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

Summary The working hypothesis was that the cocaine-insensitive component of non-exocytotic efflux of noradrenaline represents diffusion of the unprotonated amine across the axonal membrane. It was tested by examination of the effect of changing axoplasmic pH —and thus the fraction of extravesicular noradrenaline in the unprotonated form — on the overflows of endogenous noradrenaline and 3,4-dihydroxyphenylethylene glycol from rat tail arteries. The catechols were assayed by liquid chromatography with amperometric detection. To dissipate the H⁺ gradient across the axonal membrane, the tissues were incubated in media of different pH, in which Na⁺ was completely replaced with K⁺ and which were HCO₃ ^– - (and Ca²⁺-)free. Exposure of the tissues to these media produced substantial, but reversible increases in the overflow of noradrenaline. Subsequently, the overflows of both noradrenaline and the glycol kept rising, but their ratio did not change.Cocaine (0.1 mmol/1) lowered the (noradrenaline overflow: glycol overflow) ratio significantly. The ratio observed in its presence increased steeply with decreasing external and, presumably, axoplasmic pH. Addition of valinomycin and carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (1 mol/1 each) to the cocaine-containing media more than doubled the overflows without altering significantly the ratio. Under identical conditions, the overflow of noradrenaline from preparations with inactive neuronal monoamine oxidase did not decrease with decreasing pH.Since, in the presence of cocaine, the overflow ratio increased — rather than decreased — with decreasing pH, and because the overflow or noradrenaline from preparations with inactive monoamine oxidase did not decline with pH, the cocaine-insensitive component of noradrenaline efflux does not seem proportional to the axoplasmic concentration of the unprotonated amine. The data can, however, be accounted for by postulating that the component reflects the concentration of the protonated form of noradrenaline.     

Cocaine-sensitive O-methylation of noradrenaline in dental pulp of the rabbit: Comparison with the rabbit ear artery

Summary Incisor pulp from the rabbit metabolises exogenous noradrenaline in concentrations between 0.12 and 1.2 mol/l mainly to NMN.Effects of chronic sympathetic denervation indicated that in incisor pulp the NMN is extraneuronal in origin, and that DOPEG and DOMA formation, as well as a major part of the noradrenaline which accumulates in the tissue, are associated with the sympathetic nerves.NMN formation was unaffected by hydrocortisone 210 mol/l, but was strongly inhibited by cocaine 30 mol/l. These effects contrasted with those in the rabbit ear artery, where NMN formation was increased by cocaine 30 mol/l and decreased by hydrocortisone 210 mol/l.In COMT-inhibited denervated pulp, cocaine inhibited the accumulation of noradrenaline.Monoamine fluorescence histochemistry of pulp exposed to noradrenaline 50 mol/l indicated that cocaine-sensitive uptake occurred in fibroblasts.It is concluded that O-methylation of noradrenaline in dental pulp involves prior uptake of the amine by a process resembling uptake, but which is distinguished from uptake₁ by its extraneuronal location.Abbreviations DOMA 3,4-dihydroxy mandelic acid - DOPEG 3,4-dihydroxyphenylethyleneglycol - NMN normetanephrine - OMDA O-methyl deaminated metabolite fraction, comprising vanillyl-mandelic acid (VMA) plus the 3-methoxy derivative of DOPEG (MOPEG) - MAO monoamine oxidase - COMT catecholO-methyl transferase Send offprint requests to I. S. de la Lande at the above address     

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     

On the reduced retention of extravesicular noradrenaline in rabbit atria produced by potassium

The effects of KCl on the retention of extravesicular (?)-[³H]noradrenaline were investigated. Atria, from reserpine-pretreated rabbits, were exposed to tropolone and pargyline before incubation with (?)-[³H]noradrenaline. After efflux for 50–60 min, exposure of tissues to media containing 65 mM KCl resulted in increased efflux of (?)-[³H]noradrenaline. This effect was not Ca²⁺-dependent and was not altered by oxymetazoline, phentolamine, indomethacin or methacholine. The KCl-induced reduction in retention of (?)-[³H]noradrenaline was, however, inhibited by cocaine and desipramine, but not by lidocaine. Efflux was not increased by RbCl and CsCl. It is suggested that KCl may have accelerated the efflux of extravesicular (?)-[³H]noradrenaline by increasing a cocaine- and desipramine-sensitive carrier-mediated efflux process.     

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.     

