Inhibition of N-methyl-d-aspartate- and kainate-evoked noradrenaline release in human cerebral cortex slices by ethanol

Summary The effect of ethanol on the release of noradrenaline evoked by various stimuli was investigated in human cerebral cortical slices from patients undergoing neurosurgery. The slices were preincubated with [³H]noradrenaline and then superfused. Tritium overflow was stimulated by exposure to N-methyl-d-aspartate (NMDA; in slices superfused without Mg²⁺), kainic acid, veratridine or by increasing the K⁺ concentration.The NMDA evoked tritium overflow was concentration-dependently inhibited by ethanol; an inhibition by 37% occurred at 48 mmol/l ethanol. This ethanol concentration was not yet effective when kainic acid was used for stimulation, but ethanol 150 mmol/l strongly inhibited the tritium overflow evoked by kainic acid as well. The tritium overflow evoked by veratridine or high K⁺ was not affected by ethanol in the concentration range investigated.These findings are compatible with the suggestion that the NMDA receptor and, with less susceptibility, the kainate receptor are sites of action underlying the effect of ethanol in the human brain.Send offprint requests to M. Göthert at the above address     

Effect of metavanadate on the uptake and release of noradrenaline in rat brain cerebral cortex slices

The effect of vanadium (as VO3-) on the uptake and release of tritiated noradrenaline ([3H]NA) was studied in vitro in rat cerebral cortex slices. Vanadate inhibited [3H]NA uptake and the inhibition was dependent upon concentration and on incubation time. The IC50 value (20 min incubation) was 8 X 10(-5) M of vanadate. Inhibition of Na+, K+ -ATPase activity by VO3-, chelation of noradrenaline or autooxidation of catecholamine by this oxyanion might contribute to the decrease of [3H]NA uptake. Vanadate inhibited also the release of [3H]NA in a time- and concentration-dependent fashion.     

Studies on the mechanism of histamine-induced release of noradrenaline and 5-hydroxytryptamine from slices of rat cerebral cortex

The effect of histamine on the release of endogenous noradrenaline and 5-hydroxytryptamine (5-HT) has been examined in slices of rat cerebral cortex. Histamine was found to produce a marked release of both amines from rat cerebral cortex at concentrations between 0.1 and 1 mM. This response to histamine was relatively resistant to removal of calcium ions from the incubation medium when compared to the release evoked by depolarising potassium stimuli. The response to 1 mM histamine was not, however, significantly inhibited by the H1-antagonist mepyramine (1 microM) or the H2-antagonist cimetidine (100 microM). Furthermore, impromidine which is both a potent H2-agonist and a potent H3-antagonist was without effect on the basal and histamine-stimulated release of endogenous noradrenaline and 5-HT. The response to histamine was, however, significantly attenuated by nisoxetine, fluoxetine and imipramine which are inhibitors of the amine uptake systems. The results of this study show that high concentrations (0.1 to 1 mM) of histamine can produce a marked increase in the release of endogenous 5-HT and noradrenaline from rat cerebral cortex, apparently via a non-receptor mechanism. This effect will need to be borne in mind in interpreting biochemical and behavioral responses to histamine in this concentration range.     

Experimental conditions required for the enhancement by alpha-adrenoceptor antagonists of noradrenaline release in the rabbit ear artery.

1 Segments of the rabbit ear artery were preincubated with (-)-[3H]-noradrenaline and then perfused/superfused and stimulated by transmural electrical pulses. The outflow of [3H]-noradrenaline, separated from its metabolites by column chromatography, was determined. 2 Tetrodotoxin abolished, cocaine increased, and clonidine reduced the overflow of [3H]-noradrenaline elicited by 10 shocks at 0.2 Hz, 10 shocks at 2 Hz or 100 shocks at 2 Hz. 3 The effects of yohimbine (0.1 or 1 microM), phentolamine (1 microM) and piperoxan (1 or 10 microM) depended on the stimulation conditions. No antagonist increased the overflow of [3H]-noradrenaline evoked by 10 pulses at 0.2 Hz, but all markedly increased the overflow evoked by 100 pulses at 2 Hz. Only piperoxan (10 microM) slightly enhanced the overflow at 10 pulses, 2 Hz. The effects of yohimbine and piperoxan were similar in arteries not exposed to cocaine and in those that were perfused/superfused with medium containing cocaine (10 microM) after preincubation. 4 It is concluded that yohimbine, phentolamine and piperoxan increase the release of noradrenaline only when the concentration of noradrenaline in the biophase of the ear artery is sufficiently high. The effect is, hence, an anti-noradrenaline effect and due to the blockade of presynaptic alpha-adrenoceptors. A second prerequisite for the release-enhancing effect appears to be a sufficient length of the pulse train, indicating that the alpha-adrenergic auto-inhibition develops relatively slowly.     

