Environmental and cortisol-mediated control of Ca(2+) uptake in tilapia (Oreochromis mossambicus)

Abstract

Ca(2+) is a vital element for many physiological processes in vertebrates, including teleosts, which live in aquatic environments and acquire Ca(2+) from their surroundings. Ionocytes within the adult gills or larval skin are critical sites for transcellular Ca(2+) uptake in teleosts. The ionocytes of zebrafish were found to contain transcellular Ca(2+) transporters, epithelial Ca(2+) channel (ECaC), plasma membrane Ca(2+)-ATPase 2 (PMCA2), and Na(+)/Ca(2+) exchanger 1b (NCX1b), providing information about the molecular mechanism of transcellular Ca(2+) transports mediated by ionocytes in fish. However, more evidence is required to establish whether or not a similar mechanism of transcellular Ca(2+) transport also exists in others teleosts. In the present study, ecac, pmca2, and ncx1 were found to be expressed in the branchial ionocytes of tilapia, thereby providing further support for the mechanism of transcellular Ca(2+) transport through ionocytes previously proposed for zebrafish. In addition, we also reveal that low Ca(2+) water treatment of tilapia stimulates Ca(2+) uptake and expression of ecac and cyp11b (the latter encodes a cortisol-synthesis enzyme). Treatment of tilapia with exogenous cortisol (20 mg/l) enhanced both Ca(2+) influx and ecac expression. Therefore, increased cyp11b expression is suggested to enhance Ca(2+) uptake capacity in tilapia exposed to low Ca(2+) water. Furthermore, the application of cortisol receptor antagonists revealed that cortisol may regulate Ca(2+) uptake through glucocorticoid and/or mineralocorticoid receptor (GR and/or MR) in tilapia. Taken together, the data suggest that cortisol may activate GR and/or MR to execute its hypercalcemic action by stimulating ecac expression in tilapia.

Keywords: Ca2+ influx; Cortisol; ECaC; Ionocyte; Tilapia.

Fig. 1

Expression of ecac, pmca2, and ncx1 in various tissues of tilapia, as detected by RT-PCR. The gadph gene was used as internal control to evaluate the relative amounts of cDNAs. I intestine; L liver; B brain; K kidney; S spleen; T testis; H heart; HCa high-Ca²⁺ acclimated tilapia; LCa low-Ca²⁺ acclimated tilapia

Fig. 2

In situ hybridization against ecac, pmca2, and ncx1 in tilapia gills. a–c Expression of ecac, pmca2, and ncx1; d-f, immunohistochemical staining of ionocytes using Na,K-ATPase α5 antibody; g-i, co-localization of ionocytes with ecac, pmca2,or ncx1b signals. Arrows indicate cells with positive signals. Scale bar 20 μm

Fig. 3

Effects of environmental Ca²⁺ on Ca²⁺ influx and mRNA expression of branchial transporters in tilapia. a Ca²⁺ influx. b Expression of mRNA, as analyzed by qPCR; values were normalized to β-actin. Values are the mean ± SD (n = 5–8). * p < 0.05; ***p < 0.001. Student’s t test

Fig. 4

Effects of environmental Ca²⁺ on Ca²⁺ influx and mRNA expression of transporters in tilapia larvae. a Ca²⁺ influx; b expression of mRNA, as analyzed by qPCR; values were normalized to β-actin. Values are the mean ± SD (n = 6). * p < 0.05; ***p < 0.001. Student’s t test

Fig. 5

Effects of exogenous cortisol on Ca²⁺ influx and mRNA expression of transporters in tilapia larvae. Expression of mRNA was analyzed by qPCR, and values were normalized to β-actin. ^abIndicate a significant difference (p < 0.05) using Tukey’s multiple comparison test following one-way ANOVA. Values are the mean ± SD (n = 6)

Fig. 6

Effects of exogenous cortisol on ecac expression in tilapia larvae exposed to high Ca²⁺. Expression of mRNA was analyzed by qPCR, and values were normalized to β-actin. ^abIndicate a significant difference (p < 0.05) using Tukey’s multiple comparison test following one-way ANOVA. Values are the mean ± SD (n = 6)

Fig. 7

Effects of GR and MR antagonists on ecac expression in tilapia larvae treated with cortisol. Expression ofmRNA was analyzed by qPCR, and values were normalized to β-actin. ^abcIndicate a significant difference (p < 0.05) using Tukey’s multiple comparison test following one-way ANOVA. Values are the mean ± SD (n = 6)