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Biomedical subjects

E M Gallant

Publications and source records attributed to E M Gallant.

At least 37 records · Page 2Linked to original sources
PubMed1993

Perchlorate potentiation of excitation-contraction coupling in mammalian skeletal muscles.

The action of perchlorate (ClO4-), an agonist of the voltage sensor in excitation-contraction (EC) coupling, has been examined using bundles of intact muscle cells, isolated membrane vesicles [sarcoplasmic reticulum (SR) and transverse tubule (TT)], and cultured myotubes. The effect of ClO4- on mechanical parameters was investigated in isolated murine limb muscles. The presence of ClO4- (5 or 10 mM) greatly increased twitch tension ( > 250%), slightly enhanced tetanic tension, and increased K contracture tension. K contracture thresholds of extensor digitorum longus (EDL, 40 mM K+) and soleus (30 mM K+) muscles were not altered by ClO4-. However, in whole cell patch clamp studies of mouse myotubes, contractile activation was shifted by approximately -10 mV by 10 mM ClO4-. To further define the site of alteration of EC coupling by ClO4-, studies were conducted with isolated porcine SR and TT vesicles and with cultured mouse myotubes. The rate constant of Ca-induced 45Ca release from SR vesicles was significantly increased by ClO4-. However, neither the affinity nor level of [3H]PN200-110 binding to TT vesicles was significantly affected by ClO4- concentrations that increased twitch tension. Furthermore, slow plasmalemmal Ca currents of myotubes recorded in the whole cell patch-clamp mode were enhanced by 10 mM ClO4-, and the current-voltage relationship was shifted approximately -7mV. Thus, in enhancing EC coupling in mammalian muscle, ClO4- may act at multiple sites including the SR Ca release channel and the TT Ca channel-voltage sensor.

Animals↗
PubMed1992

Excitation-contraction coupling in pigs heterozygous for malignant hyperthermia.

A defect in the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel of malignant hyperthermia-susceptible (MHS) pigs greatly enhances SR calcium release in pigs homozygous for the malignant hyperthermia (MH) gene. In pigs heterozygous at this locus, rates of calcium release from isolated SR stimulated by Ca2+, ATP, or caffeine are intermediate to those of both MHS and normal SR [Mickelson et al. Am. J. Physiol. 257 (Cell Physiol. 26): C787-C794, 1989]. In this study bundles of intact muscle cells dissected from pigs of various genotypes were used to examine the effects of the MH gene on contractile responses to caffeine (direct stimulation of the SR) or to surface membrane (sarcolemma) depolarization (i.e., stimulation by way of the steps in excitation-contraction coupling). The caffeine threshold for contractures in the heterozygous muscles (5 mM) was intermediate to both types of homozygous muscles (2 mM for MHS and 10 mM for normal) as is the case with direct stimulation of calcium release from SR vesicles [Mickelson et al. Am. J. Physiol. 257 (Cell Physiol. 26): C787-C794, 1989]. Sarcolemmal depolarization was elicited by electrical stimuli or elevated extracellular potassium. Control twitch tension for MHS and heterozygous muscles did not differ and was significantly greater in both than in homozygous normal muscles. Potassium-induced contractures were significantly larger in MHS and heterozygous than in normal muscles. Thus, in heterozygous muscles, force production via sarcolemmal depolarization (twitches and potassium contractures) was enhanced as much as in homozygous MHS muscles. This could be the result of feedback from abnormal SR calcium channels producing altered (enhanced) transverse tubule to SR signal transduction.

Animals↗
PubMed1990

Halothane sensitivity of young pigs in vivo and in vitro.

