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In soluble and particulate extracts from muscle D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and D-myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] are metabolised stepwise to inositol. Ins(1,4,5)P3 is rapidly dephosphorylated to D-myo-inositol 1,4-bisphosphate then to D-myo-inositol 4-phosphate and finally inositol. In soluble extracts Ins(1,3,4,5)P4 is dephosphorylated to D-myo-inositol 1,3,4-trisphosphate then sequentially to D-myo-inositol 3,4-bisphosphate, D-myo-inositol 3-phosphate and inositol, while in particulate extracts D-myo-inositol 1,3-bisphosphate is the predominant inositol bisphosphate formed. Dephosphorylation of these inositol polyphosphates is Mg2+ dependent and inhibited by D-2,3-bisphosphoglyceric acid. Ins(1,4,5)P3 is also phosphorylated to form Ins(1,3,4,5)P4 in soluble extracts by Ins(1,4,5)P3 3-kinase. Ins(1,4,5)P3 3-kinase activity is Mg2+ and ATP dependent and is stimulated by Ca2+ and calmodulin. Particulate (sarcotubular) inositol polyphosphate 5-phosphatase (5-phosphatase) is found in membranes which are intimately involved in excitation-contraction coupling and the generation of the primary Ca2+ signal of muscle cells. Particulate 5-phosphatase had the highest specific activity in the transverse-tubule membrane, when compared to the terminal cisternae and longitudinal-tubule membranes of the sarcoplasmic reticulum. Particulate Ins(1,3,4,5)P4-3-phosphatase activity was also detected after fractionation of solubilised sarcotubular membranes by DEAE-Sephacel. Particulate 5-phosphatase activity was purified 25,600-fold to a specific activity of 25.6 mumol Ins(1,4,5)P3 hydrolysed.min-1.mg protein-1, after DEAE-Sephacel and novel affinity chromatography using D-2,3-bisphosphoglycerate/agarose and Sepharose-4B-immobilised Ins(1,4,5)P3-analog matrices. Purified particulate 5-phosphatase had apparent Km of 46.3 microM and 1.9 microM and Vmax of 115 and 0.046 mumol substrate hydrolysed.min-1.mg protein-1, for Ins(1,4,5)P3 and Ins(1,3,4,5)P4, respectively. In contrast, purified soluble type I 5-phosphatase had apparent Km of 8.9 microM and 1.1 microM and Vmax of 3.55 and 0.13 mumol substrate hydrolysed.min-1.mg protein-1, for Ins(1,4,5P3 and Ins(1,3,4,5)P4, respectively. As in other cells, muscle 5-phosphatases have a lower affinity, but a higher capacity to metabolise Ins(1,4,5)P3 than Ins(1,3,4,5)P4. Soluble type I 5-phosphatase may have a functional role in the metabolism of both inositol polyphosphates, while particulate 5-phosphatase may primarily metabolise Ins(1,4,5)P3. Purified Ins(1,4,5)P3 3-kinase had an apparent Km of 0.42 microM and a Vmax of 4.12 nmol Ins(1,4,5)P3 phosphorylated.min-1.mg protein-1. The profile of inositol polyphosphate metabolism in muscle is similar to that reported in other tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

The ability of the novel D- myo -inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] analogues, L- chiro -inositol 1,4,6-trisphosphate [L- chr Ins(1,4,6)P 3 ] and the corresponding trisphosphorothioate compound [L- chr Ins(1,4,6)PS 3 ] to inhibit soluble inositol (1,4,5)P 3 /(1,3,4,5)P 4 -polyphosphate 5-phosphatase, potently and selectively, has been investigated. L- chr Ins(1,4,6)P 3 competitively inhibited 5-phosphate specific dephosphorylation of Ins(1,4,5)P 3 and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P 4 ] with apparent K i values of 6.35 and 1.76 μM, respectively. L- chr Ins(1,4,6)PS 3 competitively inhibited hydrolysis of Ins(1,4,5)P 3 and noncompetitively inhibited dephosphorylation of Ins(1,3,4,5)P 4 with apparent K i values of 0.67 and 0.44 μM, respectively. L- chr Ins(1,4,6)PS 3 did not affect Ins(1,4,5)P 3 3-kinase activity. In the present investigation L- chr Ins(1,4,6)P 3 and L- chr Ins(1,4,6)PS 3 have been shown to be the most potent and selective inhibitors of inositol polyphosphate metabolism yet described.

