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Propofol, most frequently used as a general anesthetic due to its versatility and short-acting characteristics, is thought to exert its anesthetic actions via GABAA receptors; however, the precise mechanisms of its adverse action including angialgia remain unclear. We examined the propofol-induced elevation of intracellular calcium and morphological changes in intracellular organelles using SHSY-5Y neuroblastoma cells, COS-7 cells, HEK293 cells, and HUVECs loaded with fluorescent dyes for live imaging. Although propofol (>50 μM) increased intracellular calcium in a dose-dependent manner in these cells, it was not influenced by the elimination of extracellular calcium. The calcium elevation was abolished when intracellular or intraendoplasmic reticulum (ER) calcium was depleted by BAPTA-AM or thapsigargin, respectively, suggesting that calcium was mobilized from the ER. Studies using U-73122, xestospongin C, and dantrolene revealed that propofol-induced calcium elevation was not mediated by G-protein coupled receptors, IP3 receptors, or ryanodine receptors. We performed live imaging of the ER, mitochondria and Golgi apparatus during propofol stimulation using fluorescent dyes. Concomitant with the calcium elevation, the structure of the ER and mitochondria was fragmented and aggregated, and these changes were not reversed during the observation period, suggesting that propofol-induced calcium elevation occurs due to calcium leakage from these organelles. Although the concentration of propofol used in this experiment was greater than that used clinically (30 μM), it is possible that the concentration exceeds 30 μM at the site where propofol is injected, leading the idea that these phenomena might relate to the various propofol-induced adverse effects including angialgia.

In addition to inhibition of acetylcholine release in the neuromuscular junction botulinum toxin type A (BoNT-A) also inhibits the release of mediators involved in pain perception. We have investigated the effect of two types of BoNT-A on mechanical hyperalgesia in the rat models of carrageenan-induced hyperalgesia and of paclitaxel-induced peripheral neuropathy. A subplantar (s.p.) injection of BoNT-A in the ipsilateral hindpaw 3 days before carrageenan administration reduced hypersensitivity. Dysport® and Botox® elicited comparable antihyperalgesic effects. Dysport® up to 30 U/kg and Botox® up to 20 U/kg did not impair the rat withdrawal nociceptive reflex or the locomotor performance as assessed by the rotarod test. Intraperitoneal administration of the skeletal muscle relaxant dantrolene produced, in contrast to BoNT-A, more motor impairment than analgesia. Paclitaxel treatment resulted in a peripheral neuropathy that affected the two hindpaws. Injection of 20 U/kg (s.p.) Dysport® produced a significant antihyperalgesic effect in the injected paw of neuropathic animals 3 days after administration. Unexpectedly, a similar analgesic effect was observed in the contralateral paw. The same results were also observed when Botox® was used instead of Dysport®. In contrast, a contralateral administration of Dysport® in the carrageenan test was ineffective. We conclude that BoNT-A elicits antinociceptive effects independent of the effects on muscular relaxation. Our results suggest that different mechanisms of action are responsible for the effect of BoNT-A in inflammatory and peripheral polyneuropathic rat models.

The effects of puerarin on behaviour and brain neuronal activity in animal studies have been described previously. However, molecule mechanisms underlying these effects were poorly understood. Here, we examined the regulation of puerarin on the Ca(2+) signals in primary rat hippocampal neurons using Fura-2 based calcium imaging techniques. Application of puerarin had no effect on the basal intracellular calcium concentration ([Ca(2+)](i)), but potentiated the KCl-evoked [Ca(2+)](i) transient in 87% of recorded neurons. Dantrolene or ruthenium red, the inhibitors of ryanodine receptors, completely blocked this potentiation induced by puerarin. Moreover, in Ca(2+)-free solution, pre-application of puerarin significantly augmented the elevation of [Ca(2+)](i) evoked by caffeine (3 mM), which is a specific agent to activate the ryanodine receptors. In contrast, nifedipine failed to prevent the potentiation induced by puerarin. Similarly, in the experiments of whole-cell patch-clamp recording, puerarin did not show any effect on calcium currents generated by depolarization pulses. These data demonstrated that the potentiation induced by puerarin was attributed to the facilitation of Ca(2+)-induced Ca(2+) release (CICR) via ryanodine receptors, rather than extracellular Ca(2+) influx. Using estrogen receptor antagonist ICI 182780 and tamoxifen, we further demonstrated that the potentiation induced by puerarin was mediated by the estrogen receptor. Furthermore, the membrane-permeant inhibitor of protein kinase A (PKA) H89 completely inhibited this potentiation. However, U-73122, the inhibitor of phospholipase C (PLC) had no effect, indicating that the cyclic AMP/PKA signaling pathway was involved in the activation of CICR by puerarin.

