α2-Adrenoceptor mediated inhibition of the release of radiolabelled 5-hydroxytryptamine and noradrenaline from slices of the dorsal region of the rat brain

Summary Possible local interactions between noradrenergic and serotonergic systems in the dorsal raphe region of the rat were investigated by studying the effects of various drugs on depolarization (20 mmol/l K⁺)-induced release of [³H]5-hydroxytryptamine (5-HT) and [³H]noradrenaline (NA) in vitro using a superfusion method. Exogenous 5-HT did not influence the release of [³H]NA. However, NA (in the presence of 10 mol/l desipramine) as well as the selective ₂-adrenoceptor agonists clonidine and oxymetazoline strongly inhibited [³H]5-HT release. The selective ₁-adrenoceptor agonists phenylephrine and methoxamine did not affect the release of either [³H]5-HT or [⁵H]NA. The inhibition by NA of both [³H]5-HT and [⁵H]NA release was not affected by the -adrenoceptor antagonist sotalol nor by the selective ₁-adrenoceptor antagonist prazosin. However, phentolamine and the selective ₂-adrenoceptor antagonists yohimbine and rauwolscine competitively antagonized the inhibitory effect of NA on [³H]NA release (respective pA₂-values 7.5 and 8.3) and on [³H]5-HT release (respective pA₂-values 7.7 and 8.2). Moreover, the release of [³H]NA and also, but to a lesser extent, that of [³H]5-HT were increased by the antagonists. It is concluded that the release of both 5-HT and NA in the dorsal raphe region may be subject to presynaptic inhibition by NA via activation of ₂-adrenoceptors.Send offprint requests to A. L. Frankhuijzen     

Effect of ω-conotoxin GVIA on release of 3H-γ-aminobutyric acid from slices of rat neostriatum

Summary -Conotoxin GVIA (-CT) diminished the potassium-induced in vitro release of ³H--aminobutyric acid (³H-GABA) from slices of rat neostriatum in a manner which depended on the concentration of potassium. -CT (0.1 nmol/l) decreased the release of ³H-GABA induced by 25 mmol/l K⁺ from 11.6% to 6.1% of tissue content, ie. by 48%, while it did not affect the release of ³H-GABA caused by 20 mmol/l K⁺, which was 4.8% of tissue content. However, in the presence of a polyclonal antiserum or cysteamine (600 mol/l), both of which diminish the effects of endogenous somatostatin, 0.1–10 nmol/l -CT decreased the release of ³H-GABA induced by 20 mmoles/l K⁺ by 40%. It is concluded that -CT did not only inhibit GABA-neurones, but had an additional inhibitory effect on somatostatin neurones which are known to depress the release of ³H-GABA. It is further concluded that neuronal interactions, which are possible in brain slice preparations, may impede the interpretation of effects of drugs, especially if agents are used which affect basic mechanisms of transmitter release and thus the release of various transmitters from neurones. Send offprint requests to D. K. Meyer at the above address     

Comparison of CGRP release from rat lymphocytes and dorsal root ganglia neurons

Calcitonin gene-related peptide (CGRP), a neuropeptide contained in sensory neuron, has been demonstrated to be synthesized and released by rat lyrnphocytes in our previous studies.In this study, some release property and molecular     

Histamine H₃ receptors modulate depolarization-evoked [³H]-noradrenaline release from rat olfactory bulb slices

