Arachidonic Acid Inhibits Epithelial Na Channel Via Cytochrome P450 (CYP) Epoxygenase-dependent Metabolic Pathways

We used the patch-clamp technique to study the effect of arachidonic acid (AA) on epithelial Na channels (ENaC) in the rat cortical collecting duct (CCD). Application of 10 μM AA decreased the ENaC activity defined by NPo from 1.0 to 0.1. The dose–response curve of the AA effect on ENaC shows that 2 μM AA inhibited the ENaC activity by 50%. The effect of AA on ENaC is specific because neither 5,8,11,14-eicosatetraynoic acid (ETYA), a nonmetabolized analogue of AA, nor 11,14,17-eicosatrienoic acid mimicked the inhibitory effect of AA on ENaC. Moreover, inhibition of either cyclooxygenase (COX) with indomethacin or cytochrome P450 (CYP) ω-hydroxylation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) failed to abolish the effect of AA on ENaC. In contrast, the inhibitory effect of AA on ENaC was absent in the presence of N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH), an agent that inhibits CYP-epoxygenase activity. The notion that the inhibitory effect of AA is mediated by CYP-epoxygenase–dependent metabolites is also supported by the observation that application of 200 nM 11,12-epoxyeicosatrienoic acid (EET) inhibited ENaC in the CCD. In contrast, addition of 5,6-, 8,9-, or 14,15-EET failed to decrease ENaC activity. Also, application of 11,12-EET can still reduce ENaC activity in the presence of MS-PPOH, suggesting that 11,12-EET is a mediator for the AA-induced inhibition of ENaC. Furthermore, gas chromatography mass spectrometry analysis detected the presence of 11,12-EET in the CCD and CYP2C23 is expressed in the principal cells of the CCD. We conclude that AA inhibits ENaC activity in the CCD and that the effect of AA is mediated by a CYP-epoxygenase–dependent metabolite, 11,12-EET

Antioxidants Condition Pleiotropic Vascular Responses to Exogenous H2O2: Role of Modulation of Vascular TP Receptors and the Heme Oxygenase System

Aims: Hydrogen peroxide (H(2)O(2)), a nonradical oxidant, is employed to ascertain the role of redox mechanisms in regulation of vascular tone. Where both dilation and constriction have been reported, we examined the hypothesis that the ability of H(2)O(2) to effect vasoconstriction or dilation is conditioned by redox mechanisms and may be modulated by antioxidants. Results: Exogenous H(2)O(2) (0.1-10.0 μM), dose-dependently reduced the internal diameter of rat renal interlobular and 3rd-order mesenteric arteries (p\u3c0.05). This response was obliterated in arteries pretreated with antioxidants, including tempol, pegylated superoxide dismutase (PEG-SOD), butylated hydroxytoluene (BHT), and biliverdin (BV). However, as opposed to tempol or PEG-SOD, BHT & BV, antioxidants targeting radicals downstream of H(2)O(2), also uncovered vasodilation. Innovations: Redox-dependent vasoconstriction to H(2)O(2) was blocked by inhibitors of cyclooxygenase (COX) (indomethacin-10 μM), thromboxane (TP) synthase (CGS13080-10 μM), and TP receptor antagonist (SQ29548-1 μM). However, H(2)O(2) did not increase vascular thromboxane B(2) release; instead, it sensitized the vasculature to a TP agonist, U46619, an effect reversed by PEG-SOD. Antioxidant-conditioned dilatory response to H(2)O(2) was accompanied by enhanced vascular heme oxygenase (HO)-dependent carbon monoxide generation and was abolished by HO inhibitors or by HO-1 & 2 antisense oligodeoxynucleotides treatment of SD rats. Conclusions: These results demonstrate that H(2)O(2) has antioxidant-modifiable pleiotropic vascular effects, where constriction and dilation are brought about in the same vascular segment. H(2)O(2)-induced oxidative stress increases vascular TP sensitivity and predisposes these arterial segments to constrictor prostanoids. Conversely, vasodilation is reliant upon HO-derived products whose synthesis is stimulated only in the presence of antioxidants targeting radicals downstream of H(2)O(2)

Role of Vasopressin in Regulation of Renal Kinin Excretion in Long-Evans and Diabetes Insipidus Rats

Ab btract. To study the relationship between vasopressin and the renal kallikrein-kinin system we measured the rate of excretion of kinins into the urine of anesthetized rats during conditions of increased and decreased vasopressin level. The excretion of immunoreactive kinins in Brattleboro rats with hereditary diabetes insipidus (DI) (24±3 pg min- ' kg-') was lower than in the control Long Evans (LE) rats (182±22 pg min- ' kg-'; P < 0.05). The DI rats also exhibited negligible urinary excretion of immunoreactive vasopressin, reduced urine osmolality, and increased urine flow and kininogenase excretion. In LE rats, volume expansion by infusion of 0.45 % NaCl-2.5 % dextrose to lower vasopressin secretion reduced (P < 0.05) kinin excretion, vasopressin excretion, and urine osmolality to 41, 26, and 15 % of their respective control values, while increasing (P < 0.05) urine flow and kininogenase excretion. On the other hand, the infusion of 5 % NaCl, which promotes vasopressin secretion, increased (P < 0.05) the urinary excretion of kinins and vasopressin to 165 and 396 % of control, while increasing (P < 0.05) urine flow and kininogenase excretion. Infusion of vasopressin (1.2 mU/h, intravenous) enhanced (P < 0.05) kinin excretion by two to threefold in DI rats and in LE rats during volume expansion with 0.45 % NaCl-2.5 % dextrose, while decreasing urine flow and increasing urine osmolality. This study demonstrates that the urinary excretion of immunoreactive kinins varies in relation to the urinary level of vasopressin, irrespective of urine volume and osmolality and of the urinary excretions of sodium and kininogenase. The study suggests a role fo