The objective of this study is to probe the effects of dopamine and potential interactions with nicotine at the motor end plate. To accomplish this, we measured the amplitude of nerve-evoked muscle twitches of the isolated rat phrenic hemi-diaphragm preparation. Dopamine potentiated indirect muscle twitches in normal and gallamine-presensitized preparations amounting to a maximum of 31.14±0.71% and 69.23±1.96%, respectively. The dopamine-induced facilitation was well maintained in presence of 10 µM propranolol but greatly reduced in presence of 6 µM SCH 23390 or 3 µM dantrolene. In addition, SKF 81297 attained a plateau at 16 µM as opposed to 64 µM dopamine, with a percentage potentiation of 69.47±1.76. The facilitatory effect of dopamine was potentiated in nicotine treated rats. This study revealed for the first time that the facilitatory effect exerted by dopamine on neuromuscular transmission is mediated via the dopamine D1-like receptors. In addition, it highlighted the possible dependency of dopamine effects on intracellular calcium and signified potential interaction among dopamine and nicotine. Clinically, the findings generated by this study reveal potential targets for approaching motor deficit syndromes.

We tested the hypothesis that the negative functional effects of cyclic GMP were mediated by ryanodine receptors, and that these effects would be reduced in thyroxine (thyroxine, 0.5 mg/kg/day, 16 days)-induced hypertrophic myocytes. Using rabbit ventricular myocytes from control (n = 9) and thyroxine (n = 9) hearts, percent cell shortening (%) and maximum rate of contraction and relaxation were determined using a video edge detector at baseline and after 10− 6, 10− 5 M 8-bromo-cyclic GMP. Dantrolene 10− 6 M, ryanodine receptor inhibitor, was added alone or after 8-Br-cGMP treatment. Changes in cytosolic Ca2+ concentration were assessed in fura-2-loaded control and thyroxine myocytes. 8-Br-cGMP caused a significant decrease in percent shortening, from 5.3 ± 0.9% to 3.9 ± 0.6% at 10− 5 M in control, and 3.4 ± 0.3% to 2.6 ± 0.4% in thyroxine myocytes. Dantrolene significantly decreased percent shortening from 4.5 ± 0.8% to 3.7 ± 0.1% in control and from 3.7 ± 0.1% to 2.8 ± 0.3% in thyroxine myocytes. In 8-Br-cGMP treated control myocytes, dantrolene did not significantly change myocyte contractility, which suggested that cyclic GMP acted on ryanodine receptors. However, in 8-Br-cGMP treated thyroxine myocytes, dantrolene further reduced myocyte contractility implying that the interaction of cyclic GMP and ryanodine receptors appeared to be interrupted in thyroxine myocytes. Maximum rate of contraction data were consistent with the percent cell shortening data and Ca2+ transients changed similarly to myocyte contractility. We conclude that effects of cyclic GMP on myocytes contractility were partially mediated though interaction with ryanodine receptors and the subsequent decrease in cytosolic calcium levels. This interaction was reduced in thyroxine hypertrophic myocytes.

Most previous studies of leukotriene B4 (LTB4) pharmacology using primary leukocyte cultures and myeloid cell lines do not differentiate between leukotriene BLT1 and BLT2 receptor activation because both receptors are often expressed by these cells. Here we show that in HeLa cells expressing BLT1 but not BLT2 receptors, BLT1 receptor activation resulted in IP3 mediated calcium release from intracellular stores initially, followed by calcium influx through cell membrane channels. BLT1 calcium signalling was sensitive to the activity of protein kinase C (PKC), protein kinase A (PKA) and protein-tyrosine kinases (PTKs), as well as changes in membrane cholesterol levels and treatments that are known to disrupt normal membrane physiology and/or lipid rafts. Inhibition of MAP kinases, Rho-associated kinases, or phosphoinositol-3-kinases (PI3K) had no effect on BLT1 receptor induced calcium signalling, and the receptor was insensitive to the redox state of the extracellular compartment.

The present study was designed to investigate the effects of dantrolene on intracellular Ca 2+ ([Ca 2+ ] i ) handling and inotropy in rat infarcted myocardium. Dantrolene-treated rats with myocardial infarction were placed into two different dosage groups. The infarcted control group received placebo only. Isometric contractility and intracellular Ca 2+ transients were recorded simultaneously in isolated papillary muscles. Diastolic [Ca 2+ ] i was significantly lower in muscle preparations from infarcted rats receiving dantrolene compared to the placebo control group. Additionally, treatment with dantrolene in infarcted rats significantly improved the inotropic response to 10 −4 M isoproterenol. The protein levels of the sarcoplasmic reticulum Ca 2+ ATPase were increased in infarcted rat hearts with dantrolene treatment. We conclude that dantrolene improved the inotropic response to β-adrenoceptor stimulation in rat postinfarcted myocardium, which is related to improved intracellular Ca 2+ handling, and lowered diastolic Ca 2+ concentration.

