THE SECRETION OF ALPHA-MSH FROM XENOPUS MELANOTROPES INVOLVES CALCIUM INFLUX THROUGH OMEGA-CONOTOXIN-SENSITIVE VOLTAGE-OPERATED CALCIUM CHANNELS

The secretory activity of endocrine cells largely depends on the concentration of free cytosolic calcium. We have studied the mechanisms that are involved in supplying the calcium necessary for the secretion of alpha-melanophore-stimulating hormone (alpha-MSH) from melanotrope cells in the pituitary intermediate lobe of the amphibian Xenopus laevis. Using whole-cell voltage clamp, high-voltage activated calcium currents were observed, with a peak current between 0 and +20 mV. Two types of Ca2+-currents appeared, depending on the experimental setup. An inactivating current, which was observed after a 10 msec depolarizing prepulse, resembled currents through N-type channels as it was clearly inhibited by 1 mu M omega-conotoxin. The second type was a non-inactivating current, which was blocked up to 50% by 1 mu M nifedipine, indicating its L-type nature. Only a small component of this inactivating current could be blocked by omega-conotoxin. No evidence was found for the presence of transient, low-voltage activated currents. The spontaneous secretion of alpha-MSH from superfused neurointermediate lobes was dependent on extracellular calcium, as low calcium conditions (10(-4)-10(-8) M) rapidly inhibited this process. Under these conditions, secretion was not affected by depolarizing concentrations of potassium chloride. The calcium ionophore A23187 increased secretion under low calcium conditions, but had no effect on spontaneous alpha-MSH release. Treatment with CoCl2, a blocker of calcium channels, strongly inhibited the secretory process. These results suggest that spontaneous alpha-MSH release depends on influx of calcium through voltage-operated calcium channels. Nifedipine did not affect spontaneous secretion from lobes, nor did it affect potassium-induced alpha-MSH secretion from dispersed melanotropes. Also BAY-K8644, a specific agonist of L-type channels, did not influence alpha-MSH release, neither under normal nor under low calcium conditions. On the other hand, omega-conotoxin dose-dependently inhibited alpha-MSH release, to a maximum of 65% at a concentration of 5 mu M, and inhibited potassium-induced secretion by 40%. Thapsigargin, an agent that mobilizes calcium ions from intracellular stores, had no effect on spontaneous alpha-MSH release under normal or low calcium conditions. From these results it is concluded that the spontaneous release of alpha-MSH by melanotropes of X. laevis is effectuated by calcium influx through omega-conotoxin-sensitive, voltage-operated N-type calcium channels and that mobilization of calcium from intracellular stores does not play a major role in the regulation of this release.