The chemotherapeutic oxaliplatin alters voltage-gated Na(+) channel kinetics on rat sensory neurons

The chemotherapeutic oxaliplatin causes a sensory-motor neuropathy with predominantly hyperpathic symptoms. The mechanism underlying this hyperexcitability was investigated using rat sensory nerve preparations, dorsal root ganglia and hippocampal neurons. Oxaliplatin resulted in an increase of the amplitude and duration of compound action potentials. It lengthened the refractory period of peripheral nerves suggesting an interaction with voltage-gated Na(+) channels. Application of oxaliplatin to dorsal root ganglion neurons resulted in an increase of the Na(+) current, a block of the maximal amplitude and a shift of the voltage-response relationship towards more negative membrane potentials. The effect was detectable on 13 of 18 tested cells. This observation, together with the absence of any effect on Na(+) currents of hippocampal neurons, suggests that the interaction of oxaliplatin is restricted to one or more channel subtypes. The effect of oxaliplatin could be antagonised by the Na(+) channel blocker carbamazepine which could be used to reduce side effects of oxaliplatin therapy in patients.     

Specific H+/K(+)-ATPase inhibitors decreased contractile responses of isolated rat vas deferens.

The effect of H(+)/K(+)-ATPase inhibitors on rat vas deferens contractility was investigated in vitro. Omeprazole (100-300microM), lansoprazole (100-300microM) and SCH 28080 (10-100microM) (2-methyl-8-(phenylmethoxy)-imidazo[1,2-a]pyridine-3-acetonitrile) decreased contractile responses of vas deferens to electrical field stimulation, high K(+) (80mM) and phenylephrine in a reversible, reproducible and concentration-dependent manner. The inhibitory potency of lansoprazole on vas deferens contractility was increased in relatively acidic solution (pH 6.9), suggesting that the site of action may be related to H(+)/K(+)-ATPase. However, lansoprazole-induced inhibition on contractility was unaltered in K(+) free solution, indicating that the mechanism of action is independent from H(+)/K(+)-ATPase. Reversible nature of omeprazole and lansoprazole-induced inhibition on contractility also suggests that the effects are not due to inhibition of H(+)/K(+)-ATPase, since both compounds are irreversible inhibitors of the enzyme. Presence of ouabain (5microM) did not decrease lansoprazole-induced inhibition on contractility but potentiated the inhibitory effect of lansoprazole, suggesting that lansoprazole-induced inhibition is not mediated by the inhibition of Na(+)/K(+)-ATPase. Calcium-induced contractions in high K(+)-Ca(2+) free medium were completely antagonized by lansoprazole, implying that lansoprazole inhibits Ca(2+) entry through voltage-gated channels. In conclusion, three H(+)/K(+)-ATPase inhibitors decreased contractile responses of rat vas deferens to various stimulants in vitro. They may act on a common mechanism, which plays a crucial role in regulating rat vas deferens contractility and this mechanism is probably involved in the regulation of intracellular Ca(2+).     

Role of Ca(2+)-activated K(+) channels and Na(+),K(+)-ATPase in prostaglandin E(1)- and E(2)-induced inhibition of the adrenergic response in human vas deferens

We studied the role of K(+) channels and Na(+),K(+)-ATPase in the presynaptic inhibitory effects of prostaglandin E(1) (PGE(1)) and PGE(2) on the adrenergic responses of human vas deferens. Furthermore, we determined the effects of increasing extracellular K(+) concentrations ([K(+)](o)) and inhibition of Na(+),K(+)-ATPase on neurogenic and norepinephrine-induced contractile responses. Ring segments of the epididymal part of the vas deferens were taken from 45 elective vasectomies and mounted in organ baths for isometric recording of tension. The neuromodulatory effects of PGEs were tested in the presence of K(+) channel blockers. PGE(1) and PGE(2) (10(-8) to 10(-6)M) induced inhibition of adrenergic contractions. The presence of tetraethylammonium (10(-3)M), charybdotoxin (10(-7)M), or iberiotoxin (10(-7)M), prevented the inhibitory effects of PGE(1) and PGE(2) on the adrenergic contraction. Both glibenclamide (10(-5)M) and apamin (10(-6)M) failed to antagonize PGE(1) and PGE(2) effects. Raising the [K(+)](o) from 15.8mM to 25.8mM caused inhibition of the neurogenic contractions. Ouabain at a concentration insufficient to alter the resting tension (10(-6)M) increased contractions induced by electrical stimulation but did not alter the contractions to norepinephrine. The inhibition of neurogenic responses induced PGE(1), PGE(2) and increased extracellular concentration of K(+) was almost completely prevented by ouabain (10(-6)M). The results demonstrate that PGE(1) and PGE(2) inhibit adrenergic responses by a prejunctional mechanism that involves the activation of large-conductance Ca(2+)-activated K(+) channels and Na(+),K(+)-ATPase.     

