Phrenic Nerve-diaphragm Muscle Research Articles

Electrophysiological effects of BDNF and TrkB signaling at type-identified diaphragm neuromuscular junctions.

Previous studies show that synaptic quantal release decreases during repetitive stimulation, i.e., synaptic depression. Neurotrophin brain-derived neurotrophic factor (BDNF) enhances neuromuscular transmission via activation of tropomyosin-related kinase receptor B (TrkB). We hypothesized that BDNF mitigates synaptic depression at the neuromuscular junction and that the effect is more pronounced at type IIx and/or IIb fibers compared to type I or IIa fibers given the more rapid reduction in docked synaptic vesicles with repetitive stimulation. Rat phrenic nerve-diaphragm muscle preparations were used to determine the effect of BDNF on synaptic quantal release during repetitive stimulation at 50 Hz. An ∼40% decline in quantal release was observed during each 330-ms duration train of nerve stimulation (intratrain synaptic depression), and this intratrain decline was observed across repetitive trains (20 trains at 1/s repeated every 5 min for 30 min for 6 sets). BDNF treatment significantly enhanced quantal release at all fiber types (P < 0.001). BDNF treatment did not change release probability within a stimulation set but enhanced synaptic vesicle replenishment between sets. In agreement, synaptic vesicle cycling (measured using FM4-64 fluorescence uptake) was increased following BDNF [or neurotrophin-4 (NT-4)] treatment (∼40%; P < 0.05). Conversely, inhibiting BDNF/TrkB signaling with the tyrosine kinase inhibitor K252a and TrkB-IgG (which quenches endogenous BDNF or NT-4) decreased FM4-64 uptake (∼34% across fiber types; P < 0.05). The effects of BDNF were generally similar across all fiber types. We conclude that BDNF/TrkB signaling acutely enhances presynaptic quantal release and thereby may serve to mitigate synaptic depression and maintain neuromuscular transmission during repetitive activation.NEW & NOTEWORTHY Neurotrophin brain-derived neurotrophic factor (BDNF) enhances neuromuscular transmission via activation of tropomyosin-related kinase receptor B (TrkB). Rat phrenic nerve-diaphragm muscle preparations were used to determine the rapid effect of BDNF on synaptic quantal release during repetitive stimulation. BDNF treatment significantly enhanced quantal release at all fiber types. BDNF increased synaptic vesicle cycling (measured using FM4-64 fluorescence uptake); conversely, inhibiting BDNF/TrkB signaling decreased FM4-64 uptake.

Varespladib (LY315920) prevents neuromuscular blockage and myotoxicity induced by crotoxin on mouse neuromuscular preparations

Varespladib (LY315920) is a synthetic phospholipase A2 (PLA2) inhibitor that has been demonstrating antiophidic potential against snake venoms that present PLA2 neurotoxins. In this study, we evaluate the capacity of Varespladib to inhibit the neuromuscular effects of crotoxin (CTX), the main toxic component of Crotalus durissus terrificus snake venom, and its PLA2 subunit (CB). We performed a myographic study to compare the neuromuscular effects of CTX or CB and the mixture of these substances plus Varespladib in mice phrenic nerve-diaphragm muscle preparations. CTX (5 μg/mL), CB (20 μg/mL), or toxin-inhibitor mixtures pre-incubated with different concentration ratios of Varespladib (1:0.25; 1:0.5; 1:1; w/w) were added to the preparations and maintained throughout the experimentation period. Myotoxicity was assessed by light microscopic analysis of diaphragm muscle after myographic study. CTX and CB blocked the nerve-evoked twitches, and only CTX induced histological alterations in diaphragm muscle. Pre-incubation with Varespladib abolished the muscle-paralyzing activity of CTX and CB, and also the muscle-damaging activity of CTX. These findings emphasize the clinical potential of Varespladib in mitigating the toxic effects of C. d. terrificus snakebites and as a research tool to advance the knowledge of the mechanism of action of snake toxins.