The effects of ageing on neuronal uptake of noradrenaline in the rat

Summary We have examined the effects of ageing on the physiological function of the neuronal noradrenaline uptake system by comparing responses to cocaine in young adult (5 month) and aged (22 month) male Sprague-Dawley rats. In rat atria pre-incubated with [³H]-noradrenaline, cocaine (3–30 mol/l) significantly augmented the 2 Hz stimulation-evoked release of noradrenaline in tissues from young but not from old rats. Cocaine (1 mg/kg) produced a greater increase of the pressor response to noradrenaline in young than in old pithed rats. Cocaine significantly increased the tachycardia to noradrenaline only in young pithed rats, but in old pithed rats the duration of the response to noradrenaline was significantly increased. It is concluded that ageing in the rat is associated with a decreased function of the neuronal noradrenaline uptake system, at least in the cardiovascular system. Send offprint requests to J. R. Docherty at the above address     

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     

Effects of catechol-O-methyltransferase inhibition on the plasma clearance of noradrenaline and the formation of 3,4-dihydroxyphenylglycol in the rabbit

Summary The purpose of this study was to elucidate the finding of Friedgen et al. (1993b) that catechol-O-methyltransferase (COMT) inhibition is much more effective in increasing the plasma concentration of endogenous dihydroxyphenylglycol (DOPED) than in increasing the plasma concentration of infused DOPED. To this end, reserpine-pretreated rabbits were anaesthetized and infused with noradrenaline and/or DOPED, and the plasma clearances of infused noradrenaline (Cl_NA) and DOPED (Cl_DOPEG) as well as the plasma DOPED response to noradrenaline infusion [as reflected by the ratio of the steady-state increase in plasma DOPED (DOPEG) to that in plasma noradrenaline (NA)] were determined before and after blockade of neuronal uptake by desipramine. Experiments were carried out either under control conditions or after COMT inhibition by i.v. administration of 3,4-dihydroxy-4-methyl-5-nitrobenzophenone (Ro 40-7592). On the assumption that rates of neuronal noradrenaline uptake equal steady-state rates of neuronal DOPED formation, the desipramine-sensitive components of Cl_NA and DOPEG/NA were used to estimate the apparent plasma clearance of DOPED formed intraneuronally (Cl_f-DOPEG) in response to noradrenaline infusion.Cl_NA was 83.6 ml kg-^–1 min^–1 in the absence and 48.1 ml kg ^–1min^–1 in the presence of desipramine. Neither the former nor the latter value was altered after COMT inhibition. However, the COMT inhibitor reduced Cl_DOPEG from 47.6 to 28.5 ml kg ^–1 min^–1 (indicating a 1.7-fold increase in the plasma DOPED response to DOPED infusion) and Cl_f-DOPEG from 106.2 to 43.3 ml kg^–1 min^–1 (indicating a 2.5-fold increase in the neuronal component of the plasma DOPED response to noradrenaline infusion). DOPEG/NA increased following treatment with Ro 40-7592 from 0.381 to 1.294 in the absence and from 0.036 to 0.391 in the presence of desipramine. These increases were more pronounced than the Ro 40-7592-induced change in Cl_f-DOPEG would predict. Therefore, the values of Cl_f-DOPEG were used to calculate rates of DOPED formation. The results show that the increases in DOPEG/NA induced by COMT inhibition were not due only to the block of O-methylation of DOPED, but also to a pronounced increase in the extraneuronal DOPED formation.Hence, COMT inhibition affects neither the neuronal nor the extraneuronal removal of circulating noradrenaline from plasma. By contrast, COMT inhibition reduces the clearance of infused DOPED by about 40% and that of neuronally formed DOPEG by about 60%. The observation of Ro 40-7592 being more effective in increasing the plasma DOPEG response to noradrenaline infusion than in increasing the plasma DOPED response to DOPEG infusion is due to the fact that newly formed DOPEG is O-methylated not only while being removed from plasma, but also on the way to plasma, and that COMT inhibition increases the DOPEG formation at extraneuronal sites.This study was supported by the Deutsche Forschungsgemeinschaft (Gr 490/5). A preliminary account of the results was presented to the German Society for Pharmacology and Toxicology (Halbrügge et al. 1992)Correspondence to K. H. Graefe at the above address     

Metabolism of endogenous and exogenous noradrenaline in the rabbit perfused heart

