High-affinity inhibition of glutamate release from corticostriatal synapses by ω-agatoxin TK

To know which Ca²⁺ channel type is the most important for neurotransmitter release at corticostriatal synapses of the rat, we tested Ca²⁺ channel antagonists on the paired pulse ratio. ω-Agatoxin TK was the most effective Ca²⁺ channel antagonist (IC₅₀=127 nM; maximal EFFECT=211% (with >1 μM) and Hill COEFFICIENT=1.2), suggesting a single site of action and a Q-type channel profile. Corresponding parameters for Cd²⁺ were 13 μM, 178% and 1.2. The block of L-type Ca²⁺ channels had little impact on transmission, but we also tested facilitation of L-type Ca²⁺ channels. The L-type Ca²⁺ channel agonist, s-(−)-1,4 dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridine carboxylic acid methyl ester (Bay K 8644 (5 μM)), produced a 45% reduction of the paired pulse ratio, suggesting that even if L-type channels do not participate in the release process, they may participate in its modulation.     

Distinct effects of ω-toxins and various groups of Ca-entry inhibitors on nicotinic acetylcholine receptor and Ca channels of chromaffin cells

The effects of ω-toxins and various Ca²⁺ antagonist subtypes on the ⁴⁵Ca²⁺ entry into bovine adrenal medullary chromaffin cells stimulated via nicotinic acetylcholine receptors or via direct depolarization with K⁺, have been compared. The conditions selected to stimulate the ⁴⁵Ca²⁺ entry consisted of a 60-s period of exposure of cells to 100 μM of the nicotinic acetylcholine receptor agonist dimethylphenylpiperazinium or to 70 mM K⁺. The N-type voltage-dependent Ca²⁺ channel blockers ω-conotoxin GVIA and MVIIA (1 μM) inhibited ⁴⁵Ca²⁺ entry stimulated by dimethylphenylpiperazinium or K⁺ by around 25–30%. The P-type Ca²⁺ channel blocker ω-agatoxin IVA (10 nM) did not affect the dimethylphenylpiperazinium nor the K⁺ responses; 1 μM (Q-channel blockade) inhibited both responses by around 50%. The N/P/Q-type Ca²⁺ channel blocker ω-conotoxin MVIIC (1 μM) inhibited the K⁺ evoked ⁴⁵Ca²⁺ entry by 70%, while dimethylphenylpiperazinium was blocked by 50% (P<0.001). The L-type Ca²⁺ channel blockers nifedipine, furnidipine, diltiazem or verapamil (3 μM each) inhibited much more the dimethylphenylpiperazinium than the K⁺ response. The dimethylphenylpiperazinium signal was blocked 71, 88, 89, and 53%, respectively, by nifedipine, furnidipine, diltiazem and verapamil, and the K⁺ response by 38, 29, 22, and 10%. Combined ω-conotoxin MVIIC (1 μM) and furnidipine (3 μM) blocked 100% of the K⁺ evoked ⁴⁵Ca²⁺ entry. However, combined ω-conotoxin GVIA (1 μM), and furnidipine left unblocked 50% of the K⁺ response. The ‘wide spectrum' Ca²⁺ channel antagonists flunarizine or dotarizine (3 μM each) blocked the dimethylphenylpiperazinium and the K⁺ responses to a similar extent (50%); cinnarizine (3 μM) inhibited more the dimethylphenylpiperazinium (82%) than the K⁺ response (21%). At 3 μM, the highly lipophilic β-adrenoceptor antagonist (±)-propranolol, reduced by 68% the dimethylphenylpiperazinium signal and by 23% the K⁺ signal. Other high lipophilic β-adrenoceptor antagonists such as metoprolol and labetalol, reduced little the dimethylphenylpiperazinium and the K⁺ responses. The highly lipophilic agent penfluridol blocked the dimethylphenylpiperazinium response by 30% and the K⁺ response by 50%. One of the least lipophilic compounds tested, (+)-lubeluzole, blocked by 40% the dimethylphenylpiperazinium and the K⁺ responses. These data are compatible with the idea that the various ω-toxin peptides used to separate pharmacologically the different voltage-dependent Ca²⁺ channels expressed by neurones, do not block the neuronal nicotinic acetylcholine receptor ion channel. In contrast the L-type Ca²⁺ channel blockers do block the nicotinic acetylcholine receptor ionophore. Lipophilicity of the compounds is not a requirement for Ca²⁺ channel or nicotinic acetylcholine receptor blockade.     