Crotalus Neutralizing Factor (CNF) inhibits the toxic effects of Crotoxin at mouse neuromuscular preparations

Crotalus Neutralizing Factor (CNF) was the first phospholipase A2 inhibitor isolated from the plasma of the South American rattlesnake (Crotalus durissus terrificus). Previous biochemical and biophysical studies demonstrate an interaction of CNF with Crotoxin (CTX), the main toxic component in the venom of these snakes. CTX promotes the blockade of neuromuscular transmission by a sum of neurotoxic and myotoxic activities. However, the ability of CNF to inhibit these activities has not been shown until the present study. We performed a myographic study to compare the neuromuscular effects of CTX and the mixture CTX plus CNF in mice phrenic nerve-diaphragm muscle preparations. CTX (5 μg/mL) alone, or pre-incubated with CNF (5, 20 or 50 μg/mL) for 15 min was added to the preparations and maintained throughout the experimentation period. Myotoxicity was assessed by light microscopic analysis of diaphragm muscle after myographic study. CTX (5 μg/mL) blocked both indirectly and directly evoked twitches in neuromuscular preparations. In addition, CTX induced histological alterations in diaphragm muscle. Pre-incubation with CNF (50 μg/mL) abolished both the muscle-paralyzing and muscle-damaging activities of CTX. Therefore, the present study confirms, through functional studies, the antiophidic potential of CNF.

Effects of Two Fractions of Swietenia macrophylla and Catechin on Muscle Damage Induced by BothropsVenom and PLA₂.

Plant natural products can attenuate the myonecrosis caused by Bothrops snake venom and their phospholipases A2 (PLA2). In this study, we evaluated the effects of two fractions (F4 and F6) from Swietenia macrophylla and purified catechin on the muscle damage caused by a myotoxic PLA2 from Colombian Bothrops asper venom (BaColPLA2) in mice and by Bothrops marmoratus venom from Brazil in mouse phrenic nerve-diaphragm muscle (PND) preparations in vitro. Male mice were injected with PLA2 (50 µg) in the absence or presence of F4, F6, and catechin, in the gastrocnemius muscle and then killed 3, 7, 14, and 28 h later for histopathological analysis of myonecrosis, leukocyte infiltration, and the presence of collagen. Fractions F4 and F6 (500 µg) and catechin (90 µg) significantly reduced the extent of necrosis at all-time intervals. These two fractions and catechin also attenuated the leukocyte infiltration on day 3, as did catechin on day 14. There was medium-to-moderate collagen deposition in all groups up to day 7, but greater deposition on days 14 and 28 in the presence of F6 and catechin. Bothrops marmoratus venom (100 µg/mL) caused slight (~25%) muscle facilitation after 10 min and weak neuromuscular blockade (~64% decrease in contractile activity after a 120-min incubation). Pre-incubation of venom with F4 or F6 abolished the facilitation, whereas catechin, which was itself facilitatory, did not. All three fractions attenuated the venom-induced decrease in muscle contractions. These findings indicate that fractions and catechin from S. macrophylla can reduce the muscle damage caused by Bothrops venom and PLA2. These fractions or their components could be useful for treating venom-induced local damage.

&lt;b&gt;Atropine is more efficient than ZM241385 to reduce a drastic level of fade caused by cisatracurium at 50 Hz

The effects of atropine (non selective muscarinic antagonist) and ZM241385 (A 2A receptors antagonist) in the cisatracurium-induced drastic (100%) level of fade at 50 Hz (10 s) (100% Fade) were compared in the phrenic nerve-diaphragm muscle preparations of rats indirectly stimulated at a physiological tetanic frequency (50 Hz). The lowest dose and the instant cisatracurium caused 100% Fade were investigated. Cisatracurium induced 100% Fade 15 min after being administered. Atropine reduced the cisatracurium-induced 100% Fade, but the administration of ZM241385 separately, or combined with atropine, did not cause any effect in the cisatracurium-induced 100% Fade. Data indicate that the simultaneous blockage of M 1 and M 2 muscarinic receptors on motor nerve terminal by atropine is more efficient than the blockage of presynaptic A 2A receptors for a safer recovery of patients from neuromuscular blockade caused by cisatracurium.

