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.

Inhibitors of 20-hydroxyeicosatetraenoic acid reduce renal vasoconstrictor responsiveness.

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450-derived constrictor eicosanoid produced by the preglomerular vasculature where it contributes to regulation of tone. Removal of the tonic inhibitory influence of nitric oxide (NO) has been reported to increase renal 20-HETE release. Because inhibition of NO synthesis enhances responses to vasoconstrictor agents, we examined a contribution for increased 20-HETE generation. In the rat kidney perfused with Krebs' buffer, responses to U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy PGF2alpha), a thromboxane A2 mimetic, were compared before and after 50 microM L-nitroarginine (L-NA) to inhibit NO synthase. L-NA raised perfusion pressure (PP) from 79 +/- 3 to 190 +/- 7 mm Hg and enhanced constrictor responsiveness to U46619. U46619 (10, 30, 100, and 300 ng) increased PP by 7 +/- 1, 17 +/- 2, 50 +/- 7, and 67 +/- 7 mm Hg, respectively, before L-NA and 15 +/- 1, 37 +/- 7, 68 +/- 10, and 85 +/- 11 mm Hg, respectively, after L-NA, which did not increase 20-HETE efflux from the kidney. Nonetheless, an inhibitor of omega-hydroxylase, dibromododecencyl methylsulfonimide (DDMS), which reduced 20-HETE release, normalized the enhanced responsiveness to U46619. When PP was elevated with phenylephrine, vasoconstrictor responses to U46619 were similarly enhanced, an effect that was also prevented by DDMS. DDMS and an antagonist of 20-HETE, 20-HEDE [20-hydroxyeicosa-6(Z), 15(Z)-dienoic acid], also reduced vasoconstrictor responses to U46619 in the absence of elevation of PP. Because 20-HETE inhibits K+ channels, we examined the effects of K+ channel inhibitors on vasoconstrictor responses and showed that both tetraethylammonium (TEA) and charybdotoxin enhanced renal vasoconstrictor responses to U46619. However, the inhibitory effects of 20-HEDE on vasoconstrictor responses remained after treatment with TEA. These results support a role for 20-HETE vasoconstrictor responses but suggest an action independent of K+ channels.

The nitric oxide- and prostaglandin-independent component of the renal vasodilator effect of thimerosal is mediated by epoxyeicosatrienoic acids.

Epoxyeicosatrienoic acids (EETs) are cytochrome P450-derived metabolites of arachidonic acid that elicit vasodilation via activation of K(+) channels. They have been implicated as endothelium-derived hyperpolarizing factors (EDHFs), mediating the effect of some endothelium-dependent vasodilator agents such as bradykinin in some vascular tissues. We reasoned that an agent that increases the availability of free arachidonic acid should also elicit cytochrome P450-dependent vasodilation that is associated with increased release of EETs and attenuated by agents that inhibit the synthesis or action of EETs. Thus, we used thimerosal as an inhibitor of reacylation of arachidonic acid and determined the contribution of prostaglandins, nitric oxide, and EETs to the vasodilator effect in the isolated, perfused, preconstricted kidney of the rat. Thimerosal elicited vasodilator responses that were unaffected by inhibition of cyclooxygenase with indomethacin but were reduced by the further inhibition of nitric oxide synthesis. The vasodilator activity that remained after inhibition of cyclooxygenase and nitric oxide synthase was reduced by inhibition of K(+) channels with tetraethylammonium and was associated with increased release of EETs measured by gas chromatography-mass spectroscopy following hydrolysis to the corresponding diols. Inhibition of cytochrome P450 with miconazole or epoxygenase with N-methylsulfonyl-6-(2-propargyloxyphenyl)hexamide reduced the nitric oxide- and prostaglandin-independent vasodilator effect of thimerosal and attenuated the increase in the release of EETs. We conclude that thimerosal causes vasodilation of the isolated perfused kidney via nitric oxide-dependent and -independent mechanisms. The nitric oxide-independent component of the response involves activation of K(+) channels and is likely mediated by EETs, possibly acting as EDHFs.

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.

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.

Peroxynitrite attenuates hepatic ischemia-reperfusion injury.

