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• W2077760444 abstract "The study of signaling cascades and of functional interactions between 5-hydroxytryptamine (5-HT) receptor pathways with heterogenous brain cell populations remains an arduous task. We took advantage of a serotonergic cell line to elucidate cross-talks between 5-HT receptors and to demonstrate the involvement of two 5-HT2 receptor subtypes in the regulation of 5-HT1B/1D function. The inducible 1C11 cell line has the unique property of acquiring within 4 days a complete serotonergic phenotype (1C11* cells), including three 5-HT receptors. 5-HT1B/1D and 5-HT2B receptors are expressed since day 2 of the serotonergic differentiation while 5-HT2A receptors are induced at day 4. We first established that 5-HT2B receptors are coupled with the phospholipase A2 (PLA2)-mediated release of arachidonic acid (AA) and that the activation of 5-HT2B receptors in 1C11*d2 cells inhibits the 5-HT1B/1D receptor function via a cyclooxygenase-dependent AA metabolite. At day 4, this 5-HT2B-mediated inhibition of the 5-HT1B/1D function can be blocked upon concomitant 5-HT2A activation although a 5-HT2A/PLA2 positive coupling was evidenced. This suggests the existence in 1C11*d4 cells of pathway(s) for 5-HT2A receptors, distinct from PLC and PLA2. Finally, this study reveals the antagonistic roles of 5-HT2A and 5-HT2B receptors in regulating the function of 5-HT1B/1D, a receptor involved in neuropsychiatric disorders and migraine pathogenesis. The study of signaling cascades and of functional interactions between 5-hydroxytryptamine (5-HT) receptor pathways with heterogenous brain cell populations remains an arduous task. We took advantage of a serotonergic cell line to elucidate cross-talks between 5-HT receptors and to demonstrate the involvement of two 5-HT2 receptor subtypes in the regulation of 5-HT1B/1D function. The inducible 1C11 cell line has the unique property of acquiring within 4 days a complete serotonergic phenotype (1C11* cells), including three 5-HT receptors. 5-HT1B/1D and 5-HT2B receptors are expressed since day 2 of the serotonergic differentiation while 5-HT2A receptors are induced at day 4. We first established that 5-HT2B receptors are coupled with the phospholipase A2 (PLA2)-mediated release of arachidonic acid (AA) and that the activation of 5-HT2B receptors in 1C11*d2 cells inhibits the 5-HT1B/1D receptor function via a cyclooxygenase-dependent AA metabolite. At day 4, this 5-HT2B-mediated inhibition of the 5-HT1B/1D function can be blocked upon concomitant 5-HT2A activation although a 5-HT2A/PLA2 positive coupling was evidenced. This suggests the existence in 1C11*d4 cells of pathway(s) for 5-HT2A receptors, distinct from PLC and PLA2. Finally, this study reveals the antagonistic roles of 5-HT2A and 5-HT2B receptors in regulating the function of 5-HT1B/1D, a receptor involved in neuropsychiatric disorders and migraine pathogenesis. In view of the pivotal role of serotonin (5-hydroxytryptamine (5-HT)) 1The abbreviations used are: 5-HT, 5-hydroxytryptamine; 5-CT, 5-carboxamidotryptamine; AA, arachidonic acid; BSA, bovine serum albumin; Bt2cAMP, dibutyryl-cAMP; CCA, cyclohexanecarboxylic acid; DAG, 1,2-diacylglycerol; DMEM, Dulbecco's modified Eagle's medium; DOI, (±)-1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane; FScA, forskolin-stimulated cAMP accumulation; Gi, GTP-binding protein; HBSS, Hanks' balanced salt solution; IP, inositol phosphates; IP3, inositol 1,4,5-trisphosphate; PI, phosphatidylinositol; PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospolipase C. 1The abbreviations used are: 5-HT, 5-hydroxytryptamine; 5-CT, 5-carboxamidotryptamine; AA, arachidonic acid; BSA, bovine serum