The effect of water soluble cyanotoxin(s) produced by two species of Anabaena on the release of acetylcholine from the peripheral cholinergic nervous system of the rat airway

A water extract of the lyophilised fresh-water alga Anabaena flos-aquae enhanced substantially the release of [(3)H]acetylcholine ([(3)H]acetylcholine and [(3)H]choline) from cholinergic nerves of rat bronchi. Parallel experiments performed with the related species Anabaena lemmermannii did not demonstrate this effect. The effect on the release of [(3)H]acetylcholine by A. flos-aquae extract was concentration dependent. The A. flos-aquae induced [(3)H]acetylcholine release was not reduced by exposure to a low concentration of Ca(2+), but ω-conotoxin GVIA (1.0 μM), a blocker of N-type Ca(2+) channels reduced the release of [(3)H]acetylcholine induced by the A. flos-aquae extract. Addition of verapamil in a concentration (1.0 μM) specific for inhibition of L-type Ca(2+) channels had no effect on the neurotransmitter release. A reduction in the release was, moreover, observed with the intracellular Ca(2+) chelator BAPTA/AM (30 μM) and with the Na(+) channel blocker tetrodotoxin (3.0 μM). During patch-clamp studies of GH(4)C(1) neuronal cells, which have L- and T-type Ca(2+) channels, but no Na(+) channels, it was shown that a water extract of A. flos-aquae depolarised these cells and reduced, rather than enhanced, the influx of Ca(2+). Such an effect was not seen following exposure of GH(4)C(1) cells to water extracts of A. lemmermannii. In addition to its presynaptic activity, the water extract of A. flos-aquae showed an antimuscarinic effect by displacing [(3)H]QNB binding from muscarinic receptors in homogenates of rat bronchi. A similar but more potent effect was observed during experiments with water extract of A. lemmermannii. None of the respective water extracts showed any effects on cholinesterase activities in rat bronchial smooth muscle. The present observations suggest, therefore, that water extracts of A. flos-aquae may depolarise cells by activation of mono and divalent cation channels in cholinergic nerve cells. These channels are probably Na(+) channels and N-type, but not L- or T-type Ca(2+) channels. L- and T-type Ca(2+) channels were blocked in experiments with GH(4)C(1) cells and high concentrations of Ca(2+) channel blockers were necessary to reduce the effects of A. flos-aquae extract in cholinergic nerves in the airways. Furthermore, A. flos-aquae extract may also mobilise Ca(2+) from intracellular compartments. A. lemmermannii, on the other hand, does not contain components which alter mono and divalent cation-fluxes across cell membranes, but may rather have substances with more potent antagonistic effects on muscarinic cholinergic receptors than what is observed in experiments with A. flos-aquae.     

High affinity interaction of mibefradil with voltage-gated calcium and sodium channels

Mibefradil is a novel Ca(2+) antagonist which blocks both high-voltage activated and low voltage-activated Ca(2+) channels. Although L-type Ca(2+) channel block was demonstrated in functional experiments its molecular interaction with the channel has not yet been studied. We therefore investigated the binding of [(3)H]-mibefradil and a series of mibefradil analogues to L-type Ca(2+) channels in different tissues. [(3)H]-Mibefradil labelled a single class of high affinity sites on skeletal muscle L-type Ca(2+) channels (K(D) of 2.5+/-0.4 nM, B(max)=56.4+/-2.3 pmol mg(-1) of protein). Mibefradil (and a series of analogues) partially inhibited (+)-[(3)H]-isradipine binding to skeletal muscle membranes but stimulated binding to brain L-type Ca(2+) channels and alpha1C-subunits expressed in tsA201 cells indicating a tissue-specific, non-competitive interaction between the dihydropyridine and mibefradil binding domain. [(3)H]-Mibefradil also labelled a heterogenous population of high affinity sites in rabbit brain which was inhibited by a series of nonspecific Ca(2+) and Na(+)-channel blockers. Mibefradil and its analogue RO40-6040 had high affinity for neuronal voltage-gated Na(+)-channels as confirmed in binding (apparent K(i) values of 17 and 1.0 nM, respectively) and functional experiments (40% use-dependent inhibition of Na(+)-channel current by 1 microM mibefradil in GH3 cells). Our data demonstrate that mibefradil binds to voltage-gated L-type Ca(2+) channels with very high affinity and is also a potent blocker of voltage-gated neuronal Na(+)-channels. More lipophilic mibefradil analogues may possess neuroprotective properties like other nonselective Ca(2+)-/Na(+)-channel blockers.     

