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.     

UTP- and ATP-triggered transmitter release from rat sympathetic neurones via separate receptors.

In rat cultured sympathetic neurones, UDP, UTP and ATP at micromolar concentrations triggered Ca(2+)-dependent and tetrodotoxin-sensitive [3H]-noradrenaline release. The overflow evoked by UTP or ATP was similar at 100 mumol l-1, the concentration used in all subsequent experiments. Pre-exposure of the neurones to 100 mumol l-1 UTP significantly reduced ensuing secretory effects of UTP but not of ATP. Conversely, pre-exposure to ATP diminished the overflow due to ATP but not that due to UTP. In the presence of 10 mumol l-1 pyridoxal-5'-phosphate or 30 mumol l-1 suramin, the secretory response to ATP was reduced, but the effect of UTP was unaltered. Zn2+ (10 mumol l-1) reduced the overflow triggered by UTP, but increased the overflow due to ATP. These results indicate the presence of separate receptors for pyrimidine nucleotides and for purine nucleotides which both trigger transmitter release.     

Interactions of methylenedioxymethamphetamine with monoamine transmitter release mechanisms in rat brain slices

Summary This study investigates the effects of methylenedioxymethamphetamine (MDMA) and amphetamine on monoamine release from rat superfused brain slices in both the presence and absence of vesicular stores of transmitter. MDMA caused the release of radioactivity from slices incubated with [³H]5-hydroxytryptamine, [³H]noradrenaline or [³H]dopamine with EC₅₀ values of 1.9 mol/l (95% confidence limits 1.5–2.3 mol/l), 4.5 mol/l (2.3–8.7 mol/l), and greater than 30 mol/l, respectively. In contrast, amphetamine (0.1–300 mol/l) was more effective in releasing radioactivity from slices incubated with [³H]dopamine than [³H]noradrenaline or [³H]5-hydroxytryptamine. When Ca²⁺ was excluded from the superfusion fluid, the MDMA induced release of radioactivity from slices incubated with [³H]dopamine was unaltered, but that from slices incubated with [³H]noradrenaline or [³H]5-hydroxytryptamine was enhanced. MDMA (10 mol/l) facilitated the stimulation-induced (5 Hz, 1 min) outflow of radioactivity from slices incubated with [³H]noradrenaline or [³H]5-hydroxytryptamine to 7.5-fold and 2.1-fold of control values, respectively, but had no effect on that from slices incubated with [³H]dopamine. Amphetamine (1 mol/l) increased the stimulation-induced outflow from slices incubated with [³H]noradrenaline, but not that from slices incubated with [³H]5-hydroxytryptamine or [³H]dopamine.Inhibition of monoamine oxidase by a 30-min incubation with pargyline (100 mol/l) enhanced the releasing action of MDMA on all three monoamines. Pargyline (100 mol/l) also enhanced the facilitation caused by MDMA, of the stimulation-induced outflow of radioactivity from slices incubated with [³H]noradrenaline, [³H]5-hydroxytryptamine or [³H]dopamine.In some experiments, slices were obtained from reserpinised rats (2.5 mg/kg s.c. 24 h prior) and pre-exposed for 30 min to the monoamine oxidase inhibitor pargyline (100 mol/l). Under these conditions, electrical stimulation evoked a small residual stimulation-induced outflow of radioactivity from slices incubated with [³H]noradrenaline, and failed to evoke an outflow of radioactivity from slices incubated with [³H]5-hydroxytryptamine or [³H]dopamine. However, a Ca²⁺-dependent stimulation-induced outflow of radioactivity was evoked in the presence of either MDMA (10 mol/l) or amphetamine (1 mol/l) from slices incubated with either [³H]dopamine or [³H]noradrenaline, but not from slices incubated with [³H]5-hydroxytryptamine. The stimulation-induced outflow of radioactivity from slices incubated with [³H]noradrenaline was enhanced in the presence of desipramine (1 mol/l), however this enhancement was less than that caused by 10 mol/l MDMA or 1 mol/l amphetamine. The Ca²⁺-dependent response to electrical stimulation in the presence of MDMA from slices incubated with [³H]noradrenaline was greatly reduced when rats were pretreated with a higher dose of reserpine (10 mg/kg s.c.).This study demonstrates that MDMA and amphetamine release radioactivity from brain slices incubated with [³H]noradrenaline, [³H]dopamine and [³H]5-hydroxytryptamine, but their order or potency as releasers of brain monoamines differs. The results also suggest that MDMA and amphetamine have significant effects on the exocytotic release of monoamines and may interact with both vesicular and cytoplasmic monoamines. Correspondence to J. J. Reid at the above address     

