Transport of Catecholamines by Native and Reconstituted Rat Heart Synaptic Vesicles

Abstract

Highly purified heavy synaptic vesicles were isolated from rat heart by differential centrifugation. Because of the high intravesicular concentrations of proteins, catecholamine and ATP, resealed vesicle ghosts were prepared and used to study the detailed kinetics of catecholamine transport. ATP stimulated the uptake of l-norepinephrine and was saturable with a Km for l-norepinephrine at 3.3 .mu.M and 1.8 mM for ATP. The ghosts accumulated 5-hydroxytryptamine and l-epinephrine via an ATP-dependent mechanism. Uptake was stereospecific for the l-form. A functional catecholamine transporter could be solubilized for the detergent octyl-glucoside and incorporated into phospholipid vesicles, which, after detergent removal, generated proteoliposomes that accumulated l-norepinephrine. Reserpine- and l-propranolol-sensitive accumulation against a concentration gradient is achieved by artificially creating a pH gradient across the membrane; at least the initial phase of catecholamine transport is driven by the trans-membrane pH gradient created by the proton-translocating ATPase.