Renal protective effect of chronic inhibition of COX-2 with SC-58236 in streptozotocin-diabetic rats.

The induction of renal cyclooxygenase-2 (COX-2) in diabetes has been implicated in the renal functional and structural changes in models where hypertension or uninephrectomy was superimposed. We examined the protective effects of 3 mo treatment of streptozotocin-diabetic rats with a highly selective COX-2 inhibitor (SC-58236) in terms of albuminuria, renal hypertrophy, and the excretion of TNF-α and TGF-ß, which have also been implicated in the detrimental renal effects of diabetes. SC-58236 treatment (3 mg·kg(-1)·day(-1)) of diabetic rats resulted in reduced urinary excretion of PGE(2), 6-ketoPGF(1α), and thromboxane B(2), all of which were increased in the diabetic rat compared with age-matched nondiabetic rats. However, serum thromboxane B(2) levels were unchanged, confirming the selectivity of SC-58236 for COX-2. The renal protective effects of treatment of diabetic rats with the COX-2 inhibitor were reflected by a marked reduction in albuminuria, a reduction in kidney weight-to-body weight ratio, and TGF-ß excretion and a marked decrease in the urinary excretion of TNF-α. The protective effects of SC-58236 were independent of changes in plasma glucose levels or serum advanced glycation end-product levels, which were not different from those of untreated diabetic rats. In an additional study, the inhibition of COX-2 with SC-58236 for 4 wk in diabetic rats resulted in creatinine clearance rates not different from those of control rats. These results confirm that the inhibition of COX-2 in the streptozotocin-diabetic rat confers renal protection and suggest that the induction of COX-2 precedes the increases in cytokines, TNF-α, and TGF-ß.

Renal cytochrome P450 omega-hydroxylase and epoxygenase activity are differentially modified by nitric oxide and sodium chloride.

Renal function is perturbed by inhibition of nitric oxide synthase (NOS). To probe the basis of this effect, we characterized the effects of nitric oxide (NO), a known suppressor of cytochrome P450 (CYP) enzymes, on metabolism of arachidonic acid (AA), the expression of omega-hydroxylase, and the efflux of 20-hydroxyeicosatetraenoic acid (20-HETE) from the isolated kidney. The capacity to convert [(14)C]AA to HETEs and epoxides (EETs) was greater in cortical microsomes than in medullary microsomes. Sodium nitroprusside (10-100 microM), an NO donor, inhibited renal microsomal conversion of [(14)C]AA to HETEs and EETs in a dose-dependent manner. 8-bromo cGMP (100 microM), the cell-permeable analogue of cGMP, did not affect conversion of [(14)C]AA. Inhibition of NOS with N(omega)-nitro-L-arginine-methyl ester (L-NAME) significantly increased conversion of [(14)C]AA to HETE and greatly increased the expression of omega-hydroxylase protein, but this treatment had only a modest effect on epoxygenase activity. L-NAME induced a 4-fold increase in renal efflux of 20-HETE, as did L-nitroarginine. Oral treatment with 2% sodium chloride (NaCl) for 7 days increased renal epoxygenase activity, both in the cortex and the medulla. In contrast, cortical omega-hydroxylase activity was reduced by treatment with 2% NaCl. Coadministration of L-NAME and 2% NaCl decreased conversion of [(14)C]AA to HETEs without affecting epoxygenase activity. Thus, inhibition of NOS increased omega-hydroxylase activity, CYP4A expression, and renal efflux of 20-HETE, whereas 2% NaCl stimulated epoxygenase activity.

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.

Aspirin enhances the antihypertensive effect of captopril in spontaneously hypertensive rats.

