Anesth Analg 2004;99:1408-1412
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000132977.84091.B5
ANESTHETIC PHARMACOLOGY
The Inhibition of Aortic Smooth Muscle Cell Proliferation by the Intravenous Anesthetic Ketamine
Yousuke Shiga, MD PhD*,
Kouichiro Minami, MD PhD*,
Kayoko Segawa, MD PhD
,
Yasuhito Uezono, MD PhD
,
Munehiro Shiraishi, MD*,
Takeyoshi Sata, MD PhD*,
Chieko Yamamoto, PhD, and
Kim Sung-Teh, MD
*Department of Anesthesiology, University of Occupational and Environmental Health, Kitakyushu, Japan;
Kitakyushu Institute of Biophysics, Fukuoka, Japan; and
Department of Pharmacology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
Address correspondence and reprint requests to K. Minami, MD PhD, Assistant Professor, Department of Anesthesiology, University of Occupational and Environmental Health School of Medicine, 1–1 Iseigaoka, Yahatanishiku, Kitakyushu 807–8555, Japan. Address email to kminami{at}med.uoeh-u.ac.jp
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Abstract
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Smooth muscle cell (SMC) proliferation has been recognized as central to the pathology of both major forms of vascular disease, atherosclerosis and hypertension. Recently, we reported that ketamine inhibits rat mesangial cell proliferation, suggesting that ketamine inhibits cell growth. Although the IV anesthetic ketamine has been widely used clinically, the exact effects of ketamine on vascular SMC proliferation have not been studied. In this study, we investigated the effects of ketamine on vascular SMC proliferation. Ketamine inhibited [3H]thymidine incorporation and decreased the number of SMCs in a concentration-dependent manner (10–200 µM); neither propofol nor fentanyl inhibited [3H]thymidine incorporation into human aortic SMCs. The protein kinase C (PKC) inhibitor GF109203x abolished the ketamine-induced inhibition of [3H]thymidine incorporation into SMC, but the inhibition was not affected by either the protein kinase A inhibitor H-89 or the protein kinase G inhibitor KT5823. A histological analysis demonstrated the inhibitory effect of ketamine on the intimal thickening of the balloon-injured rat aorta. Based on these results, ketamine inhibits SMCs at clinical concentrations via the PKC pathway. Our results indicate that ketamine might prevent the proliferation of SMCs clinically.
IMPLICATIONS: Vascular smooth muscle cell (SMC) proliferation has been recognized as central to the pathology of major forms of vascular disease. Ketamine inhibited SMCs at clinical concentrations via the protein kinase C pathway. Our present results indicate that ketamine might prevent the proliferation of SMCs clinically.
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Introduction
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Recently, the number of patients requiring cardiac surgery, such as coronary artery bypass graft and aortic surgery, has increased. The major processes common to stenotic arterial lesions include damage to the endothelium and reactive proliferation, migration, and accumulation of smooth muscle cells (SMC). SMC proliferation has been recognized as central to the pathology of the two major vascular diseases, atherosclerosis and hypertension. The SMC plays a central role in vascular occlusive disorders such as atherosclerosis and restenosis after percutaneous transluminal coronary angioplasty (PTCA) or atherectomy (1,2).
We reported that ketamine inhibits rat mesangial cell proliferation, suggesting that ketamine inhibits cell growth (3). Although ketamine has been widely used clinically, the exact effects of this anesthetic on vascular SMC proliferation have not been studied.
In the present study, we investigated the effects of the IV anesthetic ketamine, as well as the effects of propofol and fentanyl, on vascular SMC proliferation. We also evaluated the effect of ketamine on the intimal thickening of the balloon-injured rat aorta.
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Methods
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Chemicals were obtained from the following sources: phosphate-buffered saline (PBS) from Nissui Seiyaku, Tokyo, Japan; fetal bovine serum (FBS), human epidermal growth factor (hEGF), human fibroblast growth factor-B (hFGF-B), insulin, gentamicin, and amphotericin B from Sanko Junyaku, Tokyo, Japan; SMC basal medium (SmBM) from Clonetics, San Diego, CA; propofol from Tokyo Kasai, Tokyo, Japan; fentanyl from Sankyo Tokyo, Japan; (±)-ketamine and ethylene diaminetetraacetic acid (EDTA) from Sigma, St. Louis, MO; trypsin 1:250 from Difco Laboratories, Detroit, MI; [3H]thymidine from Amersham, Buckinghamshire, UK; [N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquino-linesulfonamide Di-HCl salt (H-89), Bisindolylmaleimide III (GF109203x), and [2-[1-(3-Aminopropyl)-indol-3-yl]-3-(indol-3yl)maleimide][3-[1-(3-Aminopropyl)-1H-indol-3-yl]-4(1H-indol-3-yl)-1H-pyrrole-2,5-dione] (KT5823) from LC Laboratories, Woburn, MA.
