Differential control of Ca2+-dependent [3H]noradrenaline release from rat brain slices through presynaptic opiate receptors and alpha-adrenoceptors

The inhibitory actions of the Ca2+ antagonist Cd2+, morphine and noradrenaline (exogenously added + endogenously released) on electrically evoked release of [3H]noradrenaline from superfused rat neocortical slices were strongly reduced when release was enhanced by 4-aminopyridine. In the presence of 4-aminopyridine the release inhibiting effects of these drugs were restored by lowering the extracellular Ca2+ concentration. When release was enhanced by prolonging the pulse duration, only the release inhibiting effect of noradrenaline was reduced but the effects of Cd2+ and morphine were unchanged. Irrespective of the pulse duration, blockade of presynaptic alpha-adrenoceptors with phentolamine did not affect the release inhibiting effects of Cd2+ and morphine. The inhibitory effects of morphine and noradrenaline remained unchanged in Cl--free medium. Furthermore, these drugs strongly reduced the [3H]noradrenaline release induced by 20 mM K+ in the presence of tetrodotoxin. The results suggest that activation of presynaptic opiate-receptors inhibits Ca2+ entry through voltage-sensitive Ca2+ channels, whereas presynaptic alpha-adrenoceptors affect a step in the secretory process subsequent to Ca2+ influx. Moreover, the involvement of (direct) changes in Na+, K+ or Cl- permeability appears unlikely for both receptor systems.     

Presynaptic GABAB receptors and the regulation of [3H]noradrenaline release from rat anococcygeus muscle

The release of preaccumulated [3H]noradrenaline from the isolated rat anococcygeus muscle, a tissue endowed with a rich noradrenergic innervation, was investigated. Field stimulation produced a marked, calcium-dependent enhancement in release, which could be substantially inhibited by the inclusion of low concentrations of GABA or (-)-baclofen (but not the (+)-isomer) in the bathing medium. Classical GABAA site agonists such as muscimol or 3-aminopropanesulphonic acid were inactive, or only weakly so. Picrotoxin and bicuculline were unable to antagonise the effects of GABA or baclofen on [3H]noradrenaline release, although 5-aminovalerate was effective. These data provide further evidence for the existence of GABAB receptors associated with presynaptic noradrenergic terminals in the rat anococcygeus muscle.     

Paradoxical antagonism by bicuculline of the inhibition by baclofen of the electrically evoked release of [3H]GABA from rat cerebral cortex slices

The presynaptic regulation of the electrically evoked release of [3H]GABA was studied in the rat cerebral cortex. Among the GABA receptor agonists tested (GABA, SL 75102, muscimol, THIP, isoguvacine, (+/-)-baclofen), only (+/-)-baclofen inhibited the stimulation-evoked release of [3H]GABA. This effect of baclofen was stereoselective in favor of the (-) enantiomer. The inhibition by (+/-)-baclofen of the electrically evoked release of [3H]GABA was antagonized by bicuculline and picrotoxin. Our results suggest that the release of [3H]GABA in vitro can be modulated by a receptor-mediated mechanism which is sensitive to baclofen, bicuculline and picrotoxin but not to GABA, muscimol or THIP.     

The purported dopamine agonist DPI inhibits [3H]noradrenaline release from rat cortical slices but not [3H]dopamine and [14C]acetylcholine release from rat striatal slices in-vitro

The effects of the purported dopamine (DA) receptor agonist (3,4-dihydroxyphenylimino)-2-imidazolidine (DPI) upon the in-vitro K+-induced release of [3H]DA and [14C]acetylcholine from rat neostriatal slices, and of [3H]noradrenaline from rat neocortical slices have been investigated and compared with those of the DA receptor agonist TL-99 and the alpha-adrenoceptor agonist clonidine, respectively. The rapid decomposition of the catechol compounds DPI and TL-99 in the Krebs-Ringer bicarbonate superfusion medium was shown to be inhibited by both the chelating agent EDTA and the reducing agent ascorbic acid. The results suggest that in-vitro DPI is unable to stimulate striatal DA receptors, whereas it is effective in stimulating cortical alpha 2-adrenoceptors (EC50 = 61 nM). It is concluded that DPI should be considered as a mixed alpha 1/alpha 2-adrenoceptor agonist and that the designation of DPI as a DA receptor agonist should be abandoned.     

