North American malignant hyperthermia population: screening of the ryanodine receptor gene and identification of novel mutations.

BACKGROUND: Malignant hyperthermia (MH) is a disorder of skeletal muscle manifested as a life-threatening hypermetabolic crisis in susceptible individuals after exposure to inhalational anesthetics and depolarizing muscle relaxants. Mutations in the gene encoding the skeletal muscle ryanodine receptor (RYR1) are considered a common cause of the disorder, and, to date, more than 20 RYR1 mutations have been reported in European and Canadian families. Some studies suggest that differences may exist in the frequencies and distribution of mutations in the RYR1 gene between European and North American MH families the frequency and distribution of mutations in the RYR1 gene. METHODS: Skeletal muscle samples from 73 unrelated individuals diagnosed as MH susceptible according to the North American MH caffeine-halothane contracture test were studied. Genomic DNA of MH-susceptible patients was investigated by polymerase chain reaction-based restriction fragment length polymorphism, single-strand conformation polymorphism, and sequencing analysis. The majority of known RYR1 mutations were analyzed using the restriction fragment length polymorphism method, whereas new mutations were searched by single-strand conformation polymorphism in exons 12, 15, 39, 40, 44, 45, and 46 of the gene. RESULTS: Seven known RYR1 mutations (Arg163Cys, Gly248Arg, Arg614Cys, Val2168Met, Thr2206Met, Gly2434Arg, and Arg2454His) were detected at frequencies of 2.7, 1.4, 1.4, 1.4, 1.4, 5.5, and 4.1%, respectively. In addition, three novel amino acid substitutions (Val2214Ile, Ala2367Thr, and Asp2431Asn) were detected at frequency of 1.4% each. These 10 mutations account for 21.9% of the North American MH-susceptible population. CONCLUSION: Three novel candidate mutations in the RYR1 gene were identified in these MH patients. The frequency and distribution of RYR1 mutations observed in this North American MH population was markedly different from that previously identified in Europe. Larger-scale studies are necessary to clarify the type and frequency of mutations in RYR1 associated with MH in North American families.

Prolonging viability of swine muscle biopsy specimens in malignant hyperthermia testing.

In North America, the caffeine halothane contracture test (CHCT) is the standard test for the diagnosis of malignant hyperthermia (MH). Current CHCT protocol recommends that the test be completed within 5 h of muscle excision. The purpose of this study was to investigate whether the period of skeletal muscle viability could be extended to 24 h. We tested the gracilis muscle from normal (n = 8) and MH-susceptible swine (n = 8). After baseline (1-2 h after excision) CHCT, the remaining muscles were placed into one of the following four treatment groups. In Groups 1 and 2, the muscles remained under tension and were stored in Krebs buffer (pH 7.4) at 23 degrees C-25 degrees C (clamped-warm) and 4 degrees C (clamped-cold), respectively. In Groups 3 and 4, the muscle strips were dissected, and the ends were tied with silk sutures, cut from the clamp, and placed in Krebs buffer at 23 degrees C-25 degrees C (free-warm) and 4 degrees C (free-cold), respectively. The responses of the treatment groups to halothane (3%) and caffeine (0.5-32 mM) were tested 22-26 h after excision. The clamped-warm storage was the only storage method to correctly diagnose MH susceptibility in all muscle strips tested. This finding was also confirmed in muscle stored under clamped-warm conditions and shipped overnight to another testing center for a parallel CHCT.

Inhibition by midazolam of the adrenergic function in the isolated canine mesenteric vein.

