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.     

Influence of fentanyl,levorphanol and pethidine on the release of noradrenaline from rat brain cortex slices

Summary In slices of rat brain cortex preincubated with (–)-³H-noradrenaline, the influence of fentanyl, levorphanol and pethidine on the efflux of tritium was investigated. The spontaneous outflow of tritium was not changed by low, and was accelerated by high concentrations of the drugs. The overflow of tritium evoked by electrical stimulation at 3 Hz was diminished by 10^–8–10^–7 M fentanyl and by 10^–7–10^–6 M levorphanol, but was augmented by 10^–5 M levorphanol. Naloxone prevented the inhibitory effect of fentanyl and levorphanol. In contrast to fentanyl and levorphanol, pethidine did not decrease, but at concentrations of 10^–6–10^–5 M greatly increased the stimulation-induced overflow of tritium. However, the increase was abolished, and the stimulation-evoked overflow slightly reduced, after the re-uptake of noradrenaline had been blocked by cocaine. It is concluded that fentanyl, levorphanol and pethidine share with morphine the ability to inhibit the release of transmitter from cerebrocortical noradrenaline neurones evoked by nerve impulses.     

Influence of morphine and naloxone on the release of noradrenaline from rat brain cortex slices

Summary In slices of rat brain cortex preincubated with (–)-³H-noradrenaline, the influence of morphine and naloxone on the efflux of tritium was investigated. The spontaneous outflow of tritium was not changed by 10^–7–10^–5 M morphine and by 10^–6–10^–4 M naloxone, but was accelerated by 10^–4 M morphine. Electrical field stimulation augmented tritium outflow. The overflow evoked per ppulse decreased as the frequency of stimulation was increased from 0.3 to 3 Hz, but remained approximately constant when it was further increased to 10 Hz. At frequencies of 0.3, 1, and 3 Hz, but not at 10 Hz, morphine in concentrations of 10^–7–10^–5 M depressed the stimulation-induced overflow of tritium. 10^–4 M morphine did not influence the overflow induced by stimulation at 0.3 and 1 Hz and increased that evoked by stimulation at 10 Hz. Naloxone (10^–6–10^–4 M) did not change the response to stimulation. In the presence of 10^–4 M naloxone, 10^–6 M morphine did not diminish, and 10^–5 M morphine even enhanced the stimulation-induced overflow of tritium. The inhibitory effect of 10^–6 M morphine was not reduced, after tyrosine hydroxylase had been blocked by -methyltyrosine-methylester. It is concluded that morphine through an action on specific opiate receptors inhibits the release of transmitter from cerebrocortical noradrenergic neurones evoked by nerve impulses. By an action unrelated to opiate receptors, morphine at high concentrations increases the stimulation-induced overflow of noradrenaline, presumably by inhibiting its re-uptake into nerve endings.     

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     

Alpha-receptor-mediated modulation of transmitter release from central noradrenergic neurones

Summary Slices of rat cerebral cortex were preincubated with 10^–7 M (-)-³H-noradrenaline, and the outflow of tritium was determined. Oxymetazoline, phentolamine and cocaine did not change the spontaneous efflux. The overflow of tritium evoked by electrical field stimulation was decreased by oxymetazoline, and enhanced by phentolamine, phenoxybenzamine, and cocaine. Oxymetazoline did not counteract the increase of the stimulation-induced overflow caused by cocaine, but strongly antagonized the increase caused by phentolamine and phenoxybenzamine. When the stimulation-induced overflow was large under control conditions (high frequency of stimulation, addition of cocaine), the inhibitory effect of oxymetazoline was diminished. The results indicate that an -receptor-mediated feed-back control of noradrenaline release, previously demonstrated in postganglionic sympathetic nerves, also operates in central noradrenergic neurones.     

