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* From the Department of Anesthesiology, and
the Surgical Operating Center, Wakayama Medical University, Wakayama City, Japan.
Address correspondence to: Dr. Yoshio Hatano, Department of Anesthesiology, Wakayama Medical University, 811 – 1 Kimiidera, Wakayama City 641–0012, Japan. Phone: 81-73-441-0610; Fax: 81-73-447-0051; E-mail: yhatano{at}wakayama-med.ac.jp
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Abstract |
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Methods: The dose-dependent effects of sevoflurane and isoflurane on the Ang II-induced contraction of aortic rings, in the presence and absence of an intact endothelium, were investigated in normotensive Wistar-Kyoto rats (WKY) and SHR and compared using isometric force transducers.
Results: Ang II (10–9–10–6 M) induced a similar transient phasic contraction of endothelium-intact rings from the two rat strains in a dose-dependent manner. Removal of the endothelium augmented the Ang II-elicited phasic contraction, to a greater extent in the SHR group than in the WKY group. Sevoflurane and isoflurane (1–3 minimum alveolar concentration) concentration-dependently inhibited the Ang II-induced contraction of endothelium-intact rings from WKY; an effect that was greatly enhanced following removal of the endothelium. A greater degree of attenuation of the Ang II-induced contraction of both endothelium-intact and -denuded rings by the two anesthetics was observed in the SHR group. The inhibitory effects of isoflurane on the Ang II-induced contraction of aortic rings from both strains appeared to be stronger than that of sevoflurane at equipotent concentrations.
Conclusion: Our finding that the inhibitory effects of isoflurane and sevoflurane on Ang II-induced vasoconstriction are enhanced in SHR may, at least in part, account for the anesthesia-induced systemic hypotension frequently seen in hypertensive patients.
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Introduction |
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Volatile anesthetics reduce blood pressure in a concentration-dependent manner by, at least in part, direct vasodilating effects. Whether volatile anesthetics reduce blood pressure by inhibiting Ang II-induced vascular contraction and whether hypertensive pathology of the vasculature alters the possible effects of volatile anesthetics on Ang II-induced vascular contraction has not been determined to date. This study was designed to investigate: 1) the effects of sevoflurane and isoflurane on Ang II-induced contraction and endothelium-mediated relaxation of aortic smooth muscle in normotensive Wistar-Kyoto rats (WKY); 2) the effects of hypertension on relaxation by sevoflurane and isoflurane of the Ang II-induced contraction of aortic smooth muscle in spontaneously hypertensive rats (SHR).
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Methods |
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Tissue preparations
Rats were anesthetized with halothane and were euthanized by exsanguination from the common carotid artery. The descending thoracic aorta was isolated and excess fat and connective tissue were removed. The aorta was then cut into rings 3 to 4 mm in length. Special care was taken not to damage the endothelium for endothelium-intact rings. The endothelium was removed by gentle rubbing of the internal surface with a stainless steel needle for endothelium-denuded rings. Rings were mounted vertically between two stainless steel hooks in 10 mL organ baths. The lower hooks were fixed to the bottom of the organ bath, and the upper hooks were attached to a force-displacement transducer (Amplifier Case 7903, Nihondenkisanei Co., Tokyo, Japan). The organ bath contained Krebs bicarbonate solution [KBS (mM): NaCl 118.2, KCl 4.6, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 24.8 and dextrose 10]. The KBS was constantly aerated with 95% oxygen-5% carbon dioxide to keep the pH in the range of 7.35 to 7.45, and the temperature was maintained at 36.5 to 37.5°C. The changes in isometric force were amplified and displayed on ink-writing recorders (Recti-Horiz-8K, Nihondenkisanei Co., Tokyo, Japan). The resting tone was set at 3.0 g, which was optimal for inducing maximal constriction in our preliminary experiments. Before the start of the experiments, the aortic rings were allowed to equilibrate for 60 min, during which period the bathing fluid was replaced every 20 min.
Sevoflurane and isoflurane were introduced into the gas mixture using agent-specific vaporizers (Penlon Limited Abingdon, Oxon, UK). The concentration in the resulting gas mixture was monitored and adjusted using a calibrated Atom 303 anesthetic agent monitor (Atom, Tokyo, Japan). The concentrations of the anesthetic agents in KBS were measured by gas chromatography (Shimazu Seisakasho, Kyoto, Japan) and were determined to be 0.17 ± 0.05 mM, 0.35 ± 0.02 mM and 0.50 ± 0.03 mM at sevoflurane concentrations of 1.7% [1 minimum alveolar concentration (MAC)], 3.4% (2 MAC) and 5.1% (3 MAC), respectively (n = 8–12), and to be 0.19 ± 0.01 mM, 0.39 ± 0.01 mM and 0.56 ± 0.03 mM at isoflurane concentrations of 1.2% (1 MAC), 2.3% (2 MAC) and 3.5% (3 MAC), respectively (n = 8–12).
