Effects of serotonin 2 receptor agonists on skeletal muscle preparations in patients with a disposition toward malignant hyperthermia

In pigs genetically susceptible to malignant hyperthermia (MH), it has been shown that serotonin (5-HT2) receptor agonists can induce MH and "psychotic" behaviour. Both can be prevented by 5-HT2 receptor antagonists. Furthermore, free levels of serotonin in plasma increased concomitantly with clinical and laboratory parameters during halothane-induced MH in pigs. In this study the in vitro-effects of the 5-HT2 receptor agonist1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (DOI) were investigated in muscle specimens of MH-susceptible (MHS) and normal (MHN) patients. METHODS. Muscle biopsies were obtained from 37 patients aged 5-69 years (23.6 +/- 5.3 years) with clinical suspicion for MH. The patients were first classified as MHS, MHN or MHE (MH equivocal) by the in vitro contracture test (IVCT) according to the European MH protocol. After MH classification, surplus muscle specimens were subjected to the DOI study. DOI was added to the organ bath in a concentration of 0.02 mmol/l. The in vitro effects on contracture development and muscle twitch were observed for 120 min. RESULTS. Muscle specimens of all patients developed contractures after administration of DOI. However, DOI produced an earlier development of contracture in MHS (17.0 +/- 1.8 min; n = 17) than in MHN (64.7 +/- 5.9 min; n = 15) muscles. In MHS muscles, contractures were more distinct than in MHN muscles; at the end of the experiment, contractures had reached a maximum of 12.5 +/- 0.9 mN in MHS and 5.1 +/- 0.7 mN in MHN muscles. Muscle twitch following DOI administration was reduced significantly in both MHS and MHN muscles. The results of four MHE muscles were comparable with MHS. CONCLUSION. The present study supports the assumption that an altered serotonin system might be involved in the development of MH. In further studies it should investigated whether 5-HT2 receptors of skeletal muscles from MHS subjects are disordered in function or structure. 5-HT2 receptor agonists should be considered as MH-triggering agents.     

4-chloro-m-cresol-induced contractures of skeletal muscle specimen from patients at risk for malignant hyperthermia

PURPOSE: 4-chloro-m-cresol (4-CmC), commonly used as preservative, has been shown to induce contractures in skeletal muscle specimens from individuals susceptible to malignant hyperthermia (MH). It has been suggested that a defect of the calcium release channel of the skeletal muscle sarcoplasmic reticulum (ryanodine receptor) in MH susceptible (MHS) patients could be responsible for this phenomenon. 4-CmC was found to be a potent activator of ryanodine receptor-mediated Ca2+ release. The aim of this study was to determine the in vitro effects of 4-CmC on muscle specimens from MHS and normal (MHN) patients, and whether contracture testing with different concentrations of 4-CmC could result in a more precise discrimination between MHS and MHN. METHODS: In this prospective study muscle biopsies were obtained from 40 patients with clinical suspicion of MH. The patients were first classified by the in vitro contracture test (IVCT) according to the European MH protocol. After MH classification, surplus muscle specimens were subjected to the 4-CmC study. RESULTS: Cumulative administration of 4-CmC (25, 50, 75, 100, 150, and 200 mumol/l) produced contractures in a concentration-dependent manner. However, contractures developed significantly earlier and were greater in MHS (n = 17) than in MHN specimens (n = 23). After bolus administration of 50, 75, and 100 mumol/l 14-CmC MHS specimens developed distinct muscle contractures. In contrast, in MHN specimens only 100 mumol/l 4-CmC produced contractures. All contracture levels following bolus administration of 100 mumol/l 4-CmC were attained significantly earlier in MHS than in MHN. There was no overlapping in the range of times between both groups. CONCLUSION: In vitro contracture testing with 4-CmC seems to be a specific method to distinguish between MHS and MHN patients. However, the question whether 4-CmC is an MH-triggering agent is not completely solved. 4-CmC is a preservative within a large number of commercially available preparations (e.g. insulin, hormones, etc.). Regarding the results of contracture testing with 4-CmC it has been suggested that 4-CmC possibly represents a high-risk agent for MHS individuals. To reduce the risk of MH in susceptible patients due to administration of chlorocresols, we recommend avoiding preparations containing the preservative 4-CmC.     

