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Background: The function or expression of subtypes of the sodium ion (Na+) channel is altered in biopsies or cultures of skeletal muscle from many persons who are susceptible to malignant hyperthermia (MH). ATX II, a specific Na+ channel toxin from a sea anemone, causes delayed inactivation of the channel similar to that seen in cell cultures of MH muscle. ATX II was added to skeletal muscle to determine whether altered Na+ channel function could increase the sensitivity of normal skeletal muscle to agents (halothane, caffeine, ryanodine) to which MH muscle is hypersensitive., Methods: Studies were performed of fiber bundles from the vastus lateralis muscle of persons who were deemed not MH susceptible (MH-) or MH susceptible (MH+) according to the MH diagnostic test and of strips of diaphragm muscle from rats. Preparations in a tissue bath containing Krebs solution were connected to a force transducer. ATX II was introduced 5 min before halothane, caffeine, or ryanodine., Results: ATX II increased the magnitude of contracture to halothane in preparations from most MH-, but not MH+, human participants. After ATX II treatment, preparations from 9 of 24 MH- participants generated contractures to halothane, 3%, that were of the same magnitude as those from MH+ participants. Preparations from four of six ATX II-treated healthy participants also gave responses of the same magnitude as those of MH-susceptible participants to a graded halothane challenge (0.5-3%). The contractures to bolus doses of halothane in specimens from male participants were more than three times larger than the contractures in specimens from female participants. In rat muscle, ATX II increased the magnitude of contracture to caffeine (2 mM) and decreased the time to produce a 1-g contracture to ryanodine (1 microM)., Conclusions: ATX II, which causes delayed inactivation of the Na+ channel in cell cultures similar to that reported in cultures of MH+ skeletal muscle, increased the sensitivity of normal muscle to three agents to which MH+ muscle is hypersensitive. The increased sensitivity to halothane, 3%, occurred in most (79%), but not all, MH- participants, and this effect was most evident in male participants. Therefore, abnormal function of the Na+ channel, even if it is a secondary event in MH, may contribute to a positive contracture test result for MH.

Biopsies of human skeletal muscle were analyzed by an in vitro contracture test (IVCT) for responsiveness to a halothane challenge: noncontracting (nonresponsive; IVCT-) and contracting (IVCT+). A muscle biopsy that is IVCT+ indicates potential malignant hyperthermia (MH) susceptibility. Primary cultures were grown from portions of the skeletal muscle biopsies, and voltage-activated currents were measured by whole cell recording in the presence or absence of 2-5 microM intracellular arachidonic or oleic acids. In untreated IVCT- cells, Na+ currents were predominantly tetrodotoxin (TTX) insensitive, indicating that most of the current was carried through the embryonic SkM2 isoform of the Na+ channel. Inclusion of fatty acids in the recording pipette of IVCT- cells produced an increase in voltage-activated Na+ currents during 20 min of recording. Approximately 70% of currents in fatty acid-treated cells were TTX sensitive, indicating activation of the adult SkM1 isoform of the Na+ channel. In contrast to IVCT- cells, IVCT+ cells expressed Na+ currents that were predominantly TTX sensitive even in the absence of added fatty acid, thus showing a relatively large baseline functional expression of SkM1 channels. Addition of fatty acids to the recording pipette produced little further change in the magnitude or TTX sensitivity of the whole cell currents in IVCT+ cells, suggesting altered functional regulation of Na+ channels in MH muscle.

The temperature dependence of oleic acid-enhanced halothane- induced Ca2+ release and the existence of a reduced threshold of Ca(2+)-induced Ca2+ release were examined to determine their roles in human malignant hyperthermia. Halothane (8-11 mM) induced Ca2+ release from terminal cisternae-containing preparations from skeletal muscle. Oleic acid (15 microM) markedly reduced the threshold of halothane-induced Ca2+ release to < 0.5 mM at 37 degrees C, but not at 25 degrees C, consistent with the temperature dependence of halothane action in intact muscle. Two states of the threshold of Ca(2+)-induced Ca2+ release were unrelated to malignant hyperthermia in humans, in contrast to pigs. Excess fatty acid production may be an important modulator of halothane action in malignant hyperthermia.

Ca(2+)- and halothane-induced Ca(2+) release and Na+ currents are modulated by free fatty acids (FAs). FA modulation of ion currents may have important implications for general muscle physiology and skeletal muscle disorders, including malignant hyperthermia (MH).