Piglets less than 8 wk of age that are known by genotype to be malignant hyperthermia-susceptible (MHS) do not usually develop characteristic hyperthermia and limb muscle rigidity in response to a brief halothane exposure (5 min of 3%). To determine whether a malignant hyperthermia (MH) episode could nevertheless be provoked by a more rigorous challenge, both genetically MHS (Pietrain) and normal (Yorkshire) 5-wk-old piglets were exposed to a combined halothane-succinylcholine challenge. Only two of eight MHS piglets developed limb rigidity; however, all MHS piglets (and no normal piglets) developed clinical signs of MH episode initiation during the 30-min challenge. Temperatures rose from 37.4 to 38.6 degrees C in MHS piglets while falling slightly in normal piglets. In MHS piglets, venous pH fell from 7.46 +/- 0.02 to 6.88 +/- 0.07, PVCO2 rose from 36 +/- 2 to 126 +/- 17 mmHg, and plasma concentration of K+ rose from 4.0 +/- 0.1 to 7.1 +/- 0.6 mM, whereas all values remained stable in normal piglets. Muscles removed from the same piglets before the halothane-succinylcholine challenge were exposed to halothane in vitro. The muscles from genetically MHS piglets responded to halothane with characteristic depression of tetanic tension and prolonged tetanus relaxation time but did not develop halothane-induced contractures. We conclude that, in the absence of either halothane-induced limb rigidity or in vitro contractures, these young animals were still susceptible to potentially fatal MH episodes on exposure to appropriate triggering agents. The MH defect is apparently partially masked in piglets and expressed fully only in older pigs.

Animals↗
PubMed1989

Porcine malignant hyperthermia: halothane effects on force generation in skeletal muscles.

Halothane-induced malignant hyperthermia (MH) is thought to result from a defect in the regulation of cytosolic calcium concentration in MH-susceptible (MHS) skeletal muscle. Such a defect might be expected to alter the time course of contractile responses. To test this hypothesis, isolated intact cell bundles from external intercostal and common digital extensor muscles of normal and MHS pigs were stimulated electrically to elicit twitch and tetanic tension in the presence and absence of halothane (2.5%). Time intervals measured for both twitches and tetani were (1) the latent period between the stimulus and tension increase, (2) the time to peak tension, and (3) the half-relaxation time. In contrast to previous reports, halothane had no effect on any measured time course parameter of twitches of either type of normal or MHS muscle, nor did the twitches of MHS and normal muscles differ in any parameter in the absence of halothane. However, the tetanic tension relaxation in both types of MHS muscle was markedly slowed by halothane, whereas in normal muscles there was little change. The slower rate of relaxation induced by halothane in MHS muscles suggests that halothane, either directly or indirectly, enhances the release or slows the removal of calcium in intact MHS muscles following maximal activation. This slowed tetanus relaxation could be of use in identification of MHS individuals.

Animals↗
PubMed1989

Malignant hyperthermia: effects of halothane on the surface membrane.

We studied the electrical properties of intact muscle fibers from normal and malignant hyperthermia-susceptible (MHS) pigs. Resting membrane potentials, action potentials, and current-voltage relationships were measured with and without the presence of halothane. There were no changes in the resting potentials or the specific membrane conductances at any concentration of halothane in either the normal or MHS fibers. The current-voltage relationships of normal and MHS fibers did not differ. Contractures were observed in MHS muscle when the concentration of halothane was greater than or equal to 0.8%. These halothane-induced contractures were not associated with depolarization of the surface membrane. Contractures were not observed in normal muscle even at concentrations of 6.0% halothane. In contrast, halothane altered the shape of the action potentials of both MHS and normal fibers. However, these changes were significantly greater in MHS fibers, occurred at much lower concentrations, and were partially prevented by preincubation in 10 microM dantrolene.

Action Potentials↗
PubMed1989

Skeletal muscle excitation-contraction coupling. I. Transverse tubule control of peeled fiber Ca2+-induced Ca2+ release in normal and malignant hyperthermic muscles.

