Epoxyeicosatrienoic acid-mediated renal vasodilation to arachidonic acid is enhanced in SHR.

We tested the hypothesis that cyclooxygenase-independent vasodilation produced by arachidonic acid (AA) is mediated by epoxyeicosatrienoic acids (EETs) and is blunted in the spontaneously hypertensive rat (SHR). At normal perfusion pressure (PP; 70 to 90 mm Hg), AA constricted the renal vasculature in both SHR and normotensive Wistar-Kyoto rats, an effect abolished by cyclooxygenase inhibition, and converted to vasodilation when PP was raised to approximately 200 mm Hg. Unexpectedly, renal vasodilation elicited by AA was greater in the SHR at high PP; for example, 2.5, 5, and 10 microg of AA produced PP declines of 54+/-9, 92+/-10, and 112+/-5 mm Hg, respectively, in SHR compared with 26+/-3, 45+/-5, and 77+/-6 mm Hg in Wistar-Kyoto rats (P:<0.01). However, the renal vasodilator responses to acetylcholine (0.1 microg) and sodium nitroprusside (1 microg) did not differ between strains, indicating that vascular responsiveness to AA was independent of intrinsic changes in vascular smooth muscle. Hyperresponsiveness of the renal vasculature to AA may be unique for the SHR, because it did not occur in Sprague-Dawley rats with angiotensin II-induced hypertension. 5,8,11,14-Eicosatetraynoic acid (ETYA; 4 micromol/L), an inhibitor of all AA pathways, attenuated the vasodilator responses to AA, as did treatment with stannous chloride, which depletes cytochrome P450 enzymes, suggesting that a cytochrome P450 AA metabolite mediated the renal vasodilation. N:-Methylsulfonyl-12,12-dibromododec-11-en-amide (DDMS; 2 micromol/L), a selective omega-hydroxylase inhibitor, did not affect AA-induced vasodilation, whereas selective inhibition of epoxygenases with either miconazole (0.3 micromol/L) or N:-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH; 12 micromol/L) did, indicating that one or more EETs were involved in the renal vasodilator action of AA at high PP. This conclusion was supported by the demonstration that AA greatly enhanced the renal efflux of EETs at high PP but not at basal PP.

Reactive oxygen species: role in the relaxation induced by bradykinin or arachidonic acid via EDHF in isolated porcine coronary arteries.

Although endothelium-derived hyperpolarizing factor (EDHF) is thought to be a cytochrome P-450 product (arachidonic acid metabolite) in some tissues, in porcine coronary arteries (PCAs) its nature remains unclear. Because phospholipase A2 and C are involved in the synthesis and/or release of EDHF in the PCA, the arachidonic acid (AA) pathway may be involved. In the presence of the cyclooxygenase inhibitor indomethacin (10(-5) M) and the NOS inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME; 10(-4) M), both bradykinin (BK; 10(-9)-10(-6) M) and AA (10(-7)-10(-4) M) induced dose-dependent relaxation of PGF2alpha-contracted PCA rings, which was blocked by a high extracellular concentration of KCl (30 mM) or pretreatment with ouabain, a Na+/K+-adenosine triphosphatase (ATPase) inhibitor (5 x 10(-7) M). Eicosatetraynoic acid (ETYA; 20 microM), which inhibits all AA pathways, slightly affected the response to BK and AA; however, lipoxygenase or cytochrome P-450 inhibitors had no effect, suggesting that relaxation is independent of these enzymatic pathways. Because endothelial cells can generate reactive oxygen species (ROS) via metabolism of AA and independent of cyclooxygenase activity, we also studied (a) whether ROS can relax the PCA, as well as the mechanism(s) involved, and (b) the role of ROS in BK- and AA-induced relaxation. Xanthine (X; 100 microM) plus xanthine oxidase (XO; 0.02 U/ml) induced time-dependent relaxation of PGF2alpha-contracted PCA rings in the presence of indomethacin and L-NAME. Dilatation was not affected by superoxide dismutase (SOD; 500 U/ml) but was abolished by catalase (300 U/ml), suggesting that hydrogen peroxide (H2O2) is involved. When rings were contracted by depolarizing them with 30 mM KCl, X/XO failed to elicit relaxation. Ouabain abolished the response to X/XO, suggesting that X/XO may induce relaxation by hyperpolarizing vascular smooth muscle cells via stimulation of the Na+/K+-ATPase pump. We therefore questioned whether ROS might be involved in BK- and AA-induced relaxation. Because catalase combined with SOD had little or no effect, we concluded that in the PCA, the relaxation induced by BK via EDHF involves some mechanism independent of NO, AA metabolism, or ROS.