We have studied the effect of histamine H(3) receptor (H(3)R) activation on the depolarization-evoked release of labeled neurotransmitters from slices of the rat olfactory bulb (rOB). The presence of pre-synaptic H(3)Rs was evidenced by the specific binding of the H(3)R ligand N-α-[methyl-(3)H]histamine to membranes from rOB synaptosomes (maximum binding, B(max), 106 ± 19 fmol/mg protein; dissociation constant, K(d), 0.68 ± 0.11 nM) which was inhibited by selective H(3)R ligands (immepip, (R)(-)-α-methylhistamine (RAMH) and clobenpropit) with affinities similar to those previously reported for H(3)Rs expressed in other rat brain areas. Perfusion of rOB slices with the selective H(3)R agonist RAMH (0.1 and 1 μM) had no effect on the release of [(3)H]-γ-aminobutyric acid ([(3)H]-GABA), [(3)H]-d-aspartate, [(3)H]-dopamine or [(3)H]-5-hydroxytryptamine ([(3)H]-5-HT) evoked by depolarization with high K(+) (20 or 40 mM). [(3)H]-Noradrenaline release induced by 20 mM K(+) was reduced in a modest but significant manner by RAMH (94.9 ± 1.7% and 83.1 ± 2.1% of control release at 0.1 and 1 μM, respectively). The effect of 1 μM RAMH was blocked by the selective H(3)R antagonist/inverse agonist clobenpropit (5 μM). When tested alone clobenpropit and a second H(3)R antagonist/inverse agonist, ciproxifan (both at 1 μM) significantly increased K(+)-evoked [(3)H]-noradrenaline release to 119.4 ± 4.2% and 120.0 ± 3.7% of K(+) alone, respectively. Ciproxifan (1 μM) had no effect on the depolarization-evoked release of the other labeled neurotransmitters. These data indicate that H(3)Rs with constitutive activity modulate noradrenaline release in rOB, presumably through a pre-synaptic action. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.     

Response of nicotine self-administration in the rat to manipulations of mu-opioid and γ-aminobutyric acid receptors in the ventral tegmental area

Rationale: The mesolimbic dopamine system has been implicated in the reinforcing effects of nicotine, a drug which appears to act at least in part through the ventral tegmental area (VTA). Other neuronal elements in the VTA are important in drug reward. In particular, mu opioid receptors in the VTA have been shown to influence cocaine reinforcement. Objective: The aim of this study was to test whether the mu opioid receptors in the VTA also regulate the intake of nicotine. Methods: This research was carried out with animals trained to self-administer nicotine or cocaine, or to respond for food. Mu receptors were targeted with the selective agonist [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO) and γ-aminobutyric acid (GABA) receptors with the selective agonists baclofen and muscimol; each of these compounds was delivered by microinfusion into the VTA. Results: The mu-selective agonist DAMGO, tested over a dose range of 0.005–0.05 μg, had an effect at the highest dose only, where it produced a reduction in self-administration maintained by doses of either 10 μg/kg or 30 μg/kg per infusion of nicotine. Intra-VTA microinfusions of DAMGO did not reinstate extinguished responding previously established for nicotine, nor did they have prominent effects on operant behavior maintained by food. In contrast to the overall limited effects of DAMGO on nicotine self-administration, the GABA agonists muscimol and baclofen each reduced nicotine self-administration substantially when delivered into the VTA, whereas they were less effective against cocaine self-administration. Conclusions: The lesser effect of DAMGO microinfusions in the VTA on nicotine than cocaine self-administration is associated with the opposite efficacy of GABA agonists. These findings suggest that nicotine and cocaine differentially activate circuitry in which mu receptors are situated, especially GABAergic elements. Received: 20 October 1998 / Final version: 24 November 1999     

Effect of chronic treatment with the GABA transaminase inhibitors γ-vinyl GABA and ethanolamine O-sulphate on the in vitro GABA release from rat hippocampus

