title: CaR activation increases TNF production by mTAL cells via a Gi-dependent mechanism.
abstract: We evaluated the contribution of calcium-sensing receptor (CaR)-mediated G(i)-coupled signaling to TNF production in medullary thick ascending limb (mTAL) cells. A selective G(i) inhibitor, pertussis toxin (PTX), but not the inactive B-oligomer binding subunit, abolished CaR-mediated increases in TNF production. The inhibitory effect of PTX was partially reversed by using an adenylate cyclase inhibitor. CaR-mediated TNF production also was partially reversed by a cAMP analog, 8-Br-cAMP. IP(1) accumulation was CaR dependent and blocked by PI-PLC; partial inhibition also was observed with PTX. CaR increased calcineurin (CaN) activity by approximately threefold, and PTX prevented CaR-mediated increases in CaN activity, an nuclear factor of activated T cells (NFAT)-cis reporter construct, and a TNF promoter construct. The interaction between G(i) and PKC was determined, as we previously showed that CaR-mediated TNF production was CaN and NFAT- mediated and G(q) dependent. CaR activation increased PKC activity by twofold, an effect abolished by transient transfection with a dominant negative CaR construct, R796W, or pretreatment with PTX. Inhibition with the pan-specific PKC inhibitor GF 109203X (20 nM) abolished CaR-mediated increases in activity of CaN, an NFAT reporter, and a TNF promoter construct. Collectively, the data suggest that G(i)-coupled signaling contributes to NFAT-mediated TNF production in a CaN- and PKC-dependent manner and may be part of a CaR mechanism to regulate mTAL function. Moreover, concurrent G(q) and G(i) signaling is required for CaR-mediated TNF production in mTAL cells via a CaN/NFAT pathway that is PKC dependent. Understanding CaR-mediated signaling pathways that regulate TNF production in the mTAL is crucial to defining novel mechanisms that regulate extracellular fluid volume and salt balance.
authors.affiliation: Department of Pharmacology, New York Medical College, Valhalla, NY 10595, USA.
authors.forename: Huda Ismail
authors.initials: HI
authors.lastname: Abdullah
authors.forename: Paulina L
authors.initials: PL
authors.lastname: Pedraza
authors.forename: John C
authors.initials: JC
authors.lastname: McGiff
authors.forename: Nicholas R
authors.initials: NR
authors.lastname: Ferreri
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D019703:Calcineurin / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000037:antagonists & inhibitors / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D007097:Imines / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D007679:Kidney Medulla / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008301:Maleimides / Q000494:pharmacology; D008954:Models, Biological; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism; D037342:Pertussis Toxin / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D044169:Receptors, Calcium-Sensing / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D004791:Enzyme Inhibitors; D004963:Estrenes; D007097:Imines; D007211:Indoles; D007295:Inositol Phosphates; D008301:Maleimides; D050778:NFATC Transcription Factors; 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; C002647:inositol 1-phosphate; D015124:8-Bromo Cyclic Adenosine Monophosphate; C014925:RMI 12330A; D037342:Pertussis Toxin; D011493:Protein Kinase C; D019703:Calcineurin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; C070515:bisindolylmaleimide I; D002118:Calcium