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

Abstract

The effects of ATP, Mg2+, 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 (intravesicular:medium) equals 16 when the medium pH is 6.9 and is unaltered by ATP and Mg2+. In the absence of ATP and Mg2+, the steady-state intravesicular S14CN-concentration is lower than the medium concentration. ATP and Mge+ 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,Mg2+-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 Mge+. The have no effect on the H+ concentration gradient. The rate ofcatecholamine uptake into vesicles is increased 4-to 6-fold by ATP and Me+. The ATP,Mge+-stimulated uptake is inhibited by FCCP and NEM over the same concentration ranges that reduce the SCNdistribution (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. Chromaffin vesicles are the catecholaminergic storage vesicles in adrenal medullary cells. They are intimately involved in both secretion (via exocytosis) and intracellular synthesis of catecholamines. Catecholamine transport into vesicles is important for both these processes. Because the in vitro rate of uptake of catecholamines into chromaffin vesicles is increased severalfold by ATP and magnesium (1), it had been thought that the Mg2+-dependent ATPase associated with the vesicle membrane is a catecholamine transport enzyme. Although partial correlations have been obtained between ATPase activity and catecholamine transport (2, 3), lack of a simple quantitative correlation suggests that the ATPase may be indirectly rather than directly involved.Elements of a chemiosmotic system involving the H+ electrochemical gradient are present in chromaffin vesicles (Fig.

Uptake of Catecholamines by Storage Vesicles

“…The finding that the characteristics o f trans port in neuronal vesicles differ from those in adrenomedullary vesicles indicates the need to reexamine in neuronal vesicles some o f the basic premises for the energy source for trans port as established in adrenal preparations. While only a limited amount of information is (24). There is also disagreement about the charge on the species transported (24-26, 67).…”

“…Indeed, under some circumstances, adreno medullary catecholamine transport can be dem onstrated in the absence o f a pH gradient (24), and it is therefore apparent that the chemi osmotic principle provides only the energy source, and not the total means o f moving catecholamines to the interior of the vesicle. The pH gradient may, however, be directly responsible for the uptake o f certain amines, such as metaraminol, which have very low affinities for the catecholamine transport sys tem (35,52,53,55).…”

“…The current view is based primarily upon the 'chemiosmotic' principle first elabo rated for mitochondrial function (36). Ac cording to this hypothesis, the vesicles act as a mitochondrion in reverse, utilizing ATP to set up a proton and/or electrochemical gradient across the vesicle membrane (6,24,26,68), and the gradient then acts as the motive force for transport. While there is some uncertainty about specific coupling steps, there appears to be general agreement that this scheme is largely correct, and that utilization o f ATP is coupled indirectly to catecholamine transport.…”

A Molecular Description of Nerve Terminal Function

“…Isolated chromaffin granules are acidic (pH 5.2), whether measured by methylamine redistribution or protonation of the gamma phosphate of ATP (228,229). Isolated granules in the absence of ATP have a negative internal charge that reflects a diffusion potential induced by the proton gradient (228).…”

Characterization of the monoamine carrier of chromaffin granule membrane by binding of [2-3H]dihydrotetrabenazine.

“…[2-3H]Dihydrotetrabenazine binding occurred on the catecholamine carrier ofthe chromaffin granule membrane because it was clearly correlated with inhibition of norepinephrine uptake. In addition, inhibitors and substrates ofthe uptake reaction displaced [2-3H] The chromaffin granules of the adrenal medulla and the ghosts derived from their membranes accumulate catecholamines by a two-step ATP-dependent process (1-4): (i) inward translocation of protons by an electrogenic ATP-dependent H' pump (5-10), and (ii) a monoamine carrier driven by the H' electrochemical gradient (9,(11)(12)(13)(14)(15) and specifically blocked by reserpine or tetrabenazine (16). Whereas the H' pump has been functionally and structurally related to the mitochondrial ATPase complex (17,18), the catecholamine carrier has not yet been characterized.…”