Summary The outflow of noradrenaline, 3,4-dihydroxyphenylglycol (DOPEG) and 3,4-dihydroxymandelic acid (DOMA) from rabbit perfused hearts was studied by chromatography on alumina followed by high pressure liquid chromatography with electrochemical detection. In the absence of drugs and without nerve stimulation, the outflow of endogenous noradrenaline over a period of 108 min averaged 0.17 pmol×g^–1×min^–1 and the outflow of DOPEG 2.1 pmol×g^–1×min^–1. The outflow of DOMA was below the detection limit (<0.13 pmol×g^–1×min^–1). The effect of perfusion with (–)-noradrenaline 0.1, 1 or 10 mol/l for 18 min was then investigated. As the concentration of noradrenaline increased so did the outflow of DOPEG. Moreover, DOMA was found in the venous effluent during and after perfusion with noradrenaline 1 or 10 mol/l. The increase in the outflow of DOPEG and DOMA was almost abolished when cocaine 10 mol/l was present during the perfusion with noradrenaline 1 mol/l. The release of endogenous noradrenaline by sympathetic nerve stimulation or tyramine 10 mol/l, but not the release evoked by nicotine 30 mol/l, was accompanied by an increase in the outflow of DOPEG; an outflow of DOMA was not observed.It is concluded that, in the rabbit perfused heart, DOPEG is an important metabolite of endogenous noradrenaline. DOMA is at best a minor product, either when the neurones are at rest or when noradrenaline is released by sympathetic nerve stimulation, nicotine or tyramine. DOMA is formed in detectable amounts when the tissue is exposed to a high concentration of exogenous noradrenaline. Like DOPEG, it is formed intraneuronally. The results confirm and extend those obtained previously on guinea-pig incubated atria. They make it unlikely that, in these tissues at least, DOMA formation is one of the physiological pathways of noradrenaline catabolism.     

Nicotine receptors do not modulate the 3H-Noradrenaline release from the isolated rat heart evoked by sympathetic nerve stimulation

Summary Isolated rat hearts with the right sympathetic nerves attached were perfused at a constant flow rate of 7 ml/min with Tyrode's solution. (-)-³H-Noradrenaline (final concentration 10–13.9 nM) was infused for 10 min to label the noradrenaline stores. After wash-out the sympathetic nerves were stimulated electrically (3 Hz, 180 impulses, 1 ms, 20–30 mA) three times (S1–S3) at intervals of 15 min. ³H-Noradrenaline and its metabolites were determined by liquid scintillation counting according to Graefe et al. (1973).Both, nicotine 50 M and p-aminophenethyltrimethylammonium (PAPETA) 30 M, enhanced the ³H-noradrenaline overflow in the absence of nerve stimulation. The effect of PAPETA was biphasic and was still observed in the presence of N-methylatropine 0.1 M. Hexamethonium 10 M abolished the first phase only, but cocaine 10 M antagonized both phases.The decline of the stimulation-evoked overflow of ³H-noradrenaline from the first to the third stimulation period was similar in the absence and in the presence of cocaine 10 M starting before S1 and perfused throughout. Cocaine 10 M added before S2, however, enhanced the evoked overflow by 77%.PAPETA 30 M increased the stimulation-evoked overflow by 67% in the absence, and by 73% of the respective control in the presence, of hexamethonium 10 M. PAPETA 30 M failed to enhance the evoked overflow in the presence of cocaine. Hexamethonium (added before S2) did not modify the effectiveness of nerve stimulation.Nicotine, neither when perfused from 6 min before S2, nor when added to the perfusion fluid simultaneously with the onset of nerve stimulation, caused changes in the ³H-noradrenaline output upon S2.Upon stimulation a rather discrete increase in ³H-DOPEG overflow was observed. This increase was abolished by cocaine and/or PAPETA.It is concluded that nicotine and PAPETA stimulate the output of ³H-noradrenaline from the rat heart sympathetic nerves by activation of nicotine receptors. However, the amount of transmitter released is small. Neither drug appeared to modulate the output of ³H-noradrenaline upon electrical nerve stimulation via nicotine receptors.PAPETA, like cocaine, appears to block the reuptake of released transmittsrs thereby enhancing the ³H-noradrenaline overflow and reducing the overflow of ³H-DOPEG (formed intraneuronally from recaptured noradrenaline after nerve stimulation).Abbreviations used DOMA 3,4-dihydroxymandelic acid - DOPEG 3,4-dihydroxyphenylglycol - MOPEG 3-methoxy-4-hydroxy-phenylglycol - NA noradrenaline - NMN normetanephrine - OMDA O-methylated deaminated metabolites (sum of MOPEG and VMA) - PAPETA p-aminophenethyltrimethylammonium - VMA 3-methoxy-4-hydroxymandelic acid     

