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Deoxycorticosterone (5 mg) and aldosterone (0.25 mg), given to rats for 14 days, increased the urinary excretion of kallikrein and of prostaglandin E-like substance and produced polyuria, but affected neither sodium excretion nor blood pressure. These results suggest that interactions of mineralocorticoid hormones, kinins and of prostaglandins may be important in the maintenance of salt-water homeostasis.

Prostaglandins modulate the effects of vasoactive hormones by attenuating the renal actions of the renin-angiotensin system and contributing to and, perhaps, mediating some of those of the kallikrein-kinin system. A prostaglandin mechanism participates in the regulation of renin and erythropoietin release. When renal function is compromised, the circulation to the kidney is sustained by a major prostaglandin component withdrawal of which results in significant hemodynamic effects, particularly reduction of blood flow to the inner cortex and medulla.

Red blood cells (RBCs) are reservoirs of vasodilatory, antiaggregatory, and antiinflammatory lipid mediators-epoxyeicosatrienoic acids (EETs). This study addresses the formation and release of erythrocyte-derived EETs in response to ATP receptor stimulation that may represent an important mechanism regarding circulatory regulation.Erythrocyte EET formation and release were investigated by incubating rat RBCs in physiological salt solution with agents that effected ATP release via P2 receptor stimulation of phospholipase A2 and epoxygenase-like activities with activation of the ATP secretory mechanism. EETs were analyzed by gas and liquid chromatography-mass spectrometry.EETs were released from rat RBCs: 14,15-, 11,12-, 8,9- and 5,6-EETs in a ratio of 1.2:1.0:0.9:0.8. EETs were produced by epoxidation of arachidonic acid catalyzed by hemoglobin. Spontaneous release of EETs, 0.66+/-0.14 ng per 10(9) RBCs, was dose-dependently increased by an ATP analog, BzATP, and inhibited by P2X(7) receptor antagonists. 5 microM ATP increased release of EETs over 20% to 0.83+/-0.15 ng per 10(9) RBCs; 10 microM BzATP tripled the amount of EET release to 1.87+/-0.20 ng per 10(9) RBCs. EET release by ATP or BzATP was not associated with hemolysis. Carbenoxolone, a gap junction inhibitor that inhibits ATP release, and glibenclamide, an inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR), which is required for ATP release, inhibited the spontaneous and stimulated EET release from RBCs.EETs are produced and released from RBCs via a mechanism that is mediated by ATP stimulation of P2X(7) receptors coupled to ATP transporters, pannexin-1 and CFTR.

Prostacyclin (PGI2) and thromboxane (TxA2) labile cyclooxygenase (COX) products via PGH2 were identified in biological fluids by the ingenious application of the principle of parallel pharmacological assays developed by John Vane. Either organ perfusates or circulating blood superfuse bioassay tissues arranged in a cascade. Tissues were selected based on specificity of responses to targeted eicosanoids. Additionally, PGI2 inhibited platelet aggregation, a finding that led to discovery of its critical anti-thrombotic activity at the blood-endothelial interface. The biological activities of PGI2 and TxA2 were the fingerprints for tracking their isolation and ultimate chemical identification. These studies were responsible for opening the modern era of vascular biology that has facilitated the development of a rational approach to the treatment of diabetic and hypertensive complications involving the arterial circulation.

Nephron function is segmented, each segment has characteristic transport mechanisms and individual eicosanoid profiles. The transport function of the medullary thick ascending limb of Henle's loop (mTAL) establishes the osmolar gradient upon which extra cellular fluid volume (ECFV) conservation depends. The overriding importance of the mTAL to regulation of ECFV is evident in the diuretic-natriuretic potency of furosemide-like agents which target the mTAL.The mTAL has been shown to be heavily invested with cytochrome P450 monooxygenases (CYP), chiefly omega/omega-1 hydroxylase activity, that generate 19- and 20-hydroxyeicosatetraenoic acid (HETEs). However, displacement of omega hydroxylase by an inducible cyclooxygenase mechanism (COX-2) can be effected by several interventions: long-term infusion of angiotensin II (ANG II), adrenalectomy and elevated extracellular Ca2+ concentrations. This switching mechanism (CYPCOX-2) has been shown to be dependent on activation of tumor necrosis factor alpha (TNFalpha) by ANG II. It represents a long-term adaptive mechanism of the mTAL with production of PGE2 whereas in the short-term, ANG 11 increases 20-HETE synthesis by the mTAL. The effect of Ca2+ on mTAL eicosanoid-related mechanisms provides an explanation for the natriuretic response to hypercalcemia and diminished ability to concentrate urine.The expression of COX-2 in the TAL has been linked to activation of the renin-angiotensin system, glucocorticoid deficiency and hypercalcemia, all of which operate through a mechanism in which production of TNFalpha by the TAL is pivotal.