Activation of renal or vascular prostaglandin mechanisms (or both) has been proposed to contribute to the antihypertensive action of captopril. In conscious spontaneously hypertensive rats (SHR) studied in the established phase of hypertension, the blood pressure-lowering effect of captopril, 30 mg/kg/12 hr p.o. given for 7 days, was greatly enhanced by the addition of aspirin, 200 mg/kg/day s.c. Systolic blood pressure decreased from 185 +/- 6 and 182 +/- 4 to 135 +/- 3 mm Hg in rats treated, respectively, with captopril and aspirin or captopril alone, and was unaltered by either vehicle or aspirin alone. Water intake was inconsistently affected by captopril but was increased (p less than 0.01) by aspirin and was even higher after captopril-aspirin treatment (p less than 0.01). Urine volume was elevated in all 3 drug-treated groups, increasing threefold after captopril-aspirin treatment. Excretion of sodium and potassium was unchanged by any treatment regimen. In the vehicle group, prostaglandin F2 alpha excretion, measured by radioimmunoassay, ranged between 65 and 93 ng/8 hr and was twofold to fourfold higher than that of prostaglandin E2. Prostaglandin F2 alpha was unaffected during captopril treatment, whereas prostaglandin E2 excretion decreased to 12 +/- 2 ng/8 hr (p less than 0.01) by Day 7. Long-term aspirin treatment, either with or without captopril, did not cause sustained inhibition of renal prostaglandin excretion, although a transient effect occurred within the first four hours of administration. These results indicate 1) aspirin potentiates the blood pressure-lowering effect of captopril in SHR, an effect that is associated with a threefold increase in urine flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelial mechanism in the vascular action of hydralazine.

Relaxation of precontracted isolated chains of aortic rings with intact endothelium and in those with the endothelium removed was studied in response to various antihypertensive vasodilator drugs. Of the drugs tested--nitroprusside, nitroglycerin, prazosin, minoxidil, diazoxide and hydralazine--only the vascular relaxant effects of hydralazine were found to be dependent, in part, on the presence of intact endothelium. The endothelial component of the hydralazine response represented a major contribution to the net relaxant effect on the vascular smooth muscle, particularly at lower concentrations, 90 nM to 1 microM, which are also clinically relevant.

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.

Renal vascular responsiveness to arachidonic acid in experimental diabetes.

1. Isolated perfused kidneys from diabetic rats (duration 4-6 and 20-24 weeks) were more sensitive to the vasoconstrictor effects of arachidonic acid than kidneys from age-matched control rats. Sensitivity diminished with age in both control and diabetic groups. 2. The enhanced vasoconstrictor effect of arachidonic acid in diabetic rat kidneys was associated with increased conversion to prostaglandins. 3. The renal vasoconstrictor response to arachidonic acid in both groups was reduced by thromboxane A2/prostaglandin H2 receptor antagonism but not by inhibition of thromboxane synthase. 4. Diabetic rat kidneys were also more sensitive to the vasoconstrictor effects of the endoperoxide analogue, U46619, while vasoconstrictor responses to phenylephrine were not markedly different from those of control rat kidneys. 5. In conclusion, prostaglandin endoperoxides appear to mediate arachidonic acid-induced vasoconstriction in diabetic and control rat kidneys. The enhanced renal vasoconstrictor response to arachidonic acid in diabetic rats results from increased sensitivity to endoperoxides and increased formation of endoperoxides from 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 F2 alpha. 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.

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 effect 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 glibenclamide, 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 Ca(2+)-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 Ca(2+)-activated K+ channel to produce vasodilatation in the rat heart.

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 approximately 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.

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 Ca(2+)-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 Ca(2+)-activated K+ channels, respectively, were without effect on vasodilator responses to bradykinin, cromakalim or nitroprusside. 6. These results implicate K+ channels, specifically Ca(2+)-activated K+ channels of intermediate conductance, in the renal vasodilator effect of bradykinin and, thereby, support a role for a hyperpolarizing factor.

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 microM) and cyclo-oxygenase with indomethacin (2.8 microM) 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 microM) and 7-ethoxyresorufin (1 microM). 17-Octadecynoic acid (17-ODYA 2 microM), 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.

Hyperpolarizing factors.

