Inhalation Anesthetics Inhibit the Release of Endothelium-derived Hyperpolarizing Factor in the Rabbit Carotid Artery

After institutional approval had been obtained, New Zealand White rabbits of either sex (1.4-3.2 kg body weight) were anesthetized with sodium pentobarbital (60 mg kg sup -1 intravenous). After exsanguination by cutting through the aorta and vena cava, the left and right carotid arteries were removed, cleaned of adventitial adipose and connective tissue, and cut into rings 3-4 mm in width. Four rings were mounted between K30 force transducers (Hugo Sachs Elektronik, March, Germany) and a rigid support for measurement of isometric force. They were incubated in 10-ml organ chambers (made available by Hugo Sachs Elektronik) containing warmed (37 degrees Celsius) oxygenated (95% O²-5% CO²) Krebs-Henseleit solution, pH 7.4 (millimolar composition: Na sup + 144.0, K sup + 5.9, Cl sup - 126.9, Ca²+ 1.6, Mg²+ 1.2, H²PO⁴sup - 1.2, SO⁴²- 1.2, HCO³sup - 25.0, and D-glucose 11.1) to which the cyclooxygenase inhibitor diclofenac was added at a concentration of 1 micro Meter. Passive tension was adjusted over a 30-min equilibration period to 2 g, and the Krebs-Henseleit solution exchanged at 10-min intervals. Thereafter the segments were preconstricted with phenylephrine (1 micro Meter) to approximately 2 g tension, and the integrity of the endothelium was tested by applying 1 micro Meter acetylcholine. Segments showing < 60% relaxation to acetylcholine were discarded.

Desflurane, enflurane, halothane, isoflurane and sevoflurane were delivered from a calibrated vaporizer (Devapor, Vapor 19.3, Drager, Lubeck, Germany) to give appropriate concentrations of 2-4% for enflurane, halothane, isoflurane, and sevoflurane or 8-16% for desflurane in the carbogen gas (95% O²/5% CO²) aerating the Krebs-Henseleit solution (400 ml/min). Because desflurane and sevoflurane were not available at the beginning of the study, the effects of enflurane, halothane and isoflurane were tested with a different batch of rabbits than those used for desflurane and sevoflurane.

The concentrations of the anesthetics reflect the clinically relevant concentrations required to induce or maintain adequate anesthesia. The concentration in the carrier gas was monitored by infrared light spectroscopy (Capnomac-Ultima, Datex, Helsinki, Finland, from Hoyer, Bremen, Germany), which was calibrated daily with a standard calibration gas (Hoyer). In separate experiments, the concentration of desflurane, enflurane, halothane, isoflurane and sevoflurane in the Krebs-Henseleit solution was determined by gas chromatography-flame ionization detection (180 degrees Celsius) analysis with a gas chromatograph (series 8500, Perkin-Elmer, Uberlingen, Germany) equipped with a HS6 head space injector (maintained at 130 degrees Celsius). The anesthetics were separated at 75-80 degrees Celsius on steel capillary columns (1 m long, 0.32 cm in diameter, filled with 60/80 mesh graphite/0.4% Carb 1500 for halothane, enflurane, isoflurane, and sevoflurane; 1.8 m long, 0.32 cm in diameter, filled with 100/120 mesh graphite/10% SP1000/1% H³PO⁴, for desflurane; Perkin-Elmer) with Nitrogen²(120 kPa, 15 ml/min) as carrier gas. Tetrahydrofuran and dichloromethane in ethylene glycol were used as internal standards.

After 16 min equilibration with the anesthetics at 37 degrees Celsius in the organ bath, the following Krebs-Henseleit-gas partition coefficients were determined (means plus/minus SD): desflurane 0.242 plus/minus 0.012 (n = 39), enflurane 0.584 plus/minus 0.46 (n = 11), halothane 0.660 plus/minus 0.013 (n = 12), isoflurane 0.356 plus/minus 0.054 (n = 11) and sevoflurane 0.354 plus/minus 0.040 (n = 11). On the basis of these partition coefficients, the final millimolar concentrations of the anesthetics in the organ bath was calculated as shown in Table 1.

Four rings of the same carotid artery were examined simultaneously, one ring was randomly used as a control to exclude any time-dependent changes in agonist sensitivity.

Nitric Oxide-Induced PGI²-independent Relaxation. After washout of acetylcholine and phenylephrine, the rings were allowed to equilibrate for 20 min and then preconstricted again with phenylephrine (1 micro Meter). When a stable constriction was obtained, the inhalation anesthetics were administered for 15-20 min and a cumulative concentration-response curve to acetylcholine (0.03-10 micro Meter) was established in the presence of the anesthetics followed by a washout period of 30 min.

