Cocaine and neuronal uptake in the canine saphenous vein

Summary This study was designed to investigate the effects of the neuronal uptake inhibitor, cocaine on the adrenergic neuroeffector interaction in the canine saphenous vein. Tissues were incubated with ³H-noradrenaline in control solution or in presence of the cocaine. The tissue content of ³H-noradrenaline and its metabolites was determined after the incubation. As the concentration of cocaine in the incubation medium increased gradually less ³H-noradrenaline and DOPEG were detected in the tissue, while the content of DOMA, NMN, MOPEG and, in particular that of VMA increased; comparable results were obtained with high concentrations of cocaine and desmethylimipramine (DMI). Helical strips of canine saphenous veins were incubated with ³H-noradrenaline and mounted for isometric tension recording and for measurement of the efflux of labelled transmitter and its metabolites. Cocaine, but not DMI, slightly increased the spontaneous efflux of DOPEG, suggesting that cocaine enters the nerve terminals and displaces noradrenaline from its storage sites. During electrical stimulation, cocaine at 3×10^–5 mol/l increased the contractile response and the overflow of ³H-noradrenaline, DOMA, NMN and MOPEG and decreased the appearance of DOPEG. Similar results were obtained with DMI (10^–6 mol/l) except that it did not increase the overflow of DOMA and MOPEG. During electrical stimulation in presence of DMI, cocaine did not affect the contractile response and decreased the appearance of intact labelled transmitter. Electrical stimulation, cocaine and DMI did not affect the overflow of VMA. The present experiments indicate that in the canine saphenous vein: (1) DOPEG is formed intraneuronally, but DOMA, MOPEG, NMN and VMA extraneuronally; (2) VMA is retained in the tissue much longer than the other metabolites; (3) determination of total ³H-content after incubation with ³H-noradrenaline in presence of inhibitors of neuronal uptake underestimates the degree of inhibition of the neuronal amine carrier; and (4) the quantification of the effect of cocaine on the neuronal uptake of released transmitter is complicated by several other actions of the drug (local anesthetic properties, displacement of stored transmitter, activation of effector cells) and that of the effect of DMI by its inhibitory effect on monoamine oxidase, in particular at extraneuronal sites.Supported in part by grant HL 05883 from the National Institutes of Health     

Effects of S-11701 on accumulation, release and metabolism of norepinephrine in isolated canine saphenous veins.

1. The effects S-11701 ([morpholinyl-2)-methoxy]-8-tetrahydro-1,2,3,4 quinoline) on accumulation, overflow and metabolism of [3H]norepinephrine were investigated in isolated canine saphenous veins. 2. Saphenous veins were incubated with [3H]norepinephrine in the absence or the presence of S-11701; the drug caused a concentration-dependent inhibition of the tissue content of [3H]norepinephrine and its metabolites, except for 3-methoxy-4-hydroxymandelic acid (VMA). 3. In helical strips of canine saphenous veins previously incubated with [3H]norepinephrine and then suspended for isometric tension recording and measurement of the overflow of labelled transmitter and its metabolites, S-11701 (30 microM) significantly increased the spontaneous efflux of total 3H; this effect was almost exclusively due to an augmentation of the efflux of [3H]DOPEG. 4. During electrical stimulation (9 V, 1 Hz), S-11701 at 1 microM slightly increased the overflow of extraneuronal norepinephrine metabolites without affecting the contractile response. At the higher concentration (30 microM) the compound increased the contractive response and the overflow of 3H; the latter was due mainly to an increase in [3H]DOPEG and, to a lesser extent, in [3H]norepinephrine. 5. DMI (1 microM) did not interfere with the effects of S-11701 on DOPEG efflux. 6. These experiments indicate that in the canine saphenous vein, S-11701 causes a concentration-dependent inhibition of neuronal accumulation of [3H]norepinephrine. At higher concentrations, S-11701 enters the adrenergic nerve terminals independently of the neuronal amine carrier and displaces [3H]norepinephrine from its storage sites.     