albumin; Bt2cAMP, dibutyryl-cAMP; CCA, cyclohexanecarboxylic acid; DAG, 1,2-diacylglycerol; DMEM, Dulbecco's modified Eagle's medium; DOI, (±)-1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane; FScA, forskolin-stimulated cAMP accumulation; Gi, GTP-binding protein; HBSS, Hanks' balanced salt solution; IP, inositol phosphates; IP3, inositol 1,4,5-trisphosphate; PI, phosphatidylinositol; PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospolipase C. in neurologic and neuropsychiatric disorders, to identify the mechanisms that mediate the cellular functions controlled by 5-HT is a major challenge. To achieve this goal, the study of signaling cascades and of functional interactions between 5-HT receptor pathways with heterogenous brain cell populations still appears impractical. An alternative is to use a clonal cell line, such as 1C11, expressing a definite set of 5-HT receptors (1Kellermann O. Loric S. Maroteaux L. Launay J.-M. Br. J. Pharmacol. 1996; 118: 1161-1170Crossref PubMed Scopus (32) Google Scholar). The 1C11 clone was derived from F9 multipotential embryonal carcinoma cells through immortalization and differentiation (2Kellermann O. Kelly F. Differentiation. 1986; 32: 74-81Crossref PubMed Scopus (27) Google Scholar, 3Buc-Caron M.-H. Launay J.-M. Lamblin D. Kellermann O. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1922-1926Crossref PubMed Scopus (40) Google Scholar). The 1C11 clone has the properties of a neuroectodermal progenitor able to differentiate into 5-hydroxytryptaminergic cells (1C11*) by induction by dibutyryl cyclic AMP (Bt2cAMP) and cyclohexane carboxylic acid (CCA). The switch from the undifferentiated committed 1C11 cell type to the 1C11* serotonergic cells occurs within 4 days in more than 90% of the cell population. We recently demonstrated that, in addition to the onset of 5-HT metabolism, storage, and transport, 1C11* cells also acquire 5-HT1B/1D, 5-HT2A, and 5-HT2B receptors (1Kellermann O. Loric S. Maroteaux L. Launay J.-M. Br. J. Pharmacol. 1996; 118: 1161-1170Crossref PubMed Scopus (32) Google Scholar). Binding and transductional experiments excluded the functional presence of any other 5-HT receptor subtypes. On day 2 of the serotonergic differentiation, 5-HT1B/1D and 5-HT2B receptors became expressed and remained functional until at least day 4, at which time the 5-HT2A receptor was induced. The appearance of the latter receptor coincided with the onset of an active 5-HT transport system. This sequence of events in the inducible 1C11 cell line offers the possibility of exploring cross-talks between the signaling pathways of these three G-protein coupled receptors within a complete serotonergic phenotype. Many studies have established that 5-HT1B/1D receptors are negatively coupled with adenylate cyclase through Gi (GTP-binding protein) (4Hoyer D. Schoeffter P. Eur. J. Pharmacol. 1988; 147: 145-147Crossref PubMed Scopus (61) Google Scholar). Acting as terminal or somatodendritic autoreceptors, they mediate and modulate serotonergic neurotransmission (5Glennon R.A. Westkaemper R.B. Drug. News. Perspect. 1993; 6: 390-405Google Scholar). They can also function as terminal heteroreceptors by inhibiting, after agonist stimulation, the release of other neurotransmitters such as acetylcholine, glutamate, dopamine, norepinephrine, and γ-aminobutyric acid (6Raiteri M. Maura G. Bonanno G. Pittaluga A. J. Pharmacol. Exp. Ther. 1986; 237: 644-648PubMed Google Scholar, 7Galloway M.P. Suchowski C.S. Keegan M.J. Hjorth S. Synapse. 1993; 15: 90-92Crossref PubMed Scopus (67) Google Scholar, 8Iyer R.N. Bradberry C.W. J. Pharmacol. Exp. Ther. 1996; 277: 40-47PubMed Google Scholar). Thus, signals interfering with the Gi-coupled 5-HT1B/1D system may act widely on neuronal responses. In addition, 5-HT1B/1D receptors are involved in cardiovascular functions, vasospasm, and migraine (9Fozard J.R. Kalkman H.O. Curr. Opin. Neurol. Neurosurg. 