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Possible involvement of neuronal nitric oxide synthase enzyme in early-phase isoflurane-induced hypotension in rats
Life Sciences 76 (2004) 499 – 507 www.elsevier.com/locate/lifescie
Possible involvement of neuronal nitric oxide synthase enzyme in early-phase isoflurane-induced hypotension in rats Elizabeth A. Ellenbergera, Heather L. Lucasa,1, Janet L. Muellera, Peggy L. Barrington, Eunhee Chungb, Yusuke Ohgamib, Raymond M. Quockb,c,* a
Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, Rockford, Illinois, USA b Department of Pharmaceutical Sciences, College of Pharmacy, USA c Center for Integrative Biotechnology, Washington State University, Pullman, Washington, USA Received 13 November 2003; accepted 15 April 2004 This article is dedicated to the memory of Peggy L. Barrington
Abstract This study was conducted to demonstrate the involvement of nitric oxide synthase (NOS) in the early-phase isoflurane-induced hypotension and to ascertain whether this NOS is neuronal NOS (nNOS) or endothelial NOS (eNOS). Mean arterial pressures (MAPs) were directly measured from the femoral arteries of urethaneanesthetized rats. Isoflurane-induced changes in MAP were monitored in rats following pretreatment with vehicle or one of the following NOS inhibitors: L-NG-monomethyl-L-arginine (L-NMMA), which is nonselective; L-NG-nitro arginine (L-NOARG), which is more selective for nNOS and eNOS; and 7-nitroindazole (7-NI), which is selective for nNOS. Exposure to 2% isoflurane in oxygen produced a triphasic reduction in MAP, including an early phase in which mean arterial pressure (MAP) fell by 25-30% during the initial 2O min. This early hypotensive response, but not subsequent phases, was abolished by i.v. pretreatment with either L-NMMA or L-NOARG. The early-phase hypotension was also significantly attenuated by i.p. pretreatment with 7-NI; however, the blockade was not as complete as with L-NMMA or L-NOARG. Cerebella and aorta were removed from vehicle- and 7-NI pretreated rats and assayed for NOS activity by determining the conversion of [14C]L-arginine to [14C]L-citrulline. The 7-NI pretreatment significantly reduced NOS activity in the cerebellum but not the aorta. These findings indicate that the early-phase isoflurane-
* Corresponding author. Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, P.O. Box 646534, Pullman, WA 99164-6534, USA. Tel.: +1 509 335 5956; fax: +1 509 335 5902. E-mail address: [email protected] (R.M. Quock). 1 Present address: Red Bud Regional Hospital, Red Bud, Illinois 62278, USA. 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.04.059
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induced hypotension may involve nNOS as well as eNOS. The nNOS may participate in regulation of isoflurane-induced neuronal release of endogenous opioid peptide, which produces a vasodilation that is dependent on NO derived from an action of eNOS. D 2004 Elsevier Inc. All rights reserved. Keywords: Blood pressure; Isoflurane-induced hypotension; Nitric oxide; NOS-inhibitors
Introduction Isoflurane produces a potent systemic vasodilation (Eger, 1984). Recently we reported that inhalation of isoflurane by rats under basal urethane anesthesia caused a triphasic change in the mean arterial pressure (MAP) (Ellenberger et al., 2003). There was an initial sharp reduction in MAP that was followed by a transient recovery toward basal pressure then a prolonged and more gradual reduction in MAP. This early-phase isoflurane-induced hypotension was antagonized by pretreatment with peripherally active opioid antagonists as well as rabbit antisera against rat met-enkephalin. We suggested that this early hypotensive response might be due to isoflurane-stimulated release of met-enkephalin or a metenkephalin-like opioid peptide (Ellenberger et al., 2003). It is widely thought that relaxation of peripheral vascular smooth muscle may be dependent on endothelium-derived nitric oxide (NO) (Furchgott, 1983; Ignarro et al., 1999). Since research from our laboratory has also suggested that neuronal release of opioid peptide is NO-dependent (Hara et al., 1995), this study was conducted to demonstrate and characterize the involvement of NOS in the early-phase isoflurane-induced hypotension.
Materials and methods Animals Male Sprague Dawley rats, 250–350 g, (Hilltop Lab Animals, Inc., Scottdale, Pennsylvania) were used in this research, which was reviewed and approved by the Institutional Animal Care and Use Committee of the University of Illinois College of Medicine at Rockford. The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85–23, revised 1996). All animals were housed in temperature- and humidity-regulated quarters with a 12-h light:dark cycle. Food and water were available ad libitum. Measurement of blood pressure Basal anesthesia was induced in rats by intraperitoneal (i.p.) injection of urethane (1.4 g/kg). Animals were further pretreated with atropine methylbromide (1.0 mg/kg, i.p.) to reduce bronchial secretions. The femoral artery of each rat was exposed and cannulated with polyethylene tubing filled with 0.9% physiological saline solution and heparin (20 units/ml). The blood pressure was measured using a pressure transducer connected to a Gould Brush 2200 recorder (Gould, Cleveland, Ohio).
