Calcium sensing receptor (CaR)‐mediated TNF production in the medullary thick ascending limb (mTAL) is G i ‐coupled

We tested the contribution of CaR‐mediated G i ‐coupled signaling to TNF production in mTAL cells. CaR activation induced a 3‐fold increase in TNF production at 6 hr that was abolished by a selective G i inhibitor, pertussis toxin (PTX; 100 ng/ml; n=3; p<0.001). CaR increased calcineurin (CN) activity by approximately 3‐fold (n=4; p<0.01). PTX prevented CaR‐mediated increases in CN activity (n=4; p<0.01), an NFAT cis‐reporter construct (n=5; p<0.001), and a TNF promoter construct (n=3; p<0.01). The interaction between G i and PKC was determined, as we previously showed that CaR‐mediated TNF production was CN‐ and NFAT‐mediated and G q ‐dependent. CaR activation increased PKC activity by 2‐fold, an effect abolished by transient transfection with a dominant negative CaR construct, R796W (n=4; p<0.001) or pretreatment with PTX (n=4; p<0.001). Inhibition with the pan specific PKC inhibitor, GF 109203X (20 nM) abolished CaR‐mediated increases in activity of CN, an NFAT reporter (n=3; p<0.001), and a TNF promoter construct (n=3; p<0.05). PTX reversed CaR‐mediated decreases in ouabain‐sensitive O 2 consumption, suggesting that a G i ‐coupled mechanism is important in regulating mTAL transport function. Collectively, the data suggest that G i ‐coupled signaling contributes to NFAT‐mediated TNF production, in a CN‐ and PKC‐dependent manner, and is part of a CaR mechanism to regulate mTAL function.