We investigated the effect of dantrolene, an inhibitor of Ca2+ release from the sarcoplasmic reticulum, on the induction of nitric oxide (NO) synthase II by bacterial endotoxin (lipopolysaccharide) in the anaesthetised rat and on survival in a murine model of severe endotoxaemia. Injection of lipopolysaccharide (i) induced biphasic changes of rectal temperature and blood glucose: an initial increased phase (180 min after injection of lipopolysaccharide) followed by a decreased phase (at 240-360 min), (ii) caused a fall in mean arterial blood pressure from 115 +/- 3 mmHg (time 0) to 83 +/- 6 mmHg at 360 min, (iii) resulted in a substantial hyporeactivity to noradrenaline (1 microg/kg i.v.), (iv) raised plasma nitrate (an indicator of NO formation) in a time-dependent manner, (v) elicited a significant increase in NO synthase II activity in the lung and (vi) caused a 80% lethality (in mice). Pretreatment of animals with dantrolene not only attenuated the delayed circulatory failure, but also prevented the overproduction of NO and the induction of NO synthase II caused by lipopolysaccharide in the rat, and improved survival in a murine model of severe endotoxaemia. Thus, dantrolene has beneficial haemodynamic effects in animals with endotoxin shock. We propose that a decrease of free cytosolic Ca2+ levels plays an important role in the prevention of NO synthase II induction.

We investigated the effects of dantrolene sodium on intracellular Ca2+ homeostasis in normal and Ca2+ overloaded rat cardiac muscle. In isometrically contracting rat papillary muscles loaded with the Ca2+ indicator aequorin, dantrolene (50 microM) produced a mild negative inotropic effect (28 +/- 1.8 to 21 +/- 1.1 mN/mm2; mean +/- S.E.; n = 6; P0.01), which was paralleled by a decrease in peak systolic [Ca2+]i (0.81 +/- 0.04 to 0.67 +/- 0.04 microM; P0.01). In isolated cardiac sarcoplasmic reticulum, dantrolene (50 microM) increased the initial Ca2+ uptake rate by 23% as compared to control preparations (at pCa 6.2: 46.9 +/- 1.6 to 61.1 +/- 2.2 nmol/mg per min; n = 4; P0.001). Intracellular Ca2+ overload was provoked in isoproterenol-pretreated (100 microM) preparations with [Ca2+]o = 5.0 mM at a stimulation rate of 1.0 Hz (n = 12). Diastolic Ca2+ oscillations and aftercontractions increased mean diastolic [Ca2+]i (0.33 +/- 0.1 to 0.56 +/- 0.1 microM) and tension (9.5 +/- 1.8 to 15.3 +/- 2.1 mN/mm2), respectively. Addition of dantrolene (50 microM) reduced the amplitude of Ca2+ oscillations and aftercontractions; mean diastolic [Ca2+]i decreased to 0.44 +/- 0.1 microM and diastolic tension to 13.5 +/- 2.2 mN/mm2. We conclude, therefore, that dantrolene sodium modifies Ca2+ handling by the myocardial sarcoplasmic reticulum, an effect that might be useful in cardiac disorders with impaired [Ca2+]i homeostasis.

We have examined the inhibitory potencies of MK 801, memantine, dextromethorphan, Mg2+ and of strychnine-insensitive glycine site antagonists on the N-methyl-D-aspartate (NMDA)-evoked (300 microM) release of [14C]acetylcholine and [3H]spermidine or [14C] gamma-aminobutyric acid [14C]GABA and [3H]dopamine from rat striatal slices. MK 801, dextromethorphan and all glycine antagonists examined (7-chlorokynurenate, L-689,560 ((+/-)-trans-2-carboxy-5,7-dichlorotetrahydroquinoline-4-phenylure a), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 6,7-dichloroquinoxaline-2,3-dione (DNQX), and (+)-HA966 ((3-amino-1-hydroxypyrrolidin-2-one) more potently inhibited NMDA-evoked dopamine and GABA release than acetylcholine and spermidine release by a factor of 3-21. MgCl2, which does not inhibit NMDA-evoked spermidine release, and memantine which only weakly antagonised NMDA-evoked spermidine release, inhibited NMDA-evoked dopamine, acetylcholine and GABA release with similar potencies. No pharmacological differences were observed between NMDA-evoked dopamine and GABA release. These findings extend those suggesting that NMDA-evoked acetylcholine and spermidine release are mediated by different NMDA receptor subtypes in the striatum and suggest a third native subtype with a distinct pharmacology that regulates striatal dopamine and GABA release.

2,3-Butanedione monoxime (BDM), 0.5–12.5 mM, inhibited the twitches rapidly and reversibly in the rat diaphragm. A more extensive inhibition at direct than at indirect stimulation, a simultaneous effect on the electromyogram, and an attenuation of the inhibitory effect in preparations made hyperexcitable with Ca2+-free solution, myotonia or veratridine, indicated an interference with the gating mechanism of the sarcolemma. The excitation-contraction uncoupling with BDM was additive to the similar effect of dantrolene. A synergistic effect was not found, and neither agent alone nor both in combination caused a complete excitation-contraction uncoupling. Several differences between the effects of BDM and dantrolene were disclosed: Lowering the temperature increased and decreased the effect of BDM and dantrolene, respectively. Tetanic stimulation and high Ca2+ or high K+ antagonized the effect of dantrolene and did not affect the BDM-induced inhibition. Ca2+-free solution potentiated the effect of dantrolene and antagonized the effect of BDM. Caffeine and KCl contractures were inhibited by BDM, whereas the propranolol contracture due to Na,K-ATPase inhibition, which is completely inhibited by dantrolene, was unaffected by BDM.

