Stimulation-induced release of norepinephrine from rat superior cervical ganglia in vitro

Superior cervical ganglia removed from adult rats and grown in organ culture for two days develop axonal sprouts which take up increased amounts of labeled norepinephrine. We have found that the labeled catecholamine can be released by either potassium or electrical stimulation from cultured ganglia to a much greater extent than from fresh ganglia and that the release is calcium-dependent. Phenoxybenzamine, which enhances release of norepinephrine from sympathetic nerve endings, increased release of the catecholamine from cultured ganglia. Bretylium, which blocks release of the amine from sympathetic nerve endings, diminished stimulation-induced release of norepinephrine from cultured ganglia. Our observations suggest that the axonal sprouts (formed during organ culture of sympathetic ganglia), which release norepinephrine in response to electrical-field stimulation, resemble sympathetic nerve endings rather than ganglion cell bodies.     

Inhibition of [3H]norepinephrine uptake in peripheral organs of some mammalian species by ouabain

In order to demonstrate the possible involvement of (Na⁺ + K⁺)-ATPase in the high affinity uptake of [³H]-norepinephrine in the sympathetic nerve endings, the effect of ouabain on [³H]norepinephrine uptake in spleen and heart slices of five mammalian species was examined. The ouabain sensitivity of [³H]norepinehrine uptake in the heart slices from various species, as determined by the estimation of IC₅₀, was, in increasing order, lamb (2.3 μM) < calf (2.5 μM) < guinea pig (4 μM) < rabbit (10 μM) << rat (>500 μM). The IC₅₀ values in the spleen slices were: lamb (1 μM) < calf (3.2 μM) < rabbit (9.5 μM) < guinea pig (25 μM) << rat (>500μM). The IC₅₀ values for the inhibition of specific [³H]ouabain binding in the microsomal fractions of spleen and heart of the five mammalian species by ouabain were similar to the IC₅₀ values for the inhibition of [³H]norepinephrine uptake by the cardiac glycoside. Since ouabain is known to bind exclusively to (Na⁺ + K⁺)-ATPase of a microsomal fraction, these results suggest that the inhibition of [³H]norepinephrine uptake in the sympathetic nerve endings by ouabain is mediated by the inhibition of (Na⁺ + K⁺)-ATPase.     

A comparison of the inhibitory effects of new non-tricyclic amine uptake inhibitors on the uptake of norepinephrine and 5-hydroxytryptamine into synaptosomes of the rat brain

The effects of new non-tricyclic amine uptake inhibitors, FS32 and FS97, on the uptake of [³H]-norepinephrine (NE) into the hypothalamic synaptosomes and [³H]-5-hydroxytryptamine (5-HT) into whole brain synaptosomes were studied. Their effects were compared with those of tricyclic antidepressants. The uptake of [³H]-NE was inhibited competitively by FS32 and FS97 with a respective K_i value of 6.5 × 10^?7 M and 3.8 × 10^?7 M. The potency of FS32 and FS97 to inhibit this uptake was almost comparable to that of clomipramine and imipramine, respectively. In the case of [³H]-5-HT uptake, FS32 and FS97 also showed competitive inhibition with a respective K_i value of 2.9 × 10^?6 M and 5.9 × 10^?6M. The ability of FS32 to inhibit [³H]-5-HT uptake was almost equal to that of nortriptyline, while FS97 was two times more potent than iprindole in inhibiting this uptake.     

Acute sinoaortic denervation induces pre- and postsynaptic alpha-adrenergic subsensitivity in mesenteric arteries in the rat

Bilateral sinoaortic denervation induced a rapid increase in blood pressure and heart rate in the rat. Four hours after sinoaortic denervation, the dose-response curve to l-norepinephrine (NE) in perfused mesenteric arteries in vitro was shifted to the right and the maximal response reduced, compared with sham-operated controls. These changes were abolished in the presence of cocaine (10 μg/ml). Four hours after sinoaortic denervation, the dose-response curves to methoxamine and serotonin, but not to adenosine-5'-triphosphate (ATP), were significantly reduced and the muscular response induced by barium chloride was not modified. Vascular responses to stimulation with different frequencies of the sympathetic nerve were reduced 4hr after sinoaortic denervation. These changes in both the responses to l-NE and to nerve stimulation were reversed 24 hr after sinoaortic denervation. In the same preparation, 4 hr after sinoaortic denervation, the fractional release per pulse of [³H]-NE was significantly enhanced at 10 and 20 Hz frequencies of stimulation, whereas the blockade of the alpha-presynaptic receptors with phentolamine (3.1 μM) did not increase the release of [³H]-NE induced by nerve stimulation, as was observed in sham-operated controls. The results are compatible with the hypothesis that 4 hr after sinoaortic denervation, the pre- and postsynaptic alpha-adrenoceptors are desensitized. These changes could affect arterial sympathetic neurotransmission; the increase in blood pressure observed following sinoaortic denervation could therefore not be explained only by an increase in sympathetic tone.     