Effects of the 5-HT2 receptor antagonist ritanserin on halothane-induced increase of inositol phosphates in porcine malignant hyperthermia

Recent studies have shown a significant increase of inositol phosphates (IPs) in skeletal muscle during episodes of halothane-induced malignant hyperthermia (MH) in pigs. After treatment with dantrolene and disappearance of MH crisis the IP concentrations returned to basal levels. In order to examine if the increase of IPs during halothane-induced MH may be related to an enhanced IP synthesis in response to activation of 5-HT2 (5-hydroxytryptamine) receptors, the effects of ritanserin, a selective 5-HT2 receptor antagonist, on IP levels were investigated. Biopsies of skeletal muscle of the hindlimbs were obtained in random order and IPs were determined in homozygous MH-susceptible (MHS) and MH-non-susceptible (MHN) swine in the following order: (1) basal, (2) after treatment with ritanserin (2.0 mg/kg), (3) after halothane challenge (3 vol% for 20 min). Basal concentrations of all IPs were higher in MHS than in MHN swine. Ritanserin did not cause any significant changes of IP levels compared to the basal concentrations in MHS and MHN pigs. In MHS pigs, ritanserin did not prevent a halothane-induced MH-crisis. After halothane challenge, 1,3,4-IP3, 1,3,4,6-IP4 and 1,3,4,5-IP4 levels were increased in MHS (during MH crisis) vs. basal concentrations, whereas no changes were found in MHN pigs. Since the increases of IP levels in MHS pigs during MH crisis found in the present study were comparable to those without pretreatment with ritanserin, shown by recent studies, it may be concluded that ritanserin does not prevent the increase of IPs during a halothane-induced MH. Thus, the present data indicate that increases of IP levels during halothane-induced MH in swine are due to other mechanisms than 5-HT mediated enhancement of IP synthesis.     

Chlorocresol, an additive to commercial succinylcholine, induces contracture of human malignant hyperthermia-susceptible muscles via activation of the ryanodine receptor Ca2+ channel

BACKGROUND: A defect in the ryanodine (Ry1) receptor Ca2+ channel has been implicated as one of the possible underlying causes of malignant hyperthermia (MH), a pharmacogenetic disorder characterized by sustained muscle contracture. The disease is triggered by common halogenated anesthetics and skeletal muscle relaxants, such as succinylcholine. This study tested whether the functional properties of the Ry1 receptor Ca2+ channel are affected by chlorocresol, a preservative added to a commercial preparation of succinylcholine (Midarine) and other parenteral compounds. METHODS: In vitro contracture testing was carried out on muscle biopsies from malignant hyperthermia-susceptible (MHS) and -negative (MHN) individual according to the protocol of the European MH group. Ca2+ flux studies on isolated rabbit sarcoplasmic reticulum fractions were measured spectrophotometrically by following the A710-790 of the Ca2+ indicator antipyrylazo III. RESULTS: Chlorocresol causes muscle contracture in MHS muscles at a concentration of 25-50 microM and potentiates the caffeine contracture response in human MHS muscles. Sub-threshold (20 microM) concentrations of chlorocresol increase both the Kd and the Vmax of caffeine-induced Ca2+ release from isolated rabbit terminal cisternae. CONCLUSIONS: These data suggest that, in muscle from MHS individuals, the enhanced Ca2+ released from the sarcoplasmic reticulum may not be due to the effect of succinylcholine alone but rather to the action of the preservative chlorocresol added to the drug.     