Influence of stimulation on Ca(2+) recruitment triggering [3H]acetylcholine release from the rat motor-nerve endings

The influence of rat phrenic nerve stimulation frequency (5-50 Hz) and of pulse duration (0.04-1 ms) on Ca(2+) mobilization triggering [3H]acetylcholine release was investigated. The P-type voltage-dependent Ca(2+) channel (VDCC) blocker, omega-agatoxin IVA (100 nM), decreased [3H]acetylcholine release evoked by pulses of 0. 04-ms duration delivered at 5 Hz frequency. When the stimulus pulse duration was increased to 1 ms (5 Hz frequency) or the stimulation frequency to 50 Hz (0.04-ms duration), inhibition of [3H]acetylcholine release became evident after blockade of L-type VDCC, with nifedipine (1 microM), and/or depletion of thapsigargin-sensitive internal stores. The inhibitory effect of thapsigargin (2 microM) was still observed in Ca(2+)-free medium. Neither omega-conotoxin GVIA (1 microM) nor omega-conotoxin MVIIC (150 nM) modified neurotransmitter release. The results suggest that, depending on the stimulus paradigm, both internal (thapsigargin-sensitive) and external (either P- or L-type channels) Ca(2+) pools can be mobilized to promote acetylcholine release from motor nerve terminals.     

Inhibition of Endogenous Acetylcholine Release by Blockade of Voltage-dependent Calcium Channels in Enteric Neurons of the Guinea-pig Colon

Abstract— The effects on acetylcholine release from the guinea-pig colon of the N-type calcium channel blocker ω-conotoxin GVIA (ω-conotoxin), the L-type calcium channel blocker nifedipine and the putative blocker of T-type channels, flunarizine, have been investigated. Endogenous basal acetylcholine release and electrically (1 Hz, 1 ms, 450 mA)-evoked overflow in the presence of cholinesterase inhibitor were studied. ω-Conotoxin (1–10 nM) and nifedipine (0·03–3 μm ) dose-dependently inhibited basal and electrically-evoked acetylcholine release. Maximal inhibition of basal or electrically-evoked acetylcholine release was about 40% for nifedipine and about 75% for ω-conotoxin. The potency of nifedipine was inversely related to the external calcium concentration: its EC50 value in low-calcium medium (0·5 Mm ) was as low as 12 Nm . Flunarizine inhibited acetylcholine release only at concentrations higher than 0·2 μm . Our results are consistent with an involvement of N- and L-type calcium channels in the control of the endogenous acetylcholine release from the guinea-pig colon.     

Ameliorative effect of vasopressin-(4-9) through vasopressin V(1A) receptor on scopolamine-induced impairments of rat spatial memory in the eight-arm radial maze.

In order to clarify the mechanism by which pGlu-Asn-Cys(Cys)-Pro-Arg-Gly-NH(2) (vasopressin-(4-9)), a major metabolite C-terminal fragment of [Arg(8)]-vasopressin (vasopressin-(1-9)), improves learning and memory, we used several different drugs such as an acetylcholine receptor antagonist, a Ca(2+)/calmodulin-dependent protein kinase II inhibitor, vasopressin receptor antagonists and L-type Ca(2+) channel blocker to disrupt spatial memory in rats. Moreover, we examined the effect of vasopressin-(4-9) on acetylcholine release in the ventral hippocampus using microdialysis. Vasopressin-(4-9) (10 fg/brain, i.c.v.) improved the impairment of spatial memory in the eight-arm radial maze induced by scopolamine, pirenzepine and Ca(2+)/calmodulin -dependent protein kinase II inhibitor. Pirenzepine, a vasopressin V(1A) receptor antagonist, and L-type Ca(2+) channel blocker, but not a vasopressin V(2) receptor antagonist, suppressed the effects of vasopressin-(4-9) on scopolamine-induced impairment of spatial memory. Moreover, vasopressin-(4-9) did not affect acetylcholine release in the ventral hippocampus of intact rats or of scopolamine-treated rats as assessed by microdialysis. These results suggest that vasopressin-(4-9) activates vasopressin V(1A) receptors on the postsynaptic membrane of cholinergic neurons, and induces a transient influx of intracellular Ca(2+) through L-type Ca(2+) channels to interact with muscarinic M(1) receptors. The activation of these processes by vasopressin-(4-9) is critically involved in the positive effect of vasopressin-(4-9) on scopolamine-induced impairment of spatial memory.     