1. The effects of 2, 8 and 21 day oral treatment with the specific γ-aminobutyric acid transaminase (GABA-T) inhibitors γ-vinyl GABA (GVG) and ethanolamine O-sulphate (EOS) on brain GABA levels, GABA-T activity, and basal and stimulated GABA release from rat cross-chopped brain hippocampal slices was investigated.
2. Treatment with GABA-T inhibitors lead to a reduction in brain GABA-T activity by 65–80% compared with control values, with a concomitant increase in brain GABA content of 40–100%.
3. Basal hippocampal GABA release was increased to 250–450% of control levels following inhibition of GABA-T activity. No Ca²⁺ dependence was observed in either control or treated tissues.
4. GVG and EOS administration led to a significant elevation in the potassium stimulated release of GABA from cross-chopped hippocampal slices compared with that of controls. Although stimulated GABA release from control tissues was decreased in the presence of a low Ca²⁺ medium, GVG and EOS treatment abolished this Ca²⁺ dependency.
5. GABA compartmentalization, Na⁺ and Cl⁻ coupled GABA uptake carriers and glial release may provide explanations for the loss of the Ca²⁺ dependency of stimulated GABA release observed following GVG and EOS treatment.
6. Administration of GABA-T inhibitors led to increases in both basal and stimulated hippocampal GABA release. However, it is not clear which is the most important factor in the anticonvulsant activity of these drugs, the increased GABA content ‘leaking'' out of neurones and glia leading to widespread inhibition, or the increase in stimulated GABA release which may occur following depolarization caused by an epileptic discharge.

     

Sodium dependent 3H-noradrenaline release from rat neocortical slices in the absence of extracellular calcium presynaptic modulation by μ-opioid receptor and adenylate cyclase activation

Summary In Ca²⁺-free EGTA (1 mmol/l)-containing medium veratrine (3 mol/l) and ouabain (100 mol/l) strongly enhanced the efflux of ³H-noradrenaline from superfused rat brain neocortical slices prelabelled with the radioactive amine. In both cases ³H-noradrenaline release was prevented by tetrodotoxin (1 mol/l). These effects of veratrine and ouabain were virtually additive and independent of whether the noradrenaline uptake carrier was blocked with 1 mol/l desipramine or not. The adenylate cyclase activator forskolin (10 nmol/l–10 mol/l) strongly enhanced veratrine- and ouabain-induced ³H-noradrenaline release, without affecting spontaneous tritium efflux. The release induced by both stimuli was profoundly inhibited by the selective -opioid receptor agonist [d-Ala, MePhe⁴, Gly-ol⁵]enkaphalin (DAGO, 3 nmol/l–1 mol/l) in a concentration-dependent manner. The inhibitory effects of 1 mol/l DAGO were abolished by 1 mol/l naloxone. On the other hand, preincubation of the slices for 1 h with the -opioid receptor-selective irreversible ligand fentanyl isothiocyanate (1 pmol/l) did not change the inhibitory effects of DAGO.These data show that veratrine- and ouabain-induced ³H-noradrenaline release from central noradrenergic nerve terminals is facilitated by increasing intracellular cyclic AMP levels and reduced by activation of presynaptic -opioid receptors, indicating the involvement of exocytotic neurotransmitter release. The results provide further evidence for the hypothesis that under these conditions neurotransmitter release from central noradrenergic neurons is triggerred by a Na⁺-induced efflux of Ca²⁺ ions from intracellular stores.Abbreviations DAGO [d-Ala², McPhe⁴, Gly-ol⁵]enkephalin Send offprint requests to A. N. M. Schoffelmeer at the above address     

β Adrenoceptor-mediated facilitation of endogenous noradrenaline release from rat isolated trachea

Overflow of endogenous noradrenaline from rat isolated tracheae was evoked by electrical field stimulation (3 Hz, 540 pulses) in the presence of yohimbine, desipramine and tyrosine. Isoprenaline 100 nmol/l increased the evoked overflow of noradrenaline by about 65%. This effect was antagonized by propranolol (100 nmol/l) and the ₂-selective adrenoceptor antagonist ICI 118,551 (100 nmol/l), but not by the ₁-selective adrenoceptor antagonist CGP 20712 A (100 nmol/l). The ₂-selective adrenoceptor agonist formoterol (1–100 nmol/l) also facilitated the evoked overflow of noradrenaline, but maximally by only about 25% at 10 nmol/l, i.e. formoterol behaved as a partial agonist at these facilitatory -adrenoceptor. This assumption is also supported by the observation that formoterol (10 nmol/l) acted as antagonist against isoprenaline (100 nmol/l). Mechanical removal of the mucosa resulted in a 30% decrease in tissue noradrenaline and a 55% reduction of the evoked overflow of noradrenaline. In mucosa-denuded preparations isoprenaline failed to facilitate noradrenaline overflow. In the presence of indomethacin (3 mol/l) the evoked overflow of noradrenaline from mucosa containing preparations was increased by about 50%, but isoprenaline still further facilitated the evoked noradrenaline overflow by about 40%. In conclusion, the overflow of noradrenaline in the rat trachea is facilitated via ₂-adrenoceptors, an effect which requires an intact air-way mucosa. Correspondence to: K. Racké at the above address     