Calcium modulatory properties of O,O-diphenyl O-N-cyclooctyl phosphoramidate [corrected] isolated from Ptychodiscus brevis in rat atria and smooth muscle

Calcium modulatory activity of a marine toxin has been studied employing in vitro preparations. The toxin induced contracture in rat diaphragm was not modified by denervation, d-tubocurarine and tetrodotoxin (TTX). In contrast, varying concentrations of calcium, EGTA and ryanodine inhibited the contracture significantly. The toxin produced a series of repeating contractions in vas deferens. Experiments with TTX, adrenoceptor blockers and other agents exclude a release of neuromediators or direct stimulation of post synaptic receptors to account for the rhythmic effect in vas deferens. The dependence of rhythmicity on external Ca2+ concentration and inhibiting effect of Mn2+, ryanodine and nifedipine indicate a direct activation of voltage-sensitive calcium channel. The toxin also evoked a similar pattern of response in paced atria mediated through Ca2+ influx.     

Neurotoxic activity of venom from the Australian eastern mouse spider (Missulena bradleyi) involves modulation of sodium channel gating

Mouse spiders represent a potential cause of serious envenomation in humans. This study examined the activity of Missulena bradleyi venom in several in vitro preparations. Whilst female M. bradleyi venom at doses up to 0.05 microl ml(-1) failed to alter twitch or resting tension in all preparations used, male venom (0.02 and 0.05 microl ml(-1)) produced potent effects on transmitter release in both smooth and skeletal neuromuscular preparations. In the mouse phrenic nerve diaphragm preparation, male M. bradleyi venom (0.02 microl ml(-1)) caused rapid fasciculations and an increase in indirectly evoked twitches. Male venom (0.02 and 0.05 microl ml(-1)) also caused a large contracture and rapid decrease in indirectly evoked twitches in the chick biventer cervicis muscle, however had no effect on responses to exogenous ACh (1 mM) or potassium chloride (40 mM). In the chick preparation, contractile responses to male M. bradleyi venom (0.05 microl ml(-1)) were attenuated by (+)-tubocurarine (100 microM) and by tetrodotoxin (TTX, 1 microM). Both actions of male M. bradleyi venom were blocked by Atrax robustus antivenom (2 units ml(-1)). In the unstimulated rat vas deferens, male venom (0.05 microl ml(-1)) caused contractions which were inhibited by a combination of prazosin (0.3 microM) and P(2X)-receptor desensitization (with alpha,beta-methylene ATP 10 microM). In the rat stimulated vas deferens, male venom (0.05 microl ml(-1)) augmented indirectly evoked twitches. Male venom (0.1 microl ml(-1)) causes a slowing of inactivation of TTX-sensitive sodium currents in acutely dissociated rat dorsal root ganglion neurons. These results suggest that venom from male M. bradleyi contains a potent neurotoxin which facilitates neurotransmitter release by modifying TTX-sensitive sodium channel gating. This action is similar to that of the delta-atracotoxins from Australian funnel-web spiders.     

Pharmacological characterization of the presynaptic activity of Tityus serrulatus venom in the rat anococcygeus muscle.