Pentazocine Inhibits Norepinephrine Transporter Function by Reducing its Surface Expression in Bovine Adrenal Medullary Cells

(±)-Pentazocine (PTZ), a non-narcotic analgesic, is used for the clinical management of moderate to severe pain. To study the effect of PTZ on the descending noradrenergic inhibitory system, in the present study we examined the effect of [³H]norepinephrine (NE) uptake by cultured bovine adrenal medullary cells and human neuroblastoma SK-N-SH cells. (?)-PTZ and (+)-PTZ inhibited [³H]NE uptake by adrenal medullary cells in a concentration-dependent (3 – 100 μM) manner. Eadie-Hofstee analysis of [³H]NE uptake showed that both PTZs caused a significant decrease in the V_max with little change in the apparent K_m, suggesting non-competitive inhibition. Nor-Binaltorphimine and BD-1047, κ-opioid and σ-receptor antagonists, respectively, did not affect the inhibition of [³H]NE uptake induced by (?)-PTZ and (+)-PTZ, respectively. PTZs suppressed specific [³H]nisoxetine binding to intact SK-N-SH cells, but not directly to the plasma membranes isolated from the bovine adrenal medulla. Scatchard analysis of [³H]nisoxetine binding to SK-N-SH cells revealed that PTZs reduced the B_max without changing the apparent K_d. Western blot analysis showed a decrease in biotinylated cell-surface NE transporter (NET) expression after the treatment with (?)-PTZ. These findings suggest that PTZ inhibits the NET function by reducing the amount of NET in the cell surface membranes through an opioid and σ-receptor–independent pathway.     

The effects of antidepressants on the retention and metabolism of [3H]-norepinephrine in rat brain slices

Tricylic antidepressants acutely decrease the neuronal retention of [³H]-norepinehrine ([³H]-NE) by blocking neuronal membrane uptake and/or vesicular uptake and binding. To distinguish between effects upon the plasma membrane and upon the vesicular membrane, the retention, deamination, and O-methylation of [³H]-NE by rat brain slices were investigated in the presence of several antidepressant agents. The effects of antidepressants were compared to those of the prototype inhibitors, cocaine and reserpine, using slices of hypothalamus, brainstem. parietal cortex and caudate nucleus. Cocaine, which inhibits neuronal membrane uptake, decreased both the deamination and retention of [³H]-NE, while O-methylation was increased. Reserpine, which inhibits vesicular transport and binding, increased deamination, while it reduced retention without affecting the 0-methylation of [³H]-NE. The effects of desipramine, a prototype tricyclic antidepressant, were found to depend on the concentration. At low concentrations (10^?9-10^?8M), desipramine inhibited the retention and deamination of [³H]-NE in each brain region except the caudate. At higher concentrations (10^?7-10^w?4M), the retention of [³H]-NE was reduced further. However, deamination was increased in the caudate and, in the other three regions, deamination did not decrease further. Nortriptyline and protriptyline had actions similar to desipramine, whereas, iprindole did not affect [³H]-NE retention. These results suggest that tricyclic antidepressants are not specific selective inhibitors of neuronal membrane transport.     