Human aortic SMCs were provided by Sanko Junyaku (Tokyo, Japan). SMCs were cultured at 37°C with 95% O2 and 5% CO2 in SmBM containing 5% FBS, growth factors (0.5 ng/mL hEGF, 2 ng/mL hFGF-B, 5 µg/mL insulin), 30 µg/mL gentamycin, and 15 ng/mL amphotericin B. Cells from the second to third passages were used in these studies. SMCs were cultured with or without additives in SmBM containing 5% FBS in 96-well, flat-bottomed microtiter plates (FalconTM; Becton Dickinson, Oxnard, CA) in the presence or absence of drugs, e.g., ketamine. The cultures were pulsed with 0.5 µCi/well of [3H]thymidine (specific activity 6.0 Ci/mmol) for the last 12 h of culture and harvested with the aid of a semiautomatic cell harvester (Abe Kagaku, Chiba, Japan). The amount of radioactivity incorporated into the cellular DNA was measured with a liquid scintillation spectrophotometer (Aloka LSC-3500ETM, Aloka Co, Tokyo, Japan). Four separate series of experiments were performed, each with triplicate cultures.
To verify that the assay for [3H]thymidine incorporation was indicative of the effect of ketamine on actual cell growth, the cells were counted. In these experiments, 105 cells/mL were dispensed into Petri dishes and incubated with different concentrations of ketamine for 24 h; next the cells were dispersed with 0.25% trypsin and 1 mM EDTA in PBS and counted using a hemocytometer. In separate studies, the viabilities of cell preparations were assessed by trypan blue exclusion at the end of the experimental periods; viability was >95% for the cells from both control and experimental cultures. Three separate series of experiments were performed, each with triplicate cultures.
This study conformed to the Guide for the Care and Use of Laboratory Animals adopted and promulgated by the Japanese Pharmacology Society and approval has been granted by the Animal Research Committees of the University of Occupational and Environmental Health. Endothelial denudation of the aorta was performed as described previously (4). Briefly, male Wistar rats weighing 300–350 g were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally), and the descending aorta was denuded using a Fogarty 2F balloon embolectomy catheter (Baxter, Irvine, CA). The catheter was introduced into the descending aorta via the carotid artery, inflated with 0.2 mL saline, and passed 3 times to injure the endothelium; the carotid artery was ligated after this procedure. After denudation, an osmotic pump (Mini-Osmotic Pump Model 2001; Alza, Mountain View, CA) was surgically implanted subcutaneously in the back of each rat. Injured rats received pumps that dispensed saline (balloon-injured group n = 4) or ketamine at a dose of 0.5 mg · kg–1 · h–1 (ketamine-treated group, n = 4). Control rats (n = 4) received carotid arterial ligation and pumps, from which were dispensed normal saline subcutaneously in the back of each rat.
For the evaluation of cellular proliferation and histological changes, the animals were killed at 14 days after the surgery. For the evaluation of morphological changes, aortic cross sections were fixed in 3% paraformaldehyde (pH, 7.4), embedded in paraffin, sectioned, and stained with Mayer’s hematoxylin and eosin.
All data are presented as the mean ± SD. Statistical analyses of the data were performed using analysis of variance. Statistical significance was defined at the P < 0.05 level.
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Results
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We studied the effects of ketamine on [3H]thymidine incorporation into human aortic SMCs. [3H]thymidine incorporation into human aortic SMCs in SmBM containing 5% FBS and growth factors was 11280 ± 651 cpm, 5120 ± 120 cpm, and 2520 ± 89 cpm after 24, 48, and 72 h, respectively. Ketamine (100 µM) significantly decreased [3H]thymidine incorporation into the cells to 75%, 65%, and 88% of control levels after 24, 48, and 72 h, respectively (Fig. 1 A). Subsequent experiments were performed using the 24-h time point. Ketamine inhibited [3H]thymidine incorporation in a concentration-dependent manner; ketamine at 10 µM, 50 µM, and 100 µM decreased [3H]thymidine incorporation to 82% ± 6%, 72% ± 8%, and 69% ± 6% of control values, respectively (Fig. 1 B).