Nitric Oxide- and PGI²-independent Relaxation. Thereafter the segments were treated with the NO synthase inhibitor, N^G-nitro-L-arginine (0.1 mM), for 30 min followed by the same experimental protocol as described before.

Sodium Nitroprusside-Induced Relaxation. After another 30-min washout period, the same segments were again constricted with phenylephrine and the effects of the test compounds on the endothelium-independent relaxation induced by sodium nitroprusside (0.01-10 micro Meter) were investigated.

In some experiments, the NO synthase inhibitor was administered at the beginning of the experiment to directly assess the effects of the anesthetics on the NO- and PGI²-independent relaxant response to acetylcholine. Results from these experiments, however, did not differ from those obtained with the other experimental protocol. In another series of experiments, the relaxant response to acetylcholine was investigated in the presence of the cytochrome P450 inhibitor clotrimazole (3 micro Meter) or the K^Casup + -channel inhibitor tetrabutylammonium (0.3 mM) alone or in combination with enflurane (2%).

To elucidate the potential cytochrome P450-inhibitory effect of the anesthetics, we used a sensitive spectrofluorometric assay in which the O-dealkylation of 7-ethoxycoumarin to the highly fluorescent 7-hydroxycoumarin (umbelliferon) is monitored over time. [24,25]As a source for cytochrome P450 the microsomal fraction from the liver of noninduced New Zealand White rabbits was prepared essentially as previously described for NO synthase. [26]An aliquot of the microsomal protein (0.5 mg) was stirred in a quartz glass cuvette with 0.1 M tris(hydroxymethyl)aminomethane-HCl buffer (pH 7.6), containing 20 micro Meter 7-ethoxycoumarin. After a 2-min equilibration at 37 degrees Celsius, in the presence or absence of the anesthetics, the reaction was initiated by the addition of reduced nicotinamide adenine dinucleotide phosphate (0.1 mM) and monitored over a period of 10 min in a dual wavelength spectrofluorometer (PTI, Wedel, Germany) with the excitation and emission wavelengths set to 370 and 455 nm, respectively. Calibration of the assay was performed by adding known concentrations of umbelliferon (0.1-10 micro Meter) to a cuvette containing heat-denatured microsomal protein.

The activity of a semipurified rabbit cerebellar NO synthase preparation [27]was determined by monitoring the N^G-nitro-L-arginine-sensitive conversion of³Hydrogen-labeled L-arginine to L-citrulline. [26]The activity of purified soluble guanylyl cyclase isolated from bovine lungs was determined by monitoring the conversion of³²Phosphorus-labeled guanosine 5'-triphosphate to guanosine 3',5'-phosphate. [26].

Unless indicated otherwise, all data in the figures and text are expressed as means plus/minus SEM of n experiments with ring segments from different arteries. Statistical evaluation was performed by two-sided Fisher-Pitman analysis between groups followed by a Bonferroni post hoc test for multiple comparisons. A P value of < 0.05 was considered statistically significant.

Desflurane (Suprane) was obtained from Anaquest (Guayama, Puerto Rico); enflurane (Ethrane) and isoflurane (Forane) from Abbott (Wiesbaden, Germany); halothane (Fluothane) from ICI Pharma (Heidelberg, Germany); sevoflurane (Sevofrane) from Maruishi (Osaca, Japan); pentobarbital sodium (Nembutal) from Sanofi (Munchen, Germany); diclofenac (Voltaren) from Ciba-Geigy (Wehr, Germany); N^G-nitro-L-arginine (free acid) from Serva (Heidelberg, Germany); acetylcholine, clotrimazole, metyrapone, phenylephrine, tetrabutylammonium chloride, and sodium nitroprusside from Sigma (Deisenhofen, Germany); and SKF525a (proadifen) from Calbiochem (Bad Soden, Germany).

The concentration-dependent relaxant response of the endothelium-intact carotid artery segments to acetylcholine was slightly (16 and 8% inhibition respectively) but significantly attenuated by halothane and isoflurane (Figure 1(A)), whereas enflurane (Figure 1(A)), desflurane, and sevoflurane had no such effect (data not shown).

After inhibition of NO synthesis with N^G-nitro-L-arginine, the maximum relaxant response to acetylcholine was significantly reduced from 94.5 plus/minus 0.2% to 33.8 plus/minus 2.3% and from 98.3 plus/minus 0.7% to 60.5 plus/minus 3.8% in the two control groups (Figure 1(B and C)). In the presence of the inhalation anesthetics, this NO- and PGI²-independent relaxation was further attenuated (Figure 1(B and C) and Figure 2(A-C)). Increasing the concentration of halothane from 2% to 3.5% (Figure 2(B)) or isoflurane from 2% to 4% (Figure 2(C)) led to a more pronounced inhibition of the acetylcholine-induced relaxation. In contrast, the inhibitory effect of 4% enflurane was not greater than that of 2% enflurane (Figure 2(A)). On a molar basis (Table 1), isoflurane and sevoflurane appeared to be the most potent inhibitors of EDHF release followed by halothane, enflurane and desflurane.

