Maternal and fetal epoxyeicosatrienoic acids in normotensive and preeclamptic pregnancies.

BACKGROUND: Epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) are cytochrome P450 metabolites of arachidonic acid posited to act in the circulatory adaptation to pregnancy and the development of preeclampsia. Red blood cells (RBCs) may function as major contributors of cis- and trans-EETs. METHODS: We performed paired analyses of EETs, dihydroxyeicosatrienoic acids (DHETs), and 20-HETE in RBCs, plasma, and urine from preeclamptic and normotensive pregnant and nonpregnant women. Blood from fetal and maternal circulation was collected. EETs, DHETs, and 20-HETE were analyzed by gas chromatography and liquid chromatography mass spectrometry. Vascular function and inflammation indices were analyzed. RESULTS: Plasma EET is higher in normotensive (median, range; 9.9, 6.3-25.2ng/mL n = 29) and preeclamptic (10.9, 6.0-48.0ng/mL, n = 19) women than in nonpregnant controls (7.3, 3.7-10.2ng/mL, n = 19) and correlate with RBC EETs, C-reactive protein, and arterial stiffness. Renal production of EETs, measured as urinary DHETs, was reduced in preeclamptic (4.5, 1.6-24.5ng/mg creatinine) compared to normotensive (11.4, 1.6-44.5ng/mg creatinine) pregnancies. EETs are 3- to 5-fold greater in fetoplacental than in maternal circulation (RBCs 36.6, 13.1-69.4 vs. 12.5, 6.4-12.0ng/10(9) cells; plasma 31.6, 8.5-192.6 vs. 12.0, 6.8-48.0ng/mL). Both cis- and trans-EETs are present in fetal RBCs. CONCLUSIONS: RBCs contribute to elevated levels of EETs in the fetoplacental circulation. EETs may modulate systemic and fetoplacental hemodynamics in normal and preeclamptic pregnancies. Decreased renal EET generation may be associated with the development of maternal renal dysfunction and hypertension in preeclampsia.

The red blood cell participates in regulation of the circulation by producing and releasing epoxyeicosatrienoic acids.

Red blood cells (RBCs) have an important function in regulation of the circulation by producing and releasing epoxyeicosatrienoic acids (EETs) in response to a low O2 environment such as encountered in the cardiac microcirculation during exercise. RBCs, in their role as sensors of low pO2, release ATP and critical lipid mediators, the EETs. Both cis- and trans-EETs are synthesized and stored in RBCs and are hydrolyzed by soluble epoxide hydrolases (sEH). The trans-EETs differ from cis-EETs in their higher vascular potencies and more rapid metabolism by sEH. Thus, inhibition of sEH results in greater trans-EET levels and increased positive vascular effects of trans-EETs vs cis-EETs. The trans-EETs are responsible for a significant decline in the elevated blood pressure in the spontaneously hypertensive rat on treatment with a sEH inhibitor to raise EET levels. We predict that trans-EETs and cis-EETs will occupy important therapeutic roles in a broad spectrum of diseases and abnormal physiological conditions such as that resulting from high salt intake and hypertension.

Vitamin C activation of the biosynthesis of epoxyeicosatrienoic acids.

The cardiovascular effects of vitamin C (VitC) could be mediated by epoxyeicosatrienoic acids (EETs). We aimed to study the mechanism of VitC-dependent microsomal formation of cis- and trans-EETs and the regulation of EET levels in rat isolated perfused kidneys and in vivo. VitC biphasically stimulated rat kidney microsomal cis- and trans-EET formation in a ratio of 1:2, involving the participation of lipid hydroperoxides (LOOHs), Fe2+, and cytochrome P450 (CYP). Levels of LOOHs correlated with microsomal EET production. LOOH stimulation of CYP isoforms resulted in preferred trans-over cis-EET formation from arachidonic acid and was associated with the cleavage of LOOHs, which indicated a CYP peroxygenase activity. EETs contributed to VitC-induced vasodilator responses in rat isolated perfused kidneys. VitC (1 mg/ml) given in the drinking water for 9 days doubled rat urinary EET excretion, increased plasma levels of EETs, mostly trans-EETs, by 40%, and reduced plasma levels of 20-hydroxyeicosatetraenoic acid. Depletion of VitC in brain cortex and kidney tissues by more than 20- and 50-fold, respectively, in gulonolactone oxidase-knockout mice was associated with mild increases in tissue EETs. These data suggest that LOOHs are a determinant factor for EET formation in vivo in which VitC exerts a key regulatory effect. VitC-activated CYP peroxygenase activity may represent a CYP interaction with lipoxygenases and cyclooxygenases to mediate the cardiovascular effects of VitC via formation of EETs.

