Background Freshwater fish absorb Ca2+ predominantly from ambient water, and more than 97% of Ca2+ uptake is achieved by active transport through gill mitochondrion-rich (MR) cells. gene because of the plentiful genomic databases and expression sequence tag (EST) data. Results Using a strategy of BLAST from your zebrafish genome database (Sanger Institute), 6 isoforms of PMCAs (PMCA1a, PMCA1b, PMCA2, PMCA3a, PMCA3b, and PMCA4) and 7 isoforms of NCXs (NCX1a, NCX1b, NCX2a, NCX2b, NCX3, NCX4a, and NCX4b) were recognized. In the reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, 5 PMCAs and 2 NCXs were ubiquitously expressed in various tissues including gills. Triple fluorescence in situ hybridization and immunocytochemistry showed the colocalization of zecac, zpmca2, and zncx1b mRNAs in a portion of gill MR cells (using Na+-K+-ATPase as the marker), implying a subset of ionocytes specifically responsible for the transepithelial Ca2+ uptake in zebrafish gills. The gene expressions in gills of high- or low-Ca2+-acclimated zebrafish by quantitative real-time PCR analysis showed that zecac was the only gene regulated in response to environmental Ca2+ levels, while zpmcas and zncxs remained steady. Conclusion The present study provides molecular evidence for the specific isoforms of Ca2+ transporters, zECaC, zPMCA2, and zNCX1b, supporting the current Ca2+ uptake model, in which ECaC may play a role as the major regulatory target for this mechanism during environmental challenge. Background Ca2+ buy 65604-80-0 is an essential element for almost buy 65604-80-0 all organisms and plays comprehensive regulatory functions in eukaryotic cells; therefore, Ca2+ must be managed within a thin concentration range in organisms (cellular free [Ca2+]: 100 nM; plasma total [Ca2+]: 2~3 mM). In terrestrial vertebrates, the whole-body Ca2+ balance is mainly achieved by intestinal absorption and kidney reabsorption. Ca2+ uptake mechanisms in the mammalian kidney have been most extensively analyzed, while fish gills, a specialized organ in non-mammalian vertebrates, is usually another model for studying Ca2+ uptake mechanisms [1]. In fish, gills are the main site (> 97% buy 65604-80-0 of the whole body) of Ca2+ uptake from your aquatic environment to maintain the Ca2+ balance [2], and skin takes the place of gills during early developmental GXPLA2 stages when the gills are not yet developed and functioning [3-5]. Compared with terrestrial vertebrates, Ca2+ regulation mechanisms in aquatic animals are probably more complicated and challenging because of the dramatic fluctuations in ambient Ca2+ concentrations which can occur (seawater, around 10 mM; new water, 0.01~3 mM). According to the current model in mammals, active and transcellular Ca2+ transport is usually carried out as a 3-step process [1]. Following access of Ca2+ through apical epithelial Ca2+ channels (ECaC, TRPV5, and/or TRPV6), Ca2+ is bound to calbindins that facilitate diffusion to the basolateral membrane, and then it is extruded via the plasma membrane Ca2+-ATPase (PMCA) and/or buy 65604-80-0 Na+/Ca2+ exchanger (NCX). In this way, net transepithelial Ca2+ absorption from your luminal compartment (or environment) to the plasma is usually accomplished. A model of the capability of physiological regulation has also been similarly proposed in fish gills [6]. However, only very little molecular evidence is currently available to support this model in fish gills, which is a specialized organ for Ca2+ uptake. The ecac gene has recently been cloned and sequenced in fugu, zebrafish, and trout [4,7,8]. In zebrafish embryos, low-Ca2+ new water causes upregulation of the whole-body Ca2+ influx and zECaC expression in mitochondrion-rich (MR) cells of both gills and skin, providing molecular evidence for the role of ECaC in fish Ca2+ uptake [4]. On the other hand, some biochemical and physiological studies have investigated the possibility of PMCA and NCX’s involvement in fish gill Ca2+ uptake [9,10]. Nevertheless, no convincing evidence has been offered demonstrating the presence and involvement of PMCA and NCX in fish Ca2+ uptake mechanisms. Three NCX genes (SLC8A1, SLC8A2, and SLC8A3) and 4 PMCA genes (ATP2B1, ATP2B2, ATP2B3, and ATP2B4) have been recognized in mammals so far. The fundamental.