Monovalent cation dependency for the inhibition of outward transport of [3H]norepinephrine.

Rat hearts were labelled with [3H]norepinephrine in vivo. Slices of ventricles were prepared, preincubated in Krebs-HCO3 medium (KRB), and then incubated in a Na+-deficient, choline+-Krebs HCO3 (Ch+-Ca2+). The choline+-Krebs HCO3 medium induced a delayed neurosecretion which could be inhibited by either one of the blockers of the uptake of NE, cocaine or desipramine, when included in both the Krebs and the Ch+-Ca2+. When either desipramine or cocaine was present in the Na+-rich preincubation medium only, the duration of action of desipramine was more prolonged than that of cocaine. When desipramine was first added to the Ch+-Ca2+ medium at various times after incubation, the inhibitory response became smaller as the duration of preliminary Na+-deprivation was increased. After incubation for 80 min neither desipramine nor cocaine inhibited secretion. Sodium (added to Ch+-Ca2+ medium or in a Krebs replacement medium) then slowed the rate of release stimulated by Ch+-Ca2+ and facilitated an inhibitory action for both inhibitors. The inhibition did not require the continued presence of Na+. Depolarizing concentrations of K+ in the Ch+-Ca2+ medium with Na+ and either one of the inhibitors, prevented the inhibition of neurosecretion. However, K+ which was added to the preincubation medium with either inhibitor did not prevent the prolonged inhibition of neurosecretion. By contrast with the secretory response to Ch+-Ca2+, the secretory response to K+ in Na+-enriched Ch+-Ca2+ medium was weakly inhibited by the inhibitors of transport.

Vesicular site of action of lithium ion in choline-calcium stimulated adrenergic nerve endings of rat heart.

Reports from this laboratory have suggested that the secretion of norepinephrine (NE) in slices of ventricle from the rat heart, incubated in a medium of choline and Ca2+ (Ch+-Ca2+) deprived of Na+, is mediated by the outward transport of NE (blocked by cocaine) from synaptic vesicles fused or attached to the axolemma. Lithium (and reserpine), block the Mg2+-ATP-stimulated uptake of NE by isolated synaptic vesicles (Slotkin, Seidler, Whitmore, Salvaggio, and Lau, 1978). Hence, a hypothesis to be tested was that lithium ions would increase secretion of NE stimulated by Ch+-Ca2+. By contrast, amines mobilized by lithium ions (or reserpine) in control nerves would be deaminated. Experiments showed that lithium-Krebs increased the excretion of [3H]deaminated metabolites of [3H]NE. A much smaller quantity of [3H]NE was released by a process that was independent of Ca2+ and weakly inhibited by cocaine. When combined with Ch+Ca2+, lithium ions, in concentrations that were known to block uptake in isolated vesicles, induced a Ca2+-dependent secretion of [3H]amines. A small quantity of [3H]deaminated metabolites was excreted. Both processes were strongly inhibited by both cocaine and desipramine. Both reserpine (Bogdanski, 1982) and lithium ions blocked the inhibitory effect of exogenous ATP (2 mM) on secretion induced by Ch+-Ca2+. This effect indicated a proximity of vesicles to the axolemma. The effects of lithium ions could be explained by the hypothesis if lithium prevented storage of [3H]NE in vesicles by blocking the Mg2+ + ATP-dependent amine pump in vesicle membranes.

Evidence for the outward transport of norepinephrine in synaptic vesicles attached to the plasma membrane.

In a low-sodium, choline+ (Ch+) medium, rat heart slices, that were labelled in vivo with [3H]norepinephrine, exhibited a delayed Ca2+-dependent release of radioactivity, these radioactive compounds consisted of both amines and deaminated metabolites. The Ca2+-dependent release of radioactivity was largely blocked by cocaine suggesting that the release may represent an outward transport of [3H]amines. Reserpine, which is known to inhibit binding of norepinephrine to synaptic vesicles, stimulated the release of deaminated metabolites and some amines from the slices. Cocaine increased the release of [3H]amines in monoamine oxidase inhibited, reserpinized slices in the control medium. When Ch+-Ca2+ and reserpine stimulation were combined, the effects of Ch+-Ca2+ predominated. These effects were blocked by cocaine, which then permitted the response to reserpine to become established. The conclusion derived from the above experiments is that norepinephrine coming from synaptic vesicles brought in close proximity to the plasma membrane by a Ca2+-dependent system can be transported outwardly to the extraneuronal space by a cocaine-sensitive mechanism.