Development of an in vitro rat phrenic nerve–diaphragm muscle preparation

Development of an in vitro rat phrenic nerve–diaphragm muscle preparation

Investigation of Relaxative Effect of Stem-bark Extract of Erythrophleum suaveolens on rat Phrenic Nerve-diaphragm Muscle

Investigation of Relaxative Effect of Stem-bark Extract of Erythrophleum suaveolens on rat Phrenic Nerve-diaphragm Muscle

TRPV1 Modulates Acetylcholine Release From Motor Nerve Terminals

Transient receptor potential (TRP) proteins are expressed ubiquitously throughout the body. Previous studies demonstrated expression of TRPV1, the capsaicin receptor, in sensory neurons. Recently, we reported TRPV1 expression in mouse motor nerve endings (MNEs; J. Pharmacol. Exp. Ther. Aug 04. 2009) where we observed that capsaicin protected MNEs from botulinum neurotoxin A (BoNT/A). We hypothesized that capsaicin reduced clathrin coated pit (CCP) dependent endocytosis of BoNT/A and demonstrate the regulatory influence of TRPV1 in exo-endocytic processes of MNEs. Phrenic nerve diaphragm muscle preparations isolated from isoflurane anesthetized adult mice were analyzed for the nerve-evoked twitch and transmitter release (TR). Capsaicin produced a concentration-dependent decline of twitch tension (TT), an effect attributed to suppression of stimulus-evoked acetylcholine release (SEAR) since these capsaisin concentrations reduced the amplitude of endplate currents. These effects of capsaicin were antagonized by capsazepine, the TRPV1 antagonist. To understand the mechanism whereby capsaicin reduced TR, we studied cholinergic Neuro 2a cells. Acute exposure to capsaicin altered the subcellular distribution of clathrin heavy chain (CHc) and AP2, two proteins essential to CCP formation. Wortmannin, (non selective PI3K/PI5K inhibitor), inhibited the TT and SEAR of the isolated nerve-muscle preparations and delocalized CHc and AP2 in Neuro 2a cells. Chlorpromazine, an inhibitor of CCP dependent endocytic pathway [Cell Mol. Biol. Lett. 2004; 9 (3): 475-81], mimicked the effects of capsaicin on AP2 delocalization. These data suggest that endogenous TRPV1 proteins are coupled to the exo-endocytic mechanisms that regulate neuromuscular transmission and that activation of TRPV1 with high capsaicin concentrations reduces exocytosis of acetylcholine by down regulating the compensatory CCP dependent endocytic pathways.

Presynaptic M1, M2, and A1 receptors play roles in tetanic fade induced by pancuronium or cisatracurium

We investigated whether presynaptic facilitatory M1 and/or inhibitory M2 muscarinic receptors contributed to pancuronium- and cisatracurium-induced tetanic fade. Phrenic nerve-diaphragm muscle preparations of rats were indirectly stimulated with tetanic frequency (75 +/- 3.3 Hz; mean +/- SD). Doses of pancuronium, cisatracurium, hexamethonium, and d-tubocurarine for producing approximately 25% fade were determined. The effects of pirenzepine and methoctramine, blockers of presynaptic M1 and M2 receptors, respectively, on the tetanic fade were investigated. The concentrations required for approximately 25% fade were 413 microM for hexamethonium (26.8 +/- 2.4% 4% fade), 55 nM for d-tubocurarine (28.7 +/- 2.55% fade), 0.32 microM for pancuronium (25.4 +/- 2.2% fade), and 0.32 microM for cisatracurium (24.7 +/- 0.8% fade). Pirenzepine or methoctramine alone did not produce the fade. Methoctramine, 1 microM, attenuated the fade induced by hexamethonium (to 16.0 +/- 2.5% fade), d-tubocurarine (to 6.0 +/- 1.6 fade), pancuronium (to 8.0 +/- 4.0% fade), and cisatracurium (to 11.0 +/- 3.3% fade). 10 nM pirenzepine attenuated only the fades produced by pancuronium (to 5.0 +/- 0.11% fade) and cisatracurium (to 13.3 +/- 5.3% fade). Cisatracurium (0.32 microM) showed antiacetylcholinesterase activity (in plasma, 14.2 +/- 1.6%; 6%; in erythrocyt 17.2 +/- 2.66%) similar to that of pancuronium (0.32 microM). The selective A1 receptor blocker, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 2.5 nM), also attenuated the fades induced by pancuronium and cisatracurium. The tetanic fades produced by pancuronium and cisatracurium depend on the activation of presynaptic inhibitory M2 receptors; these agents also have anticholinesterase activities. The fades induced by these agents also depend on the activation of presynaptic inhibitory A1 receptors through the activation of stimulatory M1 receptors by acetylcholine.