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After cannulation, rats were maintained for at least 30 min on 95% oxygen administered through a feline facemask, while blood pressures stabilized. If a steady blood pressure was not maintained, the rat was excluded from the study. Basal (time zero) blood pressures were recorded at the end of this time then rats were exposed to 2% isoflurane in oxygen for 20 min. Blood pressures were continuously monitored during isoflurane exposure. The MAP was determined from the blood pressure (BP) tracings using the following equation: MAP = diastolic BP + 1/3 (systolic BP-diastolic BP). The MAPs calculated at 1.0 min, 1.5 min, 2.5 min, 5.0 min, 7.5 min, 10 min, and 20 min were used to determine the percent change from the basal MAP. Assay of nitric oxide synthase activity Two hr following pretreatment with either 7-NI or peanut oil vehicle, rats were euthanized by decapitation; their whole brains and aortas removed and frozen in liquid nitrogen. NOS activity was determined by measuring the conversion of [14C]L-arginine to [14C]L-citrulline, as described by Dwyer et al. (1991). The whole brain or aorta was homogenized in 2 volumes of Tris-HCl buffer (50 mM, pH 7.4) containing 2 mM EDTA and 2 mM EGTA, and centrifuged at 12,000 rpm at 48C for 5 min. Twenty ml of supernatant were added to test tubes containing 50 mM TrisHCl buffer, 10 mM NADPH, 6.0 mM CaCl2, 6.0 mM BH4, 2.0 mM FAD, 2.0 mM FMN and 0.5 ACi [14C]L-arginine monohydrochloride (Amersham Pharmacia Biotech, Piscataway, NJ) in a final volume of 40 ml at pH 7.4. Following incubation at 378C for 30 min, the reaction was terminated by the addition of 50 mM HEPES buffer containing 5 mM EDTA and resin. Then the reaction mixture applied onto 1.5 ml columns of Dowex AG50WX-8 (Bio-Rad, Hercules, California). [14C]L-citrulline was quantified by scintillation spectroscopy of 10-ml aliquots of the flow-through. Protein concentration was determined using a standard protein assay kit (Pierce Chemical Co., Rockford, Illinois). NOS activity was expressed in terms of pmol/mg protein/min and then expressed as % of control. Drugs The following drugs were used in this research: Isoflurane, U.S.P. (Abbott Laboratories, North Chicago, Illinois); Oxygen, U.S.P. (Rockford Industrial Welding, Rockford, Illinois); NG-monomethylL-arginine acetate (L-NMMA) (Tocris Cookson, Ballwin, Missouri); NG-nitro-L-arginine (L-NOARG) (Research Biochemicals Inc., Natick, Massachusetts); 7-nitroindazole (7-NI) (Alexis, San Diego, California); and urethane (Sigma Chemical Company, St. Louis, Missouri). Two percent isoflurane in oxygen was administered using a Fluotec 3 anesthesia machine (Fraser Harlake, Orchard Park, New York). In control experiments, 95% oxygen in lieu of anesthetic agent was delivered into the facemask at the same inflow rate. Exposures were conducted inside a fumehood. In all experiments, the levels of isoflurane and oxygen being delivered via the facemask were continuously monitored using a POET II anesthetic monitoring system (Criticare, Milwaukee, Wisconsin). The femoral vein was cannulated for i.v. drug administration. L-NMMA and L-NOARG were prepared in 0.9% saline solution and administered in i.v. doses of 10 and 30 mg/kg, respectively, 30 min prior to isoflurane exposure. Because of its poor solubility in aqueous solution, 7-NI was suspended in peanut oil (Planters, East Hanover, New Jersey) and administered in an i.p. dose of 100 mg/kg 2 hr prior to isoflurane exposure. In
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Fig. 1. Change in mean arterial pressure (MAP) in urethane-anesthetized rats exposed to 2% isoflurane (ISO) in oxygen following intravenous (i.v.) pretreatment with saline (SAL, n = 6), or 30 mg/kg NG-monomethyl-L-arginine acetate (L-NMMA, n = 6) or 10 mg/kg NG-nitro-L-arginine (L-NOARG, n = 6). The change in MAP was determined relative to MAP at time 0 prior to ISO exposure. Each symbol represents the mean and vertical lines indicate the S.E.M. Significance of difference: *, p b 0.05, compared to SAL + ISO group (repeated-measures ANOVA and post-hoc Bonferroni test). Basal MAPs at time zero were 92.6 F 4.2 mm Hg for the SAL + ISO group, 130.7 F 5.3 mm Hg for the L-NMMA + ISO group and 142.3 F 2.4 mm Hg for the L-NOARG + ISO group. The basal MAPs of the L-NMMA + ISO and L-NOARG + ISO groups were significantly greater than that of the SAL + ISO control group at p b 0.05 (one-way ANOVA and Dunnett’s t-test).
Fig. 2. Change in mean arterial pressure (MAP) in urethane-anesthetized rats exposed to 2% isoflurane (ISO) in oxygen following intraperitoneal (i.p.) pretreatment with peanut oil vehicle (VEH, n = 6) or 100 mg/kg 7-nitroindazole (7-NI, n = 6). The change in MAP was determined relative to MAP at time 0 prior to ISO exposure. Each symbol represents the mean and vertical lines indicate the S.E.M. Significance of difference: *, p b 0.05, compared to VEH + ISO group (repeated-measures ANOVA and post-hoc Bonferroni test). Basal MAPs at time zero were 101.2 F 3.3 mm Hg for the VEH + ISO group and 122.8 F 2.7 mm Hg for the 7-NI + ISO group. The basal MAP of the 7-NI + ISO group was significantly greater than that of the VEH + ISO control group at p b 0.05 (Student’s t-test).
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control experiments, saline was administered i.v. in lieu of L-NMMA and L-NOARG, and peanut oil was administered i.p. in lieu of 7-NI. Statistical analysis of data A repeated-measures analysis of variance (ANOVA) with a post-hoc Bonferroni multiplecomparison test (significance set at 0.05) was used to compare isoflurane-induced changes in MAP in control vs. pretreatment groups of rats. Percent changes in MAP were arcsine transformed prior to statistical analysis. A one-way ANOVA with a post-hoc Dunnett’s t-test or Student’s t-test (depending on the number of experimental groups) was used to compare basal MAPs of various treatment groups. Student’s t-test was employed to compare NOS activity levels of vehicle and 7-NI pretreatment groups.
Fig. 3. NOS enzyme activity in cerebellum (top panel) and aorta (bottom panel) of rats pretreated with vehicle (peanut oil) or 100 mg/kg 7-NI. Each symbol represents the mean and vertical lines indicate the S.E.M. Significance of difference: *, p b 0.05 between groups indicate by the brackets above (two-way ANOVA and post-hoc Bonferroni test).
