TYROSINE KINASES ARE REQUIRED FOR CATECHOLAMINE SECRETION AND MITOGEN-ACTIVATED PROTEIN-KINASE ACTIVATION IN BOVINE ADRENAL CHROMAFFIN CELLS

Nicotine-induced catecholamine secretion in bovine adrenomedullary chr omaffin cells is accompanied by rapid tyrosine phosphorylation of mult iple cellular proteins, most notably the mitogen-activated protein kin ases (MAPKs). The requirement for activation of tyrosine kinases and M APKs in chromaffin cell exocytosis was investigated using a panel of t yrosine kinase inhibitors. Genistein and tyrphostin 23, two compounds that inhibit tyrosine kinases by distinct mechanisms, were found to in hibit secretion by >90% in cells stimulated by nicotine, 55 mM KCl, or the Ca2+ ionophore A23187. Inhibition of secretion induced by all thr ee secretagogues correlated with a block in both protein tyrosine phos phorylation and activation of the MAPKs and their activators (MEKs) in situ, However, neither genistein nor tyrphostin 23 inhibited the acti vities of the MAPKs or MEKs in vitro. These results indicate that the target(s) of inhibition lie downstream of Ca2+ influx and upstream of MEK activation, This Ca2+-activated tyrosine kinase activity could not be accounted for entirely by c-Src or Fyn (two nonreceptor tyrosine k inases that are expressed abundantly in chromaffin cells), because the ir in vitro kinase activities were not inhibited by tyrphostin 23 and only partially inhibited by genistein. These results demonstrate that an unidentified Ca2+-activated tyrosine kinase(s) is required for MAPK activation and exocytosis in chromaffin cells and suggest that MAPK p articipates in the regulation of secretion.