Saiid Bina

Effects of halothane on EDRF/cGMP-mediated vascular smooth muscle relaxations.

Background:Halothane has been reported to inhibit endothelium-dependent relaxation in a variety of vessels. These studies were done to determine whether this inhibition is caused by interference with synthesis, release, or action of endothelium-derived relaxing factor (EDRF) on cyclic guanosine monophosphate (cGMP) levels within the vascular smooth muscle. Methods:Rat aortic rings were suspended in aerated Krebs solution (37° C) and were contracted to a stable plateau with EC60–70 norepinephrine (NE). Relaxations caused by acetylcholine (ACh; 1 × 10 −8 − 1 × 10 −6 M), nitric oxide (NO; 5 × 10−9 − 1 × 10−6 M), or nitroglycerin (NG; 2 × 10 −9 − 3 × 10 −7 M) in rings contracted with NE were compared in the presence and absence of halothane. Tissue cGMP contents were measured using a radioimmunoassay method. Results:In the presence of halothane (0.5, 1.0, and 2.0 MAC), the ACh-induced relaxations were significantly attenuated in a concentration-dependent manner, an effect that was reversible. Halothane (2 MAC) significantly attenuated NO-induced relaxations at all concentrations and NG-induced relaxations at low concentrations (5 × 10−9 − 3 × 10−8 M) but not at higher concentrations (1 × 10−9 − 3 × 10−7 M) in denuded vessels. Nitric oxide-stimulated (5 × 10−8 − 5 × 10−6 M) cGMP content was significantly attenuated by halothane (2 MAC) at NO concentrations between 1 × 10−7 and 5 × 10−6 M. Conclusions:Nitric oxide, either endogenous or exogenous, interacts with the enzyme guanylate cyclase to stimulate the production of cGMP. Halothane interfered with the relaxations caused by NO (in rings without endothelium) and decreased the NO-stimulated cGMP content. These results suggest that the site of action of halothane in attenuating endothelium-dependent relaxation in the rat aorta is within the vascular smooth muscle, rather than on the synthesis, release, or transit of the EDRF from the endothelium and that its action may involve an interference with guanylate cyclase activation.

Identical de novo Mutation in the Type 1 Ryanodine Receptor Gene Associated with Fatal, Stress-induced Malignant Hyperthermia in Two Unrelated Families

Background: Mutations in the type 1 ryanodine receptor gene (RYR1) result in malignant hyperthermia, a pharmacogenetic disorder typically triggered by administration of anesthetics. However, cases of sudden death during exertion, heat challenge, and febrile illness in the absence of triggering drugs have been reported. The underlying causes of such drug-free fatal “awake” episodes are unknown. Methods: De novo R3983C variant in RYR1 was identified in two unrelated children who experienced fatal, nonanesthetic awake episodes associated with febrile illness and heat stress. One of the children also had a second novel, maternally inherited D4505H variant located on a separate haplotype. Effects of all possible heterotypic expression conditions on RYR1 sensitivity to caffeine-induced Ca2+ release were determined in expressing RYR1-null myotubes. Results: Compared with wild-type RYR1 alone (EC50 = 2.85 ± 0.49 mM), average (±SEM) caffeine sensitivity of Ca2+ release was modestly increased after coexpression with either R3983C (EC50 = 2.00 ± 0.39 mM) or D4505H (EC50 = 1.64 ± 0.24 mM). Remarkably, coexpression of wild-type RYR1 with the double mutant in cis (R3983C-D4505H) produced a significantly stronger sensitization of caffeine-induced Ca2+ release (EC50 = 0.64 ± 0.17 mM) compared with that observed after coexpression of the two variants on separate subunits (EC50 = 1.53 ± 0.18 mM). Conclusions: The R3983C mutation potentiates D4505H-mediated sensitization of caffeine-induced RYR1 Ca2+ release when the mutations are in cis (on the same subunit) but not when present on separate subunits. Nevertheless, coexpression of the two variants on separate subunits still resulted in a ∼2-fold increase in caffeine sensitivity, consistent with the observed awake episodes and heat sensitivity.