1992; 5: 496-502PubMed Google Scholar). Because of both the pivotal role of 5-HT1B/1D receptors in neurotransmission and the clinical impact of 5-HT1B/1D receptors agonists like sumatriptan (10Branchek T. Audia J.E. Annu. Rep. Med. Chem. 1997; 32: 1-10Google Scholar) in the acute treatment of migraine, studying the regulatory aspects of 5-HT1B/1D receptor function is of great importance. Two distinct receptors of the 5-HT2 class are also induced during the serotonergic differentiation of 1C11 cells. 5-HT2A and 5-HT2B receptors display a strong amino acid sequence homology and share close but distinct pharmacological profiles (11Hoyer D. Clarke D.E. Fozard J.R. Hartig P.R. Martin G.R. Mylecharane E.J. Saxena P.R. Humphrey P.P.A. Pharmacol. Rev. 1994; 46: 157-203PubMed Google Scholar). Each of these receptors have been described as mediating phosphatidylinositol (PI) hydrolysis through activation of phospholipase C (PLCβ). The 1,2-diacylglycerol (DAG) thus obtained activates PKC (12Nishizuka Y. Nature. 1988; 334: 661-665Crossref PubMed Scopus (3523) Google Scholar), whereas inositol 1,4,5-triphosphate (IP3) increases intracellular Ca2+ (13Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6151) Google Scholar). Nevertheless, the 5-HT2B receptor fails to significantly stimulate PI hydrolysis in rat stomach (14Cohen M.L. Wittenauer L.A. J. Cardiovasc. Pharmacol. 1987; 10: 176-181Crossref PubMed Scopus (37) Google Scholar) as well as in rat vasculature (15Ellis E.S. Byrne C. Murphy O.E. Tilford N.S. Baxter G.S. Br. J. Pharmacol. 1995; 114: 400-404Crossref PubMed Scopus (82) Google Scholar). This suggests that signaling processes coupled to 5-HT2B receptors, but distinct from PLC, may also occur in vivo. Accordingly, Launay et al. (16Launay J.-M. Birraux G. Bondoux D. Callebert J. Choi D.-S. Loric S. Maroteaux L. J. Biol. Chem. 1996; 271: 3141-3147Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) have reported a 5-HT2B-dependent activation of the p21ras/MAP kinase signaling cascade. In addition, 5-HT2 receptor subtypes are found in regions of the hippocampus and cerebral cortex where 5-HT stimulates the neuronal secretion of arachidonic acid (AA) (17Pazos A. Cortes R. Palacios J.M. Brain Res. 1985; 346: 231-249Crossref PubMed Scopus (814) Google Scholar, 18Felder C.C. Kanterman R.Y. Ma A.L. Axelrod J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2187-2191Crossref PubMed Scopus (189) Google Scholar). Both 5-HT2A and 5-HT2C, which is a third member of the 5-HT2 receptor family, are likely to be involved in this release because the stable transfection of CHO cell lines with the corresponding human cDNAs activates the phospholipase A2 (PLA2)-mediated AA release (19Berg K.A. Clarke W.P. Sailstad C. Saltzman A. Maayani S. Mol. Pharmacol. 1994; 46: 477-484PubMed Google Scholar, 20Berg K.A. Maayani S. Clarke W.P. Mol. Pharmacol. 1996; 50: 1017-1023PubMed Google Scholar). The PLA2/AA pathway could also be sensitive to 5-HT2B as indicated by the colocalization of this receptor with the 5-HT2A and 5-HT2C ones in the hippocampus (21Palacios J.M. Waeber C. Hoyer D. Mengod G. Whitaker-Azimitia P.M. Peroutka S.J. Annals of the New York Academy of Sciences. New York Academy of Sciences, New York1990: 36-52Google Scholar, 22Choi D.S. Maroteaux L. FEBS Lett. 1996; 91: 45-51Crossref Scopus (110) Google Scholar, 23Duxon M.S. Flanigan T.P. Reavley A.C. Baxter G.S. Blackburn T.P. Fone K.C. Neuroscience. 