The action of dantrolene sodium on rat fast and slow muscle in vivo.

1. Rats, anaesthetized with urethane, were injected intravenously with dantrolene sodium in a carrier solution of 5% mannitol taken to pH 10 with NaOH. This carrier solution itself was without effect on extrafusal muscle contraction. 2. Dantrolene sodium (5 mg/kg) had a greater depressant action on the twitch contraction of the fast extensor digitorum longus (EDL) muscle than on the slow soleus (SOL) muscle. The EDL twitch was depressed to 25.9% +/- 1.2% (mean + s.e. mean, n = 7) of control whereas the SOL twitch was depressed to 31.3% +/- 0.4% (n = 9). These values were significantly different at the P less than 0.001 level. 3. The twitch contraction time to peak was reduced by approximately 35% in both EDL and SOL by dantrolene sodium. However, the drug reduced the half relaxation time of SOL by approximately 30% but that of EDL was hardly affected. 4. The effect of dantrolene sodium on contractions elicited by repetitive stimulation was dependent upon the stimulation frequency. For the SOL muscle the greater depression was produced at a stimulation frequency of 25 Hz and for EDL at 75 Hz. The minimum of depression was produced for a full fused tetanus for both muscles. 5. The significance of these findings is discussed in terms of the action of dantrolene sodium on motor control in the intact animal.     

Effects of subacute pyridostigmine administration on mammalian skeletal muscle function.

The subacute effects of pyridostigmine bromide were investigated on the contractile properties of rat extensor digitorum longus (EDL) and diaphragm muscles. The cholinesterase inhibitor was delivered via subcutaneously implanted osmotic minipumps (Alzet) at 9 micrograms h-1 (low dose) or 60 micrograms h-1 (high dose). Animals receiving high-dose pyridostigmine pumps exhibited marked alterations in muscle properties within the first day of exposure that persisted for the remaining 13 days. With 0.1 Hz stimulation, EDL twitch tensions of treated animals were elevated relative to control. Repetitive stimulation at frequencies greater than 1 Hz led a use-dependent depression in the amplitude of successive twitches during the train. Recovery from pyridostigmine was essentially complete by 1 day of withdrawal. Rats implanted with low-dose pyridostigmine pumps showed little or no alteration of in vivo twitch tensions during the entire 14 days of treatment. Diaphragm and EDL muscles excised from pyridostigmine-treated rats and tested in vitro showed no significant alterations in twitch and tetanic tensions and displayed the same sensitivity as muscles of control animals to subsequent pyridostigmine exposures. In the presence of atropine, subacutely administered pyridostigmine protected rats from two LD50 doses of the irreversible cholinesterase inhibitor, soman. In the absence of atropine, the LD50 of soman was not altered by subacute pyridostigmine treatment.     

Dantrolene effects on neuromuscular function in cat soleus muscle.

Dantrolene sodium (DS) was investigated for its effects on cat soleus muscle contractile properties and motor nerve terminal activity in particular. DS, 0.1-1.5 mg/kg i.v., caused a dose-dependent depression of indirectly elicited contractile strength which was more pronounced at lower frequencies of stimulation. Maximum tetanic strength at frequencies of 10-400 Hz was depressed to a lesser degree than contractile responses evoked by lower frequencies of stimulation; the twitch/tetanus contraction ratios were reduced with increasing dose, primarily because of diminished twitch. DS was without effect on motor nerve terminals as evidenced by normal post-tetanic repetition in the nerves following DS administration. Post-tetanic potentiation became relatively larger in amplitude as contractile strength was diminished. These data suggest that DS depresses neuromuscular function at a site other than the neural apparatus at the neuromuscular junction.     

The action of dantrolene on transmitter mobilization at the rat neuromuscular junction

Dantrolene sodium (20 microM) was found to decrease transmitter mobilization and the apparent available store of acetylcholine at frequencies of nerve stimulation of 50 and 100 Hz at the neuromuscular junction of the rat hemidiaphragm preparation treated with 2 microM (+)-tubocurarine. This effect of dantrolene sodium was not as marked at frequencies of nerve stimulation of 25 Hz or less, also no significant effect of the drug was observed on the amplitude of endplate potentials (EPPS) at any frequency of nerve stimulation. No effect of dantrolene sodium (20 microM) on mean miniature EPP amplitude or frequency was observed. It is suggested that the action of dantrolene sodium on mean EPP quantal content may be due to an action on stores of bound calcium within the motor nerve terminal. This effect of the drug is unlikely to be of physiological consequence in vivo, since it was only observed at high frequencies of nerve stimulation and did not cause a significant reduction of EPP amplitude during trains of stimuli.     