The effects of labetalol (AH 5158) on metabolism of 3H(-)-noradrenaline released from the cat spleen by nerve stimulation

The competitive α and β adrenoceptor antagonist labetalol, in concentrations up to 10^?4 M, produced a dose dependent increase in overflow of ³H and [³H]noradrenaline in the isolated blood perfused cat spleen following stimulation of the splenic nerves at a frequency of 10 Hz. Labetalol had no effect on the pattern of overflow of label following stimulation. In experiments in which the metabolism of [³H]noradrenaline released on nerve stimulation was examined, labetalol produced a concentration dependent increase in the percentage of [³H]noradrenaline and a decrease in the percentage of [³H]DOPEG in the venous blood following nerve stimulation. Production of [³H]COMT metabolites and [³H]DOMA was not affected. It is suggested that in the isolated blood perfused cat spleen labetalol produces the elevation of overflow and effects on noradrenaline metabolism by inhibition of neuronal uptake of noradrenaline. The drug has no detectable effects on the enzymes MAO or COMT or on extraneuronal uptake.     

The neuronal and extraneuronal uptake and metabolism of 3H-(−)-noradrenaline in the perfused rat heart

1. Hearts were obtained from reserpine-pretreated rats and perfused with 0.95 micron 3H(-)-noradrenaline. The rate of removal of 3H-noradrenaline from the perfusion fluid was measured (from the arterio-venous difference) as well as the rate at which the 3H-metabolites appeared in the venous effluent. 2. When either 30micron corticosterone was added under steady-state conditions during perfusion with 3H-noradrenaline (to inhibit neuronal and extraneuronal uptake, respectively), each inhibitor reduced the removal of noradrenaline by about 50%; in the presence of both inhibitors removal was abolished. 3. Dihydroxymandelic acid (DOMA) was of neuronal, normetanephrine (NMN) of extraneuronal origin; dihydroxyphenylglycol (DOPEG) and the OMDA fraction (containing methoxyhydroxyphenylglycol-MOPEG-and methoxyhydroxymandelic acid-VMA) were formed both neuronally and extra-neuronally. 4. The extraneuronal metabolism of 3H-noradrenaline was in quick equilibrium with the 3H-noradrenaline in the perfusion fluid; most of the total formation of DOPEG, MOPEG and NMN was recovered from the venous effluent. 5. Extraneuronally formed DOPEG, MOPEG and NMN distributed in the tissue with half times corresponding to their half time for efflux. 6. Inhibition of monoamine oxidase (MAO) by pargyline increased the extraneuronal formation of NMN; MAO and catechol-O-methyl transferase (COMT) appear to be contained in the same extraneuronal compartment. 7. The extraneuronal accumulation of 3H-noradrenaline required 30 min or more to reach a steady state; inhibition of one or both enzymes slowed this process. Inhibition of MAO increased the extra-neuronal accumulation of 3H-noradrenaline; inhibition of COMT failed to do so, since the enzyme inhibitor (U-0521) was a weak inhibitor of extra-neuronal uptake. 8. The rate constants for the efflux of the metabolites of noradrenaline decreased in the order of MOPEG greater than DOPEG greater than NMN greater than DOMA greater than VMA.     

Deamination of released 3H-noradrenaline in the canine saphenous vein

Summary Experiments were designed to determine the effect of monoamine oxidase (MAO) inhibitors on the release and the metabolism of noradrenaline in the canine saphenous vein. Helical strips were incubated with ³H-noradrenaline and mounted for superfusion and measurement of the efflux of labelled transmitter and its metabolites; in certain experiments the tissue content of ³H-noradrenaline and its metabolites was also determined. The MAO-A inhibitor clorgyline, and the non-specific inhibitor pargyline, but not the MAO-B inhibitor deprenyl decreased the appearance of deaminated and O-methylated deaminated metabolites under basal conditions and during electrical stimulation. The MAO-A and the non-specific MAO inhibitor did not decrease the efflux of VMA to the same extent as that of the other deaminated metabolites. During superfusion with etidocaine, an agent causing increased leakage of stored transmitter, clorgyline abolished the appearance of DOPEG. Addition of semicarbazide in preparations treated with pargyline did not affect the efflux of deaminated and O-methylated deaminated metabolites. From the measurement of tissue VMA, it appeared that the efflux of VMA poorly reflects quick changes in the rate of its formation but that formation is abolished by pretreatment with pargyline. These experiments indicate that in the canine saphenous vein: (1) DOPEG is formed mainly in intraneuronal sites, while DOMA, MOPEG and VMA are formed extraneuronally; (2) VMA is retained in the tissue after its formation; and (3) the only subtype of MAO involved in the metabolism of ³H-noradrenaline released from adrenergic nerve endings can be classified as MAO-A.Supported in part by grant HL-05883     