Evidence for carrier-mediated efflux of dopamine from corpus striatum

This study was designed to further evaluate the hypothesis that the efflux of cytoplasmic dopamine from nerve endings of the corpus striatum can occur by carrier-mediated facilitated diffusion. Since dopamine (DA) and sodium ions are thought to be cotransported by the neuronal uptake carrier, the change in DA efflux upon alterations induced in the sodium gradient was observed. Ouabain was used to inhibit Na⁺K⁺-ATPase and thus increase intracellular sodium concentration, while metabolic inhibitors resulted in the same effect by reducing the amount of ATP available for Na⁺K⁺-ATPase. Rats were pretreated with reserpine and incubated with [³H]DA in the presence of pargyline so that the efflux of cytoplasmic [³H]DA could be assessed. Under these conditions, ouabain produced a concentration-related increase in the efflux of [³H]DA from nerve endings of the corpus striatum. The ouabain-induced release of [³H]DA was reduced by 10^?M benztropine, a dopamine uptake inhibitor. Benztropine (10^?5M) also reduced the increase in release of pHjDA when the same tissue preparation was incubated with the metabolic inhibitors sodium cyanide, iodoacetic acid and dinitrophenol. Ouabain at a concentration which caused slight release of [³H]DA potentiated the efflux produced by 3 × 10^?6 M amphetamine, 3 × 10^?6M unlabeled DA and 10^?4M fenfluramine. These results suggest that release of [³H]DA by sympathomimetic amines may be a carrier-mediated process. The Q₁₀ for 10^?4 M ouabain-induced release of [³H]DA was much higher than the Q₁₀ for the above processes and, therefore, the rate-limiting step in ouabain-induced release may involve inactivation of an enzyme such as Na⁺K⁺-ATPase which is coupled to the transport of sodium. Thus, by changing the availability of sodium with ouabain or metabolic inhibitors and by changing the cytoplasmic accessibility of the membrane carrier with benztropine or phenethylamines, the efflux of cytoplasmic [³H]da can be altered markedly.     

The in vitro uptake of biogenic amines by snail (Heliz pomatia) nervous tissue.

The uptake of [³H]5-HT, [³H]dopamine, [³H]noradrenaline and [³H]octopamine into the suboesophageal ganglia of the snail, Helix pomatia, was studied. When tissues were incubated at 25° in mediums containing radioactive amines, tissue: medium ratios of about 30:1, 18: l, 4:1 and 5:1 for 5-HT, dopamine, noradrenaline and octopamine respectively were obtained after 20–30 min incubation. Tissues incubated at 25° in mediums containing radioactive amines for 20–30 min showed that 90% of the radioactivity was present as unchanged [³H]5-HT, [³H]dopamine, [³H]noradrenaline or [³H]octopamine. The high tissue : medium ratios for 5-HT and dopamine, but not for noradrenaline and octopamine, demonstrated saturation kinetics which were dependent upon temperature and sodium ions. From the Lineweaver-Burk plots, two uptake mechanisms for 5-HT at 25° were resolved, the high affinity uptake process having a K_m1valueof 8.48 × 10^?8 M and V_max1 value of 0.077 nmole/g per min, while the lower affinity process had a K_m2 value of 1.8 × 10^?6 M and a V_max2 value of 0.66 mole/g per min. At 0° a single uptake mechanism for 5-HT occurred which gave a K_m value of 0.152 × 10^?8 M and a V_m value of 0.0203 nmole/g per min. In the case of dopamine, the Lineweaver-Burk plot of 25° showed a single uptake process with values for K_m and V_max of 1.02 × 10^?7 M and 0.0673 nmole/g per min respectively. This process did not function at 0°. The effects of various agents and ions upon the accumulation processes for all amines were also studied, and the findings indicate that the same neurons may well accumulate more than one amine type. It is concluded that 5-HT and dopamine uptake in the snail ganglia is a mechanism for inactivating these substances at 25° and that an uptake mechanism for 5-HT also functions at 0°. The present results are discussed from the point of view of the monoamines having transmitter functions in the snail CNS.     

Effect of potassium depolarization and preganglionic nerve stimulation on the metabolism of [3H]-choline in rat isolated sympathetic ganglia

1 The effects of potassium depolarization and preganglionic nerve stimulation on the metabolism of [³H]-choline in the isolated superior sympathetic ganglion of the rat have been studied.

2 When unstimulated (resting) ganglia were incubated for 10 min with a low concentration (0.1 μM) of [³H]-choline (high affinity uptake), approximately 75% of the accumulated radioactivity was present as [³H]-phosphorylcholine, 11% was [³H]-acetylcholine ([³H]-ACh) and the remainder was unchanged [³H]-choline.

