Isolated Mouse Phrenic Nerve-diaphragm Research Articles

The Venom of Ornithoctonus huwena affect the electrophysiological stability of neonatal rat ventricular myocytes by inhibiting sodium, potassium and calcium current

ABSTRACTSpider venoms are known to contain various toxins that are used as an effective means to capture their prey or to defend themselves against predators. An investigation of the properties of Ornithoctonus huwena (O.huwena) crude venom found that the venom can block neuromuscular transmission of isolated mouse phrenic nerve-diaphragm and sciatic nerve-sartorius preparations. However, little is known about its electrophysiological effects on cardiac myocytes. In this study, electrophysiological activities of ventricular myocytes were detected by 100 μg/mL venom of O.huwena, and whole cell patch-clamp technique was used to study the acute effects of the venom on action potential (AP), sodium current (INa), potassium currents (IKr, IKs, Ito1 and IK1) and L-type calcium current (ICaL). The results indicated that the venom prolongs APD90 in a frequency-dependent manner in isolated neonatal rat ventricular myocytes. 100 μg/mL venom inhibited 72.3 ± 3.6% INa current, 58.3 ± 4.2% summit current and 54 ± 6.1% the end current of IKr, and 65 ± 3.3% ICaL current, yet, didn't have obvious effect on IKs, Ito1 and IK1 currents. In conclusion, the O.huwena venom represented a multifaceted pharmacological profile. It contains abundant of cardiac channel antagonists and might be valuable tools for investigation of both channels and anti- arrhythmic therapy development.

Neuromuscular activity of BaTX, a presynaptic basic PLA 2 isolated from Bothrops alternatus snake venom

We have previously isolated a Lys49 phospholipase A 2 homolog (BaTX) from Bothrops alternatus snake venom using a combination of molecular exclusion chromatography and reverse phase HPLC and shown its ability to cause neuromuscular blockade. In this work, we describe a one-step procedure for the purification of this toxin and provide further details of its neuromuscular activity. The toxin was purified by reverse phase HPLC and its purity and molecular mass were confirmed by SDS-PAGE, MALDI-TOF mass spectrometry, amino acid analysis and N-terminal sequencing. BaTX (0.007–1.4 µM) produced time-dependent, irreversible neuromuscular blockade in isolated mouse phrenic nerve-diaphragm and chick biventer cervicis preparations (time to 50% blockade with 0.35 µM toxin: 58 ± 4 and 24 ± 1 min, respectively; n = 3–8; mean ± S.E.) without significantly affecting the response to direct muscle stimulation. In chick preparations, contractures to exogenous acetylcholine (55 and 110 µM) or KCl (13.4 mM) were unaltered after complete blockade by all toxin concentrations. These results, which strongly suggested a presynaptic mechanism of action for this toxin, were reinforced by (1) the inability of BaTX to interfere with the carbachol-induced depolarization of the resting membrane, (2) a significant decrease in the frequency and amplitude of miniature end-plate potentials, and (3) a significant reduction (59 ± 4%, n = 12) in the quantal content of the end-plate potentials after a 60 min incubation with the toxin (1.4 µM). In addition, a decrease in the organ bath temperature from 37 °C to 24 °C and/or the replacement of calcium with strontium prevented the neuromuscular blockade, indicating a temperature-dependent effect possibly mediated by enzymatic activity.

Solid-Phase Chemical Synthesis and Oxidative Refolding of Hainantoxin-III

Hainantoxin-III (HNTX-III) was synthesized using solid-phase chemical method with Fmoc-protected amino acids. Reversed-phase high performance liquid chromatography (RP-HPLC) was used to monitor the oxidative refolding of linear HNTX-III. It is found that the best refolding yield for 0.1 g/L linear HNTX-III could be obtained in double-distilled aqueous solution at pH 7.5 containing 1.0 mol/L L-arginine, 1.0 mmol/L reduced glutathione (GSH), and 0.1 mmol/L oxidized glutathione (GSSG). The relative molecular mass of the refolded HNTX-III was determined to be 3 607.68 by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Only one peak was found in the RP-HPLC chromatogram for the mixed sample of native HNTX-III and refolded HNTX-III at ratio of 1 : 1, and the effect of refolded HNTX-III on neuromuscular transmission in isolated mouse phrenic nerve-diaphragm preparations was almost same as that of native HNTX-III. The results indicate that refolded HNTX-III had the same structure and biological activities as the native HNTX-III.