Effects of S9977 on adrenergic neurotransmission

1. Experiments were designed to determine whether or not the putative promnesic drug S9977 (1,3,7-trimethyl 8-[3-(4-diethylaminocarbonyl-1-piperazinyl) 1-propyl]-3,7-dihydro (1H)2,6-purinedione hydrochloride) affects peripheral adrenergic neurotransmission.2. Rings of canine saphenous veins (without endothelium) were suspended for isometric tension recording in conventional organ chambers filled with modified Krebs-Ringer bicarbonate solution. The adrenergic nerve endings were activated with electrical impulses (9 V, 2 msec, 0.25–8 Hz).3. At 10⁻⁵ M, S9977 significantly reduced the contraction to 0.25, 0.5 and 1 Hz. The compound did not affect the response to higher stimulation frequencies or to exogenous noradrenaline. The inhibitory effect of S9977 was prevented by methiothepin, and not affected by atropine or 8-phenyltheophylline.4. Helical strips of canine saphenous veins were incubated with [³H]noradrenaline and suspended for superfusion and isometric tension recording. Under basal conditions, S9977 (10⁻⁴ M) augmented, the total ³H-overflow which was due mainly to an augmented overflow of [³H]deoxyphenylglycol (DOPEG); the extraneuronal metabolites 3,4-dihydromandelic acid (DOMA) and 3-methoxy-4-hydroxymandelic acid (VMA) were reduced.5. During electrical stimulation of the adrenergic nerves, S9977 (10⁻⁴ M) augmented the total ³H-overflow but reduced the contractile response; the evoked overflow of [³H]noradrenaline was not significantly affected.6. These experiments suggest that S9977 the displacement of noradrenaline from the adrenergic varicosities; most of the displaced transmitter is metabolized by intraneuronal monoamine oxidase before reaching the junctional cleft. In addition, S9977 exerts an inhibitory effect on the extraneuronal metabolism of catecholamines. S9977 does not inhibit the exocytotic release of the adrenergic neurotransmitter.     

Role of the α-adrenoceptor in regulating noradrenaline overflow by nerve stimulation

1. A study of the actions of phenoxybenzamine on transmitter overflow, neuronal and extraneuronal uptake of noradrenaline and in causing alpha-adrenoceptor blockade was carried out using the isolated cat nictitating membrane preparation.2. Phenoxybenzamine increased transmitter overflow elicited by nerve-stimulation at 10 Hz in a concentration dependent manner in the range 10(-8) to 10(-5) g/ml.3. Neuronal uptake of [(3)H]-noradrenaline was not inhibited by concentrations lower than 10(-6) g/ml of phenoxybenzamine. With 10(-7) g/ml of phenoxybenzamine a significant increase in transmitter overflow was obtained, although neuronal uptake of noradrenaline was not affected. Higher concentrations of phenoxybenzamine (10(-6) and 10(-5) g/ml) inhibited the neuronal uptake of noradrenaline and further increased transmitter overflow.4. Extraneuronal uptake of [(3)H]-noradrenaline was inhibited only with the highest concentration of phenoxybenzamine tested (10(-5) g/ml) and therefore appears to be unrelated to the effects on transmitter overflow.5. There was a significant correlation between the degree of alpha-adrenoceptor block produced by phenoxybenzamine and the increase in transmitter overflow obtained by nerve stimulation.6. These results indicate that phenoxybenzamine, in addition to increasing overflow by preventing reuptake of noradrenaline, may increase transmitter release.7. The possibility that phenoxybenzamine acts on alpha-adrenoceptors in the adrenergic nerve terminal is discussed. These receptors would be involved in a negative feedback mechanism regulating transmitter release.     

