IntroductionHeme oxygenase 1 (HO-1) and HO-2 metabolize heme to biliverdin, free iron, and carbon monoxide (CO) (1, 2). HO-2 is constitutively expressed in most tissues, whereas HO-1 is inducible (1). Products of heme metabolism by HO possess biological activities that influence vascular function. Biliverdin and its metabolic product bilirubin are antioxidants (3). Free iron facilitates production of reactive oxygen species (3). CO stimulates soluble guanylate cyclase (4, 5) and calcium-activated potassium (K Ca ) channels (6) in vascular smooth muscle and inhibits expression of endothelin-1 and PDGF in endothelial cells (7).Arterial vessels express HO-1 and/or HO-2 (8-10). Interventions that alter the expression or activity of vascular HO bring about changes of vascular tone and/or reactivity. For example, inhibitors of HO produce constriction of pressurized rat gracilis muscle arterioles (10). On the other hand, heme elicits HO-dependent dilation of rat gracilis muscle arterioles (11), and conditions that induce vascular HO-1 reduce the responsiveness of the rat tail artery and aorta to constrictor agents (9, 12, 13). It would appear, then, that one or more products of heme metabolism by HO contribute to vasodilatory mechanisms (2, 9).The present study was designed to test the hypothesis that the reactivity of small arterial vessels to constrictor agonists is tonically inhibited by CO of vascular origin, via a mechanism that involves upregulation of K Ca channel activity in vascular smooth muscle. We conducted experiments in rat renal interlobar arteries (a) to quantify the generation of CO and determine whether it is HO-dependent, (b) to examine the effect of interventions that decrease the activity or expression of HO on vascular smooth muscle reactivity to constrictor agonists, and (c) to determine the involvement of K Ca channels in the action of CO on the reactivity of vascular smooth muscle to constrictor agonists. MethodsAnimals. All animal protocols were approved by the Institutional Animal Care and Use Committee of New York Medical College. Male Sprague-Dawley rats (250-300 g; Charles River, Wilmington, Massachusetts, USA) were anesthetized (pentobarbital sodium, 60 mg/kg, intraperitoneally) and the kidneys were removed and placed on a dish filled with ice-cold Krebs' buffer (composition in mmol/l: 118.5 NaCl, 4.7 KCl, 2.5 CaCl 2 , 1.2 KH 2 PO 4 , 1.2 MgSO 4 , 25.0 NaHCO 3 , and 11.1 dextrose). The kidneys were sectioned sagittally and the interlobar arteries were dissected out for use in studies on vascular contractility, recording of K + currents in vascular smooth muscle cells, and assessment of HO expression and CO production.Vascular contractility studies. Renal interlobar arteries with an internal diameter averaging 240 ± 4 µm were cut into ring segments 2 mm in length. Freshly prepared rings or rings pretreated as described below were mounted on 25 µm stainless steel wires in the chambers of a multivessel myograph (J.P. Trading, Aarhus, Rat renal interlobar arteries express heme oxygenase 2 (HO...

Nitrative stress has an important role in microvascular degeneration leading to ischemia in conditions such as diabetic retinopathy and retinopathy of prematurity. Thus far, mediators of nitrative stress have been poorly characterized. We recently described that trans-arachidonic acids are major products of NO(2)(*)-mediated isomerization of arachidonic acid within the cell membrane, but their biological relevance is unknown. Here we show that trans-arachidonic acids are generated in a model of retinal microangiopathy in vivo in a NO(*)-dependent manner. They induce a selective time- and concentration-dependent apoptosis of microvascular endothelial cells in vitro, and result in retinal microvascular degeneration ex vivo and in vivo. These effects are mediated by an upregulation of the antiangiogenic factor thrombospondin-1, independently of classical arachidonic acid metabolism. Our findings provide new insight into the molecular mechanisms of nitrative stress in microvascular injury and suggest new therapeutic avenues in the management of disorders involving nitrative stress, such as ischemic retinopathies and encephalopathies.

