Rat Phrenic Nerve-hemidiaphragm Preparation Research Articles

Effects of hyperthermia on the effective concentration of rocuronium and sugammadex-mediated reversal in isolated phrenic nerve hemidiaphragm preparations of rats

BackgroundHyperthermia is relatively rare during general anesthesia; however, a few studies have been conducted on hyperthermia and the neuromuscular blockade (NMB) induced by rocuronium, and the reversal of NMB by sugammadex. We investigated the effect of hyperthermia status on the NMB induced by rocuronium, and its reversal by sugammadex, in isolated phrenic nerve hemidiaphragm (PNHD) preparations of the rat.MethodsThirty-three male Sprague-Dawley rat PNHD preparations were randomly assigned to three groups at different temperatures (36 °C, 38 °C, and 40 °C; each group, n = 11, in Krebs solution). The train-of-four (TOF) and twitch height responses were checked mechanomyographically. The PNHD were treated with progressively increasing doses of rocuronium and three effective concentrations (ECs), EC50, EC90, and EC95, of rocuronium were analyzed in each group via nonlinear regression analysis. Then, sugammadex was administered in doses equimolar to rocuronium. Thereafter, the T1 height (%), TOFR (%) and the duration index were measured.ResultsThe EC of rocuronium (EC50, EC90, and EC95) decreased significantly in accordance with increasing temperature. The groups at 36 °C and 40 °C showed clear differences in all areas (all P < 0.001). Moreover, the T1 height (%) and the duration index upon sugammadex administration showed faster recovery results in the36 °C than the 38 °C and 40 °C groups.ConclusionA rise of temperature from 38 °C to 40 °C in rat PNHD preparations proportionally enhanced the NMB induced by rocuronium. In addition, equimolar doses of sugammadex to the administered rocuronium showed a slower recovery time as the temperature rises.

Isomeric Carborane Neuromuscular Blocking Agents.

We synthesized a family of neuromuscular blocking agents (NMB) based on decamethonium, but containing a carborane cluster in the methylene chain between the two quaternary ammonium groups. The carborane cluster isomers o-NMB, m-NMB, and p-NMB were tested in animals for neuromuscular block and compared with agents used clinically: rocuronium and decamethonium. All three isomers caused reversible muscle weakness in mice as determined by grip strength and inverted screen tests, with a potency rank of p-NMB > rocuronium > decamethonium > m-NMB > o-NMB. The mechanism of action of the compounds was determined by using the in vitro rat phrenic nerve hemi-diaphragm preparation and electrophysiologic measurements in cells. Neostigmine reversed hemi-diaphragm weakness caused by the three isomers and rocuronium, but not succinylcholine. In electrophysiologic recordings of currents through acetylcholine receptor channels, the carborane compounds did not activate channel activity but did inhibit channel activation by acetylcholine. These results demonstrate that the carborane neuromuscular blocking agents are non-depolarizers in contrast to the depolarizing action of the parent compound.

Effects of resveratrol, catechin and epicatechin on rat phrenic nerve hemi-diaphragm

Aim: Resveratrol, catechin and epicatechin are known for their preventive effects of skeletal muscle atrophy, anti-fatique effects and enhancement of exercise capacity. In this study, we investigate the effects these antioxidants on muscle contraction by using rat phrenic nerve hemi-diaphragm preparations. Methods: Twitch responses are investigated by giving direct and indirect and tetanic stimulation to rat phrenic nerve hemi-diaphragm preparations. Results: Resveratrol and catechin facilitated twitch responses to the indirect stimulus in short duration and suppressed in long duration. The decrease in the responses to the direct stimulation by applying epicatechin (p=0.010) was significantly higher than the responses by giving indirect stimulation. Epicatechin significantly suppressed tetanic responses (p=0.005), but not resveratrol and catechin. Conclusion: The results of the study show that these antioxidants have presynaptic and postsynaptic effects on neuromuscular junction. Resveratrol effective on muscle contraction via direct stimulus, catechin and epicatechin effects muscle contraction by direct and indirect stimulus.

