N~tnc oxide (NO) Inhibits a variety of heme-contammg enzymes, mcludmg NO synthase and cytochrome P4501Al and 2Bl The present study exammed whether NO mhlblts the production of 20-hydroxyelcosatetraenolc acid (20-HETE) by cytochrome P4504A enzymes and whether blockade of the production of this substance contributes to the vascular effects of NO Sodium mtroprusslde (SNP, lo-', lo-", and 10m3 mol/L) reduced the productlon of 20-HETE by renal mlcrosomes incubated with arachldomc acid to 71+5%, 29t4%, and 4+2% of control, respectlvely (n=S) Slmllar results were obtained with the use of 1-propanamme, 3-(2-hydroxy-2-mtroso-1-propylhydrazmo) (n=3) To determme whether mhlbitlon of 20-HETE contributes to the vasodllatory effects of NO, the effects of dlbromo-dodecenylmethylsulfinude (DDMS), a selective mhlbltor of the formatlon of 20-HETE, on the response to SNP (lo-' to 10m3 mol/L) were examined m rat renal artenoles preconstructed with phenylephrme (n=.5) SNP increased vascular diameter m a concentrafion-dependent manner to 82t4% of control After DDMS (25 ymol/L), SNP (lo-' mol/L) increased vascular diameter by only 17+3% The effects of DDMS on the mean arterial pressure (MAP) and renal blood flow (RBF) responses to mfuslon of an NO donor and a synthase mhibltor were also examined m thlobutabarbltal-anesthetrzed, Sprague-Dawley rats Infusion of MAHMA NONOate at 1, 3, 5, and 10 nmol/mm reduced MAP by 16+2, 3023,40t5, and 48?5 mm Hg and lowered renal vascular resistance (RVR) by 15+3%, 26?2%, 30?3%, and 34?4% of control After DDMS (10 mg/kg, n=7 rats), the MAP and RVR responses to I-hexamme, 6-(2-hydroxy-1 -methyl-2-mtrohydrazmo)N-methyl (MAHMA NONOate) averaged only 20% of those seen durmg control In other expenments, MAP Increased by 3224% and RBF fell to 56+5% of control after admmlstratlon of N-mtro-L-argmme (L-NArg) (10 mg/kg IV) After DDMS (10 mg/kg, n=7 rats), MAP Increased by only 19+4% and RBF fell by only 724% after L-NArg These results indicate that NO mhlblts cytochrome P4504A enzymes and that mhlbltlon of the production of 20-HETE contributes to the vasodllatory effects of NO (Hypertension. 1997;29[part 2]:320-325.)Key Words l mtnc oxide l vasculature l enzymes R ecent studies have mdlcated that the effects of many renal vasodllators are dependent on the release of NO from the endothehum Blockade of NO synthesis increases arterial pressure, decreases RBF, and potentiates tubuloglomerular feedback responses. 1 These results indicate that tonic release of NO plays an important modulatory role m the regulation of both renal and penpheral vascular tone. It 1s generally assumed that the vasodllatory effects of NO are mediated by cGMP secondary to shmulatlon of guanylyl cyclase 2.3 This concluslon 1s based on the observations that endothehum-dependent vasodilators and NO donors increase cGMP m vascular tissue and that methylene blue and other mhlbltors of guanylyl cyclase m many vessels can elm-nnate the vasodilatory response However, this generalized scheme for NO-induced vasodllatlon has been questioned ...