Seven out of eight piglets which were susceptible to malignant hyperthermia (MHS) died when subjected to a heat challenge which was well tolerated by controls. The piglets which succumbed developed the classical clinical and biochemical changes of malignant hyperthermia before they died. These results show that overheating alone can trigger malignant hyperthermia in susceptible animals. Because the biochemical basis of malignant hyperthermia is similar in both humans and pigs, these observations suggest that overheating can also trigger malignant hyperthermia in humans. The susceptibility to overheating in malignant hyperthermia susceptible humans and animals probably explains why the myopathy which predisposes to this condition has also been reported to predispose to heat-stroke and the sudden infant death syndrome. In view of this, particular care to prevent overheating should be taken in infants of parents who are susceptible to malignant hyperthermia.

A myoplasmic 3-kinase was detected in porcine skeletal muscle that phosphorylated [3H]Ins(1,4,5)P3 [D-myo-inositol(1,4,5)trisphosphate] to [3H]Ins(1,3,4,5)P4 [D-myo-inositol(1,3,4,5)tetrakisphosphate]. The Ins(1,4,5)P3 3-kinase activity was ATP- and Mg(2+)-dependent, and was activated by Ca2+ and calmodulin. Ins(1,4,5)P3 3-kinase activity was purified 2632-fold from soluble extracts of skeletal muscle by a combination of DEAE-Sephacel, heparin-Agarose and Ins(1,4,5)P3 structural-analogue affinity chromatography. The highest specific activity obtained was 10.6 nmol of Ins(1,4,5)P3 phosphorylated/min/mg protein. The partially purified enzyme had a mean Km and Vmax of 0.46 microM and 3.15 nmol/min/mg protein for Ins(1,4,5)P3 metabolism, respectively. After analytical gel filtration two forms of soluble Ins(1,4,5)P3 3-kinase were observed with M(r) of 39,000 and 62,000. As in other cell types, muscle Ins(1,4,5)P3 3-kinase was soluble, and had a higher affinity but a lower capacity to metabolize Ins(1,4,5)P3 in comparison to Ins(1,4,5)P3 5-phosphatase.

1. The calcium channel antagonists verapamil (100 microM) and nifedipine (100 microM) inhibited twitch response and KCl induced hypercontractility in malignant hyperpyrexia (MH)-susceptible porcine skeletal muscle. These calcium channel antagonists did not effect hypercontractility induced by 3% halothane or 2 mM caffeine. 2. The calcium channel agonist BAY K 8644 (50 microM) induced contracture in MH-susceptible muscle but did not potentiate contracture response induced by 2 mM caffeine or 3% halothane. BAY K 8644 did not increase the resting tension of control muscle or increase the sensitivity of control muscle to 4 mM caffeine, 3% halothane or 80 mM KCl. 3. The sarcoplasmic reticulum (SR) from MH-susceptible and control porcine skeletal muscle was separated into vesicular fractions enriched in the membrane elements of the terminal cisternae and longitudinal tubules. 4. Verapamil and diltiazem [which has been previously shown to inhibit the hypercontractility of MH-susceptible porcine muscle to caffeine, halothane and KCl (Foster and Denborough, 1989 Br. J. Anaesth. 62, 566-572)] did not effect Ca2+ uptake or Ca(2+)-dependent ATPase activities of SR longitudinal tubule membranes, from MH-susceptible or control muscle. These calcium channel antagonists did not effect Ca2+ release from terminal cisternae preparations. 5. The skeletal muscle relaxant dantrolene inhibited Ca2+ efflux and equilibrium-Ca2+ exchange associated with the terminal cisternae membrane of MH-susceptible and control skeletal muscle. 6. Calcium channel antagonists modify Ca2+ fluxes in MH-susceptible and control muscle by acting at a site distal to the SR. Calcium channel antagonists may inhibit contractile response by modifying events of excitation-contraction coupling associated with the voltage sensor molecule (dihydropyridine-receptor) of the transverse-tubule membrane, whereas dantrolene directly acts on the terminal cisternae membrane to inhibit Ca2+ efflux and equilibrium Ca2+ exchange. Different calcium channel antagonists seem to modify the voltage-sensor mechanism in different ways in MH-susceptible muscle. 7. An abnormality in the coupling mechanism of the voltage sensor-SR calcium release channel may exist in MH-susceptible muscle. This dysfunction may be an adaptation to the elevated levels of myoplasmic Ca2+ and/or the molecular defect described in the Ca2+ release channel of the SR of MH-susceptible porcine muscle. In view of these results it is unlikely that nifedipine or verapamil would be of therapeutic value for the treatment of MH.

