Journal. 2003;17:17-27.)
© 2003 FASEB

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates endozepine release from cultured rat astrocytes via a PKA-dependent mechanism

OLFA MASMOUDI, PIERRICK GANDOLFO, JEROME LEPRINCE, DAVID VAUDRY, ALAIN FOURNIER*, CHRISTINE PATTE-MENSAH, HUBERT VAUDRY1 and MARIE-CHRISTINE TONON

European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U413, UA CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France; and
* INRS-Institut Armand-Frappier, Université du Québec, Pointe-Claire, PQ, Canada H9R1G6

1Correspondence: European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U413, UA CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry{at}univ-rouen.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Astroglial cells synthesize and release endozepines, neuropeptides that are related to the octadecaneuropeptide ODN. Glial cells also express PACAP/VIP receptors. We have investigated the possible effect of PACAP on the release of ODN-like immunoreactivity (ODN-LI) by cultured rat astrocytes. Administration of PACAP27 and PACAP38 induced a concentration-dependent increase in secretion of ODN-LI whereas VIP was ~1000-fold less potent. The maximum effect of PACAP38 occurred after 5 min, then gradually declined during the next 10 min. The stimulatory effects of PACAP and VIP were abrogated by the PACAP antagonist PACAP6–38. PACAP38 stimulated cAMP formation, activated polyphosphoinositide turnover, and provoked calcium mobilization from IP3-sensitive pools. The PKA inhibitor H89 suppressed PACAP-induced secretion of ODN-LI, whereas PLC inhibitor U73122 and the PKC inhibitor chelerythrine had no effect. In contrast, U73122 restored the stimulatory action of PACAP on ODN-LI release and cAMP formation during prolonged (15 min) incubation with the peptide, and this effect was prevented by PMA. The present results demonstrate that PACAP stimulates endozepine release through activation of PAC1 receptors coupled to the AC/PKA pathway. Our data also show that activation of the PLC/PKC pathway down-regulates the effect of PACAP on endozepine release.—Masmoudi, O., Gandolfo, P., Leprince, J., Vaudry, D., Fournier, A., Patte-Mensah, C., Vaudry, H., Tonon, M.-C. Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates endozepine release from cultured rat astrocytes via a PKA-dependent mechanism.


Key Words: endozepines • PAC1 receptor • PKA/PKC • intracellular Ca2+ • astroglial cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The term endozepines designates a family of neuropeptides originally isolated from rat brain extracts as endogenous ligands of benzodiazepine receptors (1) . All endozepines derive from diazepam binding inhibitor (DBI), a 10 kDa polypeptide that can generate, through proteolytic cleavage, several biologically active peptides including the octadecaneuropeptide ODN (DBI [33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 ]) and the triakontatetraneuropeptide TTN (DBI [17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 ]) (2 , 3) . It was initially proposed that DBI acts as an inverse agonist of central-type benzodiazepine receptors (2) . Subsequently, DBI and TTN were found to interact with peripheral-type benzodiazepine receptors (4 , 5) . More recently it was shown that DBI-derived peptides can activate a metabotropic receptor positively coupled to phospholipase C (6 7 8 9 10) . Intracerebroventricular injection of endozepines induces proconflict behavior (2) , provokes anxiogenic effects (11) , and inhibits food intake (12) .

In situ hybridization experiments have shown that in the brain, the DBI gene is primarily expressed in glial cells (13 14 15 16) . The occurrence of DBI-related peptides has been visualized by immunohistochemistry in astroglial cells in the cerebral cortex (17) , in ependymocytes lining the third ventricle (17 18 19) , in tanycytes in the median eminence (17 , 18) , and in Bergmann cells in the cerebellar cortex (17 , 20) . In vitro studies have shown that cultured rat astrocytes contain and release substantial amounts of endozepines (7 , 21) and that the secretion of endozepines is cAMP dependent (21) .