BACKGROUND: Midazolam has been reported to cause hypotension or to depress sympathetic activity following intravenous injection. However, little information is available concerning the mechanism of these effects. The aim of the present study was to determine the effects of midazolam on release of noradrenaline (NA) at nerve terminals and on receptors in the venous smooth muscle. METHODS: The effect of midazolam at nerve terminals was examined by measuring the amount of NA release from superfused canine mesenteric vein helical strips during electrical stimulation (ES; 5 Hz, 2 ms, 9 V). The NA was quantified by high-performance liquid chromatography with electrochemical detection; tension development evoked by ES was also recorded simultaneously. In a separate series of experiments, ring preparations from the isolated vein were mounted in Krebs-Ringer solution for isometric tension recording to assess the effect of midazolam on alpha-adrenoceptors. RESULTS: Application of tetrodotoxin (10(-6) M) or replacement of superfusate with Ca(2+)-free solution decreased both the release of NA and the tension development evoked by ES. Yohimbine (5 x 10(-8) M) increased the ES-evoked release of NA, whereas it decreased tension development in the vein strips. Midazolam (10(-4) M) did not affect either the basal release of NA or the basal tension, but inhibited both the NA release (P < 0.01) and the tension development (P < 0.01) during ES; midazolam at 10(-5) M inhibited the tension development (P < 0.05) but had no effect on NA release. In the ring preparations, midazolam (10(-5) and 10(-4) M) attenuated responses to NA (a mixed alpha 1- and alpha 2-adrenoceptor agonist, 10(-8) to 10(-3) M), phenylephrine (the alpha 1-adrenoceptor agonist, 10(-8) to 10(-3) M) and 5-bromo-6-[2-imidazolin-2yl-amino]-quinoxaline (UK14304; the alpha 2-adrenoceptor agonist, 10(-7) to 10(-3) M) in a dose-dependent manner. CONCLUSION: The data obtained in the present study suggest that midazolam at 10(-4) M may reduce venous tone by inhibiting the release of NA from sympathetic nerve endings and both alpha 1- and alpha 2-adrenoceptor mediated smooth muscle contractions. It is also postulated that a stage of the post-receptor transduction mechanism linked to the venous smooth muscle contraction may be more sensitive to midazolam than the NA release mechanism at nerve terminals since midazolam at the low concentration tested inhibited ES-evoked tension development with no effect on the release of NA.

Factors contributing to differences in the regulation of cGMP in isolated porcine pulmonary vessels.

Guanosine 3',5'-cyclic monophosphate (cGMP) is an important second messenger in many biological systems including vascular smooth muscle where it mediates relaxation. Cellular levels of cGMP are regulated primarily by three enzymes; nitric oxide (NO) synthase, soluble guanylate cyclase, and cGMP-phosphodiesterase. Basal cGMP levels of isolated endothelium intact porcine pulmonary vein are five fold higher than in pulmonary artery. The objective of this study was to investigate possible reasons for this difference. Therefore, we compared NO synthase activity of pulmonary vein with artery and used pharmacologic approaches to compare soluble guanylate cyclase and phosphodiesterase activities in these vessels. NO synthase activities of pulmonary vein and artery were measured by monitoring the conversion of exogenous L-[14C]arginine to L-[14C]citrulline and by quantifying NO formation from endogenous L-arginine. Rates (pM/min per mg protein) of basal L-citrulline and NO formation from endothelium intact pulmonary vein (29.0 +/- 4.8 and 44 +/- 7.1, respectively) were significantly higher than from artery (8.3 +/- 2.2 and 17.1 +/- 3.3). Western blot analysis indicated higher constitutive NO synthase protein in the vein than in artery. N-nitro-L-arginine (0-100 microM), a potent inhibitor of NO synthase, induced contractions of the pulmonary vein which were significantly higher than those of the artery. N-nitro-L-arginine (5 and 20 microM) in the presence of phosphodiesterase inhibitors, decreased basal cGMP levels of endothelium intact blood vessels. In endothelium denuded pulmonary vein and artery, basal cGMP levels were not different from each other, but increased significantly following stimulation of soluble guanylate cyclase with exogenous NO. In the presence of both non-specific and specific cGMP phosphodiesterase inhibitors, exogenous NO-induced cGMP levels of endothelium denuded tissues were not significantly different from each other. However, in the absence of the phosphodiesterase inhibitors, exogenous NO-induced cGMP was significantly less in the artery than in the vein. These results suggest that (I) the intact porcine pulmonary vein contains higher levels of NO synthase activity than pulmonary artery, and that (II) the soluble guanylate cyclase activities in pulmonary vein and artery are equally responsive to NO, and finally (III) pulmonary artery expresses greater phosphodiesterase activity than vein. Higher NO synthase and lower phosphodiesterase activity may explain the greater accumulation of cGMP in the pulmonary vein compared to the artery.