Biological and Structural Characterization of Crotoxin and New Isoform of Crotoxin B PLA2 (F6a) from Crotalus durissus collilineatus Snake Venom

A new crotoxin B isoform PLA2 (F6a), from Crotalus durissus collilineatus was purified from by one step reverse phase HPLC chromatography using μ-Bondapack C-18 column analytic. The new crotoxin B isoform PLA2 (F6a), complex crotoxin, the catalytic subunit crotoxin B isoform PLA2 (F6a) and two crotapotin isoforms (F3 and F4), were isolated from the venom of Crotalus durissus collilineatus. The crotapotins isoforms F3 and F4 had similar chemical properties, the two proteins different in their ability to inhibit of isoforms of PLA2 (F6 and F6a). The molecular masses estimated by MALDI-TOF mass spectrometry were: crotoxin B: 14,943.14 Da, crotapotin F3: 8,693.24 Da, and crotapotin F4: 9 314.56 Da. The new crotoxin B isoform PLA2 (F6a) contained 122 amino acid residues and a pI of 8.58. Its amino acid sequence presents high identity with those of other PLA2s, particularly in the calcium binding loop and active site helix 3. It also presents similarities in the C-terminal region with other myotoxic PLA2s. The new crotoxin B isoform PLA2 (F6a) contained 122 amino acid residues, with a primary structure of HLLQFNKMIK FETRRNAIPP YAFYGCYCGW GGRGRPKDAT DRCCFVHDCC YGKLAKCNTK WDFYRYSLKS GYITCGKGTW CEEQICECDR VAAECLRRSL STYRYGYMIY PDSRCRGPSE TC. A neuromuscular blocking activity was induced by crotoxin and new crotoxin B isoform PLA2 (F6a) in the isolated mouse phrenic nerve diaphragm and the biventer cervicis chick nerve-muscle preparation. Whole crotoxin was devoid of cytolytic activity upon myoblasts and myotubes in vitro, whereas new crotoxin B isoform PLA2 (F6a) was clearly cytotoxic to these cells.

Structural, enzymatic and biological properties of new PLA 2 isoform from Crotalus durissus terrificus venom

We isolated a new PLA 2 from the Crotalus durissus terrificus venom that designated F15, which showed allosteric behavior with a V max of 8.5 nmol/min/mg and a K m of 38.5 mM. The incubated heparin salt of this isolated F15 act a positive allosteric effector by increasing the V max to 10.2 nmol/min/mg, with decreasing the V max value to 20.5 mM. The crotapotin, on the other hand acts as a negative allosteric effector by increasing the V max values to 58.4 mM. F15 also showed high calcium dependence for its catalysis similar to that found for other PLA 2 enzymes isolated from these snake venoms. The replacement of calcium by other divalent ions such Mg 2+, Mn 2+, Cd 2+, Sn 2+ and Cu 2+ resulted in lower enzymatic activity. The optimum pH and temperature for the enzyme was 8.5 and 18 °C, respectively. F15 alone showed moderate neurotoxic activity in isolated mouse phrenic nerve diaphragm in comparison to other strong myotoxic PLA 2 such as bothropstoxin-I (BThtx-I), but this activity was highly neurotoxic in a chick biventrer cervis preparation, whereas BthTx-I did not reveal this high neurotoxicity. This new protein showed a high bactericidal effect against both Gram-negative and Gram-positive bacterial strains. F15 contained 122 amino acid residues, with a primary structure of: HLLQFNKMIKFETRKNAVPFYAFYGCYCGWGGQRRPKDATDRCCFVHDCCYGKLTKCNTKWDIYRYSLKSGYITCGKGTWCKEQICECDRVAAECLRRSLSTYKNEYMFYPKSRCRRPSETC. Its molecular mass and isoeletric point were 14.5 kDa and 8.85, both estimated by two dimensional electrophoresis. The amino acid sequence of the F15 revealed high sequence homology with F16 and F17. F15 and the other PLA 2s revealed highly conserved amino acid sequences principally for calcium binding loop and active site helix. F15 also showed a high homology with the lysine-rich region of myotoxic PLA 2.