REM sleep and central monoamine oxidase inhibition

Our preliminary observations on the relationship of sleep and lumbar CSF acid monoamine metabolite levels suggested a greater decrease in lumbar CSF HVA as compared to 5HIAA following clinical doses of phenelzine in the presence of virtually total REM suppression. This report on nine psychiatric patients confirms these findings in a larger sample and thus supports an inhibitory role for dopamine or other catecholamines in REM sleep mechanisms. The drug-withdrawal results indicate that the four patients with REM rebound showed increases in HVA levels compared to treatment levels, while the single patient with no REM rebound also had no increase in HVA levels.This investigation was supported in part by Research Scientist Development Award KO1 MH 70012 to D.J.K. and U.S.P.H.S. grant MH 12873. We wish to thank Angelica Rozitis, Amanda Smith, Philip Rogers and Richard McPartland for technical assistance and the nursing staff of the research unit, Connecticut Mental Health Center, whose invaluable and consistent help made this study possible.     

Involvement of nitric oxide in clonidine-induced spinal analgesia

BACKGROUND: The chronic pain relieving effects following spinal administration of clonidine are probably connected to alpha2-adrenoreceptor-induced augmented synthesis of nitric oxide (NO) in the spinal cord. In contrast, when acute pain is considered, the possible role of NO is still speculative. The aim of the present study was to explore the role of NO in acute pain relief following intraspinal administration of clonidine. METHODS: We used the mouse tail-flick model of acute pain. Spinal injections of the following agents and their combinations were administered: clonidine, L-arginine (NO precursor), the NO production inhibitor nitro-L-arginine-methyl ester (L-NAME), the NO antagonist methylene blue (MB) and nitroglycerine (NO releasing agent). RESULTS: A 95% analgesic response was achieved with 2.0 microg clonidine. L-Arginine produced analgesia, and L-arginine administration followed by clonidine resulted in a pronounced synergistic analgesic effect. This synergistic effect was attenuated by L-NAME. Pre-treatment with MB decreased and nitroglycerine administration did not affect the clonidine-induced analgesia. CONCLUSIONS: NO may be involved in the mediation of the acute pain relieving effects of intraspinally administered clonidine. Further research is warranted to establish the potential benefits and possibility for incorporation of NO promoting agents in therapeutic regional pain regimens.     

Possible involvement of central adrenergic and histaminergic systems in clonidine-induced inhibition of pinna reflex

Clonidine produced dose-dependent inhibition of pinna reflex in rats, the ED50 being 1.20 +/- 0.28 mg/kg. The effect of agents affecting adrenergic, tryptaminergic and histaminergic systems were evaluated on clonidine-induced inhibition of pinna reflex. All these agents had no effect on pinna reflex. Phentolamine, phenoxybenzamine, prazosin, propranolol, haloperidol, cyproheptadine and mepyramine did not affect significantly the action of clonidine. However, reserpine, alpha-methyl-p-tyrosine, alpha-methyldopa, diethyldithiocarbamate, 6-hydroxydopamine, yohimbine and cimetidine significantly blocked clonidine-induced inhibition of pinna reflex, indicating the involvement of central adrenergic-histaminergic systems.     

