John Quilley

Cytochrome P450-dependent effects of bradykinin in the rat heart

1 Vasodilator responses to bradykinin (BK) in the rat heart are reported to be independent of NO and cyclo‐oxygenase/lipoxygenase products of arachidonic acid (AA). 2 We verified that inhibition of NO synthase with l‐nitroarginine (50 μm) and cyclo‐oxygenase with indomethacin (2.8 μm) were without effect on vasodilator responses to BK (10–1000 ng) in the Langendorff rat heart preparation. 3 l‐Nitroarginine elevated perfusion pressure, signifying a crucial role of NO in the maintenance of basal vasculature tone. 4 In hearts treated with l‐nitroarginine to eliminate NO and elevate perfusion pressure, vasodilator responses were reduced by inhibitors of cytochrome P450 (P450), clotrimazole (1 μm) and 7‐ethoxyresorufin (1 μm). 17‐Octadecynoic acid (17‐ODYA 2 μm), a mechanism based inhibitor of P450‐dependent metabolism of fatty acids, also reduced vasodilator responses to BK. 5 These results confirm that NO and prostaglandins do not mediate vasodilator responses to BK in the rat heart but suggest a major role for a P450‐dependent mechanism via AA metabolism.

Contribution of NO and cytochrome P450 to the vasodilator effect of bradykinin in the rat kidney

1 Inhibition of nitric oxide generation with Nw‐nitro‐l‐arginine (nitroarginine) reduced vasodilator responses to bradykinin and acetylcholine and enhanced those to nitroprusside in the rat isolated perfused kidney, preconstricted with phenylephrine. 2 Inhibition of cyclo‐oxygenase with indomethacin, decreased the vasodilator responses to bradykinin by ∼25% without affecting those to acetylcholine or nitroprusside. 3 BW755c, a dual inhibitor of cyclo‐oxygenase and lipoxygenase, reduced renal vasodilator responses to bradykinin, comparable to the effect of indomethacin suggesting an effect related to inhibition of cyclo‐oxygenase rather than lipoxygenase. 4 ETYA, an inhibitor of all arachidonic acid metabolic pathways, markedly reduced vasodilator responses to bradykinin but was without effect on the renal vasodilatation induced by acetylcholine or nitroprusside. 5 Clotrimazole and 7‐ethoxyresorufin, inhibitors of cytochrome P450, greatly attenuated vasodilator responses to bradykinin without affecting those to acetylcholine or nitroprusside. 6 These data suggest that the renal vasodilator response to bradykinin is subserved by arachidonic acid metabolites as well as nitric oxide, the former accounting for up to 70% of the vasodilator effect of bradykinin.

20-hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acids and blood pressure.

The properties of 20-hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acids, vasoactivity and modulation of ion transport and mediation/modulation of the effects of vasoactive hormones, such as angiotensin II and endothelin, underscore their importance to renal vascular mechanisms and electrolyte excretion. 20-Hydroxyeicosatetraenoic acid is an integral component of renal autoregulation and tubuloglomerular feedback as well as cerebral autoregulation, eliciting vasoconstriction by the inhibition of potassium channels. Nitric oxide inhibits 20-hydroxyeicosatetraenoic acid formation, the removal of which contributes to the vasodilator effect of nitric oxide. In contrast, epoxyeicosatrienoic acids are generally vasodilatory by activating potassium channels and have been proposed as endothelium-derived hyperpolarizing factors. 20-Hydroxyeicosatetraenoic acid modulates ion transport in key nephron segments by influencing the activities of sodium-potassium-ATPase and the sodium-potassium-chloride co-transporter; however, the primacy of the various arachidonate oxygenases that generate products affecting these activities changes with age. The range and diversity of activity of 20-hydroxyeicosatetraenoic acid is influenced by its metabolism by cyclooxygenase to products affecting vasomotion and salt/water excretion. 20-Hydroxyeicosatetraenoic acid is the principal renal eicosanoid that interacts with several hormonal systems that are central to blood pressure regulation. This article reviews the most recent studies that address 20-hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acids in vascular and renal tubular function and hypertension.