Purification and characterization of raventoxin-I and raventoxin-III, two neurotoxic peptides from the venom of the spider Macrothele raveni

The spider Macrothele raveni was recently identified as a new species of Genus Macrothele. The crude venom from M. raveni was found to be neurotoxic to mice and the LD 50 of the crude venom in mice was 2.852 mg/kg. Two neurotoxic peptides, raventoxin-I and raventoxin-III, were isolated from the crude venom by ion-exchange and reverse phase high performance liquid chromatography. Raventoxin-I was the most abundant toxic component in the venom, while raventoxin-III was a lower abundant component. Both toxins can kill mice and block neuromuscular transmission in an isolated mouse phrenic nerve diaphragm preparation, but have no effect on cockroaches. The LD 50 of raventoxin-I in mice is 0.772 mg/kg. The complete amino acid sequences of raventoxin-I and raventoxin-III were determined and found to consist of 43 and 29 amino acid residues, respectively. It was determined by mass spectrometry that all Cys residues from raventoxin-I and raventoxin-III are involved in disulphide bonds. raventoxin-III showed no significant sequence homology with any presently known neurotoxins in the protein/DNA databases, while raventoxin-I has limited sequence identity with δ-AcTx-Hv1 and δ-AcTx-Ar1, which target both mammalian and insect sodium channels. Both raventoxin-I and raventoxin-III only work on vertebrates, but not on insects. Moreover, raventoxin-I could exert an effect of first exciting and then inhibiting the contraction of mouse diaphragm muscle caused by electrically stimulating the phrenic nerve, but raventoxin-III could not.

Effects of cartap on isolated mouse phrenic nerve diaphragm and its related mechanism.

Cartap, a nereistoxin analogue pesticide, is reported to have no irritation to eyes in rabbits. However, we have demonstrated recently that cartap could actually cause acute death in rabbits via ocular exposure. Our preliminary study with isolated mouse phrenic nerve diaphragms has shown that instead of neuromuscular blockade, cartap caused muscular contracture. The objective of the study was to examine the effect of cartap on the neuromuscular junction in more detail and to investigate its possible underlying mechanism with isolated mouse phrenic nerve diaphragms and sarcoplasmic reticulum (SR) vesicles. Cartap or nereistoxin at various concentrations was added in the organ bath with isolated mouse phrenic nerve diaphragm and both nerve- and muscle-evoked twitches were recorded. Instead of blocking the neuromuscular transmission as nereistoxin did, cartap caused contracture in stimulated or quiescent isolated mouse phrenic nerve diaphragm. Both the cartap-induced muscular contracture force and the time interval to initiate the contracture were dose-dependent. The contracture induced by cartap was not affected by the pretreatment of the diaphragm with the acetylcholine receptor blocker alpha-bungarotoxin; the Na(+) channel blocker tetrodotoxin; or various Ca(2+) channel blockers, NiCl(2), verapamil, and nifedipine. On the contrary, the contracture was significantly inhibited when the diaphragm was pretreated with ryanodine or EGTA containing Ca(2+)-free Krebs solution or in combination. This suggested that both internal and extracellular Ca(2+) might participate in cartap-induced skeletal muscle contracture. Moreover, cartap inhibited the [(3)H]-ryanodine binding to the Ca(2+) release channel of SR in a dose-dependent manner. Additionally, cartap could induce a significant reduction in Ca(2+)-ATPase activity of SR vesicles at a relatively high dose. The results suggested that cartap might cause the influx of extracellular Ca(2+) and the release of internal Ca(2+), with subsequent induction of muscular contracture in the isolated mouse phrenic nerve diaphragm. Based on these findings, we propose that the acute death of rabbits following ocular exposure to cartap might have resulted from respiratory failure secondary to diaphragm contracture.