Fiber-type caffeine sensitivities in skinned muscle fibers from humans susceptible to malignant hyperthermia

BACKGROUND: The response to contracture tests may depend upon the relative proportion of muscle fiber types within the muscle specimen. To determine whether a difference in fiber-type caffeine sensitivities exists between malignant hyperthermia susceptible (MHS) and malignant hyperthermia-nonsusceptible (MHN) skeletal muscle, we compared the fiber-type caffeine sensitivities in chemically skinned muscle fibers dissected from vastus lateralis muscle from 15 MHS and 16 MHN patients. METHODS: Muscle fiber type was determined in each fiber by the difference in strontium-induced tension measurements and in 36 fibers, after contracture testing, by ATPase enzyme histochemistry. Caffeine sensitivity was defined as the threshold concentration inducing more than 10% of the maximal tension obtained with a calcium 1.6 x 10(-2) mM solution. RESULTS: Significant difference in the mean (+/- SD) caffeine sensitivity was found between type I MHS fibers (2.63 +/- 0.85 mM) versus type II MHS fibers (3.47 +/- 1.2 mM) and between type I MHN fibers (5.89 +/- 1.8 mM) versus type II MHN fibers (10.46 +/- 2.6 mM). The mean (+/- SD) caffeine sensitivities for a given muscle fiber type (I or II) were different between groups of MHS and MHN patients. Both type I and II MHS fibers had significantly lower caffeine sensitivities, and this increase in caffeine sensitivity was significantly smaller in type I than in type II fiber. CONCLUSIONS: The current study indicates that a truly MHS patient cannot have a false-negative result solely related to abnormal type II fibers contained in a given muscle strip. Although the occurrence of a very high proportion of type I fibers in MHN human muscle could result in a false-positive contracture outcome, such an occurrence is expected to be rare.     

Diagnosis of malignant hyperthermia susceptibility. 1. The significance of in vitro susceptibility tests

Molecular genetic findings indicate genetic heterogeneity in susceptibility to malignant hyperthermia (MH). At present the in vitro contracture test (IVCT) is still the most reliable diagnostic procedure for MH susceptibility. It must be performed in a standardized fashion. METHODS. We investigated 350 patients (233 children and 117 adults) using the protocol of the European MH Group for the IVCT. The test results were classified as susceptible to MH (MHS), non-susceptible to MH (MHS), non-susceptible to MH (MHN) and equivocal (MHE), with an abnormal caffeine result designated MHEc and an abnormal halothane result designated MHEh. Reasons for the IVCT were a positive family history for MH susceptibility (n = 94), a MH reaction (n = 157), creatine kinase elevation unknown aetiology (n = 53) and different neuromuscular diseases (NMD, n = 46). Physical, neurological and laboratory work-up included serum enzymes, nerve conduction studies, electromyography and muscle biopsy evaluated by different techniques. Thirty-one children and 11 adults were MHS, while 152 children and 80 adults were MHN. MHE findings were obtained in 50 children and 26 adults. While the MHS and MHN groups are diagnostically safe, the equivocal group is not, with possible false-negative or false-positive interpretation. The high number of MHE findings most probably is explained by the high proportion of patients with NMD (53% of the children, 69% of the adults). RESULTS. In a group of 18 boys with Duchenne or Becker muscular dystrophy, ranging in age from 1.5 to 24 years, the IVCT results were twice MHS, once MHE, and MHN in the remaining 15 cases. In seven other boys with Duchenne or Becker muscular dystrophy, proven by molecular techniques, there were anaesthetic complications with MH-like symptoms. After administration of trigger substances, five out of the seven suffered a cardiac arrest, two of whom died. In the surviving five boys the IVCT results were three times MHN, once MHE and once MHS. Most probably these boys suffered from effects of succinylcholine, possibly potentiated by other trigger substances. The adverse cardiac reactions are attributed to triggered rhabdomyolysis with associated hyperkalemia but not a primary hereditary disposition to MH. CONCLUSION: In patients with NMD, MHS and MHE test results do not indicate a hereditary, heterogeneous disposition to MH; the majority will be caused by a secondary induced disturbance of calcium homoeostasis in the diseased muscle cells. These results do, however, indicate the following: (1) Patients with NMD exposed to trigger substances are at higher risk than the general population for MH-like episodes, including sudden death. (2) NMD therefore should be diagnosed as early as possible and patients should not be exposed to trigger substances when alternatives are at hand. (3) Diagnostic procedures in patients having suffered an MH-like episode should include IVCT and special investigations to exclude or substantiate other NMD. The work-up may be changed if a family member is properly classified as MH susceptible. (4) In patients with known NMD there is no indication for performing IVCT, since the results may even be misleading.     