Effects of Ca channel blockers on Ca loading induced by metabolic inhibition and hyperkalemia in cardiomyocytes

The effects of the L-type (nifedipine and verapamil) and the T-type (mibefradil) Ca²⁺ channel blockers on the increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by NaCN metabolic inhibition and hyperkalemia were examined in chicken cardiomyocytes using fluorescence imaging with Fura-2. NaCN induced a slow and sustained rise in [Ca²⁺]_i, which was not affected by pretreating the cells for 5 min with nifedipine, verapamil, or mibefradil at 100 nM or 10 μM. Pretreatment of the cells with 10 μM nifedipine, verapamil, or mibefradil for 5 min remarkably inhibited the K⁺-induced increase in [Ca²⁺]_i. These inhibitory effects diminished after 48-h pretreatment with nifedipine or verapamil but not with mibefradil. Ryanodine also induces an increase in [Ca²⁺]_i, and this effect was enhanced by 48-h pretreatment of the cells with 10 μM verapamil but not with 10 μM mibefradil. We conclude that the NaCN-induced increase in [Ca²⁺]_i is independent of the Ca²⁺ influx though the L-type or T-type Ca²⁺ channels. Chronic inhibition of the L-type Ca²⁺ channels but not T-type channels may enhance the ryanodine receptor-mediated Ca²⁺ release, which may be responsible for the development of tolerance to their inhibitory effects on K⁺-induced increase in [Ca²⁺]_i.     

The effect of trimethyltin on acetylcholine release in the guinea-pig trachea

The purpose of the present work was to characterise the effects of trimethyltin on the release of acetylcholine from parasympathetic nerves and its effect on the postjunctional cholinergic stimulation of a smooth muscle. The guinea-pig trachea has been used as a model. Prejunctionally, trimethyltin (3.0 × 10⁻³ M) significantly enhanced in a reversible manner the high K⁺ (75 mM) evoked release of endogenous acetylcholine and [³H]acetylcholine. The evoked release of endogenous acetylcholine and [³H]acetylcholine was released from a pool of acetylcholine being independent of extraneuronal Ca²⁺ in the presence, but not in the absence of trimethyltin. The effect of trimethyltin on the release was not inhibited by low Ca²⁺ (0 mM and 1.0 × 10⁻⁴ M) or by Ca²⁺ channel blockers (verapamil, 1.0 × 10⁻⁴ M, flunarizine, 1.0 × 10⁻⁴ M, ω-conotoxin GVIA, 2.0 × 10⁻⁷ M and ω-agatoxin, 2.0 × 10⁻⁷ M). The present results also demonstrate that trimethyltin induce emptying of a non-vesicular, probably a cytoplasmic storage pool of acetylcholine, since AH5183 (2.0 × 10⁻⁵ M), an inhibitor of the translocation of acetylcholine into synaptic vesicles, and -latrotoxin (1.0 × 10⁻⁸ M), a toxin from black widow spider venom inducing vesicle depletion, had no inhibitory effects on the release of [³H]acetylcholine evoked by trimethyltin (3.0 × 10⁻³ M). The release of [³H]acetylcholine was moreover enhanced by trimethyltin when the vesicular uptake of [³H]acetylcholine was inhibited by AH5183, probably as a result of a higher cytoplasmic concentration of [³H]acetylcholine. Trimethyltin also reduced the neuronal uptake of [³H]choline and this was probably due to a depolarising effect of trimethyltin on the cholinergic nerve terminals. A similar depolarisation induced by trimethyltin was observed during patch clamping of GH₄ C₁ neuronal cells. Postjunctionally, trimethyltin had no effect by itself or on the carbachol-induced smooth muscle contraction, indicating that trimethyltin did not have a general depolarising effect on smooth muscle cells or an effect on muscarinic receptors. Furthermore, the reduced electrical field-induced contraction and the subsequent increase in the basal smooth muscle tension that was observed by addition of trimethyltin was activity-dependent, and was most probably due to emptying of a nervous non-vesicular storage pool of acetylcholine, followed by rapid hydrolysis of acetylcholine by acetyl- and pseudocholinesterases.     