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Results MAPs in control, urethane-anesthetized rats exposed to 95% oxygen alone were stable for up to 3 hr following cannulation of the femoral artery. When superimposed for 20 min on the basal urethane anesthesia, 2% isoflurane induced a triphasic change in blood pressure (Fig. 1). There was an initial sharp drop in MAP that was maximal at 1.5 min of isoflurane exposure; the average reduction in MAP at this time point was 27.4 F 2.7%, relative to the MAP just prior to induction with isoflurane. This initial drop in MAP was followed by a transient and partial recovery toward baseline MAP which was interrupted by a longerlasting and more gradual reduction in MAP that reached 36.4% at the end of the 20 min exposure period. The resting MAP at time zero was significantly elevated by pretreatment with all three NOSinhibitors. The early hypotensive phase induced by isoflurane was significantly attenuated in rats by pretreatment with all three NOS-inhibitors (Fig. 1). Specifically, a 3 7 repeated measures ANOVA showed a significant interaction (F14,105 = 9.77, p b 0.0001) for animals that were pretreated i.v. with 30 mg/kg L-NMMA or 10 mg/kg of L-NOARG 30 min prior to exposure to isoflurane. Post-hoc analysis revealed the attenuation by L-NMMA and L-NOARG at the 1.0, 1.5, 5.0, and 20 min time points. Further, L-NOARG also significantly attenuated the isoflurane reduction in MAP at 7.5 min. The early-phase isoflurane-induced hypotension was also significantly attenuated in rats (Fig. 2) that were pretreated with 100 mg/kg of 7-NI 2 hr prior to isoflurane exposure, compared to rats receiving vehicle only (F1,10 = 7.82, p b 0.05). The mean MAP responses of the early-phase isoflurane-induced hypotension were significantly different from one another at 1.5, 2.5 and 5.0 min. A comparison of the isoflurane-induced reduction in MAPs at 1.5 min shows that the antagonism of the early-phase isoflurane-induced hypotensive response by 7-NI appeared to be less complete than by the other two NOS-inhibitors (a mean reduction in MAP in L-NMMA-pretreated rats of 4.0%, L-NOARG-pretreated rats 2.1% and 7-NI pretreated rats 8.8%). Rats pretreated with 100 mg/kg 7-NI showed a 25% reduction in NOS activity in cerebellum and b 5% reduction in the aorta (Fig. 3), compared to rats receiving peanut oil vehicle only. Compared to rats that had no pretreatment, the peanut oil vehicle reduced NOS activity by 5% in the cerebellum but increased NOS activity by 27% in the aorta.
Discussion The attenuation of the isoflurane-induced early hypotension by L-NMMA implicates involvement of NO in the response. However, NOS occurs in neuronal (nNOS), endothelial (eNOS) and inducible (iNOS) isoforms (Fo¨rstermann et al., 1994) and since L-NMMA is relatively non-selective for all three isoforms (Reif and McCreedy, 1995), it is not immediately evident which isoform of NOS is involved in the early hypotensive response to isoflurane. To distinguish among these different forms of NOS, additional NOS-inhibitors were administered. While it must be acknowledged that these compounds cannot be considered absolutely selective for a given isoform, they do nonetheless demonstrate a relative selectivity or preference for one NOS isoform over another. L-NOARG effectively inhibits eNOS (Gross et al., 1990) and nNOS (Klatt et al., 1994) but is reportedly 20-fold less effective for inhibiting iNOS (Lambert et al., 1991). Like L-NMMA, L-NOARG virtually abolished the early hypotensive response. One interpretation of these findings is that the vasodilation responsible for the early-phase isoflurane-induced hypotension is NO-dependent and hence sensitive to antagonism by
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any inhibition of eNOS. This would be consistent with a report that L-NOARG attenuated the vasodilation induced by the opioid peptides met-enkephalin and leu-enkephalin (Devine and Armstead, 1995). On the other hand, 7-NI is reputedly selective for nNOS and has little activity against either iNOS or eNOS (Moore et al., 1993a,b). In the present research, 100 mg/kg 7-NI selectively antagonized the earlyphase hypotension; however, the antagonism by 7-NI was not as complete as observed with L-NMMA or L-NOARG. Some researchers have reported that 7-NI can elevate the blood pressure leading to speculation that 7-NI might not be entirely selective for nNOS and may also inhibit eNOS (Zagvazdin et al., 1996). This caveat was precluded by the NOS assays in this study showing that pretreatment with 100 mg/kg 7-NI reduced cerebellar NOS activity by 25%, compared to essentially no change in the peanut oil control group. On the assumption that cerebellar NOS activity reflects mainly that of nNOS and aortic NOS activity reflects mainly that of eNOS, these findings indicate that 7-NI was, indeed, selective for nNOS. The greater degree of antagonism of isoflurane by L-NMMA and N-LOARG might indicate the ability of these drugs to inhibit both nNOS and e-NOS. The lower degree of antagonism by 7-NI may be attributable to its ability to inhibit nNOS only. We earlier reported that early-phase isoflurane-induced hypotension was attenuated by opioid antagonists and was also blocked by rabbit antisera against the opioid peptide met-enkephalin (Ellenberger et al., 2003). We postulated that this early hypotensive response to isoflurane might involve release of either met-enkephalin or a met-enkephalin-like peptide which presumably activates A opioid receptors. In another investigation, NOS inhibition reduced the stimulated release of met-enkephalin in the rat spinal cord (Hara et al., 1995), suggesting that perhaps the neuronal release of met-enkephalin might be NO-dependent. Additional support for a link between NO and opioid peptides is the observation that the NO-donor sodium nitroprusside increased levels of met-enkephalin in the cortical periarachnoid cerebrospinal fluid of newborn pigs (Armstead, 1995). It is, therefore, plausible that the release of met-enkephalin by isoflurane might be dependent on NO produced by nNOS. At the same time, endothelium-derived NO is likely to be involved in the vascular muscle relaxation (Furchgott, 1983; Ignarro et al., 1999). Isoflurane-induced vasodilation of cerebral and coronary blood vessels are reportedly antagonized or reversed by pretreatment with L-NAME (McPherson et al., 1993; Koenig et al., 1994), L-NMMA (Park et al., 1994) or endothelial denudation (McPherson et al., 1993; Park et al., 1994), which implicates the endothelium and production of NO in vasodilation. This may corresponds to our observed NO-dependent early-phase hypotensive effect of isoflurane. On the other hand, there is also evidence that isoflurane-induced vasodilation in certain vascular tissue preparations is independent of NO (Flynn et al., 1992; Jensen et al., 1992; Crystal et al., 1996). This, in turn, possibly corresponds to the more prolonged late-phase hypotensive effect of isoflurane, which is seemingly not mediated by NO. In summary, our results from our experiments utilizing three NOS-inhibitors of varying selectivity implicate roles for NO derived from nNOS action as well as NO derived from eNOS action in earlyphase isoflurane-induced hypotension.