Voltage-dependent calcium release in human malignant hyperthermia muscle fibers

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

The G1021A substitution in the RYR1 gene does not cosegregate with malignant hyperthermia susceptibility in a British pedigree

A single base change in the RYR1 gene encoding the skeletal muscle ryanodine receptor (calcium-sensitive calcium-release channel of the sarcoplasmic reticulum), resulting in the substitution of G1021 by A, has been proposed to underlie malignant-hyperthermia (MH) susceptibility in as many as 10% of cases in the European population. As part of our mutation-screening program in MH-susceptible (MHS) individuals, we have investigated this substitution in individuals from 151 unrelated British MHS families and have detected G1021A heterozygotes in 7 families. This mutation was not found in 156 unrelated MH-negative (MHN) individuals. We also examined eight families with central core disease (CCD): the mutation did not occur in any family members of any disease status (affected or unaffected for CCD, MHS, or MHN). In one large family, the G1021A mutation was found but did not show complete cosegregation with MH susceptibility: it occurred in only 7/12 MHS individuals in the kinship, and susceptibility was inherited from parents who were G1021 homozygotes, as well as from parents who were heterozygotes. On the basis of these findings, it is clearly unreliable at present to offer presymptomatic DNA testing for MH status, even in families in which a mutation has been detected.     

Stimulatory and inhibitory effects of serotonergic hallucinogens on spinal mono- and polysynaptic reflex pathways in the rat

The effects of two 5-HT-related hallucinogens on rat spinal mono- and polysynaptic reflex pathways in the rat were investigated. 5-Methoxy-N,N-dimethyltryptamine (5-MeODMT, 1 and 100 micrograms/kg, i.v.), an indolealkylamine agent, produced a dose-dependent decrease in the monosynaptic reflex, whereas 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI, 1-100 micrograms/kg), a phenylalkylamine agent, produced a dose-dependent increase in the monosynaptic reflex. Both agents increased the polysynaptic reflex. The 5-HT2 receptor antagonists ketanserin (100 micrograms/kg) and ritanserin (100 micrograms/kg) blocked the effects of DOI on the monosynaptic reflex but only partially blocked the 5-MeODMT-induced effect on the monosynaptic reflex. These antagonists inhibited the change in polysynaptic reflex, induced by DOI but not by 5-MeODMT. Neither propranolol (1 mg/kg) nor 3-tropanyl-3,5-dichlorobenzoate (MDL 72222, 1 mg/kg) antagonized the effect of either agent. 5-Methoxy-N,N-dimethyltryptamine and DOI increased the excitability of motoneurons and this effect was inhibited by ketanserin. These results indicate that the two types of hallucinogens possess both common and distinct characteristics, with regard to their action on the spinal reflex: (1) both increase the activity of motoneurons through 5-HT2 receptors but (2) only 5-MeODMT has an inhibitory action on the pathway of the monosynaptic reflex.     

Definition of a diagnostic score of malignant hyperthermia using P-31 magnetic resonance spectroscopy

OBJECTIVES: To assess the metabolic muscular disorders associated with malignant hyperthermia (MH) using 31-P MRS, in order to develop a diagnostic tool for MH. STUDY DESIGN: Retrospective analysis of a series of case. PATIENTS: Group of 39 subjects, including 13 MH susceptible (MHS), and 26 not susceptible (MHN) members of recognized MHS families, according to the results of the in vitro contracture tests. METHODS: Each subject underwent two protocols, both including rest, exercise and recovery periods. Exercise was performed successively under aerobic and ischaemic conditions. RESULTS AND DISCUSSION: A significant early acidosis was recorded for the MHS group under both conditions of exercise (normoxia and ischaemia). However, the sensitivity of this parameter (77%) was not high enough to be considered as discriminant. Therefore a MRS score has been defined, corresponding to the sum of metabolic anomalies (acidosis, anomalies in PCr tum-over and in ATP or phosphomonoesters concentrations) recorded throughout both protocols. This score provided satisfactory results for both sensitivity (93%) and specificity (93%). CONCLUSION: 31-P MRS can act as a reliable diagnostic tool for MH.     

