ENDOTHELIUM-DERIVED relaxing factor, first discovered as a potent vasodilator produced by endothelium 
is now known as nitric oxide or a chemically related compound. 
Extensive studies have demonstrated that nitric oxide is an agonist for soluble guanylyl cyclase and that this nitric oxide-guanylyl cyclase signaling pathway is present in a variety of tissues. [3,4]
The enzymes responsible for the synthesis of nitric oxide from L-arginine in mammalian tissue are known as nitric oxide synthase. 
There are three major isoforms of nitric oxide synthase. 
Two are constitutive enzymes, one normally expressed in the endothelium and one in neurons. A third inducible isoform can be produced in a variety of cells including smooth muscle cells 
and macrophages 
only after induction by endotoxin or cytokines such as tumor necrosis factor-alpha and interferon-gamma. Both constitutive and inducible isoforms contain a heme moiety and require beta-nicotinamide adenine dinucleotide phosphate (reduced form, NADPH), flavin adenine dinucleotide, flavin mononucleotide, and tetrahydrobiopterin as cofactors. [5,8]
The constitutive isoforms also are calcium and calmodulin dependent, whereas the inducible isoform has a tightly bound calmodulin subunit and does not require calcium for activation (Figure 1
Nitric oxide is an important mediator for the excitatory synaptic transmission of N-methyl-D-aspartate, glutamate, and kainate in the brain. [10-12]
It is proposed that some anesthetics may suppress excitatory transmission to achieve anesthesia through inhibiting the formation or action of nitric oxide. Johns et al. 
have demonstrated that nitroG
-L-arginine methyl ester (L-NAME), a specific nitric oxide synthase inhibitor, dose dependently and reversibly reduces the minimum alveolar concentration of halothane anesthesia in rats, suggesting an important relationship between the nitric oxide-guanylyl cyclase signaling pathway and anesthesia or level of consciousness. In addition, inhalational anesthetics such as halothane, enflurane, isoflurane, and sevoflurane have been demonstrated to inhibit endothelium-dependent vasodilation in arterial rings. [14-16]
However, the mechanisms underlying these effects are controversial. 
Early studies suggest that the site of inhibition is proximal to soluble guanylyl cyclase activation. [14,15,18,19]
Some more recent reports, however, indicate that inhalational anesthetics may also inhibit the formation or release of nitric oxide or may work as a scavenger to inactivate nitric oxide after its formation or may even interfere with the activation of soluble guanylyl cyclase by nitric oxide. [20-23]
Our recent studies, using partially purified enzymes, however, clearly demonstrate that inhalational anesthetics neither affect the basal or agonist-stimulated soluble or particulate guanylyl cyclase activity nor directly inhibit the endothelial or brain nitric oxide synthase activity in vitro. [24,25]
Our study, using an endothelium-smooth muscle coculture model, further excluded the possibility of the activation of guanylyl cyclase by nitric oxide as the inhibitory site for inhalational anesthetics. 
In light of these observations, we hypothesized that the receptor activation or downstream signaling events leading to nitric oxide synthase activation are sites of inhibition for inhalational anesthetics on the nitric oxide-guanylyl cyclase signaling pathway. Because the signaling pathway after the activation of constitutive or inducible nitric oxide synthase is identical, the lack of inhibition of inducible nitric oxide synthase-induced vasorelaxation by anesthetics would imply that anesthetics do not affect activated nitric oxide synthase enzymatic function, nitric oxide itself, guanylyl cyclase activation, or effects of cyclic guanosine 3,5-monophosphate (cGMP) in causing vasorelaxation (Figure 1
). We therefore tested our hypothesis by comparing the effects of inhalational anesthetics on calcium-/calmodulin-dependent and calcium-/calmodulin-independent nitric oxide synthase activation in rat aortic rings preincubated with or without lipopolysaccharide (LPS), measuring the agonist-stimulated constriction and relaxation as well as cGMP changes. The effect of inhalational anesthetics on partially purified inducible nitric oxide synthase activity also was investigated to confirm the results of our aortic ring study.
