Role of ATP and β-γ-iminoadenosinetriphosphate in the stimulation of epinephrine and protein release from isolated adrenal secretory vesicles

Abstract

ATP-stimulated release of epinephrine and protein from isolated chromaffin granules of the bovine adrenal medulla has been characterized with respect to pH optimum, substrate requirements, and temperature. Chromaffin granules incubated at 37 °C under optimal conditions released virtually all of their epinephrine and soluble protein within 10 min. ATP-stimulated epinephrine release was optimal at pH 5.8–6.2 and was five times greater than at pH 7.0–7.4. Magnesium and chloride were absolutely required for this process. Magnesium stimulated release at concentrations up to 1.0 m m; however, it was moderately inhibitory at higher concentrations. The dependence of release on Cl − exhibited positive cooperativity and was nonsaturable. At 90 m m Cl − and 1.0 m m Mg 2+, ATP-stimulated epinephrine release followed Michaelis-Menten kinetics with a K 1 2 of 0.2 m m. The release of soluble chromaffin granule proteins closely paralleled epinephrine release under all conditions tested, while membrane components were not released. Analogs of ATP substituted at the β-γ position with methylene or imino groups were also capable of stimulating release from granules. These ATP analogs had reduced affinity and lower activity than ATP itself. ATP analogs substituted at the α-β position were essentially inactive but were potent inhibitors of ATP-stimulated release. We conclude that the regulation of release from granules by ATP is rapid and specific and may not depend on hydrolysis of ATP at the β-γ position. This ATP-dependent reaction may be involved in the cellular process of exocytosis.