Acknowledgements This work was supported by a grant from the American Heart Association/Midwest Consortium (R.M.Q.), NIH grant DA-10047 and Walter Rice Craig Fellowship from the University of Illinois at
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Chicago (H.L.L.). We are grateful to Dr. B.K. Slinker (Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, College of Veterinary Medicine, Washington State University) for assistance with the statistical analysis of the data and to Dr. S. Li (Department of Pharmacology, University of Washington) for her help with the figures.
References Armstead, W.M., 1995. Opioids and nitric oxide contribute to hypoxia-induced pial artery vasodilation. American Journal of Physiology 268 (1 Pt 2), H226 – H232. Crystal, G.J., Gurevicius, J., Salem, M.R., 1996. Isoflurane-induced coronary vasodilation is preserved in reperfused myocardium. Anesthesia and Analgesia 82, 22 – 28. Devine, J., Armstead, W.M., 1995. The role of nitric oxide in opioid-induced pial artery vasodilation. Brain Research 675, 257 – 263. Dwyer, M.A., Bredt, D.S., Snyder, S.H., 1991. Nitric oxide synthase: irreversible inhibition by L-NG-nitroarginine in brain in vitro and in vivo. Biochemical and Biophysical Research Communications 176, 1136 – 1141. Eger II, E.I., 1984. The pharmacology of isoflurane. British Journal of Anaesthesia 56 (Suppl. 1), 71S – 99S. Ellenberger, E.A., Lucas, H.L., Russo, J.M., Mueller, J.L., Barrington, P.L., Tseng, L.F., Quock, R.M., 2003. An opioid basis for early-phase isoflurane-induced hypotension in rats. Life Sciences 73, 2591 – 2602. Flynn, N.M., Buljubasic, N., Bosnjak, Z.J., Kampine, J.P., 1992. Isoflurane produces endothelium-independent relaxation in canine middle cerebral arteries. Anesthesiology 76, 461 – 467. Ffrstermann, U., Closs, E.I., Pollock, J.S., Nakane, M., Schwarz, P., Gath, I., Kleinert, H., 1994. Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions. Hypertension 23, 1121 – 1131. Furchgott, R.F., 1983. Role of endothelium in responses of vascular smooth muscle. Circulation Research 53, 557 – 573. Gross, S.S., Stuehr, D.J., Aisaka, K., Jaffe, E.A., Levi, R., Griffith, O.W., 1990. Macrophage and endothelial cell nitric oxide synthesis: cell-type selective inhibition by NG-aminoarginine, NG-nitroarginine and NG-methylarginine. Biochemical and Biophysical Research Communications l70, 96 – 103. Hara, S., Kuhns, E.R., Ellenberger, E.A., Mueller, J.L., Shibuya, T., Endo, T., Quock, R.M., 1995. Involvement of nitric oxide in intracerebroventricular h-endorphin-induced neuronal release of methionine-enkephalin. Brain Research 675, 190 – 194. Ignarro, L.J., Cirino, G., Casini, A., Napoli, C., 1999. Nitric oxide as a signaling molecule in the vascular system: an overview. Journal of Cardiovascular Pharmacology 34, 879 – 886. Jensen, N.F., Todd, M.M., Kramer, D.J., Leonard, P.A., Warner, D.S., 1992. A comparison of the vasodilating effects of halothane and isoflurane on the isolated rabbit basilar artery with and without intact endothelium. Anesthesiology 76, 624 – 634. Klatt, P., Schmidt, K., Brunner, F., Mayer, B., 1994. Inhibitors of brain nitric oxide synthase. Binding kinetics, metabolism, and enzyme inactivation. Journal of Biological Chemistry 269, 1674 – 1680. Koenig, H.M., Pelligrino, D.A., Wang, Q., Albrecht, R.F., 1994. Role of nitric oxide and endothelium in rat pial vessel dilation response to isoflurane. Anesthesia and Analgesia 79, 886 – 891. Lambert, L.E., Whitten, J.P., Baron, B.M., Cheng, H.C., Doherty, N.S., McDonald, I.A., 1991. Nitric oxide synthesis in the CNS, endothelium and macrophages differs in its sensitivity to inhibition by arginine analogues. Life Sciences 48, 69 – 75. McPherson, R.W., Kirsch, J.R., Moore, L.E., Traystman, R.J., 1993. NN¯-nitro-L-arginine methyl ester prevents cerebral hyperemia by inhaled anesthetics in dogs. Anesthesia and Analgesia 77, 891 – 897. Moore, P.K., Babbedge, R.C., Wallace, P., Gaffen, Z.A., Hart, S.L., 1993a. 5-Nitro indazole, an inhibitor of nitric oxide synthase, exhibits antinociceptive activity in the mouse without increasing blood pressure. British Journal of Pharmacology 108, 296 – 297. Moore, P.K., Wallace, P., Gaffen, Z., Hart, S.L., Babbedge, R.C., 1993b. Characterization of the novel nitric oxide synthase inhibitor 7-nitroindazole and related indazoles: antinociceptive and cardiovascular effects. British Journal of Pharmacology 110, 219 – 224.
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Park, K.W., Dai, H.B., Lowenstein, E., Darvish, A., Sellke, F.W., 1994. Heterogeneous vasomotor responses of rabbit coronary microvessels to isoflurane. Anesthesiology 81, 1190 – 1197. Reif, D.W., McCreedy, S.A., 1995. N-Nitro-L-arginine and N-monomethyl-L-arginine exhibit a different pattern of inactivation towards the three nitric oxide synthases. Archives of Biochemistry and Biophysics 320, 170 – 176. Zagvazdin, Y., Sancesario, G., Wang, Y.X., Share, L., Fitzgerald, M.E.C., Reiner, A., 1996. Evidence from its cardiovascular effects that 7-nitroindazole may inhibit endothelial nitric oxide synthase in vivo. European Journal of Pharmacology 303, 61 – 69.