1997; 76: 323-329Crossref PubMed Scopus (193) Google Scholar). In this report, we establish that the 5-HT2B and 5-HT2A receptors of 1C11*cells are coupled with a PLA2-mediated release of arachidonic acid. Moreover, activation of the 5-HT2B receptor inhibits the 5-HT1B/1D receptor function, via a cyclooxygenase dependent AA metabolite. This 5-HT2B-mediated inhibition of the 5-HT1B/1D function can be blocked by a concomitant 5-HT2A activation. Forskolin was purchased from Calbiochem (San Diego, CA); myo-[3H]inositol (≈ 20.9 Ci/mmol); [14C]AA (≈ 57 mCi/mmol) from NEN Life Science Products; 5-carboxamidotryptamine (5-CT), 5-HT HCl, and DOI from Research Biochemicals (Natick, MA). Indomethacin came from Biomol Research Laboratories (Plymouth Meeting, PA); and mepacrine, melittin, and hygromycin came from Sigma. Rolipram and LY266070 were synthesized in Hoffman-La Roche AG (Basel, Switzerland) according to the procedure described by Audia et al. (24Audia J.E. Evrard D.A. Murdoch G.R. Droste J.J. Nissen J.S. Schenck K.W. Fludzinski P. Lucaites V.L. Nelson D.L. Cohen M.L. J. Med. Chem. 1996; 39: 2773-2780Crossref PubMed Scopus (134) Google Scholar) for LY266070. All tissue culture reagents and HBSS (Hanks' balanced salt solution) were purchased from Life Technologies, Inc. All other drugs and chemicals (reagent grade) were produced by Sigma. 1C11 cells were grown and induced to differentiate in the presence of 1 mm Bt2cAMP and 0.05% CCA (3Buc-Caron M.-H. Launay J.-M. Lamblin D. Kellermann O. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1922-1926Crossref PubMed Scopus (40) Google Scholar). Due to the sequential onset of 5-HT receptors after differentiation (1Kellermann O. Loric S. Maroteaux L. Launay J.-M. Br. J. Pharmacol. 1996; 118: 1161-1170Crossref PubMed Scopus (32) Google Scholar), 1C11* cells were used 2 days (1C11*d2 cells) (5-HT1B/1D and 5-HT2B) or 4 days (1C11*d4 cells) (5-HT1B/1D, 5-HT2B, and 5-HT2A) after addition of the inducers. Cells were maintained in DMEM (Dulbecco's modified Eagle's medium) supplemented with 5-HT-depleted fetal calf serum (10%) (16Launay J.-M. Birraux G. Bondoux D. Callebert J. Choi D.-S. Loric S. Maroteaux L. J. Biol. Chem. 1996; 271: 3141-3147Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) and 300 μg/ml hygromycin. For all experiments, cells were seeded onto 12- or 24-well tissue culture vessels at a density of 4 × 104cells/cm2. After a 24-h plating period, cells were washed with HBSS and grown for 24-h, before all experiments, in serum-free medium (DMEM/F-12 (1:1) with 5 μg/ml insulin, 5 μg/ml transferrin, 30 nm selenium, 20 nm progesterone, and 100 μm putrescine). Cells were washed twice with HBSS (with calcium and magnesium) containing 10 mm HEPES (pH 7.4) and then preincubated in 500 μl of 10 mm HEPES (pH 7.4) (wash buffer) per well for 15 min in a CO2 incubator (5% at 37 °C). Where indicated, inhibitors were added during this preincubation period. 5-HT1B/1D receptor-mediated response was followed by measuring (15 min at 37 °C) the 5-CT-induced inhibition of cAMP accumulated in the presence of 1 μm forskolin and 0.1 mmrolipram, a phosphodiesterase inhibitor. Cellular cAMP content was measured by radioimmunoassay (1Kellermann O. Loric S. Maroteaux L. Launay J.-M. Br. J. Pharmacol. 1996; 118: 1161-1170Crossref PubMed Scopus (32) Google Scholar) and normalized to protein content as determined by the bicinchoninic acid assay (Pierce, Chichester, UK). For each experiment, data were expressed according to the response obtained with 1 μm forskolin (100%). Cells were labeled with 1 μCi/ml myo-[3H]inositol in serum-free medium for 24 h at 37 °C. Total IP (inositol monophosphate, inositol bisphosphate, and IP3 are collectively referred to as IP) accumulation in response to agonist stimulation in the presence of 20 mmLiCl for 10 min at 37 °C was determined as described previously (25Berthou L. Corvaı̈a N. Geoffroy C. Mutel V. Launay J.