11,12-EET increases porto-sinusoidal resistance and may play a role in endothelial dysfunction of portal hypertension.

CYP450-dependent epoxyeicosatrienoic acids (EETs) are potent arterial vasodilators, while 20-hydroxyeicosatatraenoic acid (20-HETE) is a vasoconstrictor. We evaluated their role in the control of portal circulation in normal and cirrhotic (CCl(4) induced) isolated perfused rat liver. Phenylephrine (PE) and endothelin-1 (ET-1) increased portal perfusion pressure, as did arachidonic acid (AA), 20-HETE, and 11,12-EET. Inhibition of 20-HETE with 12,12-dibromododecenoic acid (DBDD) did not affect basal pressure nor the responses to PE, ET-1, or AA. However, inhibition of epoxygenase with miconazole caused a significant reduction in the response to ET-1 and to AA, without affecting neither basal pressure nor the response to PE. Hepatic vein EETs concentration increased in response to ET-1, and was increased in cirrhotic, compared to control, livers. 20HETE levels were non-measurable. Miconazole decreased portal perfusion pressure in cirrhotic livers. In conclusion, 20HETE and EETs increase portal resistance; EETs, but not 20-HETE, mediate in part the pressure response to ET-1 in the portal circulation and may be involved in pathophysiology of portal hypertension.

Red blood cells (RBCs), epoxyeicosatrienoic acids (EETs) and adenosine triphosphate (ATP).

In addition to serving as carriers of O(2), red blood cells (RBCs) regulate vascular resistance and the distribution of microvascular perfusion by liberating adenosine triphosphate (ATP) and epoxyeicosatrienoic acids (EETs) upon exposure to a low O(2) environment. Therefore, RBCs act as sensors that respond to low pO(2) by releasing millimolar amounts of ATP, a signaling molecule, and lipid mediators (EETs). The release of EETs occurs by a mechanism that is activated by ATP stimulation of P2X(7) receptors coupled to ATP transporters, which should greatly amplify the circulatory response to ATP. RBCs are reservoirs of EETs and the primary sources of plasma EETs, which are esterified to the phospholipids of lipoproteins. Levels of free EETs in plasma are low, about 3% of circulating EETs. RBC EETs are produced by direct oxidation of arachidonic acid (AA) esterified to glycerophospholipids and the monooxygenase-like activity of hemoglobin. On release, EETs affect vascular tone, produce profibrinolysis and dampen inflammation. A soluble epoxide hydrolase (sEH) regulates the concentrations of RBC and vascular EETs by metabolizing both cis- and trans-EETs to form dihydroxyeicosatrienoic acids (DHETs). The function and pathophysiological roles of trans-EETs and erythro-DHETs has yet to be integrated into a physiological and pathophysiological context.

Failure to upregulate the adenosine2A receptor-epoxyeicosatrienoic acid pathway contributes to the development of hypertension in Dahl salt-sensitive rats.

