Calcium-Mediated Cox-2 Expression and Pge2 Production in the Rat Medullary Thick Ascending Limb(mTAL) Is Tnfα-Dependent
Severe hypercalcemia often is associated with polyuria in patients. We have shown that mTAL cells express cyclooxygenase-2(COX-2) when challenged with tumor necrosis factor-alpha(TNFα), an effect that was linked to the decrease in TNFα-mediated 86Rb+ uptake, an in vitro correlate of natriuresis. The mTAL expresses calcium sensing receptor(CaR), a G-protein coupled receptor that senses small changes in extracellular calcium concentration([Ca2+]o) and ultimately increases intracellular calcium concentration([Ca2+]i) and protein kinase C(PKC) activity. We assessed the effects of [Ca2+]oon COX-2 and TNFα protein expression as calcium increases TNFα synthesis in certain cell types, and PMA, a PKC activator, induces COX-2 gene expression in mTAL cells. Western blot analysis showed that COX-2 protein expression was increased two-fold after challenge for 9 hours with 1.7 mM CaCl2. ELISA analysis revealed that PGE2 formation was increased from 11 ± 1.5 to 68 ± 21 pg/μg protein (p<0.05) by 1.7 mM CaCl2; no effect was observed when cells were exposed to 3.4 mM NaCl suggesting that the increase in PGE2 synthesis was a function of increasing [Ca2+]o. TNFα synthesis increased from 1.3 ± 0.1 to 5.4 ± 0.4 pg/μg protein (p<0.05) after challenge with 2 mM CaCl2. CaR agonist poly-L-arginine (100nM) increased PGE2 production from 15 ± 0.1 to 66.3 ± 4.4 pg/μg protein (p<0.0001), and increased TNFαproduction from 0.7 ± 0.1 to 3.7 ± 0.3 pg/μg protein after a 9 hr incubation period. Pretreatment of cells with 1 μg/ml neutralizing TNFα antibody reduced COX-2 protein expression and PGE2 production by approximately 40%. These data indicate that Ca2+o, as well as CaR selective agonists, increase mTAL COX-2 expression, PGE2formation, and TNFα production in primary cultures of mTAL cells. We conclude that the increases in COX-2 expression and PGE2 production are, in part, TNFα-dependent, suggesting that Ca2+o may be a modulator of extracellular fluid volume regulation via a cytokine-mediated autocrine mechanism.