Transmitter Efflux Research Articles

Effects of Pb 2+ and Cd 2+ on acetylcholine release and Ca 2+ movements in synaptosomes and subcellular fractions from rat brain and Torpedo electric organ

In this work we examined the effects of Pb 2+ and Cd 2+ on (a) [ 3H]ACh release and voltage-sensitive Ca 2+ channels in rat brain synaptosomes, and (b) 45Ca 2+ binding to isolated brain mitochondria and microsomes, and synaptic vesicles isolated from Torpedo electric organs. Pb 2+ ( K i ≈ 1.1 μM) and Cd 2+ ( K i ≈ 2.2) competitively block the K +-evoked influx of 45Ca 2+ through the ‘fast’ calcium channels in synaptosomes. The K is obtained with synaptosomes are in good agreement with the K i values obtained from electrophysiological experiments at the frog neuromuscular junction ( K Pb:0.99 μM, K Cd: 1.7 μM) 7. The K i for the inhibition of ACh release from synaptosomes by Cd 2+ is 4.5 μM. Pb 2+ is a less effective inhibitor of transmitter release ( K i ≈ 16 μM) because it secondarily augments spontaneous transmitter efflux. Cd 2+ has no effect on spontaneous release at concentrations ≤ 100 μM. The enhancing effect of Pb 2+ on spontaneous release is (a) not abolished by omission of Ca 2+ from the bathing medium, (b) is delayed by 1–2 min after the beginning of Pb 2+ exposure, (c) is reversed upon the removal of Pb 2+. In the presence of physiological concentrations of ATP (1 mM), Mg 2+ (1 mM) and P i (2 mM), 1–10 μM Pb 2+ inhibits calcium uptake but Pb 2+ > 10μM causes a several-fold stimulation of passive binding of calcium to the organelles. This effect is associated with Pb 2+-induced enhancement of P i uptake. Cd 2+ inhibits Ca 2+ binding at all concentrations tested (1–50 μM) and reduces the Pb 2+-induced Ca 2+-binding to organelles. Neither Pb 2+ nor Cd 2+ have any discernible effects on spontaneous loss of calcium from mitochondria or microsomes preloaded with 45Ca. In summary, these data are consistent with the notion that Pb 2+ and Cd 2+ are potent blockers of presynaptic voltage-sensitive Ca 2+ channels and the evoked release of transmitter which is contingent on Ca 2+ influx through these channels. Our results are not consistent with the hypothesis that Pb 2+ augments spontaneous release by interfering with intraterminal Ca 2+-buffering by mitochondria, endoplasmic reticulum, or synaptic vesicles.

A hypothesis to explain the presynaptic effects of adrenoceptor antagonists.

The hypothesis of negative feedback regulation of transmitter release was examined in a range of tissues obtained from three species. Tissues were transmurally stimulated with 100 pulses at 2 Hz with pulse durations from 50 microseconds to 5,000 microseconds, and the efflux of [3H]-noradrenaline determined. The stimulation-induced efflux of tritium increased with increasing pulse duration, but yohimbine, a prototypal alpha 2-antagonist had an effect which was consistently contrary to expectations for a negative feedback system. Enhancement of efflux by the antagonist, supposedly correlated directly with the extent of ongoing auto-inhibition, became smaller rather than larger as the stimulation-induced efflux rose with increases in pulse duration, with all other parameters of stimulation maintained constant. Similar findings were obtained in rat spleen with the haloalkylamine antagonist, phenoxybenzamine. It is concluded that the presynaptic effects of adrenoceptor antagonists do not involve a negative feedback function nor do they relate, in any detectable way, to the extracellular concentration of transmitter. The effects on stimulation-induced tritium efflux of yohimbine, phenoxybenzamine and enlargment of the pulse duration, in a variety of tissues, support the previously described hypothesis of a common action to enhance efflux. The antagonists increased efflux to approximately the same value between 50 and 1,000 microseconds pulse durations and that value was equivalent to that obtained in each given tissue with pulses of 1,000-2,000 microseconds in the absence of the antagonist. Tetraethylammonium, an inhibitor of stimulation-induced potassium efflux from nerves had an effect on transmitter efflux in rat spleen essentially like that of the adrenoceptor antagonists. These findings provide further support for an alternative to the hypothesis of negative feedback. Yohimbine and other presynaptic antagonists may prolong the period of potassium efflux from nerve varicosities, and by this means prolong depolarization and the associated period of transmitter release, rather than act by disrupting an ongoing system sensing and responding to fluctuations in extracellular transmitter levels.