Pituitary adenylate cyclase-activating polypeptide (PACAP) was initially isolated from the ovine hypothalamus for its ability to stimulate cAMP formation in rat anterior pituitary cells (22) . PACAP exists in two biologically active forms of 38 and 27 amino acid residues, termed PACAP38 and PACAP27, respectively (22 , 23) . PACAP27 exhibits 68% sequence identity with vasoactive intestinal polypeptide (VIP), identifying PACAP as a member of the secretin/glucagon/VIP/growth hormone-releasing hormone superfamily of regulatory peptides (24 , 25) . Three distinct subtypes of receptors that exhibit differential affinities for PACAP and VIP have been identified: PAC1 receptors (PAC1-R) show high affinity for PACAP and a much lower affinity for VIP, whereas VPAC1 and VPAC2 receptors (VPAC1-R and VPAC2-R) recognize PACAP and VIP with high affinity (26 27 28) . Soon after the discovery of PACAP, the presence of specific PACAP binding sites was demonstrated on astroglial cells (29) . Subsequent studies have shown that all three PACAP receptors are expressed in rat astrocytes (30 , 31) . It has been reported that PACAP stimulates adenylyl cyclase activity in cultured astrocytes (32) . Taken together, these data suggest that astrocytes are main target cells for PACAP in the central nervous system.

Although glial cells express a large array of receptors for regulatory peptides (33) , the action of neuropeptides on the secretion of endozepines has never been examined. The aim of the present study was to investigate the effect of PACAP on endozepine release by rat astrocytes in primary culture and to determine the transduction pathways mediating the action of PACAP.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Dulbecco’s modified Eagle’s medium (DMEM), F12 culture medium, D(+)-glucose, bovine serum albumin, chelerythrine, forskolin, isobutylmetylxanthine (IBMX), trichloroacetic acid (TCA), trifluoroacetic acid (TFA), and U73122 were purchased from Sigma (St. Louis, MO). Acetonitrile was from Prolabo (Fontenay sous-Bois, France). Glutamine, N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) and the antibiotic antimycotic solution were obtained from BioWhittaker (Gagny, France). H89 was from Alexis (Laufelfingen, Switzerland). Fetal calf serum (FCS) was from Dutscher (Brumath, France). PACAP27 was from Novabiochem (Meudon, France). Rat VIP and PACAP6–38 were from American Peptide Co (Sunnyvale, CA). [4-Cl-D-Phe6, Leu17]-VIP and [Tyr°]-ODN were from Neosystem (Strasbourg, France). Myo-[3H]inositol (100 Ci/mmol) and Na125I (2000 Ci/mmol) were from Amersham International (Les Ulis, France). Fluo-4-acetoxymethyl ester was from Molecular Probes Europe (Leiden, Netherlands). Rat ODN, rat TTN and PACAP38 were synthesized by using the standard Fmoc procedure (34) .

Cell culture
Primary cultures of rat cortical type 1 astrocytes were prepared as described (6) . Cerebral hemispheres from newborn Wistar rats were collected in DMEM/F12 (2/1; v/v) culture medium supplemented with 2 mM glutamine, 1% insulin, 5 mM HEPES, 0.4% glucose, and 1% of the antibiotic antimycotic solution. Tissues were dissociated mechanically with a syringe equipped with a 1 mm gauge needle and filtered through a 100 µm sieve (Falcon, Franklin Lakes, NJ). Dissociated cells were resuspended in culture medium supplemented with 10% FCS and seeded at a density of 0.6 x 106 cells/mL. To measure endozepine secretion, cells were cultured in 60 mm dishes (Dutscher). To measure cAMP production and determine polyphosphoinositide metabolism, cells were seeded in 35 mm dishes (Dutscher). To measure intracellular calcium concentrations, astrocytes were cultured on 25 mm coverslips in 35 mm dishes. Cells were incubated at 37°C in a humid atmosphere (5% CO2) and the medium was changed twice a week. After 4 days in culture, ~90% of the cells were labeled with antibodies against glial fibrillary acidic protein and exhibited morphological characteristics of type 1 astrocytes (7) .

Measurement of endozepine secretion
Nine- to 12-day-old cultured cells were incubated at 37°C with fresh serum-free medium in the absence or presence of test substances. At the end of the incubation, culture media were collected and the cells were homogenized in 2 M ice-cold acetic acid. Culture media and cell extracts were purified on Sep-Pak C18 cartridges (Alltech Europe, Belgium). Bound material was eluted with 50% (v/v) acetonitrile/water containing 0.1% (v/v) TFA and dried by vacuum centrifugation (Speed Vac concentrator, Savant, Hicksville, NY) until HPLC analysis and radioimmunoassay (RIA).