We previously showed that primary cultures of mTAL cells express cyclooxygenase 2 (COX-2) when challenged with tumor necrosis factor alpha (TNFalpha) or phorbol myristate acetate (PMA). Moreover, expression of COX-2 was linked to decreases in TNFalpha-mediated 86Rb uptake, an in vitro correlate of natriuresis. mTAL cells in primary culture express calcium sensing receptor (CaR), a G-protein coupled receptor that senses changes in extracellular calcium concentration and ultimately increases intracellular calcium concentration ([Ca2+]i) and protein kinase C (PKC) activity. PGE2 synthesis by mTAL cells increases in a dose- and time-dependent manner after exposure of these cells to extracellular Ca2+. Similar effects were observed when cells were challenged with the CaR-selective agonist, poly-L-arginine. These data suggest that intracellular signaling mechanisms initiated via activation of CaR contribute to mTAL PGE2 synthesis. As TNF production is calcium-sensitive in some cells types, we postulate that these effects involve the regulation of COX-2 expression via a TNF-dependent mechanism. The functional implications of these studies relate to a cytokine-mediated mechanism that contributes to salt and water balance, and suggests that small changes in Ca(2+)o may contribute to the regulation of these events. The possibility that the effects of Ca(2+)o involve activation of CaR suggests that novel calcimimetic molecules might be useful in conditions, such as hypertension or other conditions, in which manipulation of extracellular fluid volume provides beneficial effects.

1. We tested the hypothesis that nitric oxide (NO) exerts a tonic inhibitory influence on cytochrome P450 (CYP450)-dependent metabolism of arachidonic acid (AA). 2. N(omega)-nitro-L-Arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), increased mean blood pressure (MBP), from 91+/-6 to 137+/-5 mmHg, renal vascular resistance (RVR), from 9.9+/-0.6 to 27.4+/-2.5 mmHg ml(-1) min(-1), and reduced renal blood flow (RBF), from 9.8+/-0.7 to 6.5+/-0.6 ml min(-1)) and GFR from 1.2+/-0.2 to 0.6+/-0.2 ml 100 g(-1) min(-1)) accompanied by diuresis (UV, 1.7+/-0.3 to 4.3+/-0.8 microl 100 g(-1) min (-1)), and natriuresis (U(Na)V, 0.36+/-0.04 to 1.25+/-0.032 micromol 100 g(-1) min(-1)). 3. 12, 12 dibromododec-enoic acid (DBDD), an inhibitor of omega hydroxylase, blunted L-NAME-induced changes in MBP, RVR, UV and U(Na)V by 63+/-8, 70+/-5, 45+/-8 and 42+/-9%, respectively, and fully reversed the reduction in GFR by L-NAME. Clotrimazole, an inhibitor of the epoxygenase pathway of CYP450-dependent AA metabolism, was without effect. 4. BMS182874 (5-dimethylamino)-N-(3,4-dimethyl-5-isoxazolyl)-1-naphthalenesulfo namide), an endothelin (ET)A receptor antagonist, also blunted the increases in MBP and RVR and the diuresis/natriuresis elicited by L-NAME without affecting GFR. 5. Indomethacin blunted L-NAME-induced increases in RVR, UV and U(Na)V. BMS180291 (1S-(1alpha,2alpha,3alpha,4alpha)]-2-[[3-[4-[(++ +pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2-yl ]methyl]benzenepropanoic acid), an endoperoxide receptor antagonist, attenuated the pressor and renal haemodynamic but not the renal tubular effects of L-NAME. 6. In conclusion, the renal functional effects of the CYP450-derived mediator(s) expressed after inhibition of NOS with L-NAME were prevented by inhibiting either CYP450 omega hydroxylase or cyclooxygenase or by antagonizing either ET(A) or endoperoxide receptors. 20-hydroxyeicosatetraenoic acid (20-HETE) fulfils the salient properties of this mediator.

