The functional expression of calcium-sensing receptors in BRL cells and related signal transduction pathway responsible for intracellular calcium elevation

The calcium-sensing receptors (CaSRs) exist in a variety of tissues and cells. In 2001, Canaff et al. first identified its expression in liver tissue and primary cultured hepatocytes, and demonstrated that GdCl(3) (a specific agonist of CaSR) can cause an increase in intracellular calcium and bile flow. However, authors did not elucidate its mechanisms. Therefore, this study sought to detect CaSR expression in BRL cell line, which is derived from buffalo rat liver, and to reveal the cellular signal transduction pathway by which the CaSR activation results in increased intracellular calcium by BRL cells. In this study, the expression and distribution of CaSR were detected by RT-PCR, Western blotting, and immunofluorescence, and the intracellular calcium concentration [Ca(2+)](i) was measured using LCSM. The results showed that CaSR mRNA and protein were expressed in BRL cells and mainly distributed in cell membrane and cytoplasm. Increased extracellular calcium or GdCl(3) could increase intracellular calcium concentration and CaSR expression. Moreover, this increase of [Ca(2+)](i) could be inhibited or even abolished by U73122 (a specific inhibitor of PLC), 2-APB (an inhibitor of IP(3) receptor), and thapsigargin (an inhibitor of endoplasmic reticulum calcium pump). In conclusion, CaSR is functionally expressed in BRL cells, and activation of CaSR involves in increased intracellular calcium through Gq-PLC-IP(3) pathway.