Presynaptic serotonin receptors regulate [3H]serotonin release from rat spinal cord synaptosomes

Superfused rat spinal cord synaptosomes were studied to determine if inhibitory serotonin (5-HT) receptors (autoreceptors) exist on spinal serotonergic nerve terminals. Exogenous 5-HT (1-50 nM) produced a concentration-dependent inhibition of K+-induced [3H]5-HT release but did not affect basal [3H]5-HT release. A 32-44% inhibition was produced by 30 nM 5-HT. The inhibitory effect of 30 nM 5-HT was effectively antagonized by 100 nM metitepine, a 5-HT autoreceptor antagonist. The results provide evidence for the existence of 5-HT autoreceptors in rat spinal cord tissue.     

K+ Concentration influences methohexitone inhibition of K+-stimulated release of [3H]noradrenaline from minislices of rat cerebral cortex

Abstract— This study investigated the effect of methohexitone, a barbiturate anaesthetic, with variation of the K⁺ concentration used to stimulate [³H]noradrenaline release from superfused minislices of rat cerebral cortex. The amount of [³H]noradrenaline released by K⁺-stimulation depended on the concentration of K⁺ used (increasing release was seen at 10–50 Mm K⁺). Methohexitone (10^?7-3 × 10^?4 m ) added to the superfusing medium did not alter the basal efflux of [³H]noradrenaline, but high concentrations inhibited K⁺-stimulated release. Using 12·5 Mm K⁺, inhibition of release was observed only at 10^?4 m methohexitone (48% inhibition); the effect of 25 Mm K⁺ was reduced by 3 × 10^?5 m (72% inhibition) and higher concentrations while release stimulated by 50 Mm K⁺ was inhibited by 10^?5 m methohexitone (30% inhibition) and higher concentrations.     

Inhibitory effect of nociceptin on [3H]-5-HT release from rat cerebral cortex slices.

1. The effect of nociceptin (NC) on 5-hydroxytryptamine (5-HT) release was studied in rat cerebral cortex slices preincubated with [3H]-5-HT and electrically stimulated (3 Hz, for 2 min) at the 45th (St1) and the 75th (St2) min of superfusion. 2. NC (0.1 - 3 microM), present in the medium from the 70th min onward, concentration-dependently reduced electrically evoked [3H]-5-HT efflux (pEC50=6.54, Emax -54%). The inhibition was not antagonized by naloxone (1 microM) ruling out the involvement of opioid receptors. 3. Phe1psi(CH2-NH2)Gly2]NC(1-13)NH2, which acts as an opioid-like receptor (ORL1) antagonist at the peripheral level, behaved as a partial agonist in cerebral cortex slices i.e. it inhibited [3H]-5-HT efflux when added before St2, however, when present in the medium throughout the whole experiment, [Phe1psi(CH2-NH2)Gly2]NC(1-13)NH2 prevented the action of NC added at the 70th min. 4. The non-selective ORL1 receptor antagonist, naloxone benzoylhydrazone (3 microM), in the presence of 10 microM naloxone, did not modify the St2/St1 ratio but completely abolished the NC effect. 5. These findings demonstrate that NC inhibits 5-HT release from rat cerebral cortex slices via ORL1 receptors, suggesting its involvement in central processes mediated by 5-HT.     