-M. Alouf J.E. Eur. J. Cell Biol. 1992; 58: 377-382PubMed Google Scholar). Radiolabeled IP was separated according to the ion exchange method of Berridge et al. (26Berridge M.J. Dawson R.M. Downes C.P. Heslop J.P. Irvine R.F. Biochem. J. 1983; 212: 473-482Crossref PubMed Scopus (1537) Google Scholar). Cells were labeled with 0.1 μCi/ml [14C] AA for 4 h at 37 °C (5% CO2). Under these conditions, more than 90% of total radioactivity was taken up by the cells. After labeling, cells were washed three times with HBSS containing calcium and magnesium supplemented with 20 mm HEPES and 0.1% BSA (bovine serum albumin) (experimental medium). For calcium-free experiments, cells were washed with the same experimental medium without calcium. Between two washes, cells were incubated for 5 min at 37 °C. After the last wash, cells in 1 ml of experimental medium were exposed to the indicated drugs, and aliquots (100 μl) were removed after 10 min for [14C] measurement. Concentration-response data were fitted by non-linear regression to the model: R = (Rmax)/[1 + (EC50/A)n], whereE is the measured response at the given agonist concentration A, Rmax is maximal response, EC50 is the concentration of agonist producing half-maximal response, and n is the slope index. The non-parametric Kolmogorov-Smirnov test (KS test) was used for statistical comparisons. 1C11 cells were preincubated with 0.1 μCi/ml [14C]AA for 4 h, washed, and later exposed either to 5-HT or to the 5-HT2 receptor agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Total [14C] released from the cells into the medium was measured after 10 min. With undifferentiated 1C11 cells, no [14C] release could be detected. In contrast, at day 2 of the serotonergic differentiation, when 5-HT2B and 5-HT1B/1D receptors had become induced, a basal level of [14C] release (48 ± 5 nm) was observed. In addition, 1C11*d2 cells released higher amounts of [14C] radioactivity in response to 5-HT or DOI (Fig. lA). As assessed by high performance liquid chromatography, [14C] AA contributes to 90% of the total [14C] release. This release increased with the drug concentration, with a maximal response to 5-HT reaching 126 ± 10% (108.5 ± 4.8 nm) above the basal release and an associated EC50 value of 1.67 μm (p EC50 = 5.78 ± 0.14; mean ± S.E., n = 4). DOI behaved as a partial agonist by eliciting a maximal response of 87 ± 9% above basal release with an EC50 value of 29 nm (pEC50 = 7.54 ± 0.12; mean ± S.E.,n = 6). These two EC50 values are similar to those previously obtained for 5-HT and DOI-mediated IP3 accumulation in 1C11*d2 cells (27Loric S. Maroteaux L. Kellermann O. Launay J.-M. Mol. Pharmacol. 1995; 47: 458-466PubMed Google Scholar). After incubation of 1C11*d2 cells for 10 min with 2.5 μg/ml melittin, a direct activator of PLA2 (28Clark M.A. Conway T.M. Schorr R.G.L. Crooke S.T. J. Biol. Chem. 1987; 262: 4402-4406Abstract Full Text PDF PubMed Google Scholar), the release of 14C radioactivity increased up to 252 ± 19% above the basal release (mean ± S.E., n = 4). To determine whether the observed [14C]AA release originated from activation of PLA2 or derived from the PLC/DAG lipase pathway, 14C-labeled 1C11*d2 cells were exposed for 15 min to 100 μm mepacrine, a PLA2 inhibitor, prior to the addition of agonists. As shown in Fig. 1 B, this completely inhibited the effect of up to 10 μm 5-HT on the release of [14C] radioactivity. The involvement in [14C] release of 5-HT2B receptors via PLA2 coupling could be further evinced by using DOI treatment (1 μm). DOI-mediated AA release was also fully blocked by mepacrine (Fig. lB). Mepacrine, however, had no significant effect on DOI-mediated PI hydrolysis. Cellular IP accumulation measured after DOI treatment in the presence