This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Tsuchida, H. Articles by Namiki, A. Search for Related Content PubMed PubMed Citation Articles by Tsuchida, H. Articles by Namiki, A.

---

CARDIOVASCULAR ANESTHESIA

Halothane Attenuates Nitroglycerin-Induced Vasodilation and a Decrease in Intracellular Ca²⁺ in the Rat Thoracic Aorta

Department of Anesthesiology, Sapporo Medical University School of Medicine, Sapporo, Japan

Address correspondence and reprint requests to Hideaki Tsuchida, MD, Department of Anesthesiology, Kanazawa Medical University, Daigaku 1-1, Uchinada, Kahoku, Ishikawa 920-0293, Japan. Address e-mail to tsuchida{at}kanazawa-med.ac.jp

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Although halothane inhibits endothelium-mediated vasorelaxation, the sites of inhibition remain controversial. Because the cytosolic concentration of Ca²⁺ ([Ca²⁺]_i) has crucial roles for tension development, we examined the effects of halothane on nitroglycerin-induced vasorelaxation from the standpoint of [Ca²⁺]_i. Isolated spiral strips of rat thoracic aorta without endothelium were suspended for isometric tension recordings in a physiologic salt solution. Muscle contraction was evoked with 10^–8 M norepinephrine, followed by endothelium-independent vasorelaxation with nitroglycerin 10^–7 and 10^–6 M. The effects of halothane 1.5% and 3% on nitroglycerin-induced vasorelaxation were evaluated along with the concomitant measurement of [Ca²⁺]_i using fura-2-Ca²⁺ fluorescence. In other muscle strips, incremental doses of norepinephrine were administered during halothane exposure to induce contractions comparable to those without halothane. Nitroglycerin dose-dependently reduced norepinephrine-induced muscle contractions, but the decrease in [Ca²⁺]_i reached a plateau at 10^–7 M, which indicates that nitroglycerin induced [Ca²⁺]_i-dependent and [Ca²⁺]_i-independent vasorelaxation. Both concentrations of halothane inhibited nitroglycerin-induced decreases in muscle tension and [Ca²⁺]_i, not only when the same dose of norepinephrine was used for contraction during halothane exposure, but also at incremental doses of norepinephrine. In conclusion, halothane inhibits nitroglycerin-induced vasorelaxation partly by suppressing Ca²⁺ changes in the smooth muscle.

Implications: We examined nitroglycerin-induced vasorelaxation in the rat thoracic aorta, along with the concomitant measurement of the cytosolic concentrations of Ca²⁺, and found that halothane attenuated endothelium-independent vasorelaxation by suppressing Ca²⁺ dynamics in the smooth muscle.

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Nitric oxide (NO) produced in vascular endothelium activates soluble guanylate cyclase in vascular smooth muscle to form 3',5'-cyclic guanosine monophosphate (cyclic GMP), leading to vasodilation (1). Although halothane has been reported to attenuate endothelium-mediated vasodilation, the mechanisms are not fully understood (2). Early studies reported by Muldoon et al. (3), Uggeri et al. (4), and Toda et al. (5) showed that halothane inhibits endothelium-dependent vasodilation but does not affect endothelium-independent vasodilation induced by either NO donor or NO itself, indicating that halothane exerts its inhibitory action in the endothelium.

In contrast, more recent studies by Hart et al. (6) and Nakamura et al. (7) showed that halothane inhibits both endothelium-dependent and endothelium-independent vasodilation. In addition, using a bioassay system with cultured bovine aortic endothelial cells and isolated denuded rabbit aortic ring, Blaise et al. (8) reported that halothane attenuated bradykinin-induced relaxation even when halothane was added to perfusate downstream to the perfusion of the endothelial cells. The latter articles indicated that the site of inhibition by halothane may also be distal to NO production. Johns et al. (9) and Zuo et al. (10) have published a series of elegant experiments regarding the site(s) of inhibition of endothelium-dependent vasodilation by volatile anesthetics. Using vascular rings and cultured cells, they showed that volatile anesthetics did not alter NO synthase activity or directly interfere with activation of guanylyl cyclase by either NO or NO donors. They speculate that the inhibitory effects of volatile anesthetics occur before NO synthase and guanylyl cyclase.

