Thiopental Elevates Steady-State Levels of Intracellular Ca2+ and Zn2+ in Rat Thymic Lymphocytes
Toxicity test of thiopental on rat thymic lymphocytes
DOI:
https://doi.org/10.30872/j.trop.pharm.chem.v5i2.189Keywords:
thiopental, intracellular Ca2, intracellular Zn2Abstract
Thiopental is an ultra-short-acting barbiturate and has been used commonly in the induction phase of general anesthesia. However, the toxic effect of thiopental is not completely clear.?The effect of thiopental on intracellular Ca2+ ([Ca2+]i) levels was investigated in non-excitable cells. Experiments were carried out using a flow-cytometric technique, rat thymic lymphocytes (as non-excitable cells), and appropriate fluorescent probes. Treatment of cells with 300 µM thiopental increased Fluo-3 fluorescence intensity, indicating elevation of [Ca2+]i. This increase was partially attenuated by a chelator of intracellular Zn2+. Thus, thiopental elevated both [Ca2+]i and intracellular Zn2+ ([Zn2+]i) levels. Under intracellular Zn2+-free conditions, 100–300 µM thiopental was still able to induce a statistically significant increase in [Ca2+]i, whereas removal of extracellular Ca2+ greatly reduced the increase in [Ca2+]i induced by this dose of thiopental. Therefore, the thiopental-induced increase in [Ca2+]i was mainly due to an increased influx of Ca2+. Treatment of cells with 300 µM thiopental increased FluoZin-3 fluorescence intensity, indicating the presence of [Zn2+]i, both in the presence and absence of extracellular Zn2+. The thiopental-induced elevation of [Zn2+]i was due to an increase in both influx of Zn2+ and intracellular Zn2+ release. Concanavalin A (10 µg/mL) augmented Fluo-3 fluorescence in the presence of an intracellular Zn2+ chelator. The combination of concanavalin A and 100–300 µM thiopental synergistically increased [Ca2+]i. Results suggest that thiopental increases [Ca2+]i in both quiescent and activated lymphocytes, possibly resulting in modulation of immune system function.
References
[1] Kanaya, N., Zakhary, D.R., Murray, P.A., Damron, D.S., 1998. Thiopental alters contraction, intracellular Ca2+, and pH in rat ventricular myocytes. Anesthesiology: The Journal of the American Society of Anesthesiologists 89, 202–214.
[2] Yamakage, M., Hirshman, C., Croxton, T., 1995. Inhibitory effects of thiopental, ketamine, and propofol on voltage-dependent calcium2+ channels in porcine tracheal smooth muscle cells. Anesthesiology: The Journal of the American Society of Anesthesiologists 83, 1274–1282.
[3] Zhan, R.Z., Fujiwara, N., Endoh, H., Yamakura, T., Taga, K., Fukuda, S., Shimoji, K., 1998. Thiopental inhibits increases in [Ca2+]i induced by membrane depolarization, NMDA receptor activation, and ischemia in rat hippocampal and cortical slices. Anesthesiology: The Journal of the American Society of Anesthesiologists 89, 56–466.
[4] Kimura, M., Shibukawa, Y., Momose, Y., Sugaya, M., Yamamura, S., Suzuki, T., Hatakeyama, N., Yamazaki, M., 2007. Effects of thiopental on Ca2+ currents and intracellular Ca2+ transient in single atrial cells from guinea pig. Pharmacology 80, 33–39.
[5] Lecharny, J.B., Salord, F., Henzel, D., Desmonts, J.M., Mantz, J., 1995. Effects of thiopental, halothane and isoflurane on the calcium-dependent and-independent release of GABA from striatal synaptosomes in the rat. Brain Research 670, 308–312.
[6] Miao, N., Nagao, K., Lynch, C., 1998. Thiopental and methohexital depress Ca2+ entry into and glutamate release from cultured neurons. Anesthesiology: The Journal of the American Society of Anesthesiologists 88, 1643–1653.
[7] Lewis, R.S., 2001. Calcium signaling mechanisms in T lymphocytes. Annual Review of Immunology 19, 497–521.
[8] Feske, S., 2007. Calcium signalling in lymphocyte activation and disease. Nature Reviews Immunology 7, 690–702.
[9] Hirano, T., Murakami, M., Fukada, T., Nishida, K., Yamasaki, S., Suzuki, T., 2008. Roles of zinc and zinc signaling in immunity: zinc as an intracellular signaling molecule. Advances in Immunology 97, 149–176.