Role of monoamine oxidase inhibition and monoamine depletion in fenfluramine-induced neurotoxicity and serotonin release

The role of both monoamine synthesis and monoamine oxidase inhibition in mediating the fenfluramine-induced damage to serotonin neurones was examined; as pretreatment agents, both alpha-methyl-para-tyrosine (AMPT) and parachlorophenylalanine (PCPA) were used to deplete dopamine and serotonin, respectively, while clorgyline and deprenyl were used to inhibit monoamine oxidase types A and B. While both AMPT and deprenyl did not alter fenfluramine induced serotonin or 5-hydroxyindoleacetic acid (5-HIAA) depletion in any area, PCPA did partially reduce the serotonin depletion in the hippocampus and hypothalamus. Although pretreatment with clorgyline did not significantly alter fenfluramine-induced serotonin depletion, it did produce a 65% mortality rate in animals treated with both drugs. Both PCPA and clorgyline significantly increased the depletion of striatal 5-HIAA concentration consequent to fenfluramine; however, these drugs also produced a long-term depletion of striatal 5-HIAA when administered alone, therefore, the changes seen after the coadministration with fenfluramine may be viewed as additive. Finally, acute PCPA pretreatment attenuated the rapid rise in 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (homovanillic acid) induced by fenfluramine, and acute clorgyline reversed the drop in serotonin and rise in 5-HIAA induced by fenfluramine. These results indicate that the rapid increase in dopamine activity induced by fenfluramine is partially dependent on serotonin concentration and release and that the mechanism of fenfluramine-induced toxicity is unlike that of the other substituted amphetamines.     

单胺转运蛋白与单胺重摄取抑制剂研究进展

5-羟色胺转运蛋白(serotonin transporter,SERT)和去甲肾上腺素转运蛋白(norepinephrine transporter,NET)是单胺类神经递质转运体,其功能是将释放到突触间隙的5-羟色胺(serotonin,5-HT)和去甲肾上腺素(norepinephrine,NE)分别转运入突触前神经细胞,以终止相应的突触信号传递。SERT、NET抑制剂可阻断5-HT和NE的重摄取,提高突触间隙单胺递质水平,从而发挥抗抑郁效应。SERT、NET作为主流抗抑郁药物的作用靶标,了解其分布与功能、分子结构和活性调节因素,以及单胺重摄取抑制剂的作用机制对抗抑郁药物研发及应用具有重要意义。     

Tyrosine hydroxylase activation and transmitter release from central noradrenergic neurons by electrical field stimulation

Summary Electrical stimulation of the noradrenergic neurons in the locus coeruleus of the rat results in a marked increase in the tyrosine hydroxylase activity of the hippocampus on the stimulated side (Roth et al., 1975). We have developed an in vitro system to further study this interesting phenomenon. Rat hippocampal slices were stimulated in an electrical field using specially built chambers which permitted a continous superfusion with Krebs Ringer Phosphate solution while stimulation was taking place. The slices were then homogenized and tyrosine hydroxylase activity and kinetic parameters were determined in the 104000 g supernate fraction. Electrical field stimulation (60 V, 4 ms, 5–20 Hz, 5 min) induced a stimulus-dependent increase in tyrosine hydroxylase activity. The increase in tyrosine hydroxylase activity was directly dependent on the number of pulses of stimulation applied. Potassium depolarization of hippocampal slices also resulted in a marked increase in the activity of the enzyme. Electrical field stimulation appears to activate tyrosine hydroxylase by causing an increase in its affinity for both substrate and pteridine cofactor and by decreasing its affinity for the endproduct inhibitor, norepinephrine. No change in V_max was observed. The superfusion system was also used to study spontaneous and electrically evoked release of labelled norepinephrine from hippocampal slices. Electrical field stimulation for 1.0 min produced a marked increase in the release of label. Absence of calcium from the superfusion solution almost completely abolished the electrically evoked release of exogenously taken up ³H-norepinephrine. The release of ³H-norepinephrine was also found to be dependent on the number of pulses of stimulation used. The results reported support the hypothesis that depolarization of central noradrenergic neurons results in an increase in transmitter synthesis mediated in part via a kinetic activation of tyrosine hydroxylase. The concomitant use of superfusion and electrical field stimulation of hippocampal slices provides a simple system to study neurotransmitter synthesis and release in central noradrenergic neurons.A preliminary account of this work was reported at the eight Meeting of the Argentinian Society for Experimental Pharmacology held November 14–18, 1975, at San Luis, Argentina     