Scorpion venoms are known to cause peripheral nerve stimulation with enhanced autonomic responses. This study, therefore, examined the effects of Tityus serrulatus venom (TSV) on adrenergic, cholinergic and nitrergic nerve fibers using the rat anococcygeus muscle. The contractile effects of TSV (1 microg/ml) and electrical field stimulation were markedly reduced by phentolamine (5 microM), prazosin (0.1 microM), guanethidine (30 microM) and tetrodotoxin (TTX, 1 microM), whereas imipramine (3 microM) enhanced these responses. The responses to tyramine (10 microM) were partially reduced by guanethidine and completely blocked by phentolamine, prazosin and imipramine. Atropine (1 microM) fully prevented carbachol (CCh, 30 microM)-induced contractions without affecting those mediated by TSV. Neostigmine significantly potentiated TSV-and ACh-evoked contractions, whereas hexamethonium had no effect. The relaxant responses induced by EFS and TSV (3 microg/ml) were completely blocked by L-NAME (100 microM), ODQ (1 microM) or TTX (1 microM). Addition of L-arginine (1 mM) reversed the effect of L-NAME. Thus, the motor and inhibitory responses of TSV in the rat anococcygeus muscle are mediated by prejunctional mechanisms dependent on Na(+) channel activation, causing the stimulation of NA and NO release from adrenergic and nitrergic nerve fibers, respectively.     

Paradoxical effects of thioridazine on electromechanical coupling in the human and rat vas deferens

The effects of thioridazine on the responses of isolated human and rat vas deferens to high [K+]0, A23187 and caffeine were examined. In the presence of Ca2+ (2.5 mM), thioridazine (1-10 microM) induced spontaneous contractions but caused a dose-related inhibition of the phasic and secondary parts of the response to high [K+]0 (136 mM). The relaxation phase of the high [K+]0 response of the human vas deferens was unaffected by thioridazine (up to 10 microM). In Ca2(+)-free/EGTA (0.5 mM) media, thioridazine caused a dose-related potentiation, shortened the latency and prolonged the duration of high [K+]0 responses. Contractions to caffeine (20 mM) and A23187 (20-50 microM) were relatively unchanged by thioridazine (10 microM). The spontaneous activity caused by thioridazine (10 microM) was sensitive to the Ca2(+)-channel blockers nifedipine (10 microM) or verapamil (10 microM). These results indicate that the action of thioridazine during electromechanical coupling in the human and rat vas deferens may involve more than its blockade of voltage gated Ca2+ channels.     

Rank-order inhibition by omega-conotoxins in human and animal autonomic nerve preparations

The inhibitory effects of the omega-conotoxins GVIA, MVIIA and MVIIC on electrically-evoked, tetrodotoxin (10(-7) M)-sensitive, autonomic nerve activity were studied using human, rat or guinea-pig vas deferens and intestinal tissues. In each preparation from each species, nM concentrations of omega-conotoxins GVIA and MVIIA prevented the neuronally-mediated contractions, whereas omega-conotoxin MVIIC was either markedly less potent (IC(50)'s 1.4 or 2.9 log units more than for omega-conotoxin GVIA in guinea-pig ileum and rat vas deferens, respectively) or was without significant activity (human vas deferens, human Taenia coli) when tested at similar concentrations. In contrast the differences in potency between omega-conotoxins GVIA and MVIIC were considerably less when assayed directly on Ca(2+) channel currents evoked from rat superior cervical ganglion neurons in culture (approximately 0.1 log unit difference) and from a stable cell line expressing rat alpha(1B), alpha(2)delta, beta(1b) Ca(2+) channel subunits (approximately 0.9 log unit). These different rank-orders of inhibitory activity of the conotoxins support the suggestion that there are pharmacologically distinct N-type Ca(2+) channels in the peripheral nervous system, and that this tissue-dependent difference is seen in man.     

Effects of several calcium channels modulators on the [3H]noradrenaline release and 45Ca influx in the rat vas deferens.

1. The effects of nifedipine (1 microM), CdCl2 (0.1 mM) and the Bay K 8644 enantiomers (1 microM) on [3H]noradrenaline release and 45Ca uptake in epididymal and prostatic rat vas deferens were investigated. 2. Nifedipine, CdCl2 and Bay K 8644 optical isomers did not affect the basal tritium release. However, the [3H]noradrenaline release evoked by high potassium (50 mM) from both portions of rat vas deferens was markedly inhibited by CdCl2, scarcely affected by nifedipine and not modified by Bay K 8644 enantiomers. 3. (-)-Bay K 8644 increased the basal and potassium (50 mM) induced 45Ca uptake whereas (+)-Bay K 8644, nifedipine and CdCl2 did not alter the basal 45Ca uptake. However, they strongly inhibited the uptake induced by potassium in both portions of rat vas deferens. 4. These results suggest that the calcium channels (mainly L type) are involved on the contractions in rat vas deferens epididymal and prostatic halves; these channels differ from those present in sympathetic nerve terminals (likely of N Type) which modulates the NA release. 5. This study also shows that Bay K 8644 optical isomers possess opposite effects on the L channels of bisected rat vas deferens smooth muscle.     