3 Depolarization of the ganglia with K (46 mM) before their incubation with [³H]-choline, increased [³H]-choline uptake by 70% and increased [³H]-ACh synthesis by more than 700%, so that [³H]-ACh represented almost 50% of the total radioactivity recovered. In contrast, the proportion of [³H]-phosphorylcholine fell to 36% of the total radioactivity recovered.

4 The striking effect of K-depolarization on [³H]-ACh synthesis in ganglia occurred at a concentration of 30 mM or above, and the maximum effect was seen at 45-50 mM.

5 Chronic denervation of the ganglia abolished all the effects of high-K on [³H]-choline metabolism. In resting ganglia, [³H]-ACh formation was reduced by over 80% but [³H]-phosphorylcholine synthesis and the level of unchanged [³H]-Ch were not affected by denervation.

6 Exposure of the ganglia to low-Na or hemicholinium-3 (HC-3) greatly reduced [³H]-ACh synthesis in control resting ganglia and almost abolished the effects of high-K on [³H]-ACh synthesis.

7 Prevention of transmitter release with high-Mg or low-Ca medium also prevented K-depolarization from stimulating [³H]-ACh synthesis.

8 Preganglionic nerve stimulation had an effect on [³H]-choline metabolism similar to that of K-depolarization. Thus, at all the frequencies studied (1-30 Hz), [³H]-ACh synthesis was greatly increased and [³H]-phosphorylcholine was reduced, the maximum effects occurring at 3 Hz.

9 When ganglia were incubated with a high concentration (100 μM) of [³H]-choline (low affinity uptake), a different pattern of metabolism was observed. Most of the radioactivity in resting ganglia was present as unchanged [³H]-choline (70%) with [³H]-phosphorylcholine and [³H]-ACh representing 23% and 6% of the total radioactivity respectively. K-depolarization decreased [³H]-choline uptake but increased the proportions of [³H]-phosphorylcholine and [³H]-ACh to 32% and 24% of the total radioactivity respectively.

10 It is concluded that in unstimulated (resting) rat sympathetic ganglia most of the [³H]-choline transport and metabolism occurs in postsynaptic structures. However, depolarization of the presynaptic nerve terminals appears to trigger a sodium-dependent, HC-3 sensitive, high-affinity uptake process, and causes a dramatic increase in presynaptic [³H]-ACh synthesis together with a fall in postsynaptic [³H]-phosphorylcholine synthesis. These changes in choline metabolism cannot be due to the depolarization of the nerve terminals per se, because they were abolished by high-Mg or low-Ca, i.e. when transmitter release was prevented. Thus, the increase in ACh synthesis may be triggered by a fall in the intraterminal concentration of ACh or by the changes in Ca flux induced by depolarization. Our experiments do not provide evidence on these possible mechanisms.

     

Binding and uptake studies with [3H]CP-93, 129, a radiolabeled selective 5-HT1B receptor ligand

3-(1,2,5,6-Tetrahydro-4-pyridyl)-5-pyrrolo[3,2-b]pyridone, CP-93, 129, is a selective agonist ligand for 5-HT_1B receptors. High affinity binding sites of [³H]CP-93, 129 were found in rat whole brain membranes, which showed K_D and B_max values similar to those for 5-HT_1B sites labeled by [³H]5-HT. Uptake of [³H]CP-93, 129 in crude rat synaptosomes was also observed, which was potently inhibited by 5-HT uptake blockers and 5-HT but not by desipramine (NE uptake blocker) or tametraline (NE and DA uptake blocker). Because of this sensitivity to 5-HT uptake inhibitors and the structural similarity of CP-93, 129 to serotonin, [³H]CP-93, 129 uptake probably occurred in 5-HT neurons.     

High-affinity [3H]desipramine binding in the peripheral and central nervous system: A specific site associated with the neuronal uptake of noradrenaline

The specific binding of [³H]desipramine to various brain regions and peripheral tissues of the rat was of high affinity, rapid and reversible. It was inhibited with high affinity only by tricyclic antidepressants and noradrenaline uptake blockers. There was a highly significant correlation between the potencies of a series of drugs for the inhibition of [³H]desipramine binding and for the inhibition of noradrenaline uptake. Substrates for the noradrenaline uptake system however inhibited the binding of [³H]desipramine only at very high concentrations. Postganglionic sympathetic denervation of the submaxillary gland and the heart both resulted in a pronounced decrease in [³H]desipramine binding sites, which paralleled the reduction in endogenous noradrenaline levels. High-affinity [³H]desipramine binding sites thus appear to be localised on noradrenergic nerve endings and are probably closely associated with the neuronal uptake system for noradrenaline.     

