Halothane Sensitivity Research Articles

Malignant hyperthermia, environmental heat stress, and intracellular calcium dysregulation in a mouse model expressing the p.G2435R variant of RYR1

BackgroundThe human p.G2434R variant of the RYR1 gene is most frequently associated with malignant hyperthermia (MH) in the UK. We report the phenotype of a knock-in mouse that expresses the RYR1 variant p.G2435R, which is isogenetic with the human variant. MethodsWe observed the general phenotype; determined the sensitivity of myotubes to caffeine-, KCl, and halothane-induced Ca2+ release; determined the in vivo response to halothane or increased ambient temperature; and determined the in vivo myoplasmic intracellular Ca2+ concentration in skeletal muscle before and during exposure to volatile anaesthetics. ResultsRYR1 pG2435R/MH normal (MHS-Heterozygous[Het]) or RYR1 pG2435R/pG2435R (MHS-Homozygous[Hom]) mice were fully viable under typical rearing conditions, although some male MHS-Hom mice died spontaneously. The normalised half-maximal effective concentration (95% confidence interval) for intracellular Ca2+ release in myotubes in response to KCl [MH normal, MHN, 21.4 (19.8–23.1) mM; MHS-Het 16.2 (15.2–17.2) mM; MHS-Hom 11.2 (10.2–12.2) mM] and caffeine (MHN, 5.7 (5–6.3) mM; MHS-Het 4.5 (3.9–5.0) mM; MHS-Hom 1.77 (1.5–2.1) mM] exhibited a gene dose-dependent decrease, and there was a gene dose-dependent increase in halothane sensitivity. Intact animals show a gene dose-dependent susceptibility to MH with volatile anaesthetics or to heat stroke. RYR1 p.G2435R mice had elevated skeletal muscle intracellular resting [Ca2+]i, (values are expressed as mean (SD)) (MHN 123 (3) nM; MHS-Het 156 (16) nM; MHS-Hom 265 (32) nM; P<0.001) and [Na+]i (MHN 8 (0.1) mM; MHS-Het 10 (1) mM; MHS-Hom 14 (0.7) mM; P<0.001) that was further increased by exposure to volatile anaesthetics. ConclusionsRYR1 pG2435R mice demonstrated gene dose-dependent in vitro and in vivo responses to pharmacological and environmental stressors that parallel those seen in patients with the human RYR1 variant p.G2434R.

Drosophila Ryanodine Receptors Mediate General Anesthesia by Halothane

Although in vitro studies have identified numerous possible targets, the molecules that mediate the in vivo effects of volatile anesthetics remain largely unknown. The mammalian ryanodine receptor (Ryr) is a known halothane target, and the authors hypothesized that it has a central role in anesthesia. Gene function of the Drosophila Ryr (dRyr) was manipulated in the whole body or in specific tissues using a collection of mutants and transgenes, and responses to halothane were measured with a reactive climbing assay. Cellular responses to halothane were studied using Ca imaging and patch clamp electrophysiology. Halothane potency strongly correlates with dRyr gene copy number, and missense mutations in regions known to be functionally important in the mammalian Ryrs gene cause dominant hypersensitivity. Tissue-specific manipulation of dRyr shows that expression in neurons and glia, but not muscle, mediates halothane sensitivity. In cultured cells, halothane-induced Ca efflux is strictly dRyr-dependent, suggesting a close interaction between halothane and dRyr. Ca imaging and electrophysiology of Drosophila central neurons reveal halothane-induced Ca flux that is altered in dRyr mutants and correlates with strong hyperpolarization. In Drosophila, neurally expressed dRyr mediates a substantial proportion of the anesthetic effects of halothane in vivo, is potently activated by halothane in vitro, and activates an inhibitory conductance. The authors' results provide support for Ryr as an important mediator of immobilization by volatile anesthetics.