Effects of magnesium chloride on rocuronium-induced neuromuscular blockade and sugammadex reversal in an isolated rat phrenic nerve-hemidiaphragm preparation: An in-vitro study.

Magnesium potentiates the effects of nondepolarising muscle relaxants. However, few studies have used magnesium chloride (MgCl2). Sugammadex reverses neuromuscular block by steroidal nondepolarising muscle relaxants. To assess the effects of MgCl2 on rocuronium-induced neuromuscular blockade and its reversal by sugammadex. In-vitro experimental study. Animal laboratory, Asan Medical Center, Seoul, South Korea, from 20 March 2016 to 3 April 2016. Forty male Sprague Dawley rats. Left phrenic nerve-hemidiaphragms from 40 Sprague Dawley rats were allocated randomly to four groups (1, 2, 3 and 4 mmol l MgCl2 group, n = 10 each). Rocuronium was administered cumulatively until the first twitch of train-of-four (TOF) disappeared completely. Then, equimolar sugammadex was administered. The effective concentration (EC) of rocuronium was obtained in each group. After administering sugammadex, recovery of the first twitch height and the TOF ratio were measured for 30 min. EC50, EC90 and EC95 significantly decreased as the concentration of MgCl2 increased (all P ≤ 0.001), except the comparison between the 3 and 4 mmol l MgCl2 groups. After administration of sugammadex, the maximal TOF ratio (%) was lower in the 4 mmol l MgCl2 group than the 1 mmol l MgCl2 group [median 91.7 interquartile range (83.4 to 95.8) vs. 98.3 interquartile range (92.2 to 103.4), P = 0.049]. The mean time (s) from sugammadex injection to achieving maximal first twitch was significantly prolonged in the 4 mmol l MgCl2 group vs. the 1 mmol l MgCl2 and 2 mmol l MgCl2 groups [1483.9 (± 237.0) vs. 1039.0 (± 351.8) and 926.0 (± 278.1), P = 0.022 and 0.002, respectively]. Increases in MgCl2 concentration reduce the ECs of rocuronium. In addition, administering sugammadex equimolar to the administered rocuronium shows limited efficacy as MgCl2 concentration is increased. The in-vitro study was not registered in a database.

Effect of Zidovudine and its interaction with rocuronium on neuromuscular transmission.

Neuromuscular weakness is often found in patients receiving zidovudine therapy due to mitochondrial damage. Effect of zidovudine was evaluated in indirectly and directly stimulated isolated rat phrenic nerve hemidiaphragm preparations, by cumulative dose response curve with square wave pulses, 0.5 ms duration of 2 Hz at every 10 s. To understand the observed effect of zidovudine, interaction studies were carried out with rocuronium. Dose response curve of rocuronium was compared in the absence and in the presence of 1.2 and 12 mmol/ml zidovudine. In another set of experiment, intact animals were treated with zidovudine 50 and 100 mg/kg for 15 days and phrenic nerve hemidiaphragm was obtained for in vitro experiment. Effect of zidovudine (30 mmol/ml) on twitch responses inhibited by dantrolene 50 μmol/ml, magnesium chloride 8 mmol/ml, rocuronium 10 μmol/ml, succinylcholine 25 μmol/ml and lignocaine 600 μmol/ml was studied. Zidovudine (3-30 mmol/ml) significantly augmented the twitch responses up to 80 and 40% in indirectly and directly stimulated preparations, respectively (P<0.05). Zidovudine did not show significant interaction with rocuronium in any group as per dose response curve and inhibitory concentration 5%, inhibitory concentration 50% and inhibitory concentration 95% of rocuronium. Zidovudine (30 mmol/ml) augmented the twitch responses inhibited by dantrolene, magnesium chloride and rocuronium. It reduced the time for complete block of succinylcholine (P<0.05). Zidovudine affects the neuromuscular transmission. No conclusive interaction between rocuronium and zidovudine was found.

