Pharmacological characterization of adrenal paraneurons: substance P and somatostatin as inhibitory modulators of the nicotinic response.

A pharmacological study was made of the effects of various muscarinic and nicotinic agonists and their antagonists on the release of [3H]noradrenaline ([3H]NA) from cultures of isolated bovine adrenal medullary cells. A study was also made of the effects of substance P and somatostatin on the release of [3H]NA evoked by nicotinic agonists. By 2 days in culture these adrenal 'paraneurons' had developed long varicose processes with growth cones and generally resembled noradrenergic neurons in culture. In the present study, adrenal paraneurons were incubated with [3H]NA which was taken up and stored in reserpine-sensitive sites. Exposure of the cultures to acetylcholine (ACh) resulted in release of [3H]NA into the external medium. High concentrations of K+ (56 mM) also evoked release of [3H]NA. The release of [3H]NA induced by ACh or K+ (56 mM) was Ca2+-dependent. Pharmacological studies with nicotinic (ACh, nicotine) and muscarinic (methacholine, pilocarpine) agonists and their antagonists (mecamylasmine, d-tubocurarine, hexamethonium; and atropine, scopolamine, respectively) showed that the adrenal paraneurons contained only nicotinic receptors. Substance P produced a dose-dependent inhibition of ACh (5 x 10(-5) M) stimulated [3H]NA release in the range of 10(-8) to 5 x 10(-5) M with an ID50 of 10(-6) M. A similar inhibition of NA release by substance P was obtained when nicotine (K X 10(-6) M) was used as the agonist, but not when K+ (50 MM) was used to depolarize the cells. Substance P (10-10) to 5 x 10(-5) M) by itself did not have a significant effect on the basal release rate of [3H]NA from these cells. Somatostatin at relatively high concentrations (10(-6)-10(-3) M; ID50 2 x 10(-5) M) inhibited the release induced by ACh, but not by K+ (56 mM). The present results provide the first direct evidence at a cellular level that substance P and somatostatin act as inhibitory modulators of the nicotinic ACh response, and support a role for these peptides as inhibitory neuromodulators at nicotinic receptor sites in the nervous system.