Association of halothane sensitivity with growth and meat quality in pigs

Previous reports have indicated that a proportion of pigs, homozygous normal for the skeletal muscle ryanodine receptor gene (RYR1), was halothane sensitive, and this was associated with poor meat quality when pigs were handled aggressively. This study was conducted to evaluate halothane sensitivity in RYR1-normal pigs, managed under simulated commercial conditions, to ascertain the association of halothane sensitivity with growth rate and meat quality. A total of 363 pigs across four farrowing groups, from seven Landrace sires and 38 Yorkshire–Landrace F1 dams, were tested at 8 weeks of age for halothane sensitivity using a closed system that delivered 5% halothane at 2 l/min for 3 (group 1) or 2 (groups 2 to 4) min. After 1 min, limb rigidity, limb tremors and abdominal discoloration were evaluated on a binomial scale with 0 indicating no reaction and 1 indicating reaction. Testing was repeated 2 days later. At 10 weeks of age, pigs were moved to finishing pens and not moved again until marketing. Within farrowing group, pigs were harvested in one of two groups, and at marketing were moved a distance of 91 m, weighed, tattooed, loaded and transported a distance of 550 km to a commercial harvest plant. After overnight rest, pigs were harvested and the pH of the loin muscle was measured at 45 min (pH45) after stunning. After an 18-h chill, loin muscle pH (pHu), International Commission on Illumination (CIE) L*, a*, b*, color (1 to 6) and marbling (1 to 10) scores and fluid loss percent were collected. Generalized linear mixed models were used to estimate repeatabilities for response to halothane challenge. Repeatabilities for limb rigidity for the front right and left legs were 0.24 and 0.31, respectively, whereas rear right and left leg repeatabilities were 0.19 and 0.17, respectively. Repeatabilities for front right and left leg tremors were 0.16 and 0.20, respectively. Growth rate was not influenced by any measure of halothane sensitivity. Carcasses from pigs exhibiting limb rigidity tended to have lower pH45 (5.88 v. 5.97; P = 0.06), similar pHu (5.47 v. 5.49; P = 0.32), less pH decline from 45 min to 18 h (−0.40 v. −0.50; P = 0.04) and a tendency for greater fluid loss percent (5.01 v. 4.55; P = 0.08) than carcasses from pigs that did not exhibit limb rigidity during halothane challenge. A proportion of pigs normal for RYR1 did exhibit limb rigidity during halothane gas challenge, and subsequently tended to have lower 45 min pH and greater longissimus muscle fluid loss post harvest.

Genetic parameters and halothane genotype effect for residual feed intake in Piétrain growing pigs

Data were collected in the frame of the French pig breeding scheme for the Piétrain breed in which the halothane sensitivity allele (n) is segregating. This data set was used to estimate: (1) genetic parameters for residual feed intake (RFI) and growth, body composition and meat quality traits and (2) effect of the three halothane genotypes on these traits. Individual feed intake of animals raised from 35 to 107kg body weight in collective pens (12 animals maximum) was recorded using single-place electronic feeders. A total of 2312 animals were tested between 2002 and 2009 in three French central test stations: 176 were homozygous non-sensitive (NN), 470 were heterozygous (Nn) and 1666 were homozygous sensitive (nn) for the halothane locus. Variance components were estimated using the REML methodology applied to a multitrait animal model, including a random litter effect and, for meat quality traits, a random slaughter day effect. RFI showed a moderate heritability (0.40±0.06), similar to the estimate for feed conversion ratio (FCR, 0.47±0.07), with a genetic correlation of 0.85±0.04 between these traits. Pairwise contrasts between halothane genotypes showed that the halothane genotype did not affect RFI, contrary to FCR (P<0.01 for all pairwise contrasts). We conclude that, in the Piétrain breed, improving feed efficiency by selecting for lower RFI would improve FCR and reduce daily feed intake with limited impacts on growth rate and carcass traits. Moreover, it is not expected to modify the frequencies of the halothane alleles in the population, contrary to selection for reduced FCR.