In the present study, we examined the effects of peroxynitrite on reperfusion injury using a rat model of hepatic ischemia-reperfusion (HI/R). The left and median lobes of the liver were subjected to 30 min of ischemia, followed by 4 h of reperfusion. Groups A and B rats were sham-operated controls that received vehicle or peroxynitrite; groups C and D rats were subjected to HI/R and received peroxynitrite or vehicle, respectively. A dose of 2 micromol/kg body wt of peroxynitrite, diluted in saline (pH 9.0, 4 degrees C), was administered as a bolus through a portal vein catheter at 0, 60, and 120 min after reperfusion. Results showed that superoxide generation in the ischemic lobes of the liver and plasma alanine aminotransferase (ALT) activity of group C were decreased by 43% and 45%, respectively, compared with group D. Leukocyte accumulations in the ischemic lobes of liver and circulating leukocytes were decreased by 40% and 27%, respectively, in group C vs. D. The ratios of mRNA of P-selectin and intercellular adhesion molecule-1 (ICAM-1) to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA extracted from the ischemic lobes of the liver of group C were decreased compared with group D. There were no differences between the groups A and B in terms of plasma ALT activity, circulating leukocytes, superoxide generation, and leukocyte infiltration in the ischemic lobes of the liver. Moreover, hemodynamic parameters (i.e., mean arterial blood pressure, cardiac index, stroke index, and systemic vascular resistance) were not significantly different among groups B, C, and D. These results suggest that administration of peroxynitrite via the portal vein only has a local effect. Exogenous peroxynitrite at physiological concentrations attenuates leukocyte-endothelial interaction and reduces leukocyte infiltration. The mechanism of the reduction of leukocyte infiltration into ischemic lobes of the liver appears because of decreased expression of mRNA of P-selectin and ICAM-1. The net effect of administration of peroxynitrite may be to reduce adhesion molecule-mediated, leukocyte-dependent reperfusion injury.

Vascular effects of arachidonic acid in the rat perfused heart. Role of the endothelium, cyclooxygenase, cytochrome P450, and K(+) channels.

The vascular effects of arachidonic acid (AA) were addressed in the rat perfused heart in terms of metabolic pathways and effector mechanisms. Under basal perfusion pressure, AA elicited dilator responses. However, in hearts treated with nitroarginine to eliminate nitric oxide and to elevate perfusion pressure, the predominant effect of AA was vasoconstriction which was converted to a vasodilator effect by inhibition of cyclooxygenase or antagonism of TP receptors. The vasodilator effect of AA in nitroarginine- and indomethacin-treated hearts was greatly attenuated by clotrimazole, an inhibitor of cytochrome P450, and by inhibition of K(+) channels with tetraethylammonium; in the absence of indomethacin, clotrimazole enhanced the vasoconstrictor effect of AA. When endothelin was used to constrict the coronary vasculature, AA also produced cyclooxygenase-dependent vasoconstriction. In hearts constricted with the endoperoxide analogue, U46619, only endothelium-dependent vasodilator effects of AA were observed that were reduced by indomethacin or clotrimazole. These results indicate that the coronary vasoconstrictor effect of AA which is expressed with elevated tone, results from its conversion by cyclooxygenase to a product(s) that activates TP receptors. The vasodilator effect exhibits two endothelium-dependent components, one mediated by cyclooxygenase products and the other by a cytochrome P450-derived product that activates K(+) channels.

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.

20-HETE and the kidney: resolution of old problems and new beginnings.

The protean properties of 20-hydroxyeicosatetraenoic acid (HETE), vasoactivity, mitogenicity, and modulation of transport in key nephron segments, serve as the basis for the essential roles of 20-HETE in the regulation of the renal circulation and electrolyte excretion and as a second messenger for endothelin-1 and mediator of selective renal effects of ANG II. Renal autoregulation and tubular glomerular feedback are mediated by 20-HETE through constriction of preglomerular arterioles, responses that are maintained by 20-HETE inhibition of calcium-activated potassium channels. 20-HETE modulates ion transport in the proximal tubules and the thick ascending limb by affecting the activities of Na+-K+-ATPase and the Na+-K+-2Cl- cotransporter, respectively. The range and diversity of activity of 20-HETE derives in large measure from COX-dependent transformation of 20-HETE to products affecting vasomotion and salt and water excretion. Nitric oxide (NO) exerts a negative modulatory effect on 20-HETE formation; inhibition of NO synthesis produces marked perturbation of renal function resulting from increased 20-HETE production. 20-HETE is an essential component of interactions involving several hormonal systems that have central roles in blood pressure homeostasis, including angiotensins, endothelins, NO, and cytokines. 20-HETE is the preeminent renal eicosanoid, overshadowing PGE2 and PGI2. This review is intended to provide evidence for the physiological roles for cytochrome P-450-derived eicosanoids, particularly 20-HETE, and seeks to extend this knowledge to a conceptual framework for overall cardiovascular function.

Pharmacological evaluation of an epoxide as the putative hyperpolarizing factor mediating the nitric oxide-independent vasodilator effect of bradykinin in the rat heart.

