title: Tumor necrosis factor α decreases nitric oxide synthase type 3 expression primarily via Rho/Rho kinase in the thick ascending limb.
abstract: Inappropriate Na(+) reabsorption by thick ascending limbs (THALs) induces hypertension. NO produced by NO synthase type 3 (NOS3) inhibits NaCl reabsorption by THALs. Tumor necrosis factor α (TNF-α) decreases NOS3 expression in endothelial cells and contributes to increases in blood pressure. However, the effects of TNF-α on THAL NOS3 and the signaling cascade are unknown. TNF-α activates several signaling pathways, including Rho/Rho kinase (ROCK), which is known to reduce NOS3 expression in endothelial cells. Therefore, we hypothesized that TNF-α decreases NOS3 expression via Rho/ROCK in rat THAL primary cultures. THAL cells were incubated with either vehicle or 1 nmol/L of TNF-α for 24 hours, and NOS3 expression was measured by Western blot. TNF-α decreased NOS3 expression by 51 ± 6% (P<0.002) and blunted stimulus-induced NO production. A 10-minute treatment with TNF-α stimulated RhoA activity by 60 ± 23% (P<0.04). Inhibition of Rho GTPase with 0.05 μg/mL of C3 exoenzyme blocked TNF-α-induced reductions in NOS3 expression by 30 ± 8% (P<0.02). Inhibition of ROCK with 10 μmol/L of H-1152 blocked TNF-α-induced decreases in NOS3 expression by 66 ± 15% (P<0.001). Simultaneous inhibition of Rho and ROCK had no additive effect. Myosin light chain kinase, NO, protein kinase C, mitogen-activated kinase kinase, c-Jun amino terminal kinases, and Rac-1 were also not involved in TNF-α-induced decreases in NOS3 expression. We conclude that TNF-α decreases NOS3 expression primarily via Rho/ROCK in rat THALs. These data suggest that some of the beneficial effects of ROCK inhibitors in hypertension could be attributed to the mitigation of TNF-α-induced reduction in NOS3 expression.
authors.affiliation: Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Detroit, MI 48202-2689, USA.
authors.forename: Vanesa D
authors.initials: VD
authors.lastname: Ramseyer
authors.forename: Nancy J
authors.initials: NJ
authors.lastname: Hong
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Garvin
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D008138:Loop of Henle / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008856:Microscopy, Fluorescence; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D061251:Primary Cell Culture; D051381:Rats; D017207:Rats, Sprague-Dawley; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: C459092:2-methyl-1-((4-methyl-5-isoquinolinyl)sulfonyl)homopiperazine; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein