Ghosts of chromaffin granules accumulate biogenic amines according to a ?pump and leak system? without contribution of carrier-mediated efflux

Abstract

Unidirectional inward transport into, accumulation by and loss of biogenic amines from ghosts of bovine chromaffin granules were studied to determine whether a carrier-mediated process contributes to the outward passage of amines across the granule membrane. In the presence of ATP-Mg2+, incubated ghosts (30 degrees C; pH 7.3) showed a reserpine-sensitive (IC50 2-5 nmol/l), unidirectional, inward transport of catecholamine (CA = 70% adrenaline/30% noradrenaline), 5-hydroxytryptamine (5-HT) and tyramine with different Km values (mumol/l: tyramine 2; 5-HT 5; CA 8) but with the same Vmax [20 nmol/(mg protein.min)] during the first 3 min of incubation. During longer incubation, the rate of unidirectional inward transport declined rapidly with time to about 30 to 40%, at which level it stayed nearly constant from 50 to 100 min of incubation. As the decline of unidirectional transport was independent of the amine accumulated in the ghosts, it is concluded that it reflects ageing of the membrane vesicles. During incubation for up to 100 min with CA, 5-HT or tyramine (10- to 30-fold Km) in the presence of ATP-Mg2+, the amine content of the ghosts increased, approaching a steady-state content (nmol amine/mg protein: CA 400-500, 5-HT 250, tyramine 60), which was negatively correlated with the lipid solubility of the amine (tyramine greater than 5-HT greater than CA), whereas the rate of approach to steady state (t1/2 in min: CA 20-30, 5-HT 7-10, tyramine less than 5) was positively correlated. Low concentrations of reserpine (greater than or equal to 25 nmol/l) caused net loss of amine from amine-loaded ghosts by inhibition of inward transport. However, reserpine did not reduce the fractional rate of loss (FRL) of CA-loaded ghosts induced by NH+4 or uncoupling agents. Accelerated exchange diffusion was not found to occur at the granule membrane, as addition of high concentrations of 5-HT or dopamine to CA-loaded ghosts did not result in a higher FRL of CA than did blockade of inward transport by reserpine. Analysis of steady-state kinetics revealed the following features of the granule transport. The approach to steady state (t1/2) was independent of the rate of inward transport. The steady-state amine content of ghosts approached a limiting value as the external concentration of the amine was increased; it was determined by the same kinetic constants (Km and the time-dependent Vmax) as found for the rate of carrier-mediated inward transport.(ABSTRACT TRUNCATED AT 400 WORDS)