1. Malignant hyperpyrexia (MH) is an inherited muscle abnormality that presents clinically as a syndrome of life-threatening complications during general anaesthesia. 2. Propofol is a new sedative hypnotic used for the induction and maintenance of anaesthesia. 3. Propofol did not induce MH in five susceptible pigs. Propofol did not induce contracture in isolated MH susceptible muscle but did modify halothane, caffeine and KCl contractures.

The isoenzyme pattern and activity of adenylate kinase was studied in muscle and red blood cells from individuals and swine shown to be susceptible to malignant hyperpyraria. No abnormality in adenylate kinase isoenzyme pattern or activity was found.

1. 1. Malignant hyperpyrexia (MH) is an anaesthetic complication which is due to an underlying muscle membrane abnormality. 2. 2. One- and two-dimensional electrophoresis of skeletal muscle proteins were carried out to study the abnormality. 3. 3. No defect in MH susceptible muscle was found.

1. 1. Because calcium antagonist drugs increase contracture in both control and malignant hyperpyrexia susceptible (MHS) skeletal muscle, the effect of these drugs on the sacroplasmic reticulum (SR) was investigated. 2. 2. The calmodulin antagonist drugs inhibited the Ca 2+ dependent ATPase activity and the ATP-dependent Ca 2+ uptake, and accelerated the efflux of Ca 2+ from isolated SR preparations from both control and MHS skeletal muscle. These effects of calmodulin antagonist drugs on SR Ca 2+ transport functions were consistent with their in vitro pharmacological effects on control and MHS muscle.

MH occurs in people who have a generalized membrane abnormality. Two clinical myopathies are known to be associated with this membrane defect. One is usually subclinical and is inherited as a mendelian dominant characteristic. The other occurs in boys who have a number of striking physical abnormalities, and is inherited as a recessive trait. When the abnormal muscle cell membrane is exposed to anesthetic agents such as halothane or succinylcholine there is a rapid, excessive release of calcium into the myoplasm. The raised myoplasmic calcium concentration leads to all the clinical features of the syndrome, predominant among which are: metabolic acidosis, a rise in body temperature, and rigidity--particularly in the muscles of the limbs. The exact site and nature of the lesion in the muscle cell membrane are still to be determined, but recent evidence suggests that it may lie in the E-C coupling mechanism. Understanding of the pathophysiology of MH has led to the development of an appropriate drug treatment for this syndrome, the aim of which is to restore the elevated myoplasmic calcium level to normal. Dantrolene sodium promises to be the most effective drug in achieving this result.

The effect of adenylate cyclase activation on the in vitro contractures of control and malignant hyperpyrexia susceptible (MHS) porcine muscle was investigated. While fluoride and molybdate ions potentiated drug-induced contractures in control muscle, other activators of adenylate cyclase (forskolin and noradrenaline) did not. Furthermore, fluoride and molybdate had no effect on MHS skeletal muscle contractility. Cyclic AMP content, basal adenylate cyclase activity and molybdate-stimulated adenylate cyclase activity of MHS skeletal muscle was not significantly different from that of control muscle. It is concluded that increased activity of adenylate cyclase does not represent the primary skeletal muscle defect which predisposes to porcine MH.

31Phosphorus-NMR spectroscopy may have the potential to help in the noninvasive diagnosis of malignant hyperpyrexia (MH). Changes in the phosphate-metabolite profile of MH-susceptible (MHS) skeletal muscle occur more readily under conditions of anoxia than in control muscle. Induction of anoxia caused a rapid fall in intracellular phosphocreatine, an elevation of inorganic phosphate, and finally a diminution of ATP in MHS muscle. The onset of metabolic change was slower in control tissue. Increased oxygen consumption may occur in anoxic MHS muscle, which leads to accelerated glycolysis and a rapid fall in the intracellular high-energy phosphates. In MHS muscle an abnormality may exist in carbohydrate metabolism linked with poor resynthesis of the high-energy phosphates, which may be precipitated under anaerobic conditions. Accelerated muscle metabolism is also observed in the presence of 2 mM caffeine and 3% halothane in MHS muscle. Changes in the concentrations of metabolites could be mapped noninvasively under anoxic conditions using topical 31P-NMR.

