A role for Q type Ca2+ channels in neurotransmission in the rat urinary bladder.

1. In isolated bladder strips of the rat, a substantial component (46%) of the Ca(2+)-dependent contractile response to electrical field stimulation (5 Hz) was resistant to combined block of both N and P type Ca2+ channels by omega-conotoxin-GVIA (300 nM) and omega-agatoxin-IVA (100 nM) respectively. 2. The resistant portion (non-N, non-P) was sensitive to omega-conotoxin-MVIIC (3 microM), which in addition to N and P also blocks Q type channels at this concentration. omega-Conotoxin-MVIIC administered alone, inhibited the neurogenic response to the same degree as that observed in the combined presence of omega-agatoxin-IVA, omega-conotoxin-GVIA and omega-conotoxin-MVIIC. 3. omega-Agatoxin-IVA (100 nM), a concentration that fully inhibits P type channels, had a negligible effect on the neurogenic response. Following blockade of N type Ca2+ channels with omega-conotoxin-GVIA (300 nM), omega-agatoxin-IVA (3 microM) (a concentration well above that used to block P channels, inhibits Q type channels, but spares N type channels), inhibited the residual response to the same degree as omega-conotoxin-MVIIC alone. 4. Results suggest that neurotransmission in rat urinary bladder is supported by both N and Q type Ca2+ channels.

Effect of ?-conotoxin GVIA on Ca(2+) influx and endogenous acetylcholine release from chicken brain preparations.

?-Conotoxin GVIA (?-conotoxin) is an inhibitor of neuronal voltage sensitive calcium channels (VSCCs). We have examined the effects of various manipulations aimed at elucidating the effects of ?-conotoxin particularly on K(+) stimulated (45)Ca(2+) influx and endogenous acetylcholine (ACh) release. The initial phase of K(+) stimulated (45)Ca(2+) influx was accompanied by a large rapid release of ACh measured by a modification of the chemiluminescent technique of Israël and Lesbats (J. Neurochem.39, 248-250, 1982). ACh release was greatly reduced (?90% ) in Ca(2+) free solution or by Cd(2+) (100 ?M) period ?-conotoxin (1 ?M) completely blocked the initial phase of (45)Ca(2+) influx (1-3 s) from synaptosomes and reduced K(+) stimulated ACh release from tissue slices. ?-Conotoxin (0.1 ?M) reduced ACh release which was reversed in the presence of high Ca(2+) concentrations. The dihydropyridine VSCC inhibitor (?)202-791 or agonist (+)202-791 had no effect on ACh release or (45)Ca(2+) influx. The pattern of responses on (45)Ca(2+) influx and endogenous ACh release was consistent with the suggestion that ?-conotoxin inhibits N-type VSCCs and that these channels are intimately related to ACh release.

Pharmacological identification of a novel Ca2+ channel in chicken brain synaptosomes.

Ca2+ influx was measured in rat and chicken brain synaptosomes in the presence of a number of pharmacological tools which have recently been used to define voltage-sensitive Ca(2+)-channel (VSCC) types. In chicken brain synaptosomes. VSCCs which, because of their sensitivity to inhibition by omega-conotoxin (omega-CgTx), are thought to be exclusively N-type, the P-type VSCC polyamine inhibitor FTX (from Agelenopsis aperta venom; 1 microliters/ml), its synthetic analogue, sFTX (1-5 mM) and the polypeptides AgaIVA (IC50 0.29 microM) and omega-CgTx MVIIC (IC50 0.0022 microM) inhibited 70-100% of the measurable K+ stimulated Ca2+ influx. The prototypical N-channel VSCC inhibitor omega-CgTx GVIA (IC50 0.014 microM), Cd2+ (50 microM) and diluted venom from Hololena curta (1:2,500) also caused complete or almost complete, inhibition of Ca2+ influx. In comparable studies using rat brain synaptosomes, sFTX (1-10 mM) caused a dose-dependent reduction of Ca2+ influx, while FTX (1 microliters/ml) and AgaIVA (IC50 0.02 microM) completely inhibited Ca2+ influx. Similar to the findings in chicken synaptosomes, Cd2+ (50 microM) and H. curta (1:2,500 dilution) both inhibited K+ stimulated influx by > 80% whereas omega-CgTx (1 microM) only caused a maximum 25% inhibition. Both sFTX and its congener spermine, inhibited [125I]omega-CgTx binding to rat and chicken synaptosomal membranes. These results strongly implicate P-type channels as the major VSCC in rat brain. The results also clearly demonstrate a heretofore unrecognized, novel, FTX/AgaIVA/omega-CgTx GVIA/omega-CgTx MVIIC-sensitive VSCC in chicken brain.

