Anesthesiology 2003; 99:666–77 © 2003 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Mechanisms of Direct Inhibitory Action of Isoflurane on Vascular Smooth Muscle of Mesenteric Resistance Arteries Takashi Akata, M.D., Ph.D.,* Tomoo Kanna, M.D.,† Jun Yoshino, M.D.,‡ Shosuke Takahashi, M.D., Ph.D.§ Background: Isoflurane has been shown to directly inhibit tractile response to norepinephrine (i.e., a neurotrans- vascular reactivity. However, less information is available re- mitter that plays a central role in sympathetic garding its underlying mechanisms in systemic resistance arteries. maintenance of vascular tone in vivo) was not inhibited Methods: Endothelium-denuded smooth muscle strips were during exposure to isoflurane. In addition, contractile prepared from rat mesenteric resistance arteries. Isometric response to KCl (i.e., contractile response mediated by force and intracellular Ca2؉ concentration ([Ca2؉] ) were mea- 2ϩ i voltage-gated Ca channels [VGCCs] that play a crucial Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/99/3/666/336698/0000542-200309000-00023.pdf by guest on 29 September 2021 sured simultaneously in the fura-2–loaded strips, whereas only ␤ role in the regulation of vascular tone in vivo) was not the force was measured in the -escin membrane–permeabi- 12 lized strips. inhibited during exposure to isoflurane. Therefore, we Results: Isoflurane (3–5%) inhibited the increases in both proposed that in subjects with intact endothelial func- 2؉ ␮ [Ca ]i and force induced by either norepinephrine (0.5 M)or tion, the direct action of isoflurane on mesenteric resis- KCl (40 mM). These inhibitions were similarly observed after tance arteries may not contribute to systemic hypoten- depletion of intracellular Ca2؉ stores by ryanodine. Regardless of the presence of ryanodine, after washout of isoflurane, its sion during isoflurane anesthesia. However, in the -2؉ absence of endothelium, contractile responses to norepi inhibition of the norepinephrine response (both [Ca ]i and force) was significantly prolonged, whereas that of the KCl nephrine and KCl were both inhibited during exposure response was quickly restored. In the ryanodine-treated strips, to isoflurane. Thus, we speculated that in subjects with 2؉ the norepinephrine- and KCl-induced increases in [Ca ]i were -؉ impaired endothelial function, the direct (i.e., endothe both eliminated by nifedipine, a voltage-gated Ca2 channel blocker, whereas only the former was inhibited by niflumic lium-independent) inhibitory action of isoflurane on acid, a Ca2؉-activated Cl؊ channel blocker. Isoflurane caused a mesenteric arterial VSM cells (VSMCs) may contribute to -rightward shift of the Ca2؉–force relation only in the fura-2– systemic hypotension during isoflurane anesthesia. Inter loaded strips but not in the ␤-escin–permeabilized strips. estingly, only the norepinephrine response, not the KCl Conclusions: In mesenteric resistance arteries, isoflurane de- response, was significantly inhibited for a while (15 min presses vascular smooth muscle reactivity by directly inhibiting -both Ca2؉ mobilization and myofilament Ca2؉ sensitivity. or more) after washout of isoflurane in either the pres Isoflurane inhibits both norepinephrine- and KCl-induced volt- ence or absence of endothelium, indicating that its en- ؉ age-gated Ca2 influx. During stimulation with norepinephrine, dothelium-independent inhibitory action on contractile 2؉ ؊ isoflurane may prevent activation of Ca -activated Cl chan- response to norepinephrine is prolonged.12 We thus -nels and thereby inhibit voltage-gated Ca2؉ influx in a pro longed manner. The presence of the plasma membrane appears proposed that the direct inhibitory action of isoflurane essential for its inhibition of the myofilament Ca2؉ sensitivity. on norepinephrine response might contribute to the previously observed prolonged systemic hypotension af- 13 ISOFLURANE produces systemic hypotension1 and sig- ter isoflurane anesthesia. nificantly alters distribution of blood flow to various Previous studies using isolated aorta and cultured aor- 4,5 organs.2 Previous studies3–11 performed in a variety of tic VSMCs have suggested that the direct inhibitory vascular beds have suggested that isoflurane causes action of isoflurane on VSM is a result of both reduction 2ϩ 2ϩ changes in vascular tone through its direct action on of the intracellular Ca concentration ([Ca ]i) and 2ϩ vascular smooth muscle (VSM) and/or endothelial cells. inhibition of the myofilament Ca sensitivity. However, In our previous study with isolated mesenteric resis- less information is available regarding its underlying tance arteries,12 in the presence of endothelium, con- mechanisms in systemic resistance arteries, which are different from conduit