.-The present study examined the effects of ANG II on the renal synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) and its contribution to the renal vasoconstrictor and the acute and chronic pressor effects of ANG II in rats. ANG II (10 Ϫ11 to 10 Ϫ7 mol/l) reduced the diameter of renal interlobular arteries treated with inhibitors of nitric oxide synthase and cyclooxygenase, lipoxygenase, and epoxygenase by 81 Ϯ 8%. Subsequent blockade of the synthesis of 20-HETE with 17-octadecynoic acid (1 mol/l) increased the ED 50 for ANG II-induced constriction by a factor of 15 and diminished the maximal response by 61%. Graded intravenous infusion of ANG II (5-200 ng/min) dose dependently increased mean arterial pressure (MAP) in thiobutylbarbitol-anesthetized rats by 35 mmHg. Acute blockade of the formation of 20-HETE with dibromododecenyl methylsulfimide (DDMS; 10 mg/kg) attenuated the pressor response to ANG II by 40%. An intravenous infusion of ANG II (50 ng ⅐ kg Ϫ1 ⅐ min Ϫ1 ) in rats for 5 days increased the formation of 20-HETE and epoxyeicosatrienoic acids (EETs) in renal cortical microsomes by 60 and 400%, respectively, and increased MAP by 78 mmHg. Chronic blockade of the synthesis of 20-HETE with intravenous infusion of DDMS (1 mg ⅐ kg Ϫ1 ⅐ h Ϫ1 ) or EETs and 20-HETE with 1-aminobenzotriazole (ABT; 2.2 mg ⅐ kg Ϫ1 ⅐ h Ϫ1 ) attenuated the ANG II-induced rise in MAP by 40%. Control urinary excretion of 20-HETE averaged 350 Ϯ 23 ng/day and increased to 1,020 Ϯ 105 ng/day in rats infused with ANG II (50 ng ⅐ kg Ϫ1 ⅐ min Ϫ1 ) for 5 days. In contrast, urinary excretion of 20-HETE only rose to 400 Ϯ 40 and 600 Ϯ 25 ng/day in rats chronically treated with ANG II and ABT or DDMS respectively. These results suggest that acute and chronic elevations in circulating ANG II levels increase the formation of 20-HETE in the kidney and peripheral vasculature and that 20-HETE contributes to the acute and chronic pressor effects of ANG II.cytochrome P-450; epoxyeicosatrienoic acids; hypertension; kidney PREVIOUS STUDIES INDICATE that ANG II enhances the activity of phospholipases to release arachidonic acid (AA) (39,42,43) and stimulates the formation of cyclooxygenase (15,27,28,37,38,40,41) and lipoxygenase metabolites of AA (5, 40, 48) in various tissues. These products modulate the vasoconstrictor response to ANG II in both the renal and peripheral circulation (27,30). In this regard, ANG II normally stimulates the formation of prostacyclin, which attenuates the vasoconstrictor response to ANG II (12,28,30,32,37,38,40). ANG II also promotes the formation of 12-hydroxyeicosatetraenoic acid (12-HETE), which potentiates the rise in intracellular calcium concentration and the vasoconstrictor and mitogenic response to ANG II in vascular smooth muscle (VSM) cells (5,16,31,48,53). In hypertensive animals, the balance between the formation of vasodilator and vasoconstrictor metabolites of AA shifts toward the formation of vasoconstrictor metabolites, i.e., thromboxane A 2 and endoperoxides, and these products potentiate the vasoconst...

The present study examined whether inhibition of P4504A enzyme activity and the formation of 20-HETE contributes to the activation of K+ channels and vasodilator effects of nitric oxide (NO) in renal arterioles. Addition of an NO donor to the P4504A2 enzyme that produces 20-HETE increased visible light absorbance at 440 nm indicating that NO binds to heme in this enzyme. NO donors also dose-dependently inhibited the formation of 20-HETE in microsomes prepared from renal arterioles. In patch-clamp experiments, NO donors increased the open-state probability of a voltage-sensitive, large-conductance (195+/-9 pS) K+ channel recorded with cell-attached patches on renal arteriolar smooth muscle cells. Blockade of guanylyl cyclase with [1H-[1,2,4]Oxadiazolo[4,3-a] quinoxalin-1-one] (ODQ, 10 micromol/L), or cGMP-dependent kinase with 8R,9S,11S-(-)-9-methoxycarbamyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cy-cloocta-(c ,d, e)-trinden-1-one (KT-5823) (1 micromol/L) did not alter the effects of NO on this channel. In contrast, inhibition of the formation of 20-HETE with 17-octadecynoic acid (1 micromol/L) activated this channel and masked the response to NO. Preventing the NO-induced reduction in intracellular 20-HETE levels also blocked the effects of NO on this channel. Sodium nitroprusside (SNP) increased the diameter of renal interlobular arteries preconstricted with phenylephrine to 80+/-4% of control. Blockade of guanylyl cyclase with ODQ (10 micromol/L) attenuated the response to SNP by 26+/-2%; however, fixing 20-HETE levels at 100 nmol/L reduced the response by 67+/-8%. Blockade of both pathways eliminated the response to SNP. These results indicate that inhibition of the formation of 20-HETE contributes to the activation of K+ channels and the vasodilator effects of NO in the renal microcirculation.