Adenosine-activated renovascular dilatation in Sprague-Dawley (SD) rats is mediated by stimulating adenosine(2A) receptors (A(2A)R), which is linked to epoxyeicosatrienoic acid (EET) synthesis. The A(2A)R-EET pathway is upregulated by high salt (HS) intake in normotensive SD rats. Because this pathway is antipressor, we examined the role of the A(2A)R-EET pathway in Dahl salt-sensitive (SS) rats. Male Dahl salt-resistant (SR) and SS rats were fed either HS (8.0% NaCl) or normal salt (NS; 0.4% NaCl) diet for 7 days. On day 8, isolated kidneys were perfused with Krebs-Henseleit buffer containing indomethacin and N(G)-nitro-l-arginine methyl ester and preconstricted with phenylephrine. Bolus injections of the stable adenosine analog 2-chloroadenosine (2-CA; 0.1-20 microg) elicited dose-dependent dilation in both Dahl SR and SS rats. Dahl SR rats fed a HS diet demonstrated a greater renal vasodilator response to 10 microg of 2-CA, as measured by the reduction in renal perfusion pressure, than that of Dahl SR rats fed a NS diet (-104 +/- 6 vs. -77 +/- 7 mmHg, respectively; P < 0.05). In contrast, Dahl SS rats did not exhibit a difference in the vasodilator response to 2-CA whether fed NS or HS diet (96 +/- 6 vs. 104 +/- 13 mmHg in NS- and HS-fed rats, respectively). In Dahl SR but not Dahl SS rats, HS intake significantly increased purine flux, augmented the protein expression of A(2A)R and the cytochrome P-450 2C23 and 2C11 epoxygenases, and elevated the renal efflux of EETs. Thus the Dahl SR rat is able to respond to HS intake by recruiting EET formation, whereas the Dahl SS rat appears to have exhausted its ability to increase EET synthesis above the levels observed on NS intake, and this inability of Dahl SS rats to upregulate the A(2A)R-EET pathway in response to salt loading may contribute to the development of salt-sensitive hypertension.

Calcium-sensing receptor signaling pathways in medullary thick ascending limb cells mediate COX-2-derived PGE2 production: functional significance.

We determined the functional implications of calcium-sensing receptor (CaR)-dependent, Gq- and Gi-coupled signaling cascades, which work in a coordinated manner to regulate activity of nuclear factor of activated T cells and tumor necrosis factor (TNF)-alpha gene transcription that cause expression of cyclooxygenase (COX)-2-derived prostaglandin E2 (PGE2) synthesis by rat medullary thick ascending limb cells (mTAL). Interruption of Gq, Gi, protein kinase C (PKC), or calcineurin (CaN) activities abolished CaR-mediated COX-2 expression and PGE2 synthesis. We tested the hypothesis that these pathways contribute to the effects of CaR activation on ion transport in mTAL cells. Ouabain-sensitive O2 consumption, an in vitro correlate of ion transport in the mTAL, was inhibited by approximately 70% in cells treated for 6 h with extracellular Ca2+ (1.2 mM), an effect prevented in mTAL cells transiently transfected with a dominant negative CaR overexpression construct (R796W), indicating that the effect was initiated by stimulation of the CaR. Pretreatment with the COX-2-selective inhibitor, NS-398 (1 microM), reversed CaR-activated decreases in ouabain-sensitive O2 consumption by approximately 60%, but did not alter basal levels of ouabain-sensitive O2 consumption. Similarly, inhibition of either Gq, Gi, PKC, or CaN, which are components of the mechanism associated with CaR-stimulated COX-2-derived PGE2 synthesis, reversed the inhibitory effects of CaR on O2 consumption without affecting basal O2 consumption. Our findings identified signaling elements required for CaR-mediated TNF production that are integral components regulating mTAL function via a mechanism involving COX-2 expression and PGE2 production.

Hydrolysis of cis- and trans-epoxyeicosatrienoic acids by rat red blood cells.

