Malignant hyperthermia mutation Arg615Cys in the porcine ryanodine receptor alters voltage dependence of Ca2+ release.

Ca2+ inward current and fura-2 Ca2+ transients were simultaneously recorded in porcine myotubes. Myotubes from normal pigs and cells from specimens homozygous for the Arg615Cys (malignant hyperthermia) mutation of the skeletal muscle ryanodine receptor RyR1 were investigated. We addressed the question whether this mutation alters the voltage dependence of Ca2+ release from the sarcoplasmic reticulum. The time course of the total flux of Ca2+ into the myoplasm was estimated. Analysis showed that the largest input Ca2+ flux occurred immediately after depolarization. Amplitude and time course of the Ca2+ flux at large depolarizations were not significantly different in the Arg615Cys myotubes. Ca2+ release from the sarcoplasmic reticulum was activated at more negative potentials than the L-type Ca2+ conductance. In the controls, the potentials for half-maximal activation V 1/2 were -9.0mV and 16.5 mV, respectively. In myotubes expressing the Arg615Cys mutation, Ca2+ release was activated at significantly lower depolarizing potentials (V = -23.5 mV) than in control myotubes. In contrast, V of conductance activation (13.5 mV) was not significantly different from controls. The specific shift in the voltage dependence of Ca2+ release caused by this mutation can be well described by altering a voltage-independent reaction of the ryanodine receptor that is coupled to the voltage-dependent transitions of the L-type Ca2+ channel.

Xenon does not trigger malignant hyperthermia in susceptible swine.

BACKGROUND: Xenon is a noble gas with anesthetic properties currently under investigation for use in humans. This study was performed to evaluate whether xenon may trigger malignant hyperthermia in susceptible swine. METHODS: Nine malignant hyperthermia-sensitive swine (Pietrain) were initially anesthetized with pentobarbital and then ventilated with 70% xenon in oxygen for 2 h. Heart rate, mean arterial pressure, cardiac output, body temperature, arterial and mixed-venous blood gases, and plasma catecholamine and lactate levels were measured every 10 min both during xenon-oxygen ventilation and after a 30-min xenon washout phase followed by subsequent administration of halothane (1% inspired) and succinylcholine (3 mg/kg intravenous). During the investigation, no malignant hyperthermia-specific therapy was instituted. RESULTS: Xenon exposure did not induce any changes in metabolic and hemodynamic parameters nor elevations of the plasma catecholamine levels indicative for an episode of malignant hyperthermia. By contrast, in all animals, within 20 min after the administration of halothane and succinylcholine, fulminant and fatal malignant hyperthermia episodes were initiated. CONCLUSIONS: The authors conclude that xenon does not trigger malignant hyperthermia in susceptible swine.

[Comparative investigations of a combined vaccine against parvovirus and erysipelas and corresponding monovaccines in different vaccination schedules. 1: Field trial]. / Vergleichende Untersuchungen über den Einsatz eines Parvovirose-Rotlauf-Kombinationsimpfstoffes sowie entsprechender Monovakzinen bei unterschiedlichen Revakzinationszeitpunkten. Teil 1: Feldversuch

In a field trial, the development of antibodies of a combined vaccine against the porcine parvovirus (PPV) as well as against swine erysipelas was compared with corresponding mono vaccines. Furthermore, these vaccines were used in different vaccination schedules. The tests were carried out on 109 gilts in three closed farms. In all gilts, a basic immunization repeated twice was carried out at the age of six months and at intervals of three weeks. The revaccination was carried out four months after the basic immunization with half of the animals, and six months after the basic immunization with the remaining gilts. Between the combined vaccine and the mono vaccine no significant differences in the development of antibodies against PPV could be found according to different vaccination schedules. The gilts having been vaccinated with the mono vaccine and boostered six months later showed significantly higher antibody titers against Erysipelothrix rhusiopathiae. Between the remaining vaccination groups no significant difference in the development of the antibodies against swine erysipelas could be found. On only one farm, a continuous decrease of antibody titers against PPV in case of altogether 238 non-vaccinated piglets until the sixth month of life could be observed. On the two other farms, an increase of antibody titers against PPV could be found at different points of time, which indicates an infection of the piglets. Between the individual vaccination groups no significant antibody titers against PPV could be measured in milk tests. With regard to the number of piglets born alive per litter, the number of piglets born dead per litter and the number of mummies, a significant difference could neither be found between the vaccination groups 1-4.

