Comparative effects of the ionophore A23187 on the mechanical responses of muscle in normal Pietrain pigs and pigs with malignant hyperthermia

Since increased intracellular Ca2+ is believed to be the main factor causing skeletal muscle contracture in human and porcine malignant hyperthermia, the potential effects of the ionophore A23187, which enhances intracytoplasmic Ca2+, were investigated in Pietrain pig muscles. These effects were compared with those of caffeine, known to induce dose-dependent contracture in vitro in isolated muscle from human subjects with malignant hyperthermia. For this purpose, the mechanical and biochemical actions of caffeine and A23187 were tested in intercostal muscle biopsies from 10 normal pigs and 10 with malignant hyperthermia. The results show that A23187 allowed very clear differentiation between the muscles of normal and pathological animals. In view of the wide spectrum of drug sensitivity characterizing subjects with malignant hyperthermia, it is suggested that exposure to A23187 be added to the halothane and caffeine tests currently used to detect this disease.     

Fatty acids modulate calcium-induced calcium release from skeletal muscle heavy sarcoplasmic reticulum fractions: implications for malignant hyperthermia

Based on studies in swine, the malignant hyperthermia syndrome has been postulated to result from an enhanced sensitivity (low threshold) of the Ca2(+)-induced Ca2(+)-release process. However, fatty acid production is elevated in homogenates of skeletal muscle from pigs and humans susceptible to malignant hyperthermia. In the present study, we demonstrate that the threshold of Ca2(+)-induced Ca2+ release is normal in susceptible humans and in susceptible swine depleted of triglycerides. Exogenously added unsaturated fatty acids decreased the threshold of Ca2(+)-induced Ca2+ release to a much greater extent in porcine and equine muscle than in human muscle. When triglyceride and free fatty acid values were reduced to about 40 and 60%, respectively, of control values, malignant hyperthermia-susceptible swine did not exhibit muscle rigidity when challenged in vivo with halothane and succinylcholine and the threshold of the Ca2(+)-induced Ca2(+)-release process in heavy sarcoplasmic reticulum fractions was normal. Despite the reduced triglyceride and fatty acid levels, these swine had a positive in vitro contracture test for malignant hyperthermia. A low Ca2(+)-induced Ca2(+)-release threshold is not essential for malignant hyperthermia susceptibility, but appears to be the result of excessive free fatty acids produced during organelle isolation.     

Malignant hyperthermia: human stress triggering

Letter to the Editor concerns the question of a discussion of awake porcine malignant hyperthermia that erroneously omits the awake human stress reaction of malignant hyperthermia.     

Genomic organization and analysis of the 5' end of the porcine ryanodine receptor gene (ryr1).

In this study we describe the isolation of genomic clones of the 5' region of the porcine ryanodine receptor gene, a candidate for malignant hyperthermia in pigs and humans. The recombinants were isolated from a porcine liver, genomic DNA library in phage EMBL3A after screening with PCR amplified DNA fragments. The exon/intron structure of the ryanodine receptor gene was determined by DNA sequencing. Based on the sequence data it was possible to develop a simple test for the detection of malignant hyperthermia susceptible and normal pigs.     

Further Studies of Porcine Malignant Hyperthermia

A non-lethal procedure for identifying pigs apt to develop malignant hyperthermia is described. Susceptible animals were exposed to a variety of anaesthetic and other agents and it was shown that thiopentone sodium and CT 1341 (Glaxo) afforded a measure of protection against the development of the syndrome. Pretreatment with procaine did not prevent the onset of the condition and the administration of procaine when muscle rigidity was present failed to prevent a fatal outcome. The syndrome was induced in susceptible animals by halothane, chloroform, and a combination of halothane with suxamethonium. The effects of cyclopropane in susceptible pigs could not be predicted, and other tests showed that suxamethonium alone would not induce muscle contracture. Pretreatment with lignocaine failed to prevent induction of the syndrome by halothane.We believe that the porcine syndrome may result from more than one defect and that in one particular type the most effective treatment is immediate cooling coupled with the administration of sodium bicarbonate.     