The renal tubular and hemodynamic effects of endothelin-1 (ET-1) were studied in the rat in terms of the participation of cytochrome P450 monooxygenases (CYP450)-derived arachidonic acid (AA) metabolites. The availability of specific mechanism-based inhibitors of CYP450-dependent AA metabolism has greatly facilitated studies designed to link AA metabolites generated by CYP450 to renal function. Eicosanoid products synthesized by cyclooxygenase (COX) and CYP450 can account for the renal functional effects of ET-1. Inhibition of COX decreased glomerular filtration rate (GFR) and potentiated the depression of GFR elicited by ET-1. In contrast, inhibition of CY-P450-dependent AA metabolism enhanced GFR and blunted ET-1 induced increase in renal vascular resistance, yet reduced the diuretic response to ET-1. Thus, CYP450-dependent AA products depress GFR and renal blood flow, while promoting sodium excretion. The effects of ET-1 on renal function correspond to those of 20-HETE, the predominant renal CYP450-derived AA metabolite.

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.

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.

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.

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.

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.

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.

5,6-Epoxyeicosatrienoic acid (5,6-EET), a cytochrome P450-dependent arachidonate product, is a substrate for cyclooxygenase (COX) and, in some vascular preparations, elicits COX-dependent vasodilation. In the blood perfused rat kidney, 5,6-EET causes COX-dependent renal vasoconstriction, whereas in the rat isolated kidney perfused with a physiological buffer, 5,6-EET produces dose-dependent vasodilation that is unaffected by indomethacin. We examined the possible contribution of platelet COX to the vasoconstrictor action of 5,6-EET. Incubation of labeled 5,6-EET with rat washed platelets yields additional products that elute between 14 to 17 min on high-performance liquid chromatography (HPLC) and cause constriction of the perfused kidney. Indomethacin decreased the formation of these products and reduced the vasoconstrictor capacity of the corresponding HPLC fractions. Thus, platelet COX can metabolize 5,6-EET to vasoconstrictor products that may contribute to the in vivo vasoconstrictor effect of this eicosanoid.

Cell volume regulation in LLC-PK1 cells was evaluated by measuring 86Rb efflux (rate constant), used as an indicator of K+ efflux. Hypotonic shock induced a transient increase in the rate constant which returned to baseline values after 15 min. ETYA, an arachidonic acid-competitive antagonist as well as the cytochrome P450 inhibitors SKF 525-A, clotrimazole and 7-ethoxyresorufin, inhibited the hypotonic shock-induced increment in the rate constant. Arachidonic acid did not change hypotonic shock-induced increments in K+ efflux. LLC-PK1 cells were unable to metabolize 14C-arachidonic acid. We concluded that although arachidonic acid inhibitors block the hypotonic shock-induced increment in K+ efflux, this effect cannot be related to inhibition of arachidonic acid metabolism.

Bradykinin stimulates phospholipases to release arachidonic acid (AA) which can be metabolized by cyclooxygenase, lipoxygenase and cytochrome P450 (P450) to yield vasoactive products that may contribute to the effect of the peptide. In the rat kidney, pharmacological evidence suggests that a substantial component of the vasodilator response is dependent on P450-AA metabolism. In the heart, the vasodilator response to bradykinin is independent of NO and prostaglandins but reduced by inhibitors of P450, including 17-ODYA, an inhibitor of fatty acid metabolism, also suggesting a role of P450-AA. Moreover, the renal and coronary vasodilator responses to bradykinin are associated with release of P450-AA products measured by gas chromatography-mass spectrometry (GC-MS). The coronary vasodilator response to bradykinin is also dependent on activation of K+ channels linking P450-AA and hyperpolarization. Formation of vasodilator eicosanoids derived via the P450 pathway may make important contributions to the control of vascular tone, local blood flow and, thereby, blood pressure.

Based on the use of inhibitors, cytochrome P450 has been implicated in endothelium-dependent relaxant responses via metabolism of arachidonic acid (AA). However, the contribution of cytochrome P450 and its AA metabolites to the regulation of vascular tone has not been established due, in part, to questions of specificity of cytochrome P450 inhibitors which have not been extensively characterized in terms of their vascular effects. Consequently, we addressed the effects of several inhibitors on vasorelaxant responses of phenylephrine-contracted, rabbit, aortic rings to agents that utilize different transduction mechanisms to determine any actions unrelated to inhibition of cytochrome P450 and/or AA metabolism. Octadecynoic acid (2.5 and 5 microM), a mechanism-based inhibitor of cytochrome P450 metabolism of fatty acids, and eicosatetrayenoic acid (10 and 20 microM), an inhibitor of AA metabolism, were without effect on vasorelaxant responses to acetylcholine, sodium nitroprusside, isoproterenol and diazoxide. 7-Ethoxyresorufin (2-10 microM), a substrate for cytochrome P450, and clotrimazole (2.5-10 microM) which binds to the heme moiety of the enzyme, concentration-dependently reduced responses to acetylcholine but not the other agonists indicating an effect on nitric oxide synthesis although neither affected the conversion of L-arginine to L-citrulline by endothelial cells. SKF 525A (50-200 microM), the prototypical inhibitor of cytochrome P450, which is metabolized to an inhibitory intermediate, also reduced responses to acetylcholine and, in addition, impaired the vasorelaxant activities of isoproterenol and diazoxide.