Ca²⁺ availability is a likely site of halothane's interaction with vascular reactivity because halothane influences Ca²⁺ release from the sarcoplasmic reticulum or Ca²⁺ influx through the sarcolemma in the vascular smooth muscle (11,12). An increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) is an essential step in the synthesis and/or release of NO in endothelial cells (1), and halothane has been reported to inhibit bradykinin-induced increase in [Ca²⁺]_i in cultured bovine aortic endothelial cells (13–15). However, there is no qualitative study concerning the effect of halothane on the NO-induced decrease in muscle tension and [Ca²⁺]_i in an isolated vascular strip. The purpose of this investigation was therefore to examine the conflicting literature with respect to the effect of halothane on endothelium-independent vasodilation from the standpoint of [Ca²⁺]_i in the smooth muscle. We simultaneously measured muscle tension and [Ca²⁺]_i in the vascular smooth muscle of the rat thoracic aorta using the Ca²⁺ indicator fura-2. We used nitroglycerin as an NO donor to activate soluble guanylate cyclase in the smooth muscle and to elicit vasorelaxation. We hypothesized that halothane would interfere with nitroglycerin-induced vasorelaxation through its action on intracellular Ca²⁺ dynamics.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
This study was approved by the animal care committee of our institute. The descending thoracic aorta was isolated from male Sprague-Dawley rats (150–200 g) under halothane anesthesia and was cut into spiral strips approximately 10 mm in length and 1 mm in width under a dissecting microscope. The endothelium was denuded completely with a cotton swab moistened with physiologic salt solution (PSS). The absence of endothelium was verified by the failure of vasorelaxing to respond to carbachol (10^–6 M) during norepinephrine-induced contraction. The muscle strips were loaded with 5 µM acetoxymethyl ester of fura-2 (fura-2/AM) solution for 3–5 h at room temperature (23–25°C). A noncytotoxic detergent, 0.05% cremophor EL, was added to solubilize the fura-2/AM in a PSS. The PSS contained (in mM): NaCl 136.9, KCl 5.4, CaCl₂ 1.5, MgCl₂ 1.0, NaHCO₃, 23.8, glucose 5.5, and EDTA 0.01. The solution was saturated with a 95% O₂/5% CO₂ mixture at 37°C and pH 7.4.

Experiments were performed with a fluorimeter designed to measure the fluorescence of living tissues (CAF-100; Japan Spectroscopic, Tokyo, Japan) as reported previously (11,12). The muscle strip was held horizontally in a temperature-controlled organ bath. One end of the muscle strip was connected to a strain-gauge transducer (TB-612T; Nihon Kohden, Tokyo, Japan) to monitor the mechanical activity. The solution was aerated with a 95% O₂/5% CO₂ mixture. A passive tension of 0.5–0.7 g was applied and allowed to equilibrate before the experiment was started. In our pilot study, this resting tension obtained the maximal contractile response to 32.8 mM KCl. The muscle strip was illuminated alternately at 50 Hz at excitation wavelengths of 340 ± 10 nm and 380 ± 10 nm. The amount of 500 ± 20 fluorescence induced by 340 nm excitation (F340) and that induced by 380 nm excitation (F380) were measured successively. We did not calculate the absolute amount of [Ca²⁺]_i because the calculation could have contained an error of approximately 10% due to endogenous fluorescent substances such as NADH (16), and because we do not know the dissociation constant of fura-2 and Ca²⁺ in the cell. Instead, the ratio of F340 to F380 (R340/380) was used to indicate the [Ca²⁺]_i because these factors are unlikely to interfere with the quantitative changes in [Ca²⁺]_i.