Yohimbine and prolongation of stimulation pulse duration alter similarly 3H-transmitter efflux in heart: an alternative to the negative feedback hypothesis.

The hypothesis of negative feedback regulation of noradrenaline release was studied in guinea-pig left atrial halves mounted in vitro. Tissues were transmurally stimulated with 30, 100 or 300 pulses at 2 Hz with pulse durations ranging from 50 mus to 2,000 mus, and the efflux of 3H-transmitter determined. The efflux of tritium increased with increasing pulse duration as was anticipated, but the effects of supposed presynaptic antagonism by yohimbine were opposite to expectations for a negative feedback system. The magnification of efflux by yohimbine, compared to untreated controls was less rather than more as stimulation-induced transmitter efflux climbed with increases in pulse duration, and with all other parameters of stimulation held constant. It is concluded that the neuronal effect of yohimbine is not linked to negative feedback or to any other system sensing the perineuronal concentration of previously released transmitter. Analysis of the effects on tritium efflux of yohimbine and of prolongation of the stimulation pulse duration, reveals a similarity in the way that they promote transmitter release. Yohimbine increased efflux to approximately the same value at all pulse durations between 50 and 1,000 mus and the value reached was equivalent to that obtained in untreated atria during stimulation with very long pulses (2,000 mus duration). It is suggested that yohimbine prolongs the outward current attributable to the efflux of potassium from axon terminals, and by this means prolongs depolarization and the period of transmitter release. Tetraethylammonium (TEA), a quaternary ion known to plug potassium efflux channels, had an effect on transmitter efflux that was, in some ways, similar to that of yohimbine but of greater magnitude. The present findings provide, for the first time, an alternative to the hypothesis of negative feedback, that might explain the presynaptic effects of adrenoceptor antagonists and possibly other compounds.

Effects of Ca-antagonists on neuromuscular transmission in the rabbit ear artery.

1. The effects of three Ca-antagonists: diltiazem, nicardipine and flunarizine have been studied on excitatory junction potentials (e.j.p.s), force development and efflux of transmitter during stimulation of perivascular nerves in the rabbit ear artery. 2. Stimulation of these perivascular nerves produces excitatory junction potentials and repetitive stimulation causes facilitation. Increasing the frequency or the number of stimuli initiates an action potential and a large contraction. Both phenomena are completely suppressed by 3 X 10(-7) M TTX. 3. Ca-antagonists at 10(-5) M do not affect the resting membrane potential, but flunarizine and nicardipine at concentrations exceeding 10(-5) M reduce the amplitude of e.j.p.s and of the action potentials and also the concomitant contraction induced by nerve stimulation. Diltiazem at concentrations below 3 X 10(-5) M has no effect on e.j.p.s and action potentials while at 10(-4) M, it largely suppresses e.j.p.s, spikes and contraction. This inhibitory effect of the Ca-antagonists increases with prolonged exposure. 4. All these Ca-antagonists induce an increased release of 3H-DOPEG from the nerve terminals, but in our experiments in vitro they do not reduce the efflux of 3H-noradrenaline induced by nerve stimulation. 5. The results indicate that Ca-antagonists might affect the excitation-contraction coupling in vivo by inhibiting the Ca influx activated by endogenous noradrenaline. They do not exert an acute effect on the noradrenaline release induced by stimulation. The increased DOPEG release by Ca-antagonists remains unexplained.

Evidence against the unitary hypothesis of agonist and antagonist action at presynaptic adrenoceptors.

1 The concept that presynaptic receptors regulate noradrenergic transmitter release via a system of inhibitory receptors mediating negative feedback relies on a supposed association between increases in stimulation-induced efflux of [3H]-noradrenaline by antagonists and blockade by them of the inhibitory effects of exogenous noradrenaline. 2 It was shown in guinea-pig ureter, that yohimbine (3 X 10(-7)M), a presumed selective presynaptic antagonist, increased transmitter efflux substantially at 1 Hz and 5 Hz with 100 pulses, purportedly representing antagonism of the inhibitory effect of locally released noradrenaline but did not reduce the inhibitory effect of exogenous noradrenaline (1.8 X 10(-6)M or 1.8 X 10(-7)M) except in one case. 3 Additionally, the inhibitory effect of oxymetazoline (1.0 X 10(-7)M or 1.0 X 10(-8)M) on stimulation-induced efflux was in no way antagonized by yohimbine (3 X 10(-7)M). 4 It is concluded that the increased efflux of [3H]-noradrenaline produced by antagonists and the decreased efflux produced by exogenous agonists may represent actions at different loci and that the hypothesis of presynaptic feedback regulatory sites is still not substantiated.