Several actions of angiotensin II have been linked to metabolism of arachidonic acid by lipoxygenases. To evaluate the importance of this interaction intrarenally we tested the effect of three different lipoxygenase inhibitors, BW755c (50 microM), a dual lipoxygenase-cyclooxygenase inhibitor, MK447 (200 microM), a nonselective lipoxygenase inhibitor which can stimulate cyclooxygenase, and baicalein (1 microM), a highly selective 12-lipoxygenase inhibitor, on angiotensin II-evoked hemodynamic changes in the rat isolated kidney, perfused with oncotic agents. Kidneys were pretreated with indomethacin (10 microM) to exclude participation of cyclooxygenase-dependent arachidonate products. Renal perfusion pressure was kept constant at 90 mm Hg by continuous adjustments in perfusate flow rate. Inhibition of cyclooxygenase alone produced a transient potentiation of the vasoconstrictor response to angiotensin II without altering GFR. On the other hand, the lipoxygenase inhibitors attenuated the angiotensin II-induced increase in renal vascular resistance by approximately 50% and promoted an increase in GFR above that of kidneys infused with angiotensin II in the presence of only indomethacin. Base-line values were essentially unchanged by lipoxygenase inhibition. Furthermore, the vasoconstrictor response to the thromboxane/endoperoxide agonist U46619 was unaffected. We conclude that products of the lipoxygenase pathway, arising within the kidney, contribute to the renal hemodynamic effects of angiotensin II.

The 5,6-epoxyeicosatrienoic acid (5,6-EET), a renal vasodilator metabolite of arachidonic acid via cytochrome P450 (P450) requires cyclooxygenase for expression of its vasoactivity as the responses are inhibited by indomethacin and other aspirin-like drugs. We now report on the metabolism of 5,6-EET by rabbit kidneys in order to characterize those metabolites that may account for its vasoactivity. The 5,6-EET was injected close-arterially into the rabbit isolated Krebs-Henseleit perfused kidney, preconstricted with phenylephrine, and the effluent collected throughout the response period. Basal collections, following injection of 100 microliters of vehicle, were made at 20-min intervals before each 5,6-EET injection. Prior to acidic extraction, deuterated 6-keto-prostaglandin (PG) F1 alpha and PGE2 were added as internal standards. The extracts were separated by TLC and prostaglandins were derivatized for gas chromatography-mass spectrometry analysis using a negative ion chemical ionization mode. Injection of 0.5, 1, 5, 10, and 20 micrograms of 5,6-EET (n = 4) resulted in dose-related decreases in perfusion pressure of 6 +/- 2, 12 +/- 4, 21 +/- 4, 26 +/- 4, and 27 +/- 7 mm Hg, respectively. Basal perfusates contained 6-keto-PGF1 alpha and PGE2, levels of which were increased by 2-fold or more by 5,6-EET. Perfusates, collected during 5,6-EET administration, also contained 5-hydroxy-PGI1 and 5,6-epoxy-PGE1, cyclooxygenase metabolites of 5,6-EET. This is the first report of the recovery and identification of these 5,6-EET metabolites from an intact organ. Since the responses to 5,6-EET are endothelial-dependent, we also studied the profile of eicosanoids formed following incubation of 5,6-EET with cultured bovine pulmonary endothelial cells. Endothelial cells metabolized 5,6-EET to products with a similar radioactive profile on reverse-phase high pressure liquid chromatography compared to kidney perfusates. We compared the vasodilator activity of 5,6-epoxy-PGE1 and 5-hydroxy-PGI1, chemically synthesized by us from PGE2 and PGF2 alpha, respectively, with PGE2 and PGI2 in the rabbit kidney. The 5,6-epoxy-PGE1 was equipotent to PGE2 as a vasodilator. The ED50 values for 5,6-EET, 5,6-epoxy-PGE1, and PGE2 were 4.69, 0.43, and 0.42 nmol, respectively. Although PGI2 was a potent vasodilator (ED50, 0.24 nmol), 5-hydroxy-PGI1 was devoid of activity. Thus, the cyclooxygenase-dependent vasoactivity of 5,6-EET in the rabbit kidney has two components: release of vasodilator prostaglandins, PGE2 and PGI2, and metabolism of 5,6-EET to a prostaglandin analog, 5,6-epoxy-PGE1.

