Evidence that catecholamine transport into chromaffin vesicles is coupled to vesicle membrane potential

The effects of ATP, Mg(2+), and various agents on pH gradient, membrane potential, and catecholamine transport across membranes of intact bovine chromaffin vesicles were investigated. Methylamine and thiocyanate (SCN(-)) distributions across the vesicle membrane were used to estimate the H(+) concentration gradient and membrane potential, respectively. The H(+) concentration ratio (intravesiculanmedium) equals 16 when the medium pH is 6.9 and is unaltered by ATP and Mg(2+). In the absence of ATP and Mg(2+), the steady-state intravesicular S(14)CN(-) concentration is lower than the medium concentration. ATP and Mg(2+) cause an increased influx and a decreased efflux of SCN(-) that results in SCN(-) being concentrated in the vesicles 6- to 8-fold over the medium. The findings are consistent with an ATP,Mg(2+)-induced potential of approximately 50 mV (intravesicular side positive). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a H(+) translocater, and N-ethylmaleimide (NEM), a sulfhydryl reagent, decrease the SCN(-) ratio and, thus, the membrane potential in the presence of ATP and Mg(2+). They have no effect on the H(+) concentration gradient. The rate of catecholamine uptake into vesicles is increased 4- to 6-fold by ATP and Mg(2+). The ATP,Mg(2+)-stimulated uptake is inhibited by FCCP and NEM over the same concentration ranges that reduce the SCN(-) distribution (membrane potential). FCCP increases and NEM decreases vesicular membrane ATPase activity. Thus, catecholamine uptake is correlated to an inside-positive membrane potential, and not to ATPase activity. If catecholamine uptake is coupled to membrane potential, then a charged species must be involved in the transport mechanism. Reserpine and rotenone inhibit catecholamine influx but have no effect on the H(+) electrochemical gradient; they probably act at a step before coupling to the membrane potential (or the H(+) electrochemical gradient). Atractyloside, an inhibitor of nucleotide transport, has no effects on catecholamine transport or the H(+) electrochemical gradient.