Trimethyltin and triethyltin differentially induce spontaneous noradrenaline release from rat hippocampal slices

The environmental contaminants trimethyltin (TMT) and triethyltin (TET) stimulated the spontaneous release of [(3)H]noradrenaline ([(3)H]NA) from hippocampal slices in a time- and concentration-dependent manner. TMT was the most potent compound, exhibiting an EC50 value 10-fold lower (3.8 microM) than that of TET (39.5 microM). Metal-evoked [(3)H]NA release did not increase in the absence of desipramine and was completely blocked by reserpine preincubation, indicating a vesicular origin of [(3)H]NA release but not a mechanism involving reversal of the transmitter transporter. The voltage-gated Na(+) channel blocker tetrodotoxin (TTX) did not affect metal-evoked [(3)H]NA release. [(3)H]NA release elicited by TMT was partially extracellular Ca(2+)-dependent, since it was significantly decreased in a Ca(2+)-free EGTA-containing medium, whereas TET induced an extracellular Ca(2+)-independent release of [(3)H]NA. Neither inhibitors of Ca(2+)-entry through Na(+)/Ca(2+)exchanger and voltage-gated calcium channels, nor agents that interfere with Ca(2+)-mobilization from intracellular stores affected [(3)H]NA release induced by TMT. TET-evoked [(3)H]NA release was reduced by ruthenium red, which depletes mitochondrial Ca(2+)stores, but was not modified by caffeine and thapsigargin, which interfere with Ca(2+)mobilization from endoplasmic reticulum. The fact that TET effect was also attenuated by DIDS, an inhibitor of anion exchange, indicates that the effect of TET on spontaneous [(3)H]NA release may be mediated by intracellular mobilization of Ca(2+) from mitochondrial stores through a Cl(-) dependent mechanism.     

Tyramine reduces glycinergic transmission by inhibiting presynaptic Ca(2+) channels in the rat trigeminal subnucleus caudalis

We have recently reported that tyramine acts on putative presynaptic trace amine receptors to inhibit glycinergic transmission in substantia gelatinosa (SG) neurons of the rat trigeminal subnucleus caudalis. However, it is still unknown how tyramine elicits presynaptic inhibition of glycine release. In the present study, therefore, we investigated cellular mechanisms underlying the tyramine-induced inhibition of glycinergic transmission in SG neurons using a conventional whole-cell patch clamp technique. Tyramine (100 μM) reversibly and repetitively decreased the amplitude of action potential-dependent glycinergic inhibitory postsynaptic currents (IPSCs), and increased the paired-pulse ratio. Pharmacological data suggest that the tyramine-induced decrease in glycinergic IPSCs was not mediated by the modulation of adenylyl cyclase, protein kinase A and C, or G-protein coupled inwardly rectifying K(+) channels. On the other hand, glycinergic IPSCs were mainly mediated by the Ca(2+) influx passing through presynaptic N-type and P/Q-type Ca(2+) channels. The tyramine-induced decrease in glycinergic IPSCs was completely blocked by ω-conotoxin GVIA, an N-type Ca(2+) channel blocker, but not ω-agatoxin IVA, a P/Q-type Ca(2+) channel blocker. The results suggest that tyramine acts presynaptically to decrease action potential-dependent glycine release onto SG neurons via the selective inhibition of presynaptic N-type Ca(2+) channels. This tyramine-induced inhibition of glycinergic transmission in SG neurons might affect the process of orofacial nociceptive signals.     