Skeletal muscle mitochondrial phospholipase A2 and the interaction of mitochondria and sarcoplasmic reticulum in porcine malignant hyperthermia

Comparative studies were carried out on the Ca²⁺-transport systems of mitochondria and sarcoplasmic reticulum from longissimus dorsi muscle of genetically selected malignant hyperthermia-prone and normal pigs in order to identify the biochemical lesion responsible for the enhanced release of Ca²⁺ in the sarcoplasm occurring in porcine malignant hyperthermia. Mitochondria isolated from longissimus dorsi muscle of malignant hyperthermia-prone pigs contained a significantly (P < 0.001) higher amount of endogenous long-chain fatty acids. Similar amounts of endogenous mitochondrial phospholipase A₂ were observed in both types of pigs, but the total activity in malignant hyperthermia-prone pigs was at least twice that of normal. Spermine, a phospholipase A₂ inhibitor, lowered the activity in both types of mitochondria to a similar final level. Mitochondria of malignant hyperthermia-prone pigs showed a significantly (P < 0.001) higher oligomycin-insensitive (Ca²⁺ + Mg²⁺)-ATPase activity, but the Mg²⁺-ATPase and the (Ca²⁺ + Mg²⁺)-ATPase activities were similar in both types of pigs. Sarcoplasmic reticulum isolated from longissimus dorsi muscle of malignant hyperthermia-prone pigs showed a significantly higher (Ca²⁺ + Mg²⁺)-ATPase activity and a lower rate of Ca²⁺ uptake; the maximal amount and the rate of Ca²⁺ uptake by sarcoplasmic reticulum of malignant hyperthermia-prone pigs were half that of normal. Mitochondria from longissimus dorsi muscle of malignant hyperthermia-prone pigs inhibited the Ca²⁺-transport system of the sarcoplasmic reticulum of longissimus dorsi from both normal and malignant hyperthermia-prone pigs, but mitochondria from normal pigs had no influence on the sarcoplasmic reticulum from either type. Experimental evidence favours the concept that long-chain fatty acids released from skeletal muscle mitochondria by endogenous mitochondrial phospholipase A₂ are responsible for the enhanced release of Ca²⁺ from mitochondria (Cheah, K.S. and Cheah, A.M. (1981) Biochim. Biophys. Acta 634, 70–84), and also additional release of Ca²⁺ from sarcoplasmic reticulum into the sarcoplasm during porcine malignant hyperthermia syndrome.     

Localization of the PGD and TGFβ-1 loci to pig chromosome 6q

The TGF beta-1 and PGD loci have been localized by in situ hybridization to the C-greater than q2.1 and q2.2 -greater than q2.5 regions of pig chromosome 6. These assignments confirm that the conversation of syntenic groups around GPI and PGD extends to pigs where these two groups are uniquely found to be linked. Our data also support the hypothesis that the porcine and human inherited malignant hyperthermia syndromes are caused by mutations in homologous genes which map to human chromosome 19q, porcine chromosome 6q and murine chromosome 7.     

Effects of halothane, caffeine, dantrolene and tetracaine on the calcium permeability of skeletal sarcoplasmic reticulum of malignant hyperthermic pigs

Preparing skeletal sarcoplasmic reticulum from both normal and malignant hyperthermia susceptible pigs, the effects of various drugs on the passive calcium permeability of these sarcoplasmic reticulum preparations were studied. It was found that, in the absence of halothane, the permeability of heavy sarcoplasmic reticulum prepared from malignant hyperthermia susceptible pigs was much higher than that of normal pigs. It was observed that halothane, at concentrations above 10 microM (well below anesthetic concentrations, which are on the order of 1 mM), increased the permeability of sarcoplasmic reticulum. The Hill coefficient of the effect of halothane ranged from 1.96 to 2.25, suggesting that some kind of cooperativity was involved in this reaction. The effects of caffeine were similar to those of halothane. Inhibitors, such as tetracaine and ruthenium red inhibited both the calcium permeability and the halothane-induced increment. The Hill coefficient of the effect of tetracaine was 1.75. The mode of inhibition suggests that tetracaine directly binds with the calcium channel to inhibit the calcium efflux. On the contrary, dantrolene did not affect the calcium permeability of the sarcoplasmic reticulum. However, it inhibited the halothane-induced and caffeine-induced increments of the permeability. The Hill coefficient of inhibition by dantrolene ranged from 2.3 to 3.9, suggesting that several molecules of dantrolene may interact cooperatively with one calcium release channel to inhibit the effect of halothane. These results suggest that dantrolene has a unique inhibitory action, which may be related to its efficacy in ameliorating the syndrome of malignant hyperthermia.     