Antibiotics and muscle relaxation

Anesthesiologists use quite a few medications during anesthesia of a patient. Those include inhaled anesthetics, intravenous anesthetics, opioids, muscle relaxants, cholinesterase inhibitors, anticholinergic drugs, adrenergic agonists and antagonists, hypotensive agents, local anesthetics, and several adjunct drugs. We are usually well-trained in the administration of these drugs and aware of their actions and side effects. How about antibiotics? The anesthesiologists do not seem to have as much interest in antibiotics as their own favorite drugs. Antimicrobial agents can be used for prophylaxis to reduce the incidence of postoperative infections or for treatment of obvious preexisting infection. It is frequently requested to the anesthetic part to administer the prescribed antibiotics perioperatively. Many institutions have much debate as to the actual administration of the antibiotics, as well as the responsibility for their actions and side effects between anesthesiologists and surgeons. The truth is, each institution has its own protocol for antibiotic administration for the prophylaxis of surgical site infections and updates the protocol occasionally according to the rapid change of the drugs and information. There are potential difficulties that anesthesiologists be made aware of in association with the use of antibiotics, such as allergic reactions, end-organ damage and prolongation of neuromuscular block [1]. Most antibiotics can cause neuromuscular blockade alone and also potentiate blockade when combined with neuromuscular blockers. These can happen by the two different mechanisms, including inhibition of the prejunctional acetylcholine release and depression of postjunctional nicotinic acetylcholine receptor sensitivity to acetylcholine. Because gentamicin covers gram-negative organisms, and clindamycin covers gram-positive and anaerobic organisms, the combination therapy of those is a common treatment for postpartum endometritis. There are few reports about the effect of gentamicin and clindamycin on neuromuscular blockade caused by rocuronium. In this edition of the Journal, Lee et al. [2] studied about the effect of gentamicin and clindamycin to rocuronium-induced neuromuscular blockade in rat phrenic nerve-hemidiaphragm preparations. So far, the authors have succeeded in finding out the effect of a variety of drugs on neuromuscular blockade in a series of in vitro studies. They have presented us with beautiful sigmoid curves to show a relationship between drug concentration and response on the single twitch and tetanic fade in their articles. Their consecutive findings are as follows: 1. Clindamycin and gentamicin interfere with neuromuscular transmission. Neostigmine and calcium antagonize the neuromuscular blockade caused by gentamicin, but augment that caused by clindamycin [3]. 2. Neostigmine partially antagonizes the neuromuscular blockade that is induced by a combination of clindamycin and rocuronium [4]. 3. Bupivacaine inhibits neuromuscular conduction and enhances rocuronium-induced neuromuscular blockade [5]. 4. Gentamicin or a combination of gentamicin and clindamycin enhances rocuronium-induced neuromuscular blockade [2]. All the above laboratory results can be applied clinically in the operating room and intensive care unit. Since anesthesiologists will perhaps not be able to avoid a responsibility in administration of antibiotic regimen as time goes on, we should have the latest knowledge concerning antibiotics, including the interaction with the muscle relaxants.

Adenosine A2A receptor antagonists are broad facilitators of antinicotinic neuromuscular blockade monitored either with 2 Hz train‐of‐four or 50 Hz tetanic stimuli

