We determined the functional implications of calcium sensing receptor (CaR)-dependent, G_q- and G_i-coupled signaling cascades, which work in a coordinated manner to regulate activity of nuclear factor of activated T cells (NFAT) and tumor necrosis factor-alpha (TNF) gene transcription that cause expression of COX-2 derived prostaglandin E2 (PGE₂) synthesis by rat medullary thick ascending limb cells (mTAL). Interruption of G_q, G_i, protein kinase C (PKC), or calcineurin (CaN) activities abolished CaR-mediated COX-2 expression and PGE₂ synthesis. We tested the hypothesis that these pathways contribute to the effects of CaR activation on ion transport in mTAL cells. Ouabain-sensitive O₂ consumption, an in vitro correlate of ion transport in the mTAL, was inhibited by approximately 70% in cells treated for 6 hr with extracellular Ca²⁺ (1.2 mM), an effect prevented in mTAL cells transiently transfected with a dominant negative CaR overexpression construct (R796W), indicating that the effect was initiated by stimulation of the CaR. Pretreatment with the COX-2 selective inhibitor, NS-398 (1 µM), reversed CaR-activated decreases in ouabain-sensitive O₂ consumption by approximately 60%, but did not alter basal levels of ouabain-sensitive O₂ consumption. Similarly, inhibition of either G_q, G_i, PKC, or CaN, which are components of the mechanism associated with CaR-stimulated COX-2-derived PGE₂ synthesis, reversed the inhibitory effects of CaR on O₂ consumption without affecting basal O₂ consumption. Our findings identified signaling elements required for CaR-mediated TNF production that are integral components regulating mTAL function via a mechanism involving COX-2 expression and PGE₂ production.