Different in vitro halothane testing procedures have been used in the European malignant hyperthermia (MH) Group Protocol (EMHGP) and the North American MH Group Protocol (NAMHGP), whereas the caffeine-testing protocols are very similar. The present study compares the two halothane-testing protocols in ten MH susceptible swine and in four control swine. Halothane contracture testing was conducted in vitro 12-52 days following the barnyard challenge that established the MH susceptibility of the swine. There was one false positive and one false negative halothane test by the EMHGP. The MH-equivocal category in the EMHGP, which is treated clinically as MH-susceptible, affords a margin of safety in such cases. In contrast, there were no false halothane tests by the NAMHGP. While some skeletal muscle strips from MH pigs were normal by both protocols (NAMHGP 30%; EMHGP 10%), the outcome of halothane testing by the NAMHGP was unaffected. The response to halothane 3% is reduced if preceded by the EMHGP, suggesting that simply adding halothane 3% to the end of the EMHGP does not permit a direct quantitative comparison to the NAMHGP. However, the diagnostic outcomes of the two approaches are similar.

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.

Anaesthetic-induced increases in cytoplasmic free Ca2+ have been reported to be greater in lymphocytes from malignant hyperthermia (MH) susceptible patients than in those from controls, suggesting that this may be the basis for a less invasive test for MH susceptibility. In the present study the cytoplasmic Ca2+ concentrations of lymphocytes were monitored with indo-1 in 14 control subjects (nine fasted and five nonfasted) and five fasted MH susceptible and three fasted nonsusceptible patients, diagnosed by the halothane and caffeine contracture tests. No relationship was observed between MH susceptibility and Ca2+ concentrations in lymphocytes in the absence or presence of halothane. There was, however, a relationship in control subjects between fasting and the response of lymphocytes to halothane, with the halothane-induced Ca2+ increase being considerably larger in nonfasted subjects.

Phospholipase A2 (PLA2) activity enhances snake venom cardiotoxin (CTX)-induced and general anesthetic (halothane)-induced hemolysis of red blood cells. In the case of halothane-induced hemolysis, this effect appears to be related primarily to free fatty acids. In the present study, the interaction between CTXs and halothane and the effects of different free fatty acids on cardiotoxin and halothane-induced hemolysis were examined. The hemolytic actions of halothane and a CTX from Naja naja kaouthia venom were examined in erythrocytes with different phospholipid and free fatty acid composition from five species. The extent of hemolysis by CTX or halothane was dependent upon the species examined and appeared to be inversely related to the amount of saturated free fatty acid in the membrane. The order of susceptibility of red blood cells from five species to hemolysis was similar for halothane- and N. n. kaouthia CTX-induced hemolysis, but very different for osmotic fragility. The slope of the hemolysis dose-response curve was considerably steeper for halothane than for CTX. Hemolysis due to N. n. kaouthia CTX was greatly increased by halothane in erythrocytes from humans and horses and to a lesser extent in erythrocytes from sheep, goats and cows. Hemolysis induced by halothane and the N. n. kaouthia or Naja naja atra CTXs was enhanced by unsaturated fatty acids. In contrast, hemolysis induced by halothane was decreased and that caused by the two CTXs was relatively unaffected by saturated fatty acids. Halothane and CTXs differ in their exact mechanisms, but appear to act upon similar fatty acid-sensitive processes.

Caffeine and halothane contracture testing is widely used to detect malignant hyperthermia (MH) susceptibility. The accuracy and reliability of the 3% halothane test and the incremental caffeine test, as recommended by the North American MH Group, were assessed in 11 swine (five MHS, six control). Nine swine were tested twice, 4-6 weeks apart. Accuracy of the in vitro diagnosis was also assessed by in vivo anesthetic challenge. Of all muscle bundles from MH-susceptible swine, 65% reacted positively to 3% halothane and 70% to 2 mM caffeine. Only 35% had a positive caffeine-specific concentration, and 25% developed an increase in baseline tension greater than or equal to 7% at 2 mM caffeine. However, when only the most positive response to 3% halothane or to 2 mM caffeine was used (a minimum of three fresh muscle strips is recommended), these two tests were highly sensitive and specific. In control swine one of 30 muscle bundles reacted positively to 3% halothane. A positive caffeine-specific concentration developed in one of 25 control muscle bundles exposed to caffeine. The variability in the results of these tests mandated that at least three muscle bundles be used for each test. Nonviable muscle bundles could not be relied upon to provide accurate results. In this porcine model, MH susceptibility could be detected by performing the Caffeine Halothane Contracture Test (CHCT) according to the guidelines of the North American MH Group. However, only the 3% halothane test and the response to 2 mM caffeine produced adequate diagnostic results in this breed of swine.