Involvement of different calcium channels in the depolarization-evoked release of noradrenaline from sympathetic neurones in rabbit carotid artery

The calcium channels coupled to noradrenaline release from sympathetic neurones in the rabbit isolated carotid artery were examined. Rings of carotid artery were preloaded with (-)-[(3)H]noradrenaline and the fractional (3)H overflow evoked by electrical-field stimulation was determined by liquid scintillation spectrometry. The N-type Ca(2+) channel blocking agent omega-conotoxin GVIA (3x10(-9)-6x10(-8) M) reduced the stimulation-evoked (3)H overflow. The maximal inhibition was seen with 3x10(-8) M. The maximal reduction was more marked at a low (2 Hz) stimulation frequency than at a high one (30 Hz). Mibefradil (10(-6) M) irreversibly reduced the (3)H overflow evoked by field stimulation (2 Hz). At 30 Hz, the reduction was more marked than at 2 Hz. Mibefradil (3x10(-6)-10(-5) M) enhanced the passive (3)H outflow. The reduction of the stimulation (30 Hz)-evoked (3)H overflow seen with omega-conotoxin GVIA (3x10(-8) M) was enhanced by mibefradil (10(-6) M) and unaffected by nimodipine (10(-5) M) and omega-agatoxin IVA (10(-8) M). We conclude that the stimulation-evoked release of noradrenaline from sympathetic neurones in rabbit carotid artery at a low frequency (2 Hz) is mediated mainly by the N-type calcium channels. At a high frequency (30 Hz), T-type Ca(2+) channels are also involved.     

用在体脑微透析技术研究大鼠纹状体中单胺递质的释放和代谢

采用脑微透析技术与高效液相色谱-电化学检测器联用测定了清醒自由活动大鼠纹状体细胞外液中多巴胺(DA)及其酸性代谢物3,4-二羟苯乙酸(DOPAC)和高香草酸(HVA)以及5-羟色胺代谢物5-羟吲哚乙酸(5-HIAA)的含量.透析液中DA为0.44 pmol/40μl,DOPAC和HVA含量较DA高约80倍.右旋苯丙胺2 mg/kg,ip使纹状体DA释放显著增加,DOPAC和HVA含量明显下降.     

A-23187 evoked transmitter release from rabbit pulmonary artery and its inhibition by reactivation of sodium-pump

1. The spontaneous [3H]-release has been measured from the isolated main pulmonary artery of the rabbit preloaded with [3H]noradrenaline in the presence of uptake blockers (cocaine, 3 x 10(-5) M; corticosterone, 5 x 10(-5) M). 2. The Ca-ionophore A-23187 (3 x 10(-7)-3 x 10(-5) M) increased the outflow of [3H] by a concentration dependent manner. 3. Inhibition of Na+-pump by removal of K+ from the external medium also increased the release of labelled noradrenaline. 4. In the absence of external K+, the applied A-23187 (3 x 10(-6) M; EC50) further increased the release of [3H]. 5. Reactivation of Na+-pump by readmission of K+ (5.9 mM) to the external medium abolished the [3H]-release which had previously been increased in "K+-free" solution. 6. The reactivated Na+-pump significantly inhibited the transmitter releasing action of A-23187. 7. This latter was antagonized by an increase of external Ca2+ (7.5 mM). 8. It is concluded that the reactivated Na+-pump caused re-establishment of Na+-gradient is capable to counteract the Ca-ionophore facilitated Ca2+-influx and release from internal stores, which can be antagonized by excess Ca2+.     

Presynaptic noradrenergic alpha-receptors and modulation of 3H-noradrenaline release from rat brain synaptosomes.