Effects of a high n-3 fatty acid diet on membrane lipid composition of heart and skeletal muscle in normal swine and in swine with the genetic mutation for malignant hyperthermia.

Knowledge concerning the genetic defects underlying malignant hyperthermia (MH) has expanded rapidly in recent years. In contrast, our understanding of the accompanying physiological changes is less clear. In this regard, the aim of this study was to determine whether normal swine and swine susceptible to MH (both heterozygous and homozygous animals) differ in their abilities to incorporate n-3 (omega 3) fatty acids into their skeletal and heart muscles. Swine of each genotype were fed either a diet rich in n-3 fatty acids (i.e., 5% fish oil) or an equal caloric diet low in n-3 fatty acids (i.e., 5% coconut oil). All dietary supplementations were given over a 13-week period. Subsequently, for each muscle type the following was determined: 1) the relative fatty acid profiles of eight different phospholipid classes and of neutral lipids, and 2) the total phospholipid and the total lipid content. The incorporation of n-3 fatty acids (i.e., eicosapentaenoic acid and docosahexaenoic acid) occurred within the various phospholipids and neutral lipids without influencing their total lipid content. The increased content of n-3 fatty acids in neutral lipids of skeletal muscle was related to a decreased content of medium-chain saturated fatty acids, whereas an increased incorporation of n-3 fatty acids into the membrane phospholipids was often related to a decreased content of linoleic acid and/or arachidonic acid. In general, the pattern of n-3 fatty acid incorporation was considerably different between the normal animals and the MH homozygous and heterozygous animals. The significant interaction between diet-induced n-3 fatty acid profiles and the stress-susceptible MH genotype may indicate an altered mechanism for fatty acid turnover and a repair mechanism to maintain cellular functions and structure.

Influences of breed, sex, and susceptibility to malignant hyperthermia on lipid composition of skeletal muscle and adipose tissue in swine.

OBJECTIVE: To determine influences of breed, sex, and susceptibility to malignant hyperthermia on composition of skeletal muscle and adipose tissue in swine. ANIMALS: 35 male and female swine of German Landrace and Pietrain breeds and of 2 genotypes, normal (MHN) and susceptible to malignant hyperthermia (MHS). PROCEDURE: Pigs were fed a standard diet ad libitum. After attaining body weight of approximately 100 kg, pigs were slaughtered and skeletal (longissimus thoracis and supraspinatus) muscle and adipose tissue (3 sites) specimens were removed. For each specimen, lipids were extracted by chloroform/methanol and fatty acid (FA) pattern, and cholesterol concentration was determined by gas chromatography. RESULTS: The overall lipid contents differed significantly between breeds and genotypes; the MHS Pietrain pigs had the lowest lipid quantities. The relative amounts of saturated FA in all tissues were highest in Landrace pigs (P < 0.05), whereas the relative contents of monoenic FA were lower. In addition, for both breeds, the MHN pigs had significantly higher saturated and lower polyunsaturated FA values in all tissues, compared with MHS pigs. More specifically, MHS females of both breeds had the highest relative amounts of polyunsaturated FA. In general, relative cholesterol contents were found to vary little between identified groups. CONCLUSIONS: These data may indicate that, not only does mutation of the calcium release channel of the sarcoplasmatic reticulum, which occurs in MHS swine, influence secondary changes in lipid composition, but so do hormone concentrations and other genotypic factors. Observed differences in lipid content and FA composition could consequently influence specific membrane properties, such as fluidity and cell signaling.