Store-operated Ca2+ Entry in Malignant Hyperthermia-susceptible Human Skeletal Muscle

In malignant hyperthermia (MH), mutations in RyR1 underlie direct activation of the channel by volatile anesthetics, leading to muscle contracture and a life-threatening increase in core body temperature. The aim of the present study was to establish whether the associated depletion of sarcoplasmic reticulum (SR) Ca(2+) triggers sarcolemmal Ca(2+) influx via store-operated Ca(2+) entry (SOCE). Samples of vastus medialis muscle were obtained from patients undergoing assessment for MH susceptibility using the in vitro contracture test. Single fibers were mechanically skinned, and confocal microscopy was used to detect changes in [Ca(2+)] either within the resealed t-system ([Ca(2+)](t-sys)) or within the cytosol. In normal fibers, halothane (0.5 mM) failed to initiate SR Ca(2+) release or Ca(2+)(t-sys) depletion. However, in MH-susceptible (MHS) fibers, halothane induced both SR Ca(2+) release and Ca(2+)(t-sys) depletion, consistent with SOCE. In some MHS fibers, halothane-induced SR Ca(2+) release took the form of a propagated wave, which was temporally coupled to a wave of Ca(2+)(t-sys) depletion. SOCE was potently inhibited by "extracellular" application of a STIM1 antibody trapped within the t-system but not when the antibody was denatured by heating. In conclusion, (i) in human MHS muscle, SR Ca(2+) depletion induced by a level of volatile anesthetic within the clinical range is sufficient to induce SOCE, which is tightly coupled to SR Ca(2+) release; (ii) sarcolemmal STIM1 has an important role in regulating SOCE; and (iii) sustained SOCE from an effectively infinite extracellular Ca(2+) pool may contribute to the maintained rise in cytosolic [Ca(2+)] that underlies MH.

Sensitivity to halothane and its relationship to the development of PSE (Pale, Soft, Exudative) meat in female lineage broilers

This work aimed to evaluate female lineage broilers for halothane sensitivity and for their susceptibility to the subsequent development of PSE meat. The halothane test was carried out in an anesthetic chamber with 3.0% halothane. The unconscious birds were examined for leg muscle rigidity. If one or both legs became extended and rigid, the birds were classified as halothane sensitive (HAL+), while unresponsive birds were classified as halothane negative (HAL-). The results showed that of 298 birds aged 42 days old, 95.6% were HAL- and 4.4% were HAL+. A sample of pectoralis major muscle was collected from HAL- (n=105) and HAL+ (n=13) birds. The pH and breast fillet color were determined at 4ºC, 24 hours post-mortem. Interestingly, only 2.5% of HAL+ birds displayed PSE meat characteristics compared to 12.7% of HAL- individuals. The halothane test demonstrated that female lineage broilers displayed very little sensitivity towards halothane, indicating that the development of PSE meat is related to other environmental factors.

Development of a gas chamber for detecting broiler chicken halothane sensitivity and PSE (Pale, Soft, Exudative) meat formation

The objective of this work was to investigate the use of a halothane to screen broiler chickens prone to develop PSE (Pale, Soft, Exudative) meat through a special gas chamber connected to a veterinarian anaesthetic apparatus developed in our laboratory. Anaesthesia was induced with approx. 3.0% halothane at a flow rate of 6.0 Lm-1 in pure oxygen for 5 min. Commercial male broilers (n=342) aged 46 days old were evaluated and classified as either halothane sensitive (HAL+) or insensitive (HAL-), depending on the leg rigidity response. Approximately 27.5% (n=94) of broilers were HAL+ and 72.5% (n=248) were HAL-. This is a simple and rapid technique to evaluate broiler sensitivity to halothane and identify broilers prone to develop PSE meat. The occurrence of PSE meat in HAL insensitive broiler chicken samples suggests that other factors are related to PSE occurrence.

Localization of the glucose phosphate isomerase gene to the p12----q21 segment of chromosome 6 in pig by in situ hybridization.