Concentrations of ODN-like immunoreactivity (ODN-LI) were measured by RIA using an antiserum raised against synthetic rat ODN (17) . [Tyr°]-ODN was iodinated using the chloramine-T procedure (35) and purified on a Sep-Pak C18 cartridge. The ODN antiserum exhibited 100% cross-reactivity with rat TTN. Dried samples were resuspended in phosphate buffer (0.1 M; pH 7.4) containing 0.1% Triton X-100. Final dilution of the ODN antiserum was 1:30,000 and the total amount of tracer was 6000 cpm/tube. After 2 days incubation at 4°C, the antibody-bound ODN fraction was precipitated by addition of 100 µL bovine gamma globulin (1%, w/v) and 2 mL polyethylene glycol (20%, w/v). After centrifugation (5000 g, 4°C, 30 min), the supernatant was removed and the pellet containing the bound fraction was counted in a gamma counter (LKB Wallac, Rockville, MI).

Characterization of immunoreactive endozepines
Dried samples were resuspended in 800 µL 0.1% TFA and analyzed on a C18 column (250x4.6 mm, Lichrosorb; Touzart et Matignon, Courtaboeuf, France) equilibrated with 20% acetontrile/water/TFA (20:79.9:0.1; v/v/v) at a flow rate of 1 mL/min by using the gradient shown in Fig. 4 . Synthetic rat ODN and rat TTN (2 µg) were chromatographed in the same conditions as the biological samples. Fractions of 1 mL were collected, evaporated, and radioimmunoassayed in duplicate.



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  		Figure 4. Reversed-phase HPLC analysis of ODN-like immunoreactivity (ODN-LI) in incubation media of cultured rat astrocytes. Cells were incubated for 5 min in the absence (A) or presence of 10-10 M PACAP38 (B). Arrows indicate the elution position of synthetic rat ODN and TTN. The dashed lines show the concentration of acetonitrile in the eluting solvent.

Measurement of cAMP
Nine- to 12-day-old cultured cells were preincubated for 30 min with serum-free medium containing 100 µM IBMX. The cells were then incubated in the absence or presence of test substances. Incubation was stopped by removing the medium and adding 10% (w/v) ice-cold TCA. Cells were homogenized and centrifuged (14,000 g, 4°C, 10 min). The supernatant was washed three times with 1 mL water-saturated diethylether, dried, and reconstituted in RIA buffer (0.05 M sodium acetate, pH 5.8). The concentration of cAMP was measured by using a cAMP RIA kit (RPA 509; Amersham International). The pellets were used to measure protein concentration by the Lowry’s method.

Measurement of polyphosphoinositide metabolism
Nine- to 12-day-old culture cells were incubated at 37°C with 10 µCi/mL myo-[3H]inositol in glucose- and serum-free medium in the absence or presence of test substances. Incubation was stopped by removing the medium and adding 10% (w/v) ice-cold TCA. The cells were homogenized and centrifuged (13,000 g, 4°C, 10 min). The supernatant containing the phosphoinositols (IPs) was washed three times with 1 mL water-saturated diethylether neutralized with 10 µL of 1 M NaHCO3. Free [3H]inositol and [3H]IPs were separated by anion exchange chromatography (on AG1-X8 resin mini columns, 100–200 mesh, formate form, Bio-Rad Laboratories, Richmond, CA) using distilled water and 0.8 M ammonium formate in 0.1 M formic acid, respectively. The radioactivity contained in each fraction was counted in a beta counter (LKB 1217 Rack Beta, EG and G Wallac, Evry, France). [3H]Polyphosphoinositides ([3H]PIPs) were extracted from the pellet with 500 µL of chloroform/methanol (2:1, v/v) and counted in a beta counter. The remaining pellets were used to measure protein concentration by the Lowry method.

Measurement of intracellular calcium concentration
Five- to 7-day-old cultured cells were incubated at 37°C for 45 min with 5 µM fluo-4 acetoxymethyl ester (fluo-4 AM) diluted in culture medium. Thereafter, the cells were washed with fresh medium. Astrocytes were mounted in a thermostatically controlled perifusion chamber maintained at 37°C and continuously perfused with culture medium in the absence or presence of test substances. The cells were examined on a Noran OZ confocal laser scanning microscope (Noran Instrument, Middleton, WI) equipped with a krypton-argon laser (excitation wavelength: 488 nm) and the fluorescence emitted was recorded using a 500 nm long-pass filter. Images were recorded as a time series (512x480 pixels at 1 image per 532 ms) and data processing was carried out using Intervision Software (Noran Instrument).