Effects of clorgyline and (−)deprenyl on the deamination of normetanephrine and noradrenaline in strips and homogenates of the canine saphenous vein

Summary The influence of specific inhibitors of MAO A (clorgyline) and MAO B [(–)deprenyl] on the metabolism of normetanephrine (NMN), in strips of canine saphenous vein was studied, both in the absence and in the presence of inhibitors of neuronal (cocaine) and extraneuronal (hydrocortisone) uptake. Moreover, the formation of metabolites of noradrenaline and of NMN by saphenous vein homogenates and the influence of clorgyline or (–)deprenyl on this formation are described.Clorgyline reduced to the same degree (by about 70%) the formation of methoxy-hydroxy-phenylglycol (MOPEG) and of vanillylmandelic acid (VMA) in strips incubated with NMN, whereas (–)deprenyl reduced by about 50% the formation of MOPEG and had no effect on VMA production. Hydrocortisone had effects very similar to those of (–)deprenyl.Saphenous vein, homogenates ()-methylation inhibited), deaminated both noradrenaline and NMN; clorgyline and (–)deprenyl reduced the formation of metabolites of both noradrenaline and NMN.It is concluded that both MAO A and B are able to deaminate noradrenaline and NMN, but that in the intact tissue the former has no access to MAO B. Even in intact tissues MAO B may play a role in the metabolism (but not in the inactivation) of noradrenaline by deaminating the NMN formed from noradrenaline and giving preferentially origin to MOPEG.Supported by Instituto Nacional de Investigação Científica (INIC, FmPl)On leave from Faculdade de Farmácia, Universidade de Coimbra, with a grant from Instituto Nacional de Investigação Científica     

Presynaptic changes promoted by alloxan diabetes in the cat isolated heart

Adrenergic presynaptic functions were evaluated in the cat isolated perfused heart preparation. The sympathetic nerve endings were labelled with [3H]noradrenaline ([3H]NA) and the effect of electric neural stimulation was determined in the presence of drugs which inhibit neuronal or extraneuronal uptake, or which antagonize alpha-adrenoceptors. [3H]NA overflow was measured in control and diabetic cats and was significantly increased by electric neural stimulation on both conditions. Perfusion with 0.1 microM phentolamine increased transmitter overflow in control hearts but failed to do so on organs obtained from alloxan-treated cats. The data provide evidence that in alloxan diabetic cats there is an abnormality of the adrenergic synapse.     

Release of noradrenaline and dopamine by nerve stimulation in the guinea-pig and rat vas deferens.

Spontaneous and nerve stimulated release of noradrenaline and dopamine from rat and guinea-pig vas deferens have been measured electrochemically after separation by high performance liquid chromatography (h.p.l.c.). In the absence of nerve stimulation both noradrenaline (NA) and dopamine were released into the bathing fluid in the rat but in the guinea-pig only noradrenaline could be detected. Drugs which block neuronal and extraneuronal uptake of catecholamines had little effect on spontaneous overflow but both tetraethylammonium and phenoxybenzamine increased overflow. Transmural nerve stimulation (5-10 Hz) increased catecholamine overflow in both species and dopamine release was now measurable from the guinea-pig vas. In the rat, the proportion of dopamine to NA was unchanged from that released spontaneously. The release of both amines was little affected by drugs that block neuronal and extraneuronal uptake and a monoamine oxidase inhibitor, but was inhibited by tetrodotoxin 0.2 microgram ml-1. In the guinea-pig tetraethylammonium 10 mM doubled overflow and phenoxybenzamine 10(-5)M increased it by five times but the dopamine percentage remained constant and equal to the control. Following nerve stimulation the amount of dopamine released expressed as a percentage of total catecholamine release was 6% for the rat and 1.3% for the guinea-pig. These values were considerably higher than the comparable figures for dopamine: NA content of the two tissues (2% and 0.5% respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of flow-stop on the metabolism of noradrenaline released by nerve stimulation in the perfused spleen