Porcine skeletal muscle fibers were studied to determine if the defect in malignant hyperthermia involves transverse tubule (TT) to sarcoplasmic reticulum (SR) communication. Peeled (mechanically skinned) skeletal muscle fibers from normal and malignant hyperthermia susceptible (MHS) pigs were stimulated with Cl- to ionically depolarize transverse tubules and thereby trigger Ca2+ release from SR. Caffeine was used to directly stimulate the Ca2+-induced Ca2+ release mechanism (CaIR) of the sarcoplasmic reticulum. Calcium released from internal fiber stores was monitored as Ca2+-activated isomeric force generation in the form of tension transients. Cl- -induced tension transients result from a primary component of Ca2+ release which triggers a secondary CaIR component; CaIR and caffeine contractures were eliminated by procaine. The primary component of Cl- -induced SR Ca2+ release was indistinguishable for MHS and normal skeletal peeled fibers at all TT resting and Cl- stimulation conditions. Only the magnitude of the secondary CaIR component was significantly larger in MHS fibers. The [Ca2+] threshold for secondary CaIR was lowered by resting TT depolarization in both normal and MHS fibers. Conditions for resting TT hyperpolarization selectively reduced the magnitude of the secondary CaIR component of MHS fibers, making them indistinguishable from normal.

Animals↗
PubMed1989

Skeletal muscle excitation-contraction coupling. II. Plasmalemma voltage control of intact bundle contractile properties in normal and malignant hyperthermic muscles.

Bundles of cells, intact from tendon to tendon, were dissected from muscles of normal and malignant hyperthermia susceptible (MHS) pigs. Intact bundles were stimulated either (1) electrically, to elicit twitches and tetani, or (2) ionically with elevated extracellular K+ (K+0), to elicit K-contractures. Maximal tetanic force was the same for MHS and normal intact bundles. In MHS muscles, when responses were elicited from control resting plasmalemma polarization (4 mM K+0), twitches and K-contractures were significantly larger and the K-contracture activation curve was shifted towards lower [K+]0 with respect to normal bundles. Resting hyperpolarization (2 mM K+0) selectively reduced MHS twitch force to normal and K-contracture force toward normal. For K-contracture and twitches, there was a range of K+0 concentrations (7-10 mM), representing resting depolarization, which enhanced subsequent twitch and K-contracture magnitude in both MHS and normal intact bundles as compared to responses elicited from control (4 mM K+0). These results are consistent with the hypothesis that resting plasmalemma voltage sets the gain of sarcoplasmic reticulum calcium release in MHS and normal intact bundles independent of type of stimulation and suggest that a defect in this mechanism may be responsible for the enhanced twitches and K-contractures of MHS muscles.

Animals↗
PubMed1989

Halothane-sensitivity gene and muscle contractile properties in malignant hyperthermia.

Malignant hyperthermia (MH) results from the presence of the halothane-sensitivity gene and is characterized by abnormalities in muscle function. Populations of genetically defined pigs were used to determine the in vivo and in vitro expression of this gene in both the homozygous and the heterozygous condition. On exposure to halothane, isolated muscle bundles from the homozygous halothane-sensitive pigs exhibited decreased tetanus tension and increased tetanus half-relaxation time and contracture and were clearly distinguished from homozygous normal muscles. The heterozygous and homozygous normal muscles were similar in contractile responses except for the occurrence of halothane-induced contractures in the heterozygotes. The heterozygous halothane-negative pigs did not exhibit the characteristic signs of an MH episode in response to halothane succinylcholine, although some metabolic responses were significantly altered (e.g., increased venous partial pressure of CO2 and arterial and venous K+ concentration). Thus the heterozygous pigs were not MH susceptible but did represent a phenotype distinct from the homozygous normal pigs both in vitro and in vivo. These data provide the first convincing evidence for the expression of the halothane-sensitivity gene in heterozygotes.

Animals↗
PubMed1989

Effects of the halothane-sensitivity gene on sarcoplasmic reticulum function.