Convulsant actions of calycanthine

The principal alkaloid of the family Calycanthaceae, calycanthine has long been recognized as a central convulsant. The alkaloid inhibited the potassium-stimulated release of [(3)H]GABA from slices of rat hippocampus with an ED(50) of approximately 21 microM. This effect appeared to be moderately selective since calycanthine at 100 microM had only a weak effect on the potassium-stimulated release of [(3)H]acetylcholine (15%) and no significant effects on the release of [(3)H]D-aspartate from hippocampal and cerebellar slices or the release of [(3)H]glycine from spinal cord slices. Calycanthine blocked the L-type calcium currents with an IC(50) of approximately 42 microM and also weakly inhibited the N-type calcium currents (IC(50) > 100 microM) from neuroblastoma X glioma cells, suggesting voltage-dependent calcium channel blockade as a possible mechanism for its inhibition of GABA and ACh release. Calycanthine was also found to directly inhibit GABA-mediated currents (K(B) approximately 135 microM) from human alpha(1)beta(2)gamma(2L) GABA(A) receptors expressed in Xenopus laevis oocytes but had no effect at 100 microM on human rho(1) GABA(c) receptors. The results indicated that calycanthine may mediate its convulsant action predominantly by inhibiting the release of the inhibitory neurotransmitter GABA as a result of interactions with L-type Ca(2+) channels and by inhibiting GABA-mediated chloride currents at GABA(A) receptors.     

Two different signaling mechanisms involved in the excitation of rat sympathetic neurons by uridine nucleotides

UTP stimulates transmitter release and inhibits M-type K(+) channels in rat superior cervical ganglion neurons via G protein-coupled P2Y receptors. To investigate the underlying signaling mechanisms, we treated the neurons with either pertussis or cholera toxin; neither treatment altered the inhibition of M-type K(+) channels by 10 microM UTP. However, pertussis toxin reduced UTP-evoked [(3)H]noradrenaline release by 66%. UTP, UDP, ATP, and ADP caused accumulation of inositol trisphosphate in a pertussis toxin-insensitive manner. Pharmacological inhibition of inositol trisphosphate-induced Ca(2+) release (by inhibition of phospholipase C, of inositol trisphosphate receptors, and of the endoplasmic Ca(2+)-ATPase) prevented the UTP-dependent inhibition of M currents but failed to alter UTP-evoked [(3)H]noradrenaline release. Chelation of intracellular Ca(2+) by 1,2-bis(2-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid also reduced the inhibition of M currents by UTP. In addition, all these manipulations attenuated the inhibition of M currents by bradykinin, but hardly affected the inhibitory action of oxotremorine M. These results demonstrate that UTP inhibits M-type K(+) channels via an inositol trisphosphate-dependent signaling cascade that is also used by bradykinin but not by muscarinic acetylcholine receptors. In contrast, the secretagogue action of UTP is largely independent of this signaling cascade but involves pertussis toxin-sensitive G proteins. Thus, UTP-sensitive P2Y receptors excite sympathetic neurons via at least two different signal transduction mechanisms.     

Calcium channel subtypes for cholinergic and nonadrenergic noncholinergic neurotransmission in isolated guinea pig trachea

The Ca²⁺ channel subtypes in the neurotransmission of isolated guinea pig trachea were elucidated by monitoring the effects of specific Ca²⁺ channel blockers on cholinergic contractions and nonadrenergic noncholinergic (NANC) relaxation elicited by electrical field stimulation (EFS). In isolated guinea pig trachea, cholinergic contractile responses to low- and high-frequency EFS were inhibited by the selective N-type calcium channel blocker, ω-conotoxin MVIIA. ω-Agatoxin IVA (a selective P-type blocker), ω-conotoxin MVIIC (a nonselective N-, Q-, and P-type blocker), and nifedipine (a selective L-type blocker) were ineffective, whereas Ni²⁺ (a T- and R-type blocker) facilitated cholinergic contractions and produced a late contracture when its concentration exceeded 30 μM. The more the concentration of Ni²⁺ increased, the greater the number of incidences and the late contracture areas which occurred. Late contracture did not seem to be due to the effects of acetylcholine, tachykinins, or other polypeptides, but disappeared in the absence of indomethacin. The NANC relaxant responses elicited by the low- and high-frequency EFS were inhibited by ω-conotoxin MVIIA or Ni²⁺, but unaffected by ω-Agatoxin IVA, ω-conotoxin MVIIC, and nifedipine. In the absence of indomethacin, Ni²⁺ did not alter the ω-conotoxin MVIIA (100 nM)-resistant component of cholinergic contraction, but significantly further inhibited that of NANC relaxation. These results suggest that in isolated guinea pig trachea, cholinergic contraction is regulated by N-type calcium channels which may mask T- and R-type calcium channels and may be co-modulated by both, while NANC relaxation is mainly and independently controlled by N-, T-, and R-type calcium channels.     