Summary The metabolic pathway of ³H-noradrenaline released spontaneously and by nerve stimulation was studied in the isolated perfused spleen of the cat. The deaminated glycol, DOPEG, (3,4 dihydroxyphenylglycol) was the main metabolite in spontaneous outflow, accounting for 62.5±1.6% of the total radioactivity (n=13). Of the total increase in radioactive products elicited by nerve stimulation at 5 Hz or 10 Hz around 30% was accounted for by the noradrenaline metabolites, particularly DOPEG and the O-methylated fraction. In the presence of 2.9×10^–6 M of cocaine the total overflow of radioactivity induced by stimulation was unchanged but DOPEG formation from released noradrenaline was abolished. These findings indicate that DOPEG formation results from the recapture of the released transmitter by adrenergic nerve endings and subsequent intraneuronal deamination. The total overflow of noradrenaline was reduced by flow-stop while the metabolism of the released transmitter was increased significantly. Cocaine, 2.9×10^–6 M, prevented the increase in DOPEG when stimulation was applied under flow-stop conditions. The decrease in noradrenaline overflow induced by flow-stop is partly due to the increase in the metabolism of the released transmitter.     

Presynaptic alpha-adrenoceptors: do exogenous and neuronally released noradrenaline act at different sites?

The effects of dopamine receptor and alpha-adrenoceptor agonists and antagonists on the stimulation-evoked overflow of radioactivity from strips of dog saphenous vein previously loaded with [3H]-noradrenaline have been examined alone and in combination. In the presence of neuronal and extraneuronal catecholamine uptake inhibitors, noradrenaline (0.1-1 X 10(-6)M) and dopamine (0.01-1 X 10(-6)M) both inhibited the stimulation-evoked overflow of radioactivity. Sulpiride (1 X 10(-6)M) was without effect and prazosin (1 X 10(-7)M) had little effect on stimulation-evoked overflow but yohimbine enhanced it approximately 2 fold; the effect of yohimbine was similar at concentrations of 1 X 10(-7) and 1 X 10(-6)M. Sulpiride abolished the inhibitory effect of dopamine on stimulation-evoked overflow, but was without effect against noradrenaline. When allowance was made for the effects of yohimbine, alone, on overflow, yohimbine (1 X 10(-7)M) had no effect against dopamine and minimal effects against noradrenaline. A similar result was obtained when the concentration of yohimbine was increased to 1 X 10(-6)M. Prazosin did not antagonize the effect of noradrenaline. In the absence of the uptake inhibitors, clonidine (0.01-1 X 10(-5)M) inhibited stimulation-evoked overflow of radioactivity. Yohimbine (1 X 10(-6)M) was without effect on its own and antagonized the effects of clonidine at a concentration of 0.1 X 10(-5)M, but not at 0.01 or 1.0 X 10(-5)M. These findings suggest that dopamine inhibits overflow by stimulating presynaptic dopamine receptors on the terminals of the noradrenergic nerves supplying the dog saphenous vein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular sodium and chloride depletion enhances nonexocytotic noradrenaline release induced by energy deficiency in rat heart

