Anesthesiology 2008; 108:251–60 Copyright © 2008, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. The Common Inhalational Anesthetic Isoflurane Induces Apoptosis via Activation of Inositol 1,4,5-Trisphosphate Receptors Huafeng Wei, M.D., Ph.D.,* Ge Liang, M.D.,† Hui Yang, M.D.,‡ Qiujun Wang, M.D.,§ Brian Hawkins, Ph.D.,࿣ Muniswamy Madesh, Ph.D.,# Shouping Wang, M.D.,** Roderic G. Eckenhoff, M.D.†† Background: Isoflurane induces cell apoptosis by an un- Conclusion: These findings suggest that isoflurane activates known mechanism. The authors hypothesized that isoflurane the ER membrane IP3 receptor, producing excessive calcium activates inositol 1,4,5-trisphosphate (IP3) receptors on the en- release and triggering apoptosis. Neurons with enhanced IP3 doplasmic reticulum (ER) membrane, causing excessive cal- receptor activity, as in certain cases of familial Alzheimer or Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/108/2/251/366216/0000542-200802000-00013.pdf by guest on 28 September 2021 cium release, triggering apoptosis. Huntington disease, may be especially vulnerable to isoflurane Methods: The authors determined isoflurane-induced cyto- cytotoxicity. toxicity by measuring caspase-3 activity, lactate dehydrogenase release, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxy- phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) reduc- THE common inhalational anesthetic isoflurane induces tion, and imaging analysis of cell damage markers (annexin V cytotoxicity in both a concentration- and time-depen- and propidium iodide staining) in different cell types. The au- dent manner in different types of cultured cells.1–8 Fur- thors used the chicken B lymphocyte with a total knock-out of ther, at clinically relevant concentrations, isoflurane IP receptors, PC12 cells with elevated IP receptor activity 3 3 caused widespread neuronal apoptosis in developing rat (transfected with L286V presenilin 1), striatal cells with a 9,10 knock-in of Q111 Huntingtin, and each cell line’s correspond- brains with subsequent persistent learning deficits. ing wild-type controls. The authors also measured the isoflu- Isoflurane also caused cognitive dysfunction persisting rane-evoked changes of calcium concentration in cytosol for several weeks after treatment in adult and aged and/or mitochondria in these cells. rats11,12 and aged mice.13 Therefore, it is possible that Results: Isoflurane induced apoptosis concentration- and general anesthesia may contribute to the recently re- time-dependently, and sequentially elevated cytosolic and then ported cognitive deficits after surgery, especially in aged mitochondrial calcium in the chicken B-lymphocyte wild-type patients.14,15 but not the IP3 receptor total knock-out cells. Thapsigargin, a calcium adenosine triphosphatase inhibitor on ER mem- The inositol 1,4,5-trisphosphate (IP3) receptors, found branes, induced apoptosis and elevations of calcium in cy- in the endoplasmic reticular (ER) membrane, play an tosol and mitochondria in both chicken B-lymphocyte wild- important role in both normal physiology16 and patho- type and IP receptor total knock-out cells. Isoflurane 17,18 3 logic neurodegeneration. Excessive calcium release induced significantly more neurotoxicity and greater calcium release from the ER in L286V PC12 and Q111 Huntingtin from the ER, via overactivation of IP3 receptors, results 2ϩ striatal cells than in their corresponding wild-type controls, in elevation of cytosolic calcium concentration ([Ca ]c), both of which were significantly inhibited by the IP3 receptor calcium overload in mitochondria, and depletion of ER antagonist xestospongin C. calcium, all of which can contribute to cell death.19,20 In addition, cytochrome c released from mitochondria due * Assistant Professor, † Research Specialist, †† Professor, Department of to calcium overload removes the negative feedback in- Anesthesiology and Critical Care, ࿣ Visiting Scholar/Postdoctoral Research Fel- hibition of IP receptors by cytosolic calcium, leading to low, # Assistant Professor, Department of Cancer Biology and Institute of Envi- 3 ronmental Medicine, University of Pennsylvania. ‡ Visiting Scholar/Postdoc- a vicious cycle of excessive calcium release from the ER toral Research Fellow, Department of Anesthesiology and Critical Care, via IP receptors.17,20 Cytochrome c release also acti- University of Pennsylvania; Department of Anesthesiology, Union Hospital, 3 Tongji Medical College, Huazhong University of Science and Technology, vates caspase 3, which in turn cleaves IP3 receptors, Wuhan, China. § Visiting Scholar/Postdoctoral Research Fellow, Department of resulting in a permanent leak of calcium from the ER.21 Anesthesiology and Critical Care, University of Pennsylvania; Department