Effects of isoprenaline on contractions of directly stimulated fast and slow skeletal muscles of the guinea-pig

1. The actions of isoprenaline on the contraction and the resting potential of the isolated extensor digitorum longus (EDL), a fast contracting muscle, and the soleus, a slow contracting muscle, of the guinea-pig were investigated. Twitch tension was elicited by direct supramaximal stimulation and recorded isometrically.2. The twitch tension of EDL elicited by pulses of 0.5-10 ms duration was increased in the presence of isoprenaline (1 mug/ml). Isoprenaline increased the twitch tension of the soleus elicited by a pulse of more than 5 ms duration, but decreased it when elicited by a pulse of less than 1 millisecond. These effects were blocked by propranolol (1-3 mug/ml) but not by phentolamine (1-5 mug/ml).3. In EDL, isoprenaline prolonged the time to peak tension and the half-relaxation time. The twitch of the soleus was shortened by isoprenaline due to an acceleration of relaxation. These findings were independent of stimulus duration.4. The potentiating effects of isoprenaline on the twitch tension of EDL and the soleus were not observed in K(+)-free Krebs solution and were abolished by ouabain (1 mug/ml) and by reduction of the temperature from 33 degrees to 18 degrees C. The effects of isoprenaline on the relaxation proces were not affected by these treatments.5. In EDL, the resting potential increased from 77.3 mV to 78.5 mV after isoprenaline, whereas in the soleus it increased from 69.1 to 74.7 mV. These effects were blocked by propranolol, K(+)-deficiency, ouabain, and cooling to 18 degrees C. Hyperpolarization by isoprenaline was increased by substitution of isethionate for the external chloride.6. There was a good correlation between the potentiation of the mechanical response and the hyperpolarization of the membrane by isoprenaline. The hyperpolarization seems to be due to activation of the Na(+)-K(+) pump.     

Studies on the mechanism of action of dantrolene sodium

Summary In rat diaphragm-phrenic nerve preparations, dantrolene sodium has been shown to have no effect on neuromuscular transmission. KCl and acetylcholine contractures in the frog rectus muscle are depressed in the presence of dantrolene sodium. The threshold for caffeine contractures in the frog sartorius and rectus muscles is raised from 2.0 to 4.0 mM. In isolated frog sartorius muscles, the tetanic fusion frequency is increased by 29%, and the twitch response is depressed more than the tetanic.According to these observations it is proposed that the muscle relaxant properties of dantrolene sodium are dependent on its direct inhibitory action on skeletal muscle. It is further hypothesized that this direct action results from an antagonism of calcium release within the muscle.     

Actions of some cholinergic antagonists on fast-twitch and slow-twitch skeletal muscle of the cat

1. The anticholinergic drug N-ethyl-2-pyrrolidylmethylcyclopentylphenyl glycollate (PMCG) has been studied for its effects on the contraction of fast-twitch (flexor hallucis longus, FHL) and slow-twitch (soleus) muscles in the cat.2. In both muscles lower doses (0.25 to 10 mg intra-arterially) potentiated and higher doses (10 mg and above) depressed twitches produced by indirect stimulation. Similar effects were obtained in directly stimulated muscles.3. The effects of the drug on muscles stimulated repetitively were dependent on both dose and frequency of stimulation. Doses which potentiated twitches also potentiated low frequency tetani (5-30 Hz for soleus; 10-50 Hz for FHL). There was a depression and non-maintenance of higher frequency tetani (80 Hz and above for soleus; 150 Hz and above for FHL).4. Both atropine and caramiphen had similar dose dependent potentiating and depressant actions. Hyoscine acted similarly in some cats; in others it had only a depressant action.5. It is suggested that the potentiating actions of PMCG, caramiphen, atropine and hyoscine are due to a direct musculotropic action. The depressant actions resulting from higher doses are due, in part, to a curare-like action and in part to a direct action on the muscle fibres.     