The cytochrome P-450 pathway is capable of metabolizing arachidonic acid to omega- and subterminal hydroxylase metabolites, 16-, 17-, 18-, 19-, and 20-hydroxyeicosatetraenoic acids (P-450 HETEs). We have quantitated, by gas chromatography-mass spectrometry (GC/MS), endogenous HETEs exiting the rabbit isolated perfused kidney elicited by hormonal stimulation. Kidneys were perfused with Krebs-Henseleit solution containing indomethacin (2.8 microM) to prevent further metabolism of HETEs by cyclooxygenase. Phenylephrine (2-3 microM) was added to the perfusate to raise perfusion pressure to approximately 80 mmHg. Angiotensin II (ANG II), arginine vasopressin (AVP), and bradykinin (BK) were injected into the renal artery and perfusates collected throughout the vasoactive response. After addition of an internal standard, deuterated 19-HETE, perfusates were extracted and purified and P-450 HETEs were derivatized for GC/MS analysis. Under basal conditions, 16-, 18-, 19-, and 20-HETEs were released (range: 50-270 pg/ml), 19-HETE being the highest and fivefold greater than 16-HETE, the lowest. Injection of 50 ng ANG II increased by two- to sixfold P-450 HETE release associated with an increase of 40 +/- 11 mmHg in perfusion pressure. An equipressor dose of AVP (50 ng) did not release P-450 HETEs nor did a 5-micrograms dose of the vasodilator peptide BK, which decreased perfusion pressure by 22 +/- 6 mmHg. Authentic 19- and 20-HETE isomers resulted in dose-dependent dilation, as did 18(R)- and 16(R)-HETEs, whereas their enantiomers and 17-HETE isomers were without effect on perfusion pressure. The vasodilator effects of 18(R)- and 16(R)-HETEs, like 20- and 19-HETEs, were inhibited by indomethacin. Furthermore, P-450 HETEs exhibited both regio- and stereoselective inhibition of proximal tubule adenosine triphosphatase (ATPase) activity. The (S) enantiomers of 16- and 17-HETE potently inhibited activity, whereas their (R) isomers and other P-450 HETEs had negligible effects on ATPase activity. The quantity of HETEs released from the kidney, either under basal conditions or when stimulated by ANG II, and their biological profile suggest that subterminal HETEs may participate in renal mechanisms affecting vasomotion and tubular transport.

Oxygen free radicals oxidize arachidonic acid to a complex mixture of metabolites termed isoeicosanoids that share structural similarity to enzymatically derived eicosanoids. However, little is known about oxidations of arachidonic acid mediated by reactive radical nitrogen oxides. We have studied the reaction of arachidonic acid with NO2, a free radical generated by nitric oxide and nitrite oxidations. A major group of products appeared to be a mixture of arachidonic acid isomers having one trans-bond and three cis-double bonds. We have termed these new products trans-arachidonic acids. These isomers were chromatographically distinct from arachidonic acid and produced mass spectra that were nearly identical with mass spectra of arachidonic acid. The lack of ultraviolet absorbance above 205 nm and the similarity of mass spectra of dimethyloxazoline derivatives suggested that the trans-bond was not conjugated with any of the cis-bonds, and the C=C bonds were located at carbons 5, 8, 11, and 14. Further identification was based on comparison of chromatographic properties with synthetic standards and revealed that NO2 generated 14-trans-eicosatetraenoic acid and a mixture containing 11-trans-, 8-trans-, and 5-trans-eicosatetraenoic acids. Exposure of human platelets to submicromolar levels of NO2 resulted in a dose-dependent formation of 14-trans-eicosatetraenoic acid and other isomers within platelet glycerophospholipids. Using a sensitive isotopic dilution assay we detected trans-arachidonic acids in human plasma (50.3 +/- 10 ng/ml) and urine (122 +/- 50 pg/ml). We proposed a mechanism of arachidonic acid isomerization that involves a reversible attachment of NO2 to a double bond with formation of a nitroarachidonyl radical. Thus, free radical processes mediated by NO2 lead to generation of trans-arachidonic acid isomers, including biologically active 14-trans-eicosatetraenoic acid, within membrane phospholipids from which they can be released and excreted into urine.