The outward transport of axoplasmic noradrenaline induced by a rise of the sodium concentration in the adrenergic nerve endings of the rat vas deferens

Summary The adrenergic nerve endings of the rat vas deferens were loaded with ³H-(–)-noradrenaline; COMT was inhibited by the presence of 10 mol/l U-0521, and all experiments were carried out with calcium-free solution. After 100 min of wash-out a neuronal efflux of tritium was obtained which remained constant with time (when expressed as fractional rate of loss; FRL); it contained more DOPEG than noradrenaline.The in vitro administration of reserpine-like drugs (reserpine and Ro 4-1284) increased the FRL of tritium, presumably because of an increase in the leakage of noradrenaline from storage vesicles; the efflux of DOPEG increased more than that of noradrenaline, and the ratio NA/DOPEG declined.Inhibition of the membrane ATPase (by omission of potassium from the medium or by the presence of 3 mmol/l ouabain) increased the FRL of tritium, presumably because of an increase in the net leakage of noradrenaline from the storage vesicles (as a consequence of the fall in the concentration of free axoplasmic noradrenaline; see below).Veratridine also increased the FRL of tritium, partly because of its known reserpine-like effect (Bönisch et al. 1983); in the presence of 1 mol/l veratridine, the efflux of DOPEG increased.Irrespective of the presence or absence of reserpine or Ro 4-1284, inhibition of the membrane ATPase or the presence of veratridine (agents or procedures which increase the axoplasmic sodium concentration) always resulted in a brisk increase of the efflux of noradrenaline that was accompanied by a simultaneous decrease in the efflux of DOPEG (see above for one exception). In all experiments the rise in internal sodium caused the ratio NA/DOPEG to increase.These results indicate that—as long as the sodium gradient is normal—the axonal membrane functions as a barrier that largely prevents any outward movement of axoplasmic noradrenaline. Consequently, the axoplasmic amine is largely deaminated, and the ratio NA/DOPEG is low. However, when the axoplasmic sodium concentration rises, axoplasmic noradrenaline is transported out of the nerve ending at such high rates that the axoplasmic noradrenaline concentration falls; the fall in the efflux of DOPEG is indicative of a fall in the intraneuronal formation of DOPEG. The results show that changes in the efflux of DOPEG (i.e., of a highly lipophilic metabolite that easily leaves adrenergic nerve endings) can serve as an index of changes in axoplasmic noradrenaline levels.Supported by the Deutsche Forschungsgemeinschaft (Tr.96)     

Selective inhibition by amezinium of intraneuronal monoamine oxidase

Summary Intra- and extraneuronal compartments of rat hearts were selectively labelled by perfusion with ³H-noradrenaline in the presence of corticosterone 87 M or cocaine 30 M, respectively. The subsequent outflow of ³H-compounds was examined. As little as 1 nM amezinium diminished the outflow of intraneuronally formed ³H-DOPEG. This effect was antagonized by cocaine. Amezinium 1 M was necessary to diminish the outflow of extraneuronally formed ³H-DOPEG. This effect was not counteracted by corticosterone. The results indicate that amezinium is both a potent and, at low concentrations, selective inhibitor of intraneuronal MAO.Abbreviations used DOMA 3,4-dihydroxymandelic acid - DOPEG 3,4-dihydroxyphenylglycol - MAO monoamine oxidase (EC 1.4.3.4) - NA noradrenaline - NMN normetanephrine - OMDA O-methylated deaminated metabolites     

Multiple effects of cocaine upon evoked secretion in bovine adrenal medullary chromaffin cells