Tributyltin-induced Ca(2+) mobilization via L-type voltage-dependent Ca(2+) channels in PC12 cells

The effects of tributyltin (TBT) on cytosolic Ca(2+) concentration ([Ca(2+)](c)) and cell viability were investigated in nerve growth factor-differentiated PC12 cells. TBT concentration dependently increased [Ca(2+)](c) with an EC(50) value of 0.07μM. This effect was markedly reduced by removal of the extracellular Ca(2+) or membrane depolarization with a high K(+) medium, but unaffected by thapsigargin causing depletion of intracellular Ca(2+) stores. The L-type voltage-dependent Ca(2+) channel (VDCC) blocker nicardipine blocked the effect of TBT, but the N-type VDCC blocker ω-conotoxin did not. TBT decreased the number of viable cells with an EC(50) value of 0.09μM. The TBT-induced cell death was prevented by nicardipine or by chelating the cytosolic Ca(2+) with BAPTA-AM, but not by ω-conotoxin. The results show that TBT causes an increase in [Ca(2+)](c) via activating L-type VDCCs, and support the idea that the organotin-induced cell death arises through Ca(2+) mobilization via L-type VDCCs.     

The facilitating effect of prucalopride on cholinergic neurotransmission in pig gastric circular muscle is regulated by phosphodiesterase 4

The influence of the selective 5-HT(4) receptor agonist prucalopride on acetylcholine release from cholinergic nerve endings innervating pig gastric circular muscle and the possible regulation of this effect by phosphodiesterases (PDEs) was investigated, as PDEs have been shown to control the response to 5-HT(4) receptor activation in pig left atrium. Circular muscle strips were prepared from pig proximal stomach and either submaximal cholinergic contractions or tritium outflow after incubation with [(3)H]-choline, induced by electrical field stimulation, were studied. Prucalopride concentration-dependently increased the amplitude of submaximal cholinergic contractions and of acetylcholine release induced by electrical field stimulation. The effect of the highest concentration tested (0.3 μM) on cholinergic contractions was antagonized by the selective 5-HT(4) receptor antagonist GR113808 but not by granisetron or methysergide; the antagonism of prucalopride by GR113808 was confirmed in the release assay. The non-selective PDE-inhibitor 3-isobutyl-methyl-xanthine (IBMX) concentration-dependently reduced the amplitude of the cholinergic contractions; 3 μM IBMX reduced the cholinergic contractions maximally by 16% but it enhanced the facilitating effect of prucalopride from 51 to 83%. IBMX (10 μM) induced and enhanced the facilitating effect of prucalopride on electrically induced acetylcholine release. The selective inhibitors vinpocetine (PDE1), EHNA (PDE2) and cilostamide (PDE3) did not influence the effect of prucalopride on acetylcholine release but the PDE4-inhibitor rolipram (1 μM) enhanced the facilitating effect of prucalopride to the same extent as IBMX. These results demonstrate that 5-HT(4) receptors are present on the cholinergic nerves towards the pig gastric circular muscle, facilitating acetylcholine release; the intracellular transduction pathway of this facilitation is regulated by PDE4. Combination of a 5-HT(4) receptor agonist with selective inhibition of the PDE involved in this regulation of transmitter release might enhance the prokinetic effect of the 5-HT(4) receptor agonist.     

On the mechanism of levosimendan-induced dopamine release in the striatum of freely moving rats

The Ca(2+) sensitizer levosimendan (LEV) improves myocardial contractility by enhancing the sensitivity of the contractile apparatus to Ca(2+). In addition, LEV promotes Ca(2+) entry through L-type channels in human cardiac myocytes. In this study, which was performed using microdialysis, infusion of LEV at 0.25 microM for 160 min increased dopamine (DA) concentrations (up to fivefold baseline) in dialysates from the striatum of freely moving rats. Ca(2+) omission from the perfusion fluid abolished baseline DA release and greatly decreased LEV-induced DA release. Reintroduction of Ca(2+) in the perfusion fluid restored LEV-induced DA release. Chelation of intracellular Ca(2+) by co-infusing 1,2-bis (o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM, 0.2 mM) did not affect basal DA release and scarcely affected LEV-induced increases in dialysate DA. In addition, co-infusion of the L-type (Ca(v) 1.1-1.3) voltage-sensitive Ca(2+)-channel inhibitor nifedipine failed to inhibit LEV-induced increases in dialysate DA, which, in contrast, was inhibited by co-infusion of the N-type (Ca(v) 2.2) voltage-sensitive Ca(2+)-channel inhibitor omega-conotoxin GVIA. We conclude that LEV promotes striatal extracellular Ca(2+) entry through N-type Ca(2+) channels with a consequent increase in DA release.     