Mechanism of the cardiovascular activity of dibenzoxazepine in cats.

Small i.v. doses of dibenzoxazepine (DBO) (50--400 microgram/kg) given to anesthetized cats resulted in dose related increases in heart rate (up to 70 beats/min) and blood pressure (up to 80 mm Hg). The pressor response was blocked by pretreatment of the animals with phentolamine; pretreatment for 3 days with 6-hydroxdopamine; with mecamylamine and spinal transection between C1 and C2 but not by propranolol or adrenalectomy. The increase in heart rate was blocked by pretreatment with propranolol, 6-hydroxydopamine, mecamylamine and spinal transection whereas adrenalectomy only affected the response slightly. DBO produced only negative effects on the isolated rabbit heart. Bioassay of arterial blood showed an increased level of circulating catecholamines corresponding to the cardiovascular stimulation. DBO had no tyramine-like activity on the isolated rabbit aortic strip but slightly potentiated the contraction induced by noradrenaline. These findings strongly suggest that the cardiovascular effects resulted from central stimulation of the sympathetic nervous system. A minor part of the observed sympathomimetic effects may also be the result of the ability of DBO to potentiate the effects of noradrenaline perhaps by blocking catecholamine uptake.

Arylazido aminopropionyl ATP (ANAPP3): interaction with adenosine receptors in longitudinal smooth muscle of the guinea-pig ileum.

Arylazido aminopropionyl ATP (ANAPP3), an ATP-receptor antagonist containing a photosensitive arylazido moiety coupled to the 3' hydroxyl of ATP, inhibited the twitch response of electrically stimulated ileal longitudinal muscle strips in a dose-dependent manner. These agonist responses to ANAPP3 were attenuated by the enzyme adenosine deaminase and antagonized by the adenosine receptor antagonist 8-phenyltheophylline. Schild analysis yielded similar pA2 values for ANAPP3 and adenosine suggesting a common receptor site. Several 3'-ribose-modified adenosine analogs were tested for agonist activity and found to be inactive. Results suggest that ANAPP3 interacts at the presynaptic adenosine receptor of the ileum following its metabolism to adenosine, which explains the lack of antagonism at adenosine receptors of ileal smooth muscle following photolysis of ANAPP3.

Evidence of omega-conotoxin GV1A-sensitive Ca2+ channels in mammalian peripheral nerve terminals.

The existence of omega-conotoxin GV1A (omega-CgTx)-sensitive, voltage-sensitive Ca2+ channels (VSCCs) in mammalian peripheral nerves was investigated in the guinea pig ileum myenteric plexus longitudinal smooth muscle preparation (GPI). omega-CgTx (0.01-1.0 microM) reduced the electrically stimulated GPI twitch height, failed to modify exogenously applied acetylcholine (ACh) contractions, and inhibited Ca2+-dependent KCl-stimulated ACh release as measured by chemiluminescence. The 1,4-dihydropyridine VSCC antagonist (-) 202-791 (0.1-1.0 microM) inhibited the GPI twitch height, reduced contractions to exogenous ACh, but failed to affect ACh release. In the rat aorta, a nerve free preparation, omega-CgTx failed to affect contractions to KCl which were inhibited by (-) 202-791 and potentiated by the VSCC agonist (+) 202-791. The results provide evidence of neuronal N type VSCCs in mammalian peripheral cholinergic nerve terminals.

The effect of neuropeptide Y on voltage-sensitive Ca2+ channels.

Neuropeptide Y (NPY) (50-1000 nM) failed to modify basal or K(+)-stimulated Ca2+ influx in cortical or hippocampal synaptosomes from rat brain, whereas the voltage-sensitive Ca2+ channel (VSCC) blocker Cd2+ (50 microM) caused major inhibition. In cortical synaptosomes from chicken brain NPY (1.0 microM) failed to modify, whereas omega-conotoxin GV1A (0.1 microM) markedly inhibited Ca2+ influx. NPY does not appear to modify synaptosomal Ca2+ influx, however it may still affect VSCCs spatially distinct or 'upstream' from the nerve terminals.

Ganglion blocking properties of some bispyridinium soman antagonists.