Lipid peroxidation, antioxidant concentrations, and fatty acid contents of muscle tissue from malignant hyperthermia-susceptible swine.

Homogenates of semitendinosus muscle from malignant hyperthermia (MH)-susceptible pigs produced threefold more pentane than those from MH-resistant pigs, indicating enhanced free radical-mediated peroxidation of n-6 fatty acids. This did not reflect a deficiency in tissue antioxidants or antioxidant-enzymes but glutathione concentrations and glutathione peroxidase activities were increased in the tissue from MH-susceptible swine, consistent with an adaptive response to a sustained oxidant stress. A lower proportion of linoleic acid (18:2 n-6) in phospholipids and neutral lipids in muscle from MHS pigs indicated increased peroxidation or metabolism (desaturation and elongation). The increased oleic acid (18:1 n-9) in the MHS muscle indicated that desaturase activity was elevated in all lipid classes. The results are consistent with the hypothesis that enhanced free radical activity and lipid peroxidation contributes to the abnormalities in Ca2+ homeostasis and polyunsaturated fatty acid metabolism in MH.     

Oxidative stresses induced the cystine transport activity in human erythrocytes

Cystine was transported into human erythrocytes in the presence of tertiary-butyl hydroperoxide (t-BH) or 1-chloro-2,4-dinitrobenzene (CDNB). The transport rate of cystine was dependent on the extracellular concentration of t-BH or CDNB, and on the incubation time. According to Dowex-1 column chromatography, the transported cystine was incorporated into fractions of glutathione disulfide (GSSG) and glutathione-S (GSH-S) conjugate. The transport of cystine was competitively inhibited by DL-homocystine and alanine. The inhibition rates by DL-homocystine and alanine were 75% and 68%, with similar Ki values of 0.7 mM and 0.6 mM, respectively. It is suggested that cystine transport is induced for glutathione synthesis when human erythrocytes are exposed to oxidative stresses. This transport system of cystine may serve as an emergency function in human erythrocytes.     

A substitution of cysteine for arginine 614 in the ryanodine receptor is potentially causative of human malignant hyperthermia.

Malignant hyperthermia (MH) is a devastating, potentially lethal response to anesthetics that occurs in genetically predisposed individuals. The skeletal muscle ryanodine receptor (RYR1) gene has been linked to porcine and human MH. Furthermore, a Cys for Arg substitution tightly linked to, and potentially causative of, porcine MH has been identified in the ryanodine receptor. Analysis of 35 human families predisposed to malignant hyperthermia has revealed the presence, and cosegregation with phenotype, of the corresponding substitution in a single family. This substitution, by analogy to the findings in pig, may be causal for predisposition to MH in this family.     

猪STCH基因的Bi-PASA遗传标记及多态性研究

微粒体应激 70蛋白三磷酸腺苷酶 (STCH)基因属于应激 70蛋白基因伴侣家族 ,在机体免疫反应和疾病抵抗力等方面起重要作用。根据人和小鼠STCH基因的保守序列设计引物 ,PCR扩增到猪STCH基因第5外显子 4 4 5bp片段。序列测定显示 ,猪STCH基因与人和小鼠STCH基因分别具有 87 13%和 80 4 5 %的同源性。通过测定和比较中国梅山猪、欧洲约克夏猪及PIC商品猪的STCH基因序列 ,发现在猪STCH基因编码区第 5外显子 10 5 0位点上存在一个单碱基突变位点。利用双向特定等位基因PCR扩增法 (Bi PASA)建立了检测猪STCH基因变异的遗传标记 ,并用该标记分析了STCH基因在中国家猪 (梅山猪、荣昌猪和金华猪 )、欧洲家猪 (约克夏猪、大白猪 )、商品猪 (PIC合成系 )以及欧洲野猪的基因频率和多态性。本研究建立的Bi PASA遗传标记和基因变异信息 ,将为进一步分析猪STCH基因变异与经济性状的相关分析提供基础资料。     