Linoleic acid induces relaxation and hyperpolarization of the pig coronary artery.

Linoleic acid, a polyunsaturated C18 fatty acid, is one of the major fatty acids in the coronary arterial wall. Although diets rich in linoleic acid reduce blood pressure and prevent coronary artery disease in both humans and animals, very little is known about its mechanism of action. We believed that its beneficial effects might be mediated by changes in vascular tone. We investigated whether linoleic acid induces relaxation of porcine coronary artery rings and the mechanism involved in this process. Linoleic acid and two of its metabolites, 13-hydroxyoctadecadienoic acid (13-HODE) and 13-hydroperoxyoctadecadienoic acid (13-HPODE), induced dose-dependent relaxation of prostaglandin (PG) F2alpha-precontracted rings that was not affected by indomethacin (10[-5] mol/L), a cyclooxygenase inhibitor, or cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC; 10[-5] mol/L), a lipoxygenase inhibitor. Removal of endothelial cells had no effect on vasorelaxation, suggesting a direct effect on the vascular smooth muscle cells (VSMC). When rings were contracted with KCl, linoleic acid failed to induce relaxation. Although tetrabutylammonium (5 x 10[-3] mol/L), a nonselective K+ channel blocker, slightly inhibited the relaxation caused by linoleic acid, glibenclamide (10[-6] mol/L), an ATP-sensitive K+ channel blocker, and charybdotoxin (7.5x10[-8] mol/L) or tetraethylammonium (5x10[-3] mol/L), two different Ca2+-activated K+ channel blockers, had no effect. However, relaxation was completely blocked by ouabain (5x10[-7] mol/L), a Na+/K+-ATPase inhibitor, or by a K+-free solution. In addition, linoleic acid (10[-6] mol/L) caused sustained hyperpolarization of porcine coronary VSMC (from -49.5+/-2.0 to -60.7+/-4.2 mV), which was also abolished by ouabain. We concluded that linoleic acid induces relaxation and hyperpolarization of porcine coronary VSMC via a mechanism that involves activation of the Na+/K+-ATPase pump.

Autacoids mediate coronary vasoconstriction induced by nitric oxide synthesis inhibition.

Inhibition of nitric oxide (NO) synthesis results in coronary vasoconstriction. Using a Langendorff rat heart preparation, we tested the hypothesis that this vasoconstriction is caused by the unopposed effect of the autacoids prostaglandin H2 (PGH2) or thromboxane A2 (TxA2) or both through a mechanism that involves oxygen free radicals. The vasoconstriction induced by NO synthesis inhibition was studied with two different NO synthase inhibitors, N(omega)-nitro-L-arginine methyl ester (L-NAME) and N(omega)-monomethyl-L-arginine (L-NMMA). We found that the decrease in coronary flow (CF) induced by L-NAME (from 19.3 +/- 0.9 to 13.2 +/- 0.9 ml/min; p < 0.001) and L-NMMA (from 20.1 +/- 0.4 to 15.0 +/- 0.3 ml/min; p < 0.001) was completely blocked by the cyclooxygenase inhibitor indomethacin. A different cyclooxygenase inhibitor (ibuprofen), a PGH2/TxA2-receptor antagonist (SQ29548), and a TxA2 synthase inhibitor (CGS 13080) also completely abolished the vasoconstrictor effect of L-NAME, suggesting that this vasoconstriction is mediated by TxA2. Two different scavengers of superoxide radical anions (O2-), the enzyme superoxide dismutase (SOD) and a cell-permeable SOD mimic, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), also blocked the vasoconstriction induced by NO synthesis inhibition. In contrast, catalase, which inactivates hydrogen peroxide (H2O2), failed to do so, indicating that O2- is needed for the vasoconstrictor effect of L-NAME, whereas H2O2 is not. To determine whether O2- acts on the conversion of PGH2 to TxA2 or at the receptor or postreceptor level, we studied whether the vasoconstriction induced by exogenous PGH2 or the TxA2 receptor agonist U46619 is blocked by scavengers of O2-. CF decreased by 50% with PGH2 (from 21 +/- 2.1 to 10.6 +/- 5.8 ml/min; p < 0.01), and this decrease was abolished by SOD and Tempol but not catalase. However, SOD had no effect on the vasoconstriction induced by U46619, which decreased CF by 45% (from 17.3 +/- 2.5 to 9.5 +/- 1.8 ml/min; p < 0.01). In addition, PGH2 increased the release of TxB2 (the stable metabolite of TxA2) in the coronary effluent (from 5.1 +/- 1.2 to 136.1 +/- 11.8 pg/ml/min). The release of TxB2 was significantly lower in hearts treated with SOD (76.8 +/- 14.2 pg/ml/min) and CGS (65.7 +/- 13.9 pg/ml/min). We conclude that the coronary vasoconstriction induced by inhibition of NO synthesis is the result of the unopposed effect of the autacoid TxA2 through activation of its receptor, and that O2- is necessary for conversion of PGH2 to TxA2.