In the rat isolated perfused kidney, 5,8,11,14-eicosatetraynoic acid, an inhibitor of all pathways of arachidonic acid (AA) metabolism, diminished endothelin-1 (ET-1)- and angiotensin II (ANG II)-induced renal vasoconstriction by approximately 60-70%. We then examined the individual contribution of each oxygenase, cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P-450 (CYP) to the vasoconstrictor effects of ET-1 and ANG II. Inhibition of COX with indomethacin reduced by 30-40% the vasoconstrictor responses to ET-1 and ANG II. Inhibition of 12-LOX with baicalein and 5- and 12-LOX with 5,8,11-eicosatriynoic acid attenuated ANG II-induced renal vasoconstriction by approximately 40-60% but did not affect responses to ET-1. In contrast, 12,12-dibromododec-11-enoic acid (DBDD), an inhibitor of the CYP omega/omega 1-hydroxylase pathway, diminished ET-1-induced renal vasoconstriction by 30-40%, an effect reproduced by depletion of CYP enzymes with CoCl2. Neither DBDD nor CoCl2 affected renal vasoconstriction elicited by ANG II. ET-1 increased efflux of 19- and 20-hydroxyeicosatetraenoic acid, an effect reduced by DBDD. Thus products of the COX and CYP pathways contribute to the renal vasoconstrictor response to ET-1, whereas COX- and LOX-derived eicosanoids contribute to the response to ANG II, accounting for > or = 80% of the vasoactivity of the peptides.

Raising extracellular Ca2+ (Ca2+o) stimulating the Ca(2+)-sensing receptor (CaR) decreased the activity of the apical 70-pS K+ channel via a cytochrome P-450-dependent mechanism in the thick ascending limb (TAL) of the rat kidney [W. H. Wang, M. Lu, and S. C. Hebert. Am. J. Physiol. 270 (Cell Physiol. 39): C103-C111, 1996]. We have now used the patch-clamp technique and fluorescent dyes to investigate the signaling mechanism by which this effect is produced. Addition of 500 microM gadolinium (Gd3+), an agent which has been shown to activate the CaR (E. M. Brown, G. Gamba, D. Riccardi, M. Lombardi, R. Butters, O. Kifor, A. Sun, M. A. Hediger, J. Lytton, and S. C. Hebert. Nature 366: 575-580, 1993), mimics the inhibitory effect of raising Ca2+o from 1.1 to 5 mM on channel activity. Effects of the high Ca2+o and Gd3+ were abolished by blockade of phospholipase A2 (PLA2) but not by inhibition of phospholipase C (PLC). Raising Ca2+o also increased 20-hydroxyeicosatetraenoic acid production significantly. To investigate the effect of stimulation of the CaR on intracellular Ca2+ (Ca2+i), we used the acetoxymethyl ester of fura 2 to monitor the Ca2+i. Raising Ca2+o from 1.1 to 5 mM increased the Ca2+i significantly from 50 to 150 nM. However, addition of thapsigargin failed to abolish the effect of 5 mM Ca2+o on Ca2+i. Also, application of Gd3+ only slightly increased the Ca2+i, suggesting that elevation of the Ca2+i by high Ca2+o was the result of an influx of Ca2+ rather than enhanced Ca2+ release from Ca2+ stores. That the increase in Ca2+ influx is not mainly responsible for the effect of stimulating the CaR on channel activity is further supported by experiments in which 500 microM Gd3+ inhibited the K+ channel in cell-attached patches in a Ca(2+)-free bath. Furthermore, addition of 500 microM Gd3+ or 5 mM Ca2+o decreased intracellular Na+ measured with fluorescent sodium indicator, suggesting inhibition of Na+ transport. We conclude that PLA2 is involved in the stimulation of the CaR-induced inhibition of apical K+ channels in the TAL.