of 100 μm mepacrine, amounted to 124 ± 18% above the basal level (mean ± S.E.,n = 5, p > 0.05). Accumulation was very similar in the absence of mepacrine (117 ± 16%). Mepacrine alone had no effect on IP accumulation, as well as on basal AA release. Unlike phospholipase C or phospholipase D, cytosolic PLA2 requires extracellular calcium for its activity (29Irvine R.F. Biochem. J. 1982; 204: 3-16Crossref PubMed Scopus (966) Google Scholar). Accordingly, when calcium was removed from the incubation medium of 1C11*d2 cells, 5-HT-mediated AA release was reduced to 11 ± 7% above the basal level (mean ± S.E., n = 4). We verified that the basal [14C] release level was not sensitive to the deprivation of calcium. All these data indicate that the 5-HT2B receptor of 1C11*d2 cells is coupled with the cytosolic PLA2 activity. 5-HT1B/1D receptors are linked to Gi-mediated inhibition of adenylate cyclase activity (11Hoyer D. Clarke D.E. Fozard J.R. Hartig P.R. Martin G.R. Mylecharane E.J. Saxena P.R. Humphrey P.P.A. Pharmacol. Rev. 1994; 46: 157-203PubMed Google Scholar) and become expressed on day 2 of 1C11 cell differentiation (1Kellermann O. Loric S. Maroteaux L. Launay J.-M. Br. J. Pharmacol. 1996; 118: 1161-1170Crossref PubMed Scopus (32) Google Scholar). 5-CT, as do other 5-HT1B/1D receptor agonists, inhibits forskolin-stimulated cAMP accumulation (FScA). In contrast, the addition of 5-HT2 receptor agonists had no effect on FScA as well as on the basal cAMP cellular level (27Loric S. Maroteaux L. Kellermann O. Launay J.-M. Mol. Pharmacol. 1995; 47: 458-466PubMed Google Scholar). On the other hand, the IP3 content of 1C11* cells does not respond to the addition of 5-HT1B/1D agonists (1Kellermann O. Loric S. Maroteaux L. Launay J.-M. Br. J. Pharmacol. 1996; 118: 1161-1170Crossref PubMed Scopus (32) Google Scholar). This suggests that the IP3 hydrolysis pathway mediated by 5-HT2B activation does not interfere with the negative coupling of adenylate cyclase with 5-HT1B/1D receptor. As shown in Fig. 2, 5-CT (5 nm) inhibits FScA by 50%. The extent of inhibition remains similar with higher 5-CT concentrations. Activation of 5-HT2B receptors by 1 μm DOI completely abolished the 5-CT-mediated inhibition of FScA (Fig. 2). This behavior could be related to PLA2 since the direct addition of melittin (2.5 μg/ml) mimicked the action of DOI on the 5-HT1B/1D receptor signaling pathway (Fig. 2). In the presence of 100 μm mepacrine, this effect of DOI was not observed any more. This indicates that the PLA2/AA signaling cascade governs the 5-HT2B-dependent inhibition of the 5-HT1B/1D receptor pathway. Since multiple pathways including cyclooxygenase, lipoxygenase, cytochrome P-450, and autooxidation (30Shimizu T. Wolfe L.S. J. Neurochem. 1990; 55: 1-15Crossref PubMed Scopus (532) Google Scholar) may couple AA metabolism with the 5-HT1B/1D receptor response, we investigated the effect of indomethacin, a cyclooxygenase inhibitor. Addition of a 2 μm amount of this substance not only blocked the action of DOI but also enhanced the 5-CT-mediated inhibition of FScA (Fig. 2). 1C11*d2 cells were treated with either 100 μm mepacrine or 2 μm indomethacin prior to the addition of melittin (2.5 μg/ml). In both cases, the 5-CT-mediated inhibition of FScA disappeared, as observed in the presence of melittin alone. Thus, by suppressing cyclooxygenase activity, indomethacin can block the inhibition of 5-HT1B/1D function triggered by either 5-HT2B receptor stimulation or the direct activation of PLA2. These data suggest that a cyclooxygenase-dependent metabolite of AA sustains the negative regulation of the 5-HT1B/1D response by 5-HT2B receptors. Between days 2 and 4 of 1C11* differentiation, the number and the pharmacological profiles of 5-HT1B/1D and 5-HT2B receptors did not vary. On day 4, functional 5-HT2A receptors became induced (1Kellermann O. Loric S. Maroteaux L. Launay J.