SEROTONERGIC REGULATION OF TYPE-II CORTICOSTEROID RECEPTOR-BINDING IN HIPPOCAMPAL CELL-CULTURES - EVIDENCE FOR THE IMPORTANCE OF SEROTONIN-INDUCED CHANGES IN CAMP LEVELS

The development of the hypothalamic-pituitary-adrenal response to stress is profoundly altered by environmental events. One target for environmental regulation within the hypothalamic-pituitary-adrenal axis is the hippocampal type II corticosterod (or glucocorticoid) receptor system, which mediates the negative-feedback effects of glucocorticoids on hypothalamic-pituitary-adrenal activity. Thus, adult rats handled early in life show increased hippocampal type II corticosteroid receptor density and increased sensitivity to the inhibitory effects of circulating glucocorticoids on post-stress hypothalamic-pituitary-adrenal activity. Both effects persist throughout life. The effects of handling on type II corticosteroid receptor development are, at least in part, mediated by changes in hippocampal 5-hydroxytryptamine turnover. Moreover, 5-hydroxytryptamine can regulate type II corticosteroid receptor density in cultured hippocampal cells, providing a paradigm for examining the neurochemical mechanisms by which environmental stimuli might regulate neural differentiation. In the present studies, we examined the intracellular mechanisms underlying the effects of 5-hydroxytryptamine on type II corticosteroid receptors ([H-3]RU 28362 binding) in hippocampal cell cultures. cAMP, but not cGMP, levels in cultured hippocampal cells were significantly increased by the addition of 5-hydroxytryptamine to the medium. The cAMP response to 5-hydroxytryptamine was biphasic: an initial increase in cAMP levels occurred in response to nanomolar 5-hydroxytryptamine concentrations (EC50 = 7.2 nM), while a second increase was apparent at low micromolar concentrations. 5-Hydroxytryptamine also increased [H-3]RU 28362 binding (EC50 = 4.5 nM) with a maximal effect at a concentration of 10 nM. There was no further increase in [H-3]RU 28362 binding even with higher, micromolar concentrations of 5-hydroxytryptamine. The effect of 10 nM 5-hydroxytryptamine on cAMP levels was apparent 1 h following treatment and peaked at two days. The increase in cAMP levels with 10 nM 5-hydroxytryptamine was completely blocked by 100 nM ketanserin, a 5-hydroxytryptamine2 receptor antagonist. The 5-hydroxytryptamine effect on cAMP levels was at least partially mimicked by the 5-hydroxytryptamine-agonist (10 nM) quipazine and 1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane. We recently reported that the effect of 5-hydroxytryptamine on [H-3]RU 28362 binding was also blocked by ketanserin and mianserin and mimicked by quipazine and 1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane. Treatment of the cell cultures with 8-bromo cAMP, a cAMP analog, resulted in a dose-dependent increase in [H-3]RU 28362 binding capacity, with the maximal effect occurring at 10-mu-M 8-bromo cAMP. As with 5-hydroxytryptamine, the maximal effect of 8-bromo cAMP required four days of treatment. Activation of endogenous adenylate cyclase by treatment with forskolin resulted in a significant increase in [H-3]RU 28362 binding capacity. Finally, the effects of a half-maximal concentration of 5-hydroxytryptamine on [H-3]RU 28362 binding were blocked with the addition of 3-mu-M (5-isoquinylsulfonyl)-2-methyl-piperazine, which at this concentration selectively inhibits cyclic nucleotide-dependent protein kinases. These data indicate that the effect of 5-hydroxytryptamine on [H-3]RU 28362 binding in hippocampal cell cultures is likely mediated by a high-affinity 5-hydroxytryptamine2- (or 5-hydroxytryptamine1C) receptor and involves an increase in cAMP levels.