Cyclosporin A disrupts bradykinin signaling through superoxide.
Cyclosporin A (CsA) is used to reduce transplant rejection rates. Chronic use, however, has a destructive toxic effect on the kidney, resulting in hypertension. In this study, we investigated the effects of CsA treatment on the bradykinin/soluble guanylate cyclase signaling cascade and the involvement of superoxide in LLC-PK1 porcine kidney proximal tubule cells. Treatment with 1 micromol/L CsA for 24 hours increased basal cGMP levels by 41%, whereas CsA inhibited bradykinin-stimulated cGMP production by 26%. Western blotting showed increased expression of eNOS, but no other protein in the bradykinin/soluble guanylate cyclase (sGC) pathway was affected. Using lucigenin-dependent chemiluminescence, we found that CsA treatment significantly increased superoxide production. Production of O2- was not significantly reduced by 10 micromol/L oxypurinol or 30 micromol/L ketoconazole. However, it was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium chloride (10 micromol/L) as well as the O2- scavenger superoxide dismutase (SOD) (100 U). On treatment with 50 micromol/L quercetin, 10 mmol/L N-acetyl-cysteine, both antioxidants, as well as the O2- scavenger Tiron (10 mmol/L), concomitant with 1 micromol/L CsA for 24 hours the activation of cGMP production, was restored in combination with a reduction in O2-. Incubation with 100 micromol/L menadione, a reactive oxygen generator, and 10 nmol/L bradykinin showed similar effects on the level of cGMP as with CsA. CsA treatment was found to increase nitrotyrosine levels. These findings suggest that CsA activates a NADPH oxidase that releases O2- and disrupts the bradykinin/soluble guanylate cyclase pathway, probably by binding with NO to form peroxynitrite (ONOO-).
Vetter
Michael
M
Department of Medicine, Division of Hypertension, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Chen
Zi-Jiang
ZJ
Chang
Geen-Dong
GD
Che
Danian
D
Liu
Shiguo
S
Chang
Chung-Ho
CH
D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt / Q000494:pharmacology; D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D015153:Blotting, Western; D001920:Bradykinin / Q000494:pharmacology*; D006152:Cyclic GMP / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D016166:Free Radical Scavengers / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D006162:Guanylate Cyclase / Q000378:metabolism; D018374:LLC-PK1 Cells / Q000187:drug effects / Q000378:metabolism; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D009861:Onium Compounds / Q000494:pharmacology; D010740:Phospholipases / Q000378:metabolism; D011794:Quercetin / Q000494:pharmacology; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000378:metabolism; D015398:Signal Transduction; D012995:Solubility; D013481:Superoxides / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013552:Swine; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism; D024483:Vitamin K 3 / Q000494:pharmacology
D000975:Antioxidants; D016166:Free Radical Scavengers; D009861:Onium Compounds; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D013481:Superoxides; C002744:3-nitrotyrosine; D014443:Tyrosine; D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; C007517:diphenyleneiodonium; D024483:Vitamin K 3; D016572:Cyclosporine; D011794:Quercetin; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019255:NADPH Oxidases; D010740:Phospholipases; D019204:GTP-Binding Proteins; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D001920:Bradykinin; D000111:Acetylcysteine