Calcium-Mediated Cox-2 Expression and Pge 2 Production in the Rat Medullary Thick Ascending Limb(mTAL) Is Tnf α -Dependent

Abstract

P89 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(TNF α ), an effect that was linked to the decrease in TNF α -mediated 86 Rb + 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([Ca 2+ ] o ) and ultimately increases intracellular calcium concentration([Ca 2+ ] i ) and protein kinase C(PKC) activity. We assessed the effects of [Ca 2+ ] o on COX-2 and TNF α protein expression as calcium increases TNF α 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 CaCl 2 . ELISA analysis revealed that PGE 2 formation was increased from 11 ± 1.5 to 68 ± 21 pg/μg protein (p<0.05) by 1.7 mM CaCl 2 ; no effect was observed when cells were exposed to 3.4 mM NaCl suggesting that the increase in PGE 2 synthesis was a function of increasing [Ca 2+ ] o . TNF α synthesis increased from 1.3 ± 0.1 to 5.4 ± 0.4 pg/μg protein (p<0.05) after challenge with 2 mM CaCl 2 . CaR agonist poly-L-arginine (100nM) increased PGE 2 production from 15 ± 0.1 to 66.3 ± 4.4 pg/μg protein (p<0.0001), and increased TNF α production 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 TNF α antibody reduced COX-2 protein expression and PGE 2 production by approximately 40%. These data indicate that Ca 2+ o , as well as CaR selective agonists, increase mTAL COX-2 expression, PGE 2 formation, and TNF α production in primary cultures of mTAL cells. We conclude that the increases in COX-2 expression and PGE 2 production are, in part, TNF α -dependent, suggesting that Ca 2+ o may be a modulator of extracellular fluid volume regulation via a cytokine-mediated autocrine mechanism.