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CARDIOVASCULAR ANESTHESIOLOGY

Sevoflurane, but Not Propofol, Prevents Rho Kinase-Dependent Contraction Induced by Sphingosylphosphorylcholine in the Porcine Coronary Artery

Hiroyuki Kinoshita, MD, PhD^*, Naoyuki Matsuda, MD, PhD, Yoshiki Kimoto, MD, PhD^*, Setsuko Tohyama, MD, Keiko Hama, MD^*, Katsutoshi Nakahata, MD, PhD^*, and Yoshio Hatano, MD, PhD^*

From the *Department of Anesthesiology, Wakayama Medical University, Wakayama, Japan; Departments of Anesthesiology and Molecular Medical Pharmacology, Toyama University School of Medicine, Toyama, Japan; and Departments of Sports Medicine and Joint Reconstruction Surgery, Hokkaido University School of Medicine, Sapporo, Japan.

Address correspondence and reprint requests to Hiroyuki Kinoshita, MD, PhD, Department of Anesthesiology, Wakayama Medical University, 811-1 Kimiidera, Wakayama, Wakayama 641-0012, Japan. Address e-mail to hkinoshi{at}pd5.so-net.ne.jp.

Abstract

BACKGROUND: Sphingosylphosphorylcholine may induce coronary vasospasm by the activation of Rho kinase. We designed the current study to examine the differential effects of anesthetics on Rho kinase activation induced by sphingosylphosphorylcholine in porcine coronary arteries.

METHODS: Rings of porcine coronary artery without endothelium were prepared in tissue bath containing modified Krebs–Ringer bicarbonate solution. Using isometric force recording, concentration–response curves in response to sphingosylphosphorylcholine were obtained in the absence or in the presence of sevoflurane, propofol, or a selective Rho kinase inhibitor Y27632, which was added 15 min before the application of sphingosylphosphorylcholine. Intracellular translocation of Rho A toward the plasma membrane and phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase were also evaluated by Western blotting.

RESULTS: Sphingosylphosphorylcholine (10^–7 to 10^–5 M) produced contraction of the porcine coronary artery, which was abolished by a selective Rho kinase inhibitor Y27632 (2 x 10^–6 M). Sevoflurane (1.7%) reduced sphingosylphosphorylcholine-induced coronary artery constriction, and the higher concentration (3.4%) abolished it (P < 0.05). In contrast, propofol (3 x 10^–6 M and 10^–5 M) had no effect on coronary artery constriction due to sphingosylphosphorylcholine. Sevoflurane, but not propofol, reduced intracellular translocation of Rho A toward the plasma membrane. Sevoflurane and Y27632, but not propofol, similarly decreased (64.4% or 70.8% reduction, respectively, P < 0.05) phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase.

CONCLUSIONS: Sphingosylphosphorylcholine induces coronary vasocontriction via activation of Rho kinase. Sevoflurane, but not propofol, inhibits this pathway, resulting in prevention of vasoconstriction.

In response to various stimuli, cells metabolize sphingomyelin from the plasma membrane to form several sphingomyelin metabolites, including sphingosylphos-phorylcholine (SPC) (1). Several studies have suggested that SPC actions are mediated by both intra- and extracellular plasma membrane targets, whereas other sphingomyelin metabolites exert their effects only intracellularly (2,3). SPC induces vasoconstriction in many vascular beds including the coronary artery and it is capable of augmenting the sensitivity of vascular smooth muscle for intracellular Ca²⁺ via activation of Rho kinase (1,4,5). Indeed, Rho kinase-induced vasoconstriction induced by SPC has been demonstrated in the coronary arteries in several animal models (4,5).

The effects of anesthetics on Rho kinase activity in the coronary artery have not been determined. In addition, it is unknown whether volatile and IV anesthetics confer differential inhibitory effects on Rho kinase activity in the coronary artery. Since it may play a crucial role in the pathogenesis of coronary vasospasm in humans (6), understanding the role of anesthetics on Rho kinase actions has perioperative relevance. The current study using an isolated porcine coronary artery model was designed to determine whether volatile and IV anesthetics differentially prevent SPC-induced coronary contraction via effects on the activity of Rho kinase.