Forskolin and the release of noradrenaline in cerebrocortical slices

Summary The role of adenosine 3,5-cyclic monophosphate (cAMP) in the release of noradrenaline from central neurones has been investigated by examining the effects of forskolin, 3-isobutyl-1-methylxanthine (IBMX), cis-6-(p-acetamidophenyl)-1,2,3,4,4a,10b-hexahydro-8,9-dimethoxy-2-methylbenzo[c] [1,6]-naphthyridine bis (hydrogenmaleinate) (AH21-132; a new phosphodiesterase inhibitor) and N⁶, O²-dibutyryl-adenosine 3,5-cyclic monophosphate (dibutyryl-cAMP) on the outflow of tritiated compounds from rat and rabbit cerebral cortex slices preincubated with [³H]-noradrenaline. Forskolin, IBMX, AH21-132 and dibutyryl-cAMP produced a concentration-dependent increase in both basal and electrically-evoked efflux of tritium from rat and rabbit cortex slices. The increase in basal tritium efflux from rabbit cortex slices elicited by forskolin and IBMX could be attributed mainly to an increase in [³H]-DOPEG although a small increase in [³H]-noradrenaline was also observed. Forskolin and (when combined with noradrenaline) IBMX and AH21-132 increased the cAMP content of rat cortex slices at similar or somewhat higher concentrations that they increased tritium efflux. Neither forskolin nor IBMX or AH21-132 had any effect on the cocaine-sensitive uptake of [³H]-noradrenaline into synaptosomes prepared from rat or rabbit cortex. The effects of forskolin, IBMX and dibutyryl-cAMP on electrically-evoked overflow of tritium from rat and rabbit cortex slices were reduced when cocaine (10 M) was present in the superfusion medium, although forskolin produced a similar increase in cAMP in the absence or presence of cocaine. It is suggested that cAMP may facilitate the normal process of noradrenaline release by nerve stimulation.     

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.     

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.     

Quantitative studies on some antagonists of N-methyl D-aspartate in slices of rat cerebral cortex.

Coronal sections of rat brain (500 micron thick) were trimmed to form 'wedges' of tissue consisting of cerebral cortex and corpus callosum. When these slices were placed in a two-compartment bath, the cortical tissue could be depolarized, relative to the corpus callosum, by superfusions of high K+, or by amino acids such as L-glutamate, L-aspartate, quisqualate, kainate and N-methyl D-aspartate (NMDA). Responses to NMDA were reduced by magnesium ions, by the organic antagonists (-)-2-amino 5-phosphonovalerate (APV) and 2-amino 7-phosphonoheptanoate (APH), and by the dissociative anaesthetic ketamine. In this preparation, all these antagonists shifted the NMDA dose-response curve to the right in a parallel manner. A Schild plot for Mg2+ had a slope significantly less than unity, indicative of a non-competitive action, whilst Schild plots for (-)-APV, APH and ketamine appeared linear and had slopes of approximately 1. Analysis of the results of combination experiments suggested that the presumed competitive antagonists, (-)-APV and APH, share a common site of action as NMDA antagonists, and that this site is distinct from that at which ketamine exerts its action. The action of Mg2+ is clearly different from that of either (-)-APV or ketamine. It is concluded that ketamine is a non-competitive antagonist of NMDA and may act at an allosteric site on the NMDA receptor complex to influence its function.     