Summary Experiments to determine the effects of the catecholamine neuronal uptake blockers cocaine and desipramine, and of the cardiac glycoside, ouabain, upon ³H(noradrenaline) efflux have been performed with bovine adrenal medullary chromaffin cells in tissue culture. Both cocaine and desipramine reduced ³H-noradrenaline uptake into chromaffin cells. Inhibitable uptake was 80% of total accumulation over 60 min; this degree of inhibition was produced by cocaine (30 mol/l) or desipramine (1 gmol/l). Cocaine (30 mol/l) had no effect upon spontaneous ³H-efflux measured over 60 min, but reduced that evoked over the same period by carbachol (EC₅₀), veratridine (EC₅₀) and by ouabain (100 gmol/l). Cocaine did not reduce that efflux evoked by raised levels of K⁺ (28 mmol/l; EC₅₀). Desipramine (1 gmol/l), like cocaine, had no effect upon spontaneous efflux of ³H, but reduced that efflux evoked by carbachol, veratridine and ouabain. Tetrodotoxin (TTX) inhibited veratridine-evoked ³H efflux (IC₅₀ 0.2 mol/l). The degree of inhibition caused by TTX (0.2 mol/l) was not increased by cocaine (30 mol/l). TTX also inhibited ouabain-evoked ³H efflux: this was reduced by 55% by a concentration of TTX (1 mol/l) sufficient to virtually abolish veratridine-evoked efflux. Cocaine (30 gmol/l) in the presence of TTX (1 mol/l) did not further inhibit ouabain-evoked efflux. Cocaine (30 mol/l) did not alter ⁸⁶Rb⁺ uptake into chromaffin cells, nor did it alter that inhibition of ⁸⁶Rb⁺ uptake produced by ouabain (100 gmol/l) indicating that cocaine has no effect upon Na,K-ATPase activity. The results are consistent with the suggestion that both cocaine and desipramine, besides inhibiting catecholamine uptake in bovine chromaffin cells, affect also the nicotinic receptor, or its associated ion-channel, and the Na⁺-channel opened by veratridine and sensitive to TTX. The data from the K⁺ experiment suggest that cocaine does not directly affect the voltage-sensitive calcium channel nor the exocytosis step. With respect to the mechanism of action of ouabain, the data show clearly that part of the efflux is brought about by a mechanism which involves the TTX-sensitive Na⁺ channel, and, that the remaining part is independent of the neurotransmitter uptake process. Send offprint requests to D. A. Powis at the above address     

The effects of quinpirole and fenoldopam on the potassium-evoked overflow of endogenous dopamine and noradrenaline in dog mesenteric arteries

Summary (1) Dopamine and noradrenaline overflow from the main trunk of the dog mesenteric artery and its proximal branches, elicited by K⁺ (52 mmol/l), was measured by high pressure liquid chromatography with electrochemical detection. (2) Quinpirole (0.1, 1 and 10 nmol/l) produced a concentration dependent reduction of dopamine and noradrenaline overflow in both segments of the mesenteric artery. The inhibitory effect of quinpirole (10 nmol/l) on amine overflow was antagonized by sulpiride (1 mol/l) but not by phentolamine (0.2 mol/l) or the selective dopamine (DA₁), antagonist SK&F 83566 (1 ol/l). (3) Fenoldopam (0.1 and 1 mol/l) did not alter dopamine and noradrenaline overflow from both segments of the mesenteric artery; only 10 mol/l fenoldopam was found to increase the overflow of dopamine and noradrenaline in both segments of the mesenteric artery. This effect of fenoldopam on amine overflow was not altered by the addition to the perifusion fluid of SK&F 83566 (1 ol/l). (4) Clonidine (100 nmol/l) significantly reduced amine overflow from both segments of the mesenteric artery and this effect was antagonized by fenoldopam (10 mol/l) (5) These results suggest that quinpirole inhibits sympathetic neurotransmission through the activation of prejunctional dopamine receptors of the DA₂ subtype. The facilitatory effect of fenoldopam (10 mol/l) on amine release appears to be mediated through the blockade of prejunctional ₂-adrenoceptors. Send offprint requests to P. Soares-da-Silva at the above address     