Erythrocytes serve as reservoirs for cis- and trans-epoxyeicosatrienoic acids (EETs). Incubation of rat red blood cells (RBCs) with cis- and trans-EETs produces threo- and erythro-dihydroxyeicosatrienoic acids, respectively. The V(max) of EET hydrolysis by rat intact RBCs (2.35 +/- 0.24 pmol/min/10(8) RBCs for 14,15-trans-EET) decreased by approximately 2 to 3-fold sequentially from 14,15-, 11,12- to 8,9-EETs for both cis- and trans-isomers. The V(max) of trans-EET hydrolysis by RBCs is approximately 2 to 3 times that of the corresponding cis-EETs. Incubation of EETs with recombinant murine soluble epoxide hydrolase (sEH) yielded the same geometric and regio preferences of EET hydrolysis as with rat intact RBCs. The principal epoxide hydrolase activity for EET hydrolysis (approximately 90%) is present in the erythrocyte cytosol. Western blots of sEH suggested a concentration of sEH protein to be approximately 2 microg/mg protein or 0.4 microg/10(9) RBCs. The apparent K(m) values of EETs were between 1 and 2 microM, close to the K(m) for purified sEH as reported. Erythrocyte hydration of cis- and trans-EETs was blocked by sEH inhibitors, 1,3-dicyclohexylurea and 4-[4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid. Erythrocyte sEH activity was inhibited more than 80% by 0.2% bovine serum albumin in the buffer. Preferred hydrolysis of 14,15-EETs and trans-epoxides characterizes sEH activity in RBCs that regulates the hydrolysis and release of cis- and trans-EETs in the circulation. Inhibition of sEH has produced antihypertensive and antiinflammatory effects. Because plasma trans-EETs would increase more than cis-EETs with sEH inhibition, the potential roles of trans-EETs and erythrocyte sEH in terms of circulatory regulation deserve attention.

Altered release of cytochrome p450 metabolites of arachidonic acid in renovascular disease.

The aim of the present cross-sectional study was to investigate whether activation of the renin-angiotensin system in renovascular disease affects the cytochrome P450 omega/omega-1 hydroxylase (20-hydroxyeicosatetraenoic acid [20-HETE]) and epoxygenase (epoxyeicosatrienoic acids [EETs]) pathways of arachidonic acid metabolism in vivo, each of which interacts with angiotensin II. Plasma concentration and urinary excretion of 20-HETE and EETs and their metabolites, dihydroxyeicosatrienoic acids, were measured in urine and plasma by mass spectrometry in 10 subjects with renovascular disease, 10 with essential hypertension, and 10 healthy normotensive subjects (control subjects), pair-matched for gender and age. Vascular and renal function were evaluated in all of the subjects. Plasma 20-HETE was highest in subjects with renovascular disease (median: 1.20 ng/mL; range: 0.42 to 1.92 ng/mL) compared with subjects with essential hypertension (median: 0.90 ng/mL; range: 0.40 to 2.17 ng/mL) and control subjects (median: 0.45 ng/mL; range: 0.14 to 1.70 ng/mL; P<0.05). Plasma 20-HETE significantly correlated with plasma renin activity in renovascular disease (r(s)=0.67; n=10; P<0.05). The urinary excretion of 20-HETE was significantly lower in subjects with renovascular disease (median: 12.9 microg/g of creatinine; range: 4.4 to 24.9 microg/g of creatinine) than in control subjects (median: 31.0 microg/g of creatinine; range: 11.9 to 102.8 microg/g of creatinine; P<0.01) and essential hypertensive subjects (median: 35.9 microg/g of creatinine; range: 14.0 to 72.5 microg/g of creatinine; P<0.05). Total plasma EETs were lowest, as was the ratio of plasma EETs to plasma dihydroxyeicosatrienoic acids, an index of epoxide hydrolase activity, in renovascular disease (ratio: 2.4; range: 1.2 to 6.1) compared with essential hypertension (ratio: 3.4; range: 1.5 to 5.6) and control subjects (ratio: 6.8; range: 1.4 to 18.8; P<0.01). In conclusion, circulating levels of 20-HETE are increased and those of EETs are decreased in renovascular disease, whereas the urinary excretion of 20-HETE is reduced. Altered cytochrome P450 arachidonic acid metabolism may contribute to the vascular and tubular abnormalities of renovascular disease.

