Vladimír Palatý

RELEASE OF NORADRENALINE FROM ISOLATED RAT TAIL ARTERY INDUCED BY BAFILOMYCIN A1

The macrolide antibiotic bafilomycin A1, a selective inhibitor of the vesicular H+-transporting ATPase, increased irreversibly the overflow of 3,4-dihydroxyphenylethylene glycol from isolated segments of the rat tail artery. Maximum increase in the overflow was produced by exposing the tissues to 0.5 μmol/l bafilomycin As. Unless the Na−-dependent neuronal amine carrier (uptake1) was inhibited, overflow of noradrenaline was below the detection limit. The bafilomycin As-induced increase in overflow of noradrenaline from tissues with inhibited uptakes was accompanied by a significant decrease in the (noradrenaline overflow:glycol overflow) ratio. Unlike reserpine and tetrabenazine, the antibiotic did not alter the (noradrenaline overflow:glycol overflow) ratio in arteries incubated in Ca2+-free, 120 mmol/1 K+ medium.Bafilomycin A1 increased overflow of noradrenaline and normetanephrine from tissues with inhibited monoamine oxidase. Inhibitors of extraneuronal catecholamine transport (uptake2), corticosterone, 3-O-methylisoprenaline and 1,1′-diethyl-2,2′-cyanine, suppressed overflow of normetanephrine while increasing that of noradrenaline. Further increase in overflow of noradrenaline was produced by concomitant inhibition of uptake1. A similar effect was observed in tissues previously exposed to phenoxybenzamine. After exposure to bafilomycin As, tyramine and (+) amphetamine (10 μmol/l) were equally effective in increasing overflow of noradrenaline from tissues with inhibited monoamine oxidase into corticosterone-containing medium.Bafilomycin A1 promotes leakage of noradrenaline from storage vesicles without affecting its conversion to 3,4-dihydroxyphenylethylene glycol. When uptake1 is inhibited, axoplasmic noradrenaline can be translocated effectively across the axonal membrane by the ‘diffusional efflux’. When uptakes is inhibited, spontaneous quantal release contributes significantly to overflow of noradrenaline into normal media. The ‘diffusional efflux’ of noradrenaline is unaffected by inhibitors of uptake2. Even at highly elevated concentrations of axoplasmic noradrenaline, the uptake1-mediated influx of noradrenaline exceeds the uptake1-mediated efflux. Enhancement of noradrenaline overflow from tissues with inhibited monoamine oxidase by indirectly acting sympathomimetic amines depends primarily on their ability to induce leakage of the transmitter from storage vesicles rather than its translocation across the axonal membrane.

Glass electrodes in studies of ion-exchange kinetics☆

Abstract Procedures for the measurement with glass electrodes of ion-exchange processes involving Na + , K + , and H + have been described. Features characteristic of the transfers of Na + and K + during rewarming of the precooled rat tail artery were shown to illustrate the application of the technique to short- and long-term metabolic studies. The measurement of the exchange of Na + for H + during perfusion of an artery with a Na + -free medium was used as illustration for the application of the method to study of ion-exchange processes.

The cocaine-insensitive component of non-exocytotic efflux of noradrenaline from adrenergic axons in the isolated rat tail artery

SummaryThe 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 Ca2+-)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/1) 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 μmol/1 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. The data can, however, be accounted for by postulating that the component reflects the concentration of the protonated form of noradrenaline.