The effects of testosterone or insulin treatment on contractile responses of the rat vas deferens following castration or streptozotocin-induced diabetes mellitus

1. Castration and streptozotocin-induced diabetes produce significant decreases in serum testosterone levels accompanied by decreased vas deferens weights, a decreased responsiveness to nerve stimulation, and altered contractile responses to carbachol and phenylephrine. 2. Treatment of castrated rats with testosterone for 8 weeks prevented the decreased vas deferens weights and contractile changes associated with castration. 3. Treatment of diabetic rats with testosterone for 8 weeks prevented the decreased vas deferens weights and the supersensitivity to contractile agonists associated with diabetes. Testosterone treatment only partially prevented the decreased response to nerve stimulation. 4. Treatment of diabetic rats with testosterone plus insulin for 8 weeks prevented the decreased vas deferens weights and decreased the sensitivity to carbachol and phenylephrine compared to controls. Testosterone plus insulin treatment prevented the decreased response to nerve stimulation. 5. There were no differences in the IC50 values for nitrendipine among any of the groups studied, suggesting that the contractile changes observed in vasa deferentia following castration or diabetes are not the result of changes in calcium movements. 6. The results suggest that decreased testosterone levels are at least partially responsible for the changes in contractility of the vas deferens of streptozotocin-diabetic rats.     

Effects of amiloride on contractions and the release of tritium from rat vas deferens preloaded with [3H]-noradrenaline.

1 The effect of amiloride was studied on contractions and tritium release from rat vas deferens preloaded with [3H]-noradrenaline. 2 Amiloride had no effect on the resting tension and maximal contractile force of the vas deferens and did not alter the ED50 of noradrenaline. 3 Amiloride (10(-4)-10(-3)M) decreased the response of vas deferens to electrical stimulation dose-dependently without inhibiting the response to KCI (60mM). 4 The effect of amiloride was not prevented by preincubation of the tissue with phentolamine, propranolol, atropine or indomethacin. 5 Amiloride did not alter the spontaneous outflow of radioactivity from [3H]-noradrenaline labelled vasa deferentia. 6 Amiloride decreased the release of tritium induced by electrical stimulation or nicotine but did not inhibit the release of radioactivity induced by KCI or tyramine. 7 It is concluded that amiloride may inhibit the contractions of rat vas deferens by inhibiting the release of noradrenaline.     

Changes in electrophysiological properties and noradrenaline response in vas deferens of diabetic rats

The purpose of this study was to study changes in the sympathetic nerves of the vas deferens in 10-week-old streptozotocin-induced diabetic rats. To assess the activity of autonomic neurons, we recorded the amplitude and frequency of spontaneous junction potentials in vasa deferentia from age-matched controls and streptozotocin-induced diabetic rats. No change in the resting membrane potential of the smooth muscle of the vas deferens was found in streptozotocin-induced diabetic rats. The frequency of spontaneous junction potentials was significantly increased in the streptozotocin-induced diabetic rats and their amplitude was also markedly increased. The dose–response curve for the contractile response of the vas deferens to noradrenaline was significantly shifted to the right and the apparent affinity (pD₂ value) was significantly decreased in streptozotocin-induced diabetic rats. These results suggest that degeneration of sympathetic neurons may occur in the vas deferens of 10-week streptozotocin-induced diabetic rats and that the greater amplitude of the spontaneous junction potentials may be related to an increase in Ca²⁺ mobilization, though the increase in Ca²⁺ mobilization does not lead to an enhanced contractile response.     

