CaR activation increases TNF production by mTAL cells via a Gi-dependent mechanism.
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.
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
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*
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