Skeletal muscle relaxant effect of Chonemorpha macrophylla in experimental animals

OBJECTIVE: To study the skeletal muscle relaxant property of Chonemorpha macrophylla (CM). MATERIALS AND METHOD: The skeletal muscle relaxant effect of the alcoholic extract of CM was studied on isolated frog rectus abdominis muscle, isolated rat phrenic nerve diaphragm muscle preparation and in intact young chicks. The parameter studied in the isolated muscle or isolated nerve muscle preparations was the extent of inhibition of acetylcholine or electrically-induced contraction of skeletal muscles. In intact chicks, the drug was administered i.v. in wing veins and the onset, duration and nature of paralysis were recorded. In all the experiments, the effect of the drug was compared with that of gallamine and succinylcholine. RESULTS: The alcoholic extract of CM reduced the acetylcholine-induced contraction of isolated frog rectus abdominis and electrically stimulated contractions of rat phrenic nerve diaphragm in a dose-dependent manner. In unanaesthetized chicks, it produced spastic type of paralysis with extension of the neck and limbs. The effects were similar to the effects of succinylcholine but different from those of gallamine. CONCLUSION: The alcoholic extract of CM possesses skeletal muscle relaxant property. It produces depolarizing type of muscle paralysis similar to that produced by succinylcholine.

Anti-sera raised in rabbits against crotoxin and phospholipase A 2 from Crotalus durissus cascavella venom neutralize the neurotoxicity of the venom and crotoxin

Crotoxin, the principal neurotoxin in venom of the South American rattlesnakes Crotalus durissus terrificus and Crotalus durissus cascavella, contains a basic phospholipase A 2 (PLA 2) and an acidic protein, crotapotin. In this work, we examined the ability of rabbit anti-sera against crotoxin and its PLA 2 subunit to neutralize the neurotoxicity of venom and crotoxin from C. d. cascavella in mouse phrenic nerve–diaphragm and chick biventer cervicis preparations. Immunoblotting showed that the anti-sera recognized C. d. cascavella crotoxin and PLA 2. This was confirmed by ELISA, with both anti-sera having end-point dilutions of 3×10 −6. Anti-crotoxin serum neutralized the neuromuscular blockade in phrenic nerve–diaphragm muscle preparations at venom or crotoxin:anti-serum ratios of 1:2 and 1:3, respectively. Anti-PLA 2 serum also neutralized this neuromuscular activity at a venom or crotoxin:anti-serum ratio of 1:1. In biventer cervicis preparations, the corresponding ratio for anti-crotoxin serum was 1:3 for venom and crotoxin, and 1:1 and 1:2 for anti-PLA 2 serum. The neutralizing capacity of the sera in mouse preparations was comparable to that of commercial anti-serum raised against C. d. terrificus venom. These results show that anti-sera against crotoxin and PLA 2 from C. d. cascavella venom neutralized the neuromuscular blockade induced by venom and crotoxin in both nerve–muscle preparations, with the anti-serum against crotoxin being slightly less potent than that against crotoxin.

Action of Micrurus dumerilii carinicauda coral snake venom on the mammalian neuromuscular junction