The depolarization (15 mM K+)-induced release of 3H-NA from superfused rat brain synaptosomes and the effects of alpha-noradrenergic drugs thereon were studied. Noradrenaline (NA; in the presence of the uptake inhibitor desipramine) reduced synaptosomal 3H-NA release. Reduction of the concentration of calcium ions in the medium during K+ stimulation greatly enhanced the sensitivity of 3H-NA release to alpha-receptor-mediated inhibition. Under these conditions NA dose-dependently inhibited 3H-NA release from synaptosomes obtained from cortex or hypothalamus, but did not affect 3H-NA release from striatal (i.e dopaminergic) synaptosomes. Adrenaline, clonidine and oxymetazoline potently inhibited 3H-NA release from cortex synaptosomes at concentrations in the nanomolar range. Phentolamine by itself did not affect synaptosomal 3H-NA release, but antagonized the inhibitory effects of both noradrenaline and adrenaline. The data obtained further substantiate the hypothesis that the alpha-receptors mediating a local negative feedback control of NA release are localized on the varicosities of central noradrenergic neurons, Furthermore, noradrenergic nerve terminals in the hypothalamus appear to be less senstive to alpha-receptor-mediated presynaptic inhibition than those in the cortex.     

DL-5-hydroxytryptophan-induced changes in central monoamine neurons after peripheral decarboxylase inhibition

The histochemical effects of 500 and 1000 mg/kg of dl -5-hydroxy-tryptophan (5-HTP), both alone and in combination with a peripheral decarboxylase inhibitor (seryl-trihydroxy benzyl hydrazine; Ro 4–4602) have been examined on central monoamine neurons of rats by the Falck-Hillarp fluorescence technique that demonstrates monoamines and their precursors. 5-HTP alone or together with Ro 4–4602 caused only weak intraneuronal accumulation of 5-HT in the central 5-HT neurons, in spite of an increased entry of 5-HTP into the brain after Ro 4–4602 treatment, as shown by an increase in the specific neuropil fluorescence and a reduction of 5-HT accumulation in the cells of the capillary walls. Ro 4–4602 markedly potentiated the effects of 5-HTP on the central dopamine neurons, many of which became clearly yellow fluorescent. The mechanism of dopamine depletion by 5-HTP is probably therefore mainly one of displacement. The effects on the noradrenaline neurons were also potentiated by Ro 4–4602 pretreatment, the neurons exhibiting a yellow-green fluorescence. This depletion may therefore also be mainly be due to amine displacement. It is concluded that the ability of the 5-HT neurons to take up and accumulate 5-HT in the presence of 5-HTP is relatively low in spite of large amounts of 5-HTP present in the brain neuropil after extracerebral decarboxylase inhibition.     

Electrophysiological effects of monoamine oxidase inhibition on rat midbrain dopaminergic neurones: an in vitro study.

1 The effects of the inhibition of monoamine oxidase (MAO) type A and B have been evaluated on the spontaneous firing activity of the dopaminergic (principal) neurones of the rat midbrain intracellularly recorded from a slice preparation. 2 The non-specific MAO inhibitor, pargyline, superfused at a concentration of 10-100 microM, decreased or abolished the spontaneous firing discharge of the principal neurons in the subtantia nigra pars compacta and ventral tegmental area. This effect had a slow onset and appeared to be sustained. 3 The administration of the dopamine D2/3 receptor antagonist, sulpiride (100-300 nM), antagonized the pargyline-induced effect, while the superfusion of the dopamine D1 receptor antagonist, SCH 23390 (1-3 microM) did not counteract the induced inhibition of the firing rate. 4 The inhibitor for the MAO A, clorgyline (30-100 microM), reduced the firing rate of the dopaminergic neurones. A similar depressant effect was also observed when a MAO B inhibitor, deprenyl (30-100 microM), was applied. Lower concentrations of both drugs (300 nM-10 microM) did not produce consistent effects on neuronal discharge. 5 Our data suggest that only the blockade of both types of MAO enzymes favours the inhibitory action of endogenous dopamine on somato-dendritic D2/3 autoreceptors.