In both control pigs and pigs susceptible to malignant hyperpyrexia (MH), the size of the calcium current (ICa) and the amount of asymmetric charge movement varied considerably between different gracilis muscle fibers but appeared to vary in parallel. The mean amount of both ICa and charge movement were slightly but significantly smaller in MH-susceptible (MHS) muscle compared with normal muscle. Halothane (1% v/v) reduced both parameters by almost 50%. One interpretation of the data is that the signal, which couples depolarization to calcium release from the sarcoplasmic reticulum, is abnormal in MHS muscle.

1. 1. The anaesthetic complication malignant hyperpyrexia (MH) is due to an elevation myoplasmic Ca2+ concentration. 2. 2. Examination of calmodulin isolated from MH susceptible swine suggests that the disorder in calcium regulation in MH is not due to an abnormality in calmodulin.

The sarcoplasmic reticulum from muscle of swine which are susceptible to malignant hyperpyrexia is deficient in inositol 1,4,5-trisphosphate phosphatase (InsP35-ase) activity, which leads to high intracellular concentrations of inositol 1,4,5-trisphosphate (InsP3) and of calcium ions. Halothane inhibits InsP35-ase and further increases myoplasmic InsP3 and calcium ion concentrations, and produces the clinical features of malignant hyperpyrexia.

Contractures induced by halothane, caffeine, suxamethonium chloride and potassium in malignant hyperpyrexia-susceptible (MHS) and control swine skeletal muscle preparations were temperature-dependent, but differed in their temperature response profiles. Increasing the temperatures from 20 degrees C to 37 degrees C increased drug-induced contractures in MHS muscle. At 5 degrees C drug-induced contractures were not inhibited by dantrolene sodium, deuterium oxide or glycerination in either MHS or control muscle. These observations give further support to the view that the abnormality in MHS skeletal muscle is associated with the mechanism of excitation-contraction coupling.

1. Landrace swine were identified as malignant hyperpyrexia susceptible (MHS) or control by the contracture responses of gracilis muscle fibre bundles to 3% halothane, 2 mmol/l caffeine and 80 mmol/l KCl. The effects of calmodulin-antagonist drugs on the contractile behaviour of control and MHS preparations were investigated in vitro. 2. Calmodulin-antagonists at micromolar concentrations induced contracture in both control and MHS muscle. Pretreatment of MHS muscle with calmodulin-antagonist drugs potentiated its response to halothane and caffeine. 3. These results suggest that calmodulin-antagonist drugs cause an increase in myoplasmic Ca2+ concentration in both control and MHS muscle.

1. 1. The anaesthetic complication malignant hyperpyrexia (MH) occurs in individuals and swine with a muscle membrane abnormality. 2. 2. In an attempt to characterize this abnormality, the lipid and protein composition of muscle membranes from MH-susceptible swine was examined and compared with controls. 3. 3. No significant abnormality in the membrane protein or lipid content of MH-susoeptible muscle was detected.

Adenylate kinase has been implicated as a key factor in malignant hyperpyrexia, a complication of general anaesthesia which is usually triggered by the anaesthetic drug, halothane. Because of this, the enzyme was purified from both malignant hyperpyrexia susceptible and control porcine muscle. Electrophoretic studies, amino acid analysis, and peptide mapping of the purified enzymes revealed no significant differences between the two preparations. Both enzymes responded similarly to halothane and to the three sulfhydryl reacting reagents which were tested and they also showed an identical affinity for the substrate AMP. It is concluded that porcine MH is not due to an abnormality in the enzyme AK.

Sarcoplasmic reticulum membranes were isolated from the skeletal muscle of pigs susceptible or resistant to the anaesthetic complication malignant hyperpyrexia. The skeletal muscle relaxant dantrolene was shown to be without effect on the Ca2+ -dependent ATPase activity of either sarcoplasmic reticulum preparation. The active transport of calcium by sarcoplasmic reticulum from both sources was also unaffected by the presence of dantrolene. These findings indicate that dantrolene does not exert its pharmacological action in skeletal muscle by stimulating calcium uptake by the sarcoplasmic reticulum.

1. 1. The sarcoplasmic reticulum (SR) from malignant hyperpyrexia susceptible (MHS) and control porcine skeletal muscle was separated into vesicular fractions enriched in the membrane elements of the terminal cisternae and longitudinal tubules. 2. 2. The two membrane preparations were highly purified and had distinctive features which were associated with their origins in the SR membraneous network. 3. 3. Calsequestrin and calcium were enriched in the terminal cisternae fraction (HSR), in comparison to longitudinal tubule preparations (LSR). 4. 4. The HSR membrane also had a greater total capacity to store Ca 2+ and Ca 2+ release was more rapid than from LSR preparations. 5. 5. No distinction could be made between the membrane morphology, Ca 2+ -fluxes or Ca 2+ -dependent ATPase activities, associated with these functionally distinct regions of MHS and control preparations.