Calmodulin sensitivity of the sarcoplasmic reticulum ryanodine receptor from normal and malignant-hyperthermia-susceptible muscle.

Ca2+ release from sarcoplasmic reticulum (SR) of malignant-hyperthermia-susceptible (MHS) muscle is hypersensitive to Ca2+ and caffeine. To determine if an abnormal calmodulin (CaM) regulation of the SR Ca(2+)-release-channel-ryanodine-receptor complex (RYR1) contributes to this hypersensitivity, we investigated the effect of CaM on high-affinity [3H]ryanodine binding to isolated SR vesicles from normal and MHS pig skeletal muscle. CaM modulated [3H]ryanodine binding in a Ca(2+)-dependent manner. In the presence of maximally activating Ca2+ concentrations, CaM inhibited [3H]ryanodine binding with no differences between normal and MHS vesicles. In the absence of Ca2+, however, CaM activated [3H]ryanodine binding with a 2-fold-higher potency in MHS vesicles. Significant differences between normal and MHS tissue were observed for CaM concentrations between 50 nM and 10 microM. A polyclonal antibody raised against the central region of RYR1 specifically inhibited this activating effect of CaM without affecting the inhibition by CaM. This indicates that the central region of RYR1 is a potential binding domain for CaM in the absence of Ca2+. It is suggested that in vivo an enhanced CaM sensitivity of RYR1 might contribute to the abnormal high release of Ca2+ from the SR of MHS muscle.

[CO2/O2 anesthesia for the castration of male piglets (preliminary results)]. / Die CO2/O2-Anästhesie zur Kastration von männlichen Ferkeln (vorläufige Ergebnisse).

Introduction of anaesthesia with CO2/O2 (60% to 40%) is possible within 90 and 120 seconds. There are moderate to excessive excitations occurring as part of state II of anaesthesia. Anaesthesia (during castration) in CO2/O2-atmosphere produces excellent analgesia and relaxation. The duration of castration surgery is much shorter under CO2-anaesthesia than without anaesthesia. Blood cortisol levels are significantly higher after castration without CO2-anaesthesia. About 5 minutes after CO2/O2-anaesthesia and castration surgery, piglets are already awake and standing.

Culture conditions for the production of porcine myotubes and myoballs.

The existence of a method for culturing porcine muscle cells would greatly facilitate the development of new breeding criteria for stress resistance and growth regulation in pig breeding. Also, many effects of nutritional or pharmacological components that influence animal performance could be studied first in muscle cell preparations. Therefore, we developed a specific procedure to culture porcine skeletal muscle cells from well-established methods for murine and human muscle cell culturing. Best results were obtained by isolating satellite cells from muscle tissue removed postmortem after normal slaughter procedure, using enzymatic dissociation. The satellite cells were allowed to proliferate for 3 to 5 d in a culture medium composed of 83% Ham's F-12 medium, 15% fetal calf serum (FCS), and 2% chick embryo extract (CEE). Well before reaching confluence, the cells were transferred to collagen-coated dishes filled with Dulbecco's modified Eagle's medium containing 5% horse serum (HS) for the differentiation to multinucleated myotubes. Also, .5% FCS can be used instead of HS. Besides the fusion to myotubes, the presence of voltage-sensitive Na+ channels is regarded as a specific feature of the muscle phenotype of the cells. To perform electrophysiological experiments of good quality, myotubes were converted into freely floating "myoballs." Voltage-clamp experiments in the whole-cell mode showed transient inward currents that had kinetics and voltage dependences very similar to those of the Na+ currents in human myoballs. The porcine Na+ currents were almost completely blocked by 1 mumol/L of tetrodotoxin, indicative of the presence of the adult form of the Na+ channel.(ABSTRACT TRUNCATED AT 250 WORDS)

A high omega 3 fatty acid diet alters fatty acid composition of heart, liver, kidney, adipose tissue and skeletal muscle in swine.

