CALCIUM STIMULATES LUTEINIZING-HORMONE (LUTROPIN) EXOCYTOSIS BY A MECHANISM INDEPENDENT OF PROTEIN KINASE-C

Using permeabilized gonadotropes, we examined whether Ca2+-stimulated luteinizing-hormone (LH) exocytosis is mediated by the Ca2+-activated phospholipid-dependent protein kinase (protein kinase C). In the presence of high [Ca2+](free) (pCa 5), α-toxin-permeabilized sheep gonadotropes secrete a burst of LH and then become refractory to maintained high [Ca2+](free). The protein kinase C activator phorbol myristate acetate (PMA) is able to stimulate further LH release from cells made refractory to high [Ca2+](free), suggesting that Ca2+ does not stimulate LH release by activating protein kinase C. Staurosporine, a protein kinase C inhibitor, inhibited PMA-stimulated (50% inhibition at 20 nM), but not Ca2+-stimulated, LH exocytosis. In cells desensitized to PMA by prolonged exposure to high PMA concentration, Ca2+-stimulated LH exocytosis (when corrected for depletion of total cellular LH) was not inhibited. Ba2+ was able to stimulate LH exocytosis to a maximal extent similar to Ca2+, although higher Ba2+ concentrations were necessary. Ba2+ and Ca2+ stimulated LH exocytosis with a similar time course, and both were inhibitory at high concentrations. Furthermore, cells made refractory to Ca2+ were also refractory to Ba2+. These data strongly suggest that Ba2+ and Ca2+ act through the same mechanism. Since Ba2+ is a poor activator of protein kinase C, these findings are additional evidence against a major role for protein kinase C in mediating Ca2+-stimulated LH exocytosis.