After an equilibrating period, the muscle strip was contracted with 10^–8 M norepinephrine in the PSS. This concentration of norepinephrine evoked approximately 60% of the maximal norepinephrine-induced contraction in our pilot study. Increases in muscle tension and the R340/380 5 min after the administration of norepinephrine were regarded as the reference values (100%). Nitroglycerin 10^–7 M and 10^–6 M was then cumulatively administered in the PSS, and the changes in muscle tension and the R340/380 were observed. This procedure was repeated twice in each muscle strip. In the first set of contractions, no anesthetics were introduced through the O₂/CO₂ mixture. In the second set of contractions, either 0% (control), 1.5%, or 3% halothane was administered through the O₂/CO₂ mixture from 6 min before the administration of norepinephrine through the end of the experiment (n = 6 animals each). Only one concentration of halothane was exposed to a strip. Changes in muscle tension and the R340/380 between the two sets were compared.

Halothane did attenuate norepinephrine-induced contraction in a concentration-dependent manner in the second set of contractions. In some other muscle strips, therefore, greater doses of norepinephrine (3 x 10^–8 M and 3 x 10^–7 M norepinephrine during 1.5% and 3% halothane, respectively) were administered during the second set of contractions to induce muscle tension comparable to that observed during the first set (n = 6 each).

The following drugs and chemicals were used: fura-2/AM (Dojindo Laboratories, Kumamoto, Japan), cremophor EL, norepinephrine, carbachol (Sigma, St. Louis, MO), and halothane (Takeda Pharmaceuticals, Tokyo, Japan). Nitroglycerin was kindly donated by Nippon Kayaku Pharmaceuticals (Tokyo, Japan). The concentration of halothane in the gas mixture was monitored continuously by using a precalibrated anesthetic monitor (Model 303; Atom, Tokyo, Japan). The concentration of halothane in the bath fluid was determined by testing 2-µL aliquots of the PSS using gas chromatography (GC-17A; Shimadzu, Tokyo, Japan) as reported previously (11).

Data are expressed as mean ± SD. Statistical evaluation was performed by using Student's t-test or analysis of variance for multiple comparison. A P value of <0.05 was considered significant. If significant variance ratios were obtained using analysis of variance, a post hoc analysis was performed using Fisher's protected least significant difference test.

	Results

Top Abstract Introduction Methods Results Discussion References

 
As shown in Figure 1, 10^–8 M norepinephrine elicited an abrupt increase in R340/380, an indicator of [Ca²⁺]_i, with the gradual development of muscle contraction. These increases in muscle tension and R340/380 were reduced by the cumulative administration of nitroglycerin. Decreases in R340/380 elicited by nitroglycerin were smaller than decreases in muscle tension. Furthermore, in contrast to the dose-dependent decrease in muscle tension, the reduction of R340/380 reached a plateau at 10^–7 M nitroglycerin in the first set of contractions; no further decrease in R340/380 was observed during the administration of 10^–6 M nitroglycerin (Fig. 2, A–C). These results indicate that nitroglycerin induced Ca²⁺-dependent and Ca²⁺-independent vasorelaxation.

View larger version (15K): [in this window] [in a new window]   Figure 1. Typical recordings of the effects of nitroglycerin and 3% halothane on norepinephrine-induced increases in [Ca2+]i (depicted as R340/380) and muscle tension.

View larger version (26K): [in this window] [in a new window]   Figure 2. Nitroglycerin-induced decreases in muscle tension (• and ) and R340/380 ( and ) in strips preconstricted with norepinephrine. Halothane 0%, 1.5%, or 3% was administered during the second set of contractions ( and ) after taking control measurements (• and ). A–C, Muscle contractions were evoked by 10–8 M norepinephrine both before and during 0%, 1.5%, and 3% halothane exposures, respectively. D and E, Muscle contractions during 1.5% and 3% halothane administration, respectively, were evoked with larger doses of norepinephrine to obtain tension development comparable to those without halothane. Halothane significantly reduced both nitroglycerin-induced vasorelaxation and decreases in R340/380. Data are expressed as mean ± SD *P < 0.05, **P < 0.01, ***P < 0.001 versus the next lower concentration of nitroglycerin. P < 0.001 versus the first set of contractions.