We have studied the ability of the calcium channel antagonist diltiazem to inhibit and reverse the porcine malignant hyperpyrexia (MH) syndrome. Pretreatment with diltiazem modified an MH response. Treatment with diltiazem was partially effective against a mild (or early) MH response. Diltiazem should not be considered to be an effective therapeutic agent for MH and should not displace the use of dantrolene.

Malignant hyperpyrexia is a dangerous complication of anaesthesia which occurs in individuals who have an underlying disease of muscle. Two predisposing myopathies have been defined. Studies in susceptible humans and in swine, with an apparently identical myopathy, have shown that when MH muscle is exposed to a precipitating anaesthetic agent there is a sudden rise in the concentration of calcium ions in the myoplasm. The precise nature of the inherited muscle membrane abnormality is not yet known, but pharmacological and biochemical studies indicate that is closely associated with the excitation-contraction coupling mechanism.

Calsequestrin was selectively solubilized from porcine skeletal muscle sarcoplasmic reticulum by incubation with HgCl 2 and deoxycholate. While deoxycholate alone caused non-specific solubilization of calsequestrin and other extrinsic proteins, HgCl 2 alone failed to extract significant amounts of extrinsic proteins. The simultaneous presence of HgCl 2 and deoxycholate was necessary for the selective and quantitative solubilization of calsequestrin. Following DEAE-Sephadex ion-exchange chromatography of the HgCl 2 /deoxycholate-solubilized material, calsequestrin was obtained in nearly homogeneous form. Porcine calsequestrin was similar to rabbit calsequestrin with respect to its amino acid composition and apparent molecular weight as determined by SDS-polyacrylamide gel electrophoresis. The calcium-binding capacity of porcine calsequestrin was 400 nmol of Ca 2+ bound/mg protein, lower than the values reported for the rabbit protein.

Pure suxamethonium chloride does not produce in vitro contracture of skeletal muscle from swine which are susceptible to malignant hyperpyrexia (MH), but does induce MH in vivo. It is suggested that suxamethonium chloride induces MH because the fasciculations which it causes lead to an increase in the myoplasmic calcium concentration.

In malignant hyperpyrexia susceptible (MHS) porcine skeletal muscle, a low concentration (100 mumol/l) of the calcium ion antagonist 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8) inhibited KCl-induced contractures, but potentiated contractures induced by halothane, caffeine and succinylcholine. Higher concentrations of TMB-8 (333 mumol/l to 1 mmol/l) contracted MHS muscle, but had little effect on muscle tension in control preparations. Treatments which inhibit excitation-contraction coupling abolished TMB-8-induced hyper-reactivity in MHS muscle. TMB-8 (50 mumol/l and 1 mmol/l) did not alter 45Ca2+ levels in actively loaded microsomal preparations from MHS swine. These results suggest that in malignant hyperpyrexia the primary abnormality occurs proximal to the release of calcium from the sarcoplasmic reticulum, probably at the level of excitation-contraction coupling.

Diltiazem inhibited and antagonized the abnormal contractures induced by halothane, caffeine and potassium chloride in isolated skeletal muscle from pigs susceptible to malignant hyperpyrexia (MHS). Contractile responses to caffeine and electrical stimulation also were suppressed by diltiazem in control tissue. Similar effects were obtained in the presence of dantrolene. In both MHS and control preparations, diltiazem antagonized caffeine-induced contractures in the presence of maximal effective concentrations of dantrolene, and the converse was true also. In MHS and control preparations detubulated by glycerol, diltiazem did not inhibit or antagonize caffeine-induced contractures while dantrolene did. Diltiazem seems to modify contractile responses at the level of the transverse tubule membrane by inhibiting the inward flow of extracellular Ca2+, while dantrolene inhibits Ca2+ release directly from the sarcoplasmic reticulum. Ca2+ influx through transverse tubules may be important in the aetiology of the MH syndrome.

1. 1. Chlorbutol, a muscle relaxant inhibits the in vitro muscle hypercontractility which is characteristic of the anaesthetic complication, malignant hyperpyrexia (MH). 2. 2. Studies on isolated sarcoplasmic reticulum vesicles have shown that this effect of chlorbutol in MH is not due to a modification of Ca 2+ -transport mechanisms.