The inhibitory effects of ketamine on SMCs, as assayed by counting cell numbers, were also concentration-dependent (Fig. 2). Cell numbers were decreased to 84% ± 5%, 76% ± 8%, and 60% ± 8% of control values by 10 µM, 50 µM, and 100 µM ketamine, respectively.
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Figure 2. Concentration-response curve of the inhibitory effects of ketamine on human aortic smooth muscle cell numbers. Cells were treated with medium containing ketamine (1–200 µM) for 24 h. Values are means ± SD of four experiments. *P < 0.05 compared with each control.
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We also studied [3H]thymidine incorporation into SMCs in the presence of fentanyl (30 nM, 10 µg/mL), propofol (100 µM, 18 µg/mL), and ketamine (100 µM, 27 µg/mL) to determine their effects on cell proliferation. Unlike ketamine, propofol and fentanyl did not inhibit [3H]thymidine incorporation into human SMCs (Fig. 3).
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Figure 3. The effects of fentanyl, propofol, and ketamine on [3H]thymidine incorporation into human aortic smooth muscle cells. The effects of fentanyl (30 nM, 10 µg/mL), propofol (100 µM, 18µg/mL), and ketamine (100 µM, 27µg/mL) on [3H]thymidine incorporation into human aortic smooth muscle cells. Values are means ± SD of four experiments. Cells were treated with medium containing propofol, fentanyl, or ketamine for 24 h. * P < 0.05 compared with each control.
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To address the mechanism of the ketamine-induced inhibition of SMC proliferation, we used the PKC inhibitor GF109203x (Ki = 20 nM) (5), the protein kinase A (PKA) inhibitor H-89 (Ki = 48 nM) (6), and the protein kinase G (PKG) inhibitor KT5823 (Ki = 2.4 nM) (7), which are reported to be specific inhibitors for each kinase; we used the inhibitors at concentrations of 10 times their Ki values. In the presence of the PKC inhibitor GF109203x and 100 µM ketamine, [3H]thymidine incorporation was 92% ± 5% of control, demonstrating that GF109203x abolished the ketamine-induced inhibition of SMC proliferation (Fig. 4). In contrast, neither H-89 nor KT5823 affected the ketamine-induced inhibition of [3H]thymidine incorporation (Fig. 4). Each inhibitor by itself at the experimental concentration did not affect [3H]thymidine incorporation (data not shown).
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Figure 4. The effects of a protein kinase A inhibitor, a protein kinase G inhibitor, and a protein kinase C inhibitor on the ketamine-induced [3H]thymidine incorporation into human aortic smooth muscle cells. Cells were treated with medium containing ketamine (100 µM) with or without the PKC inhibitor GF109203x (200 nM), the protein kinase A (PKA) inhibitor H-89 (480 nM), or the protein kinase G (PKG) inhibitor KT5823 (24 nM) for 24 h. Values are means ± SD of four experiments. *P < 0.05 compared with each control.
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To investigate whether ketamine inhibits SMC proliferation in vivo, we looked at the morphological changes in the rat aorta after vascular injury in a rat treated with ketamine (ketamine-treated group) (Fig. 5C), a rat treated with saline (balloon-injured group) (Fig. 5B), and a rat suffering no injury by balloon (control group) (Fig. 5A). Intimal thickening was observed after vascular injury (balloon-injured group), but less intimal thickening was observed in each of the four ketamine-treated rats.
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Figure 5. Histological cross-sections demonstrating the effect of ketamine on the intimal thickening of the balloon-injured rat aorta. Histological cross sections demonstrating the effect of ketamine on intimal thickening of the balloon-injured rat aorta at 14 days in a rat treated with ketamine (C), with saline (B), and no injury by balloon (control group) (A). A catheter was introduced into the descending aorta via the carotid artery, inflated with 0.2 mL saline, and passed three times to injure the endothelium. The injured rats received saline (balloon-injured group) or ketamine at a dose of 0.5 mg · kg–1 · h–1 (ketamine-treated group). Control rats received carotid arterial ligation and pumps from which were dispensed normal saline subcutaneously. Intimal thickening was observed after vascular injury (balloon-injured group), but a lesser degree of intimal thickening was observed in each of the four ketamine-treated rats.