In contrast to the acetylcholine-induced endothelium-dependent relaxation, none of the anesthetics used had a significant effect on the endothelium-independent relaxant response to sodium nitroprusside (Figure 3(A and B)).

The NO- and PGI²-independent relaxant response to acetylcholine was virtually abolished (Figure 4) in the presence of three different, structurally unrelated cytochrome P450 inhibitors, clotrimazole (0.1 mM), metyrapone (1 mM), and SKF525a (0.1 mM). Moreover, in the presence of the K^Casup + -channel inhibitor, tetrabutylammonium, the EDHF-mediated relaxation was significantly affected at 0.3 mM (Figure 5(A)) and abolished at 1-3 mM (data not shown).

At 0.3 mM, tetrabutylammonium caused a shift to the right of the concentration-response curve of acetylcholine (50% effective concentration increased from 0.2 to 0.7 micro Meter), but only weakly attenuated its maximum relaxing effect (Figure 5(A)). Clotrimazole at a concentration of 3 micro Meter, on the other hand, reduced the maximum relaxant response to acetylcholine, but did not cause a shift to the right of the concentration-response curve (50% effective concentration 0.3 micro Meter) (Figure 5(A)). Both the K^Casup + -channel antagonist and the cytochrome P450 inhibitor (Figure 5(B and C)) significantly enhanced the inhibitory effect of 2% enflurane on the NO- and PGI²-independent relaxant response to acetylcholine in an additive manner.

All of the inhalation anesthetics tested significantly inhibited the dealkylation of 7-ethoxycoumarin to umbelliferon by the cytochrome P450 enzymes present in rabbit liver microsomes in a concentration-dependent manner (Figure 6). On a molar basis (Table 1), isoflurane and sevoflurane were the most potent cytochrome P450 inhibitors followed by halothane, enflurane and desflurane. Umbelliferon formation by these microsomes was abolished in the presence of metyrapone (93.3 plus/minus 1.4% inhibition at 1 mM, n = 6) or after heating the microsomes to 95 degrees Celsius for 10 min (data not shown).

Neither 2% halothane (10.7 plus/minus 2.3 pmol *symbol* mg sup -1 *symbol* min sup -1, n = 4) nor 2% isoflurane (8.1 plus/minus 0.6 pmol *symbol* mg sup -1 *symbol* min sup -1, n = 4) significantly affected the activity of a constitutive NO synthase preparation from rabbit cerebellum (12.0 plus/minus 1.2 pmol *symbol* mg sup -1 *symbol* min sup -1, n = 4). Moreover, neither 2.5% halothane (9.6 plus/minus 1.7-fold stimulation, n = 3), 2.5% isoflurane (8.9 plus/minus 1.4-fold stimulation, n = 4), or 2.5% enflurane (12.6 plus/minus 1.5-fold stimulation, n = 4) significantly attenuated the activity of a purified soluble guanylyl cyclase preparation from bovine lung stimulated with 10 micro Meter sodium nitroprusside (10.1 plus/minus 1.5-fold stimulation, n = 4).

The current findings demonstrate that the inhalation anesthetics tested mainly interfere with the synthesis or action of EDHF. Halothane and isoflurane also marginally affect the NO-mediated relaxant response to acetylcholine. An attenuation by enflurane, halothane, isoflurane, and sevoflurane of the NO-dependent relaxant response to acetylcholine has also been described for other blood vessels. [3-8,23]This effect of the anesthetics on NO release might be due to an interference with the acetylcholine receptor signalling or a decrease in the availability of intracellular Calcium²+ resulting in an attenuation of NO synthase activity. A direct effect on NO synthase activity, however, is less likely, because in our hands neither halothane nor isoflurane significantly affected the formation of L-[sup 3 Hydrogen]citrulline from L-[sup 3 Hydrogen]arginine by a constitutive NO synthase preparation. Moreover, an interference with the NO effector cascade in the smooth muscle is also unlikely, because no anesthetic had any effect on the endothelium-independent dilator response to sodium nitroprusside. In addition, neither enflurane, halothane, or isoflurane affected the stimulation of a purified soluble guanylyl cyclase preparation by sodium nitroprusside.