The direct effects of rat endothelin (ET-3) on renal function and prostanoid levels were examined in the isolated, oncotically perfused kidney of the rat. ET-3 at 0.75 and 2.0 ng/ml produced sustained increases in perfusion pressure of 46 and 83 mm Hg, respectively, as compared with control kidneys. Glomerular filtration rate was significantly higher than control after additions of ET-3 as was the absolute and fractional excretion of water and electrolytes. ET-3 increased perfusate 6-keto-prostaglandin (PG)F1 alpha (breakdown product of PGI2) levels and stimulated greater urinary excretion of PGE2 and PGF2 alpha than 6-keto-PGF1 alpha. ET-3 did not affect urinary excretion or perfusate levels of thromboxane B2. Time-dependent increases in renin release were suppressed by ET-3. Indomethacin (10 microM) prevented ET-3-induced increases in urinary and perfusate prostanoids; however, renal vasoconstrictor and excretory responses were the same in the presence or absence of indomethacin. These results indicate that ET-3 acts directly on the perfused rat kidney to increase the release of prostanoids from the vascular and urinary compartments. A modulatory influence of prostaglandins on the acute renal hemodynamic and excretory effects of ET-3 was not observed under these conditions.

Streptozotocin-induced diabetes resulted in diminished vasodilator responses to bradykinin in the preconstricted isolated perfused kidney of the rat which were associated with decreased renal phospholipase A2 activity and reduced release of PGE2 into the renal venous effluent.

The renovascular effects of cytochrome P450-dependent arachidonic acid (P450-AA) metabolites synthesized by rat and rabbit kidneys were studied in the rabbit isolated kidney under conditions of constant flow and examined for their dependency on cyclooxygenase relative to their expression of vasoactivity. Kidneys were perfused with Krebs-Henseleit solution, and perfusion pressure was raised to levels of 90 to 110 mm Hg with the addition of 2 to 3 microM phenylephrine to the perfusate. Close arterial injection of 1 to 20 micrograms of 5,6-, 8,9- and 11,12-epoxyeicosatrienoic acid (EET) dose-dependently decreased perfusion pressure. The 5,6-EET was the most potent and the only epoxide dependent on cyclooxygenase for expression of vasoactivity, being inhibited by indomethacin (2.8 microM). In contrast, 14,15-EET resulted in dose-dependent increases in perfusion pressure. The vasodilator effects of the omega- and omega-1 oxidation products, 20-hydroxyeicosatetraenoic acid (HETE) and the stereoisomers of 19-HETE, were also inhibited by indomethacin. Furthermore, the renal vasodilator responses to 5,6-EET were not inhibited by either superoxide dismutase (10 U) or catalase (40 U) and, therefore, were unrelated to the formation of oxygen radicals generated during transformation of the epoxide by cyclooxygenase. As 5,6-EET and 19- and 20-HETE are synthesized by the renal tubules and can affect movement of salt and water, expression of vasoactivity by P450-dependent arachidonic acid metabolites, and after release from a nephron segment, may represent a mechanism that couples altered renal tubular function to appropriate changes in local blood flow.

A metabolite of arachidonic acid, 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), which can be formed either in the 12-S or 12-R configuration, has a diversity of biological actions and is generated by a number of tissues including the renal glomerulus and the vasculature. As the two isomers have been shown to differ in their effects on epithelial transport mechanisms and vascular responsiveness, we studied their direct effects on the rat isolated kidney, perfused for four consecutive 15-min clearance periods at a pressure of 90 mm Hg with a modified Krebs' buffer containing oncotic agents. At a dose of 20 nmol, both 12(S)- and 12(R)-HETE doubled urine volume (P less than .05) and sodium and potassium excretion rate in the first, postinjection clearance period. The effects of 12(R)-HETE were sustained during all three post-treatment clearance periods, whereas those of 12(S)-HETE were short-lived, excretion rates being similar to control values by the second post-treatment clearance period. At a higher dose of 40 nmol, 12(R)-HETE significantly reduced the usual rate of decline in glomerular filtration rate, characteristic of the rat isolated kidney, and caused an even greater initial increase in urine volume and sodium excretion rate than that achieved with 20 nmol. Renin concentration in the venous effluent was reduced immediately by 12(R)- and 12(S)-HETE (P less than .01), to approximately half of the control value. Again the response to the (R)-isomer was more prolonged. Thus, a 12-HETE of glomerular origin may alter renal function through direct and indirect tubular and hemodynamic effects.