Exertional rhabdomyolysis and malignant hyperthermia in a patient with ryanodine receptor type 1 gene, L-type calcium channel α-1 subunit gene, and calsequestrin-1 gene polymorphisms

THE relationship between hypermetabolic exertional stress injuries and malignant hyperthermia (MH) has been a topic of debate for almost 30 yr. Central to this debate is the idea that some MH susceptible (MHS) patients may develop awake nonanesthesia-related manifestations similar to that seen in porcine stress syndrome. Although a link has never been established by controlled clinical studies, individual case reports and a small number of clinical series support an association between unexpected exertional rhabdomyolysis (ER) and MH susceptibility, two syndromes characterized by abnormal intracellular skeletal muscle calcium regulation. An individual is identified as MHS if he or she has a well-documented clinical episode consistent with MH during exposure to any of the known anesthetic triggering agents, or if he or she has undergone a skeletal muscle biopsy with a positive diagnostic contracture test. However, none of the published reports of patients who presented first with ER and who were subsequently identified as MHS by a positive contracture test ever developed documented clinical MH episodes involving anesthesia. We present a case that is compelling for two reasons. First, it is the only documented case of an individual who presented first with ER, followed by a clinical MH episode during anesthesia, and then by a positive contracture test. Second, genetic analysis revealed the presence of variants in the ryanodine receptor type 1 gene (RYR1), the L-type calcium channel -1 subunit gene (CACNA1S), and the calsequestrin-1 gene (CASQ1). This report provides clinical evidence for an association between ER and MH and discusses the possible role for synergistic action among rare variants in the genes encoding proteins crucial to skeletal muscle calcium regulation.

Vascular effects of halothane and isoflurane: cGMP dependent and independent actions

This study investigated the effects of halothane and isoflurane on cGMP-dependent and independent regulation of vascular contraction of the isolated rat aorta and on NO-stimulated soluble guanylate cyclase (sGC) isolated from the perfused rat liver. For the studies of the aorta, isometric tension of isolated rings, with and without, endothelium was recorded and cGMP content measured. ACh was used to initiate endothelial-dependent relaxation of norepinephrine (NE)-contracted rings while NO was used to directly stimulate isolated aortic ring sGC which catalyzes the isolated aortic ring formation of cGMP. Both halothane and isoflurane interfered with ACh and NO relaxations and with NO-stimulated increases in cGMP. Halothane was more potent, having significant attenuating effects at 0.34 mM (1 MAC) and 0.72 mM (2 MAC) while isoflurane had effects only at 0.53 mM (2 MAC). For the isolated sGC studies, a soluble liver fraction was prepared from perfused rat livers. In the absence of NO stimulation, neither halothane nor isoflurane modified the activity of the sGC. However, during NO-stimulation halothane produced significant, concentration-dependent, inhibition of sGC activity over a wide range of NO concentrations. Isoflurane also inhibited sGC activity, but to a lesser extent than halothane. The mechanism whereby the anesthetics could interfere with sGC from liver and blood vessels is unknown. It could result from anesthetic interaction at hydrophobic sites that may exist in GC. However, the results of both the aorta and liver sGC enzyme studies support the suggestion that these anesthetics can compete with NO for its binding site on the ferrous heme of sGC, with chemical structural differences accounting for the potency variations. Both anesthetics also had cGMP independent effects, causing concentration dependent relaxations of NE-contracted vessels without endothelium. Isoflurane was about 5 times more effective at 1 MAC than halothane. Therefore, the net effects of these anesthetics involve the sum of two opposite effects on tension of vessels with intact endothelium: 1) interference with NO-stimulated cGMP relaxation and 2) direct stimulation of relaxation (not dependent on changes in cGMP).