Kinetics of [3H]choline uptake in abdominal ganglia of Limulus polyphemus.

The uptake of [³H]choline by isolated ganglia of the ventral nerve cord of Limulus potyphemus was studied. The uptake process was linear for 90 min and was concentration dependent. Kinetic analysis suggested the existence of a high affinity uptake process (K_t 14μM and V_max 0.19 pmole/mg/min) and a low affinity uptake process (K_t 14μM and V_max 0.89 pmole/mg/min). The high affinity uptake system showed a greater dependence on sodium ions and was more sensitive to inhibition by hemicholinium-3. Neither uptake system was greatly influenced by the absence of calcium or potassium. It is suggested that this system may be important in supplying choline for the biosynthesis of acetylcholine in cholinergic neurons.     

The effect of ketamine HC1 on the in vitro metabolism of norepinephrine in rat cerebral cortex tissue.

The effect of ketamine HCl on the in vitro metabolism of norepinephrine (NE) was investigated in rat cerebral cortex tissue. It was shown that ketamine competitively inhibited the high affinity transport system responsible for the uptake of NE into adrenergic nerve terminals. The drug also had weak NE releasing properties and potentiated the release induced by potassium chloride, a characteristic common to drugs in which the primary action is to inhibit the reuptake process. Intraneuronal deamination of NE was reduced by ketamine. This effect appeared to be primarily related to the inhibition of reuptake and a subsequent substrate deficiency for intraneuronal monoamine oxidase (EC 1.4.3.4). However, high concentrations of ketamine did slightly reduce the deamination of [³H]-NE, directly. The ability of ketamine to inhibit the high affinity transport system for NE may be partially responsible for the central nervous system excitation observed with the use of the drug in clinical anaesthesia.     

1-Ethyl-4-(isopropylidenehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester, hydrochloride (SQ 20009)--a potent new inhibitor of cyclic 3',5'-nucleotide phosphodiesterases

The properties of SQ 20009 [1-ethyl-4-(isopropylidenehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester, HCl] as a cyclic nucleotide phosphodiesterase inhibitor have been investigated. The phosphodiesterase preparations used in this study were ammonium sulfate-fractionated supernatants of homogenates of rat brain, rabbit brain, rat adrenal, rat lipocyte and cat heart; commercially available beef heart phosphodiesterase was also studied. The concentrations of SQ 200009 required to inhibit these phosphodiesterase activities 50 per cent were 2·0, 4·8, 20, 21, 27 and 60 μM, respectively, using 1·6 × 10⁻⁷ M cyclic AMP as substrate. SQ 20006 (the parent of SQ 20009 lacking the 4-isopropylidene moiety), theophylline and caffeine were also tested against all six enzyme preparations. Whereas SQ 20009 was more potent than SQ 20006 using the phosphodiesterase prepared from rat adrenal, the potencies were reversed when the lipocyte enzyme was used. SQ 20009 was approximately 60 and 75 times as potent an inhibitor of rat brain cyclic AMP phosphodiesterase as were theophylline and caffeine respectively. The kinetic properties of the phosphodiesterases prepared from rat brain, cat heart and beef heart were also investigated. Using the rat brain enzyme, two K_m values for cyclic AMP, 4·0 × 10⁻⁶ and 1·2× 10⁻⁴ M and a single K_m, 2·0 × 10⁻⁵ M, for cyclic GMP were confirmed. The K_i of SQ 20009 against the low K_m cyclic AMP phosphodiesterase was 2·0 × 10⁻⁶ M and that for cyclic GMP hydrolysis was 2·4 × 10⁻⁵ M. The inhibition by SQ 20009 of the hydrolysis of both cyclic nucleotides by both the rat brain and beef heart phosphodiesterases was competitive. The cat heart cyclic nucleotide phosphodiesterase was inhibited non-competitively by SQ 20009; the K_i for cyclic AMP hydrolysis was 6·4 × 10⁻⁵ M, and the K_i for cyclic GMP hydrolysis was 3·0 × 10⁻⁵ M. The inhibition by SQ 20009 of cyclic AMP hydrolysis by both the rat brain and cat heart preparations was reversible.     

