a-192621 and acetovanillone

It is unknown if endothelin-A and -B receptors (ET(A)R and ET(B)R) activate the production of superoxide via NAD(P)H oxidase and subsequently stimulate the formation of cyclic adenine diphosphate ribose (cADPR) in afferent arterioles. Vessels were isolated from rat kidney and loaded with fura 2. Endothelin-1 (ET-1) rapidly increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) by 303 nM. The superoxide dismutase mimetic tempol, the NAD(P)H oxidase inhibitor apocynin, and nicotinamide, an inhibitor of ADPR cyclase, diminished the response by approximately 60%. The ET(B)R agonist sarafotoxin 6c (S6c) increased peak [Ca(2+)](i) by 117 nM. Subsequent addition of ET-1 in the continued presence of S6c caused an additional [Ca(2+)](i) peak of 225 nM. Neither nicotinamide or 8-bromo- (8-Br) cADPR nor apocynin decreased the [Ca(2+)](i) response to S6c, but inhibited the subsequent [Ca(2+)](i) response to ET-1. The ET(B)R blockers BQ-788 and A-192621 prevented the S6c [Ca(2+)](i) peak and reduced the ET-1 response by more than one-half, suggesting an ET(B)R/ET(A)R interaction. In contrast, the ET(A)R blocker BQ-123 had no effect on the S6c [Ca(2+)](i) peak and obliterated the subsequent ET-1 response. ET-1 immediately stimulated superoxide formation (measured with TEMPO-9-AC, 68 arbitrary units) that was inhibited 95% by apocynin or diphenyl iodonium. S6c or IRL-1620 increased superoxide by 8% of that caused by subsequent ET-1 addition. We conclude that ET(A)R activation of afferent arterioles increases the formation of superoxide that accounts for approximately 60% of subsequent Ca(2+) signaling. ET(B)R activation appears to result in only minor increases in superoxide production. Nicotinamide and 8-Br-cADPR results suggest that ET-1 (and primarily ET(A)R) causes the activation of vascular smooth muscle cell-ADPR cyclase.

Topics: Acetophenones; ADP-ribosyl Cyclase; Animals; Arterioles; Biphenyl Compounds; Bradykinin; Calcium Signaling; Cyclic N-Oxides; Endothelial Cells; Endothelin B Receptor Antagonists; Endothelins; Enzyme Inhibitors; Kidney; Muscle, Smooth, Vascular; NADPH Oxidases; NG-Nitroarginine Methyl Ester; Niacinamide; Oligopeptides; Onium Compounds; Peptide Fragments; Piperidines; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Receptor, Endothelin B; Renal Circulation; Spin Labels; Superoxides; Vasoconstrictor Agents; Viper Venoms

Experiments were designed to test the hypothesis that elevated levels of endothelin 1 (ET-1) in the vasculature activate NADPH oxidase and/or uncoupled nitric-oxide synthase (NOS), resulting in O2-* production, and mediate increased constriction. Rat aortic rings were incubated with ET-1 or vehicle in the presence and absence of superoxide dismutase (SOD), ebselen (glutathione peroxidase mimetic), apocynin (NADPH oxidase inhibitor), L-NAME (Nomega-nitro-L-arginine methyl ester) (NOS inhibitor), tetrahydrobiopterin (BH4) (NOS cofactor), or selective ETA and ETB receptor antagonists (BQ-123 [cyclo(D-Asp-Pro-D-Val-Leu-D-Trp)] and A-192621 [[2R-(4-propoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N-(2,6-diethylphenyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid]], respectively). O2-* production was monitored by oxidized dihydroethidine staining and/or lucigenin chemiluminescence. ET-1 significantly increased O2-* production compared with vehicle. SOD, ebselen, and apocynin inhibited the ET-1-induced increase in O2-* in intact and endothelium-denuded aorta. L-NAME and BH4 inhibited the ET-1-induced increase in O2-* in intact tissue, whereas these two compounds had no effect on ET-1-induced O2-* in endothelium-denuded aorta. Preincubation with BQ-123 or A-192621, individually, had no effect on ET-1-induced O2-*; however combining both antagonists inhibited the ET-1-stimulated increase in O2-*. Rat aortic rings were incubated with ET-1 or vehicle in the presence or absence of sepiapterin (BH4 synthesis substrate) or apocynin and mounted on wire myographs to determine isometric force generation in response to increasing KCl concentrations. ET-1 increased the contractile response to KCl compared with vehicle. Treatment with either sepiapterin or apocynin attenuated the ET-1-mediated increase with no effect of sepiapterin or apocynin alone. These data support the hypothesis that ET-1 increases vascular tone, in part, through ETA/ETB receptor activation of O2-* production from NADPH oxidase and NOS uncoupling.

Topics: Acetophenones; Animals; Aorta; Azoles; Biopterins; Endothelin-1; Endothelium, Vascular; Enzyme Inhibitors; Isoindoles; Luminescence; Luminescent Measurements; Male; NADPH Oxidases; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Organoselenium Compounds; Pyrrolidines; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Superoxides; Vasoconstriction