The working hypothesis was that the cocaine-insensitive component of non-exocytotic efflux of noradrenaline represents diffusion of the unprotonated amine across the axonal membrane. It was tested by examination of the effect of changing axoplasmic pH--and thus the fraction of extravesicular noradrenaline in the unprotonated form--on the overflows of endogenous noradrenaline and 3,4-dihydroxyphenylethylene glycol from rat tail arteries. The catechols were assayed by liquid chromatography with amperometric detection. To dissipate the H+ gradient across the axonal membrane, the tissues were incubated in media of different pH, in which Na+ was completely replaced with K+ and which were HCO3(-)-(and Ca(2+)-)free. Exposure of the tissues to these media produced substantial, but reversible increases in the overflow of noradrenaline. Subsequently, the overflows of both noradrenaline and the glycol kept rising, but their ratio did not change. Cocaine (0.1 mmol/l) lowered the (noradrenaline overflow: glycol overflow) ratio significantly. The ratio observed in its presence increased steeply with decreasing external and, presumably, axoplasmic pH. Addition of valinomycin and carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (1 mumol/l each) to the cocaine-containing media more than doubled the overflows without altering significantly the ratio. Under identical conditions, the overflow of noradrenaline from preparations with inactive neuronal monoamine oxidase did not decrease with decreasing pH. Since, in the presence of cocaine, the overflow ratio increased--rather than decreased--with decreasing pH, and because the overflow or noradrenaline from preparations with inactive monoamine oxidase did not decline with pH, the cocaine-insensitive component of noradrenaline efflux does not seem proportional to the axoplasmic concentration of the unprotonated amine.(ABSTRACT TRUNCATED AT 250 WORDS)