Statistical analysis
All values presented in the figures are means ± SE. Student’s t test and ANOVA, followed by Bonferroni’s test, were applied to determine statistical differences between basal and experimental values.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of PACAP on endozepine release
Incubation of cultured astrocytes with PACAP38 (10-13-10-6 M) or PACAP27 (10-12-10-6 M) for 5 min induced a concentration-dependent stimulation of the secretion of ODN-LI with IC50 values of 1.5 x 10-12 and 4.7 x 10-11 M, respectively; the maximum effect was observed at a concentration of 10-9 M (Fig. 1 A). In contrast, VIP stimulated ODN-LI only at high concentrations with an IC50 value of 1.9 x 10-8 M and a maximum effect at 10-6 M (Fig. 1A ). Simultaneous administration of PACAP38 (10-10 M) and VIP (10-6 M) provoked an increase in secretion of ODN-LI not significantly different from that obtained with PACAP38 and VIP alone (Fig. 1B ). Preincubation of astrocytes for 15 min with the PACAP receptor antagonist PACAP6–38 (10-6 M) had no effect by itself on basal release of ODN-LI but totally abolished the stimulatory effect of PACAP38 (10-10 M) and VIP (10-6 M) (Fig. 2 A). In contrast, the specific VIP/PACAP receptor antagonist [4-Cl-D-Phe6, Leu17]-VIP (10-5 M; 15 min) did not affect PACAP- or VIP-evoked stimulation of ODN-LI release (Fig. 2B ). Time course experiments showed that PACAP38 (10-10 M) significantly enhanced the release of ODN-LI within 2 min and reached a maximum (+83%) after 5 min of incubation (Fig. 3 ). Thereafter, the effect of PACAP gradually declined and vanished 15 min after the onset of peptide administration. HPLC analysis of culture media from astrocytes incubated for 5 min in basal conditions, coupled to RIA quantification, revealed the existence of a major form of immunoreactive material that coeluted with synthetic rat TTN (Fig. 4 A). Administration of PACAP (10-10 M) enhanced the amount of immunoreactive peptide by 66% but did not provoke the appearance of additional molecular forms (Fig. 4B ).



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  		Figure 1. Effect of graded concentrations of PACAP38, PACAP27, and VIP on the release of ODN-like immunoreactivity (ODN-LI) from cultured rat astrocytes. A) Cells were incubated for 5 min with increasing concentrations of PACAP38 (10-13-10-6 M; {blacksquare}), PACAP27 (10-12-10-6 M; •), or VIP (10-12-10-6 M; {blacktriangleup}). Results are expressed as % of the control value ({diamond}). Each value is the mean (±SE) of 3 independent experiments performed in quintuplicate. B) Comparison of the effect of concomitant administration of PACAP38 (10-10 M) and VIP (10-6 M) with the effect of each peptide administrated individually. Results are expressed as % of the control value. Each value is the mean (±SE) of 3 independent experiments performed in quintuplicate. ANOVA, followed by the Bonferroni’s test: **P < 0.01; ***P < 0.001; NS, not statistically different. Mean basal level of ODN-LI release in these experiments was 440 ± 9 pg/dish.



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  		Figure 2. Effect of the PACAP receptor antagonist PACAP6–38 and the VIP/PACAP receptor antagonist [4-Cl-D-Phe6, Leu17]-VIP on release of ODN-like immunoreactivity (ODN-LI) from cultured rat astrocytes. Cells were preincubated for 15 min in the absence or presence of 10-6 M PACAP6–38 (A) or 10-5 M [4-Cl-D-Phe6, Leu17]-VIP (B). Then cells were incubated for 5 min with PACAP38 (10-10 M) or VIP (10-6 M) in the absence or presence of the antagonists. Results are expressed as % of the control value. Each value is the mean (±SE) of 3 independent experiments performed in quintuplicate. ANOVA, followed by the Bonferroni’s test: **P < 0.01; NS, not statistically different. Mean basal level of ODN-LI release in these experiments was 560 ± 2 pg/dish.



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  		Figure 3. Time course of the effect of PACAP38 on the release of ODN-like immunoreactivity (ODN-LI) from cultured rat astrocytes. Cells were incubated with PACAP38 (10-10 M) for the times indicated. Results are expressed as % of the control values. Each value is the mean (±SE) of 5 independent experiments performed in quintuplicate. ANOVA, followed by the Bonferroni’s test: *P < 0.05; ***P < 0.001. Mean basal level of ODN-LI release in these experiments was 610 ± 12 pg/dish.