Effects of acetaldehyde on contractile response to nerve stimulation in guinea-pig vas deferens

Twitch contractions of the isolated guinea-pig vas deferens induced by sympathetic nerve stimulation were augmented by acetaldehyde (0.1-10 mM). With high concentrations (5-10 mM), acetaldehyde produced a biphasic response consisting of an initial brief depression and a subsequent potentiation of the contraction. The late effect was associated with repetitive contractions that were not prevented by tetrodotoxin. A low concentration of phentolamine (27 microM) increased and a high concentration (1.3 mM) suppressed the potentiating action of acetaldehyde. Acetaldehyde did not induce contractions in surgically sympathectomized vasa or vasa pretreated with reserpine. Acetaldehyde caused a dose-dependent increase in noradrenaline release into the bathing fluid. The study shows that acetaldehyde has a dual effect on sympathetic neuroeffector transmission, and that an increase in noradrenaline secretion appears to contribute to the late facilitatory effect in the isolated vas deferens.     

Antagonism of calcium currents and neurotransmitter release by barium ions at frog motor nerve endings

1. The effects of Ba(2+) (0.1 - 2 mM) on the component of the perineural voltage change associated with nerve terminal calcium currents (prejunctional Ca(2+) currents) were compared with the effects of this ion to antagonize calcium-dependent acetylcholine (ACh) release. These experiments were made on isolated neuromuscular junctions of the frog. 2. In the presence of sufficient concentrations of K(+) channel blockers to eliminate measurable prejunctional K(+) currents, low concentrations of Ba(2+) selectively antagonized prejunctional Ca(2+) currents in normal Ca(2+) solutions. Higher concentrations of Ba(2+) also substantially reduced the Na(+) component of the perineural waveform. 3. Ba(2+) inhibited the prolonged prejunctional Ca(2+) currents that developed in the presence of higher concentrations of K(+) channel blockers. 4. Simultaneous measurements of the prejunctional Ca(2+) currents and the electrophysiological correlates of ACh release (i.e. end-plate potentials, EPPs) were made under conditions of modest K(+) channel blockade. Under these conditions, Ba(2+) generally produced simultaneous decreases in both Ca(2+) currents and EPP amplitudes. In some instances, a prolongation of prejunctional Ca(2+) currents and a transient increase in EPP amplitudes preceded the decreases in both electrophysiological events. 5. These results suggest that Ba(2+) ions can antagonize the entry of calcium into motor nerve endings and this effect is likely to be responsible for the inhibitory effects of Ba(2+) on evoked ACh release.     

Dopamine-induced depression of adrenergic nerve-mediated contraction of smooth muscle.

1 Dopamine (0.25-1.0 micrometer) applied extraluminally depressed vasoconstrictor responses of the perfused rabbit ear artery to low frequency adrenergic nerve stimulation. 2 The depressant effect of dopamine was prevented in the presence of haloperidol (0.1 micrometer) but not in the presence of phentolamine (0.03-0.14 micrometer). 3 Extraluminal dopamine (1 micrometer) or intraluminal injection of dopamine in amounts up to 0.025 micronmol had no dilator effect on arteries partially constricted by infusion of noradrenaline. 4 Dopamine also depressed contractile responses of guinea-pig vas deferens to low frequency adrenergic nerve stimulation. However, in this tissue the effect of dopamine was prevented by phentolamine but not affected by haloperidol. 5 In neither the rabbit ear artery nor the guinea-pig vas deferens did haloperidol increase the magnitude of responses to nerve stimulation or affect muscle sensitivity to noradrenaline. 6 We conclude that the adrenergic axons in the rabbit ear artery, but not the guinea-pig vas deferens, possess specific receptors for dopamine whose activation depresses axonal conduction or transmitter release. However, our results do not favour the view that these receptors are activated during normal transmission at physiological frequencies.     

The effects of calcium on adrenergic neuron blockade

The effects of increasing extracellular calcium were investigated on the responses to sympathetic nerve stimulation of three isolated organs; rabbit ileum, guinea-pig vas deferens and rabbit ear artery. A rise in the calcium concentration increased the responses of the ileum to low frequency stimulation, the maximum increase being obtained at 8.8 mM calcium. After partial blockade by guanethidine of the responses of the ileum to high frequency stimulation, raised calcium concentrations again increased the responses. The increase was similar in guanethidine-treated and untreated preparations and the maximum increase in both was obtained using 8.8 mM calcium. In the vas deferens and rabbit ear artery preparations an increase in extracellular calcium did not antagonize the blocking action of guanethidine. These experiments do not therefore support the theory that guanethidine acts by preventing the entry of calcium into the sympathetic nerve endings.     