In vivo electrophysiological characterization of 5-HT receptors in the guinea pig head of caudate nucleus and orbitofrontal cortex

The aim of the present study was to characterize in vivo the 5-HT receptor subtypes which mediate the effect of microiontophoretic applied 5-HT in the guinea pig head of caudate nucleus and orbitofrontal cortex. 5-HT and the preferential 5-HT2A receptor agonist DOI and the preferential 5-HT2C receptor agonist mCPP, suppressed the quisqualate (QUIS)-induced activation of neurons in both structures. The inhibitory effect of DOI and mCPP was not prevented by acute intravenous administration of the 5-HT1/2 receptor antagonist metergoline (2 mg/kg) and the 5-HT2A/2C receptor antagonist ritanserin (2 mg/kg) in the two regions nor by the selective 5-HT2A receptor antagonist MDL100907 (1 mg/kg) in the head of caudate nucleus. However, the inhibitory effect of DOI, but not that of mCPP, was antagonized by a 4-day treatment with metergoline and ritanserin (2 mg/kg/day; using minipumps implanted subcutaneously) in head of caudate nucleus, but not in orbitofrontal cortex. Microiontophoretic ejection of the 5-HT1A/7 receptor agonist 8-OH-DPAT and of the 5-HT1A receptor antagonist WAY100635 both suppressed the spontaneous and QUIS-activated firing activity of orbitofrontal cortex neurons. At current which did not affect the basal discharge activity of the neuron recorded, microiontophoretic application of WAY100635 and BMY7378 failed to prevent the inhibitory effect of 8-OH-DPAT. The inhibitory effect of gepirone, which is a 5-HT1A receptor agonist but devoid of affinity for 5-HT7 receptors, was also not antagonized by WAY100635. Altogether, these results suggest the presence of atypical 5-HT1A receptors in the orbitofrontal cortex. The present results also indicate that the suppressant effect of DOI may be mediated by 5-HT2A receptors in head of caudate nucleus and atypical 5-HT2 receptors in orbitofrontal cortex.     

Modulation of brainstem 5-HT1C receptors by serotonergic drugs in the rat

1. The sparse population of brainstem 5-hydroxytryptamine1C (5-HT1C) (also called 5-HT2C) receptors has received little attention despite its possible role in the serotonin syndrome and 5-HT-mediated shaking behavior. We characterized [3H]mesulergine binding in rat brainstem and, to determine if brainstem 5-HT1C sites respond to serotonergic manipulations, performed saturation studies of [3H]mesulergine binding in brainstem from rats treated chronically with 11 different 5-HT1C/2 agonists and antagonists. 2. In competition studies in vitro, the rank order of drug potency was most compatible with a 5-HT1C receptor binding site: mianserin, 5-HT, cinanserin, 1-(3-chlorophenyl)piperazine (m-CPP), 1-(2-5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), MDL 100,907, RU 24969, 5-carboxamidotryptamine (5-CT), 8-OH-DPAT, MDL 72,222. 3. Chronic treatment with the agonists quipazine and trifluoromethylphenylpiperazine (TFMPP) and the antagonists ritanserin and methiothepin significantly down-regulated brainstem 5-HT1C sites, which were 65% of [3H]mesulergine-labeled sites in brainstem. Only metergoline and ritanserin significantly increased pKD. 4. Chronic treatment in vivo with DOI, m-CPP, mianserin, methysergide, spiperone, cyproheptadine, and metergoline had no significant effect on BMAX at the dose studied. 5. These data suggest similarities in the regulation of 5-HT1C and 5-HT2 sites at which both 5-HT1C 2 agonists and antagonists also induce receptor down-regulation. 6. 5-HT1C/2 agonists and antagonists that did not down-regulate brainstem 5-HT1C sites may be more active in vivo at 5-HT2 sites, at 5-HT1C sites in other brain regions, have effects on 5-HT1C receptors not detectable at the recognition site, or differ for pharmacokinetic reasons.     