Lobeline inhibits nicotine-evoked [H]dopamine overflow from rat striatal slices and nicotine-evoked Rb efflux from thalamic synaptosomes

The present study evaluated the interaction of lobeline with neuronal nicotinic acetylcholine receptors using two in vitro assays, [³H] overflow from [³H]dopamine ([³H]DA)-preloaded rat striatal slices and ⁸⁶Rb⁺ efflux from rat thalamic synaptosomes. To assess agonist interactions, the effect of lobeline was determined and compared to S(−)-nicotine. To assess antagonist interactions, the ability of lobeline to inhibit the effect of S(−)-nicotine was determined. Both S(−)-nicotine (0.1–1 μM) and lobeline (>1.0 μM) evoked [³H] overflow from superfused [³H]DA-preloaded striatal slices. However, lobeline-evoked [³H] overflow is mecamylamine-insensitive, indicating that this response is not mediated by nicotinic receptors. Moreover, at concentrations (<1.0 μM) which did not evoke [³H] overflow, lobeline inhibited S(−)-nicotine (0.1–10 μM)-evoked [³H] overflow, shifting the S(−)-nicotine concentration–response curve to the right. S(−)-Nicotine (30 nM–300 μM) increased (EC₅₀ VALUE=0.2 μM) ⁸⁶Rb⁺ efflux from thalamic synaptosomes. In contrast, lobeline (1 nM–10 μM) did not evoke ⁸⁶Rb⁺ efflux, and the lack of intrinsic activity indicates that lobeline is not an agonist at this nicotinic receptor subtype. Lobeline completely inhibited (IC₅₀ VALUE=0.7 μM) ⁸⁶Rb⁺ efflux evoked by 1 μM S(−)-nicotine, a concentration which maximally stimulated ⁸⁶Rb⁺ efflux. Thus, the results of these in vitro experiments demonstrate that lobeline inhibits the effects of S(−)-nicotine, and suggest that lobeline acts as a nicotinic receptor antagonist.     

Adenosine A2 receptor activation facilitates Ca uptake by rat brain synaptosomes

Adenosine has been shown to increase the release of neurotransmitters by stimulation of adenosine A₂ receptors. This effect probably depends on Ca²⁺ entry into presynaptic nerve terminals. In the present work the ability of the mixed adenosine A₁/A₂ agonist, 2-chloroadenosine, to stimulate Ca²⁺ uptake into rat brain synaptosomes was investigated. ⁴⁵Ca²⁺ uptake was induced by 20 μM veratridine. In the absence of other drugs, 2-chloroadenosine (1 μM) decreased ⁴⁵Ca²⁺ uptake into synaptosomes. Blocking the adenosine A₁ receptor with 100 nM of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), 2-chloroadenosine (1 μM) increased rather than decreased the uptake of ⁴⁵Ca²⁺ into synaptosomes. The excitatory effect of 2-chloroadenosine observed in the presence of DPCPX was reversed by 200 nM of ω-agatoxin-IVA, a specific P-type Ca²⁺ channel antagonist, but not by L-type (nifedipine, 100 nM to 1 μM; methoxyverapamil 1-10 μM) or N-type (ω-conotoxin GVIA, 500 nM) Ca²⁺ channel antagonists. The adenosine A_2A selective agonist, 2-p-(2-carboxyethyl)-phenethylamino-5′-N-ethyl-carboxamido-adenosine (CGS 21680), did not significantly modify Ca²⁺ uptake induced by veratridine. In contrast, the selective adenosine A₂ receptor agonist, N⁶-(2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl)-adenosine (DPMA), in concentrations ranging from 10 nM to 1 μM increased Ca²⁺ uptake induced by veratridine. The selective adenosine A₂ receptor antagonist 3,7-dimethyl-1-propargylxanthine (DPMX) at a concentration of 10 μM antagonized the stimulatory effect of DPMA (0.1 μM) on ⁴⁵Ca²⁺ uptake. In conclusion, activation of adenosine A₂ receptors increases Ca²⁺ uptake by synaptosomes depolarized by veratridine, which could explain the increase of neurotransmitter release observed when A₂ receptors are activated.     