There is now overwhelming evidence for factors, other than nitric oxide (NO), that mediate endothelium-dependent vasodilation by hyperpolarizing the underlying smooth muscle via activation of Ca2+-activated K+ channels. Although the identity of endothelium-derived hyperpolarizing factor (EDHF) remains to be established, cytochrome P450 (CYP)-dependent metabolites of arachidonic acid (AA), namely, the epoxides, fulfill several of the criteria required for consideration as putative mediators of endothelium-dependent hyperpolarization. They are produced by the endothelium, released in response to vasoactive hormones, and elicit vasorelaxation via stimulation of Ca2+-activated K+ channels. Our studies in the rat indicate that, of the epoxides, 5,6-epoxyeicosatrienoic acid (5,6-EET) is the most likely mediator of NO-independent, but CYP-dependent coronary vasodilation in response to bradykinin. Studies in the rat kidney, however, support the existence of additional EDHFs as acetylcholine also exhibits NO-independent vasodilation that is unaffected by CYP inhibitors in concentrations that attenuate responses to bradykinin. In some blood vessels, NO may tonically suppress the expression of CYP-dependent EDHF. In the event of impaired NO synthesis, therefore, a CYP-dependent vasodilator mechanism may serve as a backup to a primary NO-dependent mechanism, although they may act in concert. In other vessels, particularly microvessels, an EDHF may constitute the major vasodilator mechanism for hormones and other physiological stimuli. EDHFs appear to be important regulators of vascular tone; alterations in this system can be demonstrated in hypertension and diabetes, conditions associated with altered endothelium-dependent vasodilator responsiveness.

Evidence against a role of arachidonic acid metabolites in autoregulatory responses of the isolated perfused kidney of the rat.

The role of arachidonic acid metabolites in renal autoregulatory responses to changes in pressure was examined in rat isolated perfused kidneys. We also studied the influence of diabetes, a condition associated with hyperfiltration and altered renal eicosanoid production, on autoregulatory responses. The perfused rat kidney demonstrated autoregulation of flow within a pressure range of 100-150 mm Hg, with no differences between diabetic and control rat kidneys. Nifedipine resulted in vasodilatation and loss of autoregulation. Inhibition of the cyclooxygenase pathway of arachidonic acid metabolism with indomethacin failed to alter autoregulatory capacity. Similarly, inhibition of lipoxygenase with BW755C or NDGA, or inhibition of cytochrome P450-dependent enzymes with NDGA, clotrimazole or 7-ethoxyresorufin were without effect on autoregulatory responses. In vivo treatment with stannous chloride to deplete renal cytochrome P450-dependent enzymes also failed to modify autoregulatory responses. These results argue against a role of arachidonic acid metabolites in autoregulation of perfusate flow in the isolated kidney.

Captopril decreases vascular reactivity independently of changes in converting enzyme activity and prostaglandin release in the rat isolated kidney.

The relationship of the vascular effect of captopril to angiotensin converting enzyme activity and prostaglandin-dependent mechanism was studied in rat isolated kidneys, perfused with Krebs-Henseleit at 20 ml/min per 2 kidneys, with basal perfusion pressures of 78 +/- 1 mm Hg (Mean +/- S.E.M.). Two doses of captopril were used; both low (0.05 microgram/ml) and high doses (5 microgram/ml) inhibited maximally the vasoconstrictor responses to 100 and 200 ng of angiotensin I. Captopril at the low dose did not affect the renal vasoconstrictor responses to norepinephrine (NE) (25-400 ng), whereas high-dose reduced the vasoconstriction to all doses of NE. Treatment with captopril tended to diminish dose-related release of prostaglandins in response to NE. Indomethacin (1 microgram/ml) prevented NE-induced release of bioassayable and radioimmunoassayable prostaglandins but did not affect the ability of captopril to reduce NE-induced vasoconstriction. High-dose captopril also decreased the vascular reactivity to angiotensin II (5 ng) and lysine vasopressin (10 mU); however, the renal vasoconstriction caused by PGE2 (80 ng) was unaffected by captopril. We conclude that high-dose captopril decreased vascular reactivity by a mechanism independent of converting enzyme inhibition and unrelated to a prostaglandin-dependent vascular mechanism.

The effect of oral antiplatelet agents on tissue plasminogen activator-mediated thrombolysis in a rabbit model of thromboembolic stroke.