Effect of chlorocresol vs caffeine on muscle contracture in malignant hyperthermia susceptible patients

The phenotype of susceptibility to malignant hyperthermia (MHS); can only be detected reliably by the in vitro caffeine-halothane contracture test (CHCT). Enhanced sensitivity of the calcium-induced calcium release mechanism is responsible for the exaggerated contracture response of skeletal muscle fibers from MHS patients to halothane and caffeine. Chlorocresol was demonstrated to be a potent activator of Ca++ release from skeletal muscle sarcoplasmic reticulum. This effect is probably mediated through action on a ryanodine sensitive Ca++ release channel known to be more sensitive in MH. We studied the effect of chlorocresol on the mechanical contracture response of skeletal muscle from patients presenting for the in vitro CHCT. Chlorocresol induces contracture response in a concentration 1/200 of that of caffeine in muscle strips from MH patients. By adding chlorocresol to the protocol of the CHCT, there is clearer discrimination between the responses of MH patients and normal subjects can be achieved.     

Nodal tumour growth in breast cancer: the time-span of its biological progression

Malignant hyperthermia (MH) is a rare autosomally dominantly hereditary and potentially life-threatening disease. The prevalence of the genetic MH predisposition is estimated as 1:10,000 to 1:20,000. In Germany no data on the regional distribution are available. Therefore, the purpose of this investigation is to summarise and present the epidemiological data of all German MH laboratories. Nine German hospitals offer the specific in vitro contracture test to diagnose the MH predisposition. All German MH laboratories carry out the examination in accordance with the standardised protocol of the European Malignant Hyperthermia Group. The laboratories were asked to provide the number of all patients investigated, excluding those suffering from other neuromuscular diseases, separated according to diagnostic groups and their places of residence, the number of the identified MH-families as well as the number of the clinically suspected and investigated MH cases with their places of residence. Eight MH laboratories provided the requested data. Until September 1997 a total of 2620 patients were investigated. In 865 patients (34%) MH suspicion was confirmed (diagnosis: MHS). 1494 patients (56%) were released by investigation from MH-suspicion (diagnosis: MHN). In 261 patients (10%) the MH-predisposition remained unsolved (diagnosis: MHE). 580 MH families were identified. Among 2620 patients 757 were clinically suspected MH cases. 35% of these suspected MH cases were classified as MHS, 10% as MHE and 55% as MHN. The documentation of the patients places of residence classified as MHS and MHE into a map of Germany demonstrates an exhaustive distribution with an increased regional prevalence in the areas of the MH laboratories. This concentration in the area of the MH laboratories becomes even more evident, when the places of residence of the MH suspected cases are demonstrated. In conclusion, the distribution of the MH predisposition is uniform and exhaustive in Germany. The presented regional concentration of clinically suspected MH cases among the MH laboratories is mainly interpreted as an expression of effective regional education and information. Considering the overall incidence of the MH predisposition as described above only 15-20% of the MH patients have so far been identified. The MH laboratories have already released about 10,000 patients from the suspicion of MH predisposition. A preliminary prevalence of at least 1:60,000 to 1:80,000 in Germany can be estimated according to the presented data.     

In vitro 31P-magnetic resonance spectroscopy of muscle extracts in malignant hyperthermia-susceptible patients