Age-related changes in neuronal uptake of noradrenaline

Summary Age-related changes in the neuronal uptake of noradrenaline were studied in the vas deferens of 4-, 12-and 20-months old rats by determining the potentiating effect of cocaine on concentration-effect curves to noradrenaline and by evaluating the kinetic parameters (V _max and apparent K _m) of neuronal ³H-noradrenaline uptake. Since the neurotransmitters released by nerve terminals and the density of noradrenergic innervation are different in the epididymal and prostatic portion of the vas, the experiments were carried out in these two portions of the organ.In the epididymal portion, the ratio beween EC₅₀ for noradrenaline in the presence and in the absence of cocaine was reduced in tissues of middle-aged (12-months) and old (20-months) animals when compared with young (4-months) animals. The pD₂ values for noradrenaline in the absence of cocaine and the kinetic parameters for neuronal ³H-noradrenaline uptake were not modified by ageing. Thus, in the epididymal portion there was no age-related change in neuronal uptake. The age-related decrease in the cocaine-induced supersensitivity to noradrenaline was due to an age-related decrease in the pD₂ value for noradrenaline observed in the presence of cocaine, suggesting postsynaptic changes.In the prostatic portion, the ratio between EC₅₀ for noradrenaline in the presence and in the absence of cocaine was also reduced in tissues of old animals (20-months) when compared with middle-aged and young animals. The pD₂ value for noradrenaline in the absence of cocaine obtained in the prostatic portion of 20-months old rats was higher than that in the younger groups (4- and 12-months). Since in the presence of cocaine no age-related changes in the pD₂ value for noradrenaline were obtained, postsynaptic changes might not occur in this portion. Thus, the reduction in the EC₅₀ ratio for noradrenaline in the prostatic portion of 20-months old rats suggested a loss of function of the neuronal uptake system. Further support for this hypothesis was that the V _max value of neuronal ³H-noradrenaline uptake tended to decline with age only in the prostatic portion.Therefore, the age-related decrease in the cocaine-induced supersensitivity was probably due to an age-related loss of function of the neuronal uptake system in the prostatic portion and to a decrease in the sensitivity to noradrenaline in the epididymal portion. The loss of function of neuronal uptake and the change in sensitivity to noradrenaline occurred at different ages.Part of the present results have been reported in the III Annual Meeting of the Federação de Sociedades de Biologia Experimental, Caxambu, MG, June 29 — July 3, 1988 Send offprint requests to R. P. Markus, Departamento de Farmacologia, Instituto de Ciencias Bimédicas, Universidade de São Paulo, Av. Prof. Linen Prestes, 1524, 05508, Sao Paulo, Brasil     

Early intraneuronal mobilization and deamination of noradrenaline during global ischemia in the isolated perfused rat heart

Summary Isolated rat hearts were perfused according to the Langendorff technique and both extraneuronal uptake of noradrenaline and COMT were inhibited. The noradrenergic neurones were first prelabelled with ³H-(–)-noradrenaline (13 nmol/1). Thereafter the hearts were submitted to global ischemia (perfusion rate reduced from 5 up to 0.5 ml/min) for 60 min and subsequently reperfused for 5 min. The coronary effluent was continuously collected and analyzed for the appearance of ³H-noradrenaline and its metabolites. 1. Global ischemia was associated with an early release of ³H-noradrenaline. At reperfusion a brisk increase in the FRL of ³H-noradrenaline was observed which may indicate that, on severe restriction in coronary flow, perfusion of the tissue became heterogenous and thus partially masked the amount of ³H-noradrenaline released from the noradrenergic nerve terminals. Gradual reduction in coronary flow also progressively reduced (but did not abolish) the total formation of ³H-DOPEG. 2. The maximal efflux of ³H-noradrenaline was observed during the 1st min of reperfusion whereafter the efflux declined rapidly, indicating a wash-out of transmitter trapped in the extracellular space. The efflux of the lipophilic metabolite ³H-DOPEG, on the other hand, continuously increased during the reperfusion. This was due to both new formation and wash-out of ³H-DOPEG retained and/or distributed into the tissue during the period of restricted flow. 3. Neither a reduction of the extracellular calcium concentration (from 2.6 mmol/l to 0.1 mmol/1) nor the presence of the calcium entry blocker verapamil (250 nmol/l) reduced the efflux of ³H-noradrenaline seen during ischemia and reperfusion. 4. Desipramine (100 nmol/l) markedly reduced the ischemia-induced release of ³H-noradrenaline and simultaneously attenuated the formation of ³H-DOPEG. 5. A moderate reduction in the ischemia-induced mobilization of ³H-noradrenaline was seen in hearts perfused with 1ol/l reserpine, whereas the formation of ³H-DOPEG from such hearts was markedly higher than in corresponding controls. Only minor deviations from this pattern was observed when desipramine was present in addition to reserpine. It is concluded that a severe restriction in myocardial perfusion rate is associated with an enhanced net leakage of vesicular noradrenaline. This results in a rise of the free axoplasmic noradrenaline concentration which, in combination with an altered transmembrane sodium gradient, induces an increased local release of noradrenaline partly mediated by a calcium-independent, carrier-mediated outward transport. Desipramine, which inhibits this transport mechanism, may have, in addition to its effect on the membrane carrier, an additional effect in reducing the net leakage of transmitter from storage vesicles. Furthermore, despite severe restriction in coronary flow, and thus oxygen delivery, DOPEG is still formed, possibly as a consequence of the elevated axoplasmic noradrenaline concentration which may in part compensate for a reduced monoamineoxidase activity.Abbreviations DOPEG dihydroxyphenylglycol - DOMA dihydroxymandelic acid - MAO monoamineoxidase - COMT catechol-O-methyltransferase - OMI 3-O-methoxy-(±)-isoprenaline - FRL fractional rate of loss This study was supported by the Deutsche Forschungsgemeinschaft (Gr 490/5-1) and AB Hässle, Mölndal, Sweden     