Effect of PACAP on cAMP production
Exposure of cultured astrocytes to PACAP38 (10-10 M) induced a significant increase in cAMP content in cultured astrocytes within 2 min. The effect of PACAP on cAMP formation reached a maximum by 5 min and gradually declined during the next 10 min (Fig. 5 A). Incubation of astrocytes with graded concentration of PACAP (10-12-10-6 M; 5 min) induced a dose-dependent stimulation of cAMP formation (Fig. 5B ).



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  		Figure 5. Effect of PACAP38 on cAMP formation in cultured rat astrocytes. A) After a 30 min preincubation with 10-4 M IBMX, cells were incubated in the absence (control; {square}) or presence of PACAP38 (10-10 M; {blacksquare}) for the times indicated. Each value is the mean (±SE) of 3 independent experiments performed in quintuplicate. B) After a 30 min preincubation with 10-4 M IBMX, the cells were incubated for 5 min in the absence or presence of graded concentrations of PACAP38 (10-12-10-6 M). Results are expressed as % of the control value (open bar). Each value is the mean (±SE) of 4 independent experiments performed in quintuplicate. ANOVA, followed by the Bonferroni’s test: *P < 0.05; **P < 0.01; ***P < 0.001; NS, not statistically different.

Effect of PACAP on polyphosphoinositide metabolism
Exposure of cultured astrocytes to PACAP38 (10-10 M) induced a significant increase in [3H]IPs formation within 2 min. The effect of PACAP38 on [3H]IPs formation reached a maximum by 5 min and gradually declined during the next 10 min (Fig. 6 A). Concurrently, PACAP38 caused a decrease in [3H]PIPs levels (Fig. 6B ). In the presence of the phospholipase C inhibitor U73122 (10-5 M), the effect of PACAP38 on [3H]IPs and [3H]PIPs was abolished (Fig. 6C, D ).



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  		Figure 6. Effect of PACAP38 on inositol phosphate formation and polyphosphoinositide breakdown in cultured rat astrocytes. A, B) Cells were incubated in the absence or presence of PACAP38 (10-10 M; {blacksquare}). C, D) After 15 min preincubation period with U73122 (10-5 M), cells were incubated in the absence or presence of PACAP38 (10-10 M) for 5 min. Results are expressed as % of the control values. Each value is the mean (±SE) of 3 independent experiments performed in quintuplicate. ANOVA, followed by the Bonferroni’s test: *P < 0.05; **P < 0.01; ***P < 0.001; NS, not statistically different.

Effect of PACAP on intracellular calcium concentration
Exposure of cultured astrocytes to PACAP38 (10-6 M) induced a rapid and transient increase in intracellular calcium concentration [Ca2+]i, which peaked at 5 s. Thereafter, [Ca2+]i returned to basal levels within 30 s after PACAP administration (Fig. 7 A, B). For concentrations ranging from 10-10-10-6 M, PACAP provoked a dose-related increase in [Ca2+]i with an IC50 value of 5.1 x 10-8 M (Fig. 7C ). Incubation of cultured astrocytes with the specific IP3 receptor antagonist 2APB (50 µM; 10 min) abolished the stimulatory effect of PACAP on [Ca2+]i (Fig. 8 A). In contrast, the calcium chelator EGTA (6 mM; 5 min) did not affect PACAP-induced [Ca2+]i increase (Fig. 8B ).



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  		Figure 7. Effect of PACAP38 on [Ca2+]i in cultured rat astrocytes. A) Time series of pseudocolor images illustrating [Ca2+]i changes in cultured astrocytes loaded with fluo-4 acetoxymethylester. PACAP38 (10-6 M) was applied 10 s after the onset of image acquisition for 20 s, then cells were rinsed with culture medium alone. Sampling rate, 1 image per 532 ms. The pseudocolor scale indicates the corresponding [Ca2+]i changes expressed in arbitrary units. B) Profiles illustrating the time course of the variation of the fluorescence ratio evoked by PACAP38 administration in 4 selected cells indicated by colored arrows in panel A. Fo is the mean fluorescence intensity before the administration of PACAP and F is the fluorescence intensity at any time. C) Effect of graded concentrations of PACAP38 (10-10-10-6 M) on [Ca2+]i in cultured rat astrocytes. Each value is the mean fluorescence ratio (±SE) calculated from at least 3 different dishes from 5 independent cultures. Student’s t test. **P < 0.01; ***P < 0.001.



