Journal of Neurochemistry Lippincott—Raven Publishers, Philadelphia © 1998 International Society for Neurochemistry Continuous Activation of Pituitary Adenylate Cyclase- Activating Polypeptide Receptors Elicits Antipodal Effects on Cyclic AMP and Inositol Phospholipid Signaling Pathways in CATH.a Cells: Role of Protein Synthesis and Protein Kinases *André Muller, Bernadette Lutz-Bucher, Pascal Kienlen-Campard, Bernard Koch, and Jean-Philippe Loeffler IPCB, Laboratoire de Neurophysiologie et de Neurobiologie des Systèmes Endocrines, UMR CNRS 7519, and * Clinique de la Douleur, Hospices Civils de Strasbourg, Strasbourg, France Abstract: Continuous exposure of cells to agonists de- velops a process that determines the extent to which the cells eventually respond to further stimuli. Here we used CATH.a cells (a catecholaminergic neuron-like cell line), which express pituitary adenylate cyclase-activating polypeptide (PACAP) receptors linked to both adenylyl cyclase and phospholipase C-ß pathways, to investigate the influence of prolonged hormonal treatment on dual signaling and gene transcription. Prolonged incubation of cells with PACAP failed to down-regulate the density and affinity of membrane binding sites and caused opposite changes in messenger systems: PACAP-stimulated cy- clic AMP accumulation was attenuated in a time- and dose-dependent fashion (t 112 = 6.7 h and IC50 = 0.1 nM), whereas phosphoinositide turnover was overstimulated. Both effects were insensitive to pertussis toxin, whereas the drop in cyclic AMP concentration was also un- changed in the presence of 3-isobutyl-1 -methylxanthine, indicating that neither G-like proteins nor cyclic nucleo- tide phosphodiesterases play a critical role in these pro- cesses. Blockade of protein synthesis with cyclohexi- mide, as well as inhibition by H89 of cyclic AMP-depen- dent protein kinase (but not by bisindolylmaleimide of protein kinase C) antagonized the influences exerted by PACAP on adenylyl cyclase activity and inositol phos- phate formation. Transcription of the chimeric GAL4- CREB construct, transiently transfected into CATH.a cells, was stimulated by PACAP, and this effect was po- tentiated as a result of chronic PACAP treatment. The results of the present investigation provide new insight into the possible differential regulation and cross-talks of transduction signals of receptors linked to multiplex signaling. They demonstrate that prolonged exposure of CATH.a cells to PACAP results in the desensitization of the cyclic AMP pathway and superinduction of the inositol phosphate signal, through protein neosynthesis and cyclic AMP-dependent protein kinase activation. At the same time, they show that desensitization of cyclic AMP signaling not only fails to hamper, but actually amplifies PACAP-stimulated CREB-regulated transcription. Key Words: CATH.a cells—Pituitary adenylate cyclase- activating polypeptide— Cyclic AMP— Phosphoinositide turnover— Desensitization — CREB expression. J. Neurochem. 70, 1431—1440 (1998). As a result of continued activation of hormone and neurotransmitter receptor systems, cells adapt them- selves by developing a gradual attenuation/desensitiza- tion (Hausdorff et al., 1990; Gershengom, 1994; Kar- oor et al., 1996) or, on the contrary, an amplification] up-regulation of their response to agonists (Penhoat et al., 1989; Thomas et al., 1992; Hettinger-Smith et al., 1996). Thus, the receptor system provides itself with a “memory“ that remembers the cell of its first en- counter with a ligand and, as a result, determines the magnitude of its subsequent response to new stimuli. Disruption of these processes, which may result in overstimulation of cells, can have deleterious effects, and malfunctioning of signaling pathways have actu- Received September 12, 1997; revised manuscript received No- vember 4, 1997; accepted November 14, 1997. Address correspondence and reprint requests to Dr. B. Koch at IPCB, Laboratoire de Neurophysiologie et de Neurobiologie des Systèmes Endocrines, UMR CNRS 7519, 21, rue René Descartes, 67084 Strasbourg Cedex, France. Abbreviations used: AC, adenylyl cyclase; Br-cAMP, 8-bro- moadenosine 3‘,S‘-cyclic monophosphate; BSA, bovine serum albu- min; CaMK, Ca 2~/calmodulin-dependent protein kinase; cAMP, cy- clic AMP; CATH.a cells, a catecholaminergic neuron-like cell line; CRE, cyclic AMP-response element; CREB, CRE-binding protein; Cx, cycloheximide; DMEMIF12, Dulbecco‘s modified Eagle‘s me- dium/Ham‘s mixture F-12; G protein, guanine nucleotide-binding protein; IBMX, 3-isobutyl- I -methylxanthine; InsP 1, inositol mono- phosphate; InsP2, inositol bisphosphate; InsP3, inositol trisphosphate; PACAP, pituitary adenylate cyclase-activating polypeptide; PDE, cyclic nucleotide phosphodiesterase; PI, phosphoinositide; PKA, cy- clic AMP-dependent protein kinase; PKC, protein kinase C; PLC, phospholipase C-ß; PMA, phorbol I 2-myristate 13-acetate; PTX, pertussis toxin; VIP, vasoactive intestinal peptide. 1431