ACTH increases noradrenaline release in the rabbit heart

Summary The effects of ACTH on the release of noradrenaline and the increase of heart rate produced by sympathetic nerve stimulation (1 Hz) were studied in isolated perfused rabbit hearts. ACTH-(1–24) 0.1–100 nmol/l increased the stimulation-evoked overflow of noradrenaline concentration-dependently, reversibly and up to two-fold. The basal outflow of noradrenaline, the basal heart rate and the stimulation-evoked increase in heart rate were not changed. Human ACTH-(1–39) also increased the evoked overflow of noradrenaline. The effect of ACTH-(1–24) 0.3 nmol/l persisted after blockade of -adrenoceptors with propranolol and blockade of neuronal catecholamine uptake by cocaine. ACTH-(1–24) 3 nmol/l did not change the removal of noradrenaline from the perfusion fluid, when hearts were perfused with medium containing 59 nmol/l noradrenaline. The results show that ACTH increases the action potential-evoked release of noradrenaline from cardiac postganglionic sympathetic neurones, probably by activating specific presynaptic ACTH receptors. The high potency of ACTH suggests that these presynaptic receptors may be activated in vivo by circulating ACTH under certain pathophysiological conditions.Send offprint requests to B. Szabo at the above address     

Protection by phentolamine against the effects of phenoxybenzamine on transmitter release elicited by nerve stimulation in the perfused cat heart

1 The effects of cocaine, phentolamine and phenoxybenzamine on neuronal uptake of [(3)H]-noradrenaline and on (3)H-transmitter and noradrenaline overflow elicited by nerve stimulation were determined in the perfused heart of the cat.2 During perfusion with cocaine 3.4 x 10(-7)M, there was a 2-fold increase in transmitter overflow while neuronal uptake of [(3)H]-noradrenaline was inhibited by 31.3 +/- 2.1%.3 After exposure to phenoxybenzamine 8.7 x 10(-7)M for 20 min and washing with drug-free solution for 165 min there was an 8-fold increase in transmitter overflow during nerve stimulation. Under these conditions neuronal uptake of [(3)H]-noradrenaline was inhibited by only 17.5 +/- 5.4%.4 There was no significant change in transmitter overflow or in neuronal uptake of [(3)H]-noradrenaline, 155 min after a 30 min exposure to phentolamine (3.2 x 10(-5)M).5 Perfusion with phentolamine (3.2 x 10(-5)M) before and during exposure to phenoxybenzamine (8.7 x 10(-7)M), prevented the increase in transmitter overflow observed after perfusion with phenoxybenzamine alone.6 Protection by phentolamine against the effects of phenoxybenzamine supports the view that the effects on transmitter release obtained after perfusion with phenoxybenzamine are due to the blockade of presynaptic alpha-adrenoceptors which regulate transmitter release through a negative feed-back mechanism.     

Indirect adrenergic effect of histamine in cat cerebral arteries

Summary Histamine (10(^–4 M) induced an increase in the tritium outflow from cat cerebral arteries preloaded with ³H-noradrenaline. Pretreatment with reserpine (3 mg/kg, i.p., total dose) or removal of both superior cervical ganglia two weeks before the experiment abolished that increase. The presence of cocaine or diphenhydramine also prevented the rise in tritium efflux induced by histamine.Histamine (10(^–8 M to 10(^–3 M) elicited dose-dependent contractions in the isolated posterior communicating artery of the cat which were reduced in the presence of diphenhydramine at all doses except the highest three. The addition of phentolamine to the bath decreased the contractile responses at the doses lower than 10(^–6 M. Pretreatment with reserpine or removal of both superior cervical ganglia also diminished the responses at doses of histamine below 10(^–6 M and 10(^–5 M, respectively. When cocaine was added to the bath there was a decrease in the contraction elicited at all doses except the last one.These results suggest the existence of an indirect adrenergic mechanism in the contractile response to histamine in cat cerebral arteries.     

Comparison between the effects produced by chronic denervation and by cocaine on the sensitivity to,and on the disposition of,noradrenaline in isolated spleen strips

