%0 Generic
%A Schweizer, Nadja
%D 2011
%F heidok:13410
%K Calciumoszillation , RTL-W1calcium oscillation , cell line RTL-W1 , rainbow trout , ecotoxicology
%R 10.11588/heidok.00013410
%T Calcium signaling in fish cells
%U https://archiv.ub.uni-heidelberg.de/volltextserver/13410/
%X One basic cellular response towards a multitude of physical and chemical factors is the modulation of intracellular Ca2+ levels. There is also considerable evidence that a number of toxicants have an impact on Ca2+ signaling processes, alter them, and may induce cell death. Given the immense versatility of Ca2+ modulation due to the complex mechanisms which help to encode information, recording of the intracellular Ca2+ signal might eventually be a useful tool for the detection and identification of environmental stressors. Notwithstanding the universal character of Ca2+ signaling and the highly conserved pathways, research on Ca2+ as a second messenger has mainly been restricted to mammalian models. Much less is known about its function and mode of action in fish and only a handful of papers deal with the question whether there is a Ca2+ response to environmental toxicants or not. The present thesis aims at closing some of the remaining gaps. Therefore, after adapting the cell culture and Ca2+ imaging protocols for the needs of this study, the reaction of intracellular Ca2+ to different “classical” agonists such as phenylephrine and ATP was investigated systematically in order to find out the basic principles of Ca2+ dynamics in teleost fish cells. Two cell types were used and compared to one another: primary hepatocytes from rainbow trout (Oncorhynchus mykiss), and the permanent fish cell line RTL-W1 derived from rainbow trout liver, both established model systems in aquatic ecotoxicology. From an ecotoxicological point of view, we tried to answer the question whether Ca2+ imaging can be applied for the early detection of environmental stress with cell death as a last consequence. Therefore, selected model environmental toxicants and stressors such as 4-nitrophenol, 3,4-dichloroaniline, and hydrogen peroxide were used to elucidate possible interactions between contaminants and Ca2+ signaling in RTL-W1 cells. Ca2+ oscillations in response to several stimuli were recorded in RTL-W1 cells and to a lesser extent in primary hepatocytes. Interestingly, these Ca2+ oscillations are amplitude-encoded in contrast to their mammalian counterpart. Moreover, Ca2+ release in rat cells during oscillations is markedly faster than the uptake, whereas this relation is more symmetric in the fish cells. Bioinformatics and computational analysis were employed to identify key players of Ca2+ signaling in fish and to determine likely causes for the experimentally observed differences between the Ca2+ dynamics in fish cells compared to those in mammalian liver cells. Different binding characteristics of the IP3R, e.g. responsible for the Ca2+-induced Ca2+ release, could be the origin of the observations. The present thesis also indicates that the fish cell line RTL-W1 is a suitable tool for the investigation of Ca2+ signals in consequence of toxicant exposure. Evidence is provided that ecotoxicologically relevant substances take influence on intracellular Ca2+. Namely hydrogen peroxide and 4-nitrophenol showed a clear response and produced marked Ca2+ oscillations at sublethal concentrations. Effect intensity and threshold varied from cell to cell; however, general effects were reproducible and dose-dependent. At concentrations below those inducing elevated cytotoxicity and apoptosis rates (assessed by the neutral red assay and the apoptosis assay with Hoechst 33342), there is a lasting, unspecific increase in the intracellular Ca2+ level which might be interpreted as a precursor of apoptotic or necrotic processes in the cell. Generally, Ca2+ signals seem to be dependent on the pathway activated or non-specifically interfered by the respective substance. The question whether specific types of Ca2+ responses are specific of and may be used to characterize different types of stressors still cannot be answered at present. However, there is evidence that Ca2+ imaging might provide a highly sensitive, yet non-specific indicator of toxic impact, since, as a second messenger, intracellular Ca2+ integrates toxic effects of multiple other sublethal parameters.