The glucose phosphate isomerase (GPI) locus is closely linked to the halothane sensitivity locus in pig. The chromosomal localization of GPI in pig was confirmed to 6p12----q21 by using in situ hybridization. Of the total grains, 25 percent were located on chromosome 6, with about 65 percent clustered in the cent----q21 segment, suggesting the presence of the GPI gene on the very proximal part of the q arm. The efficiency of hybridization was found to be affected by ultraviolet irradiation of metaphases for RBA-banding before hybridization. The irradiated metaphases had higher background grains than the non-irradiated metaphases.

UNC-1 Regulates Gap Junctions Important to Locomotion in C. elegans

In C. elegans, loss-of-function (lf) mutations of the stomatin-like protein (SLP) UNC-1 and the innexin UNC-9 inhibit locomotion [1, 2] and modulate sensitivity to volatile anesthetics [3, 4]. It was unknown why unc-1(lf) and unc-9(lf) mutants have similar phenotypes. We tested the hypothesis that UNC-1 is a regulator of gap junctions formed by UNC-9. Analyses of junctional currents between body-wall muscle cells showed that electrical coupling was inhibited to a similar degree in unc-1(lf), unc-9(lf), and unc-1(lf);unc-9(lf) double mutants, suggesting that UNC-1 and UNC-9 function together. Expression of Punc-1::DsRED2 and Punc-9::GFP transcriptional fusions suggests that unc-1 and unc-9 are coexpressed in neurons and body-wall muscle cells. Immunohistochemistry showed that UNC-1 and UNC-9 colocalized at intercellular junctions and that unc-1(lf) did not alter UNC-9 expression or subcellular localization. Bimolecular fluorescence complementation (BiFC) assays suggest that UNC-1 and UNC-9 are physically very close at intercellular junctions. Targeted rescue experiments suggest that UNC-9 and UNC-1 function predominantly in neurons to control locomotion. Thus, in addition to the recently reported function of regulating mechanosensitive ion channels [5, 6], SLPs might have a novel function of regulating gap junctions.

Genetic characterization of Calabrese and Casertana pig ancient autochthonous genetic types (AAGT) at CRC locus

Calcium Release Channel (CRC) is a “candidate DNA segment encoding protein (gene)” of high interest for pork quality. It maps on the short arm of chromosome 6 and has two allelic forms: N (normal dominant allele) and n [mutated recessive allele, also known as “halothane sensitivity” allele (Haln)]. Haln is due to a point mutation (SNP, single nucleotide polymorphism) which consists in the transition of a thymine nucleotide base for a cytosine at position 1843 in the 134 bp exon, responsible for the synthesis of a protein containing a substitution of an “arginine” for a “cysteine” at position 615; in the muscle fiber this substitution induces anomalies in the calcium flow regulation at sarcoplasmic reticulum level (ionic calcium deregulation). This deregulation in pig species is manifested as PSS (Porcine Stress Syndrome). Meat of PSS animals displays PSE (Pale Soft Exudative) myopathy, which makes meat less acceptable to consumer and poorly suitable to transformation in products of a good quality. The individuation of the point mutation allowed to set up a fast and efficient “genetic prophylaxis” test based on the genotype determination at CRC locus in order to detect heterozygous carriers of mutated allele and their incidence. In fact, the halothane test does not distinguish between the heterozygous (Nn) and homozygous normal (NN) animals, because both of these genotypes are non reactive. Furthermore, some nn subjects are halothane negative due to incomplete penetrance. In order to individuate the possible presence of mutated allele within Casertana (CT) and Calabrese (CA) AAGTs, a genetic screening was performed on 110 subjects of CT AAGT and 40 CA AAGT pigs, reared at ConSDABI experimental farm, using PCR-RFLP (Polymerase Chain Reaction Restriction Fragment Lenght Polymorphisms) method (Russo et al., 1993). The results showed that all Casertana AAGT pigs were NN (100%) while in AAGT Calabrese the frequencies of NN and Nn genotypes were 75% and 25% respectively. No nn genotype was found in the tested animals. It is opportune to specify that familiar relationships exist between Calabrese subjects, due to the reduced consistency of pig population. These results, valid within the observation field, confirm what found in a previous research (Matassino et al., 2000), in which Casertana was different from Calabrese and Nero Siciliano AAGTs for genotypic frequencies values: NN: 100,0% vs 65,5% vs 93,9%, respectively.