Metabolism of endogenous and exogenous noradrenaline in guinea-pig atria

Summary The outflow of noradrenaline, 3,4-dihydroxyphenylglycol (DOPEG) and 3,4-dihydroxymandelic acid (DOMA) from guinea-pig isolated atria was studied by chromatography on alumina followed by high pressure liquid chromatography with electrochemical detection. In the absence of drugs, the outflow of endogenous noradrenaline over a period of 3 h averaged 1.6 pmol×g^–1×min^–1 and the outflow of DOPEG 17 pmol×g^–1×min^–1. The outflow of DOMA was below the detection limit (<0.31 pmol×g^–1×min^–1). Tyramine greatly increased the outflow of noradrenaline and DOPEG, and the reserpine-like compound Ro 4-1284 selectively increased the outflow of DOPEG; DOMA remained below the detection limit. When atria were exposed to (–)-noradrenaline 1.7 or 17 M, the subsequent outflow of noradrenaline and DOPEG was enhanced. Moreover, substantial amounts of DOMA were now found. This outflow of DOMA was prevented when atria were exposed to (–)-noradrenaline in the presence of cocaine or after an initial incubation with amezinium. Exposure to (–)-noradrenaline 1.7 M mainly enhanced the formation of DOPGE, while exposure to (+)-noradrenaline 1.7 M mainly enhanced the formation of DOMA.Our experiments confirm some and qualify other conclusions drawn from studies in which exogenous ³H-noradrenaline had been used to examine the metabolism of noradrenaline in guinea-pig atria. In agreement with the isotope studies, DOPEG is a major metabolite of endogenous noradrenaline. In contrast to what the isotope studies had suggested, however, endogenous DOMA is a very minor product, at least as long as the neurones are at rest. DOMA is only formed when the tissue is exposed to high concentrations of exogenous noradrenaline. In further contrast to previous conclusions, DOMA is then formed intra- and not extraneuronally.     

Potentiation of potassium-evoked noradrenaline and neuropeptide Y co-release by cardiac energy depletion: role of calcium channels and sodium-proton exchange

Summary The role of the cardiac energy status in the potassium-evoked exocytosis of both noradrenaline and the sympathetic co-transmitter neuropeptide Y (NPY) was investigated in the guinea-pig perfused heart. The transmitter release was stimulated by potassium depolarization (10–80 mmol/l) during normoxic perfusion (pO₂ > 100 mmHg) in the presence of glucose (11 mmol/l) and at various periods (5–40 min) of cardiac energy depletion. Energy depletion was induced either by anoxia (pO₂ < 5 mmHg) or by cyanide intoxication (1 mmol/l), both in combination with glucose-free perfusion. Endogenous noradrenaline and NPY were determined in the coronary venous overflow by high-pressure liquid chromatography combined with electrochemical detection and by radioimmunoassay, respectively.Under normoxic conditions potassium depolarization evoked a co-release of both transmitters [molar ratio 862 (noradrenaline) :1 (NPY)] at a threshold concentration of 40 mmol/l potassium. This transmitter overflow was characterized by its dependence on extracellular calcium and calcium influx through voltage-dependent neuronal calcium channels of the N-type. Cardiac energy depletion was accompanied by an acceleration and an enhancement of the potassium-evoked transmitter overflow. In comparison to normoxia, a 10-fold increased transmitter overflow with a comparable molar ratio [709 noradrenaline :1 (NPY)] was evoked by 40 mmol/l potassium after 10 min of either anoxia or cyanide intoxication. This sensitization to potassium depolarization reached a peak after 10 min of energy depletion and was characterized by a markedly reduced threshold concentration (10 mmol/l potassium). The enhanced sympathetic transmitter overflow in anoxia was suppressed by addition of glucose (11 mmol/l) to the perfusion buffer, suggesting that the sensitization of the overflow of noradrenaline and NPY to potassium depolarization requires a cessation of energy metabolism. The sensitization of the potassium-evoked (20 mmol/l) sympathetic transmitter overflow by energy depletion was further characterized: Consistent with an exocytotic release mechanism, the overflow was calcium-dependent. In contrast to normoxia, however, blockade of neuronal N-type calcium channels by either co-conotoxin (100 nmol/1) or cadmium chloride (50 mol/l) failed to reduce the potassium-evoked overflow of noradrenaline and NPY. In anoxia blockade of sodium-proton exchange by amiloride (1 mmol/l) or more specifically by ethylisopropylamiloride (1 mol/l) markedly attenuated the potassium-evoked transmitter overflow. Likewise, suppression of the potassium-evoked overflow of noradrenaline and NPY from the energy-depleted heart was achieved by extracellular acidosis (pH 6.0). In contrast, during normoxia blockade of sodium-proton exchange by either ethylisopropylamiloride (1 mol/l) or by extracellular acidosis (pH 6.0) did not affect the potassium-evoked (80 mmol/l) transmitter overflow. These findings suggest that the sensitization of sympathetic nerve endings to potassium depolarization, caused by cardiac energy depletion, requires sodium entry into the sympathetic nerve ending via sodium-proton exchange.The results of the present study indicate, that the threshold concentration for the potassium-evoked exocytotic release of noradrenaline and NPY from the guinea-pig isolated perfused heart is intimately coupled to the energy status of cardiac sympathetic nerve fibres. The energy status not only determines the quantity of the transmitters released but also the mode of sodium and calcium entry triggering the depolarization-evoked transmitter overflow.Preliminary findings were reported at the 63rd Scientific Sessions of the American Heart Association, Dallas/USA (Haass et al., 1990b) and at the Annual Meeting of the European Section of the International Society for Heart Research, Leuwen/Belgium (Haass et al. 1991b)Supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 320 — Herzfunktion and ihre Regulation) Correspondence to M. Haass at the above address     