Addition of dantrolene 8.5 × 10−5 M caused a mono-exponential decay of the depolarization contractures caused by inhibition of the sarcolemmal Na,K-ATPase with propranolol 1 mM or by depolarization of the sarcolemma and T tubular membranes with KCl 100 mM. The half-times of the inhibitory effects were 6 s for the propranolol contracture and 11 s for the KCl contracture. The inhibition of both contractures was complete. Inhibition of the caffeine (10 mM) contracture was bi-exponential with half-times of 45 s and 9.5 min. Inhibition was incomplete; 29.6 ± 5.0% of the contracture tension could not be inhibited. The inhibition of twitch contractions was similar to that of the caffeine contracture, with half-times of 48 s and 9.1 min, and 20.6 ± 1.2% of the initial twitch tension could not be inhibited. The contracture tensions induced by release of Ca from the mitochondria with dicumarol, and by actin-myosin binding with the sulfhydryl inhibitor, N-ethyl-maleimide, could not be inhibited by dantrolene. The present results indicate that dantrolene inhibits depolarization signals from the sarcolemma and the T tubular membranes, in addition to inhibition of the coupling between the T tubules and the sarcoplasmic reticulum, and of the release of Ca from the sarcoplasmic reticulum. All these effects of dantrolene may contribute to its therapeutic effect in malignant hyperthermia.

We tested the hypothesis that calcium from the sarcoplasmic reticulum contributes to exercise-induced muscle damage. Dantrolene sodium (Dantrium) is a muscle relaxant that affects the flux of calcium over the sarcoplasmic membrane. Rats were treated with dantrolene sodium for a week before a 2 h run on a treadmill. We measured the total creatine kinase activity and isoenzyme composition in plasma before and after exercise. We also prepared muscle sections to correlate the biochemical data with morphological evidence of muscle damage. The treated rats showed a marked decrease (34%) in exercise-induced enzyme efflux, caused by a decrease in the muscle specific isoenzyme: instead of a 13-fold increase found in control rats we observed only a 6.5-fold increase in treated animals. The latter showed significantly less muscle damage 48 h later. It is concluded that dantrolene sodium protects the muscle against exercise-induced damage. The possible mechanism of this protection, and its applications in neuromuscular disease are discussed.

Electrical field stimulation induces taurine release in rat vas deferens. In the present study, it was investigated if this release is secondary to contraction. The influence of Ca2+ and of the stimulation conditions was also studied. Contractions evoked by electrical field stimulation (5 Hz/270 pulses, transverse or longitudinal) were recorded and released taurine was quantified by high performance liquid chromatography with fluorimetric detection. Ca2+ removal abolished contractions, but not the overflow of taurine. Overflow elicited by longitudinal electrical field stimulation was higher than that elicited by transverse electrical field stimulation. Increasing the current strength also increased taurine overflow. In Ca2+-free medium, taurine overflow was decreased by caffeine (5 mM) or ryanodine (10 μM) but increased by dantrolene (50 μM). The results indicate that taurine release evoked by electrical field stimulation is (i) independent of contraction, (ii) modulated by Ca2+, (iii) potential dependent, and may be due to a decrease in taurine affinity for the plasma membrane and/or to an increase of Na+-dependent outward transport.

We investigated the involvement of the sympatho-adrenal axis in the hyperthermia induced by methamphetamine by using a biotelemetric system. The intraperitoneal injection of methamphetamine (1 mg/kg) induced hyperthermia preceded by an increase in oxygen consumption in freely moving rats. The hyperthermic effect of methamphetamine was completely blocked by chemical sympathectomy with 6-hydroxydopamine (50 mg/kg, i.p.). Adrenalectomy, but not adrenal demedullation, prevented the hyperthermia. In adrenalectomized rats, dexamethasone supplementation (0.5 mg/kg, s.c.) restored the methamphetamine-induced hyperthermia. Furthermore, dantrolene (1 or 2 mg/kg, i.v.), which blocks Ca2+ release from the sarcoplasmic reticulum in skeletal muscle, attenuated the hyperthermia. These results suggest that methamphetamine stimulates norepinephrine release from sympathetic nerve terminals, which then enhances thermogenesis in skeletal muscle under the permissive action of glucocorticoids.