Effects of halothane sensitivity on mobility status and blood metabolites of HAL-1843-normal pigs after rigorous handling1,2

The objective of this study was to determine if HAL-1843-normal pigs that respond abnormally to halothane anesthesia were more likely to become nonambulatory (NA) when subjected to rigorous handling than pigs that exhibit a normal response to halothane. After a 1,100-km transport, pigs exhibiting low (HS-L; n = 33), intermediate (HS-I; n = 10), and high (HS-H; n = 47) sensitivity to halothane were moved through a 36.6-m long aisle that was 2.1 m wide at each end and 0.6 m wide in the middle 18.3 m. Ten groups of 8 pigs were briskly moved down the aisle and back 4 times, receiving a minimum of 1 electrical prod per pass (8 prods/pig). Before testing, rectal temperature was measured, open-mouth breathing and skin discoloration were visually evaluated, and a blood sample was collected from each pig. After the test, the pigs were returned to their pens, and the same measurements were taken immediately posttest and 1 h posttest (no blood at 1 h posttest). Pigs that were HS-H were more prone to becoming NA compared with HS-L pigs (P < 0.02). Regardless of halothane status, a greater number of pigs exhibited open-mouth breathing and skin discolorations immediately posttest than at the pretest or 1 h posttest times (P < 0.05). No differences were observed in blood metabolites between the different halothane sensitivity categories. However, pigs that became NA had elevated blood levels of creatine phosphokinase, lactate, glycerol, nonesterified fatty acids, ammonia, and urea nitrogen before testing (P < 0.05). Collectively, these data suggest HS-H pigs are more susceptible to becoming NA than HS-L. The elevated pretest blood metabolites of NA pigs suggest that they were in a hypermetabolic state that predisposed them to becoming NA.

The effects of halothane sensitivity on carcass composition and meat quality in HAL-1843-normal pigs1,2

Objectives of this study were to determine the incidence of halothane sensitivity in pigs that are homozygous normal at the ryanodine receptor nucleotide 1843 (HAL-1843-normal) and the relationships between halothane sensitivity and carcass composition or meat quality. In Exp. 1, piglets (Lines A, B, C, and D; n = 168, 170, 168, and 169, respectively) were obtained from mating a HAL-1843-normal sire line to four HAL-1843-normal dam lines. In Exp. 2, piglets from Lines A and B (n = 87 and 90, respectively) were included with piglets (Lines E, F, G, and H; n = 94, 92, 89, and 89, respectively) obtained from mating four HAL-1843-normal sire lines to a single HAL-1843-normal dam line. Pigs were subjected to 3% halothane at approximately 9 wk of age. In Exp. 1, limb rigidity, blotching of the skin, and muscle tremors were visually assessed, and based on these criteria, halothane sensitivity (HS) was observed in 48% of the pigs. To better characterize this response, a scoring system was developed and used in Exp. 2. Using this system, 25, 42, and 33% of the pigs in E and 40, 33, and 27% of the pigs in Line G were categorized as HS-low (HS-L), HS-intermediate (HS-I), and HS-high (HS-H), respectively. In Lines F and H, 13 and 18% of the pigs were HS-I, and 0 and 2% were HS-H, respectively. No consistent effects due to HS were observed in carcass composition or meat quality; however, when a subset of pigs from Exp. 2 were subjected to more extensive handling and transportation before slaughter, ultimate pH was lower and drip loss was higher in LM from HS-H compared with HS-L pigs (P < 0.05; n = 71). These results demonstrate that some pigs are sensitive to halothane anesthesia even in the absence of the known HAL-1843 polymorphism. Additionally, halothane sensitivity may be associated with inferior pork quality under adverse antemortem conditions.