Role of K+ channels in the vasodilator response to bradykinin in the rat heart

1 The role of K+ channels in the nitric oxide (NO)‐independent coronary vasodilator efffect of bradykinin was examined in the Langendorff heart preparation in which nitroarginine was used to inhibit NO synthesis and elevate perfusion pressure; cyclo‐oxygenase was inhibited with indomethacin. 2 The K+ channel inhibitors, tetraethylammonium, procaine and charybdotoxin, but not glibenc‐lamide, further increased perfusion pressure suggesting a role for K+ channels, other than ATP‐sensitive K+ channels, in the regulation of coronary vascular tone under the experimental conditions adopted here. 3 The non‐specific K+ channel inhibitors, tetraethylammonium and procaine, reduced vasodilator responses to bradykinin and cromakalim but not those to nitroprusside in the perfused heart treated with nitroarginine and indomethacin. 4 Glibenclamide, an inhibitor of ATP‐sensitive K+ channels, reduced vasodilator responses to cromakalim but did not affect those to bradykinin or nitroprusside. 5 Charybdotoxin, an antagonist of Ca2+‐activated K+ channels, inhibited responses to bradykinin but did not affect those to cromakalim or nitroprusside. 6 Nifedipine inhibited vasodilator responses to bradykinin and cromakalim without affecting those to nitroprusside. 7 Inhibition of cytochrome P450 with clotrimazole reduced responses to bradykinin but did not modify those to cromakalim or nitroprusside. 8 These results suggest that bradykinin utilizes a Ca2+‐activated K+ channel to produce vasodilatation in the rat heart.

Role of COX-2 in the Enhanced Vasoconstrictor Effect of Arachidonic Acid in the Diabetic Rat Kidney

Abstract—In the rat isolated perfused kidney, arachidonic acid elicits cyclooxygenase-dependent vasoconstriction through activation of PGH2/TxA2 receptors; responses are enhanced in kidneys from diabetic rats. This study examined the roles of cyclooxygenase-1/cyclooxygenase-2 in the enhanced renal vasoconstrictor effect of arachidonic acid in streptozotocin-diabetic rats. Release of 20-HETE was also determined, as this eicosanoid has been reported to elicit cyclooxygenase-dependent vasoconstriction. We confirmed that vasoconstrictor responses to arachidonic acid were enhanced in the diabetic rat kidney associated with a 2-fold–greater increase in the release of 6-ketoPGF1&agr;, which was used as an index of cyclooxygenase activity. One and three micrograms of arachidonic acid increased perfusion pressure by 85±37 and 186±6 mm Hg, respectively, in diabetic rat kidneys compared with 3±1 and 17±8 mm Hg, respectively, in control rat kidneys. Inhibition of both cyclooxygenase isoforms with indomethacin (10 &mgr;mol/L) abolished the vasoconstrictor response to arachidonic acid in both diabetic and control rat kidneys, whereas inhibition of cyclooxygenase-2 with nimesulide (5&mgr;mol/L) reduced perfusion pressure responses to 1 and 3 &mgr;g arachidonic acid only in the diabetic rat kidney to 15±8 and 108±26 mm Hg, respectively, consistent with a 3-fold increase in the renal cortical expression of cyclooxygenase-2. 20-HETE release from the diabetic rat kidney was reduced almost 6-fold and was not increased in response to arachidonic acid. These results demonstrate that the renal vasoconstrictor effect of arachidonic acid is solely dependent on cyclooxygenase activity, with no evidence for a contribution from 20-HETE; in the diabetic rat, cyclooxygenase-2 activity contributes to the renal vasoconstrictor effect of arachidonic acid.

Role of endoperoxides in arachidonic acid‐induced vasoconstriction in the isolated perfused kidney of the rat

1 Administration of arachidonic acid caused dose‐dependent vasoconstriction in the isolated rat kidney perfused in situ with Krebs‐Henseleit solution. 2 Inhibition of cyclo‐oxygenase with indomethacin or meclofenamate reduced the renal vasoconstrictor effect of arachidonic acid. 3 The renal vasoconstrictor effect of arachidonic acid was unaffected by CGS‐13080 at concentrations that effectively reduced thromboxane A2 (TxA2) synthesis by platelets and the kidney. 4 The endoperoxide/TxA2 receptor antagonist, SQ 29,548, abolished the renal vasoconstrictor effect of arachidonic acid and of U46619, an endoperoxide analogue. In contrast, SQ 29,548 did not affect the renal vasoconstrictor response to angiotensin II, prostaglandin E2 or F2α. 5 These data suggest that the vasoconstrictor effect of arachidonic acid in the isolated kidney of the rat is mediated by its metabolites, including the prostaglandin endoperoxides.

