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 Gi-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 Gi 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 Gi and PKC was determined, as we previously showed that CaR-mediated TNF production was CN- and NFAT-mediated and Gq-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 O2 consumption, suggesting that a Gi-coupled mechanism is important in regulating mTAL transport function. Collectively, the data suggest that Gi-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.