1. The 2 Hz train-of-four ratio (TOF(ratio)) is used to monitor the degree of patient curarization. Using a rat phrenic nerve-hemidiaphragm preparation, we showed that antinicotinic agents, such as hexamethonium, d-tubocurarine and pancuronium, but not cisatracurium, decreased contractions produced by physiological nerve activity patterns (50 Hz) more efficiently than those caused by 2 Hz trains. Uncertainty about the usefulness of the TOF(ratio) to control safe recovery from curarization prompted us to investigate the muscarinic and adenosine neuromodulation of tetanic (50 Hz) fade induced by antinicotinic agents at concentrations that cause a 25% reduction in the TOF(ratio) (TOF(fade)). 2. Tetanic fade caused by d-tubocurarine (1.1 μmol/L), pancuronium (3 μmol/L) and hexamethonium (5.47 mmol/L) was attenuated by blocking presynaptic inhibitory muscarinic M(2) and adenosine A(1) receptors with methoctramine (1 μmol/L) and 1,3-dipropyl-8-cyclopentylxanthine (2.5 nmol/L), respectively. These compounds enhanced rather than decreased tetanic fade induced by cisatracurium (2.2 μmol/L), but they consistently attenuated cisatracurium-induced TOF(fade). The effect of the M(1) receptor antagonist pirenzepine (10 nmol/L) on fade produced by antinicotinic agents at 50 Hz was opposite to that observed with TOF stimulation. Blockade of adenosine A(2A) receptors with ZM 241385 (10 nmol/L) attenuated TOF(fade) caused by all antinicotinic drugs tested, with the exception of the 'pure' presynaptic nicotinic antagonist hexamethonium. ZM 241385 was the only compound tested in this series that facilitated recovery from tetanic fade produced by cisatracurium. 3. The data suggest that distinct antinicotinic relaxants interfere with fine-tuning neuromuscular adaptations to motor nerve stimulation patterns via activation of presynaptic muscarinic and adenosine receptors. These results support the use of A(2A) receptor antagonists together with atropine to facilitate recovery from antinicotinic neuromuscular blockade.

Identification and evaluation of agents isolated from traditionally used herbs against Ophiophagus hannah venom

The aim of this study was firstly to identify active molecules in herbs, that are traditionally used for the treatment of snake bite, such as Curcuma antinaia, Curcuma contravenenum, Andrographis paniculata, and Tanacetum parthenium; secondly to test similar structurally related molecules and finally to prepare and evaluate an efficient formulation against Ophiophagus hannah venom intoxification. Three labdane based compounds, including labdane dialdehyde, labdane lactone, and labdane trialdehyde and two lactones including 14-deoxy-11,12-didehydroandrographolide and parthenolide were isolated by column chromatography and characterised. Using the isolated rat phrenic nerve-hemidiaphragm preparation, the antagonistic effect of crude extracts, isolated compounds and prepared formulations were measured in vitro on the inhibition of the neuromuscular transmission. Inhibition on muscle contraction, produced by the 5 μg/mL venom, was reversed by test agents in organ bath preparations. A labdane trialdehyde, isolated from C. contravenenum, was identified as the best antagonising agent in the low micromolar range. Tests on formulations of the most potent C. contravenenum extract showed, that the suppository with witepsol H15 was an effective medicine against O. hannah venom. This study elucidated the active compounds, accounting for the antivenin activity of traditionally used herbs and suggested the most suitable formulation, which may help to develop potent medicines for the treatment of snake bite in the future.

Presynaptic and postsynaptic sites of action of enhydrotoxin-a (EsNTx-a) isolated from Enhydrina schistosa venom

Abstract Enhydrotoxin-a (EsNTx-a), one of the three toxins isolated from the venom of Enhydrina schistosa produced an irreversible blockade of indirect stimulation of rat phrenic nerve hemidiaphragm preparation in concentration of 0.5 to 1 μg ml−1. The response to direct stimulation was not affected over a 4 h period. The neuromuscular blockade produced by EsNTx-a was antagonized in so far as twitches evoked by indirect stimulation are concerned, when the preparation was treated with approximately double the concentration of purified sea snake antivenin. The tension of tetanic contractions, although depressed in amplitude by the toxin, was sustained. However, it was converted to a twitch-like response in toxin-antivenin treated preparations. In the presence of eserine (1 × 10−6 g ml−1), the twitch-like tetanus was dramatically restored in toxin-antivenin treated rat phrenic nerve hemidiaphragm preparations. Intracellular recordings showed that the frequency of spontaneous miniature end plate potentials (m.e.p.ps) recorded in EsNTx-a poisoned (1.24 × 10−6 g ml) rat phrenic nerve hemidiaphragm preparations decreased to zero, while the m.e.p.p. amplitude was decreased in the later phase of block. The evoked end plate potentials (e.p.p.) in EsNTx-a poisoned (1.24 × 10−1 g ml−1) preparations progressively diminished to zero. The antivenin molecules may have an easy access to bind to the toxin receptor complexes at the postsynaptic site while the presynaptic binding site by the toxin may be relatively inaccessible due to their being intracellular. The presynaptic effect of the toxin was revealed only when the postsynaptic effect was abolished.