Actions of bretylium and guanethidine on the uptake and release of [3h]-noradrenaline

The effect of bretylium and guanethidine has been studied on the uptake and the spontaneous and reserpine-induced release of [³H]-noradrenaline in the rat heart and in the splenic nerve endings of the cat. Bretylium and guanethidine inhibited the uptake by the heart of circulating [³H]-noradrenaline. Bretylium blocked spontaneous and reserpine-induced release of [³H]-noradrenaline, while guanethidine caused release and partially antagonized the reserpine-induced release. Both compounds produced a transient liberation of [³H]-noradrenaline from splenic nerves, but blocked the release of the [³H]-catechol amine following stimulation of the splenic nerve.     

Characteristics of dopamine release from isolated nerve endings of the tuberoinfundibular neurones

In the present study the release of dopamine (DA) from a synaptosomal preparation obtained from the nerve endings of the tuberoinfundibular dopaminergic nerves has been analysed, utilizing a superfusion technique.The exposure of median eminence synaptosomes to increasing concentrations of K⁺ ions produced a dose-dependent efflux of preaccumulated [³H]DA. The removal of Ca²⁺ ions from the superfusion medium completely prevented the high K⁺-induced release of [³H]DA. Removal of Na⁺ ions from the superfusion medium, a condition creating a favourable outward Na⁺ gradient, produced a marked release of [³H]DA. In order to establish whether this efflux of dopamine was a carrier-mediated process nomifensine, a dopamine transport inhibitor devoid of dopamine releasing effect in the median eminence, was added to the superfusion medium. Nomifensine largely counteracted the release of dopamine induced by the lack of Na⁺.d-Amphetamine produced a dose-related stimulation of [³H]DA in synaptosomes from the median eminence.The possible existence of a direct control of dopamine release mediated by presynaptic receptors was tested by studying the effect of apomorphine on the efflux of preaccumulated [³H]DA. Apomorphine was unable to modify both the spontaneous and the K⁺-evoked release of dopamine.The present results give support to the idea that dopamine released from the nerve endings of tuberoinfundibular neurones into the primary plexus acts as a neurotransmitter. The release of dopamine in these nerves does not seem to be regulated by autoreceptors.     

Binding of [<Superscript>3</Superscript>H]mazindol to cardiac norepinephrine transporters: kinetic and equilibrium studies

The norepinephrine transporter (NET) is the carrier that drives the neuronal norepinephrine uptake mechanism (uptake₁) in mammalian hearts. The radioligand [³H]mazindol binds with high affinity to NET. In this study, the kinetics of [³H]mazindol binding to NET were measured using a rat heart membrane preparation. Results from these studies were used to set up saturation binding assays designed to measure cardiac NET densities (B_max) and competitive inhibition assays designed to measure inhibitor binding affinities (K_I) for NET. Saturation binding assays measured NET densities in rat, rabbit, and canine hearts. Assay reproducibility was assessed and the effect of NaCl concentration on [³H]mazindol binding to NET was studied using membranes from rat and canine hearts. Specificity of [³H]mazindol binding to NET was determined in experiments in which the neurotoxin 6-hydroxydopamine (6-OHDA) was used to selectively destroy cardiac sympathetic nerve terminals in rats. Competitive inhibition studies measured K_I values for several NET inhibitors and substrates. In kinetic studies using rat heart membranes, [³H]mazindol exhibited a dissociation rate constant k_off=0.0123±0.0007 min^–1 and an association rate constant k_on=0.0249±0.0019 nM^–1min^–1. In saturation binding assays, [³H]mazindol binding was monophasic and saturable in all cases. Increasing the concentration of NaCl in the assay buffer increased binding affinity significantly, while only modestly increasing B_max. Injections of 6-OHDA in rats decreased measured cardiac NET B_max values in a dose-dependent manner, verifying that [³H]mazindol binds specifically to NET from sympathetic nerve terminals. Competitive inhibition studies provided NET inhibitor and substrate K_I values consistent with previously reported values. These studies demonstrate the high selectivity of [³H]mazindol binding for the norepinephrine transporter in membrane preparations from mammalian hearts.     