Possible involvement of neuronal nitric oxide synthase enzyme in early-phase isoflurane-induced hypotension in rats Elizabeth A. Ellenbergera, Heather L. Lucasa,1, Janet L. Muellera, Peggy L. Barrington, Eunhee Chungb, Yusuke Ohgamib, Raymond M. Quockb,c,* a
Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, Rockford, Illinois, USA b Department of Pharmaceutical Sciences, College of Pharmacy, USA c Center for Integrative Biotechnology, Washington State University, Pullman, Washington, USA Received 13 November 2003; accepted 15 April 2004 This article is dedicated to the memory of Peggy L. Barrington
Abstract This study was conducted to demonstrate the involvement of nitric oxide synthase (NOS) in the early-phase isoflurane-induced hypotension and to ascertain whether this NOS is neuronal NOS (nNOS) or endothelial NOS (eNOS). Mean arterial pressures (MAPs) were directly measured from the femoral arteries of urethaneanesthetized rats. Isoflurane-induced changes in MAP were monitored in rats following pretreatment with vehicle or one of the following NOS inhibitors: L-NG-monomethyl-L-arginine (L-NMMA), which is nonselective; L-NG-nitro arginine (L-NOARG), which is more selective for nNOS and eNOS; and 7-nitroindazole (7-NI), which is selective for nNOS. Exposure to 2% isoflurane in oxygen produced a triphasic reduction in MAP, including an early phase in which mean arterial pressure (MAP) fell by 25-30% during the initial 2O min. This early hypotensive response, but not subsequent phases, was abolished by i.v. pretreatment with either L-NMMA or L-NOARG. The early-phase hypotension was also significantly attenuated by i.p. pretreatment with 7-NI; however, the blockade was not as complete as with L-NMMA or L-NOARG. Cerebella and aorta were removed from vehicle- and 7-NI pretreated rats and assayed for NOS activity by determining the conversion of [14C]L-arginine to [14C]L-citrulline. The 7-NI pretreatment significantly reduced NOS activity in the cerebellum but not the aorta. These findings indicate that the early-phase isoflurane-
* Corresponding author. Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, P.O. Box 646534, Pullman, WA 99164-6534, USA. Tel.: +1 509 335 5956; fax: +1 509 335 5902. E-mail address: [email protected] (R.M. Quock). 1 Present address: Red Bud Regional Hospital, Red Bud, Illinois 62278, USA. 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.04.059
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induced hypotension may involve nNOS as well as eNOS. The nNOS may participate in regulation of isoflurane-induced neuronal release of endogenous opioid peptide, which produces a vasodilation that is dependent on NO derived from an action of eNOS. D 2004 Elsevier Inc. All rights reserved. Keywords: Blood pressure; Isoflurane-induced hypotension; Nitric oxide; NOS-inhibitors
Introduction Isoflurane produces a potent systemic vasodilation (Eger, 1984). Recently we reported that inhalation of isoflurane by rats under basal urethane anesthesia caused a triphasic change in the mean arterial pressure (MAP) (Ellenberger et al., 2003). There was an initial sharp reduction in MAP that was followed by a transient recovery toward basal pressure then a prolonged and more gradual reduction in MAP. This early-phase isoflurane-induced hypotension was antagonized by pretreatment with peripherally active opioid antagonists as well as rabbit antisera against rat met-enkephalin. We suggested that this early hypotensive response might be due to isoflurane-stimulated release of met-enkephalin or a metenkephalin-like opioid peptide (Ellenberger et al., 2003). It is widely thought that relaxation of peripheral vascular smooth muscle may be dependent on endothelium-derived nitric oxide (NO) (Furchgott, 1983; Ignarro et al., 1999). Since research from our laboratory has also suggested that neuronal release of opioid peptide is NO-dependent (Hara et al., 1995), this study was conducted to demonstrate and characterize the involvement of NOS in the early-phase isoflurane-induced hypotension.
Materials and methods Animals Male Sprague Dawley rats, 250–350 g, (Hilltop Lab Animals, Inc., Scottdale, Pennsylvania) were used in this research, which was reviewed and approved by the Institutional Animal Care and Use Committee of the University of Illinois College of Medicine at Rockford. The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85–23, revised 1996). All animals were housed in temperature- and humidity-regulated quarters with a 12-h light:dark cycle. Food and water were available ad libitum. Measurement of blood pressure Basal anesthesia was induced in rats by intraperitoneal (i.p.) injection of urethane (1.4 g/kg). Animals were further pretreated with atropine methylbromide (1.0 mg/kg, i.p.) to reduce bronchial secretions. The femoral artery of each rat was exposed and cannulated with polyethylene tubing filled with 0.9% physiological saline solution and heparin (20 units/ml). The blood pressure was measured using a pressure transducer connected to a Gould Brush 2200 recorder (Gould, Cleveland, Ohio).