Halothane attenuates nitric oxide relaxation of rat aortas by competition for the nitric oxide receptor site on soluble guanylyl cyclase.

Endothelial cells play an important role in the regulation of vascular activity through the release of endothelium derived relaxing factor (EDRF) now believed to be nitric oxide (NO). NO and the NO donor drug nitroglycerin relax vascular smooth muscle by stimulating soluble guanylyl cyclase leading to elevation of intracellular levels of cyclic guanosine 3',5'-monophosphate (cGMP). Halothane has been shown to inhibit the action of NO on blood vessels. This study was designed to further investigate the mechanisms by which halothane attenuates NO-induced vascular relaxations. This was done by examining the effects of halothane on nitroglycerin and NO-induced relaxations in the presence and absence of the inhibitors of soluble guanylyl cyclase, methylene blue and 6-anilino-5,8-quinolinedione (LY 83583). Thoracic aortas from anesthetized male Sprague-Dawley rats were excised and cut into rings and the endothelium was removed. The aortic rings were suspended in organ baths containing Krebs solution and equilibrated at their optimal passive tension. When a stable plateau of contraction was produced by EC60 concentrations of norepinephrine, increasing concentrations of nitroglycerin or NO were added to the baths to relax the rings. This contraction-relaxation procedure was repeated three or four times. In some baths halothane was administered by a calibrated vaporizer 10 min before beginning the second procedure. Either methylene blue or LY 83583 was added to the baths 20 min before the third procedure. The combination of halothane, methylene blue or LY 83583 was added before the fourth procedure. Halothane, methylene blue or LY 83583 significantly inhibited nitroglycerin-induced relaxation individually. Halothane and LY 83583 also significantly inhibited NO-induced relaxations (5 x 10(-9)-3 x 10(-8) M and 5 x 10(-9)-3 x 10(-5) M, respectively) individually. The combination of halothane and methylene blue or halothane and LY 83583 significantly inhibited nitroglycerin-induced relaxation, also, the combination of halothane and LY 83583 significantly inhibited NO-induced relaxations. Halothane, methylene blue and LY 83583 treatment led to rightward shift in the concentration-effect curves. Halothane, in combination with methylene blue or LY 83583, produced inhibition equivalent to the sum of their individual effects. The present study demonstrates that the halothane, methylene blue and LY 83583 attenuate nitroglycerin and NO-induced relaxations of endothelium-denuded rat aortic rings. This suggests that halothane, methylene blue and LY 83583 may act through competitive antagonism at a common site of action on soluble guanylyl cyclase in the EDRF/NO relaxation pathway.

Actions of diaspirin cross-linked hemoglobin on isolated rat and dog vessels.

The objectives of these studies were to investigate the responses of isolated blood vessels from rats and dogs to the administration of diaspirin cross-linked hemoglobin (DCLHb) and to determine the mechanisms of these responses. Isolated vascular rings (3 to 5 mm) were suspended at optimal passive tension in Krebs-filled (37 degrees C) tissue baths and bubbled with 95% O2-5% CO2, and isometric tension was recorded. With the vessels under basal conditions increasing concentrations of DCLHb (10(-8)-3 x 10(-6) mol/L) were added. DCLHb addition was repeated during a submaximal contraction with norepinephrine and again during acetylcholine relaxation. The effects of the nitric oxide synthase inhibitor L-nitro arginine (10(-5) mol/L) on the responses to DCLHb were also determined. Dog vessels developed very little tension (1% to 5% of norepinephrine maximum), whereas rat arteries contracted between 9% and 15% when exposed to DCLHb under basal conditions. However, both the dog and rat vessels developed significant tension to DCLHb when they were precontracted (5% to 54%) and also when they were relaxed with acetylcholine (21% to 93%). L-nitro arginine eliminated the contractile responses to DCLHb but did not cause contraction of any of the vessels under basal conditions. We conclude that in this model the mechanism of DCLHb-induced contractions of in vitro dog and rat vessels is dependent on interference with nitric oxide. This is similar to the mechanism of DCLHb action in isolated pig vessels reported previously. Differences in responses of dog, rat, and pig vessels under basal conditions in vitro are the result of active generation of nitric oxide by pig but not by dog or rat vessels.