Summary A comparison was made between the effects of cocaine and denervation on the sensitivity to, on the rate of inactivation of, and on the roles played by iproniazid and tropolone in the inactivation of noradrenaline by cat spleen strips. For studying the rate of inactivation of noradrenaline the oil immersion technique was used. Cocaine was used in four different concentrations. In all concentrations did it enhance the sensitivity to noradrenaline. When cocaine was used in concentrations of 10 and 50×10^–6 M, the enhancement was significantly higher than that caused by denervation (11.61 and 14.81 vs. 6.42, respectively: p<0.001). Since denervation produces an enhancement of the effect of noradrenaline which is smaller than that caused by cocaine, the blockade of neuronal uptake cannot fully account for all supersensitivity induced by cocaine. On the other hand, cocaine produces no further enhancement of the effect of noradrenaline in denervated strips. It is assumed that cocaine acts in normal (control) preparations by two mechanisms: 1. blockade of neuronal uptake accounting for an enhancement like that caused by denervation (about 6 times); 2. facilitation of the action of noradrenaline by interference with a hypothetical postjunctional structure which depends on the presence of intact adrenergic nerves and which accounts for the remaining degree of enhancement (about 2 times).Strips treated with cocaine (50×10^–6 M) required for half-relaxation 7.42 times the time of the controls, whereas denervated strips only required 4.84 times the control time. On the other hand in denervated strips cocaine (50×10^–6 M) produced a further 1.55-fold prolongation of the half-relaxation time. Thus, the effect of cocaine on this parameter is concluded to be due primarily to blockade of neuronal uptake and secondarily to another factor, which could be related to uptake₂.The influence of cocaine and denervation on the role played by iproniazid and tropolone on the inactivation of noradrenaline was not significantly different; apparently, in this preparation, the monoamine oxidase involved in terminating the action of noradrenaline is predominantly if not entirely situated intraneuronally, whereas catechol-O-methyltransferase seems to be situated intra- and extraneuronally almost in equal proportions.     

The aziridinium derivative of DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine) accelerates the beating rate of isolated rat atria by enhancing the spontaneous release of noradrenaline

Summary The aziridinium derivative of the compound N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (az-DSP4) depletes endogenous noradrenaline stores and exerts neurotoxic actions on noradrenergic neurons. These effects are persistent in the central nervous system and transient in the periphery. To determine if transmitter release plays a role in the noradrenaline depletion caused by az-DSP4, the action of the compound was studied in isolated and spontaneously beating rat atria. 1. az-DSP4 enhanced atrial beating rate when present in the incubation medium at concentrations ranging from 10^–3 M to 10^–4 M but at 10^–3 s M decreased that rate below basal levels. 2. Preincubation of atria for 30 min with the noradrenaline uptake blocker desimipramine (DMI, 10^–6 M) or with the betablocker propranolol (10^–7 M), abolished the positive chronotropic action of az-DSP4. 3. The rate-accelerating effect of az-DSP4 could be prevented by pretreating the rats with reserpine (5 mg/kg i. p. 24 h) or enhanced by pargyline pretreatment (100 mg/kg i. p. 18 h). 4. az-DSP4 stimulated the spontaneous efflux of tritium from the isolated atria previously labeled with ³H-noradrenaline (4 × 10^–7 M), an increase that was mainly accounted for by DOPEG. 5. COMT and MAO activities in atria homogenates were inhibited by az-DSP4 in a concentration-dependent manner. However, MAO inhibition did not result in a change of the metabolic pattern as could be expected. 6. The results obtained indicate that az-DSP4 enhances the rate of spontaneous beating of isolated rat atria. The positive chronotropic effect of az-DSP4 requires the interaction of the compound with the noradrenaline uptake system. The mechanism of the accelerating effect of az-DSP4 most probably involves the release of noradrenaline from adrenergic nerve terminals in the atria and its subsequent interaction with adrenergic beta-receptors.Abbreviations DSP4 N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride - az-DSP4 aziridinium derivative of DSP4 - NA noradrenaline - 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) - COMT catechol-O-methyltransferase - MAO monoamineoxidase Send offprint requests to M. E. Landa     

The effect of methacholine on noradrenaline release from the rabbit heart perfused with indometacin

Summary The experiments were undertaken in order to study the effect of inhibition of prostaglandin synthesis on the muscarinic inhibition of noradrenaline release evoked by sympathetic nerve stimulation. The right sympathetic nerves of the perfused rabbit heart were stimulated electrically. The noradrenaline output was enhanced after perfusion of the hearts with indometacin 3×10^–5 M indicating blockade of the prostaglandin-mediated negative feedback control. Both in the presence and in the absence of indometacin methacholine 4×10^–5 M decreased the noradrenaline output by a similar percentage. It is concluded that the muscarinic inhibition of noradrenaline release does not require the functional integrity of the prostaglandin-mediated feedback system.     

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.     

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).     