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After cannulation, rats were maintained for at least 30 min on 95% oxygen administered through a feline facemask, while blood pressures stabilized. If a steady blood pressure was not maintained, the rat was excluded from the study. Basal (time zero) blood pressures were recorded at the end of this time then rats were exposed to 2% isoflurane in oxygen for 20 min. Blood pressures were continuously monitored during isoflurane exposure. The MAP was determined from the blood pressure (BP) tracings using the following equation: MAP = diastolic BP + 1/3 (systolic BP-diastolic BP). The MAPs calculated at 1.0 min, 1.5 min, 2.5 min, 5.0 min, 7.5 min, 10 min, and 20 min were used to determine the percent change from the basal MAP. Assay of nitric oxide synthase activity Two hr following pretreatment with either 7-NI or peanut oil vehicle, rats were euthanized by decapitation; their whole brains and aortas removed and frozen in liquid nitrogen. NOS activity was determined by measuring the conversion of [14C]L-arginine to [14C]L-citrulline, as described by Dwyer et al. (1991). The whole brain or aorta was homogenized in 2 volumes of Tris-HCl buffer (50 mM, pH 7.4) containing 2 mM EDTA and 2 mM EGTA, and centrifuged at 12,000 rpm at 48C for 5 min. Twenty ml of supernatant were added to test tubes containing 50 mM TrisHCl buffer, 10 mM NADPH, 6.0 mM CaCl2, 6.0 mM BH4, 2.0 mM FAD, 2.0 mM FMN and 0.5 ACi [14C]L-arginine monohydrochloride (Amersham Pharmacia Biotech, Piscataway, NJ) in a final volume of 40 ml at pH 7.4. Following incubation at 378C for 30 min, the reaction was terminated by the addition of 50 mM HEPES buffer containing 5 mM EDTA and resin. Then the reaction mixture applied onto 1.5 ml columns of Dowex AG50WX-8 (Bio-Rad, Hercules, California). [14C]L-citrulline was quantified by scintillation spectroscopy of 10-ml aliquots of the flow-through. Protein concentration was determined using a standard protein assay kit (Pierce Chemical Co., Rockford, Illinois). NOS activity was expressed in terms of pmol/mg protein/min and then expressed as % of control. Drugs The following drugs were used in this research: Isoflurane, U.S.P. (Abbott Laboratories, North Chicago, Illinois); Oxygen, U.S.P. (Rockford Industrial Welding, Rockford, Illinois); NG-monomethylL-arginine acetate (L-NMMA) (Tocris Cookson, Ballwin, Missouri); NG-nitro-L-arginine (L-NOARG) (Research Biochemicals Inc., Natick, Massachusetts); 7-nitroindazole (7-NI) (Alexis, San Diego, California); and urethane (Sigma Chemical Company, St. Louis, Missouri). Two percent isoflurane in oxygen was administered using a Fluotec 3 anesthesia machine (Fraser Harlake, Orchard Park, New York). In control experiments, 95% oxygen in lieu of anesthetic agent was delivered into the facemask at the same inflow rate. Exposures were conducted inside a fumehood. In all experiments, the levels of isoflurane and oxygen being delivered via the facemask were continuously monitored using a POET II anesthetic monitoring system (Criticare, Milwaukee, Wisconsin). The femoral vein was cannulated for i.v. drug administration. L-NMMA and L-NOARG were prepared in 0.9% saline solution and administered in i.v. doses of 10 and 30 mg/kg, respectively, 30 min prior to isoflurane exposure. Because of its poor solubility in aqueous solution, 7-NI was suspended in peanut oil (Planters, East Hanover, New Jersey) and administered in an i.p. dose of 100 mg/kg 2 hr prior to isoflurane exposure. In
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Fig. 1. Change in mean arterial pressure (MAP) in urethane-anesthetized rats exposed to 2% isoflurane (ISO) in oxygen following intravenous (i.v.) pretreatment with saline (SAL, n = 6), or 30 mg/kg NG-monomethyl-L-arginine acetate (L-NMMA, n = 6) or 10 mg/kg NG-nitro-L-arginine (L-NOARG, n = 6). The change in MAP was determined relative to MAP at time 0 prior to ISO exposure. Each symbol represents the mean and vertical lines indicate the S.E.M. Significance of difference: *, p b 0.05, compared to SAL + ISO group (repeated-measures ANOVA and post-hoc Bonferroni test). Basal MAPs at time zero were 92.6 F 4.2 mm Hg for the SAL + ISO group, 130.7 F 5.3 mm Hg for the L-NMMA + ISO group and 142.3 F 2.4 mm Hg for the L-NOARG + ISO group. The basal MAPs of the L-NMMA + ISO and L-NOARG + ISO groups were significantly greater than that of the SAL + ISO control group at p b 0.05 (one-way ANOVA and Dunnett’s t-test).
Fig. 2. Change in mean arterial pressure (MAP) in urethane-anesthetized rats exposed to 2% isoflurane (ISO) in oxygen following intraperitoneal (i.p.) pretreatment with peanut oil vehicle (VEH, n = 6) or 100 mg/kg 7-nitroindazole (7-NI, n = 6). The change in MAP was determined relative to MAP at time 0 prior to ISO exposure. Each symbol represents the mean and vertical lines indicate the S.E.M. Significance of difference: *, p b 0.05, compared to VEH + ISO group (repeated-measures ANOVA and post-hoc Bonferroni test). Basal MAPs at time zero were 101.2 F 3.3 mm Hg for the VEH + ISO group and 122.8 F 2.7 mm Hg for the 7-NI + ISO group. The basal MAP of the 7-NI + ISO group was significantly greater than that of the VEH + ISO control group at p b 0.05 (Student’s t-test).
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control experiments, saline was administered i.v. in lieu of L-NMMA and L-NOARG, and peanut oil was administered i.p. in lieu of 7-NI. Statistical analysis of data A repeated-measures analysis of variance (ANOVA) with a post-hoc Bonferroni multiplecomparison test (significance set at 0.05) was used to compare isoflurane-induced changes in MAP in control vs. pretreatment groups of rats. Percent changes in MAP were arcsine transformed prior to statistical analysis. A one-way ANOVA with a post-hoc Dunnett’s t-test or Student’s t-test (depending on the number of experimental groups) was used to compare basal MAPs of various treatment groups. Student’s t-test was employed to compare NOS activity levels of vehicle and 7-NI pretreatment groups.
Fig. 3. NOS enzyme activity in cerebellum (top panel) and aorta (bottom panel) of rats pretreated with vehicle (peanut oil) or 100 mg/kg 7-NI. Each symbol represents the mean and vertical lines indicate the S.E.M. Significance of difference: *, p b 0.05 between groups indicate by the brackets above (two-way ANOVA and post-hoc Bonferroni test).