Further study of prerequisites for the enhancement by α-adrenoceptor antagonists of the release of noradrenaline

Summary 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 and total tritium was determined.In the first series of experiments, stimulation periods of approximately constant length (50 s) were used (cocaine 5 M present). Thirteen pulses (0.25 Hz) elicited an overflow of ³H-noradrenaline of 0.024% of tissue tritium; 26 pulses (0.5 Hz) elicited an overflow of 0.059%, and 52 pulses (1 Hz) of 0.166%. Rauwolscine 1 M did not change the overflow evoked by 13 pulses, increased that evoked by 26 pulses and increased most markedly that evoked by 52 pulses. Phentolamine 1 M decreased the overflow at 13, did not change the overflow at 26, and increased the overflow at 52 pulses. Corynanthine 1 M decreased the overflow at 13, and did not change the overflow at 26 and 52 pulses. The effect of tetraethylammonium (TEA) 100 M was opposite to that of rauwolscine; it increased most markedly the overflow evoked by 13 pulses, increased less that evoked by 26 pulses, and least the overflow at 52 pulses.In the second series of experiments, the frequency of stimulation was kept constant (2 Hz). In the absence of cocaine, 10 pulses elicited an overflow of ³H-noradrenaline of 0.023% of tissue tritium; 20 pulses elicited an overflow of 0.043%, and 40 pulses of 0.089%. Phentolamine 1 M did not change the overflow evoked by 10 pulses, increased that evoked by 20 pulses, and increased most markedly that evoked by 40 pulses. TEA 100 M increased the evoked overflow at all pulse numbers. Similar results were obtained in the presence of cocaine 5 M.The results demonstrate that the enhancement by -adrenoceptor antagonists of the release of noradrenaline depends on the biophase concentration of noradrenaline. Under the present conditions, graded increases in biophase noradrenaline concentration led to graded increases in the effect of the antagonists. A second prerequisite for the release-enhancing effect appears to be a sufficient length of the pulse train. Under the present conditions, graded increases in train length up to about 20s led to graded increases in the effect of the antagonists, even though the average biophase concentration of noradrenaline did not change with the pulse train length. This pattern of effects of the -antagonists is not shared by at least one other release-enhancing drug, namely TEA.     

Roflumilast inhibits the release of chemokines and TNF-α from human lung macrophages stimulated with lipopolysaccharide

BACKGROUND AND PURPOSE

Lung macrophages are critically involved in respiratory diseases. This study assessed the effects of the PDE4 inhibitor roflumilast and its active metabolite, roflumilast N-oxide on the release of a range of chemokines (CCL2, 3, 4, CXCL1, 8, 10) and of TNF-α, from human lung macrophages, stimulated with bacterial lipopolysaccharide LPS.

EXPERIMENTAL APPROACH

Lung macrophages isolated from resected human lungs were incubated with roflumilast, roflumilast N-oxide, PGE₂, the COX inhibitor indomethacin, the COX-2 inhibitor NS-398 or vehicle and stimulated with LPS (24 h). Chemokines, TNF-α, PGE₂ and 6-keto PGF_1α were measured in culture supernatants by immunoassay. COX-2 mRNA expression was assessed with RT-qPCR. PDE activities were determined in macrophage homogenates.