View larger version (45K): [in this window] [in a new window]   Figure 3. Effects of 1.5% and 3% halothane on nitroglycerin-induced decreases in muscle tension and [Ca]i. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 versus 0% halothane. P < 0.05 versus 1.5% halothane. NE = norepinephrine.

	Discussion

Top Abstract Introduction Methods Results Discussion References

It is generally accepted that halothane inhibits endothelium-mediated vasorelaxation. It is, however, still controversial whether halothane inhibits smooth muscle relaxation induced by the NO donors nitroglycerin and nitroprusside or by NO itself. Halothane may inactivate NO after its production because it has been shown to generate oxygen-derived radicals (17). It may also inhibit guanylyl cyclase activity (6,7), although Johns et al. (9) and Zuo et al. (10) did not observe this inhibition.

We did not use NO itself in this experiment because it readily oxidized, even in trace amounts of oxygen (18). We also did not use nitroprusside because it has a brown color and may interfere with fura-2-Ca²⁺ signals. Instead, we used nitroglycerin as a NO donor. Nitroglycerin is metabolized and produces NO in the smooth muscle (19), so that the amount of NO produced may hardly be influenced under these oxygen-rich conditions. This drug is known to cause tolerance; increasing doses are required to obtain a given pharmacological effect after repeated administration (19). However, we did not observe tolerance in our study, probably because the duration of nitroglycerin administration was short. Our results are therefore unlikely to be explicable in terms of the amount of NO produced in the smooth muscle.

NO activates guanylyl cyclase and produces cyclic GMP in vascular smooth muscle. Cyclic GMP decreases [Ca²⁺]_i by inhibiting the L-type voltage-dependent Ca²⁺ channels and Ca²⁺ release from the intracellular storage sites and by activating the K⁺ channels and Ca²⁺ pump in the sarcolemma, resulting in vasodilation (19). It also possesses a Ca²⁺-independent mechanism to induce vasorelaxation by decreasing Ca²⁺ sensitivity of the contractile elements (19). The above facts were revealed by this study; nitroglycerin elicited vasodilation through Ca²⁺-dependent and Ca²⁺-independent mechanisms. Halothane inhibits the L-type voltage-dependent Ca²⁺ channels and the K⁺ channels (20). It can release Ca²⁺ from the intracellular storage sites in a concentration-dependent manner (12,21), so that the amount of Ca²⁺ uptake into the intracellular stores induced by nitroglycerin may be decreased. Furthermore, halothane decreases Ca²⁺ sensitivity of the contractile elements (12). As a result, the anesthetic may produce less tension change for a given change in [Ca²⁺]_i. It is therefore not surprising that halothane attenuated nitroglycerin-induced vasodilation both by Ca²⁺-dependent and Ca²⁺-independent mechanisms. This is in contrast to the interaction of 3',5'-cyclic adenosine monophosphate (cyclic AMP) and halothane in the vascular smooth muscle. We did not observe Ca²⁺-independent vasorelaxation induced by the ß-agonist isoproterenol, or the adenylyl cyclase activator forskolin, and membrane-permeable cyclic AMP, dibutylyl cyclic AMP; and halothane did not influence forskolin- or dibutylyl cyclic AMP-induced vasorelaxation (22).

The magnitude of endothelium-independent vasorelaxation depends partly on the muscle tension already developed (19). To exclude this influence, different doses of norepinephrine were administered during halothane exposure. In other words, a dose-matched study and a tension-matched study were performed to examine the effects of halothane on vasorelaxation. Halothane significantly inhibited nitroglycerin-induced vasorelaxation both in the dose-matched and tension-matched studies. Furthermore, halothane's inhibition of nitroglycerin vasorelaxation was greater in the tension-matched study than in the dose-matched study. Therefore, halothane's inhibition of nitroglycerin-induced vasorelaxation cannot be ascribed to its vasodilatory effect.