Previous studies in cat, rat, and swine have implicated fiber type as influencing the halothane and caffeine contracture test used to diagnose malignant hyperthermia (MH). The authors performed fiber type analysis using myosin ATPase stains on 31 fascicles of skeletal muscle from nine patients following contracture testing. There was no significant difference in fiber type composition between fascicles from MH negative (n = 5) and MH positive (n = 4) patients. Furthermore, examining each of the 31 fascicles, the authors found no correlation (P greater than .05) of contracture magnitude with percentage of either Type I or Type II fibers using the Pearson Product-Moment correlation calculation. The authors conclude that fiber type composition does not influence contracture test results in human biopsies.

The association of malignant hyperthermia (MH) with neuromuscular disorders has been recognized since 1970. These disorders include central core disease, Duchenne muscular dystrophy, myotonia congenita, myotonic dystrophy, nonspecific myopathies, and King-Denborough syndrome. In order to assess the anesthetic risk of MH in the neuromuscular population, we performed halothane and caffeine contracture testing for MH susceptibility on biopsied muscle removed from 25 consecutive neuromuscular patients during diagnostic evaluation. Positive contracture tests were found in 7 of 18 patients with myopathic disorders and 3 of 7 patients with neurogenic disorders. Two of our patients had anesthetic events suggesting MH. These findings suggest that myopathic and neuropathic disorders share pathogenic mechanisms with MH, resulting in positive contracture tests and possibly leading to clinical events during anesthesia. Although there is controversy regarding the interpretation of a positive contracture test, contracture testing remains the most widely accepted test for MH susceptibility. Thus, a variety of neuromuscular disorders may be associated with MH susceptibility, and caution should be exercised during anesthesia in this group of patients.

This study examines in vitro the contractures induced by halothane and succinylcholine in skeletal muscle taken as biopsy specimens from 42 patients referred to the authors' laboratory for diagnosis of malignant hyperthermia (MH) susceptibility. In addition, possible differences between the response of preparations from these same patients with and without a history of masseter muscle rigidity following succinylcholine (SCh) administration were determined to investigate the in vitro relationship of masseter muscle rigidity to MH. Halothane 3%-induced contractures in preparations from MH susceptibles were similar, whether the group had a history of masseter muscle rigidity (1.15 +/- 0.18 g; n = 10) or not (1.02 +/- 0.21 g; n = 14). Halothane did not induce significant contractures in those diagnosed as normals. Succinylcholine alone did not elicit contractures from preparations derived from MH susceptibles or nonsusceptibles. Succinylcholine induced significant contractures in all preparations preexposed to halothane. Preparations from MH-negative patients with a history of masseter muscle rigidity were rendered sensitive to halothane (contractures of 1.17 +/- 0.30 g; n = 4) when SCh was present. In contrast, halothane added in the presence of SCh did not induce contractures to the same extent in preparations from MH-negative patients without a history of masseter muscle rigidity. This is the first reported in vitro method by which to examine the clinically troublesome interaction between SCh and halothane. This approach also may prove to be important in further investigations of the relationship between masseter muscle rigidity and MH.

The effects on erythrocyte fragility of two general anaesthetic agents (halothane and ethanol) and succinylcholine were examined using preparations from 13 normal and four malignant hyperthermia susceptible patients. Erythrocyte fragility was determined by the degree of haemolysis induced in solutions of decreasing osmolarity of NaCl. Halothane caused haemolysis of erythrocytes in an isoosmolar solution, being more potent at 42 degrees C than at 32 degrees C. Haemolysis produced by an hypoosmolar medium or halothane was potentiated by exogenously added phospholipase A2. Ethanol did not markedly alter the haemolysis of erythrocytes under conditions of decreasing osmolarity. Succinylcholine 10 mM did not significantly alter the susceptibility of erythrocytes to lysis by halothane. No differences in erythrocyte fragility were observed between preparations from normal and malignant hyperthermia susceptible patients under any of the conditions tested, despite the inclusion of malignant hyperthermia triggering agents in some instances. Although sampling a larger patient population might reveal slight differences between the groups, erythrocyte fragility tests do not appear to be useful in differentiating malignant hyperthermia susceptible from normal patients.