We report the effects of two new dihydropyridine derivatives, isradipine (4-(4'-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedic arboxylic acid methylisopropylester) and niguldipine (1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinecarboxylic acid 3-(4,4-diphenyl-1-piperidinyl)-propyl methyl ester hydrochloride), and of dantrolene (1-[(5-[p-nitrophenyl]furfurylidene)-amino]hydantoin sodium, an inhibitor of Ca2+ release from intracellular stores) on the protective efficacy of antiepileptic drugs against maximal electroshock-induced seizures. It was shown that dantrolene (5-20 mg/kg), isradipine (5-10 mg/kg) and niguldipine (up to 2.5 mg/kg) did not influence the electroconvulsive threshold in mice, although a higher dose of niguldipine (5 mg/kg) significantly elevated it. Dantrolene (10-20 mg/kg) and isradipine (1 mg/kg) did not affect the anticonvulsive activity of conventional antiepileptic drugs. In contrast, niguldipine (2.5-5 mg/kg) impaired the protective action of carbamazepine and phenobarbital. No effect of niguldipine (2.5-5 mg/kg) was observed upon the anticonvulsive efficacy of diphenylhydantoin and valproate. BAY k-8644 (methyl-1,4-dihydro-2,6-dimethyl-5-nitro-4- [(2-trifluoromethyl)-phenyl]-pyridine-5-carboxylate, an L-type Ca2+ channel agonist) did not reverse the action of niguldipine alone or the niguldipine-induced impairment of the anticonvulsive action of carbamazepine and phenobarbital. Niguldipine did not influence the free plasma levels of carbamazepine and phenobarbital, so a pharmacokinetic interaction is not probable. The results suggest that in contrast to the anticonvulsive activity of niguldipine against electroconvulsions, this Ca2+ channel inhibitor significantly weakened the protective action of both carbamazepine and phenobarbital. These effects do not seem to result from the blockade of voltage-dependent Ca2+ channels. Isradipine and dantrolene did not have a modulatory action on the threshold for electroconvulsions or on the anticonvulsive activity of antiepileptic drugs. It may be concluded that the use of niguldipine, isradipine, and dantrolene in epileptic patients seems questionable.

Activation of muscarinic receptors with carbachol has no effect on intracellular Ca2+ concentration in cerebellar granule cell cultures. Only after elevating intracellular Ca2+ concentrations using either 40 mM KCl or activating glutamatergic receptors was carbachol able to increase intracellular Ca2+. The response lasted about 10 s, and the median increase in intracellular Ca2+ with either 100 microM or 300 microM carbachol was about 85 nM. Carbachol at 30 microM elicited an increase in intracellular calcium that was half maximal. After a 16 min or 32 min delay following cell depolarization, responses to carbachol were only found in about 20% of the cells studied and the median increase in intracellular Ca2+ was about 14 nM. (-)-Quinuclidinylxanthene-9-carboxylate hemioxalate (QNX) at 10 nM (a muscarinic m3 antagonist), but not methoctramine at 5 microM (a muscarinic m2 antagonist), attenuated the action of 100 microM carbachol. This carbachol-mediated response was elicited in the absence of extracellular Ca2+ and in the presence of 10 microM dantrolene, but was blocked by 1 microM thapsigargin. Lastly, treatments of cerebellar granule cell cultures with carbachol (100 microM) for up to 12 h results in a desensitization of the carbachol-mediated release of stored Ca2+. Thus, carbachol acts on muscarinic m3 receptors to induce the release of Ca2+ from IP3-sensitive Ca2+ stores that must be filled by a prior period of high intracellular Ca2+.

The effect of dantrolene on the responses to methylxanthines (caffeine and theophylline) and a beta-adrenoceptor agonist (salbutamol) was studied in isolated guinea-pig trachea. Caffeine and theophylline (1 microM-10 mM) and salbutamol (1 nM-10 microM) produced concentration-dependent relaxation of spontaneous and stimulated (acetylcholine 1 mM) tone. Responses to high concentrations (10 mM) of caffeine and theophylline added to tracheal strips either unstimulated (spontaneous tone) or indomethacin (2.8 microM)-treated were reversed to contractions in the presence of dantrolene (3 microM-0.3 mM). This effect was not observed for salbutamol or when relaxant responses to the agonists were generated in pre-contracted (acetylcholine 1 mM) strips. Dantrolene inhibited concentration dependently the contraction evoked by caffeine (1 mM) in indomethacin (2.8 microM)-treated strips at 20 degrees C. This effect of dantrolene was attenuated in a low Ca2+ (0.8 mM) medium or when the bath temperature was lowered to 10 degrees C. Although an intracellular site of action cannot be excluded these results suggest that dantrolene mainly interferes with transmembrane Ca2+ movements in the guinea-pig trachea.

The effect of dantrolene sodium (3 microM-0.3 mM) on the spontaneous tone and responses to various contractile agonists was studied in isolated guinea-pig trachea. Dantrolene produced a concentration-related inhibition of the spontaneous tracheal tone, reaching a value of 94.8 +/- 4.8% of the relaxation induced by caffeine 10 mM. Removal of the epithelium did not affect the dantrolene-induced relaxation. Dantrolene did not alter the concentration-response curve for KCl and produced only small displacements of the concentration-response curves for CaCl2, acetylcholine and histamine, without affecting their maximal effects. Dantrolene dose relatedly inhibited the contraction induced by caffeine (1 mM) in Krebs solution containing indomethacin (2.8 microM) at 20 degrees C. The spasm induced by caffeine in Ca2(+)-free Krebs solution (20 degrees C, indomethacin 2.8 microM) was slightly depressed by dantrolene. Dantrolene did not depress the Ca2+ (1 microM)-induced contraction in skinned trachea. These results suggest that besides a possible intracellular site of action, the mechanism of the inhibitory effect of dantrolene in guinea-pig trachea may be related to interference with Ca2+ entry through pathways not susceptible to calcium channel blockers.