Characteristics of the cocaine-sensitive accumulation and O-methylation of 3H-(−)-noradrenaline by rabbit endometrium

Summary 1. The extraneuronal uptake and O-methylation of 2,5,6 ³H-(–)-noradrenaline was studied in segments of uterine endometrium from rabbits pretreated with 17-oestradiol and progesterone. 2. The uptake of ³H-noradrenaline was measured in MAO- and COMT-inhibited tissues and obeyed Michaelis-Menten kinetics with an apparent K _m of 78 mol/1 and a V _max of 5.4 nmol/g min. Uptake was inhibited by low Na⁺ and by potential substrates in the order dopamine > (–)adrenaline > (–)isoprenaline = 5-hydroxytryptamine. 3. Following uptake at 1.2 mol/1, efflux of ³H-noradrenaline was slow and appeared to be from two compartments, of which the first (I) had a t1/2 of 53 min and a capacity of 1.8 nmol/g. The presence of the second compartment (II) was inferred from the tissue content of ³H after 60 min of efflux, which was 3–4 times greater than predicted if the ³H was present in compartment I only. Following incubation with ³H-noradrenaline in the presence of cocaine 30 mol/1 the ³H efflux was rapid and the combined capacities of compartments I and II were greatly decreased. 4. ³H-NMN formation, measured in MAO-inhibited tissues, obeyed Michaelis-Menten kinetics with a half-saturating outside concentration of 12 mol/1 and a V _max of 0.9 nmol/g · min. The formation was inhibited by the neuronal uptake inhibitors, desipramine 3 mol/1 and metaraminol 100 mol/1 (each by 80%), but was unaffected by the extraneuronal uptake inhibitor, NMN 100 mol/1, and by oxytetracycline 100 mol/1 and methoxamine 10 mol/1. 5. ³H-NMN formation was inhibited to a small extent (by 30%) by hydrocortisone in a concentration which inhibited ³H-NMN formation in the myometrium by 84%. 6. It is concluded that the extraneuronal uptake of exogenous noradrenaline in the endometrium (a) resembles uptake in sympathetic nerves in its sensitivities to sodium ions and uptake inhibitors, and (b) resembles corticosteroid-sensitive extraneuronal uptake in that it has a low affinity for noradrenaline, but is linked with O-methylation in a fashion which renders uptake and O-methylation a relatively high-affinity, low-capacity system for removing noradrenaline.Abbreviations DOMA 3,4-dihydroxymandelic acid - DOPEG 3,4-dihydroxyphenylglycol - COMT catechol-O-methyl transferase - MAO monoamine oxidase - NMN normetanephrine - U-0521 3,4-dihydroxy-2-methyl propiophenone Send offprint requests to J. A. Kennedy at the above address     

Release of noradrenaline from isolated rat tail artery induced by bafilomycin A1

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