Presynaptic alpha7 and non-alpha7 nicotinic acetylcholine receptors modulate [3H]d-aspartate release from rat frontal cortex in vitro

The presynaptic nicotinic modulation of glutamatergic transmission in the CNS has been associated with activation of the alpha7 subtype of nicotinic acetylcholine receptor (nAChR) in sub-cortical regions, whereas in the frontal cortex, non-alpha7 nAChRs have been implicated. The aim of this investigation was to directly characterise nAChR-evoked release of excitatory amino acids from rat frontal cortex, by monitoring the release of [3H]D-aspartate from superfused synaptosomes or minces. Co-administration of a nAChR agonist with a depolarising stimulus enhanced [3H]D-aspartate release above the effect of depolarising agent alone. This enhancement was blocked by the nicotinic antagonist mecamylamine. Other experiments revealed that in the absence of a depolarising stimulus, the nAChR agonists nicotine, epibatidine and anatoxin-a could evoke the release of [3H]D-aspartate in a Ca2+- and concentration-dependant manner. Differential sensitivity to the alpha7- and beta2*-selective nAChR antagonists alpha-bungarotoxin (alpha-Bgt) and dihydro-beta-erythroidine (DHbetaE) implicated two nAChR subtypes (alpha7 and beta2*), and this was supported by using the subtype-selective agonists choline (10 mM; alpha7 selective, blocked by alpha-Bgt but not by DHbetaE) and 5-Iodo-A-85380 (10 nM; beta2*-selective, blocked by DHbetaE but not by alpha-Bgt). Immunocytochemistry showed that alpha-Bgt labelling was associated with structures immunopositive for vesicular glutamate transporters, in both frontal cortex sections and synaptosome preparations, supporting the presence of alpha7 nAChR on glutamatergic terminals in rat frontal cortex.     

Stimulation by vanadate of [3H]noradrenaline release from rabbit pulmonary artery and its inhibition by noradrenaline

Vanadate, the +5 oxidation state of vanadium, present in mammalian tissues, even in nerve tissue, and a competitive inhibitor of NaK-ATPase, significantly enhanced the release of [3H]noradrenaline evoked from rabbit isolated perfused pulmonary artery by electrical stimulation. Its effect proved to be concentration-dependent. Noradrenaline (10(-6) M) reduced the vanadate-potentiated release of [3H]noradrenaline. The effect of noradrenaline is mediated via alpha 2-adrenoceptors as evidenced by the finding that yohimbine 3 x 10(-7) M prevented its action. The effect of vanadate was dependent on external K ions. When the effect of vanadate on [3H]noradrenaline release was studied under conditions when the NaK-ATPase enzyme activity was inhibited by removal of external K for 45 min, vanadate was ineffective. This finding indicates that the effect is related to the inhibition of NaK-ATPase activity, a condition known to result in transmitter release.     

Presynaptic alpha 2-adrenoceptors modulate the release of [3H]noradrenaline from rat spinal cord dorsal horn neurones

Slices of the dorsal half of the rat spinal cord were used to investigate the existence of a noradrenergic feedback modulation of noradrenaline release. After crude preparation of the vertebral column, the spinal cord was ejected by hydraulic pressure and transverse slices were cut. These were preincubated with [3H]noradrenaline during 0.1 Hz electrical stimulation and then superfused and stimulated electrically for two periods. The stimulation-evoked release of [3H]noradrenaline was Ca2+-dependent and tetrodotoxin-sensitive. Pretreatment of the animals with the noradrenergic neurotoxin, DSP-4, reduced the tritium content in the slices and the stimulation-evoked release to less than 10% of the controls. Clonidine (0.01-1 microM) inhibited the evoked overflow by 60% maximally and yohimbine (0.1-1 microM) enhanced it by 160% maximally. The effects of clonidine were antagonized by yohimbine. These results provide evidence that noradrenaline release from spinal cord slices is controlled by an alpha 2-adrenoceptor-mediated, negative feedback mechanism.     

Inhibitory muscarinic receptors modulate the potassium-evoked release of [3H]serotonin from rat hypothalamic slices

The effects of oxotremorine, acetylcholine and nicotine have been investigated on the potassium-evoked release of [³H]serotonin from slices of rat hypothalamus. Oxotremorine and acetylcholine in the presence of physostigmine inhibited potassium-evoked tritium release without affecting the spontaneous release. Nicotine had no effect. The response to oxotremorine was unaffected by tetrodotoxin but was blocked by atropine and hyoscine suggesting that the muscarinic receptor mediating the response to oxotremorine was located on the serotonergic nerve terminal.     