BACKGROUND: It was recently suggested that malignant hyperthermia-susceptible (MHS) patients could have an elevated peak of phosphodiesters in leg muscles using in vivo phosphorus magnetic resonance spectroscopy. In the current study, analysis of the phosphodiesters of muscle extracts of MHS and malignant hyperthermia-negative patients was performed using in vitro phosphorus magnetic resonance spectroscopy to chemically identify and to compare the muscle concentrations of water-soluble compounds between the two groups with respect to the muscle fiber type composition. METHODS: Perchloric acid extracts of the vastus medialis muscle of seven MHS patients and ten malignant hyperthermia-negative patients on the basis of the European malignant hyperthermia contracture test were subjected to in vitro phosphorus magnetic resonance spectroscopy carried out at 9.4 T. In addition, chemical identification of the phosphodiester region and histologic examination of the muscle specimens were performed. RESULTS: The peak in the phosphodiester region was assigned to glycerophosphorylcholine. Muscle perchloric acid extracts of MHS patients had a significantly (P < 0.05) higher glycerophosphorylcholine to the sum of phosphocreatine and inorganic phosphate (glycerophosphorylcholine/ [phosphocreatine +inorganic phosphate]) value than those of malignant hyperthermia-negative patients. Neither a difference in the fiber type composition between the two groups nor any specific myopathy were found. CONCLUSIONS: In the absence of histologic differences between muscle specimens of MHS and malignant hyperthermia-negative patients, these results could suggest that glycerophosphorylcholine could be a marker of an impairment in the phospholipid metabolism in the skeletal muscle of MHS patients.     

Evidence for specific involvement of 5-HT1A and 5-HT2A/C receptors in the expression of patterns of spontaneous motor activity of the rat

The 5-HT1A and the 5-HT2A/C receptor agonists 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (0.006-0.4 mg kg-1 s.c.) and (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (0.05-4.0 mg kg-1 s.c.), respectively, produced a similar stereotyped forward locomotion in rats, although the intensity of the behavioral change was considerably less with DOI. The stereotyped forward locomotion was accompanied by a slight decrease in total activity, suppression of rearing behavior and an increased activity in the periphery of the open-field arena. In support of receptor specificity, the effects of 8-OH-DPAT and DOI could be antagonised by pretreatment with the 5-HT1A/B and the 5-HT2A/C receptor antagonists (-)-pindolol (2 mg kg-1 s.c.) and ritanserin (2 mg kg-1 s.c.), respectively. In addition, (-)-pindolol, but not the selective beta-adrenoceptor antagonist betaxolol, markedly enhanced the behavioral effects produced by DOI. The nature of these specific actions and interactions in terms of pre- and post-synaptic serotonergic mechanisms remains an important question.     

Activation of two types of fibres in rat extraocular muscles

1. The contractile responses of the inferior rectus, one of the extraocular muscles of the rat, to a depolarization induced by an elevation of the potassium concentration in the external medium ([K]O) have been studied 'in vitro'. 2. The elevation of [K]O to 20 and 30 mM-K produced contractures that consisted of a sustained or tonic tension. When [K]O was increased to 50 mM or more a well-defined transient or phasic tension appeared before the tonic response. The increment of [K]O above 50 mM enhanced the phasic component and depressed the tonic tension. The maximal tonic tension, usually evoked by 50 mM-K, is about 50% of the tetanic tension, shows a gradual decline with time and lasts for hours. Control experiments performed in diaphragm showed that this muscle only responds with phasic tensions. 3. The difference in the repriming of the phasic and tonic responses when tensions were induced with salines containing low or normal [Cl] suggests that the muscle fibres responsible for the tonic tension are poorly permeable to Cl-. 4. The amplitude of the tonic tension was reduced by Ca deprivation and by an elevation of [Ca] in the saline to 10 mM. 5. It is concluded that in rat extraocular muscles, an increase in [K]O activates two types of muscle fibres: singly and multiply innervated. These appear to be functionally equivalent to the twitch and slow fibres of amphibian and avian muscle and would give rise to the phasic and tonic components of the contracture, respectively.     