Differential Mechanisms Involved in the Effect of Nicotinic Agonists DMPP and Lobeline to Release [H]5-HT from Rat Hippocampal Slices

In the present study we investigated the effect of different nicotinic agonists (dimethylphenylpiperazinium-iodide (DMPP), (−)nicotine, cytisine, (−)-lobeline, and (−)epibatidine) and antagonists (mecamylamine and dihydro-β-erythroidine) on the release of [³H]5-HT from hippocampal slices. The nicotinic agonists DMPP and lobeline and electrical field stimulation, released [³H]5-HT from the hippocampus; other nicotinic agonists, such as (−)-nicotine, cytisine, and (−)-epibatidine had no effect. Unlike lobeline-induced release of [³H]5-HT, the effect of DMPP (10 and 40 μM) was antagonized by mecamylamine (20 and 10 μM). The effect of DMPP was [Ca²⁺]_o-independent. In experiments carried out at 7°C, i.e. the membrane carrier proteins are inhibited and the release by lobeline was abolished while the DMPP-induced release of 5-HT was rather potentiated. It is proposed that the effect of DMPP and lobeline, to enhance the release of [³H]5-HT from the hippocampus, was mediated by two different mechanisms. While DMPP-induced 5-HT release can be linked to a non-classical nAChR activation ([Ca²⁺]_o-independence), the effect of lobeline was likely mediated by uptake carriers. © 1997 Elsevier Science Ltd. All rights reserved.     

The Ca channel agonist Bay K 8644 induces central respiratory depression in cats, an effect blocked by naloxone

We analyzed the respiratory effects induced by the Ca²⁺ channel agonist Bay K 8644 and its enantiomers, Bay R 5417 and Bay R 4407, applied to the ventral medullary surface of cats. Bay K 8644 (10 to 100 μg) and Bay R 5417 (50 to 200 μg) elicited a dose-dependent respiratory depression. Naloxone (0.1 mg/kg), but not D-naloxone, reversed these effects, indicating that an endogenous opioid mechanism was involved. Bay R 4407 (100 μg) was ineffective. The respiratory depressant effects induced by Bay K 8644 and its (−) enantiomer were a consequence of their agonist properties on the L-type Ca²⁺ channel, since (1) the activity of Bay K 8644 was stereospecific, and (2) nimodipine prevented the effect. We suggest that potent activation of Ca²⁺ channels or other mechanisms by high doses of Ca²⁺ agonists elicits the release of endogenous opioid peptides in medullary respiration-related structures.     

Opioid receptor-mediated inhibition of evoked catecholamine release from cultured neurons of rat ventral mesencephalon and locus coeruleus