KEY RESULTS

Expression of PDE4 in lung macrophages was increased after incubation with LPS. Roflumilast and roflumilast N-oxide concentration-dependently reduced the LPS-stimulated release of CCL2, CCL3, CCL4, CXCL10 and TNF-α from human lung macrophages, whereas that of CXCL1 or CXCL8 was not altered. This reduction by the PDE4 inhibitors was further accentuated by exogenous PGE₂ (10 nM) but abolished in the presence of indomethacin or NS-398. Conversely, addition of PGE₂ (10 nM), in the presence of indomethacin restored inhibition by roflumilast. LPS also increased PGE₂ and 6-keto PGF_1α release from lung macrophages which was associated with an up-regulation of COX-2 mRNA.

CONCLUSIONS AND IMPLICATIONS

Roflumilast and roflumilast N-oxide reduced LPS-induced release of CCL2, 3, 4, CXCL10 and TNF-α in human lung macrophages.     

Investigations of the roles of dihydropyridine and ω-conotoxin-sensitive calcium channels in mediating depolarisation-evoked endogenous dopamine release from striatal slices

Summary The relative roles of L- and N-type voltage-sensitive calcium channels (VSCC) in mediating endogenous dopamine release have been investigated by examining the effects of the dihydropyridine (DHP) agonist BAY K 8644 and the antagonist PN 200-110, as well as the VSCC-blocking peptide -conotoxin GVIA, on depolarisationevoked dopamine release from superfused rat striatal slices. Dopamine release evoked by electrical field stimulation was virtually unaffected by either of the DHP drugs, but release evoked by raising the K⁺ concentration to 25 mmol/l was significantly increased by BAY K 8644 and reduced stereospeciflcally by PN 200-110. Quantitative differences between electrically-evoked and K⁺-evoked dopamine release with respect to their dependence on extracellular calcium concentration were also observed, with electrically-evoked release requiring higher calcium concentrations. The adenylate cyclase activator forskolin itself increased dopamine release, but did not appear to influence the effectiveness of either DHP drug in altering dopamine release. In contrast to the relatively small effects of the DHP drugs, -conotoxin produced a major reduction in electrically-evoked dopamine release as well as a substantial decrease in K⁺-evoked release. Since -conotoxin is thought to block both L- and N-type neuronal VSCC whereas DHP drugs affect only L-type VSCC, these findings suggest that electrically-evoked dopamine release is mediated mainly by calcium influx through N-type VSCC, accounting for the reported lack of effect of many organic calcium antagonists on this process. In contrast, K⁺-evoked dopamine release appears to involve both L- and N-type VSCC, and can occur at lower extracellular calcium concentrations.Abbreviations DHP 1,4-dihydropyridine - HPLC high-performance liquid chromatography - VSCC voltage-sensitive calcium channels Send offprint requests to H. Herdon at the above address     

Presynaptic actions of 4-Aminopyridine and γ-aminobutyric acid on rat sympathetic ganglia in vitro

Summary Responses to bath-applications of 4-aminopyridine (4-AP) and -aminobutyric acid (GABA) were recorded intracellularly from neurones in the rat isolated superior cervical ganglion.4-aminopyridine (0.1–1.0 mmol/l) usually induced spontaneous action potentials and excitatory postsynaptic potentials (EPSPs), which were blocked by hexamethonium. Membrane potential was unchanged; spike duration was slightly increased. Vagus nerve B-and C-fibre potentials were prolonged.In 4-AP solution (0.1–0.3 mmol/l), GABA (0.1 mmol/l), 3-aminopropanesulphonic acid or muscimol evoked bursts of spikes and EPSPs in addition to a neuronal depolarization. These bursts, which were not elicited by glycine, glutamate, taurine or (±)-baclofen, were completely antagonised by hexamethonium, tetrodotoxin or bicuculline methochloride.It is concluded that: (a) 4-AP has a potent presynaptic action on sympathetic ganglia; (b) presynaptic actions of GABA can be recorded postsynaptically in the presence of 4-AP; and (c) the presynaptic GABA-receptors revealed in this condition are similar to those on the postsynaptic membrane.     