The fatty acid profiles and total lipid contents of two skeletal muscles, adipose tissue, heart, liver and kidney of swine fed a diet rich in omega 3 (n-3) fatty acids (i.e., 5% fish oil) was investigated. These values were compared to those determined for animals which were fed an equal caloric diet low in n-3 fatty acids (i.e., 5% coconut oil). All supplementations were given over a 13-week period. The lipids were extracted with chloroform-methanol, trans-esterified and the relative fatty acid methyl-esters concentrations were determined using capillary gas chromatography. The fish oil diet significantly enhanced the relative amounts of n-3 fatty acids (i.e., eicosapentaenoic acid and docosahexaenoic acid) in all tissues examined. In the heart, liver and kidney, the increases in n-3 fatty acids were compensated by decreases primarily in arachidonic acid, but in the other tissues the contents of lauric and myristic acids were also reduced. In general, the n-3 fatty acid contents were 40-165% higher in the animals fed the fish oil. Supplementation of n-3 fatty acids in swine induced a significant incorporation of these fatty acids throughout the body, however the extent of this incorporation differed between tissues perhaps due to tissue-specific metabolic pathways.

Effects of meat quality and storage on the breakdown of adenosine triphosphate in muscle from swine.

The quality of pork is dependent on animal genotype, pre-slaughter handling, processing, maturation, and storage. We investigated the pattern of adenosine triphosphate (ATP) breakdown as it related to these factors. Samples of the thoracis portion of the longissimus dorsi muscle were obtained from 19 randomly selected German Landrace-Pietrain crossbreed swine. Based on their 40 min post-mortem pH and electrical conductances, three groups were classified: (1) PSE meat, pH ≤ 5·6 and conductance >10 mS (n = 7); (2) intermediate quality, pH 5·6-6·0 and conductance between 4 and 10 mS (n = 5); and (3) normal quality, pH ≥ 6·0 and conductance < mS (n = 7). Hence, the animals investigated included those susceptible to malignant hyperthermia (porcine stress-syndrome). Twenty-four hours post mortem samples were evaluated for the following parameters: ATP metabolism, pH values, electrical conductance, meat colour, water binding capacity, shear force and general composition (i.e. total protein, fat, mineral and water contents). Muscle composition was the same in each group, but for the other parameters there were clear differences. Following different storage periods and conditions (1 or 5 days at 4°C and 27 days at -18°C), the degree of ATP metabolism as well as general meat quality (i.e. including sensory evaluation) were reassessed. Samples from the pre-selected groups became less discernible following prolonged storage. In all animals, the pattern of ATP breakdown was similar, the major metabolites including inosine monophosphate, hypoxanthine, adenosine monophosphate, and inosine. The degree of breakdown was dependent on the duration and temperature of storage, but not on animal type. The muscular samples for the intermediate and normal muscle groups, which were stored for 27 days at -18°C, were given the highest sensory evaluation scores. The simple HPLC measurement of ATP metabolism was considered as a useful means to assess appropriate storage.

Use of naturally enriched mixed food in 13C breath tests applied in young suckling calves.

Utilization of three milk diets including cream, casein or whey, each naturally labelled with 13C (1 mmol 13C excess) from C4 sources, by six young male calves of the Deutsche Fleckvieh breed was investigated in a Latin-square split-plot design. Each milk diet was examined under resting conditions and during a short period of physical exercise on a treadmill. Delta 13C values (/1000) in carbon dioxide in expired air were measured at intervals of about 1 h during 6.5 h after food intake. Expired air samples for CO2 isolation, subsequent isotopic analysis, measurement of CO2 production and respiratory quotient were taken at about hourly intervals and 13C recovery rates over 6.5 h were calculated. Feeding milk containing enriched milk casein, cream, or whey resulted in maximal significant 13C enrichments over background (delta 13C) in CO2 of +1, +2.4 and +2.2 /1000, and recovery rates of 3.6, 9.9 and 12.2% respectively. This comparison shows the different kinetic behaviour of the main nutrients during the oxidation in tissues. The short exercise period (5 min at 1 J/s per kg body-weight +5 min at 2 J/s per kg body-weight) did not influence the recovery rates significantly. However, after 10 min of muscular exercise there was a brief decrease in delta 13C value of expired air which disappeared within the first 5 min of rest. These experiments demonstrate for the first time the applicability of the 13C breath test with naturally enriched diets in animal nutrition research and that quantitative results may be obtained.