Influence of Nutrition on Malignant Hyperthermia in Pigs

In 2 experiments malignant hyperthermia susceptible Danish Landrace pigs were fed, for 2 or 4 weeks, synthetic diets containing casein as protein source or no protein. Minerals and vitamins were supplied to both groups. The animals were anaesthetized weekly for a maximum of 20 min with a halothane-oxygen mixture. In the first experiment malignant hyperthermia was equally delayed in both groups. If malignant hyperthermia developed, the appearance was at the end of the anaesthetic period. In the second experiment a deeper anaesthesia was employed. Malignant hyperthermia was delayed in both groups, but most markedly in the protein-deficient animals. Malignant hyperthermia developed faster after return to the original feed. These results provide evidence for a nutritional influence on the penetrance of malignant hyperthermia susceptibility during halothane anaesthesia in pigs.     

Hyperthermia, unlike ionizing radiation and chemical oxidative stress, does not stimulate proteolysis in erythrocytes

1. Oxidative stress by phenazine methosulfate stimulated proteolysis in erythrocytes. 2. Gamma-irradiation of erythrocytes in the range of 50-1000 Gy also resulted in the induction of proteolysis. 3. Though it has been suggested that hyperthermia imposes an oxidative stress on a cell, hyperthermic exposure of erythrocytes (30 min, 39-49 degrees C) did not stimulate proteolysis during subsequent incubation of whole cells or hemolysates. 4. Proteolytic degradation of spectrin was accelerated during incubation of membranes isolated from cells heated above 45 degrees C but this effect seems to be due rather to thermal denaturation of spectrin than to oxidative modification of cellular proteins by hyperthermia.     

Experimental porcine malignant hyperthermia: macromolecular characterization of muscle plasma membranes

The purpose of this study was to examine muscle plasmalemma which is implicated as the site responsible for the appearance of malignant hyperthermia in human and susceptible strains of animals. In pigs with malignant hyperthermia (MH) the activity of Na+/K+, Mg2+-ATPase, p-nitrophenylphosphatase and Mg2+-ATPase fell significantly during anaesthesia. In the control group the contrary occurred. In both the groups tested there was a marginal rise in the levels of sialic acid. The levels of cholesterol and lysoderivatives were abnormal before the provoking agents were administered but they changed significantly after onset of the MH syndrome. Anaesthesia reduced the phospholipids level in both tested animal groups. Before and after the provoking agents an impoverishment in the polypeptide pattern in the range between 80,000 and 30,000 daltons of mol. wt. in MH susceptible animals occurred. It is postulated that in MH the macromolecular disorganization of the muscle plasma membranes means that defence mechanisms maintaining cell gradients do not work in the presence of provoking agents.     

Voltage-dependent calcium release in human malignant hyperthermia muscle fibers.

Malignant hyperthermia (MH) results from a defect of calcium release control in skeletal muscle that is often caused by point mutations in the ryanodine receptor gene (RYR1). In malignant hyperthermia-susceptible (MHS) muscle, calcium release responds more sensitively to drugs such as halothane and caffeine. In addition, experiments on the porcine homolog of malignant hyperthermia (mutation Arg615Cys in RYR1) indicated a higher sensitivity to membrane depolarization. Here, we investigated depolarization-dependent calcium release under voltage clamp conditions in human MHS muscle. Segments of muscle fibers dissected from biopsies of the vastus lateralis muscle of MHN (malignant hyperthermia negative) and MHS subjects were voltage-clamped in a double vaseline gap system. Free calcium was determined with the fluorescent indicator fura-2 and converted to an estimate of the rate of SR calcium release. Both MHN and MHS fibers showed an initial peak of the release rate, a subsequent decline, and rapid turn-off after repolarization. Neither the kinetics nor the voltage dependence of calcium release showed significant deviations from controls, but the average maximal peak rate of release was about threefold larger in MHS fibers.     