Materials and Methods
Preparation of Vascular Rings
Male Sprague-Dawley rats (weighing 300-350 g) were killed in accordance with our institutional Research and Animal Welfare Committee standards. The descending thoracic aorta was gently removed and placed in ice-cold modified Krebs' buffer (all in mM: NaCl 111, KCl 5, NaH2
PO sub 4 1, MgCl2
2.5, dextrose 11.1). The aorta was then dissected clean of fat and surrounding connective tissue and cut into 2.5-3.0-mm ring segments. The rings were then incubated in Dulbecco's Modified Eagle's Medium (Gibco, Grand Island, NY) containing 4,500 mg/l D-glucose and L-glutamine either with or without 500 ng/ml LPS for 5 h at 37 degrees C and continuously gassed with air and 5% CO2
Isometric Tension Measurements
The rings were either left with their endothelia intact or denuded of endothelium by gentle rotation on a forceps. The rings were then mounted on Grass Ft-03 force transducers (Grass, Quincy, MA) at 2.0 g resting tension in 37 degrees C water-jacketed 25-ml tissue baths containing 10 ml modified Krebs' buffer continuously gassed with air and 5% CO2
. Indomethacin (28 micro Meter), an inhibitor of cyclooxygenase metabolism of arachidonic acid, 
was added to the buffer throughout all experiments to prevent formation of vasoactive prostanoid metabolites. The buffer was changed every 15 min during a 60-min equilibration period. Endothelial-intact status was confirmed by constricting rings with 10 sup -7 M phenylephrine followed by relaxing them with 10 sup -6 M methacholine. If they relaxed more than 40% to methacholine they were considered to be endothelium-intact rings. Endothelium-denuded rings showed no relaxation. Rings were then washed and reequilibrated to basal tension.
Eight rings of each experiment were divided into four duplicate groups (one used for the anesthetic study, the other one used as a time-control): 1) endothelium-intact, 2) endothelium-denuded, 3) LPS-preincubated and endothelium-intact, and 4) LPS-preincubated and endothelium-denuded rings. The experimental protocols were as follows: Dose-response curves for phenylephrine (10 sup -8 to 10 sup -5 M) were first obtained to individualize the EC60 dose for each ring. This EC60 dose (60% maximal contractile dose) was used to achieve active tension and the rings were then subjected to methacholine (10 sup -7 - 10 sup -5 M). The values obtained were considered as preanesthetic control and the same experimental procedure was repeated in the presence or absence (time-control experiments) of 1%, 2%, or 3% halothane or isoflurane. Halothane or isoflurane was added to the rings 5 min before the addition of phenylephrine by a calibrated vaporizer in line with the air and 5% CO sub 2 gas at a flow rate of 4 l/min. Preliminary gas chromatographic studies suggested that the concentration of halothane or isoflurane in the buffer reached plateau after 5 min of gassing under these experimental conditions. [14,24]
Postanesthetic controls were then obtained in the absence of anesthetics. The ability of L-NAME, a competitive inhibitor of nitric oxide synthase, to reverse the relaxation caused by LPS induction or by methacholine was investigated by adding 300 micro Meter L-NAME 10 min before the addition of the same EC60 dose of phenylephrine to each of the rings. These reversal experiments were done to measure the portion of relaxation due to the nitric oxide-guanylyl cyclase signaling pathway in the total relaxation caused by LPS or methacholine.
Cyclic Guanosine 3,5-Monophosphate Analysis of Rings
Denuded rat descending thoracic aortic rings were prepared and incubated with 3 x 10 sup -7 M phenylephrine for 6 min at 37 degrees Celsius in the presence or absence of 3% halothane or 3% isoflurane preincubated as described earlier. The rings were then flash-frozen in dry ice-cooled acetone. Cyclic GMP was extracted by homogenizing each ring in 1 ml of 0.1 N ice-cold hydrochloride. After centrifugation at 1000g for 10 min, the supernatant was analyzed for cGMP content by radioimmunoassay (sup 125 Iodine kit, Amersham, Buckinghamshire, UK). 
