Exocytosis of catecholamines by adrenal chromaffin cells requires a receptor-mediated influx of calcium which causes fusion of the chromaffin granule membrane with the cell membrane. We have demonstrated that metalloendoprotease activity is required for exocytosis by mast cells, for exocytosis at the neuromuscular synapse, and for the calcium-dependent membrane fusion of myoblasts to form myotubes. We are using dissociated bovine adrenal chromaffin cells as an advantageous model system to study the role of metalloendoprotease in exocytosis. The hypothesis is that metalloendoprotease in the adrenal chromaffin cell plasma membrane hydrolyzes protein on the chromaffin granule membrane, and that this hydrolyzed protein then causes membrane fusion. The basis of this hypothesis is our observation that exocytosis in other cells requires metalloendoprotease activity, and that protein in myxovirus causes membrane fusion after proteolytic processing. Preliminary results are consistent with the requirement of metalloendoprotease in adrenal chromaffin cell exocytosis, and are similar to our observations in other cells. Metalloendoprotease inhibitors and synthetic dipeptide substrates of metalloendoproteases prevent receptor-mediated exocytosis of catecholamines by dissociated bovine adrenal chromaffin cells. Adrenal metalloendoprotease activity can be assayed with a fluorogenic protease substrate which contains a Gly-Leu peptide bond hydrolyzed by metalloendoproteases. We will characterize the adrenal metalloendoprotease, identify chromaffin granule proteins which are hydrolyzed by the plasma membrane metalloendoprotease, and hope to determine whether these chromaffin granule proteins cause fusion in reconstituted liposomes.
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