Hemoglobin-induced contraction of pig pulmonary veins.

The effects of hemoglobin Ao (HbAo), alpha alpha cross-linked hemoglobin (alpha alphaHb), cyanomet alpha alpha cross-linked hemoglobin (cyanomet alpha alphaHb), and human serum albumin (HSA) were compared under basal conditions and during relaxation with acetylcholine (ACh), sodium nitroprusside (SNP), and papaverine (PAP) in porcine pulmonary veins. Isometric tension changes were recorded in isolated rings (3 to 4 mm) that were suspended in Krebs solution bubbled with 95% O2/5% CO2. Increasing concentrations of HbAo and alpha alphaHb (10(-9) - 3 x 10(-6) mol/L) caused concentration-dependent increases in tension that reached a maximum of 4.20 +/- 0.3 gm and 3.78 +/- 0.6 gm, respectively. Cyanomet alpha alphaHb and HSA (10(-9) - 3 x 10(-6) mol/L) did not cause significant increases in tension. The maximum responses to HbAo and alpha alphaHb were significantly increased during relaxation with ACh and SNP but not with PAP. In contrast, SNP (10(-4) mol/L) and PAP (10(-5) mol/L), but not ACh, reversed contractions induced by HbAo and alpha alphaHb. These studies support the concept that hemoglobin-induced vascular contraction is primarily mediated by inactivation of the vasodilator nitric oxide in vitro. We suggest that this mechanism is common to acellular hemoglobins in which the ligand binding site is unimpaired and in which the heme iron is in the ferrous (+2) state.

Diaspirin cross-linked hemoglobin does not alter isolated human umbilical artery or vein tone.

Diaspirin cross-linked hemoglobin (DCLHbTM; Baxter Healthcare Corp., Round Lake, IL, USA) is undergoing clinical trials as a blood substitute. Administration of DCLHb is associated with an increase of mean arterial pressure in vivo and contraction of selected adult isolated blood vessels of from certain species in vitro. The mechanisms of these pressor effects may be due to scavenging of the endothelium derived relaxing factor, nitric oxide (NO), by hemoglobin. Unlike adult blood vessels, prostacyclin (PGI2) rather than EDNO is the important relaxing agent in human umbilical vessels. In this study, we examined if DCLHb had vasoconstrictor effects on isolated human umbilical vessels. Human umbilical veins and arteries were excised, cut into rings and placed in organ chambers filled with 25 ml Krebs-Ringer solution (37 degrees C). 5-hydroxytryptamine (5-HT, 0.01-10 microM) increased the tension of human umbilical arteries (HUA, from 0.4 +/- 0.2 g to 2.6 +/- 0.4g) and veins (HUV, from 0.8 +/- 0.4g to 2.5 +/- 0.4g) in a dose-dependent manner. DCLHb (0.01-10 microM) did not have a significant effect on HUA and HUV. Substance P (1 microM, via prostanoid synthesis) and nitroglycerin (NG, 1 microM) but not acetylcholine (ACh, 1 microM) caused relaxation of both HUA and HUV. The NO synthase inhibitor L-NA did not have significant effects on HUA and HUV. DCLHb did not alter 5-HT preconstricted tension of HUA and HUV. The basal cGMP contents of HUA and HUV were low. These results support our previous finding that DCLHb-induced vasoconstriction in isolated vessels is dependent primarily on the binding of NO by hemoglobin.

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 microM). 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(-6) M and 2 x 10(-6) 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 microM, 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 nonpretreated rings. RESULTS: Carbon monoxide (26-176 microM) 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 microM) 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.