Protein content was determined by dissolving the homogenate in 0.66 N NaOH and analyzing the total dissolved protein with the Bio-Rad protein assay method (Richmond, CA). 
Partially Purified Inducible Nitric Oxide Synthase Assay
Mouse RAW 264.7 macrophages were cultured in RPMI 1640 (Gibco) containing 10% fetal bovine serum. The confluent macrophages were then activated to express inducible nitric oxide synthase by incubating with LPS (300 ng/ml) in the same medium for 24 h at 37 degrees C. Partially purified inducible nitric oxide synthase was prepared in a manner similar to that previously described. 
Briefly, LPS stimulated macrophages were collected and washed twice with Dulbecco's phosphate-buffered saline (pH 7.4, Gibco). The cells were then homogenized by a tissue grinder fitted with a polytetrafluorethylene pestle in 50 mM Tris-HCl (pH 7.4) containing 0.1 mM ethylenediaminetetraacetic acid, 0.1 mM EGTA, 0.5 mM dithiothreitol, 1 micro Meter pepstatin, and 2 micro Meter leupeptin at 4 degrees C. Homogenates were centrifuged at 100,000g for 60 min at 4 degrees C. The supernatant was collected and used as the source of inducible nitric oxide synthase. The protein content in the supernatant also was measured with the Bio-Rad protein assay method. 
Nitric oxide synthase activity was determined by measuring the formation of3
H-L-arginine as described. 
Enzymatic reactions were performed in the reaction mixture (final volume 250 micro liter) containing 50 mM Tris-HCl (pH 7.4), 0.1 mM L-citrulline, 0.1 mM NADPH, 10 micro Meter tetrahydrobiopterin, and 50 micro Meter3
H-L-arginine in the presence or absence of halothane (1-4%) or isoflurane (1-5%) for 10 min at 37 degrees C. Preliminary time-course data demonstrated a linear increasing activity of the inducible nitric oxide synthase over the initial 10-min period of incubation under the current experimental conditions. Enzymatic reactions were terminated by adding 2 ml ice-cold stop buffer containing 20 mM sodium acetate (pH 5.5), 1 mM L-citrulline, 2 mM ethylenediaminetetraacetic acid, and 0.2 mM EGTA. The3
H-L-citrulline produced was then separated from3
H-L-arginine by Dowex AG 50W-X8 (Sodium+ form, Bio-Rad Laboratories, Hercules, CA) column. [7,25]
Data are presented as mean+/-SEM. The percent relaxation in the isometric tension study was calculated by dividing methacholine-induced relaxation (in grams) from the stable phenylephrine plateau constriction by the phenylephrine plateau constriction (in grams) and multiplying by 100. Statistical comparisons were made using paired Student's t test when comparing isometric tension of the same aortic rings treated with or without inhalational anesthetics or using one-way analysis of variance followed by Neuman-Keuls means comparison testing between different groups of aortic rings in the isometric tension study, cGMP study or partially purified inducible nitric oxide synthase activity study. P < 0.05 was considered significant. Each data point represents the mean of the data from at least six animals.
Drugs and Chemicals
Phenylephrine, methacholine, indomethacin, L-NAME, and L-citrulline were obtained from Sigma (St. Louis, MO). Halothane was obtained from Halocarbon Laboratories (Hackensack, NJ), isoflurane from Ohmeda Caribe Inc (Liberty Corner, NJ). Dowex AG 50W-X8 (Sodium+ form) and Bio-Rad protein assay reagent were obtained from Bio-Rad. Gasses (95% air and 5% CO2) were obtained from Roberts Oxygen Company (Waynesboro, VA).