The role of lipolysis in halothane-induced contractures of human skeletal muscle was examined using three inhibitors of phospholipase A2 (PLA2) activity (quinacrine, spermine and indomethacin) and exogenously administered PLA2. In addition, we examined the effects of the same PLA2 inhibitors on contractures induced by halothane in combination with suxamethonium. The studies were conducted on directly stimulated muscle strips isolated from biopsies from patients diagnosed as MH susceptible, or not susceptible, by the halothane contracture test. The liberation of fatty acids from skeletal muscle homogenates was examined to determine whether PLA2 activity might be increased in MH susceptible patients. The three PLA2 inhibitors antagonized halothane-induced contractures of muscle from MH susceptible patients as well as induction of contractures by suxamethonium and halothane. Pre-exposing preparations that were previously unresponsive to halothane to bee venom PLA2 caused the muscle strips to respond with contractures upon subsequent challenge with halothane, but not with suxamethonium. Free fatty acid production during an in vitro incubation of skeletal muscle homogenates was greater in preparations from MH susceptible patients than in those from patients who were not susceptible. These results suggest that excess liberation of fatty acids may be involved in halothane-induced contractures of muscle from MH susceptible individuals, in the synergism observed between halothane and suxamethonium, and in human MH.

The rat diaphragm was used as an in vitro model for studies of contractures synergistically-induced by halothane and suxamethonium. The effects of three agents reported to inhibit phospholipase A2 activity (quinacrine, spermine and indomethacin), tubocurarine and dantrolene were examined on these contractures. Contractures induced by 1% halothane (0.26 +/- 0.02 g) (mean +/- SEM) were increased (0.60 +/- 0.04 g) if suxamethonium 50 mmol litre-1 was also in the bathing medium. Suxamethonium-induced contractures (0.22 +/- 0.03 g) were also enhanced when halothane was present (0.51 +/- 0.03 g). Spermine, indomethacin and dantrolene antagonized both halothane- and suxamethonium-induced contractures. Quinacrine potentiated contractures induced by either halothane or suxamethonium. Contractures induced by suxamethonium were antagonized by tubocurarine; however, contractures induced by halothane were not antagonized by tubocurarine. These results suggest that free fatty acids may be involved in contractures induced synergistically by halothane and suxamethonium. Different mechanisms are involved in the induction of contractures by suxamethonium than by halothane.

The effects of two structurally similar butyrophenones (droperidol and haloperidol) and ketamine were evaluated in an in vitro system to determine their potential for eliciting or exacerbating an episode of malignant hyperthermia. Muscle strips from patients referred for diagnostic testing for malignant hyperthermia and muscle strips from the rat diaphragm were exposed to droperidol, haloperidol, or ketamine prior to challenge with halothane, succinylcholine or caffeine. If any agent augmented the contracture response to the malignant hyperthermia triggering or diagnostic agents, then the agent was considered unsafe for use in malignant hyperthermia susceptible patients. Droperidol 10 mumol/l and ketamine 100 mumol/l did not induce contractures in human or rat skeletal muscle when added alone, nor did they augment halothane, succinylcholine or caffeine contractures. These agents appear to be safe for use in patients susceptible to malignant hyperthermia. In contrast, haloperidol 10 mumol/l augmented the response to succinylcholine about 1.5-fold and may be contraindicated in MH susceptibles.

Dantrolene is an effective antagonist of anesthesia-induced malignant hyperthermia due to a poorly understood action on skeletal muscle. The present study examines whether the red blood cell can be used as a model to investigate the mechanism of dantrolene action. Halothane (4.7 mM) caused 9% hemolysis of red blood cells. Phospholipase A2 (1 microM) alone caused less than 2% hemolysis, despite high levels (54%) of phosphatidylcholine hydrolysis. Incubation of red blood cells with halothane and phospholipase A2 caused 72% hemolysis. Halothane addition caused 100% hydrolysis of all diacylphosphoglycerides by phospholipase A2, suggesting a mutual potentiation. The major products of phospholipase A2 activity, arachidonic acid and lysophosphatidylcholine, when exogenously added, also greatly increased hemolysis induced by halothane, with arachidonic acid most closely resembling the synergism observed with phospholipase A2. Dantrolene (10 microM) and mepacrine (10 microM) significantly antagonized hemolysis induced by halothane and phospholipase A2 or halothane and exogenously added arachidonic acid and lysophosphatidylcholine. Dantrolene and mepacrine did not antagonize phospholipid hydrolysis or free fatty acid levels. Dantrolene and mepacrine antagonized the synergism between halothane and phospholipase A2 most likely by reducing the lytic action of halothane in the presence of arachidonic acid. The red blood cell is a useful model for studying the antagonism of halothane and phospholipase A2 toxicity by dantrolene and mepacrine.