Pigs heterozygous for the halothane-sensitivity gene exhibit a distinct phenotype with regard to both in vivo and in vitro muscle responses to halothane (E. M. Gallant, J. R. Mickelson, B. D. Roggow, S. K. Donaldson, C. F. Louis, and W. E. Rempel. Am. J. Physiol. 257 (Cell Physiol. 26): C781-C786, 1989). In this paper heavy sarcoplasmic reticulum (SR) preparations were isolated from the muscles of pigs of all three genotypes. The rate of calcium release from SR of pigs homozygous for the halothane-sensitivity gene was approximately twice that of SR from pigs homozygous for the normal allele. Furthermore, in the presence of 6 microM Ca2+, the binding of [3H]ryanodine to SR isolated from the homozygous halothane-sensitive pigs was of a higher affinity than was the binding to SR isolated from the homozygous normal pigs (Kd = 70-90 vs. 265 nM, respectively). The SR from pigs heterozygous for the halothane-sensitivity gene, however, demonstrated intermediate values for the rate of calcium release and the affinity for [3H]ryanodine (Kd = 192 nM). Thus the alterations in heavy SR calcium release and [3H]ryanodine binding in the pigs containing one copy of the halothane-sensitivity gene demonstrate a distinct heterozygote phenotype. These data also suggest that the protein product of this gene is closely associated with, and perhaps identical to, the SR calcium release channel-ryanodine receptor protein.

Adenosine Triphosphate↗
PubMed1988

Abnormal sarcoplasmic reticulum ryanodine receptor in malignant hyperthermia.

Previous studies have demonstrated that skeletal muscle from individuals susceptible to malignant hyperthermia (MH) has a defect associated with the mechanism of calcium release from its intracellular storage sites in the sarcoplasmic reticulum (SR). In this report we demonstrate that the [3H]ryanodine receptor of isolated MH-susceptible (MHS) porcine heavy SR exhibits an altered Ca2+ dependence of [3H]ryanodine binding at the low affinity Ca2+ site as well as a lower Kd for ryanodine (92 versus 265 nM) when compared to normal porcine SR. The Bmax of the normal and MHS [3H] ryanodine receptor (9.3-12.6 pmol/mg) was not significantly different, and analysis of MHS and normal SR proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis did not reveal a significant difference in the intensity of Coomassie Blue staining of the spanning protein/ryanodine receptor region of the gels (Mr greater than 300,000). We also find that MHS porcine muscle intact fiber bundles exhibit a 5-10-fold lower ryanodine threshold for twitch and tetanus inhibition, and contracture onset when compared to normal muscle. Since the SR ryanodine receptor is a calcium release channel as well as a component intimately involved in transverse tubule-SR communication, abnormalities in the skeletal muscle ryanodine receptor may be responsible for the abnormal SR calcium release and contractile properties demonstrated by MHS muscle.

Alkaloids↗
PubMed1987

Skeletal muscle sarcolemma in malignant hyperthermia: evidence for a defect in calcium regulation.

Sarcolemmal properties implicated in the skeletal muscle disorder, malignant hyperthermia (MH), were examined using sarcolemma-membrane vesicles isolated from normal and MH-susceptible (MHS) porcine skeletal muscle. MHS and normal sarcolemma did not differ in the distribution of the major proteins, cholesterol or phospholipid content, vesicle size and sidedness, (Na+ + K+)-ATPase activity, ouabain binding, or adenylate cyclase activity (total and isoproterenol sensitivity). The regulation of the initial rates of MHS and normal sarcolemmal ATP-dependent calcium transport (calcium uptake after 1 min) by Ca2+ (K1/2 = 0.64-0.81 microM), calmodulin, and cAMP-dependent protein kinase were similar. However, when sarcolemmal calcium content was measured at either 2 or 20 min after the initiation of active calcium transport, a significant difference between MHS and normal sarcolemmal calcium uptake became apparent, with MHS sarcolemma accumulating approximately 25% less calcium than normal sarcolemma. Calcium transport by MHS and normal sarcolemma, at 2 or 20 min, had a similar calmodulin dependence (C1/2 = 150 nM), and was stimulated to a similar extent by cAMP-dependent protein kinase or calmodulin. Halothane inhibited MHS and normal sarcolemmal active calcium uptake in a similar fashion (half-maximal inhibition at 10 mM halothane), while dantrolene (30 microM) and nitrendipine (1 microM) had little effect on either MHS or normal sarcolemmal calcium transport. After 20 min of ATP-supported calcium uptake, 2 mM EGTA plus 10 microM sodium orthovanadate were added to initiate sarcolemmal calcium efflux. Following an initial rapid phase of calcium release, an extended slow phase of calcium efflux (k = 0.012 min-1) was similar for both MHS and normal sarcolemma vesicles. We conclude that although a number of sarcolemmal properties, including passive calcium permeability, are normal in MH, a small but significant defect in MHS sarcolemmal ATP-dependent calcium transport may contribute to the abnormal calcium homeostasis and altered contractile properties of MHS skeletal muscle.