Presynaptic alpha-adrenoceptors and opiate receptors: inhibition of [3H]noradrenaline release from rat cerebral cortex slices by different mechanisms

The inhibitory effects of morphine and Cd2+ on electrically evoked [3H]noradrenaline release from superfused brain slices were unaffected when release was enhanced by increasing the pulse duration, while the inhibitory effect of noradrenaline and the enhancing effect of phentolamine were diminished. A similar enhancement of [3H]noradrenaline release by 4-aminopyridine reduced the modulatory effects of all drugs examined. Therefore there seem to be different mechanisms for the effect of presynaptic alpha-adrenoceptors and opiate receptors on the availability of Ca2+ for the stimulus-secretion coupling process in noradrenergic nerve terminals.     

Enhancement of noradrenaline release from rat cerebral cortex by neuroleptic drugs

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

Mechanism of nicotine-evoked release of 3H-noradrenaline in human cerebral cortex slices

1. The mechanism of stimulation of noradrenaline (NA) release by nicotine (NIC) was investigated in human cerebral cortex slices preloaded with 3H-noradrenaline. 2 NIC (10-1000 micro M) increased 3H-NA release in a concentration-dependent manner. 3. NIC (100 micro M)-evoked 3H-NA release was largely dependent on external Ca2+, and was attenuated by omega-conotoxin GVIA (0.1 micro M) but not by nitrendipine (1 micro M). 4. Tetrodotoxin (1 micro M) and nisoxetine (0.1 micro M) attenuated the NIC (100 micro M)-evoked release of 3H-NA. 5. Mecamylamine (10 micro M), dihydro-beta-erythroidine (10 micro M) and d-tubocurarine (30 micro M), but not alpha-bungarotoxin (alpha-BTX, 0.1 micro M), attenuated the NIC (100 micro M)-evoked release of 3H-NA. 6. NIC (100 micro M)-evoked release of 3H-NA was not affected by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 30 micro M) and D(-)-2-amino-5-phosphonopentanoic acid (D-AP5, 100 micro M), but attenuated by MK-801 (10 micro M). MK-801 (0.1-1000 micro M) displaced the specific binding of 3H-nisoxetine with K(i) values of 91.2 micro M. NIC (100, 300 and 1000 micro M) did not induce 3H-D-aspartate release in human cerebral cortex slices. 7. NIC (100 micro M)-evoked release of 3H-NA was attenuated by 7-nitroindazole (10 micro M), N(G)-nitro-L-arginine methyl ester HCl (L-NAME, 30 micro M), N(G)-monomethyl-L-arginine acetate (L-NMMA, 300 micro M). [(3)H]-NA release induced by NIC (100 micro M) was attenuated by methylene blue (3 micro M) and 1H-[1,2,4]oxadiazole[4,3-alpha]quinoxalin-1-one (ODQ, 10 micro M), and enhanced by zaprinast (30 micro M). 8. In conclusion, NIC stimulates the release of 3H-NA through activation of alpha-BTX-insensitive nicotinic acetylcholine receptors in the human cerebral cortex slices and this action of NIC is associated with modulation of the NO/cGMP pathway.     

Transmitter release modulated by alpha-adrenoceptor antagonists in the rabbit mesenteric artery: a comparison between noradrenaline outflow and electrical activity.