Cholinergic modulation of [3H]dopamine release from dendrosomes of rat substantia nigra

Summary Dendrosomes prepared from substantia nigra are able to take up and release [³H]dopamine in a Ca²⁺-dependent manner. The V_max values of [³H]dopamine uptake in substantia nigra dendrosomes was about 5 times lower than that in caudate putamen synaptosomes. The pattern of the K⁺-dependency of the [³H]dopamine release in substantia nigra dendrosomes was significantly different from that found in caudate putamen synaptosomes. The release of [³H]dopamine evoked by 15 mmol/l KCl from superfused dendrosomes was increased in a concentration-dependent manner by acetylcholine. The maximal potentiation produced by acetylcholine was about 40%. The potentiation of [³H]dopamine release by 10 µmol/l acetylcholine was insensitive to mecamylamine but antagonized by atropine and by pirenzepine. The effects of acetylcholine on the release of [³H]acetylcholine from substantia nigra nerve endings was also studied. Exogenous acetylcholine added to the superfusion medium decreased in a concentration-dependent manner the release of acetylcholine. This effect was not antagonized by mecamylamine or pirenzepine but fully antagonized by atropine. The data suggest the existence, in the substantia nigra of the rat, of two distinct muscarinic receptor subtypes regulating respectively dopamine release from dopamine dendrites and acetylcholine release from cholinergic nerve terminals.Part of this work was presented at a satellite meeting of the 11th International Congress of Pharmacology: Dopamine '90 held in Como, Italy (July 1990) Send offprint requests to M. Raiteri at the above address     

Effect of carbonyl-binding agents and oxidative phosphorylation uncouplers on the release of [3H]norepinephrine from mouse heart

Three carbonyl reagents, namely sodium bisulfite, hydroxylaminc and phenylhydrazine, increased the release of [³H]norepinephrine from sliced mouse heart during stimulation with 50 mM potassium ion. The [³H]norepinephrine was identified by binding to aluminum hydroxide and subsequent thin-layer chromatography. These carbonyl reagents at 10 ³ M increased the overflow of [³H]norepinephrine by 162 249 per cent. Semicarbazidc, another carbonyl reagent, proved to he an exception. However, semicarbazide reacted more slowly than the other carbonyl reagents with pyridoxal phosphate (which was used as a model aldehyde). Therefore, the ability of carbonyl reagents to increase overflow of [³H]norepinephrinc appeared to correlate with their ability to react with carbonyl groups. The augmented release of [³H]norepinephrine by sodium bisulfite, hydroxylamine or phenylhydrazine was not due to inhibited reuptake of released transmitter since cocaine, a strong uptake blocker, did not increase the release of [³H]norepinephrine. When animals were pretreated with 6-hydroxydopamine to destroy adrenergic nerve terminals, the stimulus-induced overflow of [³H]neurotransmitter in the presence of sodium bisulfite was virtually abolished. Similar results were obtained when cocaine was used to prevent the accumulation of [³H]norepinephrine by nerves. Thus, the adrenergic nerve endings were the source of the increased overflow of [³H]norepinephrinc. These data implicate endogenous aldehydes or ketones in a regulatory role during the release of neurotransmitter. Experiments with norepinephrine-deplcted hearts (rcserpine. 16 hr) labeled with [³H]metaraminol (not metabolized by monoamine oxidase) as neurotransmitter showed increased release of [³H-neurotransmitter in the presence of 10^?3 M sodium bisulfite. This indicated that the aldehyde derived from norepinephrine by the action of monoamine oxidase was not responsible for the action of the carbonyl-binding agents. In separate experiments, two oxidative phosphorylation uncouplers. 2.4-dinitrophenol (5 × 10^?4M) and carbonyl cyanide m-chlorophenylhydra/one (10^?6 M). similarly increased overflow of [³H]norepinephrine. Experiments with cocaine showed that the increased release of [³H]norepinephrinc in the presence of carbonyl cyanide m-chlorophenylhydrazone was derived from nerve terminals. The oxidative phosphorylation uncouplers and the carbonyl reagents may have a common site of action, perhaps at an adenosine triphosphate-dependcnt carbonyl site in the axonal membrane.