Mg2+Dependence of Halothane-induced Ca2+Release from the Sarcoplasmic Reticulum in Skeletal Muscle from Humans Susceptible to Malignant Hyperthermia

Recent work suggests that impaired Mg(2+) regulation of the ryanodine receptor is a common feature of both pig and human malignant hyperthermia. Therefore, the influence of [Mg(2+)] on halothane-induced Ca(2+) release from the sarcoplasmic reticulum was studied in malignant hyperthermia-susceptible (MHS) or -nonsusceptible (MHN) muscle. Vastus medialis fibers were mechanically skinned and perfused with solutions containing physiologic (1 mm) or reduced concentrations of free [Mg(2+)]. Sarcoplasmic reticulum Ca(2+) release was detected using fura-2 or fluo-3. In MHN fibers, 1 mm halothane consistently did not induce sarcoplasmic reticulum Ca(2+) release in the presence of 1 mm Mg(2+). It was necessary to increase the halothane concentration to 20 mm or greater before Ca release occurred. However, when [Mg(2+)] was reduced below 1 mm, halothane became an increasingly effective stimulus for Ca(2+) release; e.g., at 0.4 mm Mg(2+), 58% of MHN fibers responded to halothane. In MHS fibers, 1 mm halothane induced Ca(2+) release in 57% of MHS fibers at 1 mm Mg(2+). Reducing [Mg(2+)] increased the proportion of MHS fibers that responded to 1 mm halothane. Further experiments revealed differences in the characteristics of halothane-induced Ca(2+) release in MHS and MHN fibers: In MHN fibers, at 1 mm Mg(2+), halothane induced a diffuse increase in [Ca(2+)], which began at the periphery of the fiber and spread slowly inward. In MHS fibers, halothane induced a localized C(2+)a release, which then propagated along the fiber. However, propagated Ca(2+) release was observed in MHN fibers when halothane was applied at an Mg(2+) concentration of 0.4 mm or less. When Mg(2+) inhibition of the ryanodine receptor is reduced, the halothane sensitivity of MHN fibers and the characteristics of the Ca release process approach that of the MHS phenotype. In MHS fibers, reduced Mg(2+) inhibition of the ryanodine receptor would be expected to have a major influence on halothane sensitivity. The Mg dependence of the halothane response in MHN and MHS may have important clinical implications in circumstances where intracellular [Mg(2+)] deviates from normal physiologic concentrations.

The use of halothane and succinylcholine to identify broilers prone to developing pale, soft, exudative meat

Within the last several years, the poultry industry has seen a dramatic increase in the occurrence of pale, soft, and exudative (PSE) meat. This problem is known to be associated with a rapid decline in postmortem (PM) muscle pH, which results in inferior protein functionality similar to that found in PSE pork. Many factors such as seasonal changes have been known to influence the occurrence of PSE meat in poultry and swine. Halothane and succinylcholine have been used within the pork industry to identify animals susceptible to stress and prone to developing PSE meat. The mechanism for the triggering of the PSE gene in poultry has not been fully understood. Therefore, a study was conducted to determine the effectiveness of screening broilers with halothane to identify those prone to developing PSE meat. Succinylcholine was used before slaughter to serve as a triggering agent for the PSE condition. At 4 wk of age, broilers from 4 commercial strains (n = 1,000) were subjected to 3% halothane gas and classified as either halothane positive (HAL+) or negative (HAL-) based on muscle rigidity within the legs. Although halothane sensitivity varied slightly among the strains, approximately 14% of the birds overall were classified as HAL+. All HAL- birds (n = 163) and an equal number of HAL-birds (n = 163) in each strain were grown to market age (7 wk) and were commercially processed. At the time of processing, half of the HAL+ and HAL- birds were injected intravenously with succinylcholine and were slaughtered at 0.25 h postinjection. Pectoralis muscle samples were collected at 0.25, 2, 5, and 24 h PM for the evaluation of rigor development (muscle pH) and meat quality (L* value, moisture, drip loss, and cook loss). Halothane sensitivity had no effect on rigor development, muscle color, or water-holding capacity in the 4 broiler strains. Although birds exhibited reactions to the halothane gas, the halothane sensitivity, along with the use of succinylcholine, was not able to identify birds prone to developing PSE meat.