In the presence of NADPH cytochrome P-450-dependent monooxygenases oxidize arachidonic acid giving rise to four epoxyeicosatrienoic acids (EETs) which are hydrolyzed enzymatically to dihydroxyeicosatrienoic acids (DHETs). EETs generate vasodilators. Allylic oxidation forms hydroxyeicosatetraenoic acids, of which 12(R)HETE is an inhibitor of Na(+)-K(+)-ATPase and renin release. Finally, omega and omega-1 hydroxylation of arachidonic acid generates 20- and 19-HETEs which are involved in the development of hypertension in SHR rats.

The isolated perfused kidney of the rat was used to address the development of tachyphylaxis to the vasoconstrictor effects of arachidonic acid. Repeated administration of arachidonic acid (3 micrograms at 10-min intervals) to the isolated kidney of the rat, perfused in situ with Krebs-Henseleit solution, led to the development of tachyphylaxis to the renal vasoconstrictor effects of arachidonic acid, using perfusion pressure changes as an index. Vasoconstrictor responses to either angiotensin or the endoperoxide analog, U46619, were unaffected by repeated administration of arachidonic acid. Associated with progressively reduced renal vasoconstrictor responses to arachidonic acid were parallel decrements in the renal venous release of prostanoids, measured by radioimmunoassay. In contrast, the release of prostanoids from the kidney stimulated by angiotensin II was increased after repeated administrations of arachidonic acid. These data suggest that the sequential reduction in renal vasoconstrictor responses to arachidonic acid is due to diminished conversion to prostaglandins, possibly due to inactivation of cyclooxygenase, decreased entry of arachidonic acid into the cell or its increased esterification into phospholipids.

In the isolated Tyrode's perfused rabbit kidney, metabolism of [9-3H]prostacyclin was examined. In addition to 7,9-dihydroxy-4,13-diketo-dinor-prostanoic acid, dinor-6-keto-prostaglandin F1 alpha, and pentanorprostaglandin (PG)F1 alpha gamma-lactone, a new, previously unreported, metabolite was isolated and identified by radio-gas chromatography and gas chromatography-mass spectrometry as 5-hydroxy-6-keto-PGF1 alpha. The structure of this metabolite was further confirmed by comparison of the mass spectra to that of the synthetic standard. The formation of 5-hydroxy-6-keto-PGF1 alpha in the kidney suggested epoxidation of prostacyclin via the renal epoxygenase pathway.

In isolated perfused rabbit mesenteric arteries, prostaglandin (PG) E1 and E2, 1-5NG/ML, did not alter the basal perfusion pressure, but reduced the vasoconstrictor responses to sympathetic nerve stimulation; the responses to injected norepinephrine were reduced by PGE1 and variably affected by PGE2. In contrast, in rat mesenteric arteries PGE1 and PGE2, 1-5 ng/ml, potentiated the vasoconstrictor responses to nerve stimulation and to injected norepinephrine. In rabbit mesenteric arteries, the inhibitor of PG synthesis, indomethacin, augmented the responses to sympathetic nerve stimulation and to injected norepinephrine, whereas in rat mesenteric arteries indomethacin inhibited the responses to both adrenergic stimuli. Arachidonic acid, a PG precursor, reduced the vasoconstrictor responses to sympathetic nerve stimulation and to injected norepinephrine in rabbit, whereas in rat, potentiation of the responses to adrenergic stimuli occurred. Since these effects of arachidonic acid were abolished by the simultaneous infusion of indomethacin, they appear to be mediated through conversion of arachidonic acid to PG. We conclude that prostaglandins modulate adrenergic transmission in mesenteric arteries and this effect is species dependent.

The possibility that 6-keto-prostaglandin E1 (6-keto-PGE1) affects renin release was studied using rabbit renal cortical slices, a preparation that eliminates hemodynamic, neural, and blood-borne factors that might influence renin release. The medium used for incubating the slices was collected for renin assay at the end of each of four successive 20-minute periods. Test agents were added only once, at the beginning of Period 3 (experimental period). Between Periods 3 an 4 (recovery period), the medium was aspirated and the slices rinsed with Krebs solution before replacing the medium. Renin release did not change in vehicle-treated slices. Unlike the PGI2-induced changes, the effects of 6-keto-PGE1 on renin release were sustained in Period 4. Indomethacin potentiated renin stimulation induced by 10 microM concentrations of PGI2 and 6-keto-PGE1 in Period 3 and by 6-keto-PGE1 in Period 4. Using platelet antiaggregatory activity as an index of stability, we found that PGI2 was largely inactivated within 10 minutes under the conditions used for incubating the slices (pH 7.4, 37 degrees C), while 6-keto-PGE1 was stable. The results lend further support to the concept that 6-keto PgE1 is capable of releasing renin through a direct action.