[10] Hasan, R., Rink, L., Haase, H., 2013. Zinc signals in neutrophil granulocytes are required for the formation of neutrophil extracellular traps. Innate Immunity 19, 253–264.
[11] Jiang, S., Chow, S.C., Nicotera, P., Orrenius, S., 1994. Intracellular Ca2+
signals activate apoptosis in thymocytes: studies using the Ca2+-ATPase
inhibitor thapsigargin. Experimental Cell Research 212, 84–92.
[12] Provinciali, M., Di Stefano, G., Fabris, N., 1995. Dose-dependent opposite
effect of zinc on apoptosis in mouse thymocytes. International Journal of
Immunopharmacology 17, 735–744.
[13] Telford, W.G., Fraker, P.J., 1995. Preferential induction of apoptosis in
mouse CD4+ CD8+ ??TCRIoCD3?Io thymocytes by zinc. Journal of Cellular
Physiology 164, 259–270.
[14] McConkey, D.J., Orrenius, S., 1997. The role of calcium in the regulation of
apoptosis. Biochemical and Biophysical Research Communications 239,
357–366.
[15] Keel, M., Mica, L., Stover, J., Stocker, R., Trentz, O., & Härter, L., 2005.
Thiopental-induced apoptosis in lymphocytes is independent of CD95
activation. Anesthesiology: The Journal of the American Society of
Anesthesiologists 103, 576–584.
[16] Roesslein, M., Schibilsky, D., Muller, L., Goebel, U., Schwer, C., Humar, M.,
Schmidt, R., Geiger, K.K., Pahl, H.L., Pannen, B.H., & Loop, T., 2008.
Thiopental protects human T lymphocytes from apoptosis in vitro via the
expression of heat shock protein 70. Journal of Pharmacology and
Experimental Therapeutics 325, 217–225.
[17] Gee, K.R., Zhou, Z.L., Ton-That, D., Sensi, S.L., Weiss, J.H., 2002.
Measuring zinc in living cells: A new generation of sensitive and selective
fluorescent probes. Cell Calcium 31, 245–251.
[18] Kao, J.P., Harootunian, A.T., Tsien, R.Y., 1989. Photochemically generated
cytosolic calcium pulses and their detection by fluo-3. Journal of Biological
Chemistry 264, 8179–8184.
[19] Sakanashi, Y., Oyama, T.M., Matsuo, Y., Oyama, T.B., Nishimura, Y., Ishida,
S., Okano, Y., Oyama, Y., 2009. Zn2+, derived from cell preparation, partly
attenuates Ca2+-dependent cell death induced by A23187, calcium
ionophore, in rat thymocytes. Toxicology in Vitro 23, 338–345.
[20] Mousa, W. F., Enoki, T., Fukuda, K., 2000. Thiopental induces contraction
of rat aortic smooth muscle through Ca2+ release from the sarcoplasmic
reticulum. Anesthesia & Analgesia 91, 62–67.
[21] Henkel, C.C., Asbun, J., Ceballos, G., Castillo, M.D.C., Castillo, E.F., 2001.
Relationship between extra and intracellular sources of calcium and the
contractile effect of thiopental in rat aorta. Canadian Journal of Physiology
and Pharmacology 79, 407–414.
[22] O'Flynn, K., Linch, D.C., Tatham, P.E.R., 1984. The effect of mitogenic
lectins and monoclonal antibodies on intracellular free calcium
concentration in human T-lymphocytes. Biochemical Journal 219, 661–666.
[23] Delogu, G., Antonucci, A., Moretti, S., Marandola, M., Tellan, G., Signore,
M., Famularo, G., 2004. Oxidative stress and mitochondrial glutathione in
human lymphocytes exposed to clinically relevant anesthetic drug
concentrations. Journal of Clinical Anesthesia 16, 189–194.
[24] Dwyer, J.M., Johnson, C., 1981. The use of concanavalin A to study the
immunoregulation of human T cells. Clinical and Experimental Immunology
46, 237.
[25] Maret, W., 1994. Oxidative metal release from metallothionein via zinc-
thiol/disulfide interchange. Proceedings of the National Academy of
Sciences 91, 237–241.
[26] Taeger, K., Lueg, J., Finsterer, U., Roedig, G., Weninger, E., Peter, K., 1986.
Thiopental levels in the plasma during induction of anesthesia. Anasthesie
Intensivtherapie Notfallmedizin 21, 169–174.