Caffeine, 10(2) M, blocked the indirectly stimulated rat phrenic nerve-diaphragm preparation at 37 degrees C. Dantrolene, above 2.25 x 10(-7) M, reversed the blockade. Microelectrode recordings showed a gradual reduction of the end plate potential (EPP) amplitude following a period of facilitation. An increased frequency of miniature (EPPs was still recorded after complete EPP blockade. A presynaptic blockade of stimulus-secretion coupling caused by an effect upon Ca2+ balance is suggested. Caffeine may release and deplete Ca2+ from the smooth endoplasmic reticulum (SER) in the nerve terminals. This Ca2+ may be sequestered by the mitochondria. The blockade suggests that the SER may also supply Ca2+ to another intraterminal release site which is necessary for stimulus-secretion coupling. This site is unaffected by caffeine and dantrolene. The dantrolene reversal may result from inhibition of the caffeine-induced release from the same Ca2+ pool. The facilitatory effects of caffeine were not antagonized by dantrolene and may result from Ca2+ release from another pool.

The effect of dantrolene sodium on the spontaneous contractions of rabbit jejunum was studied in vitro. Dantrolene sodium (4.5 × 10−6 to 4.5 × 10−4 M) reversibly decreased the amplitude of contractions in a dose-dependent manner. ED50 was found to be about 7.9 × 10−5 M. Its effect was biphasic in that a period of potentiation preceded that of suppression of contractions. Lowering or increasing (2.5 fold in each direction) the calcium concentration of bathing media did not affect the suppression of contraction caused by dantrolene sodium to any significant degree. Caffeine but not quinne was found to be able to restore the activity of the intestine to normal after a 50% inhibition caused by dantrolene sodium. Dantrolene sodium, verapamil and nifedipine were able to shift the dose-response curves of calcium in potassium-depolarized rabbit jejunum to the right and pA2 values were found to be 4.18, 7.76 and 8.47 respectively. These data indicate that the effect of dantrolene on smooth muscle is mediated via inhibition of calcium movement across the membrane.

The effects of dantrolene sodium on smooth muscle were evaluated in vitro using the guinea pig ileum and the guinea pig vas deferens preparations. Dantrolene irreversibly decreased the strength of the responses of the ileum to acetylcholine, histamine, potassium chloride and electrical stimulation. Doubling the concentration of calcium in the bathing media reduced the effect of dantrolene on all agonists. Dantrolene irreversibly decreased the response of the vas deferens to epinephrine, norepinephrine, acetylcholine and electrical stimulation. Doubling the concentration of calcium in the bathing media reduced the effect of dantrolene on all agonists. Dantrolene seems to depress smooth muscle as it does skeletal muscle. The mechanism of action probably involves the reduction of calcium flux into the muscles.

Dantrolene sodium has been compared with reference antiarrhythmic agents in rodent models of cardiac arrhythmia. In a coronary-artery-ligation model in rats, dantrolene sodium (3, 10 and 20 mg/kg i.v.) significantly decreased extrasystoles, episodes of ventricular tachyarrhythmia, and frequency, duration, and total episodes of ventricular fibrillation in a dose-dependent manner. In an electrically induced fibrillation model in rats, dantrolene sodium (10 and 20 mg/kg i.v.) significantly raised ventricular fibrillation threshold in a dose- and time-dependent manner. In contrast to its activity in these models, dantrolene sodium was not active in two chemically induced models involving automaticity. Aconitine-induced arrhythmias in rats and mice and ouabain-induced arrhythmias in guinea pigs were not suppressed by i.v. (10 or 20 mg/kg) or i.p. (100-3000 mg/kg) doses of the drug. These results show that the antiarrhythmic potential of dantrolene sodium, predicted by in vitro Class III and Class IV electrophysiological effects, is expressed in whole animal models.

Dantrolene sodium (3 X 10(-4) M) exerted a biphasic effect on the mechanical and electrical activity of the isolated guinea pig left atrium. In the first phase, the drug increased the contractile force by about 200%, and prolonged the action potential duration. This transient positive inotropic effect was antagonized by D-600, a slow Ca2+ channel inhibitor. In the later phase, the drug gradually decreased the amplitude of contractions, shortened the action potential duration and induced contracture. Dantrolene sodium increased the rate of depolarization and overshoot of Ca2+ -mediated slow action potentials. The results suggest that dantrolene sodium increases the slow inward Ca2+ current, causing a positive inotropic effect in the atrial myocardium. The late negative inotropic effect of the drug could be due to a secondary inhibition of the slow inward Ca2+ current due to an increased intracellular free Ca2+ concentration as a consequence of other actions of the drug.