Peroxynitrite (ONOO؊ ) has been shown in studies on vascular relaxation and guanylate cyclase activation to react with glutathione (GSH), generating an intermediate product that promotes a time-dependent production of nitric oxide (NO). In this study, reactions of ONOO ؊ with GSH produced a new substance, which was characterized by liquid chromatography, ultraviolet spectroscopy, and electrospray tandem mass spectrometry. The mass spectrometric data provided evidence that the product of this reaction was S-nitroglutathione (GSNO 2 ) and that S-nitrosoglutathione (GSNO) was not a detectable product of this reaction. Further evidence was obtained by comparison of the spectral and chromatographic properties with synthetic standards prepared by reaction of GSH with nitrosonium or nitronium borofluorates. Both the synthetic and ONOO ؊ /GSH-derived GSNO 2 generated a protonated ion, GSNO 2 H ؉ , at m/z 353, which was unusually resistant to decomposition under collision activation, and no fragmentation was observed at collision energy of 25 eV. In contrast, an ion at m/z 337 (GSNOH ؉ ), generated from the synthetic GSNO, readily fragmented with the abundant loss of NO at 9 eV. Reactions of ONOO ؊ with GSH resulted in the generation of NO, which was detected by the head space/ NO-chemiluminescence analyzer method. The generation of NO was inhibited by the presence of glucose and/or CO 2 in the buffers employed. Synthetic GSNO 2 spontaneously generated NO in a manner that was not significantly altered by glucose or CO 2 . Thus, ONOO ؊ reacts with GSH to form GSNO 2 , and GSNO 2 decomposes in a manner that generates NO.Exposure of vascular tissue to peroxynitrite (ONOO Ϫ ) 1 results in a prolonged relaxation (1) that appears to be mediated through a glutathione (GSH)-dependent regeneration of NO (2). Peroxynitrite has also been observed to stimulate guanylate cyclase activity in a thiol-dependent manner in vascular endothelial and smooth muscle preparations (3, 4). Whereas the reaction of ONOO Ϫ with GSH has been reported to form small amounts of S-nitroso-GSH (GSNO) (3, 5), our previous studies detected a different product of this reaction, which was isolated and demonstrated to possess potent vascular relaxant activity (2). Examination of the reaction of nitrogen dioxide (NO 2 ) with GSH detected the formation of what appears to be the same product as that observed in the reaction with ONOO Ϫ (6). Because the biologically active metabolite of these reactions co-migrated on HPLC with a the product of a reaction between nitrosonium borofluorate (NO 2 BF 4 ) and GSH, the vascular relaxant detected was suggested to be a nitrated product of GSH (GSNO 2 ) (6). Thus, additional studies are needed to identify the biologically active substances derived from the reaction of GSH with ONOO Ϫ . Peroxynitrite is also known to undergo additional reactions in the presence of physiological buffered systems and GSH. One of the first observed actions of ONOO Ϫ on thiols was that it caused oxidation reactions, and an analysis of products o...

Studies were conducted on isolated rat gracilis muscle arterioles to examine the role of vascular heme oxygenase (HO)-derived carbon monoxide (CO) on myogenic constrictor responses to stepwise increments in intraluminal pressure. The arterioles express HO-2 but not HO-1 and manufacture CO. Both HO-2 protein expression and CO production are reduced in arterioles maintained for 18 h before experimentation in media containing HO-2 antisense oligodeoxynucleotides (AS-ODN). Pressurization of arterioles mounted on a myograph over the pressure range of 40--100 mmHg elicits reduction of internal diameter. At pressures >40 mmHg, the internal diameter of vessels treated with either HO-2 AS-ODN, the HO inhibitor chromium mesoporphyrin (CrMP), or the K(+) channel blocker tetraethylammonium (TEA) are smaller than the corresponding control values. The inclusion of exogenous CO, but not of biliverdin, in the superfusion buffer attenuates pressure-induced vasoconstriction in CrMP-treated vessels. However, exogenous CO does not attenuate pressure-induced vasoconstriction in vessels treated with both CrMP and TEA. Collectively, these data suggest that CO of vascular origin attenuates pressure-induced arteriolar constriction via a mechanism involving a TEA-sensitive K(+) channel.