Isometric Tension Study
Lipopolysaccharide significantly decreased the peak tension and shifted the dose-response curve of phenylephrine to the right in both endothelium-intact and -denuded aortic rings (Figure 2
). The phenylephrine EC60 was 2.20 x 10 sup -7 M and 1.54 x 10 sup -7 M, respectively, for endothelium-intact and -denuded rings without LPS treatment, which were significantly different from those of their counterparts with LPS treatment (3.88 x 10 sup -7 M and 4.14 x 10 sup -7 M, respectively, n = 24-28, P < 0.05).
Halothane and isoflurane significantly inhibited (at 2% or 3% of halothane or isoflurane) endothelium-dependent relaxation caused by methacholine in the rings without LPS treatment (Figure 3
(A and B)). This inhibition was reversible because methacholine caused the same extent of relaxation in the postanesthetic control as that in the preanesthetic control (Figure 3
(A and B)). This inhibition is not owing to the different experimental cycles because the parallel time-control experiments showed virtually identical magnitude of relaxation caused by methacholine over the five experimental cycles (Figure 3
Neither halothane nor isoflurane at concentrations of 1-3% affected the basal tension of rings in any groups studied. The LPS-exposed aortic rings developed less than 40% of the phenylephrine EC60 tension of the nonexposed counterparts (Table 1
). Halothane reversibly inhibited the phenylephrine EC60 tension in endothelium-denuded rings without LPS treatment. Thus, the phenylephrine EC60 tension of endothelium-denuded rings in the presence of 3% halothane was significantly lower than that of posthalothane control (P < 0.05). Similarly, 3% isoflurane also significantly inhibited the phenylephrine EC60 tension compared to that of the postisoflurane control in both endothelium-intact and -denuded rings without LPS treatment (P < 0.05). Isoflurane (3%) also significantly inhibited the phenylephrine EC60 tension compared to that of postisoflurane control in endothelium-denuded rings with LPS treatment (P < 0.05). However, the phenylephrine EC60 tension in the endothelium-intact rings incubated with LPS was neither affected by halothane nor isoflurane. Halothane also failed to affect the phenylephrine EC60 tension in the endothelium-intact, LPS-treated rings (Table 1
). The parallel time control experiments excluded the possibility that the phenylephrine EC60 tension changes described earlier were caused by different experimental cycles (Table 1
L-NAME (300 micro Meter) significantly increased the phenylephrine EC60 tension of both endothelium-intact and -denuded rings treated with LPS (P < 0.05; Figure 4
). However, the phenylephrine EC60 tension of these rings in the presence of 300 micro Meter L-NAME was still significantly lower than that of the rings without LPS treatment in the presence of 300 micro Meter L-NAME (Figure 4
), suggesting that 300 micro Meter L-NAME only partially reversed the effects of LPS on the phenylephrine EC60 tension of these rings, which is consistent with previous work from our laboratory. 
However, 300 micro Meter L-NAME abolished the response to methacholine of endothelium-intact rings without LPS treatment (1 micro Meter methacholine relaxed these rings preconstricted with phenylephrine EC60 only by 1.25+/-0.66% in the presence of 300 micro Meter L-NAME, n = 12, P > 0.05 comparing 1.25 +/-0.66% to 0).
Cyclic Guanosine 3,5-Monophosphate
Cyclic guanosine 3,5-monophosphate content in the endothelium-denuded rings was significantly increased by exposure to LPS (P < 0.05). However, neither 3% halothane nor 3% isoflurane significantly decreased the cGMP content in the LPS-treated rings (Figure 5
Inducible Nitric Oxide Synthase Activity
Neither halothane (1-4%) nor isoflurane (1-5%) significantly altered the inducible nitric oxide synthase activity (Figure 6
Several studies indicate that inhalational anesthetics inhibit the nitric oxide-guanylyl cyclase signaling pathway. [13-16,30,31]
However, the site(s) at which this inhibition takes place are not clear. The proposed sites include the synthesis, release, or transport of nitric oxide as well as the activation of guanylyl cyclase. 
