Effects of substance P on the long-term regulation of tyrosine hydroxylase activity and catecholamine levels in cultured adrenal chromaffin cells

Acetylcholine, released from splanchnic nerve terminals innervating adrenal chromaffin cells, is known to increase synthesis of adrenal tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. The neuropeptide substance P is also present in the splanchnic nerve innervating the adrenal medulla, and this study examined whether substance P has any long-term effects on tyrosine hydroxylase activity and catecholamine levels in cultures of adult bovine adrenal chromaffin cells. When cultures were incubated for 3 days with substance P and carbachol, a cholinergic agonist, substance P (10−6 M, and greater) completely inhibited the increase in tyrosine hydroxylase activity normally induced by carbachol. Long-term stimulation with carbachol also depleted endogenous catecholamines from the cells and substance P prevented this carbachol-induced depletion of catecholamine content. Substance P by itself, in the absence of carbachol, had only a slight effect on tyrosine hydroxylase activity. 8-Bromoadenosine 3′:5′-cyclic monophosphate, an analogue of adenosine 3′:5′-cyclic monophosphate, also increases tyrosine hydroxylase activity in chromaffin cells; however, substance P had no effect on the increase in tyrosine hydroxylase activity induced by this analogue. These results indicate that substance P's effects are relatively specific for the carbachol-induced increased in tyrosine hydroxylase activity and that the primary site of action of substance P is not a site common to the mechanism of tyrosine hydroxylase induction by carbachol and 8-bromoadenosine 3′:5′-cyclic monophosphate. During long-term incubation of chromaffin cell cultures, substance P inhibited the carbachol-induced increase in tyrosine hydroxylase activity only if peptidase inhibitors, bacitracin and captopril, were included in the medium, suggesting that chromaffin cell peptidases may be capable of terminating substance P's actions in these cells. The peptidase inhibitors bacitracin and captopril alone increased tyrosine hydroxylase activity and depleted catecholamines from the cells. The results suggest that substance P, released either from the splanchnic nerve or from the chromaffin cells themselves, may play a role in the long-term regulation of tyrosine hydroxylase activity and catecholamine levels in the mature adrenal chromaffin cell.