Various doses of several bispyridinium compounds (HS-6, HI-6, HGG-12, HGG-42, and SAD-128) known to protect animals against the irreversible cholinesterase inhibitor soman were examined to determine their effects on the cardiovascular and respiratory system of cats. Although the potency varied considerably all of the compounds tested lowered the blood pressure, which appeared to be the result of ganglion blocking properties as determined by their reduction of the pressor response to dimethylphenylpiperazinium and the blockage of the contraction of the preganglionically stimulated cat nictitating membrane. Some of the compounds caused cessation of respiration at much lower doses than others but did so at doses greater than those causing ganglion blockage.

Effect of omega-agatoxin-IVA on autonomic neurotransmission.

omega-Agatoxin-IVA, a peptide from the venom of the funnel-web spider Agelenopsis aperta and a P type Ca2+ channel inhibitor, was examined for effects on responses to nerve stimulation in isolated autonomic neuroeffector preparations from the rabbit, guinea-pig and rat. Ca(2+)-dependent, tetrodotoxin sensitive, noradrenergic excitatory responses of rabbit pulmonary artery, rat vas deferens, and anococcygeus muscles, and cholinergic guinea-pig myenteric plexus preparations (all highly sensitive to the N type Ca2+ channel inhibitor omega-conotoxin-GVIA) were unaffected by omega-agatoxin-IVA (100 nM). Similarly, the neurogenic response of rat bladder, which has cholinergic, and non-adrenergic non-cholinergic (NANC) excitatory components, and the NANC inhibitory response of rat jejunum (atropine 0.5 microM- and guanethidine 5.0 microM-treated), which are partially sensitive and insensitive to omega-conotoxin-GVIA, respectively, were unaffected by omega-agatoxin-IVA (100 nM). Neurogenic NANC inhibitory responses of the guinea-pig taenia caecum, and rat anococcygeus muscles (atropine- and guanethidine-treated, and tone raised with prostaglandin F2 alpha), were also insensitive to omega-agatoxin-IVA. These results suggest that P type Ca2+ channels, if present, play an insignificant role in supplying the Ca2+ necessary for neurotransmitter release in the peripheral autonomic nervous system.

Evidence against ATP being the nonadrenergic, noncholinergic inhibitory transmitter in guinea pig stomach.

THe adenosine-sensitive P1 purinoceptor antagonists 8-phenyl theophylline and 8-(p-bromophenyl)theophylline (4 microM) antagonized ATP-induced relaxation of spontaneous tone in guinea pig stomach, but caused no significant modification of relaxation to electrical field stimulation (0.2 - 10 Hz, 1 msec for 30 sec, atropine 1.5 microM, and guanethidine 4 microM treated). These results suggest that in fundic muscle ATP acts via hydrolysis to adenosine, with subsequent activation of P1 purinoceptors, and that ATP is not the nonadrenergic, noncholinergic inhibitory transmitter in stomach fundus.

Pharmacological characterization of voltage-sensitive Ca2+ channels in autonomic nerves.

Hololena curta venom a potent inhibitor of voltage sensitive Ca2+ channels and neurotransmitter release in mammalian brain, and synthetic funnel web spider toxin an inhibitor of P channels, were examined for their activity on autonomic nerves. Hololena curta (0.5 to 5.0 micrograms venom protein/ml) potently inhibited motor responses of the cholinergic guinea pig ileum myenteric plexus and the adrenergic rat anococcygeus muscle. Synthetic funnel web spider toxin was inactive at concentrations up to 100 microM. Hololena curta inhibited K+, and electrically evoked release of tritium from labeled superfused tissues. Furthermore, K(+)-contracted rat aorta was not relaxed by Hololena curta thereby precluding effects of Hololena curta on postjunctional L type smooth muscle Ca2+ channels. The pattern of effects of Hololena curta on peripheral autonomic nerves was similar to the N channel inhibitor omega-conotoxin GVIA. These results suggest that Hololena curta venom constituents block Ca2+ channels in peripheral autonomic nerves. The study failed to establish the presence of functional P type Ca2+ channels on these peripheral autonomic nerves and further suggests that N type channels may be exclusively responsible for supplying the Ca2+ necessary for neurotransmitter release in these nerves.

Inhibition of a dihydropyridine, omega-conotoxin insensitive Ca2+ channel in rat synaptosomes by venom of the spider Hololena curta.