Summary The effect of either extracellular sodium or extracellular celoride reduction on the release of endogenous noradrenaline and its deaminated metabolite dihydroxyphenylglycol (DOPEG) has been studied in the isolated perfused rat heart under conditions of ischaemia and cyanide intoxication. The overflow of noradrenaline and DOPEG was determined by high pressure liquid chromatography. The efflux of DOPEG, the predominant neuronal noradrenaline metabolite, served as indicator of the free axoplasmic amine concentration. A calcium-free perfusion buffer was used to avoid exocytotic noradrenaline release. Sodium and chloride in the perfusion buffer were replaced by lithium and isethionate, respectively.(1) Reduction of extracellular sodium or chloride increased noradrenaline overflow in ischaemia. The release was suppressed by the uptake, blocker cocaine indicating carrier-mediated outward transport of noradrenaline. (2) In cyanide intoxication sodium or chloride reduction accelerated the onset of DOPEG efflux reflecting increased axoplasmic noradrenaline concentrations. This was accompanied by increased noradrenaline release. The ratio of noradrenaline/DOPEG overflow was increased by reduced sodium or chloride, indicating facilitation of carrier-mediated noradrenaline net outward transport. (3) In the presence of unaltered energy metabolism overflow of both, noradrenaline and DOPEG, was not enhanced by sodium or chloride reduction.The results demonstrate that reduction of extracellular sodium or chloride has two effects on noradrenaline release from the sympathetic neuron with reduced energy supply. First, reduced sodium or chloride induces increased axoplasmic noradrenaline concentrations by interference with vesicular storage function. Second, both interventions enhance carrier-mediated noradrenaline release.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 320) Send offprint requests to A. Schömig at the above address     

Sympathetic denervation caused by long-term noradrenaline infusions; prevention by desipramine and superoxide dismutase.

1. The effects of continuous intravenous infusion of noradrenaline (0.01 and 0.1 microgram kg-1 h-1) were studied in both the infused lateral saphenous vein and the contralateral saphenous vein of normal dogs. Noradrenaline, saline, noradrenaline + desipramine or noradrenaline + superoxide dismutase were infused using Alzet osmotic minipumps. 2. After a 5 day infusion period, the noradrenaline content in plasma and in both saphenous veins was determined, and the venous tissues submitted to light microscope morphometry and ultrastructural study and used for the determination of their O-methylation capacity (with [3H]-isoprenaline as a substrate). 3. Noradrenaline caused dose-dependent damage to the sympathetic nerve endings of the lateral saphenous veins. Concomitant changes in extraneuronal structure and function were observed (hypertrophy of smooth muscle cells, nuclear dysmorphy, thickening of the vessel wall, impairment in O-methylation capacity). 4. Desipramine and superoxide dismutase prevented or reduced the effects of noradrenaline on both the morphological and the biochemical parameters; the protection afforded by superoxide dismutase was more marked than that by desipramine. 5. It is concluded that moderately high doses of noradrenaline exert a 6-hydroxydopamine-like effect and that this chemical sympathectomy is partially or totally prevented by desipramine or superoxide dismutase. The data suggest that a substance derived from noradrenaline, in the formation of which free oxygen radicals are involved and which is subject to neuronal uptake, is the chemical entity responsible for the neurotoxic effect observed.     

The termination of action of catecholamines in the isolated venous tissue of the dog

Summary The termination of responses to noradrenaline and adrenaline was studied, using the oil immersion technique, in helically cut strips obtained from the lateral saphenous vein of the dog. Inhibitors of catechol-O-methyltransferase (tropolone), monoamine oxidase (iproniazid) or neuronal uptake (cocaine and imipramine-N-oxide) slowed the relaxation rate of strips contracted by noradrenaline or adrenaline (10^–5 and 10^–6 M) and immersed in oil. From these experiments it is concluded that, at the concentrations used, oxidative deamination represents the major inactivation pathway for both catecholamines, and that O-methylation plays only a minor role in the disposition of the amines; however, the inactivation of adrenaline is more dependent on catechol-O-methyltransferase than that of noradrenaline. Tissue uptake and storage occupied an intermediate position between the enzymatic pathways referred to: iproniazid inhibited 79%, cocaine 54% and tropolone 27% of the inactivation capacity when 10^–5 M noradrenaline was used. However, the combination of the three drugs inhibited only 88% of the inactivation capacity. This is due to the marked overlap of the effects of cocaine and iproniazid, which is attributed to the blockade by cocaine of the access of the amine to intraneuronal sites of oxidative deamination.Cocaine caused marked potentiation of responses to noradrenaline and moderate prolongation of relaxation time, while iproniazid, which had much more marked effects on the termination of action of noradrenaline, induced only a slight augmentation of the contractile responses. It is concluded that potentiation is an unreliable index of altered disposition of noradrenaline in this preparation.Experiments with perfused venous segments showed that, following the concentration gradient, noradrenaline diffuses freely through the vein wall, and that there is no marked difference in responses to adventitial or intimal application of the amine. Radioautographic studies gave evidence of two different types of accumulation (neuronal and extraneuronal), which appeared to be in equilibrium.Supported in part by a grant from III Plano de Fomento (Actividades, 1970).     