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  		Figure 8. Effects of the IP3 receptor antagonist 2APB and the calcium chelator EGTA on PACAP-evoked [Ca2+]i increase in cultured rat astrocytes. Cells were perfused with PACAP38 (10-6 M) for 20 s, then rinsed with culture medium alone. After a 10 min incubation with 50 µM 2APB (A) or 6 mM EGTA (B), the same cells were perfused with PACAP38 (10-6 M) for 20 s. The profiles are representative recordings from at least 3 independent cultures.

Involvement of the adenylyl cyclase-PKA pathway in the PACAP-evoked stimulation of ODN-LI release
Forskolin (10-5 M; 30 min) mimicked the stimulatory effect of PACAP38 (10-10 M; 5 min) on ODN-LI release (Fig. 9 A, B). The selective protein kinase A (PKA) inhibitor H89 (2x10-5 M; 30 min) significantly reduced the basal release of ODN-LI and totally suppressed forskolin-evoked stimulation of secretion of ODN-LI (Fig. 9A ). H89 abolished the stimulatory effect of PACAP on ODN-LI release (Fig. 9B ). Incubation of astrocytes with the phospholipase C (PLC) inhibitor U73122 (10-5 M; 15 min) or the protein kinase C (PKC) inhibitor chelerythrine (10-5 M; 30 min) did not affect basal and PACAP (5 min) -stimulated ODN-LI release (Fig. 9C, D ). Although U73122 (10-5 M) had no effect by itself, this PLC inhibitor induced a 45% increase in ODN-LI release within 15 min after PACAP administration (Fig. 10 ). Addition of PMA (10-5 M) suppressed the stimulatory effect on ODN-LI observed after a 15 min incubation with PACAP plus U73122 (Fig. 10) . Similarly, U73122 restored the stimulatory action of PACAP on cAMP production after a 15 min incubation with the peptide, and this effect was prevented by PMA (Fig. 11 ).



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  		Figure 9. Effects of the PKA inhibitor H89, the PLC inhibitor U73122, and the PKC inhibitor chelerythrine on basal and forskolin- or PACAP-stimulated release of ODN-like immunoreactivity (ODN-LI) from cultured rat astrocytes. A, B) Cells were preincubated for 30 min in the absence or presence of 2 x 10-5 M H89, then incubated with medium alone or forskolin (10-5 M; 30 min; A) or PACAP38 (10-10 M; 5 min; B). C) Cells were preincubated for 15 min in the absence or presence of U73122 (10-5 M), then incubated for 5 min with medium alone or PACAP38 (10-10 M). D) Cells were preincubated for 30 min in the absence or presence of chelerythrine (10-6 M), then incubated for 5 min with medium alone or PACAP38 (10-10 M). Results are expressed as % of control values. Each value is the mean (±SE) of 3 independent experiments performed in quintuplicate. ANOVA, followed by the Bonferroni’s test: **P < 0.01; ***P < 0.001; NS, not statistically different. Mean basal level of ODN-LI release in these experiments was 420 ± 11 pg/dish.



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  		Figure 10. Effect of the PLC inhibitor U73122 and the PKC activator PMA on the release of ODN-like immunoreactivity (ODN-LI) from cultured rat astrocytes during prolonged administration of PACAP38. Cells were incubated for 15 min with PACAP38 (10-10 M) in the absence or presence of U73122 (10-5 M), PMA (10-5 M), or U73122 plus PMA (10-5 M each). Results are expressed as % of the control value. Each value is the mean (±SE) of 3 independent experiments performed in quintuplicate. ANOVA, followed by the Bonferroni test: **P < 0.01; NS, not statistically different. Mean basal level of ODN-LI release in these experiments was 540 ± 6 pg/dish.



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  		Figure 11. Effect of the PLC inhibitor U73122 and the PKC activator PMA on cAMP formation in cultured rat astrocytes during prolonged administration of PACAP38. Cells were preincubated with IBMX (10-4 M; 30 min), then incubated for 15 min with PACAP38 (10-10 M) in the absence or presence of U73122 (10-5 M), PMA (10-5 M), or U73122 plus PMA (10-5 M each). As a control, cell were preincubated for 30 min with forskolin plus IBMX (10-5 M and10-4 M, respectively), then incubated for 15 min in the absence or presence of PMA (10-5 M). Results are expressed as % of the control value. Each value is the mean (±SE) of 3 independent experiments performed in quintuplicate. ANOVA, followed by the Bonferroni’s test: **P < 0.01; NS, not statistically different.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It has been shown that endozepines are synthesized and released by cultured rat astrocytes (7 , 21) and demonstrated that astroglial cells express all three isoforms of PACAP and VIP receptors (29 , 36 , 37) . The present study reveals that PACAP stimulates the release of endozepines from rat astrocytes through activation of PAC1 receptors coupled to adenylyl cyclase (AC) and PLC and that only the adenylyl cyclase/PKA transduction pathway is involved in the effect of PACAP on endozepine secretion.