The ryanodine receptor type 1 gene variants in African American men with exertional rhabdomyolysis and malignant hyperthermia susceptibility

It has been suggested that exertional rhabdomyolysis (ER) and malignant hyperthermia (MH) are related syndromes. We hypothesize that patients with unexplained ER harbor mutations in the ryanodine receptor gene type 1 (RYR1), a primary gene implicated in MH, and therefore ER patients are at increased risk for MH. Although there are reported cases of MH in individuals of African descent, there are no data available on molecular characterization of these patients. We analyzed RYR1 in six, unrelated African American men with unexplained ER, who were subsequently diagnosed as MH susceptible (MHS) by the Caffeine Halothane Contracture Test. Three novel and two variants, previously reported in Caucasian MHS subjects, were found in five studied patients. The novel variants were highly conserved amino acids and were absent among 230 control subjects of various ethnic backgrounds. These results emphasize the importance of performing muscle contracture testing and RYR1 mutation screening in patients with unexplained ER. The MHS‐associated variant Ala1352Gly was identified as a polymorphism predominant in individuals of African descent. Our data underscore the need for investigating RYR1 across different ethnic groups and will contribute to interpretation of genetic screening results of individuals at risk for MH.

Patients with Malignant Hyperthermia Demonstrate an Altered Calcium Control Mechanism in B Lymphocytes

Background Altered Ca2+ homeostasis in skeletal muscle is a key molecular event triggering malignant hyperthermia (MH) in malignant hyperthermia-susceptible (MHS) individuals. Genetic studies have shown that mutations in the type 1 ryanodine receptor (RYR1) are associated with MH susceptibility. Because human B lymphocytes express the RYR1, it is hypothesized that Ca2+ homeostasis in B lymphocytes is altered in MHS individuals. Methods This study investigated the Ca2+ response of B cells to caffeine and 4-chloro-m-cresol in 13 MHS and 21 MH-negative (MHN) individuals who had been diagnosed by caffeine halothane contracture test (CHCT) and 18 healthy volunteers. Changes in [Ca2+]i in B cells were measured directly in fluo-3 loaded cells using a dual-color flow cytometric technique. Further, B cell phenotype was correlated with CHCT results in a family with the Val2168Met (G6502A) mutation. Results Caffeine-induced (50 mm) increases in [Ca2+]i in B cells were significantly greater in MHS than in MHN (P = 0.0004), control (P = 0.0001) or non-MHS (MHN and control) individuals (P < 0.0001). The 4-chloro-m-cresol-induced (400 &mgr;m) increases in [Ca2+]i were also significantly different between MHS and controls (P = 0.003) or between MHS and non-MHS (MHN and control) individuals (P = 0.0078). A study of a family with the Val2168Met mutation demonstrated expression of the RYR1 mRNA mutant in B cells from the family members with MHS phenotype and a clear segregation of genotype with B-cell phenotype. Conclusion The Ca2+ responses to caffeine or 4-chloro-m-cresol in B lymphocytes showed significant differences between MHS and MHN (or control) individuals. Although the molecular mechanisms of these alterations are currently undetermined, the results suggest that the enhanced Ca2+ responses are associated with mutations in the RYR1 gene in some MHS individuals.

Serum Ropivacaine Concentrations and Systemic Local Anesthetic Toxicity in Trauma Patients Receiving Long-Term Continuous Peripheral Nerve Block Catheters