Adenosine Triphosphate↗
PubMed1987

Increased skeletal muscle acetylcholinesterase activity in porcine malignant hyperthermia.

The content and distribution of acetylcholinesterase (AChE) activity in the skeletal muscle disorder malignant hyperthermia (MH) was examined. The AChE activity of sarcolemma membranes isolated from MH-susceptible (MHS) swine was increased twofold when compared with normal sarcolemma. The total AChE activity of muscle extracts was also doubled in MHS tissue; however, the relative distribution between low-salt extractable (globular forms) and high-salt extractable (asymmetric forms) AChE activities were similar in MHS and normal muscle. Our results suggest that, for as yet unexplained reasons, both the sarcolemmal and total AChE activity of skeletal muscle is increased in porcine MH.

Acetylcholinesterase↗
PubMed1987

Porcine malignant hyperthermia: false negatives in the halothane test.

Purebred Pietrain pigs presumed (on the basis of pedigree) to be homozygous for malignant hyperthermia (MH) susceptibility were subjected to a 3% halothane challenge test. A few (6%) pigs that should have been MH susceptible on the basis of parental genotype did not develop muscle rigidity in response to repeated halothane tests. Three of these animals were brought into the laboratory, and muscle biopsy specimens were obtained for in vitro analysis. Bundles of intact muscle cells dissected from biopsy specimens were electrically stimulated, and mechanical responses were monitored during exposure to halothane. In all instances, the muscle bundles from the halothane-negative (ie, not sensitive to halothane), but genetically susceptible, pigs gave in vitro responses that were similar of those of halothane-positive MH-susceptible pigs in that tetanic tension was depressed, tetanus relaxation was slowed, and small contractures were produced upon halothane exposure. Thus, the presence of a halothane-sensitive abnormality in the skeletal muscles, in and of itself, is not always sufficient for development of in vivo muscle rigidity during a brief halothane test. Furthermore, when the halothane testing of pigs is conducted by recommended techniques, false negatives still occur in a small percentage of the genetically MH-susceptible animals.

Animals↗
PubMed1986

Porcine malignant hyperthermia: cell injury enhances halothane sensitivity of biopsies.

A possible relationship between muscle cell injury or deterioration and enhanced halothane sensitivity was studied by monitoring mechanical responses of skeletal muscles from normal pigs and pigs susceptible to malignant hyperthermia (MH). Increased time postbiopsy and decreased maximum control tetanic tension both correlated significantly with enhanced sensitivity to halothane. In both normal and MH-susceptible (MHS) muscles, greater halothane sensitivity was observed in cut cell than in intact cell bundles and in low tetanic tension as compared to high tension preparations. Subsequent to halothane exposure, twitches of high tension (greater than or equal 1.75 kg . cm-2) intact bundles of both normal and MHS muscles were potentiated. Tetani of normal intact bundles were not altered, whereas those of MHS bundles were depressed after halothane exposure. Control twitch-to-tetanus ratios (twitch ratios) were higher in MHS (0.23) than in normal (0.12) intact bundles. According to discriminant analysis, the best distinction between normal and MHS muscles, either cut or intact, was obtained by comparing halothane-induced changes in tetanic tension and control twitch ratios.

Animals↗
PubMed1985

Effects of calcium antagonists on mechanical responses of mammalian skeletal muscles.

