Role of a transmembrane pH gradient in epinephrine transport by chromaffin granule membrane vesicles.

Abstract

ABSTRACr ATP-driven transport and accumulation of epinepbrine in chromaffin granule membrane vesicles isolated from vine adrenal medulla is inhibited by the proton ionophores carbonylcyanide p-trifluoromethoxyphenylhydrazone and nigercin, but not by valinomycin. Moreover, an artificially imposed pH gradient (interior acid) is able to drive this reserpine-sensitie tran rt system in the absence of ATP. Diacan inactivator of the chromaffin granye membrane-bound ATPase, completely inhibits ATP-dependent epinephrine accumulation, but has much less effect when an imposed pH gradient is the driving force for epinephrine transport. The findings provide a strong indication that a pH gradient (interior acid) is the immediate driving force for epinephrine uptake in these storage granules and suggest that ATP-driven epinephrine transport is the result of two processes: (i) generation of a proton electrochemical gradient (interior acid and positive) by the membrane-bound, proton-translocating ATPase; and (ii) pH gradient-driven accumulation of the catecholamine.Chromaffin granules concentrate, store, and release most of the catecholamines in the adrenal medulla (1-4). Isolated granules catalyze ATP-dependent uptake of massive amounts of epinephrine (5, 6), and the process is inhibited by proton conductors (7). ATPase activity (8-11), ATP-dependent proton translocation (12), and ATP-dependent changes in the fluorescence of 1-anilino naphthalene-8-sulfonate (ANS) (11) have been detected in intact granules or membrane vesicles prepared from chromaffin granules by osmotic shock. Moreover, it has been demonstrated that an uncoupler-sensitive pH gradient (ApH, interior acid) is maintained across the chromaffin granule membrane (13-15). These findings have led to the proposal that the membrane-bound ATPase in chromaffin granules is a proton-translocating enzyme that generates a proton electrochemical gradient (interior acid and positive) and that the low internal pH may have a dual role in the mechanism of catecholamine uptake and storage: (i) it may provide the driving force for catecholamine accumulation (putatively, the catecholamine is translocated across the membrane in its unprotonated form and accumulation results from protonation in the intragranular space); and (Ui) it may prevent oxidation of intragranular catecholamines.In this communication, we demonstrate that imposition of a ApH (interior acid) across the membrane of isolated chromaffin granule membrane vesicles induces the accumulation of epinephrine by a transport system that is inhibited by reserpine. This phenomenon is independent of ATP, relatively mildly inhibited by dicyclohexylcarbodiimide (DCCD), an inactivator of the membrane-bound ATPase, and markedly inhibited by carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and nigericin. The findings support the contention that ATP hydrolysis in chromaffin granules leads to the generation of a ApH (interior acid) that provides the immediate driving force for epinephrine accumulation.MATERIALS AND METHODS Prepa...

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“…Chromaffin granules were isolated from bovine adrenal glands by differential sedimentation as described [17]. Membrane vesicles were obtained by osmotic shock, frozen, and stored at -70°C.…”

The molecular mechanism of "ecstasy" [3,4-methylenedioxy-methamphetamine (MDMA)]: serotonin transporters are targets for MDMA-induced serotonin release.

“…Reactions (200 1.l per assay) were stopped by dilution, filtration, and washing, and radioactivity in filtered vesicles was measured as described (28). The standard error of replicate assay values was typically <5% of the mean.…”

“…Platelet plasma membrane vesicles were isolated by the method of Barber and Jamieson (26) with the modifications described (27). Chromaffin granule membrane vesicles were prepared as described by Schuldiner et al (28) by repeated osmotic lysis of bovine adrenal medullary chromaffin granules isolated by differential sedimentation.…”

Homology of a vesicular amine transporter to a gene conferring resistance to 1-methyl-4-phenylpyridinium.

“…Chromaffin granules were prepared from bovine adrenal glands essentially as described by Kirshner (13). Membrane vesicles were obtained by osmotic shock, frozen, and stored in liquid air (14). Membranes (5 mg of protein per ml) were solubilized with Triton X-100 (1.5%) and fractionated using anion-(DE-52) and cation-(phosphoAbbreviations: VAT, vesicular amine transporter; MPP+, 1-methyl-4-phenylpyridinium; CHO, Chinese hamster ovary.…”