BACKGROUND: Ropivacaine is a long-acting local anesthetic used frequently for peripheral nerve blocks and continuous peripheral nerve block catheters. Combat trauma patients at Walter Reed Army Medical Center often receive continuous peripheral nerve block catheters as part of their pain regimen. These catheters remain in situ for several days to weeks. In this study, we evaluated the free ropivacaine drug levels over time in trauma patients by measuring the serum concentration of bound and unbound local anesthetic. The corresponding &agr;1-acid glycoprotein concentration in patients with prolonged ropivacaine infusions was also measured. METHODS: Fifteen patients were enrolled in the study; 2 patients were excluded because only a single ropivacaine level was obtained. Of the remaining 13 patients in the study, 2 had peripheral nerve catheters placed at the time of enrollment; the remaining 11 patients had catheters placed before enrollment. These patients were already receiving 0.2% ropivacaine infusions for a period of 18–126 h before the first assessment of local anesthetic level. Catheters infused 0.2% ropivacaine at a rate of 6–14 mL/h; catheter boluses were administered with 0.5% ropivacaine. Local anesthetic blood concentrations were scheduled to be measured on Days 1, 3, 5, 7, and 10 and every 3 days thereafter until all catheters were removed, although not all patients underwent each assessment. Specimens were assayed using high-performance liquid chromatography for total and free serum ropivacaine concentrations. &agr;1-Acid glycoprotein was also measured. RESULTS: Thirteen patients remained in the study, for a total of 59 blood samples. The median number of days catheters remained in situ for the duration of acute pain therapy was 7 days (range: 6–27 days). The median number of days catheters remained in situ after enrollment into the study was 7 days (range: 4–25 days). The median number of blood samples collected per patient was 4 (range: 2–10 samples). Two patients had isolated increased concentrations of free ropivacaine into a previously identified toxic range with no obvious mitigating factors; both patients had received a 300-mg bolus of 0.5% ropivacaine approximately 24 h before that blood collection. The median ropivacaine concentration over the length of the study was 0.11 mg/L (range: undetectable to 0.63 mg/L). During the first week of the study, the median change in ropivacaine concentration per patient was 0.00 mg/L (range: −0.35 to 0.47 mg/L). CONCLUSION: Although 2 patients demonstrated isolated serum ropivacaine concentration spikes into a previously identified toxic range, continuous peripheral nerve block catheter management and local anesthetic doses as practiced at Walter Reed Army Medical Center did not result in clinically evident systemic ropivacaine toxicity. There was no correlation between free ropivacaine concentration and &agr;1-acid glycoprotein concentration except in patients who had already been receiving ropivacaine infusions before entering the study. Despite this lack of correlation, the total duration of local anesthetic infusion did not seem to influence the free concentration of the drug.

Ryanodine receptor type 1 gene variants in the malignant hyperthermia-susceptible population of the United States.

BACKGROUND:Mutations in the ryanodine receptor type 1 gene (RYR1) that encodes the skeletal muscle–specific intracellular calcium (Ca2+) release channel are a cause of malignant hyperthermia (MH). In this study, we examined RYR1 mutations in a large number of North American MH-susceptible (MHS) subjects without prior genetic diagnosis. METHODS:RYR1 was examined in 120 unrelated MHS subjects from the United States in a tiered manner. The &agr;-1 subunit of the dihydropyridine receptor gene (CACNA1S) was screened for 4 variants in subjects in whom no abnormality was found in ≥100 exons of RYR1. RESULTS:Ten known causative MH mutations were found in 26 subjects. Variants of uncertain significance in RYR1 were found in 36 subjects, 16 of which are novel. Novel variants in both RYR1 and CACNA1S were found in the 1 subject who died of MH. Two RYR1 variants were found in 4 subjects. Variants of uncertain significance were found outside and inside the hotspots of RYR1. Maximal contractures in the caffeine–halothane contracture test were greater in those who had a known MH mutation or variant of uncertain significance in RYR1 than in those who did not. CONCLUSIONS:The identification of novel RYR1 variants and previously observed RYR1 variants of uncertain significance in independent MHS families is necessary for demonstrating the significance of these variants for MH susceptibility and supports the need for functional studies of these variants. Continued reporting of the clinical phenotypes of MH is necessary for interpretation of genetic findings, especially because the pathogenicity of most of these genetic variants associated with MHS remains to be elucidated.

Effects of halothane and isoflurane on carbon monoxide-induced relaxations in the rat aorta