-M. Br. J. Pharmacol. 1996; 118: 1161-1170Crossref PubMed Scopus (32) Google Scholar). Thus, with 1C11*d4 cells, it was possible to study the combined effects of these two members of the 5-HT2 receptor class on 5-HT1B/1D receptor function and signaling. Incubation during 10 min of [14C]AA-labeled 1C11*d4 cells with 10 μm 5-HT induced a release of [14C] radioactivity equal to 204 ± 19% above the basal release (mean ± S.E., n = 4) (Fig. 3). The released radioactivity is markedly greater than that measured at day 2 (144 ± 12%) (Fig. 1). However, the release could also be fully antagonized by pretreatment with mepacrine (8 ± 9% above basal release; mean ± S.E., n = 6). According to previous reports (18Felder C.C. Kanterman R.Y. Ma A.L. Axelrod J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2187-2191Crossref PubMed Scopus (189) Google Scholar, 20Berg K.A. Maayani S. Clarke W.P. Mol. Pharmacol. 1996; 50: 1017-1023PubMed Google Scholar), the larger increase in [14C] release observed at day 4 can be partly attributed to an activation of PLA2 by the newly expressed 5-HT2A receptor. A contribution of the PLC pathway may be ruled out by verifying that the pretreatment of 1C11* d4 cells with mepacrine did not change the DOI-mediated PI hydrolysis. Finally, an involvement in AA release of both 5-HT2 receptor subtypes could be firmly established by using 5-HT2 antagonists with different specificities. LY266070 (1 nm), a selective antagonist of the 5-HT2B receptor (24Audia J.E. Evrard D.A. Murdoch G.R. Droste J.J. Nissen J.S. Schenck K.W. Fludzinski P. Lucaites V.L. Nelson D.L. Cohen M.L. J. Med. Chem. 1996; 39: 2773-2780Crossref PubMed Scopus (134) Google Scholar), reduced by 52% the AA release measured at day 4, whereas it abolished PLA2 activation at day 2 by nearly 100% (Fig. 3). Ketanserin (5 nm), which has only a small effect on the 5-HT2B receptor response at day 2, reduces the [14C]AA release at day 4 by 54% (Fig. 3). These findings are consistent with the preferential 5-HT2A receptor antagonist property of ketanserin, as already described (11Hoyer D. Clarke D.E. Fozard J.R. Hartig P.R. Martin G.R. Mylecharane E.J. Saxena P.R. Humphrey P.P.A. Pharmacol. Rev. 1994; 46: 157-203PubMed Google Scholar). In contrast with the situation at day 2, addition of DOI (1 μm) at day 4 had no effect any more on the 5-CT-mediated inhibition of FScA (Fig. 4) (48 ± 5% and 47 ± 4%, in the presence or absence of DOI, respectively; mean ± S.E.,n = 4). This behavior suggests that the newly expressed 5-HT2A receptor can antagonize the negative regulation exerted by 5-HT2B on the 5-HT1B/1D function. This was reasserted because the effect of DOI on the 5-CT-mediated inhibition of FScA was restored in the presence of 5 nm ketanserin (not shown). To know whether the blocking by 5-HT2A of the 5-HT2B-dependent regulation occurred through the PLA2 pathway, 1C11* d4 cells were at first treated with melittin (2.5 μmol/ml) for 10 min. As shown in Fig. 4, this only induced a small relative decrease (16 ± 4%) of the 5-CT-mediated inhibition of FScA. In contrast, when the cells were pretreated with both melittin and DOI, the inhibition of cAMP accumulation increased by 46 ± 9% above controls (100%) (mean ± S.E., n = 4) (Fig. 4). These results strongly indicate that the 5-HT2A-mediated DOI effect can only be partly accounted for by the PLA2 signaling cascade and that additional transduction pathway(s) must also be involved in the control of 5-HT1B/1D receptor function within the 1C11 serotonergic cell line. So far, the diversity of 5-HT receptors observed in vivo or in heterogeneous primary