Newly synthesized noradrenaline mediates the alpha 2-adrenoceptor inhibition of [3H]5-hydroxytryptamine release induced by beta-phenylethylamine in rat hippocampal slices

In slices of the rat hippocampus, alpha 2-adrenoceptors located presynaptically on serotonergic nerve terminals modulate the electrically evoked calcium-dependent release of [3H]serotonin [( 3H]5HT). We have investigated the effects of a naturally occurring trace amine, beta-phenylethylamine (beta-PEA), on noradrenergic transmission in the rat hippocampus. Under experimental conditions in which MAO of type B is inhibited by deprenyl-exposure to beta-PEA (0.1-10 microM) facilitates the spontaneous outflow of [3H]noradrenaline and inhibits the electrically evoked release of [3H]5HT. The inhibitory effect of beta-PEA (3 microM) on the evoked release of [3H]5HT was antagonized by the alpha 2-adrenoceptor antagonist idazoxan at 1 microM, and by pretreatment with alpha-methyl-p-tyrosine (alpha-MpT, 300 mg/kg i.p., 2 h). The inhibition of tyrosine hydroxylase activity by alpha-MpT does not modify the inhibition of the evoked release of [3H]5HT by the alpha 2-adrenoceptor agonist, 6-fluoronoradrenaline, or the serotonin receptor agonist, 5-methoxytryptamine. Pretreatment with the neurotoxin DSP4 (50 mg/kg i.p., 10 days) markedly antagonized the inhibitory action of beta-PEA on [3H]5HT release. These results indicate that the noradrenaline-releasing action of beta-PEA inhibits the electrically evoked release of [3H]5HT through the activation of alpha 2-adrenoceptors. This inhibitory effect appears to be mediated exclusively through the release of newly synthesized noradrenaline, and does not involve the direct activation by beta-PEA of the inhibitory 5HT autoreceptors which modulate [3H]5HT release in the rat hippocampus.     

Histamine H3 receptors modulate the release of [3H]-acetylcholine from slices of rat entorhinal cortex: evidence for the possible existence of H3 receptor subtypes.

1. The effect of agents which interact with the histamine H3 receptor on potassium-stimulated tritium release from slices of rat entorhinal cortex preloaded with [3H]-choline is described. We have examined the effects of the selective H3 receptor agonist, (R)-alpha-methylhistamine (RAMH), and a number of H3 receptor antagonists, including the selective compound thioperamide, on the potassium-stimulated release of tritium. 2. In the presence of mepyramine and ranitidine, RAMH (0.01-10 microM) inhibited potassium-stimulated tritium release in a concentration-dependent manner, EC50 = 0.11 microM. The maximum inhibition was approximately 50%. 3. Thioperamide displaced the RAMH concentration-response curve to the right yielding a pKB value of 8.4. There was no change in the maximum response to RAMH. 4. Other H3 receptor antagonists, including impromidine and burimamide, also caused rightwards displacement of the linear portion of the RAMH concentration-response curve. However, phenylbutanoylhistamine and betahistine, which are reported to be relatively potent H3 receptor antagonists, showed very low affinity. 5. Thioperamide (0.001-1 microM) alone enhanced the potassium-stimulated release of tritium in a concentration-dependent manner. Maximum effects were observed at 0.1-1 microM thioperamide, enhancing release by approximately 20%. 6. Results are discussed in terms of the regulatory role of H3 receptors on acetylcholine release and the possible existence of H3 receptor subtypes.     

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.     

Dicyclomine- and pirenzepine-sensitive muscarinic receptors mediate inhibition of [3H]serotonin release in different rat brain areas

The effects of acetylcholine (ACh) on the release of [3H]5-hydroxytryptamine ([3H]5-HT) were investigated in synaptosomes prepared from rat cerebral cortex, hypothalamus and hippocampus and depolarized with 15 mM KCl under superfusion conditions. ACh inhibited the release of [3H]5-HT in all three brain areas. This effect was not modified by hexamethonium but was antagonized by atropine and by the non-classical antagonists pirenzepine and dicyclomine.     