Excitation-contraction uncoupling in skeletal muscle by dantrolene sodium

Summary Dantrolene sodium, a skeletal muscle relaxant, inhibits the twitch response by direct action on the muscle. The action potentials from dantrolene sodium depressed muscles have been examined by taking their first derivative, and membrane capacitance has been measured with cable analysis techniques. It is concluded that dantrolene sodium has no effect on the electrical excitability of the muscle and uncouples excitation-contraction mechanisms.     

ACTIONS OF DANTROLENE SODIUM ON CONTRACTIONS OF THE TIBIALIS ANTERIOR AND SOLEUS MUSCLES OF CATS UNDER CHLORALOSE ANAESTHESIA

SUMMARY 1. Dantrolene depresses the tension of directly or indirectly elicited twitches and tetaní of the tíbíalís anteríor and soleus muscles of cats under chloralose anaesthesia, without affecting the gross muscle action potentials.
2. The decrease in twitch tension is associated with slowed rates of rise of tension and of relaxation, but there is no change in the time to peak tension. The decrease in maximal tetanic tension is associated with a slowed rate of rise of tension, but there is no change in the rate of relaxation. It is concluded that dantrolene decreases the intensity of the active state, possibly by impairing the release of Ca²⁺ from the sarcoplasmic reticulum.
3. The soleus muscle is slightly more resistant than the tibialis anterior muscle to the action of dantrolene, and maximal tetani of both muscles are much more resistant than twitches.
4. Adrenaline, theophylline and quazodine produce effects on the dantrolene-depressed twitches that are proportionately the same as those produced on the control twitches; there is no evidence of a specific antagonistic effect of any of these drugs.
5. Tetanic stimulation of the tibialis anterior muscle but not the soleus muscle causes a temporary relief of the twitch depression produced by dantrolene.     

Effects of the sea anemone Anemonia sulcata toxin II on skeletal muscle and on neuromuscular transmission

Effects of anemone toxin II (ATX II) have been analysed on the neuromuscular junction of the frog and different twitch muscles. Amplitudes of evoked endplate potentials and endplate currents are increased by ATX II, without effects on the amplitudes of miniature endplate potentials and endplate currents resulting from ionophoretically applied transmitter. The increase in evoked transmitter release is due to an increase in quantal content caused by an effect of the toxin on the presynaptic action potentials. ATX II is also effective on muscle fibers. The action potentials of frog twitch muscles are reversibly prolonged by ATX II. Their rate of rise and amplitudes are increased, while there is no effect on resting membrane potential. Similarly, action potentials of fast twitch muscle (extensor digitorum longus, EDL) of the mouse are reversibly prolonged by ATX II. In slow twitch muscle (soleus, SOL) of the mouse the toxin induces repetitive action potentials following the generation of a single action potential. Tetrodotoxin resistant action potentials of both denervated EDL and SOL are greatly and irreversibly prolonged by ATX II. The effects on muscle are due to a Na+ channel specific action of ATX II. Na+ current inactivation is slowed with the time constant tau h increasing towards positive membrane potentials. The steady state inactivation curve hoo was shifted to more positive potentials and its slope reduced.     

Changes of isolated cardiac muscle function in response to extracellular sodium reduction

The effects of low extracellular sodium concentration [( Na+]0, 76 mmol/l) on force of contraction, transmembrane action potentials and on calcium, sodium and potassium contents were studied in guinea-pig heart muscle using sucrose or lithium as substitutes for sodium. In papillary muscle, the positive inotropic response to low [Na+]0 was accompanied by shortening in action potential duration, in atrial muscle, prolongation at 90% of repolarization was observed. After 30 min. in low [Na+]0 solution (Ca2+ 1.8 mmol/l), the net uptake of calcium in left atria was larger in sucrose- than in lithium-substituted solution, i.e. 0.45 and 0.2 mmol Ca2+/kg wet weight, respectively. The net sodium content decreased monophasically; but the potassium content did not change consistently. The uptake of lithium by the atria was at least partially compensated for by a loss in potassium and by a transient decrease in sodium content. Increase in stimulation frequency from 0.1 to 1 Hz accelerated the time course of change by a factor of 2. In low [Na+]0, post-rest adaptation of twitch tension appeared abolished in atria because of high amplitude contractions. The recovery pattern of post-rest twitch amplitude was greatly accelerated in papillary muscle. In conclusion, low [Na+]0 elevates the cellular calcium content; this extra calcium is probably located in cellular stores that are involved in the regulation of twitch amplitude.     