METHODS

The institutional animal care and use committee (Wakayama University, Wakayama, Japan) approved this study and the experimental design was in accordance with the National Institutes of Health guidelines for the care of experimental animals. Adult pig hearts were obtained from a slaughter house immediately after death and put in ice-cold modified Krebs–Ringer bicarbonate solution (control solution, pH 7.4) with the following composition: 119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.17 mM MgSO₄, 1.18 mM KH₂PO₄, 25 mM NaHCO₃, and 11 mM glucose. The left descending coronary artery was dissected and cut into 2-mm rings in length. Endothelial cells were removed mechanically by abrasion with a needle to avoid the involvement of endothelium-derived vasodilator substances induced by SPC (7,8). Sevoflurane was introduced into the gas mixture (see below) using an agent-specific vaporizer (Penlon; Sigma, Abingdon, Oxon, UK). The concentration of the resulting gas mixture was monitored and adjusted using an Atom 303 anesthetic agent monitor (Atom, Tokyo, Japan). The concentrations of sevoflurane in modified Krebs–Ringer bicarbonate solution were confirmed by gas chromatography as previously described (9). Bradykinin, dimethyl sulfoxide, SPC, U46619 and Y27632, and sevoflurane were purchased from Sigma-Aldrich Fine Chemicals (St. Louis, MO) or Abbott Japan (Osaka, Japan), respectively. Propofol, which is prepared in dimethyl sulfoxide (3 x 10^–4 M), was a generous gift from Astra-Zeneca Pharmaceutical (Södertälje, Sweden), and each concentration of propofol was calculated according to the volume of Krebs–Ringer bicarbonate solution in an organ chamber.

Organ Chamber Experiments
Each artery ring was connected to an isometric force transducer and suspended in an organ chamber filled with 10 mL control solution (37°C) insufflated with 95% O₂ and 5% CO₂. The artery was gradually stretched to the optimal point of its length–tension curve as determined by the contraction to a prostaglandin H₂/thromboxane receptor agonist, U46619 (10^–7 M). Optimal tension was achieved with approximately 3.0 g. Several rings from same artery were studied in parallel and the order of study was randomized using a table of random numbers. Preparations were equilibrated for 90 min. Endothelial removal was confirmed by the absence of relaxation induced by bradykinin (10^–6 M). Concentration– response curves of the ring preparations to cumulative concentrations of SPC from 10^–7 to 10^–5 M were obtained in the absence or in the presence of sevoflurane (1.7% or 3.4%), propofol (3 x 10^–6 or 10^–5 M), or a selective Rho kinase inhibitor Y27632 (2 x 10^–6 M), which was added 15 min before the application of SPC. To express the vasoconstrictor response induced by SPC as a percent, maximal contraction in response to KCl (60 mM) was obtained for each ring before SPC exposure. In all experiments, the different interventions were performed in separate coronary artery rings, and data collection was performed during steady-state contraction.

Translocation of Rho A and Rho Kinase Assay
To evaluate the effects of SPC on intracellular translocation of Rho A and Rho kinase activity, coronary artery rings were incubated with SPC for 30 min in the modified Krebs–Ringer bicarbonate solution (37°C) insufflated with 95% O₂ and 5% CO₂ gas mixture, in the absence or in the presence of sevoflurane (3.4%), propofol (10^–5 M), or Y27632 (2 x 10^–6 M). Rings were quickly frozen (–80°C) after the incubation. The blood vessels were powdered under liquid nitrogen and solublized in 1 mL of ice-cold sterile water containing 0.1% Triton X-100. The lysate was centrifuged at 600g_max for 15 min at 4°C and the supernatant fluid was used as a total protein fraction in the Rho kinase assay and Western blot analysis. Total protein levels were determined by the method of Lowry et al. (10) using BCA protein assay kit (Pierce, Rockfold, IL). A portion of the supernatant fluid was centrifuged at 100,000g_max for 30 min at 4°C and the pellet was used as a membrane fraction. To detect Rho kinase activity, 100 µg of the supernatant fluid was incubated for 10 min at 30°C with 10 µg of myosin-targeting subunit of myosin light chain phosphatase (MYPT1) (Upstate, Lake Placid, NY). After adding Laemmli buffer (125 mM Tris-HCl, 10% 2-mercaptoetanol, 4% SDS, 10% sucrose, 0.004% Bromphenol blue), the sample (20 µg for total fraction, 2 µg for the membrane fraction) was subjected to SDS-polyacrylamide gel electrophoresis, using 7.5% polyacrylamide gel and electrotransferred to polyvinylidine difluoride filter membrane. To reduce nonspecific binding, the membrane was blocked for 60 min at room temperature in PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, and 1.5 mM KH₂PO₄), containing 1% bovine serum albumin. The membrane was then incubated for 60 min at 4°C with primary antibodies of anti-mouse Rho A antibody (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-rabbit phospho-MYPT1 polyclonal antibody (Thr 853) (Upstate Biotechnology, Lake Placid, NY) or anti-rabbit MYPT1 antibody (Upstate Biotechnology, Lake Placid, NY) diluted at 1 µg/mL in PBS buffer. After an extensive washing for 30 min with PBS containing 0.05% Tween 20, the membrane was incubated with horseradish peroxidase-conjugated anti-mouse (eBioscience, San Diego, CA) or anti-rabbit antibody (eBioscience, San Diego, CA) diluted at 1:1000 in PBS-Tween buffer at room temperature for 60 min. The blots were washed twice for 15 min in PBS-Tween buffer and developed using the enhanced chemiluminescence detection system (Amersham, Little Chalfont, Buckinghamshire, UK), followed by exposure to radiographic film. Thereafter the samples were analyzed with NIH Image software produced by Wayne Rasband (National Institutes of Health, Bethesda, MD). To determine loading/transfer variations of protein, all blots were stained with Ponceau Red (washable, before incubation with antibodies) as well as Coomassie Brilliant Blue (permanent, after the enhanced chemiluminescence detection system). Intensity of total protein bands per lane was evaluated by densitometry. Negligible loading/transfer variation was observed among samples.