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Results MAPs in control, urethane-anesthetized rats exposed to 95% oxygen alone were stable for up to 3 hr following cannulation of the femoral artery. When superimposed for 20 min on the basal urethane anesthesia, 2% isoflurane induced a triphasic change in blood pressure (Fig. 1). There was an initial sharp drop in MAP that was maximal at 1.5 min of isoflurane exposure; the average reduction in MAP at this time point was 27.4 F 2.7%, relative to the MAP just prior to induction with isoflurane. This initial drop in MAP was followed by a transient and partial recovery toward baseline MAP which was interrupted by a longerlasting and more gradual reduction in MAP that reached 36.4% at the end of the 20 min exposure period. The resting MAP at time zero was significantly elevated by pretreatment with all three NOSinhibitors. The early hypotensive phase induced by isoflurane was significantly attenuated in rats by pretreatment with all three NOS-inhibitors (Fig. 1). Specifically, a 3 7 repeated measures ANOVA showed a significant interaction (F14,105 = 9.77, p b 0.0001) for animals that were pretreated i.v. with 30 mg/kg L-NMMA or 10 mg/kg of L-NOARG 30 min prior to exposure to isoflurane. Post-hoc analysis revealed the attenuation by L-NMMA and L-NOARG at the 1.0, 1.5, 5.0, and 20 min time points. Further, L-NOARG also significantly attenuated the isoflurane reduction in MAP at 7.5 min. The early-phase isoflurane-induced hypotension was also significantly attenuated in rats (Fig. 2) that were pretreated with 100 mg/kg of 7-NI 2 hr prior to isoflurane exposure, compared to rats receiving vehicle only (F1,10 = 7.82, p b 0.05). The mean MAP responses of the early-phase isoflurane-induced hypotension were significantly different from one another at 1.5, 2.5 and 5.0 min. A comparison of the isoflurane-induced reduction in MAPs at 1.5 min shows that the antagonism of the early-phase isoflurane-induced hypotensive response by 7-NI appeared to be less complete than by the other two NOS-inhibitors (a mean reduction in MAP in L-NMMA-pretreated rats of 4.0%, L-NOARG-pretreated rats 2.1% and 7-NI pretreated rats 8.8%). Rats pretreated with 100 mg/kg 7-NI showed a 25% reduction in NOS activity in cerebellum and b 5% reduction in the aorta (Fig. 3), compared to rats receiving peanut oil vehicle only. Compared to rats that had no pretreatment, the peanut oil vehicle reduced NOS activity by 5% in the cerebellum but increased NOS activity by 27% in the aorta.
Discussion The attenuation of the isoflurane-induced early hypotension by L-NMMA implicates involvement of NO in the response. However, NOS occurs in neuronal (nNOS), endothelial (eNOS) and inducible (iNOS) isoforms (Fo¨rstermann et al., 1994) and since L-NMMA is relatively non-selective for all three isoforms (Reif and McCreedy, 1995), it is not immediately evident which isoform of NOS is involved in the early hypotensive response to isoflurane. To distinguish among these different forms of NOS, additional NOS-inhibitors were administered. While it must be acknowledged that these compounds cannot be considered absolutely selective for a given isoform, they do nonetheless demonstrate a relative selectivity or preference for one NOS isoform over another. L-NOARG effectively inhibits eNOS (Gross et al., 1990) and nNOS (Klatt et al., 1994) but is reportedly 20-fold less effective for inhibiting iNOS (Lambert et al., 1991). Like L-NMMA, L-NOARG virtually abolished the early hypotensive response. One interpretation of these findings is that the vasodilation responsible for the early-phase isoflurane-induced hypotension is NO-dependent and hence sensitive to antagonism by
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any inhibition of eNOS. This would be consistent with a report that L-NOARG attenuated the vasodilation induced by the opioid peptides met-enkephalin and leu-enkephalin (Devine and Armstead, 1995). On the other hand, 7-NI is reputedly selective for nNOS and has little activity against either iNOS or eNOS (Moore et al., 1993a,b). In the present research, 100 mg/kg 7-NI selectively antagonized the earlyphase hypotension; however, the antagonism by 7-NI was not as complete as observed with L-NMMA or L-NOARG. Some researchers have reported that 7-NI can elevate the blood pressure leading to speculation that 7-NI might not be entirely selective for nNOS and may also inhibit eNOS (Zagvazdin et al., 1996). This caveat was precluded by the NOS assays in this study showing that pretreatment with 100 mg/kg 7-NI reduced cerebellar NOS activity by 25%, compared to essentially no change in the peanut oil control group. On the assumption that cerebellar NOS activity reflects mainly that of nNOS and aortic NOS activity reflects mainly that of eNOS, these findings indicate that 7-NI was, indeed, selective for nNOS. The greater degree of antagonism of isoflurane by L-NMMA and N-LOARG might indicate the ability of these drugs to inhibit both nNOS and e-NOS. The lower degree of antagonism by 7-NI may be attributable to its ability to inhibit nNOS only. We earlier reported that early-phase isoflurane-induced hypotension was attenuated by opioid antagonists and was also blocked by rabbit antisera against the opioid peptide met-enkephalin (Ellenberger et al., 2003). We postulated that this early hypotensive response to isoflurane might involve release of either met-enkephalin or a met-enkephalin-like peptide which presumably activates A opioid receptors. In another investigation, NOS inhibition reduced the stimulated release of met-enkephalin in the rat spinal cord (Hara et al., 1995), suggesting that perhaps the neuronal release of met-enkephalin might be NO-dependent. Additional support for a link between NO and opioid peptides is the observation that the NO-donor sodium nitroprusside increased levels of met-enkephalin in the cortical periarachnoid cerebrospinal fluid of newborn pigs (Armstead, 1995). It is, therefore, plausible that the release of met-enkephalin by isoflurane might be dependent on NO produced by nNOS. At the same time, endothelium-derived NO is likely to be involved in the vascular muscle relaxation (Furchgott, 1983; Ignarro et al., 1999). Isoflurane-induced vasodilation of cerebral and coronary blood vessels are reportedly antagonized or reversed by pretreatment with L-NAME (McPherson et al., 1993; Koenig et al., 1994), L-NMMA (Park et al., 1994) or endothelial denudation (McPherson et al., 1993; Park et al., 1994), which implicates the endothelium and production of NO in vasodilation. This may corresponds to our observed NO-dependent early-phase hypotensive effect of isoflurane. On the other hand, there is also evidence that isoflurane-induced vasodilation in certain vascular tissue preparations is independent of NO (Flynn et al., 1992; Jensen et al., 1992; Crystal et al., 1996). This, in turn, possibly corresponds to the more prolonged late-phase hypotensive effect of isoflurane, which is seemingly not mediated by NO. In summary, our results from our experiments utilizing three NOS-inhibitors of varying selectivity implicate roles for NO derived from nNOS action as well as NO derived from eNOS action in earlyphase isoflurane-induced hypotension.