Effects of alpha-adrenoceptor blockers (prazosin, yohimbine, phentolamine and phenoxybenzamine) on the outflow of noradrenaline (NA) and 3,4-dihydroxyphenylglycol (DOPEG) during perivascular nerve stimulation were observed in relation to electrical events in the rabbit mesenteric artery. Cocaine or imipramine increased the NA outflow and reduced the DOPEG outflow induced by nerve stimulation. In the absence of stimulation, cocaine and imipramine did not significantly modify the NA and DOPEG outflows. The alpha-adrenoceptor blockers we used enhanced the NA and DOPEG outflow during nerve stimulation, in a dose-dependent manner; the potency of the enhancement was higher for phentolamine and phenoxybenzamine than for prazosin and yohimbine. Higher concentrations (10(-5) M) of yohimbine reduced the NA and DOPEG outflows induced by nerve stimulation. Prazosin increased the DOPEG outflow in the absence of stimulation, and this effect was not inhibited by pretreatment with cocaine. Guanethidine increased the NA and DOPEG outflow in the absence of stimulation, and the NA outflow was reduced during nerve stimulation. These effects of guanethidine were prevented by pretreatment with cocaine or imipramine. Perivascular nerve stimulation evoked excitatory junction potentials (e.j.ps) and with high frequency stimulation, slow depolarization and spike potentials. Application of phentolamine, phenoxybenzamine or yohimbine enhanced, and of prazosin had no effect, on the amplitude of the e.j.p. Spike potentials were not affected by these alpha-adrenoceptor blockers. Slow depolarization ceased in the presence of prazosin, phentolamine or phenoxybenzamine, and was slightly inhibited by yohimbine. Guanethidine blocked all of these electrical responses induced by perivascular nerve stimulation. Application of prazosin, phentolamine or phenoxybenzamine did not alter the resting membrane potential of the smooth muscle cells. Depolarizations of smooth muscle membrane produced by exogenously applied NA were inhibited by prazosin, phentolamine or phenoxybenzamine. Yohimbine itself depolarized the membrane and the inhibitory effects on the NA-induced depolarization were weaker. We conclude that the smooth muscle membrane of the rabbit mesenteric artery possesses alpha 1-adrenoceptors. Increase in NA outflow by alpha-adrenoceptor antagonists during nerve stimulation was not always consistent with increase in e.j.p. amplitude, presumably due to involvement of actions other than alpha-adrenoceptor blockade with each of these antagonists.     

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.     

Adrenaline hypothesis: effect of formoterol on noradrenaline release

1 Originally, the so-called 'adrenaline hypothesis' related the release of noradrenaline (NA) to stimulation of presynaptic beta2-receptors in nerve endings; now it confers a possible role to adrenaline taken up then released by nerves endings. It represents a potentially useful therapeutic pathway. The present study aims to investigate the effects of formoterol, a highly selective beta2-adrenoceptor agonist. 2 It was carried out in freely moving rats, the isotope dilution technique being used to measure the NA spill over rate (NA-SOR) and metabolic clearance rate (MCR). 3 A series of three results are reported. (a) When compared with adrenaline on equimolar basis, formoterol (2.3 micro kg-1 min-1) increased NA-SOR while mean arterial blood pressure was decreased and heart rate increased. Thus, it was difficult to separate a direct presynaptic effect from indirect baroreflex-dependent activation of the sympathetic system. (b) When formoterol was infused at 1 ng kg-1 min-1, a dose empirically defined to induce no haemodynamic effect, NA-SOR was significantly increased, while NA-MCR remained unchanged. (c) The NA-SOR response to formoterol was not amplified by the presynaptic alpha2-adrenoceptor blocker, yohimbine, in contrast to the NA-SOR response to adrenaline. 4 In conclusion, formoterol, a beta2-adrenoceptor agonist, is shown to increase the release and plasma concentration of NA while its clearance was not changed.     

Adrenaline hypothesis: effect of formoterol on noradrenaline release

1 Originally, the so‐called `adrenaline hypothesis' related the release of noradrenaline (NA) to stimulation of presynaptic β₂‐receptors in nerve endings; now it confers a possible role to adrenaline taken up then released by nerves endings. It represents a potentially useful therapeutic pathway. The present study aims to investigate the effects of formoterol, a highly selective β₂‐adrenoceptor agonist. 2 It was carried out in freely moving rats, the isotope dilution technique being used to measure the NA spill over rate (NA‐SOR) and metabolic clearance rate (MCR). 3 A series of three results are reported. (a) When compared with adrenaline on equimolar basis, formoterol (2.3 μg kg^?1 min^?1) increased NA‐SOR while mean arterial blood pressure was decreased and heart rate increased. Thus, it was difficult to separate a direct presynaptic effect from indirect baroreflex‐dependent activation of the sympathetic system. (b) When formoterol was infused at 1 ng kg^?1 min^?1, a dose empirically defined to induce no haemodynamic effect, NA‐SOR was significantly increased, while NA‐MCR remained unchanged. (c) The NA‐SOR response to formoterol was not amplified by the presynaptic α₂‐adrenoceptor blocker, yohimbine, in contrast to the NA‐SOR response to adrenaline. 4 In conclusion, formoterol, a β₂‐adrenoceptor agonist, is shown to increase the release and plasma concentration of NA while its clearance was not changed.