Statistical Analysis
The data are expressed as mean ± sd. Statistical analysis was performed using repeated measures analysis of variance, followed by Student–Newman–Keuls test for multiple comparisons. Differences were considered to be statistically significant when P was <0.05.

RESULTS

The concentrations of sevoflurane in Krebs–Ringer bicarbonate solution determined by gas chromatography were 0.17 ± 0.02 mM (n = 5) and 0.28 ± 0.01 mM (n = 5) at sevoflurane concentrations of 1.7% or 3.4%, respectively.

Organ Chamber Experiments
SPC (10^–7 to 10^–5 M) produced contraction of the porcine coronary arteries that was abolished by Y27632 (2 x 10^–6 M) (Fig. 1). Maximal vasocontraction induced by KCl (60 mM) was 5.2 ± 1.0 g for control rings and 5.0 ± 1.0 g for rings treated with Y27632 (2 x 10^–6 M) (P = 0.88). Sevoflurane (1.7%) reduced constriction in response to SPC (P < 0.05) and the higher concentration (3.4%) completely abolished contraction (Fig. 2). Maximal vasocontraction induced by KCl (60 mM) was 4.5 ± 1.3 g, 4.3 ± 1.2 g, or 3.6 ± 1.3 g for control rings and rings treated with sevoflurane (1.7% or 3.4%), respectively (P = 0.43). In contrast, propofol (3 x 10^–6 M and 10^–5 M) had no effect on SPC-induced constriction (P = 0.72, Fig. 2). Maximal KCl-induce vasoconstriction was 5.4 ± 1.9 g, 4.8 ± 3.5 g, or 4.8 ± 2.1 g for control rings and rings treated with propofol (3 x 10^–6 or 10^–5 M), respectively (P = 0.88).

View larger version (13K): [in this window] [in a new window]   Figure 1. Concentration–response curves to sphingosylphosphorylcholine (SPC, 10–7 to 10–5 M) in the absence or in the presence of Y27632 (2 x 10–6 M), obtained in the porcine coronary artery without endothelium. *Difference between the control ring and the ring treated with Y27632 is statistically significant (P < 0.05). Whiskers represent standard deviations.

View larger version (12K): [in this window] [in a new window]   Figure 2. Concentration–response curves to SPC (10–7 to 10–5 M) in the absence or in the presence of sevoflurane (1.7% and 3.4%) or propofol (3 x 10–6 M and 10–5 M), obtained in the porcine coronary artery without endothelium. *Difference between the control ring and the ring treated with sevoflurane is statistically significant (P < 0.05). Whiskers represent standard deviations.

Translocation of Rho A and Rho Kinase Assay
Sevoflurane (3.4%), but not propofol (10^–5 M), reduced intracellular translocation of Rho A toward the plasma membrane in porcine coronary arterial smooth muscle cells (Fig. 3). Sevoflurane (3.4%) and Y27632 (2 x 10^–6 M), but not propofol (10^–5 M), produced inhibition of MYPT1 phosphorylation to similar extent (64.4% or 70.8% reduction, respectively) seen in Rho A translocation (Fig. 4).

View larger version (21K): [in this window] [in a new window]   Figure 3. Intracellular translocation of Rho A toward the plasma membrane in rings incubated with SPC for 30 min in the absence or in the presence of sevoflurane (3.4%) or propofol (10–5 M). *Difference between the ring treated with SPC and the ring treated with SPC in combination with sevoflurane is statistically significant (P < 0.05). Whiskers represent standard deviations.

View larger version (20K): [in this window] [in a new window]   Figure 4. The ratio of MYPT1 phosphorylation in rings incubated with SPC for 30 min in the absence or in the presence of sevoflurane (3.4%), propofol (10–5 M), or Y27632 (2 x 10–6 M). *Differences between the ring treated with SPC and the ring treated with SPC in combination with sevoflurane or Y27632 is statistically significant (P < 0.05). Whiskers represent standard deviations.