The venoms of coral snakes (mainly Micrurus species) have pre- and/or postsynaptic actions, but only a few of these have been studied in detail. We have investigated the effects of Micrurus dumerilii carinicauda coral snake venom on neurotransmission in rat isolated phrenic nerve-diaphragm muscle and chick biventer cervicis preparations stimulated directly or indirectly. M. d. carinicauda venom (5 or 10 μg/ml) produced neuromuscular blockade in rat (85–90% in 291.8±7.3 min and 108.3±13.8, respectively; n=5) and avian (95.0±2.0 min; 5 μg/ml, n=5) preparations. Neostigmine (5.8 μM) and 3,4-diaminopyridine (230 μM) partially reversed the venom-induced neuromuscular blockade in rat nerve-muscle preparations. In neither preparation did the venom depress the twitch response elicited by direct muscle stimulation. The contractures induced by acetylcholine in chick preparations were inhibited by the venom (95–100%; n=4; p<0.05). In rat preparations, the venom produced a progressive decrease in the amplitude of miniature end-plate potentials (m.e.p.ps control frequency=69.3±5.0/min and control amplitude=0.4±0.2 mV) until these were abolished. Neostigmine (5.8 μM) and 3,4-diaminopyridine (230 μM) partially antagonized this blockade of m.e.p.ps. The resting membrane potential was not altered with the venom (10 μg/ml). M. d. carinicauda venom produced dose-dependent morphological changes in indirectly stimulated mammal preparations. Twenty-five per cent of muscle fibers were affected by a venom concentration of 5 μg/ml, whilst 60.7% were damaged by 10 μg of venom/ml. In biventer cervicis preparations, the morphological changes were slower in onset and were generally characterized by undulating fibers and, to a lesser extent, by zones of disintegrating myofibrils. A venom concentration of 5 μg/ml damaged 52.2% of the fibers. These findings indicate that M. d. carinicauda venom has neurotoxic and myotoxic effects and that the neuromuscular blockade involves mainly a postsynaptic action.

Counter Effects of Higenamine and Coryneine, Components of Aconite Root, on Acetylcholine Release from Motor Nerve Terminal in Mice

The counter effects of higenamine and coryneine, components of aconite root, on acetylcholine (ACh) release from motor nerve terminals in the mouse phrenic nerve–diaphragm muscle preparation were studied by a radioisotope method. Both nerve-evoked release and spontaneous release of [3H]-ACh from the preparation preloaded with [3H]-choline were measured. The change in the tetanic tension of muscle was simultaneously recorded in the same preparation. Higenamine (10 μM) augmented both the nerve-evoked and spontaneous ACh releases, and the muscle tension. The effects were inhibited by pretreatment with propranolol (10 μM), a β-adrenoceptor antagonist. Coryneine reduced the nerve-evoked release of ACh, accelerated the decay of tetanic tension (tetanic fade) at 30 μM, and it depressed the peak amplitude of tetanic tension at a higher concentration of 100 μM. These results suggest that of the two components contained in aconite root, higenamine increases ACh release via activation of β-adrenoceptor, and conversely coryneine depresses ACh release by preferentially acting at motor nerve terminal.

Contractile and fatigue properties of the rat diaphragm musculature during the perinatal period.

The following two hypotheses regarding diaphragm contractile properties in the perinatal rat were tested. First, there is a major transformation of contractile and fatigue properties during the period between the inception of inspiratory drive transmission in utero and birth. Second, the diaphragm muscle properties develop to functionally match changes occurring in phrenic motoneuron electrophysiological properties. Muscle force recordings and intracellular recordings of end-plate potentials were measured by using phrenic nerve-diaphragm muscle in vitro preparations isolated from rats on embryonic day 18 and postnatal days 0-1. The following age-dependent changes occurred: 1) twitch contraction and half relaxation times decreased approximately two- and threefold, respectively; 2) the tetanic force levels increased approximately fivefold; 3) the ratio of peak twitch force to maximum tetanic force decreased 2.3-fold; 4) the range of forces generated by the diaphragm in response to graded nerve stimulation increased approximately twofold; 5) the force-frequency curve was shifted to the right; and 6) the propensity for neuromuscular transmission failure decreased. In conclusion, the diaphragm contractile and phrenic motoneuron repetitive firing properties develop in concert so that the full range of potential diaphragm force recruitment can be utilized and problems associated with diaphragm fatigue are minimized.

The neuromuscular action of Ancylometes sp. spider venom in the rat phrenic nerve–diaphragm preparation

The effects of Ancylometes sp. venom on muscle contraction and bioelectrical potentials were investigated in the rat phrenic nerve–diaphragm muscle preparation. The venom (50 μg/ml) depolarized the diaphragm muscle fiber membranes. This effect was abolished by tetrodotoxin and by reduction of the sodium concentration of the Tyrode solution. The increase in the frequency of miniature end plate potentials induced by the venom was also suppressed by tetrodotoxin (3 μM). These results indicate that the venom may activate voltage-dependent sodium channels in cell membranes. All of the effects of Ancylometes sp. venom on this nerve–muscle preparation (i.e. increase in twitch tension, spontaneous small phasic contractions and increase in the frequency of miniature end-plate potentials) may be explained in terms of its action on sodium channels.