Halothane inhibits an intermediate conductance Ca2+-activated K+ channel by acting at the extracellular side of the ionic pore.

Actions of volatile anesthetics on ligand-gated ion channels, such as gamma-aminobutyric acid type A receptors, have been studied extensively. However, actions on other types of channels, such as K+ channels, are poorly understood. The authors previously showed that a Ca2+-activated K+ channel, IK, is sensitive to halothane, whereas SK1, another Ca2+-activated K+ channel, is insensitive. To explore how halothane acts on Ca2+-activated K+ channels, chimeras between IK and SK1 were constructed, and halothane sensitivity was analyzed. IK, SK1, and chimera channels were expressed in Xenopus laevis oocytes. Currents of expressed channels were measured in the presence of 10 microm Ca2+ by excised patch clamp analysis. Time constants of inhibition by halothane were compared between inside-out and outside-out patch configurations. Currents from chimera channels possessing the pore domain derived from IK were inhibited by halothane, whereas those possessing the SK1 pore domain were insensitive. Time constants of inhibition by halothane were significantly smaller in the outside-out patches than in the inside-out patches of both wild-type IK and a chimera with pore domain of IK. It is suggested that halothane interacts with the extracellular part of the ionic pore of IK. Whether this type of interaction is involved in the mechanism of anesthetic actions on ligand-gated ion channels warrants further investigation.

Anesthetic-sensitive 2P domain K+ channels.

Anesthetic-sensitive 2P domain K+ channels.

Blockage of one class of potassium channel alters the effectiveness of halothane in a brain circuit of Drosophila.

At concentrations comparable to those used in the clinic, halothane has profound effects on a neuronal pathway devoted to the escape reflex of the fruit fly, Drosophila melanogaster. We studied the influence of the potassium channel that is encoded by the Shaker gene on the halothane sensitivity of this circuit. Shaker channels were specifically inactivated either by genetic means, using strains with two different severe Shaker mutations, or by pharmacologic means, using ingestion of millimolar concentrations of 4-aminopyridine. In all cases, halothane potency decreased substantially. To ensure that the genetic alteration was specific, both mutations were studied as stocks that had been repeatedly backcrossed to a control strain. The specificity of the pharmacologic inhibition was demonstrated by the fact that 4-aminopyridine had no effect on halothane potency in a Shaker mutant. Quantitative differences in the effects of channel inhibition between males and females suggested a sexual dimorphism in the functional brain anatomy of the reflex circuit.

Blockage of One Class of Potassium Channel Alters the Effectiveness of Halothane in a Brain Circuit of Drosophila

At concentrations comparable to those used in the clinic, halothane has profound effects on a neuronal pathway devoted to the escape reflex of the fruit fly, Drosophila melanogaster. We studied the influence of the potassium channel that is encoded by the Shaker gene on the halothane sensitivity of this circuit. Shaker channels were specifically inactivated either by genetic means, using strains with two different severe Shaker mutations, or by pharmacologic means, using ingestion of millimolar concentrations of 4-aminopyridine. In all cases, halothane potency decreased substantially. To ensure that the genetic alteration was specific, both mutations were studied as stocks that had been repeatedly backcrossed to a control strain. The specificity of the pharmacologic inhibition was demonstrated by the fact that 4-aminopyridine had no effect on halothane potency in a Shaker mutant. Quantitative differences in the effects of channel inhibition between males and females suggested a sexual dimorphism in the functional brain anatomy of the reflex circuit.