A cytochrome P450-derived metabolite of arachidonic acid, namely an epoxyeicosatrienoic acid (EET), has many of the properties of a hyperpolarizing factor that mediates endothelium-dependent, nitric oxide-independent vasodilation. As there are four EET regioisomers, we used pharmacological criteria, based on previous observations with bradykinin (BK), to evaluate which, if any, of the EETs could be considered a potential mediator of vasodilator responses to BK in the rat isolated heart treated with indomethacin and nitroarginine to eliminate prostaglandin and nitric oxide components of the response. Nifedipine, used as a probe for dilator mechanisms dependent on closure of voltage-dependent Ca++ channels, almost abolished the vasodilator effect of cromakalim and attenuated those of BK and 5,6 EET. The vasodilator effects of the other EETs were not reduced and were excluded from consideration as mediators of BK-induced vasodilation. The vasodilator effect of 5,6 EET, as with that of BK, was markedly reduced by charybdotoxin but not iberiotoxin, suggesting the contribution of a similar type K+ channel to the vascular response to both agents. As expected for a putative endothelium- and cytochrome P450-derived mediator, the coronary vasodilator effect of 5,6 EET was not affected by either removal of the endothelium or inhibition of cytochrome P450 with clotrimazole, interventions that virtually abolished the vasodilator activity of BK. Thus, of the four EET regioisomers, 5,6 EET is the most likely mediator of the vasodilator effect of BK in the isolated heart under these experimental conditions.

Evidence against anandamide as the hyperpolarizing factor mediating the nitric oxide-independent coronary vasodilator effect of bradykinin in the rat.

The mediator of nitric oxide-(NO) independent vasodilation attributed to endothelium-derived hyperpolarizing factor remains unidentified although there is evidence for a cytochrome P450-derived eicosanoid. Anandamide, the ethanolamide of arachidonic acid and an endogenous ligand for cannabinoid receptors, was proposed as an endothelium-derived hyperpolarizing factor-mediating mesenteric vasodilation to acetylcholine and the hypotensive effect of bradykinin. Using pharmacological interventions that attenuate responses to bradykinin, we examined the possibility of anandamide as a mediator of the NO-independent vasodilator effect of bradykinin in the rat perfused heart by determining responses to anandamide and arachidonic acid. Hearts were treated with indomethacin to exclude prostaglandins and nitroarginine to inhibit NO synthesis and elevate perfusion pressure. The cannabinoid receptor antagonist, SR 141716A (2 microM), reduced dose-dependent vasodilator responses to anandamide (1-10 microgram) but was without effect on responses to AA (1-10 microgram), bradykinin (10-1000 ng) or cromakalim (1-10 microgram). Inhibition of voltage-dependent Ca++ channels with nifedipine (5 nM) attenuated vasodilation to anandamide and arachidonic acid whereas inhibition of Ca++-activated K+ channels with charybdotoxin (10 nM) reduced responses to arachidonic acid but had no effect on vasodilation induced by anandamide. Inhibition of cytochrome P450 with clotrimazole (1 microM) greatly reduced vasodilator responses to bradykinin with less effect on those to anandamide. Finally, the time course of the coronary vasodilator responses to anandamide and bradykinin were dissimilar. These results argue against a role of anandamide in the vasodilator effect of bradykinin in the rat heart.

Vascular and excretory effects of angiotensin II in the rat isolated perfused kidney: influence of an AT1 and a nonselective AT receptor antagonist.

Angiotensin II (AII) is a potent vasoconstrictor which, at physiological plasma concentrations, produces antinatriuresis, whereas high intrarenal concentrations cause natriuresis and diuresis. We examined the effects of a selective AT1 receptor antagonist, losartan, and a nonselective AT receptor antagonist, Sar1Thr8AII, on the response to infusion of AII in the isolated rat kidney perfused at constant pressure with a recirculating modified Krebs-Henseleit buffer. AII increased renal vascular resistance (RVR), glomerular filtration rate (GFR) and urinary volume (UV) and sodium excretion (UNaV) without changing the fractional excretion of water or electrolytes. Thus, changes in GFR can account for the natriuresis/diuresis. Both AII receptor antagonists prevented the increase in RVR. However, losartan was without effect on angiotensin-induced increases in GFR, UV or UNaV, whereas Sar1Thr8 AII also prevented the increases in GFR, UV and UNaV. The angiotensin receptor mediating the increase in GFR can be dissociated from that mediating the increase in RVR, providing functional evidence of angiotensin receptor subtypes in the rat kidney.

A method for the determination of 5,6-EET using the lactone as an intermediate in the formation of the diol.