The effects of selective opioid agonists on the evoked release of [³H]dopamine and [³H]noradrenaline were studied in cultured dopaminergic neurons of the ventral mesencephalon (containing the substantia nigra and ventral tegmental area) and in cultured neurons of the noradrenergic locus coeruleus, respectively. The cultures were prepared from embroyonic day 15 rat brains. After 9 days in culture, the calcium-dependent release of [³H]dopamine from dopaminergic substantia nigra/ventral tegmental aera neurons induced by 23 mM k⁺ appeared to be inhibited exclusively by activation of κ-opioid receptors, as [³H]dopamine release was inhibited selectively by the κ- agonists U69,593 and dynorphin-(1–13) (EC₅₀ 8 and 5 nM, respectively), and this inhibitory effect was antagonized by the κ-selective antagonist nor-binaltorphine (K_i 0.07 nM). In contrast, cultured noradrenergic locus coeruleus neurons appeared to contain release-inhibitory μ-opioid receptors only, as evoked [³H]noradrenaline release was inhibited selectively by the μ agonist [D-Ala², MePhe⁴, Gly-ol⁵]enkephalin (EC₅₀ 45 nM), a response that was antagonized by the preferential μ antagonist naloxone (K_i = 0.7 nM). The δ-opioid receptor agonist [D-Ser²(O-butyl), Leu⁵]enkephaly-Thr⁶ did not affect catecholamine release. Dopamine release from cultured ventral mesencephalic neurons, induced by 100 μM N-Methyl-D-Aspartate (NMDA), also appeared to be subject to κ receptor-mediated inhibition, whereas NMDA-induced noradrenaline release from cultured locus coeruleus neurons was under the inhibitor control of μ receptors. It is therefore concluded that in rat brain neurotransmitter release from dopaminergic and noradrenergic neurons, originating from the substantia nigra/vental tegmental area and the locus coeruleus, is liable to inhibition by homogenous populations of κ- and μ-opioid receptors, respectively, independent of the input of non-opioid neurons from distict nuclei.     

The action of trelibet, a new antidepressive agent on [H]noradrenaline release from rabbit pulmonary artery

Trelibet, a new antidepressant, used at 10⁻⁷–10⁻⁴ M failed to affect the [³H]noradrenaline ([³H]NA) release evoked from the isolated main pulmonary artery of the rabbit low frequency (2 Hz) nerve stimulation whether the neuronal uptake inhibitor cocaine (3 × 10⁻⁵ M) was present or not. Its metabolite (EGYT-2760) however, potentiated the nerve-evoked release of [³H]NA. In the absence of cocaine both the resting and the stimulation-evoked release of ³H increased in response to EGYT-2760. These effect were accompanied by muscle contraction. The EGYT-2760-potentiated transmitter release was inhibited either by exogenously applied 1-noradrenaline (10⁻⁶ M) or clonidine (10⁻⁶ M), preferential agonists of presynaptic ₂-adrenoceptors. The 1-noradrenaline-induced inhibition of transmitter release potentiated by EGYT-2760 was antagonized by 3 × 10⁻⁷ M yohimbine, a preferential ₂-adrenoceptor inhibitor. In the absence of cocaine, Ca²⁺ removal from the external medium failed to affect the ³H outflow-increasing effect of EGYT-2760 but abolished the nerve-evoked release-potentiating action of this compound. It is concluded that the metabolite of trelibet exerts a ‘yohimbine-like’ action, as well as a ‘tyramine-like’ effect in peripheral sympathetic nerve fibres.     

Sarcoplasmic reticulum Ca2+ release channel ryanodine receptor (RyR2) plays a crucial role in aconitine-induced arrhythmias

The present study established a model of RyR₂ knockdown cardiomyocytes and elucidated the role of RyR₂ in aconitine-induced arrhythmia. Cardiomyocytes were obtained from hearts of neonatal Sprague–Dawlay rats. siRNAs were used to down-regulate RyR₂ expression. Reduction of RyR₂ expression was documented by RT-PCR, western blot, and immunofluorescence. Ca²⁺ signals were investigated by measuring the relative intracellular Ca²⁺ concentration, spontaneous Ca²⁺ oscillations, caffeine-induced Ca²⁺ release, and L-type Ca²⁺ currents. In normal cardiomyocytes, steady and periodic spontaneous Ca²⁺ oscillations were observed, and the baseline [Ca²⁺]_i remained at the low level. Exposure to 3 μM aconitine increased the frequency and decreased the amplitude of Ca²⁺ oscillations; the baseline [Ca²⁺]_i and the level of caffeine-induced Ca²⁺ release were increased but the L-type Ca²⁺ currents were inhibited after application of 3 μM aconitine for 5 min. In RyR₂ knockdown cardiomyocytes, the steady and periodic spontaneous Ca²⁺ oscillations almost disappeared, but were re-induced by aconitine without affecting the baseline [Ca²⁺]_i level; the level of caffeine-induced Ca²⁺ release was increased but L-type Ca²⁺ currents were inhibited. Alterations of RyR₂ are important consequences of aconitine-stimulation and activation of RyR₂ appear to have a direct relationship with aconitine-induced arrhythmias. The present study demonstrates a potential method for preventing aconitine-induced arrhythmias by inhibiting Ca²⁺ leakage through the sarcoplasmic reticulum RyR₂ channel.     