Acknowledgements This work was supported by a grant from the American Heart Association/Midwest Consortium (R.M.Q.), NIH grant DA-10047 and Walter Rice Craig Fellowship from the University of Illinois at
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Chicago (H.L.L.). We are grateful to Dr. B.K. Slinker (Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, College of Veterinary Medicine, Washington State University) for assistance with the statistical analysis of the data and to Dr. S. Li (Department of Pharmacology, University of Washington) for her help with the figures.
References Armstead, W.M., 1995. Opioids and nitric oxide contribute to hypoxia-induced pial artery vasodilation. American Journal of Physiology 268 (1 Pt 2), H226 – H232. Crystal, G.J., Gurevicius, J., Salem, M.R., 1996. Isoflurane-induced coronary vasodilation is preserved in reperfused myocardium. Anesthesia and Analgesia 82, 22 – 28. Devine, J., Armstead, W.M., 1995. The role of nitric oxide in opioid-induced pial artery vasodilation. Brain Research 675, 257 – 263. Dwyer, M.A., Bredt, D.S., Snyder, S.H., 1991. Nitric oxide synthase: irreversible inhibition by L-NG-nitroarginine in brain in vitro and in vivo. Biochemical and Biophysical Research Communications 176, 1136 – 1141. Eger II, E.I., 1984. The pharmacology of isoflurane. British Journal of Anaesthesia 56 (Suppl. 1), 71S – 99S. Ellenberger, E.A., Lucas, H.L., Russo, J.M., Mueller, J.L., Barrington, P.L., Tseng, L.F., Quock, R.M., 2003. An opioid basis for early-phase isoflurane-induced hypotension in rats. Life Sciences 73, 2591 – 2602. Flynn, N.M., Buljubasic, N., Bosnjak, Z.J., Kampine, J.P., 1992. Isoflurane produces endothelium-independent relaxation in canine middle cerebral arteries. Anesthesiology 76, 461 – 467. Ffrstermann, U., Closs, E.I., Pollock, J.S., Nakane, M., Schwarz, P., Gath, I., Kleinert, H., 1994. Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions. Hypertension 23, 1121 – 1131. Furchgott, R.F., 1983. Role of endothelium in responses of vascular smooth muscle. Circulation Research 53, 557 – 573. Gross, S.S., Stuehr, D.J., Aisaka, K., Jaffe, E.A., Levi, R., Griffith, O.W., 1990. Macrophage and endothelial cell nitric oxide synthesis: cell-type selective inhibition by NG-aminoarginine, NG-nitroarginine and NG-methylarginine. Biochemical and Biophysical Research Communications l70, 96 – 103. Hara, S., Kuhns, E.R., Ellenberger, E.A., Mueller, J.L., Shibuya, T., Endo, T., Quock, R.M., 1995. Involvement of nitric oxide in intracerebroventricular h-endorphin-induced neuronal release of methionine-enkephalin. Brain Research 675, 190 – 194. Ignarro, L.J., Cirino, G., Casini, A., Napoli, C., 1999. Nitric oxide as a signaling molecule in the vascular system: an overview. Journal of Cardiovascular Pharmacology 34, 879 – 886. Jensen, N.F., Todd, M.M., Kramer, D.J., Leonard, P.A., Warner, D.S., 1992. A comparison of the vasodilating effects of halothane and isoflurane on the isolated rabbit basilar artery with and without intact endothelium. Anesthesiology 76, 624 – 634. Klatt, P., Schmidt, K., Brunner, F., Mayer, B., 1994. Inhibitors of brain nitric oxide synthase. Binding kinetics, metabolism, and enzyme inactivation. Journal of Biological Chemistry 269, 1674 – 1680. Koenig, H.M., Pelligrino, D.A., Wang, Q., Albrecht, R.F., 1994. Role of nitric oxide and endothelium in rat pial vessel dilation response to isoflurane. Anesthesia and Analgesia 79, 886 – 891. Lambert, L.E., Whitten, J.P., Baron, B.M., Cheng, H.C., Doherty, N.S., McDonald, I.A., 1991. Nitric oxide synthesis in the CNS, endothelium and macrophages differs in its sensitivity to inhibition by arginine analogues. Life Sciences 48, 69 – 75. McPherson, R.W., Kirsch, J.R., Moore, L.E., Traystman, R.J., 1993. NN¯-nitro-L-arginine methyl ester prevents cerebral hyperemia by inhaled anesthetics in dogs. Anesthesia and Analgesia 77, 891 – 897. Moore, P.K., Babbedge, R.C., Wallace, P., Gaffen, Z.A., Hart, S.L., 1993a. 5-Nitro indazole, an inhibitor of nitric oxide synthase, exhibits antinociceptive activity in the mouse without increasing blood pressure. British Journal of Pharmacology 108, 296 – 297. Moore, P.K., Wallace, P., Gaffen, Z., Hart, S.L., Babbedge, R.C., 1993b. Characterization of the novel nitric oxide synthase inhibitor 7-nitroindazole and related indazoles: antinociceptive and cardiovascular effects. British Journal of Pharmacology 110, 219 – 224.
E.A. Ellenberger et al. / Life Sciences 76 (2004) 499–507
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Park, K.W., Dai, H.B., Lowenstein, E., Darvish, A., Sellke, F.W., 1994. Heterogeneous vasomotor responses of rabbit coronary microvessels to isoflurane. Anesthesiology 81, 1190 – 1197. Reif, D.W., McCreedy, S.A., 1995. N-Nitro-L-arginine and N-monomethyl-L-arginine exhibit a different pattern of inactivation towards the three nitric oxide synthases. Archives of Biochemistry and Biophysics 320, 170 – 176. Zagvazdin, Y., Sancesario, G., Wang, Y.X., Share, L., Fitzgerald, M.E.C., Reiner, A., 1996. Evidence from its cardiovascular effects that 7-nitroindazole may inhibit endothelial nitric oxide synthase in vivo. European Journal of Pharmacology 303, 61 – 69.