Calcium-sensing receptor-mediated TNF production in medullary thick ascending limb cells

Abstract

Medullary thick ascending limb (mTAL) cells in primary culture express the Ca 2+ -sensing receptor (CaR), a G protein-coupled receptor that senses changes in extracellular Ca 2+ (Ca[Formula: see text]) concentration, resulting in increases of intracellular Ca 2+ concentration and PKC activity. Exposure of mTAL cells to either Ca[Formula: see text] or the CaR-selective agonist poly-l-arginine increased TNF-α synthesis. Moreover, the response to Ca[Formula: see text] was enhanced in mTAL cells transfected with a CaR overexpression vector. Transfection of mTAL cells with a TNF promoter construct revealed an increase in reporter gene activity after exposure of the cells to Ca[Formula: see text], suggesting that intracellular signaling pathways initiated by means of activation of a CaR contribute to TNF synthesis by a mechanism that involves transcription of the TNF gene. Neutralization of TNF activity with an anti-TNF antibody attenuated Ca 2+ -mediated increases in cyclooxygenase-2 (COX-2) protein expression and PGE 2 synthesis, suggesting that TNF exerts an autocrine effect in the mTAL, which contributes to COX-2-mediated PGE 2 production. Preincubation with the PKC inhibitor bisindolylmaleimide I inhibited Ca 2+ -mediated TNF production. Significant inhibition of COX-2 protein expression and PGE 2 synthesis also was observed when cells were challenged with Ca[Formula: see text] in the presence of bisindolylmaleimide I. The data suggest that increases in TNF production subsequent to activation of the CaR may be the basis of an important renal mechanism that regulates salt and water excretion.