Some pharmacological properties of piperazine

1. The action of piperazine on mammalian smooth, cardiac and skeletal muscles has been studied.2. Piperazine increased tone and produced a dose dependent contraction of isolated smooth muscle, which was antagonized by atropine.3. With cardiac muscle, piperazine depressed both the rate and force of contraction and was antagonized by atropine. At higher concentrations, a non-specific depression of cardiac muscle was found. The intravenous injection of piperazine produced a transient decrease in both heart rate and blood pressure and this was followed by an increase.4. In about half of the mammalian skeletal muscle preparations, piperazine potentiated the twitch of the muscle evoked by electrical stimulation.5. On the frog neuromuscular preparation, piperazine produced potentiation of the twitch but this was followed first by blockade of the effects of nerve stimulation and then by depression of the effects of direct muscle stimulation.     

An examination of the pharmacology of two substance P antagonists and the evidence for tachykinin receptor subtypes.

The effects of intravenous injection of the muscle relaxant dantrolene sodium on ventilation, integrated EMG of external intercostal muscles and motor unit discharge in these muscles were measured in anaesthetized rats. Dantrolene sodium was administered in a carrier solution at pH 10. The carrier was not responsible for the effects seen on dantrolene sodium infusion. Dantrolene sodium (5 mg kg-1 body weight) reduced inspired ventilation (VI) on average from 308 +/- 106 ml min-1 (s.d., n = 8) in the control period to 247 +/- 118 ml min-1 (n = 8) after dantrolene sodium. Expired ventilation was reduced likewise from 274 +/- 132 ml min-1 (n = 8) to 229 +/- 109 ml min-1 (n = 8). These changes are significant at P less than 0.05. The integrated EMG was increased by 40.0 +/- 29.3% (n = 9), a change significant at P less than 0.001. Dantrolene sodium not only decreased the mean interspike interval of individual motor units by 16.2 +/- 11.1% (n = 13), a change significant at P less than 0.001, but also increased the duration of the burst of activity of each individual motor unit on average by 36.2 +/- 52.4% (n = 13), a change significant at P less than 0.05. The recruitment of additional motor units was also effected by dantrolene sodium. These units did not behave in any noticeably different manner from units previously active. It is concluded that the nervous system compensates for the effect of dantrolene sodium only to a limited extent by increasing the frequency of discharge of active motor units.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of dantrolene sodium on rat skeletal muscle in relation to the plasma concentration.

Dantrolene sodium is a muscle relaxant used in the treatment of spasticity. It has been shown to interfere with calcium release from the sarcoplasmic reticulum and thus to inhibit excitation--contraction coupling. The effect of dantrolene sodium on the twitch tension of the tibialis anterior muscle of the rat was measured after 2 mg/kg i.v. or 25 mg/kg orally. Plasma concentrations were estimated at maximum twitch depression and during recovery from the block. In a separate series of experiments the half-life of labelled dantrolene sodium was measured in blood plasma, skeletal muscle and heart muscle of rats. Dantrolene sodium 2 mg/kg i.v. gave a maximal block of approximately 47%, the mean dantrolene sodium concentration was then 5.8 microgram/ml. A half-life for distribution of 1.1 min and an elimination half-life of 31 min after intravenous administration were observed, elimination rate constants in skeletal and heart muscle were comparable. Recovery from the block went much slower, the half-time of the process being approximately 80 min. Dantrolene sodium 25 mg/kg orally gave a maximal block of approximately 38% at a mean plasma concentration of 3.6 microgram/ml after 14 min. The recovery was again very slow. These experiments demonstrated that dantrolene sodium acts according to a two-compartment pharmacokinetic model. There was a discrepancy between duration of effect and plasma concentration of dantrolene sodium in the rat. This suggests that the receptor for dantrolene sodium is not located in the central compartment.     