In-vitro interaction between H2 antagonists and vecuronium.

The interaction between histamine H2 antagonists and the neuromuscular blocking drug vecuronium was investigated in the rat phrenic nerve-hemidiaphragm preparation. Cimetidine alone, in the concentration range 800-4000 microM produced between 14 and 74% neuromuscular paralysis with an EC50 (mean +/- s.e.) of 2900 +/- 100 microM. Ranitidine augmented the indirectly-evoked muscle response at concentrations between 30 and 160 microM but at higher concentrations, between 300 and 1800 microM, produced neuromuscular paralysis. Famotidine produced negligible and statistically insignificant (0-5%) neuromuscular paralysis at concentrations between 0.3 and 300 microM. Cimetidine (800 microM) shifted the neuromuscular concentration-effect curve of vecuronium to the left in a parallel manner, while ranitidine (160 microM) shifted it to the right. The potentiation ratio was 1.90 +/- 0.14 for cimetidine and 0.62 +/- 0.05 for ranitidine. Famotidine (30 microM) did not alter the response to vecuronium. These data indicate that higher than clinically relevant concentrations of cimetidine and ranitidine produce neuromuscular paralysis and may potentiate the action of vecuronium. Low concentrations of ranitidine may antagonize the action of vecuronium. Famotidine, in contrast, lacks significant neuromuscular effects.

Methylmercury and the skeletal muscle receptor.

Methylmercury at bath concentration of 2 X 10(-5) M was capable of inhibiting muscular contractions of the isolated rat phrenic-nerve hemidiaphragm preparation. At the height of inhibition, nerve action potential could still be recorded and the muscles continued to respond to direct stimulation. The inhibition was not reversible with L-cysteine or D-penicillamine but limited protection was possible by prior treatment with (+)-tubocurarine. Treatment of frog rectus muscles with methylmercury (0-2 mM for 15 min) resulted in a shift to the right of 1 log unit in the dose response curve to acetylcholine and a reduction in the maximum response of the tissue. The observed inhibitory action of methylmercury on neuromuscular transmission may be explained by an action on the disulphide bond believed to be present on a cholinergic receptor.

In-vitro and in-vivo antivenin activity of 2-[2-(5,5,8a-trimethyl-2-methylene-decahydro-naphthalen-1-yl)-ethylidene]-succinaldehyde against Ophiophagus hannah venom