Activation of presynaptic α2-adrenoceptors attenuates the inhibitory effect of μ-opioid receptor agonists on noradrenaline release from brain slices

Summary ³H-noradrenaline release from rat neocortical slices induced by 15 mM K⁺ was concentration-dependently inhibited by morphine, [D-Ala²-D-Leu⁵] enkephalin (DADLE) and the calcium entry blocker Cd²⁺. Blockade of presynaptic ₂-adrenoceptors with phentolamine, almost doubling K⁺-induced ³H-noradrenaline release, slightly enhanced the relative inhibitory effects of morphine and DADLE, whereas that of Cd²⁺ remained unaffected. In contrast, activation of presynaptic ₂-adrenoceptors with clonidine (1 M) or TL-99 (1 M), inhibiting release by about 50%, completely abolished the inhibitory effects of morphine and DADLE without affecting that of Cd²⁺. When in the presence of 1 M clonidine adenylate cyclase was activated with forskolin (10 M), which restored release to the drug-free control level, the opioids still did not display their inhibitory effects. Therefore, -opioid receptor efficacy appears to be dependent on the degree of activation of ₂-adrenoceptors in central noradrenergic nerve terminals, probably through a local receptor interaction within the nerve terminal membrane.     

Different effects of reducing agents on ω-conotoxin GVIA inhibition of [3H]-acetylcholine release from rat cortical slices and guinea-pig myenteric plexus

1. The effect of reducing reagents on ω-conotoxin GVIA (ω-CgTX) inhibition of the release of [³H]-acetylcholine ([³H]-ACh) induced by tityustoxin, K⁺ 50 mM and electrical stimulation was investigated in rat brain cortical slices.
2. In cortical slices the inhibition of tityustoxin or electrically-stimulated [³H]-ACh release by ω-CgTX was dramatically increased by reducing reagents ascorbate or β-mercaptoethanol. Dehydroascorbic acid did not substitute for ascorbate
3. Depolarization induced by K⁺ 50 mM caused [³H]-ACh release from cortical slices which was not inhibited by ω-CgTX, even in the presence of ascorbate.
4. In the guinea-pig myenteric plexus, ω-CgTX inhibition of the tityustoxin induced release of [³H]-ACh was independent of ascorbate.
5. It is suggested that N-type-like calcium channels in guinea-pig myenteric plexus may have pharmacological/biochemical diversity from similar channels of rat cerebral cortex.

     

The nature of the stimulatory action of γ-aminobutyric acid in the isolated perfused dog adrenals

Summary The effect of -aminobutyric acid (GABA) on catecholamine (CA) release from adrenal medulla was investigated. GABA and GABA agonists, 3-amino-1-propanesulfonic acid and imidazole-4-acetic acid caused CA release from isolated perfused dog adrenals in a dose-dependent manner, and no tachyphylaxis to GABA was observed. CA release elicited by GABA was antagonized by bicuculline and picrotoxin. This antagonism was specific for GABA-and GABA agonist-induced responses, response to acetylcholine being unaffected. Pretreatment with atropine plus hexamethonium did not affect the response to GABA. GABA-induced CA release was abolished by the removal of Ca²⁺ from perfusion medium, but not by the removal of Na⁺ or Cl^–. Verapamil, CoCl₂ and dibucaine blocked the effect of GABA. A Na⁺ channel blocker, tetrodotoxin did not reduce GABA-evoked CA release. These results suggest that GABA may interact with its receptor to evoke CA release from adrenal medulla in a fashion of Ca²⁺-dependence and independence on external Na⁺ or Cl^–.     