ALTERATION OF NOREPINEPHRINE RELEASE FROM [3]-NOREPINEPHRINE PRELOADED BASILAR ARTERY BY NAPHTHALENESULFONAMIDES

AbstractThe effects of W-7, W-5, No. 233, and chlorpromazine on sympathetic nerve transmitter efflux were compared in superfused canine basilar arterial preparations preloaded with [3H]-norepinephrine. In vitro experiments suggest that these agents are selective calmodulin antagonists. The electrical transmural stimulation-induced efflux of tritium was reduced by W-7 and W-5, although they were unexpectedly equipotent since W-5 is a chloride-deficient derivative of W-7 and has a lower affinity for calmodulin than does W-7. The median inhibitory concentration (IC50) of W-7 for stimulation-induced efflux was 3.4×10−6 M. The addition of No. 233 at relatively high concentrations (3×10−5 M and 5×10−5 M) caused a reduction in stimulation-induced efflux. Chlorpromazine produced a dual effect on the efflux: enhancement at low concentrations (below 1 ×10−6 M) and reduction at high concentrations. The IC50 values of No. 233 and chlorpromazine were 3.5×10−5 M and 2.5 × 10−5 M, respectively. The additions of these four agents also caused a significant elevation in the spontaneous basal efflux of tritium from the preparations. The concentrations of the agents that elevated the spontaneous efflux to the level of half the stimulation -induced efflux were closely fitted to the IC50 values for stimulation-induced efflux. This finding indicates that the elevation in spontaneous efflux is directly proportional to the reduction in electrical stimulation-induced efflux. From these findings, it is concluded that naphthalenesulfonamides including W-7 have a direct effect on sympathetic nerve terminals which is independent of the effect on calmodulin.

Alteration of norepinephrine release from [3h]-norepinephrine preloaded basilar artery by naphthalenesulfonamides.

The effects of W-7, W-5, No. 233, and chlorpromazine on sympathetic nerve transmitter efflux were compared in superfused canine basilar arterial preparations preloaded with [3H]-norepinephrine. In vitro experiments suggest that these agents are selective calmodulin antagonists. The electrical transmural stimulation-induced efflux of tritium was reduced by W-7 and W-5, although they were unexpectedly equipotent since W-5 is a chloride-deficient derivative of W-7 and has a lower affinity for calmodulin than does W-7. The median inhibitory concentration (IC50) of W-7 for stimulation-induced efflux was 3.4 X 10(-6) M. The addition of No. 233 at relatively high concentrations (3 X 10(-5) M and 5 X 10(-5) M) caused a reduction in stimulation-induced efflux. Chlorpromazine produced a dual effect on the efflux: enhancement at low concentrations (below 1 X 10(-6) M) and reduction at high concentrations. The IC50 values of No. 233 and chlorpromazine were 3.5 X 10(-5) M and 2.5 X 10(-5) M, respectively. The additions of these four agents also caused a significant elevation in the spontaneous basal efflux of tritium from the preparations. The concentrations of the agents that elevated the spontaneous efflux to the level of half the stimulation-induced efflux were closely fitted to the IC50 values for stimulation-induced efflux. This finding indicates that the elevation in spontaneous efflux is directly proportional to the reduction in electrical stimulation-induced efflux. From these findings, it is concluded that naphthalenesulfonamides including W-7 have a direct effect on sympathetic nerve terminals which is independent of the effect on calmodulin.

The effects of (+)- and (-)-propranolol on 3H-transmitter efflux in guinea-pig atria and the presynaptic beta-adrenoceptor hypothesis.