The 5,6 epoxyeicosatrienoic acid (5,6-EET) exhibits a range of biological activities but the functional significance of this labile eicosanoid is unknown due, in part, to difficulties of quantitation in biological samples. We have developed a sensitive and specific method to measure 5,6-EET utilizing its selective capacity to form a lactone. The initial conversion of 5,6-EET and 5,6-dihydroxyeicosatrienoic acid (5,6-DHT) to 5,6-delta-lactone is followed by selective purification using reverse phase high performance liquid chromatography (HPLC), reconversion to 5,6-DHT and quantitation by gas chromatography-mass spectrometry (GCMS). In oxygenated Krebs' buffer, 5,6-EET degrades to 5,6-delta-lactone and 5,6-DHT with a t1/2 approximately 8 min. In the presence of camphorsulfonic acid, 5,6-EET and 5,6-DHT convert to a single HPLC peak (lambda = 205) comigrating with 5,6-delta-lactone. Incubation of 5,6-delta-lactone with triethylamine resulted in a single HPLC peak with the retention time of 5,6-DHT. In the perfusate from the isolated kidney, release of 5,6-EET (20 +/- 5 pg/ml), measured indirectly via conversion to 5,6-DHT, was approx. 6-fold less than that reported for prostaglandin E2 (PGE2) and 20-HETE. The coronary perfusate concentration of 5,6 EET was 9 +/- 2 pg/ml. 5,6-EET recovered from renal and coronary perfusates was increased 2-fold to 45.5 +/- 5.5 pg/ml and 21.6 +/- 6.3 pg/ml, respectively, by arachidonic acid.

NO-independent vasodilation to acetylcholine in the rat isolated kidney utilizes a charybdotoxin-sensitive, intermediate-conductance Ca(++)-activated K+ channel.

The role of K+ channels in the nitric oxide-independent renal vasodilator effect of acetylcholine (Ach) was examined to address the hypothesis that the mechanism underlying this response was different from that of bradykinin, because an earlier study indicated the possibility of different mediators. We used the rat isolated, perfused kidney that was constricted with phenylephrine and treated with nitroarginine and indomethacin to inhibit nitric oxide synthase and cyclooxygenase, respectively. The nonspecific K+ channel inhibitors, procaine and tetraethylammonium (TEA), reduced vasodilator responses to Ach and cromakalim, but not those to nitroprusside. Glibenclamide, an inhibitor of ATP-sensitive K+ channels, reduced vasodilator responses to cromakalim but did not affect those to Ach or nitroprusside. Charybdotoxin, an inhibitor of Ca(++)-activated K+ channels, reduced vasodilator responses to Ach without affecting those to cromakalim or nitroprusside. Iberiotoxin and apamin, inhibitors of large- and small-conductance Ca(++)-activated K+ channels, respectively, did not reduce vasodilation induced by Ach, cromakalim or nitroprusside. The inhibitor of cytochrome P450, clotrimazole, reduced the renal vasodilator effects of Ach and bradykinin but not those of nitroprusside or SCA 40, an agonist for Ca(++)-activated K+ channels. These results suggest that in the rat kidney, Ach, like bradykinin, utilizes a charybdotoxin-sensitive Ca(++)-activated K+ channel of intermediate conductance to elicit vasodilation and that this effect may be dependent on cytochrome P450 activity.

Analysis of eicosanoid mediation of the renal functional effects of hyperchloremia.

Depression of GFR and antinatriuresis in response to high chloride has been linked to a cyclooxygenase (COX)-dependent mechanism involving thromboxane A2 (TxA2) and prostaglandin endoperoxide (PGH2), because inhibition of COX prevented the fall in GFR and antinatriuresis produced by hyperchloremia. However, hyperchloremia did not increase, but unexpectedly decreased, renal prostaglandin and TxA2 efflux (Yin et al., 1995). To resolve questions regarding the role of eicosanoids in mediating the renal functional effects of high chloride (117 mM), by stimulating either TxA2 synthesis or TxA2/PGH2 receptors, we compared the ability of indomethacin to block high-chloride effects in the rat isolated kidney with that of BMS 180291 and SQ 29548, antagonists of the TxA2/PGH2 receptor. These antagonists differ in terms of their selectivity and their capacity to inhibit isoforms of the TxA2/PGH2 receptor. Indomethacin and SQ 29548 had identical actions, preventing the decrease of GFR and antinatriuresis evoked by hyperchloremia, e.g., sodium excretion rate in the SQ 29548 and indomethacin groups increased to 7.2 +/- 1.3 and 7.1 +/- 1.2 microEq/min, respectively, compared with 2.6 +/- 0.7 microEq/min in the control group. In contrast, neither BMS 180291 nor the TxA2 synthase inhibitors, OKY 046 and CGS 13080, modified the negative effects of high chloride on GFR or sodium excretion. These results argue against either TxA2 or PGH2 acting as mediator of the effects of high chloride on renal function and suggest a product of COX activity such as a 20-HETE analog of prostaglandin endoperoxide. Evidence to support this proposal was obtained: 1) Hyperchloremia increased 20-HETE release from the rat kidney by 2-fold when compared with low-chloride conditions of renal perfusion. 2) The renal vasoconstrictor action of 20-HETE was shown to be dependent on COX activity and to be antagonized by blockade of the TxA2/PGH2 receptor.