Induction of skeletal muscle contracture and calcium release from isolated sarcoplasmic reticulum vesicles by sanguinarine.

The benzophenanthrine alkaloid, sanguinarine, was studied for its effects on isolated mouse phrenic-nerve diaphragm preparations. Sanguinarine induced direct, dose-dependent effects on muscle contractility. Sanguinarine-induced contracture was partially inhibited when the extracellular Ca(2+) was removed or when the diaphragm was pretreated with nifedipine. Depletion of sarcoplasmic reticulum (SR) internal calcium stores completely blocked the contracture. Sanguinarine induced Ca(2+) release from the actively loaded SR vesicles was blocked by ruthenium red and dithiothreitol (DTT), consistent with the ryanodine receptor (RyR) as the site of sanguinarine action. Sanguinarine altered [(3)H]-ryanodine binding to the RyR of isolated SR vesicles, potentiating [(3)H]-ryanodine binding at lower concentrations and inhibiting binding at higher concentrations. All of these effects were reversed by DTT, suggesting that sanguinarine-induced Ca(2+) release from SR occurs through oxidation of critical SH groups of the RyR SR calcium release channel.

Purification and characterization of huwentoxin-II, a neurotoxic peptide from the venom of the Chinese bird spider Selenocosmia huwena.

A neurotoxic peptide, huwentoxin-II (HWTX-II), was purified from the venom of the Chinese bird spider Selenocosmia huwena by ion exchange chromatography and reversed phase HPLC. The toxin can reversibly paralyse cockroaches for several hours, with an ED50 of 127 +/- 54 microg/g. HWTX-II blocks neuromuscular transmission in an isolated mouse phrenic nerve diaphragm preparation and acts cooperatively to potentiate the activity of huwentoxin-I. The complete amino sequence of HWTX-II was determined and found to consist of 37 amino acid residues, including six Cys residues. There is microheterogeneity (Ile/Gln) in position 10, and mass spectrometry indicated that the two isoproteins have a tendency to dimerize. It was determined by mass spectrometry that the six Cys residues are involved in three disulphide bonds. The sequence of HWTX-II is highly homologous with ESTX, a toxin from the tarantula Eurypefina californicum.

Studies on Neuromuscular Blockade by Boldine in the Mouse Phrenic Nerve-Diaphragm

The effects of boldine [(S)-2,9-dihydroxyl-l,10-dimethoxy-aporphine], a major alkaloid in the leaves and bark of Boldo (Peumus boldus Mol.), on neuromuscular transmission were studied using a muscle phrenic-nerve diaphragm preparation. Boldine at concentrations lower than 200 μM preferentially inhibited, after an initial period of twitch augmentation, the nerve-evoked twitches of the mouse diaphragm and left the muscle-evoked twitches unaffected. The twitch inhibition could be restored by neostigmine or washout with Krebs solution. The twitches evoked indirectly and directly were both augmented initially, suggesting that the twitch augmentation induced by boldine was myogenic. Boldine inhibited the acetylcholine-induced contraction of denervated diaphragm dose-dependently with an IC50 value of 13.5 μM. At 50 μM, boldine specifically inhibited the amplitude of the miniature end plate potential. In addition, boldine was similar to d-tubocurarine in its action to reverse the neuromuscular blocking action of α-bungarotoxin. These results showed that the neuromuscular blockade by boldine on isolated mouse phrenicnerve diaphragm might be due to its direct interaction with the postsynaptic nicotinic acetylcholine receptor.