Background Halothane and isoflurane previously were reported to attenuate endothelium‐derived relaxing factor/nitric oxide‐mediated vasodilation and cyclic guanosine monophosphate (cGMP) formation in isolated rat aortic rings. Carbon monoxide has many chemical and physiologic similarities to nitric oxide. This study was designed to investigate the effects of halothane and isoflurane on carbon monoxide‐induced relaxations and cGMP formation in the isolated rat aorta. Methods isometric tension was recorded continuously from endothelium denuded rat aortic rings suspended in Krebs‐filled organ baths. Rings precontracted with submaximal concentrations of norepinephrine were exposed to cumulative concentrations of carbon monoxide (26–176 micro Meter). This procedure was repeated three times, with anesthetics delivered 10 min before the second procedure. Carbon monoxide responses of rings contracted with the same concentration of norepinephrine (10‐8 M and 2 x 10‐8 M) used in the anesthetic‐exposed preparations also were examined. The concentrations of cGMP were determined in denuded rings using radioimmunoassay. The rings were treated with carbon monoxide (176 micro Meter, 30 s) alone, or carbon monoxide after a 10‐min incubation with halothane (0.34 mM or 0.72 mM). To determine whether the sequence of anesthetic delivery influenced results, vascular rings pretreated with halothane were compared with non‐pretreated rings. Results Carbon monoxide (26–176 micro Meter) caused a dose‐dependent reduction of norepinephrine‐induced tension, with a maximal relaxation of 1.51+/‐0.07 g (85+/‐7% of norepinephrine‐induced contraction). Halothane (0.34 mM and 0.72 mM) significantly attenuated the carbon monoxide‐induced relaxations, but only the highest concentration of isoflurane (0.53 mM) significantly attenuated the carbon monoxide‐induced relaxations. Carbon monoxide (176 micro Meter) significantly increased cGMP content (+88.1+/‐7.1%) and preincubation of the aortic rings with halothane (0.34 mM and 0.72 mM) inhibited this increase (‐70.7 +/‐6.8% and ‐108.1+/‐10.6%, respectively). When aortic rings and carbon monoxide were added simultaneously to Krebs solution equilibrated with halothane (0.72 mM), no inhibition of cGMP formation occurred. Conclusion Carbon monoxide‐induced endothelium‐independent relaxations of rat aortic rings were decreased by clinically relevant concentrations of halothane and isoflurane. The carbon monoxide‐induced elevations of cGMP were attenuated by halothane only when the anesthetic was incubated with aortic rings before carbon monoxide treatment. The possible clinical significance of the actions of the anesthetics on this endogenous vasodilator is yet to be determined.

Effects of caffeine, halothane, and 4-chloro-m-cresol on skeletal muscle lactate and pyruvate in malignant hyperthermia-susceptible and normal swine as assessed by microdialysis.

Background:Skeletal muscle fibers from malignant hyperthermia (MH)–susceptible humans and swine are markedly more sensitive to ryanodine receptor (RyR1) agonists than those from normal individuals. Reproducible shifts in the dose–response of skeletal muscle to caffeine and halothane are the basis of the current in vitro diagnostic caffeine–halothane contracture test. In an attempt to develop a less invasive MH diagnostic test, the authors determined the effects of RyR1 agonists (caffeine, 4-chloro-m-cresol [4CmC], and halothane) on the adductor muscle with respect to the lactate–pyruvate (L/P) system that was percutaneously dialyzed using a microdialysis technique in homozygous MH-susceptible compared with normal swine. Methods:Animals were anesthetized (ketamine–propofol) and artificially ventilated. Sets of six CMA/20 microdialysis catheters were implanted; each catheter was perfused with different RyR1 agonist concentrations. After a 30-min equilibration after implantation, one of the catheters was perfused (2 &mgr;l/min) with vehicle (0.9% saline or lipid emulsion), and the other five were perfused with caffeine (1–64 mm), 4CmC (0.1–8 mm), or halothane (prepared in lipid emulsion; 10–500 mm). Outflow dialysate fractions collected at 10-min intervals and L/P parameters were measured enzymatically. Results:Only in the MH-susceptible group did all RyR1 agonists increase dialysate L/P in a dose-dependent manner. The dose–effect relations were most prominent with 4CmC. With the halothane lipid emulsion, data scatter was high compared with that of the caffeine group and especially the 4CmC group. There were no signs of global muscle rigidity, systemic hypermetabolism, or a clinical MH episode during microdialysis RyR1 perfusion. Conclusions:The authors data demonstrate that the in vivo muscle microdialysis of the porcine L/P system reveals distinct differences between MH-susceptible and MH-normal muscle, especially in response to highly specific RyR1 agonists such as 4CmC. The microdialysis L/P technique seems to have an MH diagnostic potential in the clinical setting.