Rat astrocytes express the PACAP-specific receptor PAC1-R and the two VIP/PACAP mutual receptors VPAC1-R and VPAC2-R (31 , 38) . Our data show that subnanomolar concentrations of PACAP38 and PACAP27 stimulated the release of ODN-LI dose dependently whereas VIP was at least 1000-fold less potent. The PACAP antagonist PACAP6–38 totally suppressed the stimulatory effect of PACAP whereas the selective VPAC1/2 receptor antagonist [4-Cl-D-Phe6, Leu17]-VIP was ineffective. Although high concentrations of VIP could stimulate the release of ODN-LI, the effects of PACAP and VIP were not additive. Altogether, these observations demonstrate that PACAP stimulates endozepine secretion from rat astrocytes and that this effect is mediated exclusively through activation of PAC1-R.

It has been reported that in cultured rat astrocytes, intracellular processing of DBI generates TTN but not ODN, and it has been found that the cleavage of TTN to produce ODN occurs exclusively in the extracellular medium (21) . In the present study, reversed-phase HPLC analysis of culture media confirmed that the ODN immunoreactive peptide released by rat astrocytes coeluted with synthetic rat TTN and revealed that PACAP, though stimulating the secretion of TTN, did not affect processing of the peptide.

Alternative splicing of the primary transcript encoding PAC1-R has the potential to generate a series of variants exhibiting distinct pharmacological properties. Insertion of various cassettes (hip, hop1, hop2, or hip-hop1) in the third intracellular loop region determines the coupling efficacy of PAC1-R to AC and PLC (25 , 39) . RT-PCR analysis has recently shown that rat astrocytes express the short isoform and the hop2 variant of PAC1-R (30 , 38) , both coupled simultaneously to AC and PLC (40) . We therefore investigated which signaling cascade was involved in PACAP-induced endozepine release. Several lines of evidence indicate that the stimulatory effect of PACAP is mediated through activation of the AC/PKA pathway. 1) In agreement with previous reports (31 , 32 , 41) , we found that PACAP provoked a dose-dependent increase in cAMP production in cultured rat astrocytes; 2) the time course of the effect of PACAP on cAMP formation paralleled that observed for the release of ODN-LI; 3) we earlier found that the cell-permeant cAMP analog dbcAMP provokes the release of endozepines from cultured astrocytes (21) , and we now show that forskolin mimics the stimulatory effect of PACAP on ODN-LI secretion; and 4) the specific PKA inhibitor H89 abolished PACAP-induced endozepine release but the PKC inhibitor chelerythrine did not affect PACAP-evoked ODN-LI secretion. Taken together, these data indicate that the effect of PACAP on endozepine secretion can be ascribed solely to activation of the AC/PKA pathway.

The absence of a typical signal sequence in the primary structure of DBI (14 , 42) hampers its targeting to the endoplasmic reticulum (17 , 43) , and the mechanism by which endozepines are secreted from astroglial cells is currently unknown. It has been shown that release of interleukin-1ß (another leaderless secretory protein) from macrophages in monolayers requires activation of the ATP binding cassette (ABC) ABC1 transporter (44) . Several observations suggest that members of the ABC transporter family may play a role in endozepine release from astrocytes. 1) Expression of functional P-glycoprotein and multidrug resistance-associated proteins of the ABC transporter family have been identified in astrocytes (45 , 46) ; 2) ABC transporters possess consensus sites for PKA-dependent phosphorylation on their regulatory domain (47 , 48) ; and 3) cAMP has been shown to activate ABC1 transporters in Xenopus laevis oocytes (49) .