Stimulus-evoked efflux of GABA from preloaded slices of the rabbit oviduct

The effect of electrical and chemical stimulation on the efflux of [³H]-γ-aminobutyric acid (GABA) from preloaded slices of the rabbit oviduct was examined. Electrical field stimulation significantly increased the outflow of [³H]GABA. This effect could not be prevented by tetrodotoxin or by the removal of Ca²⁺ from the medium. High K⁺ concentrations, veratrine and ethylenediamine also evoked a remarkable elevation in the efflux. The release induced by veratrine was completely abolished in a Ca²⁺-free medium or in the presence of tetrodotoxin, while the release evoked by high K⁺ or ethylenediamine was resistant to both conditions. These findings indicate that GABA can be released from the oviduct under the effect of depolarizing stimuli, raising the possibility of a physiological interaction between oviductual GABA and its receptors. The characteristics of oviductal GABA efflux differ from those of neuronal and glial GABA release.     

Effect of calcium agonists and antagonists on cerebellar granule cells

In cerebellar cultures, comprising predominantly granule neurones, dihydropyridine (DHP) Ca²⁺ agonists were potent stimulators of voltage-sensitive ⁴⁵Ca²⁺ uptake. Their effect was maximal in partially depolarized cells; at 15 mM K_e⁺ half maximal stimulation occurred at about 5 × 10⁻⁸ M BAY K 8644 and 10⁻⁷ M(+)-(S)-202 791. Organic Ca²⁺ antagonists were effective inhibitors of voltage-sensitive calcium entry into granule cells: the order of potency in blocking uptake induced by sub-maximal concentration of K⁺ and BAY K 8644 was nifedipine > (−)-202 791 > D600. BAY K 8644 also stimulated the release of glutamate, the transmitter of the granule cells, from depolarized cells. Granule cells are therefore a class of neurones whose responsiveness to organic Ca²⁺ effectors is similar to that of cardiac and smooth muscle. The discrepant findings on the effect of calcium effectors in various preparations of nervous tissues may thus reflect a differential distribution of voltage-sensitive Ca²⁺ channels in different neuronal cell types.     

Mechanisms of pinacidil-induced vasodilatation

The mechanism of the vasodilator effect of pinacidil was examined. Pinacidil (0.1–100 μM) inhibited the increases in cytosolic Ca²⁺ ([Ca²⁺]_i) and muscle tension due to norepinephrine in rat aorta. In contrast, a Ca²⁺ channel blocker, verapamil, inhibited the norepinephrine-stimulated [Ca²⁺]_i more strongly than the contraction. Higher concentrations of pinacidil (3–100 μM) inhibited the verapamil-insensitive portion of the contraction and [Ca²⁺]_i. An inhibitor of ATP-sensitive K⁺ channels, glibenclamide, antagonized the inhibitory effect of low concentrations ( 10 pM) of pinacidol. Pinacidil did not change the contraction induced by Ca²⁺ in vascular smooth muscle permeabilized with Staphylococcus aureus -toxin. Norepinephrine (in the presence of GTP), 12-deoxyphorbol 13-isobutyrate (in the absence of GTP), and treatment with GTP_γS potentiated the contraction of permeabilized smooth muscle induced by the addition of Ca²⁺. Pinacidil (100 μM) inhibited the potentiation due to GTP_γS or noepinephrine but not to phorbol ester. These results suggest that pinacidil has dual effects on vascular smooth muscle contraction. At lower concentrations (>0.1 μM), it decreases [Ca²⁺]_i, possibly by activating ATP-sensitive K+ channels. At higher concentrations (> 3 μM), it may additionally inhibit the receptor-mediated, GTP-binding protein-coupled phosphatidyl inositol turnover.     