In addition to NO, 30-60% of the acetylcholine-induced relaxation in the rabbit carotid artery, similar to findings obtained with other vascular preparations, [12-14]appears to be mediated by the release of EDHF. This EDHF release was unmasked by the blockade of the endothelial NO synthase with 0.1 mM N^G-nitro-L-arginine. This concentration of the NO synthase inhibitor is sufficient to completely abrogate both the basal and agonist-stimulated release of NO from endothelium-intact arterial segments, as judged by both bioassay and stimulation of purified soluble guanylyl cyclase with the effluate from these segments. [13,28,29].

Recently, EDHF has been characterized as a cytochrome P450-derived arachidonic acid metabolite. [13,16,17]Prime candidates for this metabolite are the four region-specific epoxides of arachidonic acid, [19]one of which elicited a relaxant response in endothelium-denuded coronary artery segments that was sensitive to the blockade of K^Casup + channels. [13]By opening these Potassium sup + channels, EDHF has been shown to hyperpolarize, hence relax different types of vascular smooth muscle. [9-12]In contrast to K^Casup + -channel inhibitors such as apamin or charybdotoxin, the inhibitor of K sup +^ATP-dependent channels, glibenclamide has no effect on the EDHF-mediated relaxation. [9,13]There are differences, however, in the type of K^Casup + channels mediating the effect of EDHF in different vascular beds. [12-14,16,17].

What is the mechanism underlying this partial inhibitory effect of the inhalation anesthetics on the EDHF-mediated relaxation? One possibility is that they interfere with the K^Casup + channel(s) mediating the effect of EDHF on the smooth muscle. In human red blood cells for example, halothane has been shown to decrease the conductance of K^Casup + channels with consequent inhibition of membrane hyperpolarization. [30]In rat glioma cells, as well as in rat neurons, enflurane, halothane and isoflurane also attenuated the open probability of charybdotoxin-sensitive K^Casup + channels at clinically relevant concentrations. [31,32]These effects on Potassium sup + -channel conductance may be due to an increase in membrane fluidity caused by the anesthetics or an increase in the lateral membrane pressure, [33,34]which could inhibit the opening or accelerate the closure of these channels. [35].

On the other hand, inhalation anesthetics are known to be metabolized by several cytochrome P450 isoenzymes. [36]It was not clear, however, whether the anesthetics can also inhibit the activity of these enzymes and therefore potentially also the cytochrome P450-dependent synthesis of EDHF. This notion is strongly supported by our finding that all five inhalation anesthetics inhibited the cytochrome P450 activity of rabbit liver microsomes in a concentration-dependent manner. Moreover, the rank order of potency established for this inhibitory effect on a molar basis matched that established for the inhibition by these anesthetics of the EDHF-mediated relaxant response to acetylcholine. Linear regression analysis thus revealed a correlation coefficient of r = 0.7755 with a P value of 0.04.

The hypothesis that the inhibition by the inhalation anesthetics of the release of EDHF is based on their cytochrome P450-inhibitory properties is further supported by the characteristics of the inhibitory action of enflurane in the presence of either clotrimazole or tetrabutylammonium. Thus, the pharmacologic profiles of enflurane and clotrimazole were very similar: both compounds attenuated the maximum relaxant response to acetylcholine but did not shift the concentration-response curve to the right. In contrast, tetrabutylammonium caused a rightward shift of the concentration-response curve, but did not significantly affect the maximum response. Moreover, the combination of enflurane with clotrimazole or tetrabutylammonium produced an additive effect. An increase in the concentration of isoflurane or halothane, on the other hand, resulted in an inhibitory effect that closely resembled that of the combination of enflurane and clotrimazole.

Taken together, these findings suggest that the inhalation anesthetics most likely interfere with the synthesis of EDHF by the endothelium rather than with its effect on K^Casup + -channel activity in the smooth muscle. It should be emphasized, however, that on the basis of the current experiments we cannot entirely rule out an additional effect of the inhalation anesthetics on the activity of these channels. Because the release of EDHF may play an important role as a compensatory or reserve mechanism for the maintenance of vascular tone in situations where NO synthesis is reduced, such as atherosclerosis, hypertension, hypercholesterolemia, [14]or ischemia, the potential adverse effect of these anesthetics on the perfusion of vital organs during or after surgical procedures should be considered.

In summary, we have shown that inhalation anesthetics selectively attenuate the NO-independent, EDHF-mediated relaxant response to acetylcholine in the rabbit carotid artery. This effect appears to be based on an inhibition of the cytochrome P450-dependent synthesis of EDHF by the endothelium rather than an interference with its action at the level of the K^Casup + channels in the vascular smooth muscle. Further studies are needed to verify the inhibitory effects of inhalation anesthetics on EDHF formation in the human vasculature.

The authors thank Dr. Agnieszka T. Bara and Harald Rosenberg for help with the organ bath studies and gas chromatography analysis, respectively, and Gabriele Hoch for expert technical assistance.