Renal microsomal cytochrome P-450-dependent arachidonic acid metabolism was correlated with the level of cytochrome P-450 in the rabbit kidney. Cobalt, an inducer of haem oxygenase, reduced cytochrome P-450 in both the cortex and medulla in association with a 2-fold decrease in aryl-hydrocarbon hydroxylase, an index of cytochrome P-450 activity, and a similar decrease in the formation of cytochrome P-450-dependent arachidonic acid metabolites by renal microsomes (microsomal fractions). Formation of the latter was absolutely dependent on NADPH addition and was prevented by SKF-525A, an inhibitor of cytochrome P-450-dependent enzymes. Arachidonate metabolites of cortical microsomes were identified by g.c.-m.s. as 20- and 19-hydroxyeicosatetraenoic acid, 11,12-epoxyeicosatrienoic acid and 11,12-dihydroxyeicosatrienoic acid. The profile of arachidonic acid metabolites was the same for the medullary microsomes. Induction of cytochrome P-450 by 3-methylcholanthrene and beta-naphthoflavone increased cytochrome P-450 content and aryl-hydrocarbon hydroxylase activity by 2-fold in the cortex and medulla, and this correlated with a 2-fold increase in arachidonic acid metabolites via the cytochrome P-450 pathway. These changes can also be demonstrated in cells isolated from the medullary segment of the thick ascending limb of the loop of Henle, which previously have been shown to metabolize arachidonic acid specifically via the cytochrome P-450-dependent pathway. The specific activity for the formation of arachidonic acid metabolites by this pathway is higher in the kidney than in the liver, the highest activity being in the outer medulla, namely 7.9 microgram as against 2.5 micrograms of arachidonic acid transformed/30 min per nmol of cytochrome P-450 for microsomes obtained from outer medulla and liver respectively. These findings are consistent with high levels of cytochrome P-450 isoenzyme(s), specific for arachidonic acid metabolism, primarily localized in the outer medulla.

The effects of three angiotensin converting enzyme (ACE) inhibitors - captopril, MK-421 diacid, and teprotide - on renal vascular responses to graded (50, 100, 200 ng) injections of norepinephrine (NE) were examined in rat isolated perfused kidneys, having a mean basal perfusion pressure of 78 +/- 10 mm Hg. The minimum dose of captopril (0.05 microgram/ml, low dose) that abolished the vasoconstrictor responses to 100 and 200 ng angiotensin I did not affect NE-induced renal vasoconstriction, whereas a dose 100 times greater (high-dose captopril, 5 micrograms/ml) reduced the vasoconstrictor action of NE. MK-421 diacid also at high dose (1 microgram/ml), caused similar reduction in renal vasoconstrictor responses to NE. In contrast, a high dose of teprotide (50 micrograms/ml) did not affect renal vascular responsiveness to NE. The threshold dose of NE that released prostaglandins, measured by bioassay, was 50 ng. Indomethacin (1 microgram/ml) prevented NE-induced release of prostaglandins but did not affect the ability of captopril to attenuate NE-induced vasoconstriction. We conclude that captopril and MK-421 diacid decreases vascular reactivity in the rat isolated kidney by a mechanism independent of ACE inhibition and unrelated to a prostaglandin-dependent vascular mechanism. Moreover, the presence of mercapto function in the ACE inhibitor is not essential since captopril, which has a sulfhydryl group, and MK-421 diacid, which lacks this group, have similar effects on renal vascular responsiveness.

Changes in renal perfusion pressure and eicosanoid release in response to arginine vasopressin (AVP; 1-10 ng) and angiotensin II (ANG II; 1-10 ng) were determined 5 days, 2 wk, and 8-12 wk after the induction of diabetes with streptozotocin (STZ) in male Wistar rats. Renal perfusion pressure responses to AVP and ANG II were reduced at 2 and 8-12 wk, but not at 5 days, after the induction of diabetes. However, AVP- and ANG II-stimulated release of prostaglandins into the renal venous effluent was depressed at all times tested. Inhibition of cyclooxygenase with indomethacin did not significantly influence the perfusion pressure responses to ANG II and AVP. Likewise, raising perfusate glucose levels to 400 mg/dl or adding insulin (180 microU/ml) to the perfusate failed to modify responses to ANG II. In contrast, administration of 0.3 microgram arachidonic acid (AA), a dose approaching threshold in control rat kidneys, to the kidney of the diabetic rat resulted in a marked increase in perfusion pressure. Associated with the increase in renal perfusion pressure to AA in the diabetic rat were significant increases in renal venous efflux of prostaglandin E2 and prostacyclin compared with control. These data suggest a defect in renal deacylation-reacylation of AA associated with an increase in cyclooxygenase activity in the diabetic rat.