We investigated the possible inhibitory sites using rat aortic rings treated with or without LPS.
Lipopolysaccharide has been demonstrated to induce expression of the inducible nitric oxide synthase isoform in endothelium and vascular smooth muscle cells as well as macrophages. [7,32,33]
In the current study, L-NAME, a specific nitric oxide synthase inhibitor, significantly increased the phenylephrine EC60 of the LPS-treated aortic rings, suggesting the induction of inducible nitric oxide synthase. Because inducible nitric oxide synthase has calmodulin tightly bound in its resting state, it is continuously activated without additional calcium. [5,9]
The observation that neither halothane nor isoflurane significantly increased the phenylephrine EC60 tension in the LPS-treated rings suggests that neither inhalational anesthetic inhibits the nitric oxide production of these vascular rings. The cGMP data further suggest that halothane and isoflurane do not inhibit the inducible nitric oxide synthase activity because the cGMP increase caused by inducible nitric oxide synthase was not affected by either anesthetic. Consistent with this, neither halothane nor isoflurane significantly inhibited the partially purified inducible nitric oxide synthase activity. Therefore, direct inhibition of nitric oxide synthase enzymatic function or any distal point in the nitric oxide-guanylyl cyclase-cGMP pathway is not the major site at which these two anesthetics inhibit the nitric oxide-guanylyl cyclase signaling pathway.
This is consistent with the results of a study conducted in our laboratory that demonstrated that inhalational anesthetics at concentrations ranging from 1% to 4% produced no significant effect on either endothelial or brain nitric oxide synthase activity in vitro under a variety of experimental conditions. 
However, a study by Tobin et al. 
showed that halothane and isoflurane at clinically relevant concentrations (0.5-2%) inhibited isolated rat brain nitric oxide synthase activity. The reason for these controversial results is not known. However, consistent with our results, Tagliente 
recently reported that halothane at different concentrations caused no significant change in the Michaelis constant (Km) for L-arginine or maximum velocity (Vmax
) of nitric oxide synthase, suggesting that the mechanism of anesthetic action of halothane is not mediated by direct alteration of nitric oxide synthase activity.
Alternatively, guanylyl cyclase has been proposed as the site for inhalational anesthetic inhibition of the nitric oxide-guanylyl cyclase signaling pathway. This has been suggested by arterial ring studies using sodium nitroprusside, nitroglycerin, or nitric oxide as the vessel relaxants 
and by evaluating the effect of anesthetics on a partially purified guanylyl cyclase enzyme system. [22,23,36]
However, a variety of studies using similar models have not confirmed these observations. [14,24,36]
We prepared partially purified soluble and particulate guanylyl cyclases from rat brain and demonstrated that halothane, enflurane, or isoflurane at a very wide range of concentrations did not affect the basal or agonist-stimulated activity of partially purified guanylyl cyclase in vitro. 
Consistent with these results, another study employing endothelium smooth muscle cell coculture methods, using intact cells, also strongly suggested that halothane and isoflurane did not affect the activation of guanylyl cyclase by sodium nitroprusside, nitroglycerin, or nitric oxide. 
The current study provides further evidence that halothane and isoflurane do not inhibit guanylyl cyclase or the subsequent actions of cGMP in eliciting vascular relaxation. If the activation of guanylyl cyclase or the action of cGMP is the site of inhibition, the increase of cGMP in the LPS-treated rings should be significantly inhibited by halothane or isoflurane and the decrease in constriction to phenylephrine of the LPS-treated rings should be reversed by these two anesthetics. These two effects have not been observed in this study; therefore, current evidence strongly suggests that the inhibitory sites for inhalational anesthetics on the nitric oxide-guanylyl cyclase signaling pathway are proximal to guanylyl cyclase.