The effects of isoprenaline and of the (+)- and (-)-isomers of propranolol on the stimulation-induced overflow of 3H-transmitter was assessed in guinea-pig atria to evaluate the hypothesis of presynaptic beta-adrenoceptors. 2 Isoprenaline (1.2 x 10(-8) M) enhanced the efflux of tritium at 2 and 5 Hz with 100 pulses and did so to a similar extent at both frequencies. 3 The (-)-isomer of propranolol (1.0 x 10(-7) M) blocked the enhancing effect of isoprenaline but did not by itself modify transmitter efflux. 4 The (+)-isomer of propranolol, almost devoid of beta-adrenoceptor blocking properties, was also effective at 1.0 x 10(-7) M in blocking the enhancement of tritium efflux by isoprenaline. 5 The (-)-isomer of propranolol (1.0 x 10(-7) M) blocked almost entirely the inotropic response to isoprenaline (3 x 10(-7) M) but even 3.0 x 10(-6) M (+)-propranolol was ineffective in antagonizing the beta-adrenoceptor-mediated contractile responses to the catecholamine. 6 It is concluded that the presynaptic site of isoprenaline action does not show the requisite stereo-specificity of beta-adrenoceptors and that a 'non-specific' action of the antagonist probably accounts for its reduction of the effect of isoprenaline.

Inhibition by dopamine of the stimulation-induced efflux of [3H]noradrenaline in renal arteries: limitations of the unitary hypothesis of presynaptic regulation of transmitter release.

The supposition was tested that a pattern of agonist inhibition of 3H-labelled transmitter efflux which shows a decline in intensity as the frequency of stimulation rises provides major evidence for the existence of a functional autoinhibitory feedback loop mediated by released transmitter and located on adrenergic nerve terminals. For these experiments dopamine was used, as it is not released in significant quantities in the ordinary course of sympathetic nerve activation and it appears to act presynaptically in several tested tissues at a locus discrete from that acted on by noradrenaline. Dopamine (3 X 10(-7) and 3 X 10(-6) M) inhibited the stimulation-induced efflux of [3H]noradrenaline from cattle renal arteries and did so to a declining extent with increasing frequency (1-15 Hz). Known antagonists of dopamine action, pimozide and metoclopromide, antagonized this effect of dopamine but did not by themselves enhance stimulation-induced transmitter efflux or block the inhibiting effects of exogenous noradrenaline on efflux, establishing the specificity of dopamine action in renal artery and indicating the absence of an operative negative feedback loop mediated by dopamine. This interpretation was substantiated by the finding that although dopamine reduced the magnitude of contractile responses to nerve stimulation neither pimozide nor metoclopromide enhanced the amplitude of nerve-induced contractions. It thus appears that a pattern of agonist effect on transmitter efflux which manifests a diminution in intensity as the frequency of stimulation climbs is not derived from the operation of an ongoing autoinhibitory feedback system regulating transmitter release but by a yet to be established factor.

Enhancement of noradrenaline release from rat cerebral cortex by neuroleptic drugs.

The effect of different neuroleptic drugs (levomepromazine, haloperidol, thioridazine, clozapine and sulpiride) on (-)-3H-noradrenaline uptake and release by parieto-occipital slices of the rat cerebral cortex was investigated. 1. All neuroleptic drugs tested increased the 3H-efflux from electrically stimulated cortical slices preincubated in (-)-3H-noradrenaline already at 1 microM, clozapine was the most potent compound followed by haloperidol, thioridazine, levomepromazine and sulpiride. The enhanced 3H-efflux due to field stimulation was found at concentrations, which did not increase the basal 3H-efflux. Only haloperidol raised the basal 3H-efflux at 1 microM. 2. All neuroleptic drugs failed to inhibit (-)-3H-noradrenaline (10(-7) M) accumulation by cortical slices at 1 microM. Sulpiride was inactive in concentrations up to 100 microM. Clozapine again proved to be most potent at 10-100 microM. 3. Clozapine was able to enhance the stimulated transmitter overflow when noradrenaline uptake was already blocked by cocaine thus indicating a different mode of action. 4. Clozapine and levomepromazine antagonized the presynaptic alpha-adrenergic effect of clonidine. 5. The antidepressant drug amitriptyline increased the transmitter efflux at the same concentrations and to a similar extent as neuroleptic agents. It is concluded that neuroleptics enhance the stimulation induced noradrenaline release mainly by acting on presynaptic alpha-adrenoceptors. The effect of clozapine, however, includes a noradrenaline uptake inhibition. These findings may explain the increased noradrenaline turnover produced by neuroleptic drugs and the weak antidepressant action of low potent neuroleptics as well.