Solid-phase synthesis of huwentoxin-I and its structure and bioactivity analysis

Huwentoxin-I, a neurotoxic peptide from the spiderSelenocosmia huwena, was synthesized by solid-phase method with Fluorenylmethoxycarbonyl amino acid pentafluorophenyl esters (Fmoc-AA-OPfp). The carboxyl and the hydroxy groups were protected by tBu; the side chains of Lys and His were protected by Boc; the guanidine group of Arg was protected by Mtr and the mercaptan group of Cys was protected by Trt. The solid-phase carrier was ethylene diamine-polyethylene-polystyrene (DEA-PEG-PS) resin. The synthetic peptide was cleaved from the resin and deprotected by a 90 % TFA solution containing 5 % thioanisole, 3% ethanedithiol and 2 % anisole. The product was reduced with DTT and then incubated with GSSG and GSH to form the correct disulfide bond linkages. The synthetic peptide was purified by HPLC and then characterized by amino acid composition and sequence analysis, peptide mapping and NMR. The biological activity of the synthetic product was tested by electrophysiological method using the isolated mouse phrenic nerve diaphragm preparation. The results indicated that the synthetic huwentoxin-I has the same chemical and conformational structure and biological activity as those of the native huwentoxin-I from the spider.

Novel effects of guanidine on the neuromuscular junction

1. 1. The effects of guanidine on the isolated mouse phrenic nerve diaphragm (MPND) and chick biventer cervicis (CBC) neuromuscular preparations were determined by myographic and electrophysiological methods. 2. 2. Guanidine at concentrations of 5–10 mM induced an initial facilitation followed by neuromuscular blockade in both preparations. In the isolated MPND such blockade was associated with the abolition of miniature end-plate potentials (MEPPs), but in the CBC the acetylcholine-induced contracture remained unimpaired. After guanidine removal, a heretofore undescribed pronounced facilitation of neuromuscular transmission associated with an increase in MEPP frequency was observed. Simultaneously, the muscular contractions exhibited delayed relaxation and aftercontractions. 3. 3. The K + channel opener, cromakalim (100–200 μM) inhibited both the well-described initial and the novel postremoval facilitatory effects of guanidine in a concentration-dependent manner. These findings are consistent with the proposal that guanidine blocks K + channels in motor nerve endings. 4. 4. The guanidine-induced NMB was reverted by increasing the Ca 2+ concentration (1.8–5 mM) in the nutritive solution. 5. 5. Tetrodotoxin (TTX, 1.56 μM) did not influence the increase in MEPPS frequency induced by guanidine (10 mM) but did reduce the rise in MEPPS frequency observed after guanidine removal. 6. 6. The present findings indicate that the effects of guanidine on the neuromuscular junction are more complex than currently described because they include a neuromuscular blockade and a postremoval facilitation previously unreported in the literature.

The enterotoxin of Clostridium perfringens type a binds to the presynaptic nerve endings in neuromuscular junctions of mouse phrenic nerve-diaphragm

The enterotoxin of Clostridium perfringens type A, a channelpore forming protein toxin, inhibited neuromuscular transmission in isolated mouse phrenic nerve-diaphragm preparation at low concentrations of calcium. We investigated immunohistochemically the localization of the binding sites of the enterotoxin in the preparation under the conditions in which the enterotoxin reduced maximally the amplitudes of the twitch tension elicited by electrical stimulations to the phrenic nerve. Under the conditions, double immunohistochemical staining of the preparation with (1) rabbit anti-enterotoxin IgG-rhodamine-labeled goat anti-rabbit IgG and (2) mouse anti-synaptophysin (one of the synaptic vesicle-specific membrane proteins)-fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG showed that the enterotoxin binds specifically to most of the sites which were stained with anti-synaptophysin exactly in the same configurations having the shapes of the nerve endings in the endplates. The thin section electron micrographs of the enterotoxin-intoxicated preparation showed no alterations in the ultrastructure of the neuromuscular junction and the nerve endings filled with numerous synaptic vesicles. The present results, together with our previous electrophysiological findings, indicate that the enterotoxin binds specifically to the presynaptic nerve endings and inhibits neurotransmitter release at the neuromuscular junction.