We did not obtain absolute amounts of [Ca²⁺]_i because of the limitations of this technique when applied to the smooth muscle tissue (see Methods). In the present experiments, we instead measured changes of muscle tension and R340/380. To determine whether halothane really inhibits nitroglycerin-induced vasorelaxation in a [Ca²⁺]_i-independent manner, further research is required.

At least two laboratories have reported conflicting results regarding whether halothane inhibits vasorelaxation induced by NO donor or by NO itself (3,5–7). This is not surprising considering the review by Ahlner et al. (19) These authors showed that although NO donors increase cyclic GMP content in vascular smooth muscle regardless of the type of drug used and vascular type, the absolute relationship between the degree of vascular relaxation evoked and the increase in cyclic GMP level varies greatly, even when the same vessel and the same drug are used. The type of contractile drug used to induce tension, the type of material(s), and the age of individual from which the vessel specimens were obtained may all influence the experimental results.

Zuo et al. (10) reported an interesting experiment in which they introduced inducible nitric oxide synthase by incubating rat aortic rings with lipopolysaccharide. Because halothane did not influence muscle tension and cyclic GMP production in the lipopolysaccharide-treated rat aortic rings preconstricted with phenylephrine, they suggested that neither the activation of guanylyl cyclase nor the action of cyclic GMP was the site of inhibition by halothane. However, 3% halothane reduced phenylephrine-induced muscle contraction in their rings without lipopolysaccharide pretreatment, which indicates that halothane interfered with the ₁-adrenergic system in the smooth muscle. Therefore, even if 3% halothane did not influence muscle tension and cyclic GMP content in the lipopolysaccharide-treated rings, this may not indicate that the action of cyclic GMP is not inhibited by 3% halothane. Conversely, 3% halothane may inhibit both phenylephrine-induced muscle contraction and cyclic GMP-induced muscle dilation.

In conclusion, in the present study, we clearly demonstrated that halothane inhibited nitroglycerin-dependent vasorelaxation and a decrease in [Ca²⁺]_i in the rat thoracic aorta. In terms of Ca²⁺ mobilization, therefore, our study indicates that the site of halothane's inhibition of endothelium-mediated vasorelaxation also lies in the smooth muscle.