Evidence against a cytochrome P450-derived reactive oxygen species as the mediator of the nitric oxide-independent vasodilator effect of bradykinin in the perfused heart of the rat.

The coronary vasodilator effect of bradykinin (BK) in the rat is independent of NO but dependent on activation of phospholipases with involvement of cytochrome P450 mono-oxygenase (P450) and stimulation of Ca++-activated K+ channels, implicating an unidentified hyperpolarizing factor generated via P450 metabolism of arachidonic acid (AA). Because P450 activity also generates free radicals, such as superoxide, which can lead to the formation of hydrogen peroxide and hydroxyl radicals, which are vasoactive, we addressed the contribution of superoxide to the vasodilator effect of BK in the rat heart. Using rat renal microsomes as a source of P450, we verified that P450-dependent metabolism of AA generated superoxide, as detected by chemiluminescence with lucigenin. The signal was almost abolished by inhibition of P450 with clotrimazole and the superoxide scavenger 4,5-dihydroxy-1,3-benzene sulfonic acid. However, base-line superoxide formation, detected by chemiluminescence, in cardiac slices and perfused hearts was unchanged in response to BK or AA. Furthermore, in perfused hearts treated with nitroarginine and indomethacin to eliminate NO and prostaglandins and elevate perfusion pressure, dose-dependent vasodilator responses to BK were unaffected by superoxide dismutase plus catalase, a combination that abolished dilator responses to hydrogen peroxide. Similarly, the superoxide scavengers 4,5-dihydroxy-1,3-benzene sulfonic acid and 4-hydroxy-2,2,6,6-tetramethylpiperidine-noxyl were without effect on vasodilator responses to BK. Thus, the coronary vasodilator action of BK is independent of superoxide or its derivatives, which can be excluded as hyperpolarizing factors mediating NO-independent vasodilation in the rat.

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.

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.

Role of phospholipase C and phospholipase A2 in the nitric oxide-independent vasodilator effect of bradykinin in the rat perfused heart.

The cytochrome P450-dependent component of the coronary vasodilator action of bradykinin which requires activation of K+ channels was examined in terms of the contribution of phospholipases in the rat Langendorff heart preparation. This component was isolated by inhibition of nitric oxide synthase with nitroarginine and cyclooxygenase with indomethacin, neither of which affects the coronary vasodilator action of bradykinin. However, nitroarginine elevated coronary perfusion pressure from approximately 40 to 130 mm Hg. The phospholipase C inhibitor, U73122 {1-(6-((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl)-1H-pyrrole-2,5-dione}, reduced coronary vasodilator responses to bradykinin by greater than 80%. U73122 also diminished the coronary vasodilator action of cromakalim which activates ATP-sensitive K+ channels. The maleimide moiety of U73122 that has the capacity to affect K+ channels inhibited cromakalim-induced coronary vasodilation, but did not affect that to bradykinin. Inhibition of diacylglycerol lipase with RHC 80267 {1,6-bis-(cyclohexyloximinocarbonylamino)-hexane} was without an overall effect on coronary vasodilator responses to bradykinin. The cytosolic phospholipase A2 inhibitor, AACOCF3 {arachidonyl trifluoromethyl ketone¿} decreased responses to bradykinin by up to 90% whereas inhibitors of the secretory form of phospholipase A2 oleyloxyethyl phosphorylcholine and ONO-RS-082 {2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid} were less effective than either AACOCF3 or U73122. The phospholipase inhibitors demonstrated selectivity as they did not affect the coronary vasodilator responses to nitroprusside. We obtained additional evidence for the antiphospholipase activity of the inhibitors by demonstrating their capacity to suppress bradykinin-stimulated increases in the release of prostacyclin, measured as 6-keto prostaglandin F1 alpha. The phospholipase inhibitors did not affect cyclooxygenase activity as the ability of arachidonic acid to stimulate prostaglandin formation was unimpaired. These results indicate that the coronary vasodilator action of bradykinin is linked to the activities of both phospholipase C and A2.