Noradrenaline inhibition of transmitter efflux in a renal artery preparation and the presynaptic receptor theory.

The effect of noradrenaline on the stimulation-induced efflux of tritium in cattle renal arteries preincubated with [3H]noradrenaline was determined. Preparations were stimulated transmurally, with 300 shocks over a range of frequencies (1--15 Hz) in the presence and absence of noradrenaline (3 X 10(-6) M). The agonist inhibited the efflux most at 1 Hz but the extent of the inhibition did not vary with frequency between 2, 5, and 15 Hz. It is concluded that a negative feedback system, modulating neurotransmitter release, and increasingly activated by endogenously released noradrenaline as the frequency of stimulation rises, cannot account for the pattern of efflux inhibition induced by exogenous noradrenaline.

Interaction between Neuronal Amine Uptake and Prejunctional Alpha-Adrenergic Receptor Activation in Smooth Muscle from Canine Blood Vessels and Spleen

Experiments were performed to determine the conditions in which norepinephrine release from adrenergic nerve terminals in smooth muscle from canine blood vessels and spleen might be inhibited by prejunctional alpha-adrenergic receptor activation. Strips of aorta, mesenteric and splenic arteries, splenic capsule and portal and saphenous veins were labeled with 7-3H-norepinephrine and mounted for superfusion. In the portal vein, an inhibitory effect of prejunctional receptor activation with exogenous norepinephrine (1.2 X 10(-6) M) on transmitter efflux could be demonstrated during electrical stimulation (9 V, 2 Hz) of the nerve terminals. By contrast, in the other tissues, inhibition of transmitter release during electrical stimulation or depolarization of the nerve terminals with K+ (40 mEq/l) could only be demonstrated aftet blockade of the neuronal uptake mechanism. That activation of prejunctional alpha-adrenergic receptors in blood vessels inhibits the exocytotic process is suggested by the failure of exogenous norepinephrine to affect either the basal efflux of 3-H-norepinephrine or the displacement of 3H-norepinephrine by tyramine.

Effects of pimozide on noradrenergic transmission in rabbit isolated ear arteries

In the rabbit ear artery both dopamine and noradrenaline inhibit stimulation-induced (S-I) transmitter noradrenaline efflux. Pimozide, which is supported to be a specific dopamine receptor antagonist, was used to further study the efects of dopamine on transmitter efflux. In a concentration of 0.2 μM pimozide blocked the inhibition of S-I efflux produced by 0.5 μM dopamine but not that produced 0.5 μM noradrenaline. In a concentration of 10 nM, pimozide enhances transmitter release and vosoconstriction responses to sympathetic nerve stimulation; this may be due to blockade of feedback inhibition of transmitter release by endogenous dopamine. In a concentration of 1 μM, pimozide reduced transmitter release and vasoconstrictor responses to sympathetic nerve stimulation. Vasoconstrictor responses to noradrenaline and histamaine are antogonized by pimozide in a noncompetitive manner.

EFFECTS OF THE MUSCARINIC AGONIST McN‐A‐343 ON THE RELEASE BY SYMPATHETIC NERVE STIMULATION OF [3H]‐NORADRENALINE FROM RABBIT ISOLATED EAR ARTERIES AND GUINEA‐PIG ATRIA

1 McN-A-343 (4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride) in concentrations of 10(-5) and 10(-4)M inhibits the stimulation-induced efflux of radioactivity from segments of rabbit ear artery that have previously been incubated with (-)-[(3)H]-noradrenaline, and also decreases the contractile response.2 The inhibitory effects of McN-A-343 (10(-5)M) on the efflux of radioactivity and the contractions induced by low frequencies of stimulation (2 and 5 Hz) are reversed by atropine, but atropine does not modify these effects with high frequencies of stimulation (20 and 50 Hz).3 McN-A-343 (10(-4)M) enhances the stimulation-induced efflux of radioactivity from guinea-pig atria that have previously been incubated with (-)-[(3)H]-noradrenaline, and prolongs the positive inotropic response. These effects are not modified by atropine.4 It is concluded that McN-A-343 has different effects on adrenergic transmitter release in the two tissues. In the artery, it acts as an agonist on muscarinic receptors of adrenergic terminals to inhibit transmitter release at low frequencies of stimulation. In the atria it enhances transmitter efflux from the tissue, largely by inhibiting re-uptake.