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Discussion
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Ketamine inhibited SMC proliferation in a concentration-dependent manner; propofol and fentanyl did not affect SMC proliferation. This is the first report of the inhibitory effects of ketamine on SMC proliferation. In clinical situations, it was also reported that the plasma concentration of ketamine on awakening from general anesthesia is in the range 0.6–1.1 µg/mL and analgesic concentrations are approximately 0.1–0.2 µg/mL (8). The free plasma concentration of ketamine at 5 min after the IV injection of 2.5 mg/kg was approximately 10.5 µM (9); however, a much larger plasma concentration of ketamine (60 µM) has been demonstrated in patients at 5 min after the IV injection of 2 mg/kg (10). Although the concentrations of ketamine that inhibited SMC proliferation in this study are comparable to the peak blood concentrations of ketamine used for clinical anesthesia, the present results suggest that ketamine may have inhibitory effects on SMC proliferation.
In the present study, the PKC inhibitor GF109203x eliminated the inhibitory effect of ketamine on SMC proliferation, but the PKG and PKA inhibitors had little effect. These findings indicate that ketamine may inhibit SMC proliferation via the activation of the PKC pathway. Kudoh et al. (11) reported that ketamine suppresses norepinephrine-induced IP3 production and that the inhibition occurs through pathways that include PKC and a decrease in intracellular Ca2+ concentrations; they suggested that ketamine seems to activate PKC in cardiomyocytes. The report of Kudoh et al. (11) supports our present results. We previously reported that ketamine inhibits rat mesangial cell proliferation via the PKA pathway (3), but the PKA inhibitor had no effect on the ketamine-induced inhibition of SMC proliferation. Recently, we have reported that substance-P receptor mediated Ca2+-activated Cl– currents are inhibited by ketamine at pharmacological concentrations without PKC pathway, probably as the result of competitive inhibition of substance-P receptor binding (12). Moreover, ketamine had little effect on 5HT2A receptor functions that share the same signaling steps as substance-P receptor in Xenopus oocytes (13), suggesting ketamine had little effect on PKC activity in Xenopus oocytes. These differences might be attributable to cell type variations such that PKC, rather than PKA, might be dominant in regulating SMC proliferation.
The proliferation of SMCs plays a central role in vascular occlusive disorders. Based on our present results, ketamine would have inhibitory effects on SMC proliferation and might thus be useful for the prevention of SMC proliferation. Our present results also showed that intimal thickening was less in the injured rats receiving ketamine. It would be interesting to study the inhibitory effects of ketamine on SMC proliferation clinically. However, Bowdle et al. (14) have reported a linear relation between psychedelic effects of ketamine and steady-state plasma concentrations between 50 and 200 ng/mL, a range of plasma concentration that is clinically relevant for healthy volunteers receiving ketamine. It will be necessary to study the effect of small concentration of ketamine on SMC proliferation. We are now planning to investigate the effects of long-term administration of ketamine at a subanesthetic concentration on intimal thickening in injured rats.
In conclusion, we studied the effects of ketamine on human aortic SMC proliferation and found that ketamine inhibited SMC proliferation via the PKC pathway. Our present results indicate that ketamine might prevent the proliferation of SMC in clinical situations.
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Acknowledgments
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Supported, in part, by Grants-in-Aid for Scientific Research (10770539, 11671532, 10770778, 11770878, 14704040, and 16059429) from the Japan Society for the promotion of Science and from the Japanese Foundation for Cardiovascular Research.
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References
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Accepted for publication May 5, 2004.
Top
Abstract
Introduction
) or without (
) medium containing 100 µM ketamine during the incubation period. Values are mean ± SD of four experiments. *P < 0.05 compared with each control. B, Concentration-response curves of the ketamine-induced inhibitory effects of [3H]thymidine incorporation into human aortic smooth muscle cells. Cells were treated with medium containing ketamine (0–200 µM) for 24 h. Values are mean ± SD of four experiments. *P < 0.05 compared with each control.
