Activation of gill Ca2+-sensing receptor as a protective pathway to reduce Ca2+-induced cytotoxicity

The expression of the Ca(2) (+)-sensing receptor (Casr) in the endocrine gland known as the corpuscle of Stannius (CS) regulates the secretion of the hypocalcemic hormone stanniocalcin-1 (STC1) to inhibit gill Ca(2) (+) uptake. Although numerous studies have reported the branchial expression of Casr and Stc1, the functions of these proteins in gills have not been elucidated yet. On the basis of recent findings regarding the autocrine/paracrine functions of STC1 in mammalian models, we proposed the hypothesis that branchial CaSR has an in situ 'sensing' function to regulate STC1 that maintains local Ca(2) (+) homeostasis. In this study, we investigated Casr-mediated signaling and its regulation of Stc1 and cyclooxygenase-2 (Cox2) expression/function using a primary gill-cell culture model. The biochemical responses of gill cells isolated from Japanese eels to an increasing concentration of extracellular Ca(2) (+) (0.1-1 mM) were tested. This stimulation led to a transient increase in phosphatidylcholine-phospholipase C (PC-PLC) activity, followed by activation of ERK and inositol 1,4,5-trisphosphate-Ca(2) (+)/calmodulin-dependent protein kinase 2 (CaMK2) signaling pathways. Cotreatment with the calcimimetic R467 caused synergistic effects on Ca(2) (+)-stimulated PC-PLC activity, ERK signaling, and CaMK2 signaling. The activation of the CaSR-PLC-ERK pathway was associated with increased expression levels of Stc1 and Cox2 as confirmed by the inhibition of Erk using a chemical inhibitor, PD98059. Functionally, Ca(2) (+)/R-467 pretreatment was found to protect cells from thapsigargin-induced cell death. Inhibition of COX2 activity using NS398 abolished this protection, while transduction of STC1 lentiviral particles in the gill cells increased the protective effects. Collectively, our data revealed the expression of functional CaSR in gill tissues. The identification of the CaSR-STC1/COX2-mediated protective pathway in gill cells sheds light on a possible cellular protective mechanism against an increase in intracellular Ca(2) (+) levels associated with transepithelial Ca(2) (+) transport.