Phospholipids as adjuncts for calcium ion stimulated release of chromaffin granule contents: implications for mechanisms of exocytosis.

Abstract

Structure-function relationships for the lipid component of chromaffin granules isolated from the bovine adrenal medulla have been investigated by employing 31P nuclear magnetic resonance (NMR), freeze-fracture, and spectrophotometric techniques. Two aspects have been studied in detail, namely, the structural preferences of lipids in the isolated granule membrane and derived liposomal model membrane systems as well as the influence of exogenous lipid (in the form of sonicated vesicle systems) on the Ca2+-stimulated release of granule contents. It is shown that at least 90% of endogenous granule membrane phospholipids assume a liquid-crystalline bilayer configuration at physiological temperatures. Liposomal dispersions of total granule lipid also exhibit bilayer structure, consistent with a structural role of phospholipids in vivo. Incubation of intact isolated granules in the presence of up to 10 mM Ca2+ does not induce significant release of contents above background levels. However, it is shown that incubation of granules in the presence of sonicated phospholipid systems which undergo structural transitions in the presence of Ca2+ can cause immediate and total release of granule contents at Ca2+ levels of 2 mM or more. This behavior is attributed to disruption of granule membrane integrity due to fusion of the vesicle systems with the chromaffin granules. Direct evidence for such fusion is obtained by freeze-fracture electron microscopy. On the basis of this information and with the assumption that the inner leaflet of the adrenal cell plasma membrane is composed predominantly of phosphatidylethanolamine and phosphatidylserine, a mechanism of Ca2+-stimulated exocytotic release of catecholamines in vivo is proposed.