An investigation into the effects of 5-HT agonists and receptor antagonists on ethanol self-administration in the rat

Pharmacological manipulation leading to altered 5-HT function has been widely demonstrated to reduce ethanol intake in free choice tests. The aim of the present study was to examine the effects of a range of compounds known to influence 5-HT neurotransmission, including selective 5-HT receptor agonists and antagonists, on ethanol ingestion and maintained behaviour in an operant self-administration paradigm. Female Sprague-Dawley rats were trained to respond for 8% ethanol (v/v) in a 60-min test by a previously described technique. The number of responses and ethanol reinforcers (dipper deliveries), ethanol consumption (g/kg of body weight), and locomotor activity (LMA) were measured following administration of 5-HT agonists (5-HT, d-fenfluramine, fluoxetine, buspirone, TFMPP, and DOI) and antagonists (metergoline, ritanserin, and ondansetron) 30 min prior to testing. d-Fenfluramine, fluoxetine, buspirone, TFMPP, and DOI all produced a reduction in ethanol ingestion and maintained behaviour at doses that failed to reduce LMA. Conversely, metergoline and ritanserin only reduced ethanol self-administration at doses that concomitantly reduced LMA. 5-HT and ondansetron were without effect on any measure. These results demonstrate that, under the present experimental conditions, activation of central 5-HT1A, 5-HT1B, and 5-HT2 receptors reduced ethanol intake and reinforced behaviour in an operant paradigm.     

cAMP-signaling pathway acts in selective synergism with glucose or tolbutamide to increase cytosolic Ca2+ in rat pancreatic beta-cells

The effect of repeated cocaine administration on serotonin2 (5-HT2) receptor function was examined in male rats. Rats were fitted with indwelling jugular catheters and subsequently received cocaine (15 mg/kg, i.p., b.i.d.) or saline for 7 days. Rats were challenged with the 5-HT2 agonist DOI (25, 100, 400 micrograms/kg, i.v.) or saline 42 hr and 8 days after cessation of chronic treatment. Serial blood samples were collected at various times after DOI challenge and analyzed for prolactin levels. DOI-induced head shakes and skin jerks were examined concurrently in the same subjects. After 42 hr of withdrawal, the stimulatory effects of DOI on prolactin release and shaking behavior were significantly enhanced in cocaine-treated rats. Conversely, the skin jerk response to DOI was not altered by prior cocaine exposure. After 8 days of withdrawal, the prolactin and head shake responses to DOI were still potentiated in cocaine-treated rats, but this effect was no longer statistically significant. The data indicate that chronic cocaine enhances the sensitivity of 5-HT2 receptor mechanisms. Our findings further suggest the possibility that altered 5-HT2 receptor function may be involved in the mood disturbances experienced by abstinent cocaine addicts.     

Reduced inhibitory effect of Mg2+ on ryanodine receptor-Ca2+ release channels in malignant hyperthermia

Malignant hyperthermia (MH) is a potentially fatal, inherited skeletal muscle disorder in humans and pigs that is caused by abnormal regulation of Ca2+ release from the sarcoplasmic reticulum (SR). MH in pigs is associated with a single mutation (Arg615Cys) in the SR ryanodine receptor (RyR) Ca2+ release channel. The way in which this mutation leads to excessive Ca2+ release is not known and is examined here. Single RyR channels from normal and MH-susceptible (MHS) pigs were examined in artificial lipid bilayers. High cytoplasmic (cis) concentrations of either Ca2+ or Mg2+ (>100 microM) inhibited channel opening less in MHS RyRs than in normal RyRs. This difference was more prominent at lower ionic strength (100 mM versus 250 mM). In 100 mM cis Cs+, half-maximum inhibition of activity occurred at approximately 100 microM Mg2+ in normal RyRs and at approximately 300 microM Mg2+ in MHS RyRs, with an average Hill coefficient of approximately 2 in both cases. The level of Mg2+ inhibition was not appreciably different in the presence of either 1 or 50 microM activating Ca2+, showing that it was not substantially influenced by competition between Mg2+ and Ca2+ for the Ca2+ activation site. Even though the absolute inhibitory levels varied widely between channels and conditions, the inhibitory effects of Ca2+ and Mg2+ were virtually identical for the same conditions in any given channel, indicating that the two cations act at the same low-affinity inhibitory site. It seems likely that at the cytoplasmic [Mg2+] in vivo (approximately 1 mM), this Ca2+/Mg2+-inhibitory site will be close to fully saturated with Mg2+ in normal RyRs, but less fully saturated in MHS RyRs. Therefore MHS RyRs should be more sensitive to any activating stimulus, which would readily account for the development of an MH episode.