	Acknowledgments

 
We are very grateful to Paul A. Murray, PhD, Center for Anesthesiology Research, Cleveland Clinic Foundation, for reviewing the manuscript.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide : physiology, pathology, and pharmacology. Pharmacol Rev 1991;43:109–42.[Web of Science][Medline]
2. Johns RA. Endothelium, anesthetics, and vascular control. Anesthesiology 1993;79:1381–91.[Web of Science][Medline]
3. Muldoon SM, Hart JL, Bowen KA, Freas W. Attenuation of endothelium-mediated vasodilation by halothane. Anesthesiology 1988;68:31–7.[Web of Science][Medline]
4. Uggeri MJ, Proctor GJ, Johns RA. Halothane, enflurane, and isoflurane attenuate both receptor- and non-receptor-mediated EDRF production. Anesthesiology 1992;76:1012–7.[Web of Science][Medline]
5. Toda H, Nakamura K, Hatano Y, et al. Halothane and isoflurane inhibit endothelium-dependent relaxation elicited by acetylcholine. Anesth Analg 1992;75:198–203.[Abstract/Free Full Text]
6. Hart JL, Jing M, Bina S, et al. Effects of halothane on EDRF/cGMP-mediated vascular smooth muscle relaxations. Anesthesiology 1993;79:323–31.[Web of Science][Medline]
7. Nakamura K, Terasako K, Toda H, et al. Mechanisms of inhibition of endothelium-dependent relaxation by halothane, isoflurane, and sevoflurane. Can J Anaesth 1994;41:340–6.[Web of Science][Medline]
8. Blaise G, To Q, Parent M, et al. Does halothane interfere with the release, action, or stability of endothelium-derived relaxing factor/nitric oxide? Anesthesiology 1994;80:417–26.[Web of Science][Medline]
9. Johns RA, Tichotsky A, Muro M, et al. Halothane and isoflurane inhibit endothelium-derived relaxing factor-dependent cyclic guanosine monophosphate accumulation in endothelial cell-vascular smooth muscle co-cultures independent of an effect on guanylyl cyclase activation. Anesthesiology 1995;83:823–34.[Web of Science][Medline]
10. Zuo Z, Tichotsky A, Johns RA. Halothane and isoflurane inhibit vasodilation due to constitutive but not inducible nitric oxide synthase. Anesthesiology 1996;84:1156–65.[Web of Science][Medline]
11. Tsuchida H, Namba H, Yamakage M, et al. Effects of halothane and isoflurane on cytosolic calcium ion concentrations and contraction in the vascular smooth muscle of the rat aorta. Anesthesiology 1993;78:531–40.[Web of Science][Medline]
12. Namba H, Tsuchida H. Effect of volatile anesthetics with and without verapamil on intracellular activity in vascular smooth muscle. Anesthesiology 1996;84:1365–74.
13. Loeb AL, Longnecker DE, Williamson JR. Alteration of calcium mobilization in endothelial cells by volatile anesthetics. Biochem Pharmacol 1993;45:1137–42.[Web of Science][Medline]
14. Simoneau C, Thuringer D, Cai S, et al. Effects of halothane and isoflurane on bradykinin-evoked Ca²⁺ influx in bovine aortic endothelial cells. Anesthesiology 1996;85:366–79.[Web of Science][Medline]
15. Pajewski TN, Miao N, Lynch C III, Johns RA. Volatile anesthetics affect calcium mobilization in bovine endothelial cells. Anesthesiology 1996;85:1147–56.[Web of Science][Medline]
16. Ozaki H, Sato K, Satoh T, Karaki H. Simultaneous recordings of calcium signals and mechanical activity using fluorescent dye fura-2 in isolated strips of vascular smooth muscle. J Pharmacol 1987;45:429–33.
17. Shayevitz JR, Varani J, Ward PA, Knight PR. Halothane and isoflurane increase pulmonary artery endothelial cell sensitivity to oxidant-mediated injury. Anesthesiology 1991;74:1067–77.[Web of Science][Medline]
18. Tracey WR, Linden J, Peach MJ, Johns RA. Comparison of spectrophotometric and biological assays for nitric oxide (NO) and endothelium-derived relaxing factor (EDRF) : nonspecificity of the diazotization reaction for NO and failure to detect EDRF. J Pharmacol Exp Ther 1990;252:922–8.[Abstract/Free Full Text]
19. Ahlner J, Andersson RGG, Torfgard K, Axelsson K. Organic nitrate esters : clinical use and mechanisms of actions. Pharmacol Rev 1991;43:351–423.[Web of Science][Medline]
20. Bosnjak ZJ. Ion channels in vascular smooth muscle. Anesthesiology 1993;79:1392–401.[Web of Science][Medline]
21. Kakuyama M, Hatano Y, Nakamura K, et al. Halothane and enflurane constrict canine mesenteric arteries by releasing Ca from intracellular Ca stores. Anesthesiology 1994;80:1120–7.[Web of Science][Medline]
22. Tanaka S, Tsuchida H. Effects of halothane and isoflurane on beta adrenoceptor-mediated responses in the vascular smooth muscle of rat aorta. Anesthesiology 1998;89:1209–17.[Web of Science][Medline]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Tsuchida, H. Articles by Namiki, A. Search for Related Content PubMed PubMed Citation Articles by Tsuchida, H. Articles by Namiki, A.