Norepinephrine uptake dependent upon apparent Mg++-ATPase activity and proton transport in storage vesicles in axoplasm.

A Ca++-dependent secretion of norepinephrine ([3H]NE) was evoked in adrenergic nerve endings in rat heart ventricle slices incubated in a modified Krebs-HCO3 medium containing choline Cl as the replacement for NaCl (Ch+-Ca++). Exogenous ATP inhibited secretion and lithium ion, a known inhibitor of NE uptake dependent upon Mg++-ATPase activity in vesicles (but not ouabain) prevented the response to ATP (Bogdanski, 1983,1986). It was suggested that vesicles attached to the axolemma recaptured [3H]NE from the extracellular fluid. This report indicates that other known inhibitors of uptake in isolated vesicles also inhibited the response to ATP in the attached vesicles. Included were two inhibitors of Mg++-ATPase activity, N-ethylmaleimide (NEM) and dicyclohexylcarbodiimide (DCCD), and the proton transporters 2,4-dinitrophenol (2,4-DNP 1.0 mM) and chlorpromazine (CPZ). Potassium ionophores (valinomycin with 2,4-DNP 0.1 mM, and nigericin) and a pH neutralizing reagent for vesicles (NH3 from ammonium sulfate in solution) were also effective. The uptake inhibitors, except 2,4-DNP, could also increase the rate of depletion of stored NE and its deamination in nonsecreting nerve endings incubated in Krebs-HCO3 (KRB) medium. Valinomycin by itself stimulated uptake in the presence of ATP. It is suggested that mechanisms of uptake and retention of NE in isolated vesicles (symposium (1982) Fed. Proc. 41:2742-2780) apply to the axoplasmic vesicles as well. Thus, the activity of Mg++-ATPase drives proton transport to establish the electrochemical gradients of H+, which drive the transport of NE. A lowering of the gradients can mobilize amines and evoke secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of exogenous adenosinetriphosphate on the choline-calcium stimulated release of 3H-norepinephrine in rat heart ventricle slices.

Evidence in this and other reports from this laboratory suggest that adrenergic nerves in rat heart ventricle slices incubated in a Na+-deprived (choline+) medium containing Ca++ (Ch+--Ca++), transport (by a cocaine-sensitive mechanism) 3H-norepinephrine outwardly from synaptic vesicles attached or fused to the plasma membrane. The 3H-amine secretion was not inhibited by probenecid, an anion transport inhibitor which may prevent exocytosis. The 3H-amine release was rapidly inhibited by exogenous nucleotides ATP, UTP, and GTP greater than ADP greater than AMP greater than the nucleoside adenosine. Magnesium++ tended to increase and reserpine to decrease the effect of ATP. Neither increasing the [Ca++] nor [Mg++] (to compete with Ca++ for ATP) decreased the effect of 3 mM ATP. After secretion began, lowering the Ca++ concentration by ommission, or by the inclusion of either a low concentration of EDTA or the Ca++-binding, but non-energy-conserving synthetic analogs of ATP: AMP--PCP and AMP--PNP, gradually lowered the rates of secretion. By comparison, the rapid effects of the energy-conserving nucleotides suggested that their effects were at least partially independent of chelation, and were energy dependent. ATP, unlike cocaine, did not inhibit the uptake of NE in a Krebs HCO3 medium. Inhibition of (Na+ + K+)-ATPase by ouabain neither inhibited the release by Ch+--Ca++, nor antagonizes the release inhibiting effect of ATP. Hence, ATP did not increase apparent retention of NE by stimulating the uptake of released NE. The ATP-inhibited secretion was not increased by theophylline.