arteries in many of their proper- ties, including Ca2ϩ-mobilization processes and respon- * Lecturer, † Research Associate, ‡ Postgraduate Student, § Professor and siveness to pharmacological agents.14–16 We previously Chair. proposed that the direct inhibitory action of isoflurane Received from the Department of Anesthesiology and Critical Care Medicine, 2ϩ Faculty of Medicine, Kyushu University, Fukuoka, Japan. Submitted for publica- on VSM is primarily a result of reduction of the [Ca ]i in tion February 17, 2003. Accepted for publication May 28, 2003. Supported in mesenteric resistance arteries.8 However, our proposal part by a Grant-in-Aid (B-09470330) from Ministry of Education, Science, Sports, and Culture, Japan (1997-1999), and Grants-in-Aid for Scientific Research (B- was based on the results obtained in experiments per- 09470330, C-13671590) from Japan Society of the Promotion of Science (1999- formed at approximately 22°C in the cell membrane– 2004, Tokyo, Japan). Presented in part at the 49th annual meeting of the Japanese 8 Society of Anesthesiologists, Fukuoka, Japan, April 18–20, 2002; to be presented permeabilized condition, in which we might have failed at the Annual Meeting of the American Society of Anesthesiologists, San Fran- to detect significant effects of isoflurane on myofilament cisco, California, October 11–15, 2003. Ca2ϩ sensitivity that require the intact cell membrane. In Address reprint requests to Dr. Akata: Department of Anesthesiology and Critical Care Medicine, Faculty of Medicine, Kyushu University, Fukuoka 812- addition, little information is available regarding the 8582, Japan. Address electronic mail to: [email protected]. In- mechanisms behind the aforementioned prolonged in- dividual article reprints may be purchased through the Journal Web site, www.anesthesiology.org. hibitory action on norepinephrine response in mesen- Anesthesiology, V 99, No 3, Sep 2003 666 DIRECT ACTION OF ISOFLURANE ON VASCULAR SMOOTH MUSCLE 667 teric resistance arteries.12 Furthermore, although there is Our method on the fura-2 fluorometry was also detailed direct evidence to indicate that isoflurane influences Ca2ϩ previously.22,23 Briefly, to allow loading of the fura-2 into mobilization from the intracellular stores in VSMCs of iso- the VSMCs, the strips were incubated in normal physio- 17 lated resistance arteries, no direct evidence is currently logic salt solution (PSS) containing 10 ␮M acetoxymethyl available to indicate that isoflurane inhibits plasmalemmal ester of fura-2 (fura-2/AM) and 2% albumin for approxi- Ca2ϩ influx in VSMCs of systemic resistance arteries. mately2hatapproximately 35°C. After this period, the In this study, using the fura-2 fluorometry and thereby solution containing fura-2/AM was washed out with nor- 2ϩ measuring force and [Ca ]i simultaneously, we further mal PSS for approximately1htoensure sufficient ester- investigated the mechanisms behind the direct inhibi- ification of fura-2/AM in the cells and to equilibrate the tory action of isoflurane on VSM in membrane-intact strips before the measurements.22,23 Changes in the flu- mesenteric resistance arteries. Specifically, we tested orescence intensity of the fura-2–Ca2ϩ complex were Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/99/3/666/336698/0000542-200309000-00023.pdf by guest on 29 September 2021 two major hypotheses, i.e., (1) the hypothesis that isoflu- measured by a fluorometer equipped with a dual-wave- rane inhibits myofilament Ca2ϩ sensitivity of VSMCs in length excitation device (CAM-230; Japan Spectroscopic, the presence of intact cell membrane in mesenteric Tokyo, Japan) connected to the microscope with optical resistance arteries and (2) the hypothesis that isoflurane fibers. The VSM tissue was illuminated with ultraviolet directly inhibits plasmalemmal Ca2ϩ influx in VSMCs of lights at wavelengths of 340 and 380 nm alternatively mesenteric resistance arteries. Our experiments to test limited to a frequency of 1000 Hz. The fura-2 fluores- the latter hypothesis were performed in the presence of cence signals induced by excitation at 340 and 380 nm ryanodine, which has been shown to deplete the intra- were collected through the 10ϫ objective lens (Plan cellular Ca2ϩ stores of mesenteric arterial VSMCs used in Fluor; Nikon, Tokyo, Japan) and measured through a this study.17 We also tested the hypothesis that in mes- 500-nm filter with a photomultiplier. The background enteric resistance arteries, isoflurane causes the pro- fluorescence as excited by 340- and 380-nm ultraviolet longed inhibition of contractile response to norepineph- light was obtained after completion of each experiment rine by inhibiting Ca2ϩ mobilization in VSMCs. In this by breaking the cell membranes with Triton X-100