Isometric force measurement
After equilibration, rings were incubated with 3 x 10–2 M KCl to determine the integrity of VSMCs, and to assess their overall contractile responsiveness. Rings that did not develop at least 2.0 g contractile active force were discarded. Removal of the endothelium was confirmed in 3 x 10–7 M phenylephrine-precontracted rings by the lack of relaxation to 10–5 M acetylcholine. The presence of an intact endothelium was also confirmed by acetylcholine (10–6 M)-induced relaxation reaching more than 60% of phenylephrine-produced constriction.
To avoid the occurrence of tachyphylaxis to Ang II, each ring was challenged only once with a single concentration of Ang II and was treated with a single concentration of anesthetic agents. In preliminary experiments, aortic rings (with and without an intact endothelium) from each of the two strains were contracted with 10–9 M, 10–8 M, 10–7 M or 10–6 M Ang II, in order to determine the Ang II-induced dose-dependent contractile response. The results showed that Ang II induced an almost maximal response at the concentration of 10–7 M. Therefore, the concentration of 10–7 M was selected for the following anesthetic-challenge experiment. Rings from each of the two strains were incubated to the equivalent of 1, 2 or 3 MAC sevoflurane/isoflurane for 15 min prior to application of 10–7 M Ang II, to examine the dose-effect on Ang II-induced contraction.
In order to represent the different mechanisms of contraction, both phasic and tonic tensions were calculated and analyzed. The phasic contraction to Ang II was considered as the tension from baseline to the peak amplitude and the tonic contraction was the tension from baseline to the plateau amplitude.
Materials
WKY and SHR were supplied by the Shizuoka Laboratory Animal Center (Shizuoka, Japan). Ang II was obtained from Sigma-Aldrich Fine Chemicals (St. Louis, Missouri, USA). Sevoflurane and isoflurane were purchased from Maruishi Pharmaceutical Company Limited (Osaka, Japan). All other reagents used in the experiments were of analytical grade.
Statistical analysis
The contraction elicited by Ang II was expressed as a percentage of that induced by 3 x 10–2 M KCl. The data are presented as the mean ± SD. The sample sizes (n values) are equal to the number of rats from which aortic rings were taken. Statistical analysis was made by Scheffe’s F test, after analysis of variance (one- or two-factor), and unpaired Student’s t test (unpaired), where appropriate, using the software program StatView (SAS Institute Inc. Cary, NC, USA). P values of < 0.05 were considered significant.
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Results |
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). Removal of the endothelium enhanced the phasic and tonic contractile response to Ang II in both the strains. Potentiation of the contractile response, especially tonic contraction, was greater in SHR than in WKY (Figures 1C and 1D).
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). In the absence of intact endothelium, inhibitory effects of anesthetics on the phasic contraction were potentiated in both strains. Sevoflurane and isoflurane significantly attenuated the contraction at all of the concentrations tested (Figure 2B). At higher concentrations (3 MAC in WKY and 2 and 3 MAC in SHR), inhibitory effect of isoflurane was greater than that of sevoflurane when compared at equipotent concentrations. When comparing WKY and SHR, the inhibitory effects of both anesthetics on the phasic contractile response to Ang II were greater in SHR (Figures 2A and 2B).
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). In endothelium-denuded rings, the attenuation of the contraction by these anesthetics was potentiated and reached a statistical significance at 2 and 3 MAC in WKY and all concentrations tested in SHR (Figure 3B). Similar to the phasic contraction, isoflurane inhibited the tonic contraction to a greater extent than sevoflurane in SHR. When comparing WKY and SHR, the inhibition of the tonic contraction by anesthetics was greater in endothelium-denuded rings from SHR (Figure 3B).
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Discussion |
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It is well known that volatile anesthetics reduce blood pressure to a different extent in different clinical situations and in a concentration-dependent manner. It is also known that hypertensive patients are more sensitive to anesthetic-induced hypotension. We speculated that the deranged smooth muscle and endothelium associated with hypertension likely alters the response of the vasculature to anesthetics.
VSM contraction and endothelium-mediated vasodilation by Ang II, and the increased sensitivity of the vasculature to Ang II in hypertension were confirmed in the current study. The phasic and tonic contractions are mainly mediated by increases in intracellular Ca2+ and enhanced Ca2+ sensitivity, respectively. Increased contractile responsiveness of the vasculature to Ang II, especially the tonic tension, was observed in SHR compared to WKY in the present study. This hyper-responsiveness may likely be attributed to the enhanced Ang II receptor density,8 the increased protein kinase activation,5,6 and altered Ca2+ handling.8 The decrease of the release of EDRFs, such as nitrous oxide9 and the increase in production of vasoconstrictors such as endothelin in the endothelium9 are also involved in the high sensitivity of the vasculature to Ang II in hypertension.