Therapeutically relevant concentrations of neomycin selectively inhibit P-type Ca2+ channels in rat striatum

The effects of neomycin on voltage-activated Ca(2+) channels (VACCs) were studied by Ca(2+)-dependent K(+)- and veratridine-evoked [3H]dopamine release from rat striatal slices. Neomycin (0.01-1 mM) concentration dependently reduced K(+)-evoked [3H]dopamine release (IC(50) approximately 25 microM), producing approximately 98% inhibition at 1 mM. Contribution of N-, P- and Q-type Ca(2+) channels to this neomycin-sensitive [3H]dopamine release was tested by the combined application of 100 microM neomycin and selective Ca(2+) channel blockers. The effects of neomycin combined with 1 microM of omega-conotoxin GVIA (N-type Ca(2+) channels) or with 100 nM of omega-conotoxin MVIIC (Q-type Ca(2+) channels) were additive, excluding involvement of N- and Q-type Ca(2+) channels. However, the combined effects of neomycin with 30 nM of omega-agatoxin-IVA (P-type Ca(2+) channels) were not additive, suggesting involvement of P-type Ca(2+) channels in neomycin-induced inhibition of [3H]dopamine release. On the other hand, veratridine-evoked [3H]dopamine release was shown to be mediated by Q-type Ca(2+) channels only. In addition, neither the inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase thapsigargin (500 nM) nor the blocker of sarcoplasmic reticulum ryanodine Ca(2+) channels ryanodine (30 microM) modulate veratridine-evoked [3H]dopamine release, suggesting no contribution of intracellular Ca(2+) stores. Neomycin (up to 100 microM) did not affect veratridine-evoked [3H]dopamine release, suggesting that intracellular Ca(2+) stores are not a prerequisite for the action of neomycin. Lack of inhibitory effect of neomycin is taken as additional indirect evidence for the involvement of P-type Ca(2+) channels. In conclusion, therapeutically relevant concentrations of neomycin preferentially block P-type Ca(2+) channels which regulate dopamine release in rat striatum. This block could be responsible for aminoglycoside-induced toxicity.     

Inositol 1,4,5-trisphosphate receptor-mediated initial Ca(2+) mobilization constitutes a triggering signal for hydrogen peroxide-induced apoptosis in INS-1 β-cells

Reactive oxygen species, including hydrogen peroxide (H(2)O(2)), are known to induce β-cell apoptosis. The present study investigated the role of Ca(2+) in H(2)O(2)-induced apoptosis of the β-cell line INS-1. Annexin V assay with flow cytometry and DNA ladder assay demonstrated that treatment of INS-1 cells with 100 μM H(2)O(2) for 18 h significantly increased apoptotic cells. A comparable level of apoptosis was also observed after 18 h when the cells were treated with 100 μM H(2)O(2) only for initial 30 min. The H(2)O(2)-induced apoptosis was abolished by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA/AM), a chelator of intracellular Ca(2+), by 2-aminoethoxydiphenylborate (2-APB), a blocker of inositol 1,4,5-trisphosphate (IP(3)) receptors and cation channels, and by xestospongin D, a blocker of IP(3) receptors, and was partially blocked by SKF-96365, a non-selective cation channel blocker. However, nicardipine, an L-type voltage-dependent Ca(2+) channel blocker, or N-(p-amylcinnamoyl)anthranilic acid (ACA), a TRPM2 blocker, had little effect on the apoptosis. The inhibitory effect of BAPTA/AM or 2-APB on the H(2)O(2)-induced apoptosis was largely attenuated when the drug was added 30 min or 1 h after start of the treatment with H(2)O(2). These results suggest that the initial intracellular Ca(2+) elevation induced by H(2)O(2), which is mediated via IP(3) receptors and store-operated cation channels, plays an obligatory role in the induction of β-cell apoptosis.     