[Pteridine pattern in the urine of swine of different breeds and halothane genotypes in relation to nutrition and stress]. / Untersuchungen zum Pteridinmuster im Harn von Schweinen verschiedener Rassen und Halothan-Genotypen in Abhängigkeit von Ernährung und Belastung.

Urinary pteridine patterns and creatinine concentrations were determined in pigs of the breeds Piétrain, Deutsche Landrasse and their crossbreds. Also the influence of the halothane genotypes, of different feeding and of treadmill exercise was examined. We conclude that creatinine concentration in pig urine is not a reliable reference parameter for the pteridine excretion. Creatinine values were significantly influenced by breed and feeding. The pteridine pattern of pigs' urine depends on the breed and correlates with other metabolic parameters. Stress is reflected by altered pteridine concentrations.

Malignant hyperthermia: an altered phospholipid and fatty acid composition in muscle membranes.

There is thought to be a genetic defect within the calcium release channel of the sarcoplasmic reticulum in malignant hyperthermia (MH). This primary alteration is hypothesized to influence the function and/or structure of various muscle membrane systems; e.g., to have a direct effect on the composition of the lipid matrix. Therefore, in striated muscle samples, we determined the quantity and fatty acid composition of the various types of membrane phospholipids. German Landrace pigs were classified as normal or susceptible to MH. Total lipid content from longissimus dorsi, semi-membranosus muscle, and heart left ventricular (HLV) samples were extracted with chloroform/methanol and subsequently separated by high performance liquid chromatography. The single phospholipid fractions were collected and, following derivatization, the quantities of individual fatty acids were determined using a capillary gas chromatographic method. In general, samples from the susceptible pigs contained lower absolute amounts of individual phospholipids. The most notable differences occurred in the HLV, where phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and sphingomyelin were all significantly less (P less than or equal to 0.05). The muscle from the susceptible animals also contained decreased amounts of the polyunsaturated phospholipid-bound fatty acids (P less than or equal to 0.05). These differences in phospholipid and fatty acid concentrations of membranes isolated from swine susceptible to MH may relate to their apparently increased sensitivities to halothane (e.g., fluidizing effects) or elevated temperature.

Characterization of swine susceptible to malignant hyperthermia by in vivo, in vitro and post-mortem techniques.

We investigated German Landrace pigs from a special breeding program producing animals which were of three genotypes with respect to in vivo halothane inhalation (i.e., exposure to 3% halothane for up to 3 min): (1) Hal NN, i.e. homozygous normal exhibiting no response; (2) Hal Nn, i.e. heterozygous, also responding with a normal reaction; and (3) Hal nn, i.e. homozygous for the 'halothane gene n' which exhibited signs of malignant hyperthemia (MH). Additional characteristics of these three groups of animals were studied using accepted methodology from the fields of animal science, clinical testing, and food science. The following characteristics of group (2) and (3) were different from those of the normal animals: 1) creatine kinase levels; 2) in vitro sensitivities of muscles to caffeine and halothane administration (contracture test) and 3) postmortem muscle properties. In humans, results of the in vitro contracture test are indicative of susceptibility to MH. In humans, MH is considered to be inherited as an autosomal dominant trait. Similarly the results of the in vitro contracture test described here also indicate that MH is inherited as an autosomal dominant trait in German Landrace swine.

Enhanced mobilization of intracellular Ca2+ induced by halothane in hepatocytes isolated from swine susceptible to malignant hyperthermia.