Depolarizing neuromuscular blocking action of coryneine derived from aconite root in isolated mouse phrenic nerve-diaphragm muscles.

The mode of the neuromuscular blocking action of coryneine (a quaternary ammonium derivative of dopamine) derived from aconite root was investigated in isolated phrenic nerve-diaphragm muscles and denervated diaphragm muscles of mice. Coryneine (20-150 microM) blocked the nerve-evoked twitch response without affecting the contraction evoked by electrical stimulation of the muscle. The blocking effect was reversed by neostigmine, a cholinesterase inhibitor. The electrical charge-response curve on depolarization produced by iontophoretically applied acetylcholine (ACh) at the endplate regions in normal muscles was shifted to the right on decreasing the maximal response by 40 microM coryneine. The double-reciprocal plot revealed that coryneine reduced the apparent affinity of ACh for its receptor on decreasing the maximal response. Coryneine (20 microM-2mM) itself depolarized the endplate membrane and this effect was reversibly suppressed by 1 and 5 microM pancuronium. Coryneine 30 microM-10mM) produced contractions of denervated muscles in a concentration-dependent manner and the effects were reduced by 70nM pancuronium. These results indicate that coryneine is a depolarizing agent and a mixed-type competitive and noncompetitive neuromuscular blocker.

Improvement by diazepam of neuromuscular transmission blocked by anticholinesterase agents in mouse diaphragms

Effects of diazepam on the neuromuscular transmission blocked by neostigmine were studied in isolated mouse phrenic nerve-diaphragm preparations. Diazepam, in the absence of neostigmine, had no significant effect on the neuromuscular transmission at concentrations lower than 100 μM, except to enhance muscle contractility. Single and train pulses-evoked endplate potentials (e.p.p.s) and miniature e.p.p.s (m.e.p.p.s) were also unaffected. At concentrations of 175 μM or higher, diazepam caused an axonal conduction block. However, neostigmine-induced twitch potentiation, spontaneous fasciculation and tetanic fade were antagonized by diazepam at 3.5–35 μM. Diazepam did not decrease the amplitude of neostigmine-augmented e.p.p.s and m.e.p.p.s but slightly reduced their decay time. The incidence of regenerative depolarization of endplates induced by repetitive stimulation in the presence of neostigmine, was decreased from 92% to 35% junctions and the duration shortened from 650 ms to 230 ms. The amplitude of train e.p.p.s was increased. It is suggested that diazepam reverses the muscle paralysis induced by anticholinesterase agents by inhibiting the regenerative release of acetylcholine.

Properties and amino acid sequence of huwentoxin-I, a neurotoxin purified from the venom of the Chinese bird spider Selenocosmia Huwena

S-p. Liang, D-y. Zhang, X. Pan, Q. Chen and P-A. Zhou. Properties and amino acid sequence of huwentoxin-I, a neurotoxin purified from the venom of the Chinese bird spider Selenocosmia huwena. Toxicon31, 969–978, 1993.—By means of reverse phase and ion-exchange high performance liquid chromatography, a neurotoxic peptide named huwentoxin-I was purified from the venom of the Chinese bird spider Selenocosmia huwena. The intraperitoneal and intracisternal ld50 in mice of the toxin were 0.70 mg/kg and 9.40 μg/kg, respectively. This toxin at the concentration of 1 × 10−5g/ml can irreversibly block the neuromuscular transmission of the isolated mouse phrenic nerve-diaphragm preparation in 13.4 ± 1.3 min (mean ± S.D., n = 5). The isoelectric point is 8.95 determined by isoelectric focusing electrophoresis. It consists of 33 amino acids including 6 Cys and 6 Lys determined by amino acid analysis. The complete amino sequence of huwentoxin-I was determined. The N-terminal and C-terminal residues were Ala and Leu, respectively. The primary structure showed partial homology with that of μ-agatoxins from the funnel-web spider Agelenopsis aperta.