CaR activation increases TNF production by mTAL cells via a Gi-dependent mechanism.

We evaluated the contribution of calcium-sensing receptor (CaR)-mediated G(i)-coupled signaling to TNF production in medullary thick ascending limb (mTAL) cells. A selective G(i) inhibitor, pertussis toxin (PTX), but not the inactive B-oligomer binding subunit, abolished CaR-mediated increases in TNF production. The inhibitory effect of PTX was partially reversed by using an adenylate cyclase inhibitor. CaR-mediated TNF production also was partially reversed by a cAMP analog, 8-Br-cAMP. IP(1) accumulation was CaR dependent and blocked by PI-PLC; partial inhibition also was observed with PTX. CaR increased calcineurin (CaN) activity by approximately threefold, and PTX prevented CaR-mediated increases in CaN activity, an nuclear factor of activated T cells (NFAT)-cis reporter construct, and a TNF promoter construct. The interaction between G(i) and PKC was determined, as we previously showed that CaR-mediated TNF production was CaN and NFAT- mediated and G(q) dependent. CaR activation increased PKC activity by twofold, an effect abolished by transient transfection with a dominant negative CaR construct, R796W, or pretreatment with PTX. Inhibition with the pan-specific PKC inhibitor GF 109203X (20 nM) abolished CaR-mediated increases in activity of CaN, an NFAT reporter, and a TNF promoter construct. Collectively, the data suggest that G(i)-coupled signaling contributes to NFAT-mediated TNF production in a CaN- and PKC-dependent manner and may be part of a CaR mechanism to regulate mTAL function. Moreover, concurrent G(q) and G(i) signaling is required for CaR-mediated TNF production in mTAL cells via a CaN/NFAT pathway that is PKC dependent. Understanding CaR-mediated signaling pathways that regulate TNF production in the mTAL is crucial to defining novel mechanisms that regulate extracellular fluid volume and salt balance.

Characterization of a long-term rat mTAL cell line.

A medullary thick ascending limb (mTAL) cell line, termed raTAL, has been established from freshly isolated rat mTAL tubules and cultured continuously for up to 75 passages; it retains characteristics of mTAL cells even after retrieval from storage in liquid nitrogen for several months. The cells express Tamm-Horsfall glycoprotein (THP), a TAL-specific marker, grow to confluence, exhibit a polygonal morphology characteristic of epithelial cells, and form "domes." Detection of THP, Na(+)-K(+)-2Cl(-) cotransporter (NKCC2), Na(+)-K(+)-ATPase, and renal outer medullary K(+) channel (ROMK) was achieved using indirect immunofluorescence and confocal microscopy. Western blot analysis of NKCC2 expression using two different antibodies revealed a band of approximately 160 kDa, and RT-PCR analysis demonstrated the presence of NKCC2 isoforms A and F, which was confirmed by DNA sequencing; transport of Cl(-) into raTAL cells was inhibited by furosemide. Ouabain- and bumetanide-sensitive oxygen consumption, an index of ion transport activity in the mTAL, was observed in raTAL cells, and the number of domes present was reduced significantly when cells were incubated in the presence of ouabain or bumetanide. The specific activity of Na(+)-K(+)-ATPase activity was determined in raTAL cells (0.67 +/- 0.18 nmol P(i).microg protein(-1).min(-1)), primary cultures of mTAL cells (0.39 +/- 0.08 nmol P(i).microg protein(-1).min(-1)), and freshly isolated mTAL tubules (1.10 +/- 0.29 nmol P(i).microg protein(-1).min(-1)), and approximately 30-50% of total cellular ATPase activity was inhibited by ouabain, in accord with other mTAL preparations. This cell line will be used in studies that address biochemical, molecular, and physiological mechanisms in the mTAL.

Rat mesenteric arterial dilator response to 11,12-epoxyeicosatrienoic acid is mediated by activating heme oxygenase.

