CAMP regulation of vasopressin mRNA content in hypothalamo-neurohypophysial explants CELIA D. SLADEK, KIRSTEN Y. FISHER, HANNA E. SIDOROWICZ, AND JOANNE R. MATHIASEN Department of Physiology, Finch University of Health Sciences/ The Chicago Medical School, North Chicago, Illinois 60064 Sladek, Celia D., Kirsten Y. Fisher, Hanna E. Sidoro- wicz, and Joanne R. Mathiasen. CAMP regulation of vasopressin mRNA content in hypothalamo-neurohypophy- sial explants. Am. J. Physiol. 271 (Regulatory Integrative Comp. Physiol. 40): R554-R560, 1996.-Stimulation of vaso- pressin (VP) gene expression by adenosine 3’,5’-cyclic mono- phosphate (CAMP) has been observed in dispersed hypotha- lamic cultures, in VP-expressing cell lines, and in cells transfected with reporter genes regulated by the VP gene promoter. However, treatment of hypothalamo-neurohypophy- sial system (HNS) explants with forskolin (25 FM), an activator of adenyl cyclase, and 3-isobutyl-1-methylxanthine (IBMX; 500 FM), a phosphodiesterase inhibitor, resulted in a decrease in VP mRNA. Time course analysis revealed that IBMX and forskolin reduced the VP mRNA content to 50% of control explants after 8 and 12 h despite a dramatic stimula- tion of VP release. This effect was due to the activation of adenyl cyclase by forskolin, because neither IBMX alone nor the inactive analogue of forskolin, 1,9-dideoxyforskolin, de- creased VP mRNA content. In contrast, 8-bromoadenosine 3’,5’-cyclic monophosphate and the Di dopamine receptor agonist, SKF-38393, increased VP mRNA content, but these agents were less potent in stimulating VP release, suggesting a concentration dependency of the forskolin effect. This was confirmed when forskolin (10 uM) was found to increase VP mRNA content. Thus receptor-mediated activation of adenyl cyclase results in an increase in VP mRNA content. supraoptic nucleus; dopamine; oxytocin VASOPRESSIN (VP) mRNA content of the supraoptic (SON) and paraventricular nucleus (PVN) is signifi- cantly increased in response to stimuli for VP release. This is observed following chronic saline drinking (2, 10,15,26), water deprivation (1, ll), and acute hypoten- sive hemorrhage (17). VP mRNA content of explants of the hypothalamo-neurohypophysial system (HNS) also is increased following exposure to a slow, sustained increase in osmolality of the perifusion medium (24). However, the intracellular mechanisms responsible for this increase in VP mRNA content remain to be eluci- dated. An increase in intracellular adenosine 3’,5’-cyclic monophosphate (CAMP) as a result of activation of adenyl cyclase has been suggested as one mechanism that may be involved in regulation of VP mRNA con- tent. CAMP is elevated in the SON and PVN of rats given saline to drink for 2-7 days (4,25), and treatment of dispersed hypothalamic cultures with drugs that elevate intracellular CAMP increases VP gene expres- sion as well as VP release (14, 19). Additional support for a role for CAMP in the regulation of VP mRNA content comes from evidence for the presence of CAMP responsive elements in the VP gene promoter. Se- quence analysis of 1 kb of the VP gene promoter detected a CAMP response element and four AP2 bind- ing sites (12), and CAMP has been shown to upregulate the VP gene promoter in homologous and heterologous expression systems (22). In contrast to this evidence for stimulation of VP gene expression by CAMP, VP mRNA content of HNS explants was decreased following a 24-h exposure to forskolin, an activator of adenyl cyclase, and 3-isobutyl- l-methylxanthine (IBMX), a phosphodiesterase inhibi- tor (20). This occurred despite a massive and sustained stimulation of VP release. The current studies were performed to evaluate this surprising observation. The time course of the effect of IBMX and forskolin (IF) together was evaluated as well as the response to independent exposure to IBMX and forskolin and the response to lower concentrations of forskolin. In addi- tion, the response to %bromoadenosine 3’,5’-cyclic monophosphate (8-BrcAMP) was evaluated as another mechanism of directly elevating intracellular CAMP. SKF-38393 (SKI?), an agonist at the D1 dopamine receptor, was also evaluated, because Di receptors are linked to adenyl cyclase (9), and dopamine has been shown to excite VP neurons and facilitate osmotically stimulated VP release (13, 21). Therefore, this agent provided a means of evaluating the effect on VP mRNA content of increasing intracellular CAMP through a membrane receptor-mediated mechanism. MATERIALS AND METHODS Explant Preparation The HNS explants were obtained from decapitated male Sprague-Dawley rats (125-150 g), as described previously (24). These explants included the VP neurons of the supraop- tic nuclei with their axonal projections extending through the median eminence and terminating in the neural lobe. They also included the organum vasculosum of the lamina termina- lis, the suprachiasmatic nucleus, and the arcuate nucleus. The PVN was not included. Perifusion Cultures of HNS Explants HNS explants were perifused individually in closed culture chambers (0.5 ml) at 37°C with oxygenated culture medium at a rate of 2.0 ml/h. The medium consisted of F12 nutrient mixture (Grand Island Biochemical) fortified with 20% fetal calf serum, 1 mg/ml glucose, 2.5 U/ml penicillin, 2.5 ug/ml streptomycin, 2.5 pg/ml fungizone, and 1 X 10m4 M bacitracin. Bacitracin was added to prevent degradation of VP in the culture medium (18). The osmolality of the basal medium was 305 mosmol/kgHzO. Six explants were perifused simulta- neously Outflow from the microchambers was collected indi- R554 0363-6119196 $5.00 Copyright o 1996 the American Physiological Society