To further explore the mechanism by which PACAP regulates endozepine secretion in astroglial cells, we studied the effect of the peptide on calcium mobilization. Using a video imaging confocal microscopy technique, we found that PACAP consistently provoked a rapid and robust increase in [Ca2+]i. Incubation of astrocytes with the IP3 receptor antagonist 2APB suppressed the effect of PACAP on [Ca2+]i whereas reduction of calcium concentration in the extracellular medium by EGTA did not affect the Ca2+ response, indicating that PACAP causes mobilization of intracellular calcium pools. The observation that PACAP stimulated polyphosphoinositide turnover provides additional evidence that the PACAP-evoked Ca2+ wave resulted from mobilization of IP3-sensitive Ca2+ pools. Whereas PACAP induced a dose-dependent increase of [Ca2+]i in type 1 astrocytes from postnatal day 1 rat (this study), the peptide was found to have no effect on type 1 astrocytes from embryonic day 17 rat (50 , 51) . Consistent with this observation, it was recently shown that different sets of PAC1 receptor isoforms are expressed in rat astrocytes during development (38) . Although the [Ca2+]i increase induced by PACAP can be accounted for by activation of a PLC, the PLC inhibitor U73122 did not affect basal and PACAP-stimulated endozepine secretion, indicating that intracellular calcium is not involved in PACAP-evoked endozepine release. The physiological role of PACAP-evoked intracellular calcium mobilization is currently unknown. It was recently reported that glutamate and the neuropeptide precursor secretogranine II are released from rat astrocytes in a calcium-dependent manner (52 , 53) . Calcium may thus serve as an intracellular messenger mediating the effect of PACAP on the release of vesicular neurotransmitters and/or neuropeptides by astroglial cells. Alternatively, PACAP has been shown to stimulate astrocyte proliferation (41) , suggesting that PACAP-evoked [Ca2+]i increase may mediate the neurotrophic activity of the peptide.

Time course studies revealed that PACAP provoked a rapid and transient stimulation of ODN-LI release. The maximum stimulatory effect occurred within 5 min and the secretion of endozepines returned to basal level within 15 min after the onset of PACAP administration, suggesting that subnanomolar concentrations of PACAP induce rapid desensitization of the glial cell response. In support of this hypothesis, homologous desensitization after exposure to low concentrations of PACAP has been reported in {alpha}T3–1 gonadotroph-derived and Y-79 retinoblastoma cell lines (54 , 55) . Two observations indicate that in glial cells, the down-regulation of the response observed during prolonged (15 min) exposure to PACAP can be ascribed to activation of the PLC/PKC pathway. First, the PLC inhibitor U73122 partially restored the stimulatory effect of PACAP on endozepine release and cAMP formation. Second, these effects of U73122 were totally abolished by the phorbol ester PMA. Since PMA did not affect forskolin-evoked cAMP production, it appears that the desensitization process can be accounted for by PKC-dependent phosphorylation of PACAP receptors. Consistent with this notion, it was recently shown that in Y-79 retinoblastoma cells, activation of PKC by PMA causes heterologous desensitization of PAC1-R (55) . Although during the first 5 min PACAP induced a substantial rise in ODN-LI, after a 15 min exposure to PACAP, the amount of ODN-LI released by cultured astrocytes returned to control level. This observation indicates that endozepines released in the incubation medium are continuously eliminated. The clearance of ODN-LI can likely be ascribed to accumulation of proteolytic enzymes that are responsible for the degradation of ODN-related peptides. Consistent with this hypothesis, it has been shown that cultured astrocytes produce several endopeptidases involved in the proteolytic degradation of various neuropeptides (56 57 58) .

In conclusion, the present study has shown that PACAP, acting through PAC1 receptors positively coupled to adenylyl cyclase and phospholipase C, stimulates the release of endozepines. Even though the effect of PACAP on endozepine secretion can be ascribed solely to activation of the AC/PKA signaling cascade, activation of the PLC/PKC transduction pathway is responsible for the rapid desensitization of PAC1 receptors and thus for down-regulation of the effects of PACAP on endozepine release.


   ACKNOWLEDGMENTS
 
The authors wish to thanks Mrs. Catherine Buquet, Mrs. Hugette Lemonnier, and Mr. Gérard Cauchois for skillful technical assistance. This study was supported by INSERM (U413), an INSERM-FRSQ exchange program, and the Conseil Régional de Haute-Normandie. O.M. was the recipient of a scholarship from the French and Tunisian Ministry of Research. H.V. is Affiliated Professor at the INRS-Institut Armand Frappier (Montréal, Canada).

Received for publication May 8, 2002. Accepted for publication September 9, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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