11,12-Epoxyeicosatrienoic acid (11,12-EET), a potent vasodilator produced by the endothelium, acts on calcium-activated potassium channels and shares biological activities with the heme oxygenase/carbon monoxide (HO/CO) system. We examined whether activation of HO mediates the dilator action of 11,12-EET, and that of the other EETs, on rat mesenteric arteries. Dose-response curves (10(-9) to 10(-6) M) to 5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, and ACh (10(-9) to 10(-4) M) were evaluated in preconstricted (10(-6) mol/l phenylephrine) mesenteric arteries (<350 microm diameter) in the presence or absence of 1) the cyclooxygenase inhibitor indomethacin (2.8 microM), 2) the HO inhibitor chromium mesoporphyrin (CrMP) (15 microM), 3) the soluble guanylyl cyclase (GC) inhibitor ODQ (10 microM), and 4) the calcium-activated potassium channel inhibitor iberiotoxin (25 nM). The vasodilator response to 11,12-EET was abolished by CrMP and iberiotoxin, whereas indomethacin and ODQ had no effect. In contrast, the effect of ACh was attenuated by ODQ but not by CrMP. The vasodilator effect of 8,9-EET, like that of 11,12-EET, was greatly attenuated by HO inhibition. In contrast, the mesenteric vasodilator response to 5,6-EET was independent of both HO and GC, whereas that to 14,15-EET demonstrated two components, an HO and a GC, of equal magnitude. Incubation of mesenteric microvessels with 11,12-EET caused a 30% increase in CO release, an effect abolished by inhibition of HO. We conclude that the rat mesenteric vasodilator action of 11,12-EET is mediated via an increase in HO activity and an activation of calcium-activated potassium channels.

Adenosine2A receptor vasodilation of rat preglomerular microvessels is mediated by EETs that activate the cAMP/PKA pathway.

Dilation of rat preglomerular microvessels (PGMV) by activation of adenosine A2A receptors (A2AR) is coupled to epoxyeicosatrienoic acid (EET) release. We have investigated the commonality of this signal transduction pathway, i.e., sequential inhibition of G(salpha), adenylyl cyclase, PKA, and Ca2+-activated K+ (KCa) channel activity, to the vasoactive responses to A2AR activation by a selective A2A agonist, CGS-21680, compared with those of 11,12-EET. Male Sprague-Dawley rats were anesthetized, and microdissected arcuate arteries (110-130 microm) were cannulated and pressurized to 80 mmHg. Vessels were superfused with Krebs solution containing NG-nitro-L-arginine methyl ester (L-NAME) and indomethacin and preconstricted with phenylephrine. We assessed the effect of 3-aminobenzamide (10 microM), an inhibitor of mono-ADP-ribosyltranferases, on responses to 11,12-EET (3 nM) and CGS-21680 (10 microM) and found that both were inhibited by approximately 70% (P<0.05), whereas the response to SNP (10 microM) was unaffected. Furthermore, 11,12-EET (100 nM), like cholera toxin (100 ng/ml), stimulated ADP-ribose formation in homogenates of arcuate arteries compared with control. SQ-22536 (10 microM), an inhibitor of adenylyl cyclase activity, and myristolated PKI (14-22) amide (5 microM), an inhibitor of PKA, decreased activity of 11,12-EET and CGS-21680. Incubation of 11,12-EET (3 nM-3 microM) with PGMV resulted in an increase in cAMP levels (P<0.05). The responses to both 11,12-EET and CGS-21680 were significantly reduced by superfusion of iberiotoxin (100 nM), an inhibitor of KCa channel activity. Thus in rat PGMV activation of A2AR is coupled to EET release upstream of adenylyl cyclase activation and EETs stimulate mono-ADP-ribosyltransferase, resulting in Gsalpha protein activation.

NFAT regulates calcium-sensing receptor-mediated TNF production.

Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca(2+) (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca(2+) were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.

Exaggerated response to adenosine in kidneys from high salt-fed rats: role of epoxyeicosatrienoic acids.