5,6-Epoxyeicosatrienoic Acid Mediates the Enhanced Renal Vasodilation to Arachidonic Acid in the SHR

Abstract—We have shown a cytochrome P450–dependent renal vasodilator effect of arachidonic acid in response to inhibition of cyclooxygenase and elevation of perfusion pressure, which was enhanced in the spontaneously hypertensive rat (SHR) and linked to increased production of and/or responsiveness to epoxyeicosatrienoic acids (EETs). In the SHR, vasodilation elicited by low doses of arachidonic acid was attenuated by the nitric oxide synthase inhibitor Nw-nitro-l-arginine (50 &mgr;mol/L), whereas the responses to high doses were unaffected. Inhibition of epoxygenases with miconazole (0.3 &mgr;mol/L) in the presence of Nw-nitro-l-arginine greatly reduced the renal vasodilator response to all doses of arachidonic acid. Tetraethylammonium (10 mmol/L), a nonselective K+ channel blocker, abolished the nitric oxide–independent renal vasodilator effect of arachidonic acid as well as the vasodilator effect of 5,6-EET, confirming that EET-dependent vasodilation involves activation of K+ channels. Under conditions of elevated perfusion pressure (200 mm Hg) and cyclooxygenase inhibition, 5,6-EET, 8, 9-EET, and 11,12-EET caused renal vasodilatation in both SHR and Wistar-Kyoto rats (WKY), whereas 14,15-EET produced vasoconstriction. 5,6-EET was the most potent renal vasodilator of the EET regioisomers in the SHR by a factor of 4 or more. In the SHR, 5,6-EET- and 11,12-EET–induced renal vasodilatation was >2-fold greater than that registered in WKY. Thus, the augmented vasodilator responses to arachidonic acid in the SHR is through activation of K+ channels, and 5,6-EET is the most likely mediator.

Contribution of calcium-activated potassium channels to the vasodilator effect of bradykinin in the isolated, perfused kidney of the rat

1 NO‐ and prostaglandin‐independent, endothelium‐dependent vasodilator responses to bradykinin are attributed to release of a hyperpolarizing factor. Therefore, the contribution of K+ channels to the renal vasodilator effect of bradykinin was examined in rat perfused kidneys that were preconstricted with phenylephrine and treated with NG‐nitro‐L‐arginine (L‐NOARG) and indomethacin to inhibit NO and prostaglandin synthesis. 2 The non‐specific K+ channel inhibitors, TEA and TBA reduced vasodilator responses to bradykinin and cromakalim but not those to nitroprusside. 3 Glibenclamide, an inhibitor of ATP‐sensitive K+ channels, blocked the vasodilator response to cromakalim without affecting responses to bradykinin. 4 Charybdotoxin, a selective inhibitor of Ca2+‐activated K+ channels, greatly attenuated vasodilator responses to bradykinin without affecting those to cromakalim or nitroprusside. 5 Iberiotoxin and leiurotoxin, inhibitors of large and small conductance Ca2+‐activated K+ channels, respectively, were without effect on vasodilator responses to bradykinin, cromakalim or nitroprusside. 6 These results implicate K+ channels, specifically Ca2+‐activated K+ channels of intermediate conductance, in the renal vasodilator effect of bradykinin and, thereby, support a role for a hyperpolarizing factor.

The antihypertensive effect of captopril in essential hypertension: relationship to prostaglandins and the kallikrein-kinin system

Two groups, each with nine essential hypertensive patients, were maintained on 10 mmol sodium daily over 14-17 days and treated in this sequence: placebo; captopril (25 or 50 mg given thrice daily) or indomethacin (50 mg given thrice daily) alone; captopril plus indomethacin, and (4) captopril alone. The initial fall in mean blood pressure induced by captopril (118 +/- 1 to 102 +/- 1 mmHg) was unaffected by the addition of indomethacin. However, if indomethacin treatment preceded captopril, the antihypertensive effect was attenuated (116 +/- 4 to 109 +/- 4), and was associated with significant reductions in urinary prostaglandin and kinin excretion. Addition of captopril to indomethacin returned kinin excretion to placebo levels but did not affect indomethacin-induced reduction in prostaglandin excretion. Captopril alone stimulated plasma renin activity (PRA) fivefold; aldosterone excretion was lowered by 25% and further reduced by indomethacin. Thus, when captopril and indomethacin are administered together, the order of administration is critical to the antihypertensive effect of captopril.

Missing links: Cytochrome P450 arachidonate products: A new class of lipid mediators

Three major enzyme systems-cyclooxygenases, lipoxygenases, and cytochrome P450 monooxygenases (CYP)-generate biological mediators from arachidonic acid (AA). A distinct profile of arachidonate products characterizes each renal tubular segment and each section of the renal vascular tree from arteries to microvessels to veins. The most recent discoveries on renal mechanisms subserved by arachidonate metabolites relate to CYP pathways of AA metabolism. These arachidonate products have prominent effects on blood vessels and ion transport, including modulation and mediation of the actions of vasoactive hormones. The diverse properties of these AA metabolites and the wide distribution of the CYP system make them prime candidates for participation in regulatory mechanisms that affect the circulation and transporting epithelia. CYP-AA products are as important to renal and circulatory control as are nitric oxide and prostaglandins.