The marine polyether gambierol enhances muscle contraction and blocks a transient K current in skeletal muscle cells

Gambierol is a complex marine toxin first isolated with ciguatoxins from cell cultures of the toxic dinoflagellate Gambierdiscus toxicus. Despite the chemical complexity of the polycyclic ether toxin, the total successful synthesis of gambierol has been achieved by different chemical strategies. In the present work the effects of synthetic gambierol on mouse and frog skeletal neuromuscular preparations and Xenopus skeletal myocytes have been studied. Gambierol (0.1-5 μM) significantly increased isometric twitch tension in neuromuscular preparations stimulated through the motor nerve. Less twitch augmentation was observed in directly stimulated muscles when comparing twitch tension-time integrals obtained by nerve stimulation. Also, gambierol induced small spontaneous muscle contraction originating from presynaptic activity that was completely inhibited by d-tubocurarine. Gambierol slowed the rate of muscle action potential repolarization, triggered spontaneous and/or repetitive action potentials, and neither affected action potential amplitude nor overshoot in skeletal muscle fibers. These results suggest that gambierol through an action on voltage-gated K⁺ channels prolongs the duration of action potentials, enhances the extent and time course of Ca²⁺ release from the sarcoplasmic reticulum, and increases twitch tension generation. Further evidence is provided that gambierol at sub-micromolar concentrations blocks a fast inactivating outward K⁺ current that is responsible for action potential prolongation in Xenopus skeletal myocytes.     

The rat external urethral sphincter an in vitro model to evaluate the activity of drugs on the smooth and striated components of the urinary bladder outlet.

Electrical field stimulation (EFS, 2.5 Hz, 60 V, 1 ms, trains of 5 sec every 5 min) of the rat external urethral sphincter (EUS) produced contractile responses characterized by a "slow" tonic contraction on which was superimposed a series of phasic "twitch-like" contractions. Both responses were abolished by tetrodotoxin (0.6 microM), and their amplitude was significantly lower in samples taken from denervated (15 days before) sphincters. The tonic contraction showed duration, voltage, and frequency dependency, whereas the twitches were markedly duration dependent. No correlation was found between the amplitude of the tonic and that of the twitch-like contractions. Phentolamine (3 microM) reduced by 95% the amplitude of the tonic contraction produced by maximal parameters, whereas it did not affect the twitches. On the other hand, hexamethonium (10 microM) was ineffective on both components. Dantrolene (10 microM) inhibited the twitch response, whereas it did not influence the tonic component. Preincubation with d-tubocurarine (0.2 mM) or succinylcholine (2 mM) significantly inhibited the amplitude of twitches produced by EFS (0.1 Hz, 60 V) up to a duration of 50 microseconds. Stimulus width higher than 50 microseconds, resulted in twitches that were resistant to neuromuscular blocking agents but sensitive to dantrolene (10 microM). Our results indicate that the rat external urethral sphincter is a reliable and easy "in vitro" model for studying the activity of drugs capable of interfering with the nerve-mediated activity of the striated and smooth muscle portion of the urinary bladder outlet.     

SKELETAL MUSCLE RESTING METABOLISM IN COLD-ACCLIMATED RATS: EFFECT OF AGE, NORADRENALINE AND HYPEROSMOLARITY

1. A myothermic technique has been used to measure the resting metabolism of small bundles of a fast twitch muscle, extensor digitorum longus (EDL), and a slow twitch muscle, soleus (SOL), in 7-week-old rats. At 27°C, mean (± SEM) resting heat rates were 2.33 ± 0.41 and 2.09 ± 0.37 mW/g in EDL and SOL, respectively (n= 16). 2. Seven-week-old rats were cold acclimatized at 4°C for 1–4 weeks and the metabolic rates of the fast and slow twitch muscles were monitored and compared with 7- and 11-week-old controls. There was a 160% increase in metabolic rate from week 7 to week 11, but the increase also occurred in the control group. 3. In accordance with several literature reports, noradrenaline at concentrations of 10”⁷ and 10”⁶ mol/L had no effect on either the control or cold-acclimatized resting heat rate. 4. The osmolarity of the physiological solution bathing the muscle bundles was increased by lOOmosmol using sodium sulphate. Basal metabolism increased by similar amounts (approximately 250%) in both the fast and slow muscle bundles. Periods of cold exposure had no significant effect on the magnitude of the increment. 5. Bumetanide, a potent inhibitor of Na⁺-Cl^? co-transport, produced only a slight reduction in the heat increments caused by hyperosmolar challenge.     

