Expression of genes encoding Ca 2+ exporting proteins in freshwater crayfish Procambarus clarkii during cold exposure Yongping Gao a , Christopher M. Gillen b , Daniel R. Whalen a , Francieli M. Vigo c , Ashkahn E. Golshani a , Michele G. Wheatly a,Ã a Department of Biological Sciences, Wright State University, Dayton, OH 45435, USA b Department of Biology, Kenyon College, Gambier, OH 43022, USA c Universidade Federal do Rio Grande do Sul, Porto Alegre 90040-060, Brasil article info Article history: Received 21 July 2008 Accepted 5 January 2009 Keywords: Cold exposure Calcium export Plasma membrane calcium ATPase (PMCA) Sodium/calcium exchanger (NCX) Crayfish Procambarus clarkii Antennal gland Axial abdominal muscle abstract 1. This study examined expression of two primary transmembrane Ca 2+ export proteins (plasma membrane Ca 2+ ATPase, (PMCA); Na + /Ca 2+ exchanger, sodium/calcium exchanger (NCX)) in epithelial (antennal gland, kidney) and non-epithelial (axial abdominal muscle) tissues of the freshwater crayfish Procambarus clarkii following exposure (28 days) to 4 1C (compared with control 23 1C). 2. Crayfish tissues responded to cold exposure within 5–7 days by upregulating the mRNA (determined through real-time polymerase chain reaction (PCR)) for both PMCA and NCX. After 28 days, levels were 4–5  higher in antennal gland and 2–3  higher in muscle. In situ hybridization localized the mRNA increase to the nephridial canal in antennal gland sections and to the extensor muscles in the tail. Western blotting confirmed corresponding increases in PMCA protein expression; however, expression of NCX protein was unchanged. & 2009 Elsevier Ltd. All rights reserved. 1. Introduction The concentration of cytosolic (intracellular (IC)) free calcium (Ca 2+ ) of animal cells is maintained in the micromolar range except, briefly, during periods of signaling or activation of certain processes or following damage. This state depends upon the interaction and balance of numerous mechanisms including transmembrane proteins (channels, pumps and exchangers) on apical, basolateral and endomembranes that import or export Ca 2+ as well as Ca 2+ -binding and regulatory proteins. Our lab has employed the freshwater crayfish (Procambarus clarkii) molting cycle as a non-mammalian model system to study mechanisms of Ca homeostasis. Specifically, the organismal need to obtain (branchial uptake) and retain (renal reabsorption) Ca 2+ in the postmolt stage to effect cuticular remineralization (Wheatly, 1999) has been correlated with upregulation in epithelia (with attenuated changes in non-epithelial tissues) of basolateral Ca 2+ export proteins including Na + /Ca 2+ exchanger (NCX), the ‘‘work horse’’ mediating vectorial transfer and the plasma membrane Ca 2+ ATPase (PMCA), which ‘‘fine tunes’’ cytosolic Ca 2+ level. Both proteins have been characterized physiologically (Wheatly et al., 1999, 2002) and from the molecular perspective (Stiner et al., 2002; Gao and Wheatly, 2004). While the molting cycle serves as a useful model for eliciting significant changes in vectorial Ca 2+ transfer at epithelial tissues, understanding the regulation of expression of the genes that code for the target proteins would benefit from an experimental variable that is more amenable for experimental purposes. Molting is an exceptional event in the life of the organism (a typical crayfish undergoes about 10 molts during its life), is difficult to precipitate and is under complex regulatory control. Our team has recently been exploring the effect of environ- mental (abiotic) stressors on Ca 2+ -associated proteins (Wheatly et al., 2009). For poikilotherms such as crustaceans, an environmen- tally relevant stressor is temperature change which has been reported to affect multiple physiological systems (Hand, 1993). At the cellular level, temperature affects protein degradation and metabolism as well as membrane biophysics (Cossins, 1983; Portner, 2002). Studies on fish have shown that exposure to low temperature affects expression and kinetics of transmembrane Ca-motive proteins (Shiels et al., 2000; Nelson et al., 2003). Cold exposure has environmental relevance to the temperate crayfish (P. clarkii) whose range extends from freezing to 35 1C(Huner and Barr, 1991). In the present study, we investigated the effect of transferring to ARTICLE IN PRESS Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/jtherbio Journal of Thermal Biology 0306-4565/$ - see front matter & 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2009.01.002 Ã Corresponding author. Tel.: +1937 775 2611; fax: +1937 775 3068. E-mail address: michele.wheatly@wright.edu (M.G. Wheatly). Journal of Thermal Biology 34 (2009) 144–151