of Anesthesiology, The Third Clinical Hospital, Hebei Medical University, Shijiazhuang, Predictably, cells deficient in IP3 receptors demonstrate China. ** Visiting Scholar/Postdoctoral Research Fellow, Department of Anesthesi- resistance to apoptosis induced by a variety of injuries.22 ology and Critical Care, University of Pennsylvania; Department of Anesthesia, Second Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China. Over activation of IP3 receptors is implicated in the Received from the Department of Anesthesiology, University of Pennsylvania, neurodegeneration seen in diseases such as Alzheimer Philadelphia, Pennsylvania. Submitted for publication May 17, 2007. Accepted disease and Huntington disease (HD) and in conditions for publication October 2, 2007. Supported by grant No. 1-K08-GM-073224-01 18,19,23,24 from the National Institute of General Medical Science, National Institutes of such as stroke. Health, Baltimore, Maryland (to Dr. Wei); grant No. 12-FY05-62 from March of The mechanisms of isoflurane neurotoxicity are un- Dimes Birth Defects Foundation Research, White Plains, New York (to Dr. Wei); and the Research Fund at the Department of Anesthesiology and Critical Care, known. Our previous work has demonstrated that University of Pennsylvania, Philadelphia, Pennsylvania (to Dr. Wei). Presented as dantrolene, an ER ryanodine receptor antagonist, in- an oral presentation at the 54th Annual Meeting of the Association of University 1 Anesthesiologists, Chicago, Illinois, April 27, 2007. hibited isoflurane-induced apoptosis, suggesting a Address correspondence to Dr. Wei: Department of Anesthesiology and Crit- role for abnormal calcium release from the ER in ical Care, University of Pennsylvania School of Medicine, 305 John Morgan Building, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104. weih@ isoflurane neurotoxicity. In neurons, isoflurane may uphs.upenn.edu. Information on purchasing reprints may be found at www.an- induce calcium release from the ER, although it is not esthesiology.org or on the masthead page at the beginning of this issue. ANESTHE- clear whether this is the result of actions on the IP or SIOLOGY’s articles are made freely accessible to all readers, for personal use only, 3 25 6 months from the cover date of the issue. the ryanodine receptor. We hypothesized in the Anesthesiology, V 108, No 2, Feb 2008 251 252 WEI ET AL. current study that isoflurane induces apoptosis by Imaging Analysis of Annexin V and Propidium excessive calcium release from ER via overactivation Iodide of IP3 receptor. To test this hypothesis, we studied Translocation of membrane phospholipid phosphati- apoptosis and calcium release from the ER in a variety dylserine from the inner to the outer leaflet of the plasma of cell types with varying IP3 receptor expression or membrane is an early indication of cell damage. Annexin activity. V, a phospholipid binding protein with a high affinity for phospholipid phosphatidylserine, can bind to phospho- lipid phosphatidylserine once it is exposed to the extra- cellular environment. Propidium iodide (PI) can bind to Materials and Methods nucleic acid after penetrating a breached plasma mem- Cell Cultures brane, as occurs in the later stages of cell damage. We Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/108/2/251/366216/0000542-200802000-00013.pdf by guest on 28 September 2021 Cells of wild-type (WT) chicken B lymphocyte (DT40) treated DT40 cells, grown floating in the medium, with different concentrations of isoflurane (0.6, 1.2, and cell line, and its total IP3 receptor knock-out (DT40 IP3R TKO) type, were cultured in RPMI 1640 with 10% fetal 2.4%) for 24 h, as well as with 2.4% isoflurane for differ- calf serum, 1% chicken serum, 50 ␮M 2-mercaptoethanol, ent times (6, 12, and 24 h). Immediately after treatment, ML we determined annexin V– or PI–positive cells by the 4m -glutamine, and antibiotics in a 95% air, 5% CO2 26 humidified atmosphere at 38°C as previously described.26 methods described previously. Cells were dropped Rat pheochromocytoma (PC12) cells transfected with onto 25-mm cover slips and stained with annexin V or PI. The stained cells were visualized and counted by two WT presenilin 1 (PS1) or point mutated PS1 (L286V) persons blinded to the treatments. The percentage of were cultured in Dulbecco’s Modified Eagle’s Medium annexin V– or PI–positive cells averaged from four areas supplemented with 10% heat-inactivated horse serum, on each cover slip was then calculated and compared. 5% fetal calf serum, 200 ␮g/ml G418, and antibiotics in a 95% air, 5% CO humidified atmosphere at 37°C as de- 2 Detection of Caspase-3 Activity scribed.1,27 The transfection of the WT and mutant PS1 has 28,29 Increased caspase-3 activity