Intravenous lecithin-coated microcrystals of dantrolene are effective in the treatment of malignant hyperthermia: an investigation in rats, dogs, and swine.

Dantrolene effectively treats malignant hyperthermia (MH) hut the current form, Dantrium, must be dissolved to a 0.33 mg/mL, pH 9.5 solution. This study describes lecithin-coated microcrystal formulations of sodium dantrolene (MC-NaD) and neutral dantrolene (MC-D) which reconstitute to 200 mg/mL within 1 min. In rats, the pharmacokinetics and pharmacodynamics of MC-NaD and Dantrium were similar: half-lives of 3.1 h, volume distributions of 0.54 and 0.59 L/kg, and 95% effective dose (ED95) values for depression of skeletal muscle twitch height (ED95T) of 2.6 +/- 0.7 and 2.8 +/- 0.5 mg/kg. In swine, the ED95T values for MC-NaD and Dantrium were also similar (2.8 +/- 0.4 vs 2.7 +/- 0.6 mg/kg), but MC-D and Dantrium were only similar at doses more than 2.5 mg/kg (ED95T: 3.5 +/- 0.4 vs 2.7 +/- 0.5 mg/kg). In susceptible swine, MC-NaD successfully treated five of six MH episodes and prevented MH in three of four swine. However, MC-NaD caused marked pulmonary hypertension in swine, while MC-D caused only a mild response that was eliminated by filtration. Likewise, MC-D caused no pulmonary response in dogs. These observations suggest that MC-D has potential to improve the treatment of MH.

Effect of halothane on phenylephrine-induced vascular smooth muscle contractions in endotoxin-exposed rat aortic rings.

OBJECTIVES: a) To determine the response of endotoxin-exposed vascular smooth muscle to exogenous vasoconstrictors during concomitant exposure to an inhaled anesthetic (halothane); and b) to determine if excess nitric oxide production is responsible for any altered response. DESIGN: In vitro, prospective, repeated-measures, dose-response study. SETTING: University/medical school experimental physiology laboratory. SUBJECTS: Adult male Sprague-Dawley rats, whose aortae were studied in an in vitro preparation. INTERVENTIONS: Thoracic aortae were excised from anesthetized animals and cut into 3-mm rings. After incubation in aerated organ baths containing a modified essential medium with or without Escherichia coli lipopolysaccharide (100 micrograms/mL) at 37 degrees C for 5 hrs, the rings were removed and suspended in separate baths for isometric tension recording. Phenylephrine dose-response data (10(-10) to 10(-5) M) were determined for lipopolysaccharide- and nonlipopolysaccharide-treated rings. After washout and equilibration, two vessels (one each lipopolysaccharide- and nonlipopolysaccharide-treated) were additionally exposed to 2% halothane and phenylephrine dose-response determinations were repeated for all vessels. This procedure was repeated for 1% halothane in a separate experiment. In some experiments, the nitric oxide synthase inhibitor, N omega-nitro-L-arginine (3 x 10(-4) M), was added to the bath after the washout from the second phenylephrine dose-response determination. Then, a third phenylephrine dose-response determination was performed, with and without 2% halothane. MEASUREMENTS AND MAIN RESULTS: Dose-response curves were evaluated using a logistic regression analysis. In addition, absolute and percentage changes in tension were compared between the first and second contractions. Exposure to lipopolysaccharide resulted in a decrease in the maximum tension from 2.07 +/- 0.03 (controls) to 1.24 +/- 0.04 g/mg of vessel dry weight and an increase in the dose at which the contraction is 50% of maximum (ED50) from 3.78 x 10(-8) to 2.05 x 10(-7) M (p < .05). Exposure to 2% halothane produced significant reductions in the maximum tensions in both groups. The lipopolysaccharide-treated vessels showed not only a proportionately larger decrease (-51 +/- 5% vs. -18 +/- 2% in the control plus halothane group), but also a significantly greater absolute decrease (0.59 +/- 0.09 vs. 0.34 +/- 0.04 g/mg in the control plus halothane group). The addition of 1% halothane produced less pronounced decreases in tension, with only an additive effect in the lipopolysaccharide-treated vessels. The addition of N omega-nitro-L-arginine resulted in a reversal of the lipopolysaccharide-induced decrease in tension. However, 2% halothane still had a significantly greater effect on the lipopolysaccharide-exposed rings. CONCLUSIONS: Exposure of rat aortic rings to lipopolysaccharide in vitro decreased the contractile response to phenylephrine. The addition of 2% halothane resulted in a more than additive decrease in tension in the lipopolysaccharide-treated vessels. Patients in septic or endotoxic shock are sensitive to most anesthetic regimens, and some of this sensitivity may be due to an altered vasoconstrictive response induced by lipopolysaccharide exposure. The inability of nitric oxide synthase inhibition to reverse this response completely suggests that induction of nitric oxide synthase and increased production of nitric oxide are not solely responsible for this finding.

