Calcium-sensing receptor signaling pathways in medullary thick ascending limb cells mediate COX-2-derived PGE2 production: functional significance.
We determined the functional implications of calcium-sensing receptor (CaR)-dependent, Gq- and Gi-coupled signaling cascades, which work in a coordinated manner to regulate activity of nuclear factor of activated T cells and tumor necrosis factor (TNF)-alpha gene transcription that cause expression of cyclooxygenase (COX)-2-derived prostaglandin E2 (PGE2) synthesis by rat medullary thick ascending limb cells (mTAL). Interruption of Gq, Gi, 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 h 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 microM), 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 Gq, Gi, 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.
Abdullah
Huda Ismail
HI
Department of Pharmacology, New York Medical College, Valhalla, NY 10595, USA.
Pedraza
Paulina L
PL
McGiff
John C
JC
Ferreri
Nicholas R
NR
D000818:Animals; D019703:Calcineurin / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000378:metabolism*; D015232:Dinoprostone / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000037:antagonists & inhibitors / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D008138:Loop of Henle / Q000166:cytology / Q000201:enzymology*; D008297:Male; D008301:Maleimides / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D010101:Oxygen Consumption / Q000187:drug effects / Q000502:physiology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D044169:Receptors, Calcium-Sensing / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D004791:Enzyme Inhibitors; D004963:Estrenes; D007211:Indoles; D008301:Maleimides; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D044169:Receptors, Calcium-Sensing; D014409:Tumor Necrosis Factor-alpha; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D010042:Ouabain; D051546:Cyclooxygenase 2; C489691:Ptgs2 protein, rat; D011493:Protein Kinase C; D019703:Calcineurin; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D015232:Dinoprostone; C070515:bisindolylmaleimide I