Cytochrome P-450 (CYP)-dependent epoxyeicosatrienoic acids (EETs) dilate rat preglomerular microvessels when adenosine(2A) receptors (A(2A)R) are stimulated. As high salt (HS) intake increases epoxygenase activity and adenosine levels, we hypothesized that renal adenosine responses would be greater in HS-fed rats. Male Sprague-Dawley rats were fed either HS (4.0% NaCl) or normal salt (NS; 0.4% NaCl) diet. On day 8, isolated kidneys were perfused with Krebs' buffer containing indomethacin (10 microM) and L-NAME (200 microM) and preconstricted to approximately 150 mmHg with infusion of phenylephrine (10(-7) M). Renal effluents were extracted for analysis of eicosanoids by gas chromatography-mass spectrometry. Bolus injections of the stable adenosine analog 2-chloroadenosine (2-CA; 0.1-10 microg) resulted in dose-dependent dilation; at 10 microg, perfusion pressure (PP) was lowered to a greater extent in the kidneys of HS rats compared with NS rats (-60 +/- 4 vs. -31 +/- 8 mmHg; P < 0.05) and the area of response was increased (27 +/- 6 vs. 9 +/- 4 mm(2); P < 0.05), as was EET release (132 +/- 23 vs. 38 +/- 18 ng; P < 0.05). HS treatment increased A(2A)R and CYP2C23 protein expression. A selective epoxygenase inhibitor, MS-PPOH (12 microM), significantly reduced the response to 2-CA in HS rats; PP, area of response, and EET release decreased by 40, 70, and 81%, respectively, whereas lesser changes were evident in NS kidneys. Thus the greater vasodilator response to 2-CA seen in kidneys obtained from HS-fed rats was mediated by increased EET release. As EETs are renal vasodilator and natriuretic eicosanoids, interactions between adenosine and EETs may contribute to the adaptive response to HS intake.

Red blood cells: reservoirs of cis- and trans-epoxyeicosatrienoic acids.

Epoxyeicosatrienoic acids (EETs) are candidate endothelium-derived hyperpolarizing factors that demonstrate a wide range of biological effects. The presence of both cis- and trans-EETs in rat plasma was identified with HPLC-electrospray ionization tandem mass spectrometry in this study. The total EETs in plasma are 38.2 ng/ml with cis-EETs representing 21.4 +/- 0.4 ng/ml and trans-EETs 16.8 +/- 0.4 ng/ml. EETs in RBCs were estimated to be 20.2 ng/10(9) RBCs, which corresponds to 200 ng in RBCs contained in 1 ml blood. RBC incubation with 10 mM tert-butyl hydroperoxide resulted in 4.4-fold increase of total cis-EETs (from 9.2 to 40.2 ng/10(9) RBCs) and 5.5-fold increase of total trans-EETs (from 11.0 to 60.8 ng/10(9) RBCs). EETs were released (2 ng/ml) from RBCs after incubation at 37 degrees C for 10 min even after being washed 3 times, indicating that RBCs are reservoirs of plasma EETs. The identification of cis- and trans-EETs in RBCs and in plasma as well as their release from RBCs suggest a vasoregulatory role of RBCs in view of their potent vasoactivity.

Purinoceptors in renal microvessels: adenosine-activated and cytochrome P450 monooxygenase-derived arachidonate metabolites.

Cytochrome P450 (CYP)-dependent epoxyeicosatrienoic acids (EETs) dilate rat preglomerular microvessels (PGMVs) when adenosine 2A receptors (A(2A)R) are stimulated. As high salt intake increases epoxygenase activity and adenosine levels, we hypothesized that renal adenosine responses would be greater in high salt-fed rats. We have obtained evidence supporting this hypothesis in rats fed a high salt diet for 7 days. Stimulation of adenosine receptors with 2-chloroadenosine in kidneys obtained from rats on high salt (4%) intake produced an increase in EET release that was several-fold greater than in kidneys of rats on normal salt (0.4% NaCl) diets, which was associated with a sharp decline in renovascular resistance. Under conditions of high salt intake, an associated upregulation of A(2A)R and 2C23 protein expression was observed. As EETs are renal vasodilator and natriuretic eicosanoids, the antipressor response to salt loading may operate through an A(2A)R - EET mechanism. These findings expand the role of adenosine-related mechanisms in protecting renal function.