Endothelial nitric oxide synthase is a constitutive form of nitric oxide synthase, which requires calcium for activation 
). Methacholine acts on the muscarinic receptor on the endothelial cell surface, resulting in a receptor-mediated increase in cytosolic calcium from both extracellular and intracellular sources and a subsequent increase in production of nitric oxide. 
Methacholine may also cause the release of endothelium-derived hyperpolarizing factor to induce vasorelaxation, mainly in small blood vessels. 
The contribution of hyperpolarizing factor to the vasorelaxation caused by methacholine in our current experiments is minimal because 300 micro Meter L-NAME abolished the vasorelaxation by methacholine. Our results demonstrate that both halothane and isoflurane reversibly inhibited the vascular ring relaxation caused by methacholine. This inhibition occurred in the presence of indomethacin, which inhibits the production of vasoactive prostanoid metabolites; the production of which may be stimulated by methacholine as well as by inhalational anesthetics, 
confirming the previous vascular ring studies in the absence of indomethacin. [14,15,18,19]
The results also showed that neither halothane nor isoflurane affected the basal endothelial nitric oxide synthase activity because neither of them affected the basal tension in those endothelium-intact rings. Therefore, agonist-stimulated receptor activation and/or subsequent events leading to an increase in cytosolic calcium and nitric oxide synthase activation may be important sites for the inhalational anesthetic inhibition of the nitric oxide-guanylyl cyclase signaling pathway (Figure 1
Inhalational anesthetics have been demonstrated to have significant effects on cytosolic calcium concentration in multiple cell types, including endothelial cells, through an effect on calcium movement into the cells, either by changing calcium influx through receptor- or voltage-activating membrane calcium channels or by an alteration in calcium release from or uptake into the sarcoplasmic reticulum. [38,39]
Using fluorescent dye, Uhl et al.* and Loeb et al. 
reported that halothane significantly inhibited the endothelial cell calcium transient stimulated by the agonists bradykinin and adenosine triphosphate. Inhalational anesthetics also have been shown to impair receptor activation. Halothane has been shown to shorten acetylcholine receptor kinetics, 
and isoflurane has been shown to cause flickering of the acetylcholine receptor. 
Many inhalational anesthetics (such as halothane, enflurane, and isoflurane) have been shown to interfere with the coupling between muscarinic receptors and their G proteins. [43-45]
Therefore, it is clear from the literature that inhalational anesthetics can impair receptor activation and the cytosolic calcium responses caused by agonists. Consistent with this idea, a study from our laboratory demonstrated that inhalational anesthetics inhibited the receptor-mediated and nonreceptor-mediated but calcium-dependent nitric oxide synthase activation in rat aortic rings. 
Apart from the inhibition of endothelium-dependent relaxation, both halothane and isoflurane are also shown to have vasorelaxant effects in this isolated vessel preparation because the phenylephrine EC60 tension in the presence of 3% halothane or 3% isoflurane was significantly less than in controls. Consistent with previous reports, this vasorelaxation was endothelium-independent. 
In summary, both halothane and isoflurane produced a reversible inhibition of agonist-stimulated, nitric oxide-mediated vasorelaxation of rat aortic rings. Neither halothane nor isoflurane, at the tested concentrations, affected the basal endothelial nitric oxide synthase or inducible nitric oxide synthase vasorelaxation, isolated inducible nitric oxide synthase activity, or the increase of cGMP caused by inducible nitric oxide synthase in the LPS-treated rings. Therefore, the receptor activation and/or downstream signaling events that lead to increases in intracellular calcium and nitric oxide synthase activation or interactions with other cofactors or regulatory mechanisms of nitric oxide synthase activity may be primary sites for inhalational anesthetics to inhibit the nitric oxide-guanylyl cyclase signaling pathway.
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© 1996 American Society of Anesthesiologists, Inc.