OBJECTIVE: The success of thrombolytic therapy in acute stroke relies on timely reperfusion. The current study examines the efficacy of antiplatelet agents as adjuvants for thrombolytic therapy. METHODS: Using an established rabbit model of clot embolization and a randomized blinded design, rabbits (n = 8 in each group) were orally pretreated daily for 5 days with adjuvant aspirin (1 mg/kg of body weight or 20 mg/kg), ticlopidine (100 mg/kg), or vehicle (sodium carbonate). On the 6th day, tissue plasminogen activator (6.3 mg/kg administered intravenously over 2 h), was initiated 1 hour after embolization. RESULTS: In all groups, cerebral blood flow (CBF) was reduced to < 10 ml/100 g/min immediately after clot embolization. After the initiation of tissue plasminogen activator (t-PA), there was significant restoration of CBF in the control (t-PA only) and ticlopidine groups (P < 0.05) only. Restoration of CBF generally correlated with brain infarct size (percent hemisphere, mean +/- standard error of the mean), which was 18.0 +/- 7.0 in the t-PA only group versus 11.0 +/- 3.3, 26.5 +/- 5.8, and 21.5 +/- 3.4 in the ticlopidine, low-dose aspirin, and high-dose aspirin groups, respectively (ticlopidine versus aspirin, P < 0.05). Clot lysis was identical in the control and ticlopidine groups, with 6 of 8 animals demonstrating complete clot lysis. Aspirin antagonized clot lysis in a dose-related manner, with low-and high-dose aspirin groups noting clot lysis in four of eight and two of eight animals, respectively. CONCLUSIONS: Pretreatment with ticlopidine significantly reduced brain infarct size when compared with aspirin treatment (P < 0.05). Moreover, whereas ticlopidine treatment did not affect clot lysis or CBF relative to t-PA alone, aspirin therapy resulted in antagonism of clot lysis and was associated with a more modest restoration of blood flow. This study provides a background for a more comprehensive understanding of the balance of thrombogenicity and thrombolysis and may assist in the development of novel therapies to expedite cerebrovascular patency and reduce ischemic and reperfusion-mediated neuronal injury.

Hypotensive effects of eicosapentaenoic acid (EPA) and its influence on eicosanoid metabolism in spontaneously hypertensive rats.

Oral administration of pure eicosapentaenoic acid (EPA) ethyl ester caused a reversible hypotensive effect in spontaneously hypertensive rats. Treatment with EPA also lowered the platelet response to ADP and inhibited the conversion of leucotrienes (LTs) A4 and LTB4 and other sulphidopeptide leucotrienes. Therefore, diet supplemented with EPA may lower blood pressure in hypertensive rats and affect cardiovascular functions which may be mediated through eicosanoid metabolism.

Apamin/charybdotoxin-sensitive endothelial K+ channels contribute to acetylcholine-induced, NO-dependent vasorelaxation of rat aorta.

BACKGROUND: Activation of endothelial K+ channels and the subsequent increase in intracellular Ca2+, may be an important step in the release of relaxant factors in response to endothelium-dependent vasodilator agents. However, the type of K+ channel involved in hyperpolarization of the endothelium and the subsequent release of relaxing factors remains to be defined. MATERIAL AND METHODS: Rat aortic rings precontracted with U46619 were used to address the effects of inhibitors of K+ channels on the vasorelaxant response to acetylcholine (Ach). As responses to Ach were mediated solely by endothelium-derived NO and responses to NO derived from nitroprusside were unaffected by inhibition K+ channels, any effect of K+ channel inhibitors could be attributed to actions on endothelial K+ channels to modify NO release. RESULTS: Tetraethylammonium (TEA) and elevated K+ attenuated the relaxant effect of Ach, indicating a role for K+ channels in NO release. The Ca2+-activated K+ channel inhibitors, apamin, charybdotoxin and iberiotoxin as well as glibenclamide and BaCl2, inhibitors of ATP-sensitive K+ channels and inwardly rectifying K+ channels, respectively, did not affect the response to Ach. However, a combination of apamin and charybdotoxin, but not apamin and iberiotoxin, attenuated the vasorelaxant response to Ach. CONCLUSIONS: The results of this study indicate that NO release in response to Ach involves activation of an endothelial K+ channel that is inhibited by a combination of apamin and charybdotoxin.