Activation of Fyn tyrosine kinase upon secretagogue stimulation of bovine chromaffin cells.

Abstract

Adrenal medullary chromaffin cells derive from the neural crest during embryogenesis and differentiate into dedicated secretory cells that release catecholamines in response to acetylcholine in vivo or nicotinic agonists in vitro. Previous studies have indicated that tyrosine kinases participate in early secretagogue-induced events in these cells and are required for exocytosis. Abundant levels of the cytoplasmic tyrosine kinases, c-Src and c-Yes, have been detected in chromaffin cells, thereby implicating them as kinases relevant to these events. However, c-Src has been found to undergo a decrease in activity following secretagogue-stimulation, and c-Yes appears to exist in a constituitively low activity state, suggesting that other tyrosine kinases are involved. Furthermore, other members of the Src family of tyrosine kinases have been implicated as playing roles in secretion in a variety of cell types. Therefore, we sought to determine if other Src family members were present in chromaffin cells, and if so, to examine them for subcellular localization and changes in activity following treatment with nicotinic agonists. To this end, antibodies for Fyn, Lck, Lyn, and Fgr were assembled and used in immunoprecipitation, in vitro autokinase, and Western immunoblotting assays. Of these four kinases, only Fyn was found to be expressed at detectable levels. Differential centrifugation studies revealed that Fyn resides predominantly (> 95%) in the crude plasma membrane fraction and undergoes nicotinic-and carbachol-induced activation. This activation is reduced by the nicotinic antagonist, mecamylamine, is not elicited by muscarine, and is dependent upon the presence of extracellular Ca2+. These results suggest that Fyn is involved in signalling through the nicotinic receptor and may be one of the relevant kinases responsible for at least some of the tyrosine phosphorylations detected after stimulation.