Inhibition of the N and L type Ca2+ channels with omega conotoxin GVIA (omega-CgTx) together with the dihydropyridine (-)-202-791 produces slight reduction (congruent to 25%) of K(+)-evoked Ca2+ influx in mammalian synaptosomes. These results and others suggest the existence of a third high threshold voltage sensitive calcium channel (VSCC) responsible for the majority of influx. Venom from the funnel web spider Hololena curta potently and persistently inhibited Ca2+ influx in rat cortical synaptosomes (IC50 1:10,000 or 4.21 micrograms/venom protein/ml of synaptosomes). Also Ca2+ influx in cerebellar synaptosomes was inhibited in a similar manner. K(+)-evoked tritium release from synaptosomes labeled with [3H]noradrenaline was inhibited by Hololena venom (congruent to 60% reduction at 10 micrograms/venom protein). Inhibition of Ca2+ influx by venom was unaffected by combined omega-CgTx and (-)-202-791 pretreatment (both 1 microM). Hololena venom and its active constituent should provide useful tools to investigate the role of this novel Ca2+ channel in neuronal function.

Pharmacological evidence for an omega-conotoxin, dihydropyridine-insensitive neuronal Ca2+ channel.

Inactivation of N-type voltage-sensitive Ca2+ channels (VSCC) with omega-conotoxin (omega-CgTx) in tissue obtained from chicken brain produces a concentration dependent (0.01-0.1 microM) inhibition of K(+)-stimulated Ca2+ influx (delta K+), the rise in [Ca2+]i and acetylcholine (ACh) release. In identical preparations from rat brain, Ca2+ influx and the rise in [Ca2+]i were only marginally affected by much higher (1-10 microM) concentrations of omega-CgTx. The release of ACh, however, was inhibited to the same degree with similar amounts of omega-CgTx as those used in chicken brain. An L-type VSCC inhibitor failed to affect any of these parameters alone, or to augment the effect of omega-CgTx. The results suggest that almost all the VSCC in chicken brain are of the N type and that these channels regulate neurotransmitter release. In rat brain, on the other hand, Ca2+ channels resistant to N- or L-type blockers account for almost 75% of the measurable Ca2+ influx and rise in [Ca2+]i. The conspicuous dissociation between the regulation of Ca2+ influx and ACh release demonstrated in rat brain by using omega-CgTx, suggest that neurotransmitter release is governed by only a small proportion of strategically located N-type, omega-CgTx sensitive, VSCC in the presynaptic terminal.

Evidence of mammalian Ca2+ channel inhibitors in venom of the spider Plectreurys tristis.

Plectreurys tristis venom inhibited K(+)-stimulated Ca2+ influx in a concentration-dependent manner in rat (0.5-4.0 micrograms venom protein/ml) and chicken (1.0-64.0 micrograms venom protein/ml) brain synaptosomes. In contrast to Hololena curta venom or omega conotoxin GVlA which both show selectivity for avian synaptosomes, inhibition of Ca2+ influx by the venom appeared to be relatively selective for rat synaptosomes. Plectreurys tristis venom also inhibited K(+)-evoked release of [3H](-)-noradrenaline from labeled rat cortical synaptosomes. Responses to electric field stimulation of the sympathetically innervated rat vas deferens in vitro were inhibited by Plectreurys tristis venom at dilutions similar to those which inhibited Ca2+ influx in synaptosomes. Inhibition persisted following washout of the venom. K(+)-evoked contractions of rat aortic rings were relaxed by the dihydropyridine antagonist (-)-202-791, but not by Plectreurys tristis venom, thus precluding an effect on K(+)-depolarized smooth muscle L-type channels. Contractions to exogenous (-)-noradrenaline in rat aorta were not inhibited by Plectreurys tristis venom, ruling out an effect on alpha 1-adrenergic receptors, and further suggesting a prejunctional site of action. The results suggest that this venom inhibits N-type Ca2+ channels, as well as unclassified Ca2+ channels, which are neither N- nor L-type.

Identification of noradrenaline in venom from the funnel-web spider Hololena curta.

Venom from the funnel-web spider Hololena curta was added to the Krebs-Henseleit solution bathing isolated ring preparations of rat thoracic aorta, suspended in water-jacketed organ baths, for the purpose of tension recordings. Hololena curta venom at dilutions of 1:100,000 to 1:1000 caused a marked vasoconstriction, which was completely inhibited by the alpha 1 adrenoceptor antagonist prazosin (1 microm). The vasoconstriction appears to be due to the direct effects on alpha 1 adrenoceptors of a venom constituent, which we have identified using HPLC/ECD as the catecholamine, noradrenaline.