The critical importance of dystrophin to cardiomyocyte contraction and sarcolemmal and myofibers integrity, led us to test the hypothesis that dystrophin reduction/loss could be involved in the pathogenesis of doxorubicin-induced cardiomyopathy, in order to determine a possible specific structural culprit behind heart failure. Rats received total cumulative doses of doxorubicin during 2weeks: 3.75, 7.5, and 15mg/kg. Controls rats received saline. Fourteen days after the last injection, hearts were collected for light and electron microscopy, immunofluorescence and western blot. The cardiac function was evaluated 7 and 14days after drug or saline. Additionally, dantrolene (5mg/kg), a calcium-blocking agent that binds to cardiac ryanodine receptors, was administered to controls and doxorubicin-treated rats (15mg/kg). This study offers novel and mechanistic data to clarify molecular events that occur in the myocardium in doxorubicin-induced chronic cardiomyopathy. Doxorubicin led to a marked reduction/loss in dystrophin membrane localization in cardiomyocytes and left ventricular dysfunction, which might constitute, in association with sarcomeric actin/myosin proteins disruption, the structural basis of doxorubicin-induced cardiac depression. Moreover, increased sarcolemmal permeability suggests functional impairment of the dystrophin-glycoprotein complex in cardiac myofibers and/or oxidative damage. Increased expression of calpain, a calcium-dependent protease, was markedly increased in cardiomyocytes of doxorubicin-treated rats. Dantrolene improved survival rate and preserved myocardial dystrophin, calpain levels and cardiac function, which supports the opinion that calpain mediates dystrophin loss and myofibrils degradation in doxorubicin-treated rats. Studies are needed to further elucidate this mechanism, mainly regarding specific calpain inhibitors, which may provide new interventional pathways to prevent doxorubicin-induced cardiomyopathy.

The effects of dantrolene sodium on the spontaneous and oxytocin-induced contractions of guinea-pig uterus were studied in vitro. Dantrolene reversibly increased the spontaneous contraction frequency of guinea-pig uterus. The effect was not dose-related and was not antagonized by atropine, methysergide or indomethacin. Dantrolene dose dependently and reversibly reduced the contractile responses of guinea-pig uterus to oxytocin. The varying effects of dantrolene on the uterus probably involve inhibition of calcium movement at more than one site of the muscle.

Administration of succinylcholine (SCh) to chicks produces rigid paralysis and death due to respiratory impairment. The mechanism of the SCh effect is probably related to the multiple innervation of muscle fibres, leading to excessive intracellular accumulation of calcium. This situation may be similar to that in malignant hyperpyerxia (MH) occurring in mammals. Dantrolene sodium, phenytoin and procain, drugs used against MH, were found to afford protection against SCh rigidity and death in chicks. It is suggested that the chick can be used as a convenient model for a rapid screening of drugs potentially active against MH.

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.

The effect of dantrolene-Na (DAN) on electrical and mechanical responses was investigated to single fibers of frog semitendinosus. Twitch tension was potentiated over 2--4 min by intracellular application of DAN and was rapidly decreased thereafter. Extracellular DAN depressed the tension with all dosages used. Although DAN had no effect on membrane potential, it exerted a biphasic action on membrane excitability. From these results, a mechanism is postulated for the release of trigger Ca2+ from T-tubules.

Verapamil is known to reduce Ca2+ entry in a variety of cells. At 10(-5) M it produces a small reduction in MEPP frequency at the frog neuromuscular junction, whereas the rate of spontaneous release rises following treatment at a concentration of 10(-4) M. This latter effect is augmented by raising [Ca2+]0 to 9 mM or, more especially, by raising the temperature from 17 to 23 degrees C. It is argued that both these opposing effects are related to the action of verapamil in modifying [Ca2+]i at the presynaptic terminals and it is suggested that the drug can affect both inward Ca2+ flux (so reducing the steady-state position of [Ca2+]i) and also, at higher concentration, either inhibit the membrane Ca2+ pump or cause the release of Ca2+ from intracellular Ca2+ stores (so raising [Ca2+]i).

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.

Dantrolene sodium (DaNa) markedly depresses the frequency of spontaneous miniature potentials at the frog neuromuscular junction. Its action is still observed at low [Ca2+]o and is little affected by temperature; its effect is not readily reversible. Theophylline antagonises the action of DaNa. DaNa prevents the stimulatory effect of the ionophore A23187 at 25°C, but is ineffective in antagonising the action of X537A. DaNa and theophylline do not affect the functioning and integrity of mitochondria in vitro and it is suggested that DaNa acts at intracellular Ca2+ stores (other than the mitochondria) in the presynaptic terminals.

The potency of putative blockers (lidocaine, dantrolene Na, DAN, SKF 525A) for various forms of Ca 2+ release from sarcoplasmic reticulum (SR) against two types of spontaneous contractions induced by 0.5 mM caffeine (caffeine response) or myocardium Ca 2+ overload (after-contractions) was tested in rat papillary muscle. There were differences in the sensitivity of spontaneous contractions to the agents employed. The caffeine response was depressed by DAN but not affected by lidocaine. Conversely, conventional diastolic after-contractions were eliminated by lidocaine and SKF 525A but DAN failed to affect them. The two types of spontaneous contractions were suggested to be triggered by different Ca 2+ release processes supposedly originating from different SR compartments.