Pharmacological activity and structure-activity relationship of (+/-)-erythro-mefloquine and related compounds on the isolated mouse phrenic nerve diaphragm preparation.

The effects of the antimalarial agent, (+/-)-erythro-mefloquine and related compounds [(+/-)-threo-mefloquine, (+/-)-erythro-N-methylmefloquine and its N-oxide, quinine, WR 184806 and halofanthrine] on the isolated mouse phrenic nerve diaphragm preparation were investigated. Based on their pharmacological effects, these compounds may be divided into two groups. The group I compounds, comprising (+/)-erythro-mefloquine, (+/-)-threo-mefloquine and WR 184806, were found to exert a contractile effect on the muscle and also to inhibit the indirectly (nerve) stimulated and directly (muscle) stimulated (after alpha-bungarotoxin) twitch responses. The group II compounds, comprising the other compounds except halofanthrine, lacked a contractile effect on muscle but potentiated the directly stimulated twitch responses (after alpha-bungarotoxin). Halofanthrine did not elicit any response from the preparation. The minimum energy conformations of these compounds were determined using an interactive molecular modelling program which incorporates MMX force field for molecular mechanics calculations. Conformational analyses of the erythro and threo isomers of mefloquine hydrochloride were also undertaken using 1H-NMR. The 1H-NMR data supported the proposal made on the basis of MMX calculations that the erythro isomer exists in solution as one predominant conformer whereas the threo isomer is present in solution as a mixed population of two stable conformers. The structure-activity relationship of the compounds is discussed.

Inhibition of neuromuscular transmission in isolated mouse phrenic nerve-diaphragm by the enterotoxin of Clostridium perfringens type A

The enterotoxin of Clostridium perfringens type A, a channel forming protein toxin, inhibited neuromuscular transmission under conditions of low calcium. Twitch tension of isolated phrenic nerve-diaphragm preparations elicited by electrical stimulations to the phrenic nerve was recorded isometrically, and the preparations were exposed to the purified enterotoxin. In Krebs solution containing 0.5 mM calcium, the enterotoxin (20 μg/ml) reduced within 10 min the amplitude of the twitch tension to 34 ± 7% (mean ± S.D., n = 11) of that recorded before the treatment. The effects of the enterotoxin on the twitch tension were irreversible and proceeded independently of stimulation. The reduction of the twitch tension by the enterotoxin was apparent in Krebs solution containing less than 0.6 mM calcium and the degree of reduction was inversely related to the concentration of calcium. The reduction of the twitch tension by the enterotoxin was also dependent on temperature and concentration of the toxin. At temperatures below 20°C, no obvious reduction of twitch tension was observed with 20 μg/ml of the enterotoxin. Enterotoxin at a concentration of 0.4 μg/ml caused 16 ± 2% (mean ± S.D., n = 4) reduction of twitch tension, and the degree of the reduction in twitch tension increased with toxin concentration, reaching a plateau of 65 ± 4% (mean ± S.D., n = 7) at 6.5 μg/ml of the enterotoxin. The effects of the enterotoxin were antagonized by 2 μM physostigmine. Unlike curare, pretreatment of the preparation with enterotoxin did not antagonize the neuromuscular block by decamethonium. Neither the tension of muscular twitch elicited by direct electrical stimulation to the muscle nor the resting membrane potentials of muscle fibers recorded intracellularly were affected by the enterotoxin. The enterotoxin (2.2 μg/ml) reduced the frequency, but not mean amplitude or amplitude distribution, of miniature end-plate potentials, from 0.91 ± 0.07/sec to 0.72 ± 0.07 (mean ± S.E., n = 5). The results suggest that the enterotoxin will provide a novel tool for the studies on the mechanism of the neuromuscular transmission because of the unique characteristics of the inhibition and of the known mechanism of its action on the cell membrane.