Neuropeptide Y differentiates between exocytotic and nonexocytotic noradrenaline release in guinea-pig heart

Summary The overflow of neuropeptide Y (NPY; radioimmunoassay), noradrenaline and dihydroxyphenylethylenglycol (DOPEG; high pressure liquid chromatography) from guinea-pig perfused hearts was investigated in relationship to exocytotic and nonexocytotic release mechanisms. Exocytotic release: Electrical stimulation of the left stellate ganglion (12 Hz; 1 min) evoked a calcium-dependent overflow of noradrenaline and NPY, that was accompanied by a minor and prolonged increase in DOPEG overflow. This increase in DOPEG overflow was attenuated by blockade of neuronal amine re-uptake. In the presence of calcium, a closely related co-release of noradrenaline and NPY was also observed during administration of veratridine (10 M); it was completely prevented by tetrodotoxin (1 M). Nonexocytotic release: In the absence of extracellular calcium, veratridine (30 M) induced noradrenaline overflow only when combined with the reserpine-like agent Ro 4-1284 (10 M). This overflow was accompanied by efflux of DOPEG, but not of NPY. Similarily, tyramine (1–100 M) induced a calcium-independent concomitant overflow of both noradrenaline and DOPEG, but not of NPY. During anoxic and glucose-free perfusion a predominantly calcium-independent overflow of noradrenaline was observed; only in the presence of extracellular calcium was this overflow accompanied by a minor overflow of NPY. Noradrenaline overflow, induced by veratridine plus Ro 4-1284 (in the absence of calcium), by tyramine, or by anoxia, was suppressed by blockade of neuronal amine re-uptake, and was, therefore, mediated by reversed transmembrane amine transport by the neuronal uptake₁ carrier.The results indicate that NPY is co-released with noradrenaline only during calcium-dependent exocytosis. On the other hand, whenever, noradrenaline is released by non-exocytotic (calcium-independent and carrier-mediated) release mechanisms, no substantial NPY overflow is observed. The simultaneous determination of noradrenaline and NPY overflow, therefore, allows a differentiation between exocytotic and nonexocytotic noradrenaline release, and NPY may be utilized as a marker of exocytotic noradrenaline release.This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 320 — Herzfunktion und ihre Regulation)Presented in part at the 62nd Scintific Sessions of the American Heart Association, New Orleans/USA, November 1989     

Single pulse stimulation of guinea-pig vas deferens and the presynaptic receptor hypothesis.

1 The effect of phenoxybenzamine on the efflux of [3H]-noradrenaline and the mechanical response to single pulse excitation of superfused guinea-pig vas deferens was determined to examine the validity of the currently accepted hypothesis of a presynaptic negative feedback system on adrenergic nerve terminals. 2 The adrenoceptor antagonist enhanced both the outflow of tritium and the mechanical response to single pulse stimulation. The efflux of labelled material and the responses to 4 pulses were also enhanced, as expected. 3 Blockade of neuronal and extraneuronal uptake did not by itself increase nerve-induced outflow or the mechanical response nor dated within the framework of a hypothesis that proposes that the enhancement of response and tritium efflux by phenoxybenzamine results from blockade of a feedback system whereby noradrenaline released by previous impulses inhibits its own subsequent release.     

