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.

The expression of P-450 4A isoforms responsible for the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) was examined using the reverse transcription and polymerase chain reaction in various nephron segments and preglomerular arterioles microdissected from the kidneys of Sprague-Dawley rats. Expression of cytochrome P-450 4A1, 4A2, 4A3, and 4A8 mRNA could be detected in RNA extracted from the whole kidney. The expression of P-450 4A1, 4A3, and 4A8 mRNA was similar in the kidney of male and female rats, whereas the expression of 4A2 mRNA was fourfold greater in the kidney of male vs. female rats. At the single-nephron level, P-450 4A1 mRNA could not be detected in either preglomerular arterioles or any nephron segments. P-450 4A2 mRNA was readily detected in preglomerular arterioles, glomeruli, proximal convoluted tubule (PCT), proximal straight tubule (PST), medullary thick ascending limb (MTAL), cortical thick ascending limb (CTAL), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD). P-450 4A3 mRNA was also detected in every nephron segment, but the expression of this isoform was barely detectable in preglomerular arterioles. The expression of P-450 4A8 mRNA was detected in the glomerulus, PCT, PST, CTAL, and CCD. It was not detectable in preglomerular arterioles, MTAL, OMCD, or IMCD. Immunoblot analysis using a P-450 4A antibody exhibited a strong signal for P-450 4A protein in the proximal tubule. Smaller signals were also observed in glomerulus, MTAL, and preglomerular arterioles, but no signal could be detected in the IMCD. A similar pattern of P-450 4A protein expression was seen in kidney sections immunostained with this antibody. These results indicate that the expression of P-450 4A isoforms in the kidney of rats is sex dependent and that different P-450 4A isoforms are expressed throughout various nephron segments and the renal vasculature of rats.

The present study examined the effects of a series of 20-hydroxyeicosatetraenoic acid (20-HETE) derivatives on the diameter of renal arterioles to determine the structural requirements of the vasoconstrictor response to 20-HETE. The vascular responses to 5-, 8-, 12-, 15-, 19-, 20-, 21-HETEs, arachidonic acid (AA), and saturated, partially saturated, dimethyl, carboxyl, and 19-carbon derivatives of 20-HETE (10(-8) to 10(-6) M) were assessed in rat renal interlobular arteries (65-125 micrometer). 20-HETE, 21-HETE, dimethyl-20-HETE, and a partially saturated derivative of 20-HETE, 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid, reduced vessel diameter by 19 +/- 3, 17 +/- 3, 16 +/- 2, and 28 +/- 2%, respectively. In contrast, 5-, 8-, 12-, 15-, and 19-HETE, AA, saturated, partially saturated, carboxyl, and the 19-carbon derivatives of 20-HETE had no effect on vessel diameter. Pretreatment with 5-, 15-, and 19-HETE, the 19-carbon derivative or 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (1 microM) completely blocked the vasoconstrictor response to 20-HETE in renal arterioles. Pretreatment with AA, carboxyl, saturated 19-carbon, and saturated 20-HETE derivatives (1 microM) partially blocked the response, whereas 8- and 12-HETE (1 microM) had no effect on the vasoconstrictor response to 20-HETE. These findings suggest that 20-HETE agonists and antagonists require a carboxyl or an ionizable group on carbon 1 and a double bond near the 14 or 15 carbon. 20-HETE agonists also require a functional group capable of hydrogen bonding on carbon 20 or 21, whereas antagonists lack this reactive group.

4. In summary, the available evidence indicates that CYP metabolites of AA play a central role in the regulation of renal, pulmonary and vascular function and that abnormalities in this system may contribute to the pathogenesis of cardiovascular diseases.