DISCUSSION

The Role of SPC in Coronary Circulation
Several studies have suggested that SPC acts from outside as well as inside the cellular membrane, whereas other sphingomyelin metabolites exert their effects only on intracellular targets (2,3). In the current study, extracellular SPC induced marked contraction of porcine coronary artery, consistent with previous studies using isolated coronary arteries (5). Therefore, these results support the concept that SPC affects vascular smooth muscle function when it is applied from outside blood vessels. However, it has not been determined whether receptors targeted by SPC are expressed in the intact arterial smooth muscle.

In rabbits, plasma levels of SPC are <1.3 x 10^–7 M and in the current study, SPC above 10^–6 M was capable of inducing coronary arterial contraction, suggesting that the concentrations of SPC which induce coronary vasoconstriction, are beyond the physiological range (11). However, since cells metabolize sphingomyelin from the plasma membrane to form SPC depending on various stimuli, it is possible that several disease states should enhance local production of SPC (1). It is also important to note that SPC is one of the major constituents of blood plasma, indicating upon enhanced production, this substance may be capable of inducing its effects at a remote place from its production (1). It is unknown, though, whether SPC plays a crucial role in coronary vasospasm in humans because local levels of SPC during vasospastic angina have not been determined.

Effects of Anesthetics on Rho Kinase Activity
Rho kinases, the immediate downstream targets of Rho A, are ubiquitously expressed serine-threonine protein kinases that are involved in diverse cellular functions, including vascular smooth muscle contraction (12). Rho A migrates from cytosol to plasma membrane on stimulation, and therefore, an increased membrane fraction of Rho A indicates increased Rho A activity (12). Previous studies demonstrated that Rho kinase inhibits MYPT1 activity through phosphorylation of MYPT1, resulting in smooth muscle contraction (13–16). The micromolar concentration of Y27632 is reportedly a selective inhibitor of Rho kinase targeting on adenosine triphosphate-dependent kinase domains (12). In addition, it is capable of decreasing MYPT1 phosphorylation to levels seen in quiescent vascular smooth muscle cells (17). Therefore, the concentration of Y27632 used in the current study should be appropriate.

We have documented in the current study that sevoflurane, in a concentration-dependent manner, decreased SPC-induced vasoconstriction, whereas the constriction due to SPC was completely abolished by a selective Rho kinase inhibitor. In contrast, propofol (3 x 10^–6 M and 10^–5 M) did not affect coronary arterial constriction induced by SPC. To confirm the involvement of Rho A or the activity of Rho kinase in the inhibitory effect of sevoflurane on coronary contraction induced by SPC, total and membrane fractions of Rho A and MYPT1 phosphorylation levels were determined. In the present study, sevoflurane and Y27632, but not propofol, similarly decreased MYPT1 phosphorylation, and sevoflurane, but not propofol, reduced translocation of Rho A toward plasma membrane. These results suggest that increased activity of Rho kinase induced by SPC is counter-regulated by sevoflurane but not by propofol. Since the degree of inhibition induced by sevoflurane is quite similar between Rho A translocation and Rho kinase activity, this volatile anesthetic probably affects the upstream site at least before the translocation of Rho A within the axis of Rho kinase activation induced by SPC. These results are in agreement with our previous study on the rat aorta reporting that sevoflurane similarly reduces Rho A and Rho kinase translocations (9).

Recent studies have shown Rho kinases play pivotal roles in cardiovascular diseases, such as vasospastic angina, heart failure, hypertension, ischemic stroke, cerebral vasospasm after subarachnoid hemorrhage, and metabolic syndrome, including diabetes mellitus (6,12,18–21). In our study, the targeted plasma levels of propofol were in the range used during anesthesia in humans (up to 3 x 10^–6 M) (22,23). Considering that this IV anesthetic substantially binds to plasma proteins, the free plasma concentration reached would be less (22,23). We further used sevoflurane in a dose range similar to that used during induction and maintenance of anesthesia. Our data thus suggest that, in clinical situations where these anesthetics are used, sevoflurane (above one minimum alveolar concentration), but not propofol, may attenuate or prevent coronary vasoconstriction that results from Rho kinase activation via increased plasma SPC levels. Nonetheless, in our experiment the modulating effects of sevoflurane and propofol on coronary artery vasoconstriction from SPC were performed on vessels lacking endothelial cells, indicating that we have to be cautious when applying our results to situations in which endothelial function is intact.

ACKNOWLEDGMENTS

Drs. Kinoshita, Matsuda, and Kimoto contributed equally to this study.

Footnotes

Accepted for publication May 1, 2007.

This work was presented in part at the annual meeting of the American Society of Anesthesiologists, Chicago, IL, October 14–18, 2006.

Supported in part by Grant-in-Aid, 16390458 (H.K.), 18659462 (H.K.), 17791052 (Y.K.), 18791101 (K.H.), 18689038 (K.N.), 17390432 (Y.H.), and 17659493 (Y.H.) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Tokyo, Japan.

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