Curcuma zedoaroides A. Chaveerach & T. Tanee, locally known as Wan-Paya-Ngoo-Tua-Mia, is commonly used in the North-Eastern part of Thailand as a 'snakebite antidote'. The aim of this study was to isolate the active compound from the rhizome of C. zedoaroides, to determine its structure and to assess its antagonistic activity in vitro and in vivo against King cobra venom. The active compound was obtained from C. zedoaroides by extraction with acetone followed by purification using column chromatography; its X-ray structure was determined. Its inhibition of venom lethality was studied in vitro in rat phrenic nerve-hemidiaphragms and in vivo in mice. The acetone extract of the Curcuma rhizomes contained a C20 dialdehyde, [2-(5,5,8a-trimethyl-2-methylene-decahydro-naphthalen-1-yl)-ethylidene]-succinaldehyde, as the major component. The isolated curcuma dialdehyde was found active in vitro and in vivo for antivenin activity against the King cobra venom. Using isolated rat phrenic nerve-hemidiaphragm preparations, a significant antagonistic effect on the inhibition of neuromuscular transmission was observed in vitro. Inhibition on muscle contraction, produced by the 4 microg/ml venom, was reversed by 2-16 microg/ml of Curcuma dialdehyde in organ bath preparations over a period of 2 h. Mice intraperitoneally injected with 0.75 mg/kg venom and dialdehyde at 100 mg/kg had a significantly increased survival time. Injection of Curcuma dialdehyde (100 mg/kg) 30 min before the subcutaneous injection of the venom resulted in a 100% survival time after 2 h compared with 0% for the control group. The in vitro and in vivo evaluation confirmed the medicinal use of traditional snake plants against snakebites. The bioactivity is linked to an isolated molecule and not a result of synergistic effects of a mixture. The active compound was isolated and the structure fully elucidated, including its stereochemistry. This dialdehyde is a versatile chemical building block and can be easily obtained from this plant source.

Effect of pantoprazole and its interactions with vecuronium on the neuromuscular junction

Objective:To study the effect of pantoprazole on neuromuscular transmission and its interactions with vecuronium at the neuromuscular junction (NMJ).Materials and Methods:Effect of pantoprazole on neuromuscular transmission (2 μM – 16 mM) and reversal of neuromuscular blockade by pantoprazole and vecuronium with neostigmine (3.3 μM), 3,4-diaminopyridine (0.25 mM), KCl (6 mM), and CaCl2 (10 mM) were studied by the indirect and direct stimulated preparation of rat phrenic nerve hemidiaphragm. Cumulative reponse curves (CRC) of vecuronium (1 μM to 32 μM) were studied in the absence and presence of 32 μM, 64 μM, and 128 μM pantoprazole. Time for head drop by vecuronium infusion was recorded in the absence and presence of acute and chronic administration of pantoprazole (1.9 mg/kg) in rabbits.Results:Pantoprazole potentiated the basal contractile twitch responses at a lower concentration followed by neuromuscular blockade at a higher concentration. The neuromuscular blockade was not reversed by neostigmine (3.3 μM), 3,4-diaminopyridine (0.25 mM), KCl (6 mM), and CaCl2 (10 mM). Pantoprazole potentiated the vecuronium-induced neuromuscular blockade. It decreased the total time for complete blockade in rat phrenic nerve hemidiaphragm preparation (P < 0.05) and decreased the time for the head drop in rabbits with vecuronium infusion (P < 0.0001).Conclusion:Pantoprazole has a direct neuromuscular blocking action. It has the potential to interact with vecuronium.

P.l.c.045 Effects of catechin and epicatechin on rat phrenic nerve-hemidiaphragm preparation

P.l.c.045 Effects of catechin and epicatechin on rat phrenic nerve-hemidiaphragm preparation

Negative crosstalk between M 1 and M 2 muscarinic autoreceptors involves endogenous adenosine activating A 1 receptors at the rat motor endplate

