Calcium-sensing receptor expression and regulation by extracellular calcium in the AtT-20 pituitary cell line

A 120 kDa, G protein-coupled calcium-sensing receptor (CaR) was recently identified and cloned from bovine parathyroid and rat kidney. We report here that a similar calcium-sensing receptor is also present in rat and mouse pituitary as well as in the mouse pituitary cell line, AtT-20. Fragments (383-bp) of the extracellular domain of the calcium-sensing receptor from the AtT-20 cells and mouse pituitary were amplified by RT-PCR, sequenced, and found to be identical. By Northern blot analysis, AtT-20 cells expressed a major CaR mRNA transcript of 7.5 kb and three minor transcripts of 9.5, 4.0, and 1.5 kb. Except for the 9.5-kb species, these CaR transcripts were also found to be present in mouse kidney, where the 7.5-kb transcript was again the predominant form. The presence of the CaR protein in AtT-20 cells was documented directly by fluorescence immunocytochemistry using an antibody directed against the extracellular domain of the CaR. Exposure of AtT-20 cells to increasing extracellular calcium concentrations from 0.3 to 3 mM for 24 h resulted in a 2- to 4-fold increase in the levels of CaR mRNA, but not of the RNAs for beta-actin or POMC. The CaR appeared to be functional in AtT-20 cells, since acute increases in extracellular calcium between 2 and 5 mM induced increases in the cellular content of total inositol phosphates, cytosolic calcium, and cAMP. This report suggests that pituitary cells respond to changes in extracellular calcium via a G protein-coupled CaR.