Accumulation of amantadine by isolated chromaffin granules

Abstract

The accumulation of amantadine and the effect of this pharmacologic amine upon the magnitude of the transmembrane proton gradient (ΔpH) and of the transmembrane potential gradient (ΔΨ) were investigated in bovine adrenal chromaffin granules isolated in isotonic sucrose. Freshly isolated chromaffin granules have an intragranular pH of 5.5, as measured by [ 14C]methylamine distribution [R. G. Johnson and A. Scarpa, J. gen. Physiol. 68, 601 (1976)]. The addition of amantadine (1–50 mM) to well-buffered suspensions of granules at pH 7.0 resulted in a dose-related alkalinization of the granule interior. Similar results were obtained with equivalent external concentrations of ammonia. When the time-resolved influx of labeled amines into the granules was studied radiochemically, using low external amine concentrations, the accumulation of [ 3H]amantadine was quite similar to that of [ 14C]methylamine with regard to rate and extent over a wide range of magnitudes of the transmembrane proton gradient. However, unlike biogenic amine accumulation into the chromaffin granule, which is driven by both transmembrane proton and potential gradients, the accumulation of [ 3H] amantadine was not stimulated by the existence of a transmembrane potential, nor was it inhibited by reserpine. Moreover, low concentrations of amantadine did not competitively inhibit biogenic amine accumulation in the isolated granules. These results indicate that amantadine can distribute across the membrane of chromaffin granules according to the magnitude of the endogenous ΔpH, and suggest that in vivo amantadine may be concentrated and stored as a pharmacologic agent in amine containing granules.