Acetylcholine sensitivity of biphasic Ca2+ mobilization induced by nicotinic receptor activation at the mouse skeletal muscle endplate

1. Acetylcholine (ACh) was locally applied onto the endplate region in a mouse phrenic nerve-diaphragm muscle preparation to measure intracellular free calcium ([Ca2+]i) entry through nicotinic ACh receptors (AChRs) by use of Ca2+-aequorin luminescence. 2. ACh (0.1-3 mM, 20 microl) elicited biphasic elevation of [Ca2+]i (fast and slow Ca2+ mobilization) in muscle cells. The peak amplitude of the slow Ca2+ mobilization (not accompanied by twitch tension) was concentration-dependently increased by ACh, whereas that of the fast component (accompanied by twitch tension) reached a maximum response at a lower concentration (0.1 mM) of applied ACh. 3. A pulse of nicotinic agonists, (-)-nicotine (10 mM) and 1,1-dimethyl-4-phenyl-piperazinium (10 mM), but not a muscarinic agonist pilocarpine (10 mM), also elicited a biphasic Ca2+ signal. 4. Even though ACh release from motor nerve endings was blocked by botulinum toxin (5 microg, bolus i.p. before isolation of the tissue), the generation of both a fast and slow Ca2+ component caused by ACh application was observed. 5. These results strongly suggest that ACh locally applied onto the endplate region of skeletal muscle induces a slow Ca2+ signal reflecting Ca2+ entry through a postsynaptic nicotinic AChR, which has a low sensitivity to transmitter ACh.

Effects of feeding condition after birth on the sensitivity of neuromuscular transmission to d-tubocurarine In vitro in mice

1. 1. Effects of feeding condition from birth were examined on the sensitivity of neuromuscular transmission to d-tubocurarine (dTc) in vitro in male mice of the ddY strain. 2. 2. Mice were trained to climb two separated cylindrical steel-wire tubes for feeding and drinking, respectively, from 16 days of age. Some mice were conventionally fed, from 99 days of age. Nerve-muscle preparations were made from the left phrenic nerve diaphragm muscle (DPH), the sciatic nerve soleus muscle (SOL), and the sciatic nerve extensor digitorum longus muscle (EDL) of 99-day-old and 155-day-old mice. The nerve trunk was electrically activated with trains of four pulses and tetanic pulses. 3. 3. The sensitivity to the effects of dTc decreased in the order EDL, SOL, and DPH. This result held true in all mice tested. 4. 4. This sensitivity was significantly potentiated by the compulsory movement. 5. 5. The supersensitivity remained even when mice were conventionally fed after 99 days of age. 6. 6. The compulsion rendered EDL antifatigable on tetanic stimulation. This property was also retained after a return to conventional feeding. 7. 7. These results suggest that the effects of feeding condition from birth might remain on neuromuscular functions after termination of the conditioning.

Slow Ca2+ (RAMIC) mobilization operated by postsynaptic neuronal nicotinic receptor regulates synaptic function at the mouse neuromuscular junction

We have found that non-contractile slow Ca2+ mobilization (RAMIC; Receptor-Activity Modulating Intracellular Ca2+) is generated by motor nerve stimulation with anti-cholinesterase at the skeletal muscle, and desensitizes muscle nicotinic receptor (nAChR). To confirm this Ca2+ mobilization without anti-cholinesterase, acetylcholine (ACh) was locally applied by N2-gas pressure onto endplate region at the mouse phrenic nerve-diaphragm muscle preparation. ACh (0.1-3 mM, 20 microliters) elicited bi-phasic elevation of [Ca2+]i (fast and slow Ca2+ mobilization measured as Ca(2+)-aequorin luminescence) in muscle cells. The peak amplitude of slow Ca2+ mobilization (not accompanied by contraction) was increased by ACh concentration-dependently, whereas that of fast component (accompanied by contraction) reached a maximum response at a lower concentration of ACh. The slow Ca2+ mobilization was blocked by lower concentrations of competitive nAChR antagonists which did not affect the fast Ca2+ transients. Moreover, the slow Ca2+ signal was selectively depressed by a neuronal nAChR antagonist methyllycaconitine. Neither Ca2+ channel blockers nor a Na+ channel blocker tetrodotoxin prevented the generation of the slow Ca2+ mobilization. These results suggest that RAMIC is mobilized through postsynaptic neuronal nAChR subtype to desensitize muscle nAChR at the neuromuscular junction.