Comparison of the effects of phenoxybenzamine and uptake blockade on noradrenaline efflux from rabbit ear arteries evoked by field stimulation and propagated nerve impulses

1 A comparison has been made of the effects of blockade of prejunctional α-adrenoreceptors and blockade of transmitter noradrenaline uptake in segments of rabbit ear arteries subjected to field stimulation or neuronally propagated impulses. 2 The relationship between evoked release and frequency of stimulation differed in artery segments subjected to field stimulation and those receiving propagated nerve impulses. However, the effectiveness of phenoxybenzamine in increasing stimulation-induced efflux of radioactivity decreased as the frequency of stimulation increased in artery segments subjected to either field stimulation or neuronally propagated impulses. 3 Blockade of neuronal and extraneuronal uptake had no effect on evoked efflux from field-stimulated artery segments but it did produce a marked and significant enhancement of release evoked by propagated nerve impulses.     

Differences between the neuronal handling of 3H-7- and 3H-7,8-(−)noradrenaline: implications for the release of the labelled neurotransmitter by nerve stimulation

Neighbouring rabbit aortic strips were exposed to a tracer concentration of 3H-7- or 3H-7,8-(-)noradrenaline (bearing 30-35% of its label in position 8) and to 0.5 mumol/l unlabelled (-)noradrenaline for 60 min and then washed in amine-free Krebs solution. Catechol-O-methyl transferase and extraneuronal amine uptake were inhibited throughout. After 114 min of wash-out, the tissue contained less tritium when loaded with 3H-7-(-)noradrenaline than when loaded with 3H-7,8-(-)noradrenaline, and the fractional rate of loss of tritium was greater for the former than for the latter tissues. In the presence of cocaine (to prevent neuronal re-uptake), the same percentage of tissue tritium was released by nerve stimulation (six consecutive periods of stimulation at 1 Hz for 5 min each) in spite of the above difference between tissue tritium levels of the two differently labelled amines. In the absence of cocaine, a higher percentage of tissue tritium was released by nerve stimulation (1 or 3 Hz, 5 min each) for 3H-7- than 3H-7,8-(-)noradrenaline. Unchanged 3H-(-)noradrenaline amounted to 35% of tritium in the stimulation-evoked overflow for 3H-7- and to 50% for 3H-7,8-(-)noradrenaline (frequency of stimulation, 1 Hz). When monoamine oxidase (MAO) was inhibited, no differences were observed between the neuronal handling of 3H-7- and 3H-7,8-(-)noradrenaline, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the noradrenaline metabolites on tyrosine hydroxylase activity in guinea-pig atria

Summary The effects of noradrenaline, its five metabolites and metanephrine, were studied on tyrosine hydroxylase activity in guinea-pig atria. The deaminated metabolite, (±)-3,4-dihydroxyphenylglycol (DOPEG), was equipotent with (±)-noradrenaline in its inhibitory action on tyrosine hydroxylase activity in the homogenates of guinea-pig atria. The inhibition by DOPEG was competitive with the cofactor, reduced pteridine. The deaminated acid, 3,4-dihydroxymandelic acid (DOMA) and the O-methylated deaminated acid, 3-methoxy, 4-hydroxymandelic acid (VMA) had 1/50th and 1/30th, respectively, the potency of noradrenaline in inhibiting tyrosine hydroxylase. The rest of the metabolites did not inhibit tyrosine hydroxylase in homogenates in concentrations up to 1.0 mM. In intact guinea-pig atria noradrenaline was considerably more potent than DOPEG in inhibiting tyrosine hydroxylase. Normetanephrine 1.4×10^–4 M inhibited tyrosine hydroxylase in the intact tissue but failed to inhibited the enzyme in the homogenate even in higher concentrations. The effect of normetanephrine in the intact tissue is related to the ability of this compound to release endogenous noradrenaline.A reserpine-like agent, Ro 4-1284, did not inhibit tyrosine hydroxylase activity in the homogenate but in the intact tissue the inhibition was more than 50%. This effect of Ro 4-1284 in the intact tissue appears to be related to the releasing effects of this agent and to an increase in the axoplasmic levels of DOPEG.Since the formation of the deaminated glycol, DOPEG, represents the main metabolic pathway for the neurotransmitter in adrenergic nerve endings, the present results are compatible with the view that, in addition to the pool of extravesicular noradrenaline, the cytoplasmic concentration of DOPEG could also participate in the regulation of the activity of tyrosine hydroxylase.