The effects of intravenous dantrolene sodium on contractions of the tibialis anterior, the flexor digitorum longus, the soleus and the diaphragm muscles, and on respiratory parameters, blood pressure and heart rate were recorded in cats under chloralose anaesthesia. Dantrolene maximally depressed twitches of the tibialis anterior and flexor digitorum loncles to a significantly greater extent (ca. 90%) than it depressed those of the soleus and diaphragm muscles (ca. 72%). When the muscles were indirectly stimulated at frequencies high enough to produce complete fusion of responses, the depressant action of dantrolene was largely masked. The onset of this “break through” effect was evident with paired stimuli, and was maximal with trains of about 6 stimuli. Dantrolene was without effect on blood pressure and heart rate in all experiments; the slight effects that accompanied each injection were entirely attributable to the solvent (propylene glycol). In 8 out of 12 experiments, dantrolene was without any detectable effect on breathing. In one cat, however, which happened to be unusually deeply anaesthetised, dantrolene produced a decrease in the frequency of breathing and the animal tended to hold each breath for longer than usual. In 3 out of the 12 cats, dantrolene produced a slight increase in transpulmonary pressure, a slight reduction in tidal volume and a slight shortening of the duration of each respiratory cycle with no alteration in the frequency of breathing. It is proposed that the lack of effect of dantrolene on the respiratory parameters in most experiments was the result of a reflex increase in the frequency of discharge from the respiratory centre to the extent that resistant tetanic contractions of the respiratory muscles were evoked.

In rat ventricular muscles, ryanodine (10–30 nM) evoked tension oscillation in the relaxation phase of an isometric twitch (relaxation oscillations) and an increase of tonic tension (ryanodine contracture). Both events were more pronounced in Ca 2+ -loaded rat muscles due to the addition of 0.5 μM adrenaline, an increase in the Ca 2+ concentration in the solution and high muscle activity. The ryanodine-induced tension oscillations were comparable to those triggered by caffeine in this species. In both cases, blockers of the release of Ca 2+ from the sarcoplasmic reticulum, namely tetracaine and dantrolene, abolished the relaxation oscillations and the ryanodine contracture. The results suggest that the ability of low concentrations of ryanodine to facilitate the release of Ca 2+ from the sarcoplasmic reticulum, as shown recently in biochemical experiments, makes a direct contribution to triggering the relaxation oscillations and the ryanodine contracture in intact ventricular muscles. The Ca 2+ load of the sarcoplasmic reticulum appears to be essential for the manifistation of the ryanodine Ca 2+ -releasing activity in rat ventricular muscles.

To determine the mechanism by which Ag+ induces a transient contracture in skeletal muscle, the effect of dantrolene sodium on the Ag+ contracture was examined and the findings compared with those for the twitch, tetanus and caffeine contracture. The inhibition of twitch by dantrolene was equivalent to that of the Ag+ contracture at concentrations of 1, 2 or 5 microM of dantrolene. The tetanus tension was slightly inhibited by dantrolene, but not the caffeine contracture. These observations suggest that the Ag+ contracture may be governed by the same mechanism as that involved in the development of twitch tension.

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.

Dantrolene sodium in different concentrations was administered to the spontaneously beating heart placed in a modified Langendorff apparatus. Heart frequency and contractility were recorded. Dantrolene sodium was also administered to the rat diaphragm. Twitch tension after indirect supramaximal stimulation was recorded. Dantrolene sodium produced a long lasting dose-dependent reduction of the contractility of the isolated rat heart up to 75% of control values. It had no effect on the heart frequency. The drug also decreased the force of contraction of the rat skeletal muscle in vitro to the same extent. The diaphragm appeared to be more sensitive to low concentrations of dantrolene sodium than was heart muscle i.e. the dose-response curve on rat diaphragm was flatter. It may be concluded however that higher concentrations of dantrolene sodium may effect the contractility of heart muscle as well and that this may have clinical implications.

The spasmogenic activity of caffeine (10 mM) was evaluated in tracheal strips obtained from control and sensitized guinea-pigs then pretreated with indomethacin (2.8 microM) and cooled to 20 degrees C. The contraction elicited by caffeine was inhibited by verapamil (100 microM), trifluoperazine (100 and 500 microM) and dantrolene (50 and 500 microM) in the control and the sensitized tissues but was unaffected by disodium cromoglycate (39 microM). However, the same concentration of verapamil produced significantly less inhibition of the caffeine-induced contraction in sensitized compared to control tissues while the reverse was found for trifluoperazine and dantrolene. Exposure to a Ca2+-free, EGTA-containing medium resulted in 33% inhibition of the response to caffeine in control tissues but no inhibition in sensitized tissues. These results suggest the existence of differences in calcium movements in response to caffeine between control and sensitized tissues that may reflect abnormalities in calcium handling by the sensitized tissue.