Effect of ethanol on function of the rat heart and skeletal muscles.

The present study was undertaken to evaluate the acute effects of ethanol on responses of the rat heart and skeletal muscles both in vivo and in vitro. In the anesthetized rat, intravenous infusion of ethanol at 0.1-0.5 g/kg body weight (33-167 mM) decreased the breathing rate by 8-83%, heart rate by 4-52%, and QRS amplitude by 5-27%, and increased the P-R interval by 1-49%. In the anterior tibialis muscle subjected to repetitive nerve stimulation at 100 Hz for 0.5 sec, ethanol at 0.1 g/kg increased the amplitude of the muscle action potential (AP) by 7%, whereas at 0.5 g/kg it decreased the muscle AP by 32%. The nerve-evoked tetanic tension was reduced by 7-34% at 0.1-0.5 g/kg ethanol. In the isolated rat heart, perfusion of ethanol at 0.1-3.0% (22-651 mM) decreased the heart rate by 8-48% and QRS amplitude by 10-39%, and increased the P-R interval by 5-61%. Left ventricular pressure was increased by 10% at 0.1% ethanol, and decreased by 80% at 3.0% ethanol. In the isolated rat phrenic nerve-diaphragm muscle preparation subjected to repetitive nerve stimulation at 100 Hz for 0.5 sec, 0.1-3.0% ethanol decreased the amplitude of the nerve AP by 5-89%, nerve-evoked muscle AP by 2-96%, and peak tetanic tension by 1-87%. On repetitive direct muscle stimulation at 100 Hz for 0.5 sec, 0.1-3.0% ethanol decreased the amplitude of the muscle-evoked muscle AP by 8-65%, and muscle-evoked tetanic tension by 2-65%. These studies indicate that ethanol causes smaller reduction in responses of the heart and skeletal muscles at clinical concentrations, but marked reduction in these responses at higher concentrations due to direct action on excitability of these tissues. At higher concentrations, ethanol causes greater reduction in excitability of the skeletal muscle than of the heart.

Potentiating effects of beta-eudesmol-related cyclohexylidene derivatives on succinylcholine-induced neuromuscular block in isolated phrenic nerve-diaphragm muscles of normal and alloxan-diabetic mice.

beta-Eudesmol, a sesquiterpenoid alcohol contained in Atractylodes lancea, potentiates succinylcholine (SuCh)-induced neuromuscular blockade. The potentiating effect is greater in diabetic muscles than in normal ones. As a ligand for affinity chromatography to study the potentiating mechanism, we designed and synthesized newly beta-eudesmol-related cyclohexylidene derivatives (2-(3-hydroxy-3-methylbutyl)cyclohexylidene; KTE-13, 2-(3-hydroxy-3-methylbutyl)-4-cyclohexylidene carboxylic acid; KTE-32 and 4-tert-butoxycarbonyl-2-(3-hydroxy-3-methylbutyl) cyclohexylidene; KTE-33). We examined the potentiating effects of those compounds in phrenic nerve-diaphragm muscle preparations of normal and alloxan-diabetic mice. KTE-33 (100 microM) potentiated more greatly SuCh-induced neuromuscular blockade in diabetic muscles than in normal ones (the potentiating ratios in normal and diabetic muscles were 6.7 and 10.6, respectively), while KTE-13 (100 microM) and -32 (200 microM) potentiated weakly. These results suggest that the ester group in KTE-33 rather than a carboxyl group in KTE-32 is important in inducing the potentiation of SuCh-induced neuromuscular blockade in diabetic state.