Involvement of adenylate cyclase and protein kinase C in the α2-adrenoceptor-mediated inhibition of noradrenaline and 5-hydroxytryptamine release in rat hypothalamic slices

Summary In superfused rat hypothalamic slices prelabelled with [³H]-noradrenaline, the ₂-adrenoceptor agonist UK 14304 inhibited in a concentration-dependent manner the electrically-evoked release of tritium. This inhibition was antagonized by the ₂-adrenoceptor blocking agent idazoxan, which by itself increased the electrically-evoked tritium overflow. Exposure to forskolin, an adenylate cyclase activator, increased the electrically-evoked release of [³H]-noradrenaline. In the presence of forskolin (1 mol/l), both the inhibitory effect of UK 14304 and the increasing effect of idazoxan on the electrically-evoked release of [³H]-noradrenaline were less pronounced than in the absence of the adenylate cyclase activator. Exposure to forskolin and to the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine shifted to the right the concentration-effect curve for UK 14304 in a similar manner as that observed in the presence of forskolin alone. Exposure to phorbol-12,13-dibutyrate (0.01–10 mol/l), a drug which activates protein kinase C, increased the electrically-evoked release of [³H]-noradrenaline. In the presence of phorbol-12,13-dibutyrate (0.1 and 1 mol/l), the concentration effect curve for UK 14304 on tritium overflow was significantly shifted to the right. The increasing effect of idazoxan on tritium overflow was significantly less pronounced in the presence of 1 mol/l phorbol-12,13-dibutyrate.In superfused rat hypothalamic slices prelabelled with [³H]-5-hydroxytryptamine, the ₂-adrenoceptor agonist UK 14304 significantly inhibited the electrically-evoked release of tritium. Exposure to forskolin increased in a concentration-dependent manner [³H]-5-hydroxytryptamine overflow, but did not modify the UK 14304-mediated inhibition. Exposure to 3-isobutyl-1-methylxanthine enhanced the electrically-evoked release of [³H]-5-hydroxytryptamine. In the presence of both forskolin (1 mol/l) and 3-isobutyl-l-methylxanthine (1 mmol/l), the concentration-response curve for UK 14304 was significantly shifted to the right. Exposure to phorbol-12,13-dibutyrate (0.01–10 mol/l) enhanced in a concentration-dependent manner the electrically-evoked overflow of [³H]-5-hydroxytryptamine. In the presence of phorbol-12,13-dibutyrate (0.1 and 1 mol/l), UK 14304 was significantly less potent to inhibit tritium release than in the absence of the protein kinase C activator.It is concluded that both cyclic AMP and phosphoinositide turnover are involved in the modulation of noradrenaline and 5-hydroxytryptamine release by presynaptic ₂-adrenoceptors in rat hypothalamic slices. However, these interactions do not represent definitive proof for a cause-effect relationship for the second messengers mediating the ₂-adrenoceptor induced inhibition of transmitter release either as autoreceptor or as heteroreceptor.Send offprint requests to S. Z. Langer at the above address     

Subunit-dependent interaction of the general anaesthetic etomidate with the γ-aminobutyric acid type A receptor