Physiological properties of rat hind limb muscles after 15 days of simulated weightless environment

Weightlessness during space mission results in atrophic changes in those muscles which have maximum weight bearing function and consist primarily of slow twitch fibres. In the present study an animal model was designed to evaluate the effects of 15 days of hindlimb unloading (HU) in rats by tail suspension on the (i) weight of gastrocnemius (G), plantaris (P), both predominantly having fast twitch fibres and soleus (S) muscle, predominantly having fast twitch fibres and (ii) contractile properties viz peak twitch contraction (Pt) and peak tetanic contraction (Po) of GPS muscle. HU rats showed significant weight reductions of G (-17.9%), P (-13.3%) and S (-41.2%) muscles. Pt and Po were also reduced in HU group but when these were expressed per gm of GPS muscle, no significant changes in Pt and Po were observed. These findings confirm that HU in rats result in maximum atrophic change in those muscles which have predominantly slow twitch fibres and reductions in contractile properties of muscles are in proportion to reduction in muscle weight. Also, HU by tail suspension provides a good ground based model for developing the deconditioning of muscles as applicable to weightlessness of space and offers a scope for the development of various countermeasures.     

The pharmacology of clodanolene sodium, a new skeletal muscle contraction antagonist

The pharmacology of hydrated 1 less than ([5-(3,4-dichlorophenyl)-2-furanyl]methylene) amino greater than-2,4,-imidazolidinedione sodium salt (clodanolene sodium), as skeletal-muscle contraction antagonist, is presented. Clodanolene sodium is remarkable in that it has no measurable direct effect on the peripheral or central nervous systmes. Skeletal muscle relaxation can be achieved with this drug at doses that do not affect motor coordination. Rats receiving clodanolene sodium for up to 30 days evidenced a downward trend in gross observation score of skeletal muscle relaxation, but the extent of twitch inhibition was the same on day 30 as on day 1. In an animal model of muscle spasticity (Straub-tail mouse), clodanolene sodium has been shown to be more efficacious for induction of skeletal muscle relaxation than neuromuscular blocking agents, local anesthetics, or centrally-acting muscle relaxants. Clodanolene sodium's mode of action has been identified as specific for skeletal muscle. It has no measurable effect on neuromuscular transmission or on the electrically excitable surface membrane. Indirect evidence indicates that the site of action of clodanolene sodium, like that of dantrolene sodium, is within the muscle cell and is related to caffeine-sensitive calcium stores. Its skeletal-muscle relaxant activity, we suggest results from a decrease in the release of calcium from the sarcoplasmic reticulum.     

Effects of dantrolene on force development in slow- and fast-twitch muscle of euthyroid, hypothyroid, and hyperthyroid rats.

1 The effects of dantrolene on twitch and tetanic force development were determined in soleus and gastrocnemius muscle of euthyroid, hypothyroid, and hyperthyroid rats. 2 Maximum twitch force of the gastrocnemius muscle was significantly more depressed by dantrolene than that of the soleus muscle in euthyroid and hyperthyroid rats. In hypothyroid rats, the effect of dantrolene on maximum twitch force was similar in soleus and gastrocnemius muscle. 3 Maximum tetanic force in soleus and gastrocnemius muscle was less depressed by dantrolene than the twitch force in either thyroid state. The effect of dantrolene on maximum tetanic force increased in both muscles in the direction hypothyroid----euthyroid----hyperthryoid. 4 The results are discussed in terms of an effect of thyroid hormones on Ca2+ -cycling during force development, as a result of thyroid hormone-induced proliferation of the sarcoplasmic reticulum.