Bradykinin regulates cyclooxygenase-2 in rat renal thick ascending limb cells.

Cyclooxygenase-2 (COX-2) is constitutively expressed in a subset of thick ascending limb cells in the cortex and medulla and increases when the renin-angiotensin and kallikrein-kinin systems are activated. Although the contribution of angiotensin II to the regulation of COX-2 is known, the effects of bradykinin on COX-2 expression have not been determined in this nephron segment. We evaluated expression of B2 bradykinin receptors in thick ascending limb cells containing COX-2 and the effect of bradykinin on COX-2 expression in primary cultured medullary thick ascending cells. The presence of B2 receptors was studied in renal sections by immunohistochemistry with antibodies against B2, COX-2, and Tamm-Horsfall glycoprotein. B2 receptors were detected on the apical and basolateral portion of the thick ascending cells. These cells also contained COX-2, suggesting that COX-2 expression may be regulated via B2 receptor. Incubation of cultured medullary thick ascending cells with bradykinin (10(-7) to 10(-5) mol/L) induced a significant increase on COX-2 protein expression. Maximal expression of COX-2 was observed 4 hours after exposure to bradykinin (10(-7) mol/L), effect abolished by a B2 receptor antagonist (HOE-140; 10(-6) mol/L). Prostaglandin E2 production increased when these cells were challenged with bradykinin for 4 hours, indicating that COX-2 was enzymatically active. We have demonstrated (1) the presence of B2 receptors in thick ascending limb cells expressing COX-2 and (2) the stimulatory effect of bradykinin on COX-2 protein expression, via B2 receptors, in cultured medullary thick ascending cells. We suggest that bradykinin can affect ion transport in the thick ascending limb via a COX-2-mediated mechanism.

Identification of 5,6-trans-epoxyeicosatrienoic acid in the phospholipids of red blood cells.

A novel eicosanoid, 5,6-trans-epoxy-8Z,11Z,14Z-eicosatrienoic acid (5,6-trans-EET), was identified in rat red blood cells. Characterization of 5,6-trans-EET in the sn-2 position of the phospholipids was accomplished by hydrolysis with phospholipase A(2) followed by gas chromatography/mass spectrometry as well as electrospray ionization-tandem mass spectrometry analyses. The electron ionization spectrum of 5,6-erythro-dihydroxyeicosatrienoic acid (5,6-erythro-DHET), converted from 5,6-trans-EET in the samples, matches that of the authentic standard. Hydrogenation of the extracted 5,6-erythro-DHET with platinum(IV) oxide/hydrogen resulted in an increase of the molecular mass by 6 daltons and the same retention time shift as an authentic standard in gas chromatography, suggesting the existence of three olefins as well as the 5,6-erythro-dihydroxyl structure in the metabolite. Match of retention times by chromatography indicated identity of the stereochemistry of the red blood cell 5,6-erythro-DHET vis à vis the synthetic standard. High pressure liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of the phospholipase A(2)-hydrolyzed lipid extracts from red blood cells revealed match of the mass spectrum and retention time of the compound with the authentic 5,6-trans-EET standard, providing direct evidence of the existence of 5,6-trans-EET in red blood cells. The presence of other trans-EETs was also demonstrated. The ability of both 5,6-trans-EET and its product 5,6-erythro-DHET to relax preconstricted renal interlobar arteries was significantly greater than that of 5,6-cis-EET. In contrast, 5,6-cis-EET and 5,6-trans-EET were equipotent in their capacity to inhibit collagen-induced rat platelet aggregation, whereas 5,6-erythro-DHET was without effect. We propose that the red blood cells serve as a reservoir for epoxides which on release may act in a vasoregulatory capacity.