1. The GABA modulating and GABA-mimetic actions of the general anaesthetic etomidate were examined in voltage-clamp recordings performed on Xenopus laevis oocytes induced, by cRNA injection, to express human recombinant γ-aminobutyric acid_A (GABA_A) receptor subunits.
2. Currents mediated by recombinant receptors with the ternary subunit composition α_xβ_yγ_2L (where x=1,2,3 or 6 and y=1 or 2), in response to GABA applied at the appropriate EC₁₀, were enhanced by etomidate in a manner that was dependent upon the identity of both the α and β subunit isoforms.
3. For the β₂-subunit containing receptors tested, the EC₅₀ for the potentiation of GABA-evoked currents by etomidate (range 0.6 to 1.2 μM) was little affected by the nature of the α subunit present within the hetero-oligomeric complex. However, replacement of the β₂ by the β₁ subunit produced a 9–12 fold increase in the etomidate EC₅₀ (6 to 11 μM) for all α-isoforms tested.
4. For α₁, α₂ and α₆, but not α₃-subunit containing receptors, the maximal potentiation of GABA-evoked currents by etomidate was greater for β₂- than for β₁-subunit containing receptors. This was most clearly exemplified by receptors composed of α₆β₁γ_2L compared to α₆β₂γ_2L subunits, where a maximally effective concentration of etomidate potentiated currents evoked by GABA at EC₁₀ to 28±2% and 169±4% of the maximal GABA response, respectively.
5. For α₁ subunit-containing receptors, the potency and maximal potentiating effect of either pentobarbitone or propofol was essentially unaffected by the β subunit isoform contained within the receptor complex. The potency of the anaesthetic neurosteroid 5α-pregnan-3α-ol-20-one was marginally higher for β₁ rather than the β₂ subunit-containing receptor, although its maximal effect was similar at the two receptor isoforms.
6. The GABA-mimetic action of etomidate was supported by β₂- but not β₁-subunit containing receptors, whereas that of pentobarbitone or propofol was evident with either β isoform. For β₂-subunit containing receptors, both the agonist EC₅₀ and the maximal current produced by etomidate were additionally influenced by the α isoform.
7. It is concluded that the subtype of β-subunit influences the potency with which etomidate potentiates GABA-evoked currents and that the β isoform is a crucial determinant of the GABA-mimetic activity of this compound. The nature of the α-subunit also impacts upon the maximal potentiation and activation that the compound may elicit. Such pronounced influences may aid the identification of the site that recognises etomidate. More generally, these results provide a clear example of structural specificity in anaesthetic action.

     

Frequency-dependence of serotonin autoreceptor but not α2-adrenoceptor inhibition of [3H]-serotonin release in rat hypothalamic slices

Summary The overflow of tritium from stimulated rat hypothalamic slices preincubated with [³H]-serotonin (5-HT) was significantly enhanced by reducing the frequency of stimulation from 3 Hz to 1 Hz while keeping the number of impulses constant. The 5-HT receptor agonist 5-methoxytryptamine inhibited in a concentration-dependent manner the electrically-evoked release of [³H]-5-HT with IC₅₀ values of 560 nmol/l and of 34 nmol/l when the stimulations were delivered at 3 Hz and 1 Hz, respectively. The terminal 5-HT autoreceptor antagonist methiothepin enhanced in a concentration-dependent manner the electrically-evoked release of [³H]-5-HT and this effect was greater at a frequency of stimulation of 3 Hz than at 1 Hz. In the same paradigm, the 5-HT reuptake inhibitors citalopram and paroxetine did not alter the overflow of radioactivity elicited by stimulation at 3 Hz but significantly decreased it at 1 Hz. In the presence of 5-HT autoreceptor blockade achieved with methiothepin, citalopram increased the overflow of [³H]-5-HT to the same extent at 1 Hz and at 3 Hz. The IC₅₀ values for inhibition of [³H]-5-HT release by the selective ₂-adrenoceptor agonist UK 14.304 were 35 nmol/l at 3 Hz and 30 nmol/l at 1 Hz. It is concluded that modulation of 5-HT release by 5-HT autoreceptors, but not by ₂-adrenoceptors is dependent on the synaptic concentration of 5-HT as a function of the frequency of depolarization. Send offprint requests to S. Z. Langer at the above address     

Effect of anesthetic doses of γ-hydroxybutyrate on the acetylcholine content of rat brain

Summary Gamma-hydroxybutyrate administered in anesthetic doses produces a time dependent increase in the levels of rat brain acetylcholine. A maximal increase in whole brain and subcortical levels of acetylcholine is observed about 15 min after administration of the lactone form of the drug. A similar GHB-induced increase in acetylcholine is observed in the striatum and a 75% increase in the hippocampus 15 min after administration of the drug. A good temporal correlation was not obtained between the increase in acetylcholine and the depth of anesthesia produced by the drug. Gamma-hydroxybutyrate did not cause a significant change in the striatal or hippocampal levels of choline. Possible mechanisms involved in the production of this increase in acetylcholine are discussed.A partial account of this paper was presented at the fall meeting of the American Society for Pharmacology and Experimental Therapeutics held at Montreal, Quebec on August 18–22, 1974 [Pharmacologist, 16, 463 (1974)]