cultures has rendered difficult the determination of their precise role in the signaling networks that mediate the 5-HT physiological functions. Consequently, most of the possible functional coupling(s) associated with one given receptor subtype could be defined only by using cDNA-transfected heterologous cell lines. With newly established neuronal cell lines expressing a definite set of receptors, studies may be carried out in conditions closer to in vivo physiological circumstances, particularly concerning active stoichiometries and pathways of G protein coupling. The 1C11 neural-like stem cell is at present a uniquein vitro clonal model able to acquire three 5-HT receptors during its serotonergic differentiation program (1Kellermann O. Loric S. Maroteaux L. Launay J.-M. Br. J. Pharmacol. 1996; 118: 1161-1170Crossref PubMed Scopus (32) Google Scholar). Therefore, this model appears suitable to define the transductional pathways related to the activation of all 5-HT1B/1D, 5-HT2B, and 5-HT2A receptors within the same serotonergic context. The sequential onset of these three receptors along the differentiation process makes it possible to follow the functional interaction(s) between their associated signaling cascades at day 2, when 5-HT1B/1D and 5-HT2B receptors are coexpressed, and at day 4, when 5-HT2A has also been induced. The in vivo signaling pathways that mediate the physiological functions of 5-HT2B receptors remain largely unknown (14Cohen M.L. Wittenauer L.A. J. Cardiovasc. Pharmacol. 1987; 10: 176-181Crossref PubMed Scopus (37) Google Scholar,15Ellis E.S. Byrne C. Murphy O.E. Tilford N.S. Baxter G.S. Br. J. Pharmacol. 1995; 114: 400-404Crossref PubMed Scopus (82) Google Scholar). In the present study, we have investigated the PLA2/AA pathway because 5-HT was already known to stimulate the direct release of AA via 5-HT2 receptor subtypes in hippocampal neurons (18Felder C.C. Kanterman R.Y. Ma A.L. Axelrod J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2187-2191Crossref PubMed Scopus (189) Google Scholar, 31Axelrod J. Burch R.M. Jelsema C.L. Trends Neurosci. 1988; 11: 117-123Abstract Full Text PDF PubMed Scopus (423) Google Scholar). As early as day 2 of the 1C11 differentiation, the activation of 5-HT2B receptors with 5-HT or DOI stimulates the release of AA. The PLA2 inhibitor mepacrine completely inhibits this behavior while having no effect on the 5-HT2B-mediated PI hydrolysis (Fig. 1). Furthermore, the 5-HT2B receptor-mediated AA release observed here may be responsible for the direct activation of the PLA2 activity and does not derive from the phospholipase C- or D-induced PLA2 activation. Indeed, it was totally dependent on extracellular calcium. Thus, inside the 1C11* cells, in addition to its role in activation of PI hydrolysis (27Loric S. Maroteaux L. Kellermann O. Launay J.-M. Mol. Pharmacol. 1995; 47: 458-466PubMed Google Scholar), p21ras/MAP kinase signaling (16Launay J.-M. Birraux G. Bondoux D. Callebert J. Choi D.-S. Loric S. Maroteaux L. J. Biol. Chem. 1996; 271: 3141-3147Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), and NO signaling, 2J. M. Launay, unpublished data. the 5-HT2B receptor can also control AA release through the activation of PLA2. All members of the 5-HT2 receptor family have been detected in those regions of the cerebral cortex and hippocampus where 5-HT stimulates the release of AA (17Pazos A. Cortes R. Palacios J.M. Brain Res. 1985; 346: 231-249Crossref PubMed Scopus (814) Google Scholar, 18Felder C.C. Kanterman R.Y. Ma A.L. Axelrod J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2187-2191Crossref PubMed Scopus (189) Google Scholar, 23Duxon M.S. Flanigan T.P. Reavley A.C. Ba" @default.
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