Halothane, in a dose-dependent manner, induced the release of intracellular Ca2+ in hepatocytes prepared from swine. The magnitude of the release induced by halothane was greater for hepatocytes prepared from animals susceptible to malignant hyperthermia (MH) than for those from normal swine. Two different methods were used to ascertain the release of Ca2+ induced by halothane: 1) the release of 45Ca2+ from nonmitochondrial stores of saponin-permeabilized hepatocytes was measured; and 2) changes in luminescence from intact hepatocytes loaded with the Ca2(+)-sensitive photoprotein aequorin were recorded. It was also observed that, although 1,4,5-inositol trisphosphate (IP3), guanosine-5-triphosphate, and arachidonic acid all induced a significant release of 45Ca2+ from permeabilized swine hepatocytes, only the quantities of 45Ca2+ released by IP3 were significantly greater for the hepatocytes prepared from the animals susceptible to MH. These data indicate an abnormal Ca2+ homeostasis in hepatocytes isolated from swine susceptible to MH, which supports the hypothesis that membrane systems from multiple organs may be affected in this genetic disorder.

D-3-deoxy-3-substituted myo-inositol analogues as inhibitors of cell growth.

A number of unnatural D-3-deoxy-3-substituted myo-inositols were synthesized and their effects on the growth of wild-type NIH 3T3 cells and oncogene-transformed NIH 3T3 cells were studied. The compounds were found to exhibit a diversity of growth-inhibitory activities and showed selectivity in inhibiting the growth of some transformed cells as compared with wild-type cells. Remarkably, D-3-deoxy-3-azido-myo-inositol exhibited potent growth-inhibitory effects toward v-sis-transformed NIH 3T3 cells but had little effect on the growth of wild-type cells. The growth-inhibitory effects of the myo-inositol analogues were antagonized by myo-inositol. Since [3H]-3-deoxy-3-fluoro-myo-inositol was shown to be taken up by cells and incorporated into cellular phospholipids, we suggest that these unnatural myo-inositol analogues may act as antimetabolites in the phosphatidylinositol intracellular signalling pathways. Because cells transformed by oncogenes often exhibit elevated phosphatidylinositol turnover, the inhibition of signalling pathways that mediate oncogene action could offer new opportunities for controlling the growth of cancer cells.

Separation of major phospholipid classes by high-performance liquid chromatography and subsequent analysis of phospholipid-bound fatty acids using gas chromatography.

A sensitive high-performance liquid chromatographic method for the separation of major phospholipid (PL) classes in biological materials is described. Using this method it was easy to separate P-cholin, P-ethanolamine, P-serine, P-inositol, cardiolipin, sphingomyelin, lyso-P-choline and lyso-P-ethanolamine from skeletal and cardiac muscle samples. The method is based on the simultaneous use of a pH gradient and a polarity gradient. This procedure can easily be modified to optimize the separation of PLs from very different tissues. Subsequent analysis of the PL-bound fatty acids (FAs) by gas chromatography resulted in a well separated FA pattern. Following this FA separation it was possible to recalculate the specific PL content in the original sample.

Epinephrine and norepinephrine related to cardiorespiratory and metabolic changes in calves during physical exercise.

Experiments were designed to study changes of blood levels of norepinephrine (NE) and epinephrine (E) and their possible role in metabolic adaptation to short-lasting physical exercise in calves. After a resting period for 5 min (I), animals walked on a treadmill for 10 min at a speed of 60 m/min, first horizontally for 5 min (II), then at a slope of 6 degrees for another 5 min (III), followed by a recovery period for 5 min (IV). Levels of NE and E increased within minutes during walking and then decreased. Changes were closely related to respiration rate, ventilation volume, O2-uptake, heart rate and blood lactate levels. Blood triiodothyronine and protein slightly increased only during period III, whereas glucose, non-esterified fatty acids and the respiration quotient increased throughout the experiment. Blood insulin levels were decreased during walking and rapidly increased afterwards. Blood glucagon did not change significantly. Work load affected all parameters except glucagon and triiodothyronine. There were individually significant differences for all parameters, except for NE and the respiration quotient. The data demonstrate rapid reactions of the cardiorespiratory system and of blood insulin and lactate levels on submaximal work load, normally in close association with plasma E and NE.