1. Studies were made of the effects on responses to vasoconstrictor agents of prostaglandins released from Krebs perfused isolated kidneys of genetic hypertensive and normotensive rats. 2. Prostaglandin E-like activity, detected by bio-assay, was released from kidneys of both groups of rats during the vasoconstriction produced by noradrenaline, angiotensin or prostaglandin F2α. 3. In preparations obtained from hypertensive rats, responses to higher doses of noradrenaline or angiotensin were initially greater than those from normotensive rats and these were then reduced to a greater extent by infusion of indomethacin, which abolished release of prostaglandin E-like activity. Thereafter, in kidneys of either group, vasoconstriction to noradrenaline was potentiated by infusion of prostaglandin E2. 4. We conclude that, in rats, renal prostaglandins released in response to vasoconstrictor agents could augment the effect of such agents and in genetic hypertensive rats release of renal prostaglandins could contribute to the disease.

In the anesthetized dog, acute arteriovenous fistulae sufficient to increase the cardiac output by from 16 to 130% resulted in an increase in the coronary blood flow even in the presence of a definite and even marked drop in the mean arterial blood pressure. The arteriovenous fistulae also resulted in an increase of the cardiac work and oxygen consumption as well as the cardiac efficiency.

Renal prostaglandins (PC s ) might mediate an antihypertensive function of the kidney. The blood-superfused organ technique possesses the sensitivity (threshold < 0.4 ng/ml blood) and specificity required for identification of PGs in blood. Induction of unilateral renal ischemia in 14 chloraloseanesthetized dogs reduced renal blood flows from a mean value of 257 to 109 ml/min on the ischemic side and from 250 to 209 ml/min on the contralateral side. Concomitantly, PG-like substances were detected by assay organs in the venous blood of ischemic (13 experiments) and contralateral (11 experiments) kidneys. In one experiment, in a spontaneously hypertensive dog, PGs were not detected during renal ischemia. Renal venous blood and renal medullary tissue were extracted for acidic lipids and assayed for PG-like substances. Extracts of venous blood collected during renal ischemia and extracts of renal medulla yielded substances with biological activity indistinguishable from PG-like substances or PG standards. Chromatographic characterization of PG-like substances suggests that they are predominantly a mixture of PGE 2 and PGF 2α .

SummaryThe effect of Isuprel on MABP, CO and TPR has been investigated in the anesthetized dog in which the effect of the drug on HR is allowed to occur, as well as in the anesthetized dog in which electrically induced atrial or ventricular tachycardia is initiated before and maintained throughout period of action of the drug to prevent the increase in heart rate normally induced by the drug. Under the circumstances of drug dosage, rate of the induced tachycardia as well as control values of HR and CO, the effect of the drug on CO and TPR is essentially the same, whether the effect of the drug on HR is permitted or prevented.

In the isolated rabbit kidney perfused with Tyrode's solution, we examined the metabolism of radiolabeled prostacyclin. [9-3H]Prostacyclin was infused into the kidney and the radiolabeled products from the renal venous effluent were separated by thin layer chromatography and identified by gas chromatography-mass spectrometry. The major products were 7,9-dihydroxy-4,13-diketo-dinor-prostanoic acid and dinor-6-keto-prostaglandin F1alpha. They represented 25% and 10% of the total radioactivity, respectively. Metabolism of prostacyclin by the kidney may be an important determinant of the effects of prostacyclin on renal function.

WE describe here the release from the kidney by angiotensin II and noradrenaline of substances which have the properties of prostaglandins. This observation may help to account for the antihypertensive function of the kidney first suggested by Fasciolo1, who demonstrated that removal of the kidney contralateral to an ischaemic kidney produced sustained hypertension in dogs. We have adapted the bioassay method of Vane2, the super-fused blood-bathed organ technique, for the detection of prostaglandins in renal venous blood in response to infusions of arigioteiisin II or noradrenaline into the renal artery.