Cholecystokinin-8 increases K-evoked [H]γ-aminobutyric acid release in slices from various brain areas

[³H]γ-Aminobutyric acid (GABA) release was studied in rat brain slices in the absebce or presence of cholecystokinin-8 (CCK-8). [³H]GABA release under the conditions used was Ca²⁺-dependent and insensitive tot he presence of the glial uptake blocker β-alanine. While the basal release of [³H]GABA was not affected by CCK-8, the K⁺-stimulated release of [³H]GABA wasm significantly enhanced by 300 nM of CCK-8 in the caudate putamen, the substantia nigra, the hippocampal formation and the parietofrontal cortex. In the cerebral cortex the CCK-8 enhancement of [³H]GABA release was concentration-dependent and abolished by the CCK_B receptor antagonists PD135,158 (1.0 nM) and L-365,260 (100 nM). A significant counteraction of the CCK-8 action was also found with the CCK_A receptor antagonist L-364,718 (100 nM) but only in concentrations at which both CCK_A and CCK_B receptors are blocked. No CCK-8 effects on [³H]GABA release were observed when tetrodotoxin was superfused 5 min before the K⁺-induced [³H]GABA release. It is suggested that the enhancing actions of CCK-8 on K⁺-stimulated [³H]GABA release is mainly related to an activation of CCK_B receptors.     

Mechanisms for resin acid effects on membrane currents and GABA(A) receptors in mammalian CNS

Resin acids from bleached wood pulp are toxic to fish. 12,14-Dichlorodehydroabietic acid (12,14-Cl₂DHA) raises cytoplasmic Ca²⁺ in synaptosomes and blocks neural GABA_A receptors; however, the underlying mechanism remains unclear in these earlier rodent studies. 12,14-Cl₂DHA (50 μM) almost completely blocked native GABA_A currents (rat cortical cultures) but had no significant effect on picrotoxin-sensitive recombinant human receptors in oocytes (1, β2 and γ2L: the most prevalent isoforms in mammalian brain). In oocytes, 12,14-Cl₂DHA failed to produce a calcium-activated chloride current, in contrast to the calcium ionophore ionomycin (10 μM). However, in cultured cortical pyramidal cells, both ionomycin and 12,14-Cl₂DHA produced chloride-selective currents of similar magnitude (presumably secondary to Ca²⁺ release). 12,14-Cl₂DHA was unable to stimulate phosphate labelling of [³H]-inositol in mouse synaptosomes, indicating that the study compound does not cause Ca²⁺ release via an IP₃ mechanism. Calcium pump ATPase inhibition also seems unlikely since thapsigargin did not elevate free calcium in synaptosomes. 12,14-Cl₂DHA clearly blocks GABA_A currents indirectly: we infer that its toxicity may be secondary to the elevations in cytoplasmic Ca²⁺ via an unidentified recognition site (or receptor) found in neuronal cells.     

Selective effects of [D-Ser(O-t-butyl),Leu]enkephalyl-Thr and [D-Ser(O-t-butyl),Leu]enkephalyl-Thr(O-t-butyl), two new enkephalin analogues, on neurotransmitter release and adenylate cyclase in rat brain slices

The selectivity and potency of two new enkephalin-derived δ-opioid receptor agonists, DSTBULET ([D-Ser²(O-t-butyl), Leu⁵]enkephalyl-Thr⁶) and BUBU ([D-Ser²(O-t-butyl),Leu⁵]enkephalyl-Thr⁶(O-t-butyl)) were determined with functional tests in vitro of μ-, δ-, and κ-opioid receptor activation in the rat brain. Both peptides concentration dependently (1 nM-1 μM) inhibited the release of radiolabeled acetylcholine (ACh) from striatal slices (pD₂ 7.6–7.9), an effect exclusively mediated by δ-opioid receptor activation. Fentanyl isothiocyanate (FIT), an irreversible δ-antagonist, completely blocked the inhibitory effects of DSTBULET and BUBU. Up to a concentration of 1 μM, the peptides did not affect striatal [³H]dopamine (DA) release nor cortical [³H]noradrenaline (NA) release, processes which are known to be inhibited by opioids activating κ and μ-receptors, respectively. Furthermore, both DSTBULET and BUBU caused a strong inhibition (pD₂ 8.2–8.3) of D-1 dopamine receptor-stimulated cyclic AMP efflux from striatal slices, an effect known to be mediated by μ- and/or δ-opioid receptor activation. However, the peptides were without effect when D-1 and D-2 dopamine receptors were stimulated simultaneously, a situation in which only μ-agonists are able to inhibit the resulting cAMP efflux. In conclusion, DSTBULET and BUBU appear to display a high selectivity and potency toward functional δ-opioid receptors in the brain.     