Rodent muscles were exposed to several organic calcium antagonists, and mechanical responses to direct electrical stimulation were recorded. Verapamil and D600, at 25 microM, depressed twitch and tetanus tension and caused fading of the tetanus plateau. These effects increased with frequency of stimulation, and were not reversed by doubled extracellular calcium. Depression of tension progressed to complete paralysis after 60-90 min exposure to verapamil. Bepridil and diltiazem both caused depression of tension and tetanus fade. Nifedipine caused marked, and nitrendipine caused slight, potentiation of twitch tension but did not alter tetanic tension. The magnitude of the observed effects on tension (either depression or potentiation) correlated with neither the relative calcium antagonist potencies of the drugs in other tissues nor with the ability of the drugs to cross the cell membrane. The continued decline in tension observed on prolonged exposure indicates that chronic exposure to low levels in vivo might lead to significant muscle weakness.

Animals↗
PubMed1985

Verapamil is not a therapeutic adjunct to dantrolene in porcine malignant hyperthermia.

We have investigated the hypothesis that the calcium antagonist verapamil might be useful for prevention or treatment of malignant hyperthermia (MH) in MH-susceptible (MHS) swine. MH episodes were triggered in four groups of four swine with halothane alone or combined with succinylcholine (SCh) and, with and without verapamil. MH episodes were reversed by therapy with dantrolene and NaHCO3 in all groups. Verapamil did not alter MH episodes triggered by halothane alone or combined with SCh. The dantrolene-NaHCO3 requirements for reversal of MH were greater for the groups receiving halothane-SCh, but did not differ in groups pretreated with and without verapamil. In vitro verapamil (25 microM) did not reduce responses of intact muscle fibers to halothane and, in fact, exaggerated some halothane-induced responses. High concentrations of verapamil (0.5 mM) caused contractures in MHS but not in normal muscles. Neither our in vivo nor in vitro results support the use of verapamil in the treatment of MH. Further, doses of dantrolene used to reverse these MH episodes, although admittedly small (1-2 mg/kg), did not produce myocardial depression when used in combination with verapamil.

Anesthesia, Inhalation↗
PubMed1983

Barium-treated mammalian skeletal muscle: similarities to hypokalaemic periodic paralysis.

In vivo, Ba2+ produces flaccid paralysis of mammalian skeletal muscle and lowers serum K+ concentration. An episode of hypokalaemic periodic paralysis (h.p.p.) produces similar changes. Reduced K+ permeability may be involved in the onset of h.p.p., and Ba2+ is known to block K+ channels in some types of excitable cells. I have investigated the mechanism of Ba2+ action in mammalian skeletal muscle (mouse and pig) in vitro. Ba2+ (1-10 mM) initially potentiated twitch force but subsequently depressed both twitches and tetani. These effects were enhanced by low external K+ concentration (0 mM) and diminished by doubling the external K+ (8 mM). Muscle paralysed by Ba2+ responded to caffeine (25 mM) or K+ (100-200 mM) with a contracture. The Ca2+ channel blocker verapamil did not prevent paralysis and, in fact, enhanced Ba2+ depression of tetani. Mouse long digital extensor (e.d.l.) muscles were depolarized by approximately 35 mV after 5 min in the presence of 1 mM-Ba2+. Action potentials from porcine muscles exposed to Ba2+ for 30 min rose and fell more slowly than controls and thus had a longer duration. Continuous recordings of membrane potential from individual cells revealed that 5 mM-Ba2+ depolarized mouse e.d.l. muscle at about 0.05 times the rate with high K+ (200 mM). I conclude that Ba2+ acts relatively slowly to block K+ channels, to decrease K+ fluxes and to induce depolarization. The rate of spontaneous inactivation of the contraction was apparently faster than the rate of activation. This would account for the Ba2+-induced flaccid paralysis without an initial contracture. These results with Ba2+-treated muscle illustrate similarities to h.p.p. and indicate that Ba2+ may create a useful model for studies relevant to h.p.p. Furthermore, Ba2+ may provide an appropriate means for evaluating K+ channel function in other muscle disorders.

Action Potentials↗