The development of pale, exudative meat in two genetic lines of turkeys subjected to heat stress and its prediction by halothane screening

Previous research has indicated that seasonal-type heat stress (HS) can contribute to the development of pale, soft, exudative (PSE) meat in fast-growing turkeys and that halothane exposure may identify stress-susceptible animals. This study evaluated the ability of halothane screening to identify stress-susceptible birds prone to developing pale, exudative meat when reared to market age. Two lines of turkeys (n = 292), one selected for rapid overall growth (BODY) and the other for large breast muscle yield (BREAST), were exposed to 3% halothane for 5 min at 2 to 4 wk of age and were raised together until 16 wk of age. Approximately 10% of both BODY and BREAST birds were sensitive to halothane. Between 16 and 20 wk, all of the halothane sensitive (HAL+) and half of the halothane nonresponders (HAL-) were exposed to an HS environment of 30 to 36 C (night/day), whereas the other half of the HAL- birds were kept at an ambient temperature of 13 to 21 C (night/day). All birds were slaughtered at 20 wk of age, and samples were collected for pH, L* value, drip loss, cooking loss, and shear value. The BREAST strain had 5% greater breast percentage than the BODY strain, and there were no differences in ready-to-cook yields between any treatments. The HAL+ HS birds had significantly lower muscle pH (0 h) and significantly higher L* values at 2 h postmortem compared with HAL- HS birds in the BREAST strain; however, there was no difference in L* value at 24 h postmortem. The HAL- HS birds had significantly lower muscle pH (0 h and 2 h) and significantly higher L* values at 2 h postmortem compared with HAL- controls in the BODY strain. The HAL- HS BREAST birds had significantly higher drip loss than HAL- controls. No differences in shear value were found among any treatments. The incidence of PSE (2-h L* values >52) was significantly higher in HAL+ HS birds (34.7%) compared with HAL- HS birds (13.4%). These results suggest that halothane sensitivity early in life is associated with HS susceptibility and the development of pale meat when birds are slaughtered at market age. These results also suggest that halothane screening may be better at predicting the development of PSE meat during HS in the strain selected for large breast yield rather than rapid overall growth.

Halothane induces calcium release from human skinned masseter muscle fibers.

An increase in masseter muscle tone in response to halothane or succinylcholine anesthesia (or both) can be observed in healthy persons. Thus the authors compared the fiber-type halothane and succinylcholine sensitivities in human masseter and vastus lateralis muscles. Masseter and vastus lateralis muscle segments were obtained from 13 and 9 healthy persons, respectively. After chemical skinning of a single fiber and loading the sarcoplasmic reticulum with Ca++ 0.16 microM solution, halothane (0.5-4 vol% bubbled in the incubating solution), succinylcholine (0.1 microM to 10 mM), or both sensitivities were defined as the concentration inducing more than 10% of the maximum tension obtained by application of 16 microM Ca++ solution. The myofilament response to Ca++ was studied with and without halothane by observing the isometric tension of skinned masseter fibers challenged with increasing concentrations of Ca++. Muscle fiber type was determined by the difference in strontium-induced tension measurements. A significant difference in halothane sensitivity was found between type 1 masseter fibers (0.6+/-0.2 vol%; mean +/- SD) versus type 1 (2.7+/-0.6 vol%) and type 2 vastus lateralis muscle (2.5+/-0.4 vol%). Succinylcholine did not induce Ca++ release by the sarcoplasmic reticulum. In the masseter muscle, 0.75 vol% halothane decreased the maximal activated tension by 40% but did not change the Ca++ concentration that yields 50% of the maximal tension. The very low halothane threshold for Ca++ release from the masseter muscle usually could be counteracted by a direct negative inotropic effect on contractile proteins. However, halothane may increase the sensitivity of the sarcoplasmic reticulum Ca++ release to succinylcholine-induced depolarization, leading to an increase in masseter muscle tone.