Influence of drugs with affinity for α-adrenoceptors on noradrenaline release by potassium,tyramine and dimethylphenylpiperazinium

The influence of oxymetazoline and phentolamine on the overflow of noradrenaline evoked by potassium, tyramine and dimethylphenylpiperazinium (DMPP) was investigated in isolated perfused rabbit hearts.Oxymetazoline decreased, and phentolamine increased, the outflow of noradrenaline evoked by potassium. In hearts previously perfused with (?)-³H-noradrenaline, oxymetazoline reduced, and phentolamine enhanced, potassium-induced overflow of both ³H-noradrenaline and total tritium. These actions closely resemble previously described effects on noradrenaline overflow evoked by electrical stimulation of sympathetic nerves. At concentrations which modified the response to potassium, oxymetazoline and phentolamine did not influence the overflow evoked by tyramine. Both drugs diminished DMPP-induced overflow.It is concluded that oxymetazoline depresse noradrenaline release evoked by potassium or orthodromic action potentials through activation of neuronal α-adrenoceptors, followed by inhibition of electro-secretory coupling. Phentolamine blocks the analogous inhibitory effect of liberated noradrenaline and thus enhances release. The action of tyramine does not involve electro-secretory coupling and therefore is not changed. The influence of oxymetazoline and phentolamine on noradrenaline release by DMPP is not related to α-adrenoceptors.     

Studies on alpha adrenoceptors in guinea-pig peripheral lung strips

Summary Contractile responses of guinea-pig peripheral lung strips to noradrenaline were determined in the presence of propranolol (2.5 × 10^–6 mol/l). All strips (n = 44) contracted to noradrenaline and a geometric mean EC₅₀ of 1.4 × 10^–6 mol/l (1.1 × 10^–6 mol/l, 1.8 x 10^–6 mol/l 95% confidence limits) was obtained. Contractions were antagonised by phentolamine (5 × 10^–7–10^–5 mol/l) and by prazosin (10–10^–7 mol/l). Dose-ratios (DR) were calculated and log (DR-1) was plotted against log concentration of antagonist to yield slopes (± SE) of 0.84 ± 0.14 and 0.73 ± 0.22 respectively which were not significantly different from unity. A pA₂ value (± SE) of 6.7 ± 0.2 was obtained for phentolamine and 7.5 ± 0.1 for prazosin. Yohimbine (10^–7–10^–5 mol/l) did not significantly affect the maximal tension generated or the EC₅₀ values for noradrenaline. These results suggest that ₁ adrenoceptors are mediating the contractile responses to noradrenaline. In the presence of cocaine (10^–5 mol/l, n=18), normetanephrine (2 × 10^–5 mol/l, n = 15), hydrocortisone (2.5 × 10^–5 mol/l, n = 15) and normetanephrine (2 × 10^–5-5 mol/l) plus cocaine (10^–6 5 mol/l, n=15) pA₂ values for phentolamine of 6.9, 6.7, 6.6, and 6.3 respectively were obtained. Since these pA₂ values are not significantly different from 6.7, it is unlikely that this original pA₂ value, which is lower than values obtained with phentolamine at -adrenoceptors in other tissues, may be explained by neuronal or extraneuronal uptake of noradrenaline. A possible explanation may be that more than one population of -adrenoceptors contribute to changes in tension in peripheral lung strips. Send offprint requests to J. P. Seale at the above address     

Influence of morphine and naloxone on the release of noradrenaline from rat cerebellar cortex slices

Summary In slices of rat cerebellar cortex preincubated with (-)-³H-noradrenaline, the influence of morphine and naloxone on the efflux of tritium was investigated. The spontaneous outflow was not changed by 10^–5 M of either morphine or naloxone. On the other hand, morphine caused a concentration-dependent decrease of the overflow, of tritium evoked by electrical field stimulation. Naloxone did not change the stimulation-induced overflow, but prevented its inhibition by morphine. It is concluded that morphine, through an action on opiate receptors located on cerebellar noradrenergic neurones, inhibits the secretion of the transmitter in response to nerve impulses.     