H3 receptor-mediated inhibition of intestinal acetylcholine release: pharmacological characterization of signal transduction pathways

The present study investigates the mechanisms through which prejunctional histamine H3 receptors modulate intestinal cholinergic neurotransmission. The experiments were performed on longitudinal muscle-myenteric plexus preparations of guinea pig ileum, preincubated with [3H]choline, superfused with physiological salt solution containing hemicholinium-3, and subjected to electrical field stimulation. The stimulation-induced outflow of radioactivity was taken as an index of endogenous acetylcholine release. The electrically induced [3H]acetylcholine release was inhibited by histamine (EC50)=33.5 nM) or the H3 receptor agonist R-alpha-methylhistamine (EC50=41.6 nM), whereas it was not affected by pyridylethylamine (H1 agonist), impromidine (H2 agonist), pyrilamine (H1 antagonist), cimetidine (H2 antagonist), thioperamide or clobenpropit (H3 antagonists). The inhibitory effects of histamine or R-alpha-methylhistamine were antagonized by thioperamide (pKd= 8.31 and 8.53, respectively) or clobenpropit (pKd=9.44 and 9.32, respectively), but not by pyrilamine or cimetidine. The modulatory action of histamine on the evoked tritium outflow was attenuated by pertussis toxin and abolished by N-ethylmaleimide, two selective blockers of Gi/Go proteins. Tetraethylammonium or 4-aminopyridine, acting as inhibitors of voltage-dependent K+ channels, enhanced the evoked tritium outflow when tested alone, and apparently counteracted the inhibitory effect of histamine. However, the blocking actions of tetraethylammonium and 4-aminopyridine were no longer evident when their enhancing actions were compensated by appropriate reductions of Ca2+ concentration in the superfusion medium. Histamine-induced inhibition of evoked tritium output was enhanced by omega-conotoxin, a selective blocker of N-type Ca2+ channels, or low Ca2+ concentration, whereas it was not modified by nifedipine, an antagonist of L-type Ca2+ channels. In addition, the inhibitory effect of histamine was not significantly affected by forskolin (activator of adenylyl cyclase), 8-bromo-cyclic AMP (a stable analog of cyclic AMP), rolipram (a selective blocker of type IV phosphodiesterase), phorbol myristate acetate (activator of protein kinase C), H-89 (N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide, inhibitor of protein kinase A), Ro-31-8220 (2-(1-[3-(amidinothio)propyl]-1H-indol-3-yl)-3-(1-methylindol-3-yl)-maleimide, inhibitor of protein kinase C), KT5823 (N-methyl-(8R*,9S*,11S*)-(-)-9-methoxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo [a,g]cycloocta[c,d,e]-trinden-1-one, inhibitor of protein kinase G), or lavendustin A (inhibitor of tyrosine kinase). The present results indicate that histamine inhibits intestinal cholinergic neurotransmission through presynaptic H3 receptors coupled to Gi/Go proteins. It is suggested that adenylyl cyclase, serine-threonine protein kinase and tyrosine kinase pathways are not implicated in this regulatory action, and that Gi/Go proteins modulate the activity of N-type Ca2+ channels through a direct link, thus causing a reduced availability of extracellular Ca2+ at the level of ileal cholinergic nerve terminals.     

Suppression by cholinesterase inhibition of a Ca(2+)-independent efflux of [3H]acetylcholine from the neuromuscular junction of the isolated rat diaphragm.

Endplate preparations of the left rat hemidiaphragm were incubated with [3H]choline to label neuronal acetylcholine stores. Elevation of the concentration (13.5-135 mmol/l) of extracellular potassium chloride (KCl) stimulated the release of [3H]acetylcholine in a concentration-dependent manner. KCl (27 mmol/l) still caused a significant efflux of [3H]acetylcholine in a Ca(2+)-free medium. Inhibitors of cholinesterase (physostigmine, diisopropylfluorophosphate) suppressed by 80% this Ca(2+)-independent efflux of [3H]acetylcholine. Vesamicol (10 mumol/l), the blocker of the vesicular acetylcholine carrier, also suppressed the stimulated, Ca(2+)-independent efflux of [3H]acetylcholine. The inhibitory effect of physostigmine was not prevented by muscarine or nicotine receptor antagonists, but the inhibitory effect was lost when the stimulus strength was increased (81 mmol/l KCl). The present experiments showed cholinesterase inhibition to suppress a Ca(2+)-independent efflux of [3H]acetylcholine, probably by interference with a membrane-bound acetylcholine carrier.     