Comparison of several oximes against poisoning by soman, tabun and GF.

Three oximes currently being evaluated for adoption as replacement nerve agent therapy by various countries were compared for therapeutic efficacy against the toxic organophosphate inhibitors soman and tabun under a standard set of conditions. These oximes together with PAM-Cl and toxogonin, were also compared for efficacy against GF, an agent weaponized by Iraq. The order of effectiveness against soman was HI-6 greater than HLö-7 greater than pyrimidoxime. HLö-7 was very effective against tabun poisoning while HI-6 and pyrimidoxime were of moderate value. Against GF, HI-6 and HLö-7 were extremely effective, toxogonin was moderately effective, and PAM-Cl and pyrimidoxime were the least effective. HI-6 provided a high level of protection against all of the agents tested as did HLö-7 to a slightly lesser degree. The other oximes suffered from their lack of effects against one or more of the organophosphates.

Effect of arylazido aminopropionyl ATP (ANAPP3), on ATP responses of isolated guinea pig smooth muscle.

Arylazido aminopropionyl ATP (ANAPP3), a photoaffinity analogue of adenosine 5'-triphosphate, photoactivated with visible light (+hv), specifically and irreversibly antagonized ATP contractions of the guinea pig vas deferens. ANAPP3 (30 microM) antagonized responses to exogenously added ATP in untreated, and in tissues pretreated with indomethacin (2.9 microM) and 6-(2-hydroxy-5-nitrobenzyl)-thio guanosine (10 microM). It was of interest to see if this pharmacological antagonist of ATP could be used to assess the validity of the purinergic nerve hypothesis by allowing a differentiation between, or proof of the identity of, responses to ATP and the non-adrenergic inhibitory transmitter in guinea pig stomach fundus. After photoactivation (+hv) in the organ bath and subsequent washout, ANAPP3 (30 and 100 microM) failed to antagonize relaxant responses to ATP (1.0 - 1000 microM) in fundic strips. In addition ANAPP3 failed to antagonize ATP-induced inhibition of the twitch response in electrically stimulated guinea pig ileum longitudinal muscle strips. We conclude that ANAPP3 does not antagonize all actions of ATP, which may limit its usefulness in assessing the above hypothesis. Results with this compound suggest that ATP excitatory receptors may differ from those mediating relaxation and other ATP actions.

Modification of cytosolic free calcium concentrations in human keratinocytes after sulfur mustard exposure.

Exposure of confluent cultures of human skin keratinocytes to sulfur mustard (SM) induced an immediate and irreversible rise in internal free Ca(2+) levels that was independent of external Ca(2+) concentrations. The response was rapid, beginning within 1min after addition of SM to the cells, and sensitive, with significant effects observed at 100 mum. The rise in [Ca(2+)](INT) was unaffected by zero external Ca(2+) but was blocked by prior incubation with thapsigargin. The sensitivity to and irreversibility of the effects of SM on Ca(2+) levels was paralleled by cellular toxicity as assessed using three different cell viability assays. In addition, the time course of the onset of irreversible toxicity in our cultures coincides with the time course of effects on [Ca(2+)](INT). SM was also found to displace specifically bound ATP from purinergic (P(2)) receptors. These results suggest that therapies aimed at protecting internal stores of Ca(2+) from disruption by SM, perhaps at P(2) receptors, may provide substantial benefit in protecting human skin cells from the toxic effects of this vesicant.

A comparison of the effects of sarin and succinylcholine on respiratory parameters in anesthetized domestic swine.

Differences in the "respiratory paralysis" caused by sarin (GB) and succinylcholine (SDC) were observed in a domestic swine model using a bedside pulmonary dynamics monitor. GB was administered intravenously (9 micrograms/kg/30 min) and compared with SDC administered intravenously (20 mg/30 min). All animals developed respiratory insufficiency indicated by decreased respiratory frequency. Minute ventilation was relatively maintained in animals that received GB by increasing tidal volume, whereas both of these parameters decreased in animals that received SDC. GB animals showed an increase in airway resistance and work of breathing. The former was unchanged and the latter was decreased in animals that received SDC. Mouth occlusion pressure at 100 milliseconds and tidal volume were relatively maintained in GB animals but decreased in SDC animals, suggesting a central mechanism for respiratory paralysis with GB and a peripheral mechanism for respiratory paralysis with SDC.