At the rat motor nerve terminals, activation of muscarinic M 1 receptors negatively modulates the activity of inhibitory muscarinic M 2 receptors. The present work was designed to investigate if the negative crosstalk between muscarinic M 1 and M 2 autoreceptors involved endogenous adenosine tonically activating A 1 receptors on phrenic motor nerve terminals. The experiments were performed on rat phrenic nerve-hemidiaphragm preparations loaded with [ 3H]-choline (2.5 μCi/ml). Selective activation of muscarinic M 1 and adenosine A 1 receptors with 4-( N-[3-clorophenyl]-carbamoyloxy)-2-butyryltrimethylammonium (McN-A-343, 3 μM) and R- N 6 -phenylisopropyladenosine (R-PIA, 100 nM), respectively, significantly attenuated inhibition of evoked [ 3H]-ACh release induced by muscarinic M 2 receptor activation with oxotremorine (10 μM). Attenuation of the inhibitory effect of oxotremorine (10 μM) by R-PIA (100 nM) was detected even in the presence of pirenzepine (1 nM) blocking M 1 autoreceptors, suggesting that suppression of M 2-inhibiton by A 1 receptor activation is independent on muscarinic M 1 receptor activity. Conversely, the negative crosstalk between M 1 and M 2 autoreceptors seems to involve endogenous adenosine tonically activating A 1 receptors. This was suggested, since attenuation of the inhibitory effect of oxotremorine (10 μM) by McN-A-343 (3 μM) was suppressed by the A 1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (2.5 nM), and by reducing extracellular adenosine with adenosine deaminase (0.5 U/mL) or with the adenosine transport blocker, S-( p-nitrobenzyl)-6-thioinosine (NBTI, 10 μM). The results suggest that the negative crosstalk between muscarinic M 1 and M 2 autoreceptors involves endogenous adenosine outflow via NBTI-sensitive ( es) nucleoside transport system channelling to the activation of presynaptic inhibitory A 1 receptors at the rat motor endplate.

Inhibition of SNARE-driven neuroexocytosis by plant extracts

Neuronal soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins mediate membrane fusion between synaptic vesicle and presynaptic membrane, resulting in neurotransmitter release. SNARE proteins are specific substrates of botulinum neurotoxins (BoNT) which are now widely used for therapeutic and cosmetic purposes. While BoNT blocks neuroexocytosis by cleaving SNAREs, inhibiting SNARE assembly process might exert the same effect on neurotransmission. In the present study, some extracts of 100 plants reduced neurotransmitter release by inhibiting SNARE complex formation in neuronal cells. The extracts effectively paralyzed muscle of rat phrenic nerve-hemidiaphragm preparation. Our results raise the possibility that SNARE folding inhibitors from natural resources might replace some special BoNT application fields.

Effects of 5-hydroxytryptamine on rocuronium-induced neuromuscular blockade in the rat phrenic nerve-hemidiaphragm preparation

Department of Anesthesiology and Pain Medicine, Asan Medical Center, College of Medicine, University of Ulsan, Songpa-gu, Republic of Korea

L‐Citrulline inhibits [3H]acetylcholine release from rat motor nerve terminals by increasing adenosine outflow and activation of A1 receptors

Nitric oxide (NO) production and depression of neuromuscular transmission are closely related, but little is known about the role of L-citrulline, a co-product of NO biosynthesis, on neurotransmitter release. Muscle tension recordings and outflow experiments were performed on rat phrenic nerve-hemidiaphragm preparations stimulated electrically. L-citrulline concentration-dependently inhibited evoked [(3)H]ACh release from motor nerve terminals and depressed nerve-evoked muscle contractions. The NO synthase (NOS) substrate, L-arginine, and the NO donor, 3-morpholinosydnonimine chloride (SIN-1), also inhibited [(3)H]ACh release with a potency order of SIN-1>L-arginine>L-citrulline. Co-application of L-citrulline and SIN-1 caused additive effects. NOS inactivation with N(omega)-nitro-L-arginine prevented L-arginine inhibition, but not that of L-citrulline. The NO scavenger, haemoglobin, abolished inhibition of [(3)H]ACh release caused by SIN-1, but not that caused by L-arginine. Inactivation of guanylyl cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) fully blocked SIN-1 inhibition, but only partially attenuated the effects of L-arginine. Reduction of extracellular adenosine accumulation with adenosine deaminase or with the nucleoside transport inhibitor, S-(p-nitrobenzyl)-6-thioinosine, attenuated the effects of L-arginine and L-citrulline, while not affecting inhibition by SIN-1. Similar results were obtained with the selective adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine. L-citrulline increased the resting extracellular concentration of adenosine, without changing that of the adenine nucleotides. NOS catalyses the formation of two neuronally active products, NO and L-citrulline. While, NO may directly reduce transmitter release through stimulation of soluble guanylyl cyclase, the inhibitory action of L-citrulline may be indirect through increasing adenosine outflow and subsequently activating inhibitory A(1) receptors.