Algogen-specific pain processing in mouse spinal cord: differential involvement of voltage-dependent Ca(2+) channels in synaptic transmission

1. The effects of intrathecal (i.t.) administration of N-, P/Q- or L-type voltage-dependent Ca(2+)-channel blockers were tested in two pain models involving bradykinin (BK)- and alpha,beta-methylene ATP (alpha,beta meATP)-induced activation of primary afferent neurons in mice. 2. The nociceptive response (amount of time spent licking and biting the hindpaw) induced by intraplantar injection of BK (500 pmol mouse(-1)) was significantly attenuated by both omega-conotoxin GVIA (N-type blocker) and calciseptine (L-type) but not by omega-agatoxin IVA (P/Q-type). 3. The nociceptive response induced in a similar way by alpha,beta meATP (100 nmol) was significantly inhibited by both the above N- and P/Q-type Ca(2+)-channel blockers but not by the L-type blocker. 4. The nociceptive responses elicited by BK and alpha,beta meATP were dose-dependently inhibited by a tachykinin-NK1-receptor antagonist (L-703,606) and an N-methyl-D-aspartate (NMDA)-receptor antagonist (D-AP5), respectively. 5. Intrathecal administration of substance P (SP) (1.8 nmol) or NMDA (350 pmol) elicited algesic responses, such as licking, biting and scratching of the hindquarters. The SP-induced algesic behaviour was significantly inhibited by the L-type blocker but not by the N-type. The NMDA-induced response was not affected by either the N- or the P/Q-type blocker. 6. These findings suggest that BK and ATP most likely excite different types of sensory neurons in the periphery and that within the spinal cord the former stimulates peptidergic transmission regulated by presynaptic N- and postsynaptic L-type Ca(2+) channels, while the latter stimulates glutamatergic transmission regulated by presynaptic N- and P/Q-type channels.     

Influence of nitric oxide donors and of the alpha(2)-agonist UK-14,304 on acetylcholine release in the pig gastric fundus

This study in circular muscle strips of the pig gastric fundus aimed to measure the release of acetylcholine directly and to investigate whether NO and alpha(2)-adrenoceptor agonists can modulate acetylcholine release from cholinergic neurones. After incubation of the tissues with [(3)H]-choline, basal and electrically induced release of tritium and [(3)H]-acetylcholine were analyzed in a medium containing physostigmine (10(-5) M) as well as atropine (10(-6) M). The NO synthase inhibitor L-N(G)-nitroarginine methyl ester (3x10(-4) M), and the NO donors sodium nitroprusside (10(-5) M) and 3-morpholino-sydnonimine (10(-5) M) did not influence the basal release nor the electrically evoked release, indicating that NO does not modify [(3)H]-acetylcholine release. The alpha(2)-adrenoceptor agonist UK-14,304 (10(-5) M) significantly inhibited the electrically evoked release of [(3)H]-acetylcholine, and this effect was prevented by the alpha(2)-adrenoceptor antagonist rauwolscine (2x10(-6) M), suggesting that presynaptic alpha(2)-adrenoceptors are present on cholinergic neurones of the pig gastric fundus.     

Functional proteins involved in regulation of intracellular Ca(2+) for drug development: chronic nicotine treatment upregulates L-type high voltage-gated calcium channels

Neurochemical mechanisms underlying drug dependence and withdrawal syndrome remain unclear. In this review, we discuss how chronic nicotine exposure to neurons affects expression of diazepam binding inhibitor (DBI), an endogenous anxiogenic neuropeptide supposed to be a common substance participating drug dependence, and function of L-type high voltage-gated Ca(2+) channels (HVCCs). We also discuss the functional interaction between DBI and L-type HVCCs in nicotine dependence. Both DBI levels and [(45)Ca(2+)] influx significantly increased in the brain from mice treated with nicotine for long term, which was further enhanced after abrupt cessation of nicotine and was abolished by nicotinic acetylcholine receptor (nAChR) antagonists. Similar responses of DBI expression and L-type HVCC function were observed in cerebral cortical neurons after sustained exposure to nicotine. In addition, increased DBI expression was inhibited by antagonists of nAChR and L-type HVCCs. Sustained exposure of neurons to nicotine significantly enhanced expression of alpha(1) and alpha(2)/delta(1) subunits for L-type HVCCs and caused an increase in the B(max) value of [(3)H]verapamil binding to the particulate fractions. Therefore, it is concluded that the alterations in DBI expression is mediated via increased influx of Ca(2+) through upregulated L-type HVCCs and these neurochemical changes have a close relationship with development of nicotine dependence and/or its withdrawal syndrome.