Effects of enflurane on adrenergic function in canine mesenteric artery and vein.

We examined the pre- and postsynaptic effects of enflurane on the superior mesenteric artery and vein. We measured the release of norepinephrine (NE) from sympathetic nerve terminals caused by electrical stimulation (ES) or potassium and changes in vascular smooth muscle tension. The effect of enflurane was also examined in the presence of alpha 1- and alpha 2-adrenoceptor antagonists. Enflurane (1.4 to 1.6 minimum alveolar anesthetic concentration [MAC] in the dog) did not alter basal tension, but it significantly inhibited tension development caused by 40 mM KCl and ES in the canine mesenteric artery and vein. Enflurane inhibited the KCl- and ES-induced release of NE from sympathetic nerve terminals in the canine mesenteric vein. The alpha 1- and alpha 2-adrenoceptor antagonists, corynanthine or yohimbine, did not modify the effect of enflurane on the ES-induced frequency response curve of the mesenteric artery and vein. These data suggest that enflurane has at least two sites of action in mesenteric vascular beds. One site is presynaptic where it inhibits NE release, and the other is a postsynaptic site distal to the adrenergic receptor where it inhibits the smooth muscle contraction process.

Effects of halogenated and non-halogenated anesthetics on diaspirin cross-linked hemoglobin induced contractions of porcine pulmonary veins.

Diaspirin crosslinked hemoglobin (DCLHb) is a resuscitative fluid presently undergoing clinical trials. Administration of DCLHb is associated with an elevation of mean arterial pressure in vivo and contraction of isolated blood vessels in vitro. The mechanisms for the vascular actions are unknown but may be due to inhibition of nitric oxide (NO). Halothane has been reported to inhibit NO induced relaxation. We examined the effect of anesthetics on DCLHb induced contraction of blood vessels. Porcine pulmonary veins were excised, cut into rings and placed in organ chambers filled with 25 ml Krebs-Ringer solution (37 degrees C). Following equilibration at their optimal length the rings were exposed to increasing concentrations of serotonin(10(-8)M-10(-5)M). Endothelial activity was confirmed by relaxation greater than 80% with ACh (10(-6)M). DCLHb (1.5 x 10(-8)M to 6 x 10(-7)M) contracted porcine pulmonary veins (1.04 +/- 0.17g to 3.45 +/- 0.22g), and halothane (0.5% and 1%) significantly inhibited these DCLHb induced contractions in a dose-related manner (-41.6 +/- 8.1% and -73.3 +/- 8.2%, respectively). At equi-molar concentrations, isoflurane had no inhibitory activity. The relative effect of these volatile anesthetics is consistent with their inhibitory actions on other heme containing proteins. Propofol (10(-5)M) only has inhibitory effects on lower concentrations of DCLHb. Fentanyl did not have inhibitory effects. These results suggest that halogenated anesthetics may interact with the heme iron of DCLHb and inhibit its binding with NO.

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).

Comparative effects of exogenous nitrovasodilators on cGMP levels in different canine blood vessels.