Accumulating evidence suggests that anesthetic agents alter agonist-evoked vasoconstriction by changing Ca2+ mobilization and/or by modulating protein kinase C- or other kinase-mediated Ca2+ sensitization of contractile proteins.10–16 In addition to the inhibition of contraction of VSMCs, volatile anesthetics also attenuate endothelium-mediated vasodilation by inhibiting the release or action of EDRFs.17,18 The net effect of volatile anesthetics on vascular tone depends upon the relative extent of inhibition on both smooth muscle contraction and endothelium-mediated vasorelaxation. Potentiation of inhibitory effect by anesthetics on Ang II-induced contraction in rings without endothelium in the present study suggests that these anesthetics can inhibit not only smooth muscle contraction but also endothelium-dependent relaxation induced by Ang II. The present study also demonstrated that the inhibition of contraction of rat aortic smooth muscle is greater by isoflurane than by MAC-equal sevoflurane. This may be due to differing mechanisms of action on the contractile response between sevoflurane and isoflurane. Isoflurane (0.5–3%) decreases the Ang II-induced mobilization of Ca2+ by directly altering the tubular network organization of cultured rat aortic smooth muscle,19 while sevoflurane (1.7–5.1%) has been demonstrated to depress the Ang II-induced activation of Ca2+-dependent protein kinase C.20
Since the functional and structural alterations of VSM and endothelium by hypertension increase the reactivity to agonists, the inhibitory effects of anesthetics on agonist-induced vascular contraction can be altered in the hypertensive state. It has been demonstrated that propofol at clinically relevant concentrations attenuates the norepinephrine-induced contraction of aortae from SHR to a greater extent than that from WKY.21 In the present study, the inhibitory effects of sevoflurane and isoflurane on the Ang II-elicited contraction of both endothelium-intact and -denuded rat aortic rings from SHR was greater than that from WKY. Due to the altered function and structure of VSMCs and vascular endothelium in hypertension, the mechanisms by which anesthetics affect Ang II-induced contraction are likely due to changes in the SHR. Propofol has been found to decrease the Ang II-induced Ca2+ influx through voltage-independent channels in SHR, to a greater extent than in WKY, without altering Ca2+ release from internal stores in aortic VSMCs.22 On the other hand, isoflurane has been proven to be less sensitive in attenuating the Ang II-induced Ca2+ mobilization in cultured aortic VSMCs from SHR than that from WKY.19 The mechanisms by which anesthetic agents attenuate VSMCs contraction appear to differ in WKY and SHR, and it seems that the mechanisms of Ca2+ sensitivity (mainly responsible for tonic contraction) are more susceptible to attenuation by anesthetics in SHR than in WKY, according to the findings of our study.
At clinically relevant concentrations (1–2 MAC), isoflurane and sevoflurane did not significantly inhibit Ang II-induced contraction of endothelium-intact rings from WKY, but significantly suppressed the contraction of those from SHR; inhibition by isoflurane was greater than by sevoflurane. Although the findings in the present in vitro study, using a conductance vessel in a hypertensive animal model, cannot directly elucidate the effects of volatile anesthetics on the human vascular system in vivo, we consider that these results will provide a valuable reference for clinical anesthesia research, particularly in hypertensive patients. Our study supports the observation that hypertensive patients are more susceptible to volatile anesthetics than normotensive patients during clinical anesthesia. In a prospective, randomized, multicentre study,23 hemodynamic instabilities including hypotension requiring therapeutic intervention occurred in 14.6% and 20.8% during sevoflurane and isoflurane anesthesia, respectively. When only patients over 50 yr old were compared, the incidence of hemodynamic side effects increased with isoflurane anesthesia (29.1%) compared to sevoflurane anesthesia (15.2%). This study suggested that patients over 50 yr of age show a higher risk for hemodynamic instability, especially when receiving isoflurane anesthesia. The patho-physiological alterations in the vasculature secondary to hypertension and atherosclerosis in these older patients likely accounts, at least in part, for the higher incidence of hemodynamic side effects during anesthesia. In another clinical study,24 sevoflurane (8%) was used for induction of anesthesia in 25 hypertensive patients (SBP 156 ± 3 mmHg). Hypotension occurred in as many as 22 patients (88%) and SBP decreased to less than 100 mmHg in 20 patients. This finding indicates the concentration-dependent incidence of hemodynamic depression by anesthetics, especially in hypertensive patients. According to the findings of the current study, we recommend that close attention be paid to hypertensive patients during sevoflurane or isoflurane anesthesia. Sevoflurane may be the better choice to maintain stable hemodynamics.
In summary, the present study demonstrates that Ang II-induced vasoconstriction of rat aortic rings was greater in SHR than in WKY. The findings that the inhibitory effects of isoflurane and sevoflurane on vaso-constriction were enhanced in SHR may, at least in part, account for the anesthesia-induced systemic hypotension frequently observed in hypertensive patients.
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Footnotes |
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Source of financial support: This research was supported in part by grant-in-aid No. 13470327 and No. 13671607 for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.
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