Effect of high salt intake in mutant mice lacking bradykinin-B2 receptors.

Renal kinins release prostaglandins and nitric oxide via the B2 receptor, promoting diuresis and natriuresis; hence, they may also contribute significantly to blood pressure regulation. We hypothesized that mutant mice lacking the gene encoding for the bradykinin-B2 receptor (B2-KO) become hypertensive when placed on a long-term high-salt diet. To test this, B2-KO and control mice were placed on either a normal (0.2%) or high-Na+ diet (3.15% in food plus 1% saline as drinking water) for 8 weeks. Systolic blood pressure was determined during weeks 6 and 8 by a computerized tail-cuff system. At the end of the 8-week period, mice were anesthetized for determination of mean blood pressure, renal blood flow, and renal vascular resistance. In B2-KO mice maintained on high Na+, systolic blood pressure was 15 mm Hg higher than in knockout animals on normal Na+ (P < .01). In contrast, there was no difference in blood pressure in control mice fed either a normal or a high-Na+ diet. Consistent with the systolic blood pressure data, direct mean arterial pressure revealed that B2-KO mice on high Na+ were hypertensive (115 +/- 6 in B2-KO on high-Na+ diet versus 79 +/- 2.8 in B2-KO on normal Na+, P < .0001); renal blood flow was reduced by 20% (P < .05) and renal vascular resistance was doubled (P < .0001) compared with B2-KO mice on normal Na+. In contrast, control mice on high Na+ were normotensive and tended to have increased renal blood flow and decreased renal vascular resistance compared with control mice on a normal Na+ diet. These findings indicate that kinins play an important role in preventing salt-sensitive hypertension; this may be achieved by maintaining renal blood flow under conditions of high salt intake.

Nitric oxide inhibits ADH-stimulated osmotic water permeability in cortical collecting ducts.

Nitric oxide (NO) reduces blood pressure in vivo by two mechanisms, vasodilation and increasing urinary volume: however, the exact mechanism by which it increases urinary volume is not clear. We hypothesized that NO inhibits antidiuretic hormone (ADH)-stimulated fluid reabsorption (J(r)) by the isolated rat cortical collecting duct (CCD) by decreasing water permeability (Pf) and sodium reabsorption (Jna). In the presence of 10(-11) MADH, Jv was 0.15 +/- 0.04 nl.min-1.mm-1; after 10(-6) M spermine nonoate (SPM) was added to the bath. Jv decreased to 0.06 +/- 0.03 nl.min-1.mm-1 (P < 0.03). To investigate whether the inhibition of Jv was the result of decreased Pf and/or Jna, we first tested the effect of SPM on ADH-stimulated Pf. Basal Pf was stimulated to 289.2 +/- 77.3 microns/s after 10(-11) M ADH was added to the bath (P < 0.01). SPM decreased Pf to 159.8 +/- 45.0 microns/s (P < 0.05). To ensure that this effect on Pf was due to NO release, we used another NO donor, nitroglycerin (NTG). Pf was initially -25.8 +/- 18.3 microns/s and increased to 133.9 +/- 30.5 microns/s after addition of 10(-11) M ADH (P < 0.002). NTG, 20 microM, lowered Pf to 92.4 +/- 18.4 microns/s (P < 0.02). In the presence of 10(-9) M ADH, NTG also decreased Pf(P < 0.04). Next we investigated the effect of SPM on ADH-stimulated JNa. In the presence of ADH, JNa was 37.8 +/- 7.3 pmol.min-1.mm-1. After SPM was added, it dropped to 24.3 +/- 5.1 pmol.min-1.mm-1 (P < 0.05). Time controls exhibited no change in ADH-stimulated Jv, Pf, or Jna. We concluded that 1) NO decreases ADH-stimulated water and sodium transport in the isolate CCD, and 2) water reabsorption is inhibited by a primary effect on Pf. A direct effect of NO on the CCD may explain its natriuretic and diuretic effects observed in vivo.