Abstvuct-Inducing renal cytochrome P4504A (P4504A) activity with clofibrate prevents the development of hypertension m Dahl salt-sensitive (Dahl S) rats To determine if this also occurs with other antlhpldemlc agents, we compared the effects of a related drug, fenofibrate, with those of an unrelated agent, pravastatm, on blood pressure, renal histology, and P4504A activity Dahl S rats were pretreated with fenofibrate (95 mg/kg per day), pravastatm (70 mg/kg per day), or vehicle for 7 days before and after bemg switched from a low-salt (0.1% NaCl) to a high-salt (8 0% NaCl) diet. After 3 weeks on the high-salt diet, mean artenal pressures averaged 183+13 (n=9), 126+10 (n=9), and 148% 11 mm Hg (n=8), respectively, m vehicle-, fenofibrate-, and pravastatm-treated animals Both drugs reduced the degree of protemurla and glomerular injury P4504A protein levels and the synthesis of 8,11, were increased m the liver and kidney of fenofibrate-treated, but not pravastatm-treated rats We also administered these agents to Dahl S rats m which hypertension had previously been induced by a high-salt diet Mean arterial pressures averaged 164+ 10, 113+23, and 160+ 15 mm Hg m rats treated with vehicle, fenofibrate, or pravastatm for 3 weeks Fenofibrate-treated rats exhibited a natrmresls Protemuna and glomerular injury were reduced by pravastatm but not by fenofibrate These results indicate that fenofibrate prevented the development of hypertension and reduced subsequent glomerular injury m Dahl S rats, probably secondary to increased renal production of 20-HETE Although pravastatm did not induce renal P4504A activity m these animals, it reduced the seventy of hypertension and renal damage through some other mechanism (Hy~evtension.1998;31[part 2]:225-231.)Key Words: salt sensltlvlty n cytochrome P450 m hydroxyelcosatetraenolc acids n glomerulosclerosls n antlhpldemlc drugs w pravastatm n fenofibrate T he Dahl S rat IS the most widely studied genetic model of salt-sensmve hypertension ' When these animals are fed a high-salt diet, mean arterial pressure typically increases by 20 mm Hg within 24 hours and continues to nse to a plateau of 170 mm Hg or higher, around 2 weeks '-' The mltlal rise m artenal pressure appears to be tnggered by sodium retention The ammals gain about 7% m body weight; plasma volume and cardiac output mcrease slgmficantly * Later, cardiac output returns toward control values, and the hypertension IS mamtamed by increased penpheral vascular resistance 4Previous studies have indicated that the pressure-natnuretlc relation 1s reset to a higher level of renal perfiuslon pressure m Dahl S rats 5 ' This resetting IS largely due to a marked elevation in sodium reabsorption m the thick ascending limb of the loop of Henle5,"" and IS associated with a deficiency of the production of 20-HETE,"' which IS a potent inhibitor of Na+,K+,2Ci-cotransport m this segment of the nephron " Indeed, exogenous admmlstratlon of 20-HETE has been reported to normalize chloride transport m the loop of Henle of Dahl S rats, and mhlbltors...

The present study examined the contribution of elevations in cGMP versus inhibition of cytochrome P-4504A enzymes and the production of the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) to the vasodilator actions of NO in renal arterioles. The NO donor sodium nitroprusside (SNP) at 10−5, 10−4, and 10−3 M reduced the production of 20-HETE in microsomes prepared from renal arterioles to 80 ± 2, 43 ± 5, and 7 ± 1% of control, respectively ( n = 4). In other experiments, the vasodilator response to SNP (10−7 to 10−3 M) was examined in rat renal interlobular arteries (<90 μm ID), preconstricted with phenylephrine (1 μM) under control conditions and after blockade of the cGMP and P-4504A pathways. Inhibition of guanylyl cyclase with 1H-[1,2,4]oxadiazole[4,3- a]quinoxalin-1-one (ODQ) (10 μM, n = 6) or of cGMP-dependent protein kinase with 8 R,9 S,11 S-(−)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7 b,11 a-trizadibenzo-( a, g)-cycloocta-( c, d, e)-trinden-1-one (KT-5823, 1 μM; n = 5) attenuated the vasodilator response to SNP by 26 and 30%, respectively. In contrast, inhibition of the endogenous production of 20-HETE with a suicide substrate, irreversible inhibitor [17-octadecynoic acid (17-ODYA), 1 μM, n = 5], or a selective, competitive inhibitor of 20-HETE formation (dibromo-dodecenyl-methylsulfimide, 25 μM, n = 5) markedly impaired the vasodilator response to SNP by 76 and 78%, respectively. Similarly, when 20-HETE levels were fixed at 100 nM ( n = 6), the response to SNP was attenuated by 73%. Blockade of both pathways with ODQ and 17-ODYA completely abolished the response to SNP ( n = 6). These results indicate that the vasodilator response to NO is largely cGMP independent and that inhibition of 20-HETE formation contributes to the cGMP-independent effects of NO in the renal microcirculation.