Fatty acids markedly lower the threshold for halothane-induced calcium release from the terminal cisternae in human and porcine normal and malignant hyperthermia susceptible skeletal muscle

Malignant hyperthermia is caused by an abnormal increase in Ca2+ levels in skeletal muscle in response to anesthetics, including halothane. Since fatty acid production is elevated in skeletal muscle from individuals with malignant hyperthermia, the effects of fatty acids on the threshold of halothane-induced Ca2+ release were examined. In the absence of fatty acids halothane caused Ca2+ release from porcine and human heavy sarcoplasmic reticulum fractions, but only at concentrations above the clinically relevant range. Oleic acid (20 microM), an unsaturated fatty acid, reduced the threshold at which halothane induced Ca2+ release to concentrations used for anesthesia. Stearic acid, a saturated fatty acid had considerably less effect on the threshold of halothane action. The greater sensitivity of malignant hyperthermia muscle to halothane can be explained by elevated fatty acid production.     

Identification of glucose and nucleoside transport proteins in neonatal pig erythrocytes using monoclonal antibodies against band 4.5 polypeptides of adult human and pig erythrocytes

Cytochalasin B and nitrobenzylthioinosine (NBMPR), which inhibit membrane transport of glucose and nucleosides, respectively, have served as photoaffinity ligands that become covalently linked at inhibitor binding sites on transporter-associated proteins. Thus, when membranes from erythrocytes of neonatal pigs with site-bound [3H]cytochalasin B or [3H]NBMPR were irradiated with uv light, two labeled membrane polypeptides (peak Mr values: 55,000 and 64,000, respectively) were identified. Treatment of the photolabeled membranes with endoglycosidase F increased the mobility of [3H]cytochalasin B- and [3H]NBMPR-labeled material (peak Mr values: 44,000 and 57,000, respectively) and limited digestion with trypsin yielded different polypeptide fragments (Mr values: 18,000-23,000 and 43,000, respectively). Identification of the photolabeled polypeptides as transporter components was established using monoclonal antibodies (MAbs) raised against partially purified preparations of band 4.5 from erythrocytes of adult pigs and humans. MAbs 65D4 and 64C7 (anti-human band 4.5), raised in this study, reacted with [3H]cytochalasin B-labeled material from membranes of human erythrocytes and bound to permeabilized erythrocytes but not to intact cells. MAb 65D4 also bound to erythrocytes of mice and neonatal pigs and to a variety of cultured cells (mouse, human, rat), including AE1 mouse lymphoma cells, which lack an NBMPR-sensitive nucleoside transporter. Also employed was MAb 11C4 (anti-pig band 4.5), which recognizes the NBMPR-binding protein of erythrocyte membranes from adult pigs. When membrane proteins from neonatal and adult pigs were subjected to electrophoretic analysis and blots were probed with different MAbs, MAb 65D4 (anti-human band 4.5) bound to material that comigrated with [3H]cytochalasin B-labeled polypeptides (band 4.5) from neonatal, but not adult, pig erythrocytes, whereas MAb 11C4 (anti-pig band 4.5) bound to material that comigrated with [3H]NBMPR-labeled band 4.5 polypeptides of erythrocytes from both neonatal and adult pigs. These results, which indicate structural differences in the cytochalasin B- and NBMPR-binding proteins of pig erythrocytes, establish the presence of both proteins in erythrocytes of neonatal pigs and suggest that only the NBMPR-binding protein is present in erythrocytes of adult pigs.     

Whole body hyperthermia of rats decreases insulin binding to erythrocytes

125I-insulin binding to rat erythrocytes was studied to investigate the effect of whole body hyperthermia on the insulin receptor. Heat treatment of rats at 42 degrees C for 15 min caused a significant decrease (48.7% of control) in 125I-insulin binding to rat erythrocytes. Scatchard analysis showed that the decreased binding resulted from a decrease in the number of the insulin receptors rather than from a decrease in receptor affinity. The decreased receptor number for insulin showed no evidence of recovery, 2 h and 8 h after the hyperthermia. Plasma insulin levels remained lower than the control, up to 8 h after the hyperthermia, whereas plasma glucose, which decreased immediately after the hyperthermia, increased higher than the control, 8 h after the hyperthermia. The low plasma insulin level and decreased number of insulin receptor are believed to be possible factors for the elevation of plasma glucose.