Elsevier

Brain Research

Volume 1037, Issues 1–2, 10 March 2005, Pages 139-147
Brain Research

Research report
Isoflurane and sevoflurane affect cell survival and BCL-2/BAX ratio differently

https://doi.org/10.1016/j.brainres.2005.01.009Get rights and content

Abstract

Depletion of calcium from the neuronal endoplasmic reticulum (ER) induces apoptosis. Isoflurane depletes calcium from sarcoplasmic reticulum (SR) of muscle, an analogue of ER in neurons, while sevoflurane maintains or increases SR calcium. We hypothesized that isoflurane, but not sevoflurane, induces apoptosis by depleting the ER calcium. Rat PC12 pheochromocytoma cells and primary cortical neurons were treated with equipotent doses of isoflurane and sevoflurane. Isoflurane, but not sevoflurane, at equipotent doses induced cell damage determined by both LDH release and MTT reduction assays, dose and time dependently, in both types of cells. Isoflurane at 2.4% for 24 h induced cytotoxicity in both cell types, which was characterized by nuclear condensation and fragmentation and activation of caspases 3 and 9. Isoflurane cytotoxicity was suppressed by dantrolene, a ryanodine receptor antagonist that inhibits abnormal calcium release from the ER. Isoflurane decreased the Bcl-2/Bax ratio by as much as 36% (P < 0.05). However, sevoflurane did not cause neuronal damage by apoptosis nor did it decrease the Bcl-2/Bax ratio. These results suggest that isoflurane and sevoflurane differentially affect the Bcl-2/Bax ratio and cell survival. At equipotent concentrations, isoflurane, but not sevoflurane, induces cytotoxicity in both PC12 cells and primary cortical neurons and decreases the Bcl-2/Bax ratio.

Introduction

Maintenance of a sufficient amount of calcium inside the endoplasmic reticulum (ER) plays a critical role in normal ER structure, protein synthesis, cell growth, and death [9], [10], [49]. Excessive calcium release, from the ER by over activation of the ryanodine receptor and/or reduced calcium uptake by inhibition of calcium ATPase on the ER membrane, leads to ER calcium depletion and subsequent cell death by apoptosis, a programmed cell death [9], [31], [44], [52], [53]. Meanwhile, inhibition of excessive calcium release from the ER by dantrolene, a selective ryanodine receptor antagonist, has been shown to inhibit neuronal toxicity evoked by glutamate and NMDA excitotoxicity, trauma peptide, HIV coat protein, beta-amyloid, and ER calcium depletion in vitro [14], [16], [37], [45], [52], [53]. Dantrolene also inhibits cell damage after muscle or cerebral ischemia, seizure, and sepsis in animal studies [8], [23], [28], [34], [46], [52].

The Bcl-2 family members are active mediators of apoptosis that either inhibit (e.g., Bcl-2) or facilitate (e.g., Bax) apoptotic cell death [1], [42]. Bcl-2 may inhibit apoptosis by suppressing abnormal Ca2+ release from the ER and by preventing ER Ca2+ depletion [15], [22], [31], [53]. The balancing effect of Bcl-2 and Bax on the ER Ca2+ homeostasis plays an important role in determining the fate of the cells to either undergo proliferation or apoptosis [4], [5]. Any agent that decreases the Bcl-2/Bax ratio may promote apoptosis.

Volatile anesthetics seem to affect calcium homeostasis differently. Isoflurane induces Ca2+ release from the ER reversibly in cerebrocortical and hippocampal neurons [27]. In mouse cerebral neurons, sevoflurane (0.5 mM) increased base line and NMDA-induced cytosolic calcium elevation, but in a less degree than isoflurane (0.35 mM) [17]. Isoflurane also induces Ca2+ release from the sarcoplasmic reticulum (SR), analogous to the ER in neurons, via activation of the ryanodine receptor [2], [47], or by inhibition of Ca2+ uptake into SR [47]. Isoflurane has been shown to decrease the SR Ca2+ content [13], [26]. However, sevoflurane has either no effect [6], or inhibits Ca2+ release from the SR [13]. Thus, sevoflurane either maintains [13] or increases the SR Ca2+ content [19] in muscle cells. Isoflurane inhibits protein synthesis and secretion, and induces cytotoxicity in both human and rat hepatocytes [32], and induces apoptosis in human lymphocytes [33]. In developing rat brains, isoflurane at clinical concentrations (0.75% isoflurane for 6 h) causes widespread neurodegeneration and persistent learning deficits [25]. Although blockade of NMDA receptors and/or activation of GABA receptors have been proposed for isoflurane-induced neurodegeneration in developing brain [24], [25], it is plausible that isoflurane neurotoxicity is caused by depletion of ER Ca2+. In contrast, sevoflurane does not seem to deplete the ER Ca2+ or may in fact elevate the ER Ca2+ content and therefore may have less potency to induce cytotoxicity as compared to isoflurane. The current study aims at answering the following questions: (1) Will isoflurane cause cell death by apoptosis in different type of cells? (2) Will sevoflurane have different effects on cytotoxicity based on its different effects on the ER calcium content? (3) What are the effects of volatile anesthetics on changes in the ratio of apoptotic/antiapoptotic proteins (e.g., Bcl-2 and Bax)?

Section snippets

Rat pheochromocytoma cells (PC12) culture

PC12 cells were cultured as described [54], [55]. Briefly, PC12 cells were maintained in RPMI 1640 medium (Invitrogen, Grand Island, NY) supplemented with 10% horse serum (Invitrogen Life Technologies, Carlsbad, CA), 5% fetal calf serum (Hyclone Laboratories, Logan, UT), and penicillin/streptomycin (Invitrogen Life Technologies, Carlsbad, CA). Monolayer cultures at a density of 0.1–0.3 × 106 cells/cm2 were incubated in plastic flasks precoated with 0.01% poly-l-ornithine (Sigma-Aldrich, St.

Isoflurane, but not sevoflurane, induce cytotoxicity in both PC12 cells and primary cortical neurons

The cytotoxic effects of isoflurane and sevoflurane at equipotent doses were studied and compared in two cell types. As shown in Fig. 1, isoflurane exposure caused a concentration-dependent increased release of LDH (Fig. 1A) and reduction of MTT (Fig. 1B) in PC12 cells. In addition, isoflurane also caused PC12 cell damage in a time-dependent manner (Fig. 2). PC12 cells treated with isoflurane at 2.4%, (approximately twice the minimum alveolar concentration; MAC) for 24 h caused significant cell

Discussion

We have demonstrated that isoflurane, but not equipotent dose of sevoflurane, induced cytotoxicity in rat PC12 pheochromocytoma cells and primary cortical neurons. PC12 cells treated with 2 MAC of isoflurane for 24 h demonstrated cytotoxicity, while an equipotent dose of sevoflurane did not have similar cytotoxic effects. Such differential cytotoxic effects also existed with the primary cortical neurons. These results suggest that isoflurane and sevoflurane may affect cell survival differently.

Acknowledgments

The study was supported by the departmental research start fund at the University of Pennsylvania. The authors acknowledge intellectual assistance and support by Dr. Randall Pittman, Department of Pharmacology, University of Pennsylvania, and Drs. Maryellen F. Eckenhoff and James Hecker, Department of Anesthesiology, University of Pennsylvania.

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