Inhibition of noradrenaline release in the rat brain cortex via presynaptic H3 receptors

Summary The effects of histamine and related drugs on the evoked tritium overflow from superfused rat brain cortex slices preincubated with³H-noradrenaline were determined. Tritium overflow was stimulated electrically (3 Hz; slices superfused with normal physiological salt solution) or by introduction of CaCl₂ 1.3 mmol/l (slices superfused with Ca²⁺-free medium containing K⁺ 20 mmol/l).Histamine slightly decreased the electrically evoked^H overflow in slices superfused in the presence of desipramine. The degree of inhibition obtained with histamine was doubled when both desipramine and phentolamine were present in the superfusion medium (pIC₁₅ 6.46). Under the latter condition, the evoked overflow was inhibited by the H₃ receptor agonist R-(–)--methylhistamine and its S-(+) enantiomer (pIC₁₅ 7.36 and 5.09, respectively), but was not affected by the H₂ receptor agonist dimaprit and the H₁ receptoragonist 2-thiazolylethylamine (both at up to 32 µmol/l). The concentration-response curve of histamine was shifted to the right by the H3 receptor antagonists thioperamide, impromidine and burimamide (apparent pA₂ 8.37, 6.86 and 7.05, respectively), by the H₂ receptor antagonist ranitidine (apparent pA₂ 4.27) and was not affected by the H₁ receptor antagonist dimetindene (32 µmol/l). The inhibitory effect of R-(–)--methylhistamine on the evoked overflow was also counteracted by thioperamide. Given alone, none of the five histamine receptor antagonists affected the evoked overflow. In the absence of desipramine plus phentolamine, impromidine and burimamide facilitated the electrically evoked³H overflow whereas thioperamide had no effect. The facilitatory effects of impromidine and burimamide were abolished by phentolamine, but not affected by desipramine. The concentration-response curve of noradrenaline for its inhibitory effect on the evoked overflow was shifted to the right by impromidine and burimamide, but not influenced by thioperamide (apparent pA₂ 5.24, 5.04 and <6.5, respectively; experiments carried out in the presence of desipramine). In slices superfused with Ca²⁺-free K⁺-rich medium containing tetrodotoxin, desipramine plus phentolamine, the tritium overflow evoked by introduction of Ca²⁺ was inhibited by histamine; the concentration-response curve of histamine was shifted to the right by thioperamide.The present study shows that the inhibitory effect of histamine on noradrenaline release in the rat brain cortex involves presynaptic H₃ receptors and that the degree of inhibition is increased in the presence of phentolamine. The H₃ receptor antagonists impromidine and burimamide are weak ₂-adrenoceptor antagonists. Send offprint requests to E. Schlicker at the above address     

Gamma-aminobutyric acid enhancement of potassium-stimulated release of [3H]norepinephrine by multiple mechanisms in rat cortical slices

Gamma-Aminobutyric acid (GABA) and GABAA agonists enhance stimulated release of [3H]norepinephrine [( 3H]NA) in several regions of the rat brain. In this study, the mechanisms by which GABA and GABAergic agonists augment potassium-stimulated release of [3H]NA from rat frontal cortical slices were examined. GABA enhanced potassium-stimulated [3H]NA release, but did not alter release of [3H]NA evoked by the calcium ionophore A23187, 10(-5) M, either in the presence or the absence of extracellular calcium. The effect of GABA on potassium-stimulated [3H]NA release was apparently reduced by the GABAA antagonist bicuculline methiodide, 10(-4) M, and by the selective inhibitor of GABA uptake SKF 89976A, 10(-5) M, but was abolished only when bicuculline methiodide and SKF 89976A were present in combination. The GABAA agonist muscimol enhanced potassium-stimulated release of [3H]NA in a manner similar to GABA. In addition, nipecotic acid, a substrate for GABA uptake, enhanced potassium-stimulated [3H]NA release. Thus, GABA appears to enhance potassium-stimulated [3H]NA release by acting upon both GABA uptake and GABAA receptors. The GABAA receptors involved in this effect may be a subtype of GABAA receptors since they are not modulated by benzodiazepines. These results support the involvement of the GABA uptake carrier and the GABAA receptor in mediating the enhancement by GABA of potassium-stimulated [3H]NA release in the cortex of the rat.