R. A. Van Dyke

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.

Isoflurane causes endothelium-dependent inhibition of contractile responses of canine coronary arteries.

The authors sought to determine if isoflurane would attenuate effects of three different types of vasoconstrictors on isolated segments of canine epicardial coronary arteries removed from healthy dogs. As the endothelium has a major role in regulating epicardial coronary artery tone, and as it modulates the effect of many vasoactive substances, experiments were conducted both on normal rings and on rings whose endothelium had been mechanically removed. In addition, the endothelium is thought to be damaged in human atherosclerosis. Rings were suspended in organ chambers filled with modified Krebs-Ringer bicarbonate solution, aerated with 95% oxygen and 5% carbon dioxide, and connected to strain gauges for the measurement of isometric tension. Isoflurane 2.3% (1.5 MAC in the dog) was added to the aerating gas mixture in half the preparations, while the other rings served as control. The vasoconstrictors serotonin, phenylephrine, or prostaglandin F2α were added in increasing concentrations to the bath solution. In the presence of endothelium, vasoconstrictor evoked contractions were attenuated by isoflurane. Maximal tension generated by prostaglandin F2α in untreated rings was 114 ± 18% (mean ± SEM) of a reference contraction, while, following isoflurane, it was 46 ± 8% (P < 0.005). In the absence of endothelium, isoflurane attenuated neither prostaglandin F2α nor serotonin evoked contraction, and had decreased effectiveness against phenylephrine mediated contraction (P < 0.001). It is concluded that isoflurane attenuates vasoconstrictor-evoked contraction of isolated canine epicardial coronary arteries, and that this effect is mediated by the endothelium.

The Effects of Volatile Anesthetics on Ca++ Mobilization in Rat Hepatocytes

This study provides direct evidence that in hepatocytes, intracellular Ca++ is released from internal stores by halothane, enflurane, and isoflurane. Hepatocytes isolated from rat livers were used fresh or treated with saponin and then incubated in 45Ca++ media. The uptake of 45Ca++ by hepatocytes was maximal following 13-16 min of incubation (untreated or saponin-treated) and the effects of various agents on the release of 45Ca++ was studied following maximal loading. The agents used included halothane, enflurane, isoflurane, and several putative intracellular second messengers. The anesthetics, to various degrees, all stimulated a significant release of 45Ca++ from internal stores at concentrations that were at or less than clinical concentrations. The release of intracellular 45Ca++ by each of the anesthetic agents was dose-dependent with halothane and enflurane being equally potent at concentrations equivalent to 1 MAC exposure. The halothane-induced release was only somewhat suppressed by preincubation in either 2 mM LaCL3 or 10 microM dantrolene, both suggested Ca++ channel blockers. Transient increases in intracellular Ca++ regulates a number of enzyme systems, including glycogenolysis, while prolonged elevation in Ca++ concentrations have been implicated in the mechanism of hepatotoxicity.

IS ISOFLURANE A CALCIUM ANTAGONIST

The vasodilating and myocardial depressant effects of isoflurane have been allocated to calcium channel blockade. The present study aimed to test this hypothesis by assessing the effect of isoflurane on the contractile response to potassium stimulation of vascular smooth muscle. Seventy two left anterior descending and circumflex coronary artery rings were removed in twelve dogs and mounted in organ chambers filled with Krebs-Ringer bicarbonate solution and aerated with 95% O2-5% CO2. Rings were pretreated with either 3.8% isoflurane (2.5 MAC in the dog) or 10(-8) mol.l-1 nifedipine, a calcium entry blocker. They were stimulated by addition of 10 to 150 mmol.l-1 potassium chloride. At 70 mmol.l-1 K+, the tension generated by the untreated rings was 119 +/- 4.25% of control, while in the isoflurane treated group the tension was 99 +/- 2.4% of control. In the opposite, the tension was 25 +/- 7.11% in the nifedipine treated rings. Likewise, when isoflurane was added to rings preconstricted with 40 mmol.l-1 potassium chloride, no relaxation occurred, while nifedipine produced relaxation. Isoflurane, unlike nifedipine, had a weak effect on ring tension. The calcium-entry blockade effect of isoflurane appeared weak, dose-dependent and virtually absent at clinical concentrations. Therefore, the vasodilation seen with clinical concentrations of isoflurane is mediated by mechanisms other than calcium-entry blockade.

Transient increases of intracellular Ca2+ induced by volatile anesthetics in rat hepatocytes

The affects of volatile anesthetics on mobilization of intracellular Ca2+ was monitored in primary cultures of rat hepatocytes using the fluorescent Ca2+ probe Fura-2. The use of Fura-2 was limited by several factors which complicated the quantitative analysis of the results, such as: (i) a high rate of dye leakage; (ii) changes in the redox state of the hepatocytes which interfered with the fluorescence produced by the dye at various excitation wavelengths; (iii) compartmentalization of the dye producing high local intracellular concentrations; and, of particular importance for this study, (iv) enhanced photobleaching of the dye in the presence of halothane. To aid in the interpretation of the Fura-2 data, the Ca2(+)-sensitive photoprotein aequorin was also used to monitor changes in [Ca2+]i. The aequorin and Fura-2 techniques qualitatively yielded the same result, that the volatile anesthetic agents halothane, enflurane, and isoflurane induce an immediate and transient increase of [Ca2+]i. The durations of these transients were approximately between 5 and 10 min and were not related to any evident acute cell toxicity. The [Ca2+]i increases induced by the volatile anesthetic agents were dose-dependent, with halothane the most potent. The exact mechanism governing these increases in [Ca2+]i induced by these anesthetics in rat hepatocytes is unknown, but is likely to involve effects on both the cell surface membrane and endoplasmic reticulum components of the signal transducing system.

Effect of halothane on hypoxic toxicity and glutathione status in cultured rat hepatocytes.

BackgroundIn hypoxic rats, halothane causes hepatotoxicity at oxygen levels that would cause minimal hepatotoxicity in the absence of halothane. Using a model that excludes systemic and extrahepatic effects of halothane, the authors tested the hypothesis that halothane hepatotoxicity in the wholerat model is caused by a direct hepatotoxic effect of halothane, which is mediated by halothane-derived free radicals. MethodsRat hepatocyte monolayer cultures were exposed to defined gas phases for 2 h. Three experimental variables were present or absent: hypoxia (1% O2), halothane (2%), and cytochrome P-450 induction (by phenobarbital). Two experimental outcomes were measured: aspartate aminotransferase release, a measure of cell death, and reduced glutathione, an endogenous free radical scavenger whose levels are decreased by physiologically significant free radical injury. ResultsAs anticipated, hypoxia increased cell death. Cytochrome P-450 induction by itself increased cell death during hypoxia. However, halothane had no effect on cell death during hypoxia, with or without cytochrome P-450 induction. Halothane had no toxic effect, even when glutathione was depleted before the onset of hypoxia. Glutathione was decreased moderately by hypocia alone. Neither halothane nor cytochrome P-450 induction had any effect on glutathione levels. ConclusionsHalothane was not toxic, and it did not generate a physiologically significant free radical insult during hypoxia in the isolated rat hepatocyte under the experinmental conditions used in testing.