Effect of the dihydropyridine Bay K 8644 on the release of [3H]-noradrenaline from the rat isolated vas deferens.

The effects of Bay K 8644 on the release of [3H]-noradrenaline evoked by potassium, electrical stimulation or tyramine from the rat isolated vas deferens labelled with [3H]-noradrenaline were investigated. Bay K 8644 (1 microM) by itself did not affect the spontaneous release of tritium from the rat isolated vas deferens. However, it increased the calcium-dependent release of tritium elicited by both high potassium (59 mM) and electrical field stimulation. The exposure of rat vas deferens to phentolamine (10 microM) increased the release of tritium induced by potassium (59 mM) and electrical field stimulation. Bay K 8644 (1 microM) failed to increase further the release of tritium elicited by both stimuli in preparations previously treated with phentolamine (10 microM). The calcium-independent release of [3H]-noradrenaline evoked by tyramine (10 microM) was not affected by Bay K 8644 (1 microM). The results of our study support the view that alpha2-adrenoceptors modulate noradrenaline release by restricting calcium influx into sympathetic nerve terminals through voltage-dependent channels.     

Methysergide induces selective potentiation in cholinergic contractions of the guinea-pig vas deferens by facilitating acetylcholine release.

Methysergide (3 x 10(-6) M) enhanced the contractile responses of the isolated stripped vas deferens of guinea-pig to acetylchline(ACh) and arecoline, but not those to noradrenaline, tyramine and bradykinin. Methysergide (3 x 10(-5) M) suppressed the contraction elicited by noradrenaline or histamine. The methysergide-induced potentiation of the response to ACh was prevented by pre-addition of hemicholinium but not by tetrodotoxin or morphine. The augmentation of the response to ACh by physostigmine was unaffected by hemicholinium. The phasic contraction of the tissue elicited by 30 mM KCl was also enhanced by methysergide, and this enhancement was prevented by the pre-addition of atropine (1.4 x 10(-7) M). In the depolarized vas deferens after exposure to 30 mM KCl, methysergide occasionally induced a sustained tonic contraction which was inhibited by atropine. These findings suggest that methysergide facilitates a release of ACh by acting on the cholinergic nerve terminals and selectively potentiates the cholinergic response.     

Effect of normorphine and enkephalin on spontaneous potentials in the vas deferens.

Spontaneous excitatory junction potentials were recorded intracellularly from the smooth muscle of the mouse vas deferens. Exposure of the vas deferens to either normorphine (1 muM) or methionine-enkephalin (0.5 muM) did not alter the frequency or amplitude distribution of the spontaneous potentials. The possible mechanisms by which the narcotic agonists depress the release of noradrenaline on nerve stimulation without inhibiting the spontaneous release of noradrenaline are discussed.     

Excitatory effect of a new polypeptide (anthopleurin-B) from sea anemone on the guinea-pig vas deferens.

1 Anthopleurin-B (AP-B, greater than 3 x 10(-9) M), a newly isolated polypeptide from sea anemone (Anthopleura xanthogrammica), caused powerful rhythmic contractions in the guinea-pig isolated vas deferens. The other polypeptides anthopleurin-A from A. xanthogrammica and anthopleurin-C from A. elegantissima, elicited similar effects but in higher concentrations ( less than 5 x 10(-8) M). Toxin II (10(-6) M) isolated from the sea anemone, Anemonia sulcata, had no effect. 2 The rhythmic contractions induced by AP-B were inhibited by phentolamine, bretylium, guanethidine, reserpine, 6-hydroxydopamine, tetrodotoxin (TTX) and verapamil. Mecamylamine, atropine, methysergide, chlorpheniramine, and indomethacin had no effect. 3 AP-B (10(-8) M approximately 10(-5) M) caused a dose-dependent increase in the amount of endogenous noradrenaline (NA) released from the vas deferens. AP-B (10-5M) increased the amount of NA released to approximately 310 times (12 micrograms/g tissue) that of untreated tissues. 4 The AP-B-induced release of NA was inhibited or abolished by TTX, verapamil, or incubation in Ca-free medium. 5 These results suggest that the AP-B-induced rhythmic contraction of the vas deferens is mediated through the release of NA from adrenergic nerve endings; AP-B is one of the most potent substances in stimulating NA release from the vas deferens.