Mechanisms of the Synergistic Interactions between Organic Calcium Channel Antagonists and Various Neuromuscular Blocking Agents

The effects of Mn2+, neomycin and four organic Ca2+-channel antagonists (OCA): nicardipine, nifedipine, diltiazem and verapamil on the neuromuscular blocking activities of tubocurarine, succinylcholine (SCh), decamethonium and neomycin were studied in isolated mouse phrenic nerve-diaphragm preparations. The effective concentration of SCh for 50% inhibition (IC50) of single indirect twitch responses were reduced markedly by more than 3-fold when the preparations were pretreated with OCA at 10 μM; the latter alone did not appreciably affect the indirect twitch response or the amplitude of miniature endplate potentials. The neuromuscular blocking effect of decamethonium was also enhanced synergistically by OCA to a similar extent. On the other hand, under the comparable condition, the combined uses of OCA plus tubocurarine or neomycin, neomycin plus tubocurarine or SCh, and Mn2+ plus tubocurarine, SCh or neomycin all resulted in insignificant potentiation. These results suggest that OCA have a specific effect to enhance the agonist effect of depolarizing agents on nicotinic acetylcholine receptors. Nicardipine at 2 μM non-competitively inhibited depolarizations of endplates elicited by SCh and decamethonium and abolished them completely at 10 μM nicardipine. The IC50’s in inhibiting endplate potentials and miniature endplate potentials by SCh and decamethonium were also reduced 2 to 3.5-fold by nicardipine. It is inferred that OCA are endowed with a unique capability to allosterically affect the postsynaptic nicotinic acetylcholine receptor, promoting its desensitization liability, hence synergistic interaction with depolarizing agents. Presynaptic effects of OCA are probably not involved.

Nicardipine inhibits axon conduction but causes dual changes of acetylcholine release in the mouse motor nerve

The effects of nicardipine, a dihydropyridine Ca2(+)-channel antagonist, on neuromuscular transmission and impulse-evoked release of acetylcholine were compared with those of nifedipine. In the isolated mouse phrenic nerve diaphragm, nicardipine (50 microM), but not nifedipine (100 microM), induced neuromuscular block, fade of tetanic contraction, and dropout or all-or-none block of end-plate potentials. Nicardipine had no significant effect on the resting membrane potential and the amplitude of miniature end-plate potentials but increased the frequency and caused the appearance of large size miniature potentials. The quantal contents of evoked end-plate potentials were increased. In the presence of tubocurarine, however, nicardipine depressed the amplitude of end-plate potentials. The compound nerve action potential was also decreased. It is concluded that nicardipine blocks neuromuscular transmission by acting on Na+ channels and inhibits axonal conduction. Nicardipine appeared to affect the evoked release of acetylcholine by dual mechanisms, i.e., an enhancement presumably by an agonist action on Ca2+ channels, like Bay K 8644 and nifedipine, and inhibition by an effect on Na+ channels, like verapamil and diltiazem. In contrast with its inactivity on the amplitude of miniature end-plate potentials, depolarization of the end plate in response to succinylcholine was greatly depressed. The contractile response of baby chick biventer cervicis muscle to exogenous acetylcholine was noncompetitively antagonized by nicardipine (10 microM), but was unaffected by nifedipine (30 microM). These results may implicate that nicardipine blocks the postsynaptic acetylcholine receptor channel by enhancing receptor desensitization or by a use-dependent effect.