Nitrovasodilators, by releasing nitric oxide (NO) in vascular smooth muscle, activate soluble guanylate cyclase (sGC) in vascular smooth muscle. However, there is little information on their relative effectiveness, concentration ranges, or on the incubation times required to produce maximum sGC stimulation. To determine the optimal concentrations and incubation times we measured 3', 5'-cyclic guanosine monophosphate (cGMP) levels in response to different concentrations of NO, S-nitroso-L-cysteine (SNC), and S-nitroso-N-acetylpenicillamine (SNAP), in canine aorta, femoral, and carotid arteries incubated in Krebs. Production of cGMP following incubation of endothelium denuded tissues with NO, SNC, and SNAP peaked close to 20 +/- 5, 90 +/- 20, and 120 +/- 60 seconds respectively. Results indicate that cGMP levels vary with concentration of nitrovasodilators and time of incubation. SNAP was the least effective in increasing cGMP levels among the three nitrovasodilators used. In different vascular beds, the production of cGMP in the presence of nitrovasodilators may depend on variations in the levels of guanylate triphosphate (GTP) and/or sGC.

A clinical grading scale to predict malignant hyperthermia susceptibility.

BACKGROUND: The diagnosis of an acute malignant hyperthermia reaction by clinical criteria can be difficult because of the nonspecific nature and variable incidence of many of the clinical signs and laboratory findings. Development of a standardized means for estimating the qualitative likelihood of malignant hyperthermia in a given patient without the use of specialized diagnostic testing would be useful for patient management and would promote research into improved means for diagnosing this disease. METHODS: Using the Delphi method and an international panel of 11 experts on malignant hyperthermia, a multifactor malignant hyperthermia clinical grading scale comprising standardized clinical diagnostic criteria was developed for classification of existing records and for application to new patients. RESULTS: This scale ranks the qualitative likelihood that an adverse anesthetic event represents malignant hyperthermia (malignant hyperthermia event rank) and that, with further investigation of family history, an individual patient will be diagnosed as malignant hyperthermia susceptible (malignant hyperthermia susceptibility rank). The assigned rank represents a lower bound on the likelihood of malignant hyperthermia. The clinical grading scale requires the anesthesiologist to judge whether specific clinical signs are appropriate for the patient's medical condition, anesthetic technique, and surgical procedure. CONCLUSIONS: The malignant hyperthermia clinical grading scale is recommended for use as an aid to the objective definition of this disease. It use may improve malignant hyperthermia research by allowing comparisons among well-defined groups of patients. This clinical grading system provides a new and comprehensive clinical case definition for the malignant hyperthermia syndrome.

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 degrees C) and were contracted to a stable plateau with EC60-70 norepinephrine (NE). Relaxations caused by acetylcholine (ACh; 1 x 10(-8)-1 x 10(-6) M), nitric oxide (NO; 5 x 10(-9)-1 x 10(-6) M), or nitroglycerin (NG; 2 x 10(-9)-3 x 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 x 10(-9)-3 x 10(-8) M) but not at higher concentrations (1 x 10(-9)-3 x 10(-7) M) in denuded vessels. Nitric oxide-stimulated (5 x 10(-8)-5 x 10(-6) M) cGMP content was significantly attenuated by halothane (2 MAC) at NO concentrations between 1 x 10(-7) and 5 x 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.

Neurovascular effects of reactive oxygen intermediates produced by photoradiation.

These experiments examined the effect of reactive oxygen intermediates, produced by laser illumination of the photosensitizer hematoporphyrin derivative, on the accumulation and release of norepinephrine from sympathetic nerve terminals. Using an isolated, spirally cut, superfused caudal artery of the rat, basal overflow of norepinephrine (NE) was significantly increased both during and after generation of reactive oxygen intermediates. Generation of reactive oxygen intermediates increased overflow of NE in vascular preparations in which release of NE had previously been elevated by the continuous superfusion of Krebs' solution, containing high concentrations of potassium (40 mM). Calcium free solutions did not block the overflow of norepinephrine augmented by reactive oxygen intermediates. This increase in overflow was due both to an increase in release of NE and an inhibition of accumulation of NE.