Release of vesicular noradrenaline in the rat tail artery induced by cocaine

Summary The effects of cocaine on overflows of endogenous noradrenaline and DOPEG from isolated rat tail arteries were examined. 1. Both overflows increased progressively with increasing concentration of cocaine, while the (NA overflow)/(DOPEG overflow) ratio first increased and then decreased. The changes in the overflows induced by cocaine (0.1 mmol/l) appeared reversible. 2. Exposure of the tissue for 30 min to cocaine, 1 mmol/l, resulted in a significant decrease in the proportion of storage vesicles containing electron-dense cores. 3. The changes in overflows of noradrenaline and DOPEG induced by cocaine (0.1 mmol/l) were unaffected by the presence of desipramine (0.1 mol/l) or removal of extracellular Ca²⁺. The effect of cocaine on the overflow of noradrenaline was potentiated by prior inhibition of MAO with clorgyline. 4. Exposure of segments to a Ca²⁺-free, high K, low Na incubation medium was accompanied by increased overflow of noradrenaline. Cocaine (0.1 mmol/l) reduced the overflow of noradrenaline to about a half, and substantially increased the overflow of DOPEG. 5. The increase in the overflow of DOPEG from segments bathed in HEPES-buffered solutions, the pH of which ranged from 6.80 to 7.38, was approximately proportional to the calculated concentration of unprotonated (uncharged) cocaine. 6. Quantitatively similar changes in the overflows were observed when norcocaine was substituted for cocaine. Ecgonine methyl ester was much less potent than cocaine, and O-benzoyl ecgonine was ineffective. 7. The small increases in the overflow of noradrenaline observed at relatively low concentration (<30 mol/l) of cocaine can be attributed primarily to inhibition of reuptake of the released transmitter by the cocaine- and desipramine-sensitive amine carrier. The overflows of NA and DOPEG in the presence of higher concentrations of the alkaloid exhibit features compatible with the following hypothesis: (A) Cocaine is translocated across the axonal membrane mainly in the form of the unprotonated species, a large fraction of which is reprotonated upon the entry into the axon. (B) Cocaine releases noradrenaline from storage vesicles into the extravesicular space, where the bulk of the amine is converted to DOPEG. (C) Efflux of the remaining noradrenaline from the axon is not mediated by the Na⁺-dependent, cocaine- and desipramine-sensitive neuronal amine carrier. It seems to represent uncoupled efflux of the protonated form of noradrenaline.Abbreviations DOPEG 3,4-dihydroxyphenylethylene glycol - DOMA 3,4-dihydroxymandelic acid - HEPES N-(2-hydroxyethyl)piperazine-N-ethanesulfonic acid - MAO monoamine oxidase - MOPEG 3-methoxy-4-hydroxyphenylethylene glycol - NA (–)noradrenaline - pH_j pH in the extravesicular space of the axon - pH_o pH of the bathing solution - pK_a negative logarithm of the dissociation constant This study was supported by the British Columbia Heart Foundation Send of fprint requests to V. Palaty at the above address     

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.     

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     

Serotonin (5-HT) enhances hippocampal noradrenaline (NA) release: Evidence for facilitatory 5-HT receptors within the CNS

Summary Slices of rabbit hippocampus were preincubated with ³H-noradrenaline (³H-NA), then superfused continuously in the presence of the noradrenaline (NA) uptake inhibitor (+)oxaprotilin and twice stimulated electrically. The stimulation induced tritium overflow was increased by the 5-HT receptor agonists, 5-HT, 2-methyl-5-HT and 5-carboxamidotryptamine in a concentration dependent manner; a tyramine-like displacement of NA by the 5-HT agonists was prevented by (+)oxaprotilin. The 5-HT M-receptor antagonists, MDL 72222 and ICS 205-930, inhibited the facilitatory effects of 5-HT agonists as well as the enhanced tritium overflow due to the selective 5-HT uptake inhibitor, 6-nitroquipazine: in each case, concentrations much higher than those required to block M-receptors of the periphery were necessary. At high concentrations MDL 72222, in contrast to ICS 205-930, seems to have -adrenoceptor antagonistic activity. The 5-HT₂ receptor antagonist, ketanserin, had no effect on 5-HT-induced facilitation of transmitter release; metitepin facilitated stimulationevoked transmitter release per se both in the absence and presence of phentolamine.From our results we conclude that, as on peripheral nerve endings, also on central noradrenergic terminals, facilitatory 5-HT receptors are present that modulate NA release. The enhanced tritium overflow following 6-nitroquipazine may be due to an increased release of endogenous 5-HT, a suggestion which supports the hypothesis of a physiological innervation of these facilitatory 5-HT receptors on NA terminals.This study was supported by the Deutsche Forschungsgemeinschaft (SFB 70 and 325)