Effects of divalent cations on snake venom cardiotoxin-induced hemolysis and H-deoxyglucose-6-phosphate release from human red blood cells

, and . Effects of divalent cations on snake venom cardiotoxin-induced hemolysis and ³H-deoxyglucose-6-phosphate release from human red blood cells. Toxicon 27, 1297–1305, 1989.—At a low concentration of Naja naja kaouthia cardiotoxin (3 μM) Ca²⁺, Sr²⁺ and Ba²⁺ (2 mM), had little to no effect on ³H-deoxyglucose-6-phosphate (³H-dGlu-6-p) or hemoglobin release. At higher concentrations of N. n. kaouthia cardiotoxin (≥ 10 μM), Ca²⁺ (2 mM), but not Sr²⁺ or Ba²⁺, significantly enhanced ³H-dGlu-6-p and hemoglobin release. Mn²⁺ (2 mM) almost completely inhibited ³H-dGlu-6-p release and hemolysis at both the 3 μM and 10 μM concentrations of cardiotoxin. At a fixed concentration of N. n. kaouthia cardiotoxin (3 μM), Ca²⁺ at low concentrations (0.5 mM) enhanced ³H-dGlu-6-p and hemoglobin release, but at higher concentrations caused a dose-dependent inhibition of cardiotoxin action. The cardiotoxin from N. n. kaouthia venom (3 μM) induced ³H-dGlu-6-p release and hemolysis release with similar time courses and to similar extents. ³H-dGlu-6-p release induced by cardiotoxin was greatly enhanced as the pH of the medium was increased from 7.0 to 8.5. Similarities between ³H-dGlu-6-p and hemoglobin release do not support opening of pores in the plasmalemma of all red blood cells as the mode of action of cardiotoxins, but suggests that complete lysis of a subpopulation of cells occurs. Cardiotoxins have two components of lysis, only one of which is Ca²⁺-dependent. The Ca²⁺-dependent lysis is only evident at higher cardiotoxin concentrations and is likely due to trace phospholipase A₂ contamination in the toxin fraction. Mn²⁺ is an effective antagonist of cardiotoxin action.     

5-Hydroxytryptamine induces transient Ca influx through Ni-insensitive Ca channels in rat vascular smooth muscle cells

The effects of Ni²⁺, a non-selective cation channel inhibitor, on 5-hydroxytryptamine (5-HT)- and angiotensin II (Ang II)-induced intracellular Ca²⁺ dynamics in rat aortic smooth muscle cells were investigated. Ni²⁺ (1 mM) significantly inhibited the transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by Ang II (100 nM) in aortic smooth muscle cells, as measured using fura-2. However, Ni²⁺ did not suppress the transient increase in Ca²⁺ influx induced by 5-HT (10 μM), while significantly suppressed the sustained increase. Ca²⁺ influx evoked by high KCl (80 mM), thapsigargin (TG) (1 μM) or depletion of intracellular Ca²⁺ store was almost completely suppressed by Ni²⁺. Ni²⁺ had no effect on 5-HT-induced inositol triphosphate production and Ca²⁺ release from the intracellular store(s). These results suggest that 5-HT, but not Ang II, induces transient Ca²⁺ influx through Ni²⁺-insensitive Ca²⁺ channels, which are distinguishable from the voltage-dependent or store-operated Ca²⁺ channels.