Reserpic acid as an inhibitor of norepinephrine transport into chromaffin vesicle ghosts.

L Chaplin,A H Cohen,P Huettl,M Kennedy,D Njus,S J Temperley

doi:10.1016/s0021-9258(17)39134-2

Abstract

Reserpic acid, a derivative of the antihypertensive drug reserpine, inhibits catecholamine transport into adrenal medullary chromaffin vesicles. Since it does not affect the membrane potential generated by the H+-translocating adenosine triphosphatase but inhibits ATP-dependent norepinephrine uptake with a Ki of about 10 microM, reserpic acid must block the H+/monoamine translocator. Because reserpic acid is much more polar than reserpine, it does not permeate the chromaffin vesicle membrane, nor is it transported into chromaffin vesicle ghosts in the presence of Mg2+-ATP. Although it inhibits norepinephrine transport when added externally, reserpic acid does not inhibit when trapped inside chromaffin vesicle ghosts. Therefore, reserpic acid must bind to the external face of the monoamine translocator and should be a good probe of the translocator's structural asymmetry.

Highlights

Reserpic acid, a derivative of the antihypertensive drug reserpine, inhibits catecholamine transport into adrenal medullary chromaffin vesicles
A derivative of the antihypertensive drug reserpine, inhibits catecholamine transport into adrenal medullary chromaffin vesicles. Since it does not affect themembranepotential generated by the H*translocating adenosine triphosphatase but inhibits ATP-dependent norepinephrine uptake with a Ki of about 10 PM, reserpic acid mustblock the H+/monoamine translocator
We show here that reserpic acid is animpermeant inhibitor of norepinephrine transport in membrane vesicles prepared from bovine adrenal medullary chromaffin vesicles

Summary

MATERIALS AND METHODS

Reserpine is an antihypertensive drugthat blocks the storage of biogenic amines in the secretory vesicles of the adrenal medulla, adrenergic neurons, and platelets (Stitzel, 1977). Reserpine is apparentlya competitive inhibitor of the monoamine translocator; it exhibits a Kiof about 10 nM in chromaffin vesicles from bovine adrenal medulla (Kanner et al, 1979;Deupree and Weaver, 1984)and in synaptic vesicles from bovine caudate nucleus (Near and Mahler, 1983). For assays of reserpic acid content, ghosts were collected either by centrifugation (Table I and Fig. 2) or by filtration (Table I and Fig. 3). A sample from the supernatant (0.8 ml) was diluted to 2.0 ml with ethanol and assayed In the latter case, ghosts were collected on cellulose acetate filters (0.45-pm pore size) and washed with -2 mlof0.3 M NaCl, 10 mM Hepes, pH 7.0. Filters were incubated in 5 mlof ethanol overnight, and a 2.0-ml aliquot of the ethanolextract was assayed for reserpic acid. Protein was assayed using biuret reagent (Casey et al, 1976)

RESULTS

DISCUSSION

P bance changes measured 4 min after