Effects of 5-hydroxytryptamine on Rocuronium-induced Neuromuscular Blockade in a Rat Phrenic Nerve-hemidiaphragm Preparation

Background: The 5-hydroxytryptamine 3 receptor (5-HT3R) is a member of a superfamily of ligand-gated ion channels which has structural similarities and common evolutionary origin to those of the nicotinic acetylcholine receptor (nAChR). 5-hydroxytryptamine (5-HT) and muscle relaxants may have cross reaction. Rocuronium is a non-depolarizing neuromuscular blocking agent which has a rapid onset. The aim of this study was to examined the effects of 5-HT on rocuronium-induced neuromuscular blockade in a rat phrenic nerve-hemidiaphragm preparation. Methods: Institutional approval was obtained for the experimental procedure. Fifty male Sprague-Dawley rats (150－200 g) were divided into 5 groups; the control, and 0.1, 1, 10, and 20μg/ml of 5-HT. The animals were injected with phentobarbital at 40 mg/kg into the peritoneal cavity. The hemidiaphragm with the phrenic nerve was dissected and then mounted in a bath containing 100 ml Krebs solution at room temperature. The phrenic nerve was stimulated at the supramaximal intensity using a Grass® S88 stimulator via an SIU5 isolation unit. The twitch height was measured and recorded using a precalibrated Grass® FT88 force displacement transducer and recorded with a Grass® 79 polygraph, respectively. In the cumulative dose-response study, the rocuronium 100μg/dl and each dose of 5-HT were administered simultaneously administered, with additional 50μg/dl incremental doses of rocuronium were added to obtain grteater than 95% neuromuscular twitch inhibition. The ED5, ED50, ED90, and ED95 of rocuronium in each group were calculated using a probit model. Results: The ED50, ED90, and ED95 of rocuronium were significantly reduced in 5-HT 20μg/ml group (P ＜ 0.05), but no differences were observed with the other 5-HT groups compared to the control groups. Conclusions: 5-HT at 20μg/ml enhanced the neuromuscular blockade of rocuronium.

Effects of Ondansetron and Granisetron on Rocuronium-induced Neuromuscular Blockade in a Rat Phrenic Nerve-hemidiaphragm Preparation

Background: Both ondansetron and granisetron exert their antiemetic effects via a blockade of the 5-hydroxytryptamine 3 receptor (). Because the is a member of a superfamily of ligand-gated ion channels and has structural similarities to the nicotinic acetylcholine receptor (nAChR), a antagonist may also inhibit the nAChR. This study examined the effects of antagonists, ondansetron and granisetron, on rocuronium-induced neuromuscular blockade in vitro. Methods: Rat phrenic nerve-hemidiaphragm preparations were isolated and allocated randomly into seven groups (control, 1, 10, 100/ml of ondansetron, 0.1, 1, 10/ml of granisetron). Two studies were carried out using single twitch responses. In the cumulative dose-response study, rocuronium 1/ml and each doses of ondansetron or granisetron were administered simultaneously, and incremental 0.5/ml doses of rocuronium were added to obtain more than 95% neuromuscular twitch inhibition. , , , and of rocuronium in each group were calculated using a logistic model. In the partial curarization study, the twitch heights were measured after administering ondansetron or granisetron (10 minutes after administering 3/ml rocuronium) and were measured 10 minutes later. The 2 twitch heights were then compared. Results: In the cumulative dose-response study, ondansetron 100/ml and granisetron 10/ml significantly reduced the of rocuronium (P ＜ 0.05). There were no intergroup differences in the partial curarization study. Conclusions: High concentration of ondansetron and granisetron enhanced the neuromuscular blockade of rocuronium. Granisetron enhanced the neuromuscular blockade of rocuronium more potently than ondansetron.