A chemical description of the action of phospholipase A2 (PLA2) can now be inferred with confidence from three high-resolution x-ray crystal structures. The first is the structure of the PLA2 from the venom of the Chinese cobra (Naja naja atra) in a complex with a phosphonate transition-state analogue. This enzyme is typical of a large, well-studied homologous family of PLA2S. The second is a similar complex with the evolutionarily distant bee-venom PLA2. The third structure is the uninhibited PLA2 from Chinese cobra venom. Despite the different molecular architectures of the cobra and bee-venom PLA2s, the transition-state analogue interacts in a nearly identical way with the catalytic machinery of both enzymes. The disposition of the fatty-acid side chains suggests a common access route of the substrate from its position in the lipid aggregate to its productive interaction with the active site. Comparison of the cobra-venom complex with the uninhibited enzyme indicates that optimal binding and catalysis at the lipid-water interface is due to facilitated substrate diffusion from the interfacial binding surface to the catalytic site rather than an allosteric change in the enzyme's structure. However, a second bound calcium ion changes its position upon the binding of the transition-state analogue, suggesting a mechanism for augmenting the critical electrophile.

The mechanisms that promote an inflammatory environment and accelerated atherosclerosis in diabetes are poorly understood. We show that macrophages isolated from two different mouse models of type 1 diabetes exhibit an inflammatory phenotype. This inflammatory phenotype associates with increased expression of long-chain acyl-CoA synthetase 1 (ACSL1), an enzyme that catalyzes the thioesterification of fatty acids. Monocytes from humans and mice with type 1 diabetes also exhibit increased ACSL1. Furthermore, myeloid-selective deletion of ACSL1 protects monocytes and macrophages from the inflammatory effects of diabetes. Strikingly, myeloid-selective deletion of ACSL1 also prevents accelerated atherosclerosis in diabetic mice without affecting lesions in nondiabetic mice. Our observations indicate that ACSL1 plays a critical role by promoting the inflammatory phenotype of macrophages associated with type 1 diabetes; they also raise the possibilities that diabetic atherosclerosis has an etiology that is, at least in part, distinct from the etiology of nondiabetic vascular disease and that this difference is because of increased monocyte and macrophage ACSL1 expression.

Functional and biochemical data have suggested a role for the cytochrome P450 arachidonate monooxygenases in the pathophysiology of hypertension, a leading cause of cardiovascular, cerebral, and renal morbidity and mortality. We show here that disruption of the murine cytochrome P450, family 4, subfamily a, polypeptide 10 (Cyp4a10) gene causes a type of hypertension that is, like most human hypertension, dietary salt sensitive. Cyp4a10 -/-mice fed low-salt diets were normotensive but became hypertensive when fed normal or high-salt diets. Hypertensive Cyp4a10 -/-mice had a dysfunctional kidney epithelial sodium channel and became normotensive when administered amiloride, a selective inhibitor of this sodium channel. These studies (a) establish a physiological role for the arachidonate monooxygenases in renal sodium reabsorption and blood pressure regulation, (b) demonstrate that a dysfunctional Cyp4a10 gene causes alterations in the gating activity of the kidney epithelial sodium channel, and (c) identify a conceptually novel approach for studies of the molecular basis of human hypertension. It is expected that these results could lead to new strategies for the early diagnosis and clinical management of this devastating disease. IntroductionPrevalence, complexity, and multiple medical and socioeconomic consequences make hypertension a major health challenge for most of the Western world (1). While environmental factors and coexist ing conditions play a role in the development and progression of hypertension, segregation and linkage analyses indicate that mul tiple genetic factors contribute to its complex etiology (2-7). Fur thermore, clinical studies show that the cardiovascular and renal morbidity and mortality resulting from hypertension are markedly reduced by timely diagnosis and early clinical intervention (1). As the kidneys play a central role in the control of body salt and fluid balance, they are frequent targets for the treatment of hypertension, especially those forms sensitive to dietary salt (2-5). However, since the molecular basis of prevalent forms of the disease remains uncer tain, its early diagnosis and treatment are largely symptomatic. It is expected that the identification of novel pathways/genes involved in blood pressure variations (3, 6, 7) will lead to new therapeutic targets and to improved diagnosis and prevention. Indeed, early detection and treatment are urgently needed to prevent the dangerous and profound consequences of untreated chronic hypertension.The metabolism of endogenous arachidonic acid (AA) to epoxy eicosatrienoic acids (EETs) and 20hydroxyeicosatetraenoic acid

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...

We used the patch-clamp technique to study the effect of arachidonic acid (AA) on epithelial Na channels (ENaC) in the rat cortical collecting duct (CCD). Application of 10 μM AA decreased the ENaC activity defined by NPo from 1.0 to 0.1. The dose–response curve of the AA effect on ENaC shows that 2 μM AA inhibited the ENaC activity by 50%. The effect of AA on ENaC is specific because neither 5,8,11,14-eicosatetraynoic acid (ETYA), a nonmetabolized analogue of AA, nor 11,14,17-eicosatrienoic acid mimicked the inhibitory effect of AA on ENaC. Moreover, inhibition of either cyclooxygenase (COX) with indomethacin or cytochrome P450 (CYP) ω-hydroxylation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) failed to abolish the effect of AA on ENaC. In contrast, the inhibitory effect of AA on ENaC was absent in the presence of N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH), an agent that inhibits CYP-epoxygenase activity. The notion that the inhibitory effect of AA is mediated by CYP-epoxygenase–dependent metabolites is also supported by the observation that application of 200 nM 11,12-epoxyeicosatrienoic acid (EET) inhibited ENaC in the CCD. In contrast, addition of 5,6-, 8,9-, or 14,15-EET failed to decrease ENaC activity. Also, application of 11,12-EET can still reduce ENaC activity in the presence of MS-PPOH, suggesting that 11,12-EET is a mediator for the AA-induced inhibition of ENaC. Furthermore, gas chromatography mass spectrometry analysis detected the presence of 11,12-EET in the CCD and CYP2C23 is expressed in the principal cells of the CCD. We conclude that AA inhibits ENaC activity in the CCD and that the effect of AA is mediated by a CYP-epoxygenase–dependent metabolite, 11,12-EET.