Cytochrome P450 Eicosanoids Research Articles

20-HETE, Blood Pressure, and Vascular Stiffness in Young Adults.

Cytochrome-P450 eicosanoids from arachidonic acid, including vasodilator epoxyeicosatrienoic acids (EETs) and the vasoconstrictor 20-HETE, are important in regulating blood pressure, vascular tone, and cardiac and renal function. This study examined plasma 20-HETE and EETs in relation to blood pressure and vascular stiffness in a cohort of 1054 community-dwelling young adults from the Raine Study at 27 years. Plasma 20-HETE, EETs, and their hydroxylated products were measured by liquid chromatography-tandem mass spectrometry. Pulse wave velocity and aortic distensibility were measured to assess vascular stiffness. Sex differences were assessed using univariate analysis. Multiple regression models assessed the relationship between 20-HETE and systolic blood pressure (SBP) and measures of vascular stiffness. The regression model for SBP showed a positive relationship with plasma 20-HETE (β, 0.092; P<0.0001), after adjusting for sex (P<0.0001), body mass index (P<0.0001), (ln)triglycerides (P<0.0001), and (ln)HOMA-IR (P=0.015), and accounted for 29% of the variance in SBP. 20-HETE was positively related to pulse wave velocity (β, 0.059; P=0.021); after adjusting for sex (P<0.0001), SBP (P<0.0001), (ln)triglycerides (P=0.014), (ln)HOMA-IR (P=0.0001), (ln)high-sensitivity C-reactive protein (P=0.005), and age (P=0.002), the model accounted for 34% of the variance in pulse wave velocity. 20-HETE was inversely associated with aortic distensibility (β, -0.051; P=0.029), independent of sex (P<0.0001), SBP (P<0.0001), and (ln)HOMA-IR (P<0.0001); the model accounted for 25% of the variance in aortic distensibility. Plasma EETs were not significant predictors of SBP or vascular stiffness. In young adults, 20-HETE may play a fundamental role in regulating blood pressure and vascular stiffness independent of numerous cardiometabolic risk factors and cytochrome 450-derived EETs. URL: https://www.anzctr.org.au; Unique identifier: CTRN12617001599369.

CYP eicosanoid pathway mediates colon cancer-promoting effects of dietary linoleic acid.

Human and animal studies support that consuming a high level of linoleic acid (LA, 18:2ω-6), an essential fatty acid and key component of the human diet, increases the risk of colon cancer. However, results from human studies have been inconsistent, making it challenging to establish dietary recommendations for optimal LA intake. Given the importance of LA in the human diet, it is crucial to better understand the molecular mechanisms underlying its potential colon cancer-promoting effects. Using LC-MS/MS-based targeted lipidomics, we find that the cytochrome P450 (CYP) monooxygenase pathway is a major pathway for LA metabolism in vivo. Furthermore, CYP monooxygenase is required for the colon cancer-promoting effects of LA, since the LA-rich diet fails to exacerbate colon cancer in CYP monooxygenase-deficient mice. Finally, CYP monooxygenase mediates the pro-cancer effects of LA by converting LA to epoxy octadecenoic acids (EpOMEs), which have potent effects on promoting colon tumorigenesis via gut microbiota-dependent mechanisms. Overall, these results support that CYP monooxygenase-mediated conversion of LA to EpOMEs plays a crucial role in the health effects of LA, establishing a unique mechanistic link between dietary fatty acid intake and cancer risk. These results could help in developing more effective dietary guidelines for optimal LA intake and identifying subpopulations that may be especially vulnerable to LA's negative effects.

Eicosanoids and Oxidative Stress in Diabetic Retinopathy.

Oxidative stress is an important factor to cause the pathogenesis of diabetic retinopathy (DR) because the retina has high vascularization and long-time light exposition. Cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) enzymes can convert arachidonic acid (AA) into eicosanoids, which are important lipid mediators to regulate DR development. COX-derived metabolites appear to be significant factors causative to oxidative stress and retinal microvascular dysfunction. Several elegant studies have unraveled the importance of LOX-derived eicosanoids, including LTs and HETEs, to oxidative stress and retinal microvascular dysfunction. The role of CYP eicosanoids in DR is yet to be explored. There is clear evidence that CYP-derived epoxyeicosatrienoic acids (EETs) have detrimental effects on the retina. Our recent study showed that the renin-angiotensin system (RAS) activation augments retinal soluble epoxide hydrolase (sEH), a crucial enzyme degrading EETs. Our findings suggest that EETs blockade can enhance the ability of RAS blockade to prevent or mitigate microvascular damage in DR. This review will focus on the critical information related the function of these eicosanoids in the retina, the interaction between eicosanoids and reactive oxygen species (ROS), and the involvement of eicosanoids in DR. We also identify potential targets for the treatment of DR.

19-Hydroxyeicosatetraenoic acid analogs: Antagonism of 20-hydroxyeicosatetraenoic acid-induced vascular sensitization and hypertension.

19-Hydroxyeicosatetraenoic acid (19-HETE, 1), a metabolically and chemically labile cytochrome P450 eicosanoid, has diverse biological activities including antagonism of the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE, 2). A SAR study was conducted to develop robust analogs of 1 with improved in vitro and in vivo efficacy. Analogs were screened in vitro for inhibition of 20-HETE-induced sensitization of rat renal preglomerular microvessels toward phenylephrine and demonstrated to normalize the blood pressure of male Cyp4a14(-/-) mice that display androgen-driven, 20-HETE-dependent hypertension.

A Randomized Trial of Effects of Alcohol on Cytochrome P450 Eicosanoids, Mediators of Inflammation Resolution, and Blood Pressure in Men.

Cardiovascular effects of alcohol consumption may be influenced by both pro- and anti-inflammatory mechanisms. We previously showed that chronic alcohol consumption increased blood pressure (BP), oxidative stress, and 20-hydroxyeicosatetraenoic acid (20-HETE), a vasoconstrictor and pro-inflammatory eicosanoid synthesized by cytochrome P450 (CYP450) enzymes from arachidonic acid. This study in men examined the effect of consuming red wine (RW) on BP in relation to changes in 20-HETE, oxidative stress (F2 -isoprostanes), markers of inflammation, anti-inflammatory CYP450 epoxyeicosatrienoic acids (EETs), and specialized pro-resolving mediators of inflammation (SPMs) derived from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Normotensive men (n=22) were randomly allocated to drink RW (375ml/d) or the equivalent volume of dealcoholized red wine (DRW) or water for 4weeks in a 12-week, 3-period crossover trial. BP, heart rate, 20-HETE, F2 -isoprostanes, and SPM were measured at baseline, 4, 8, and 12weeks. Drinking RW increased BP (p<0.05), plasma and urinary 20-HETE (p<0.05), plasma F2 -isoprostanes (p<0.0001), and the SPMs 18-hydroxyeicosapentaenoic acid (18-HEPE) from EPA, and resolvin D1 (RvD1) and 17R-resolvin D1 (17R-RvD1) from DHA (all p<0.05) compared with DRW and water. EETs and high-sensitivity C-reactive protein were unaffected by RW. Plasma 18-HEPE was positively related to urinary 20-HETE (p<0.008) only after RW. This study has shown that men consuming moderate-to-high alcohol as RW for 4weeks had increased BP, 20-HETE, and oxidative stress, as well as specific SPM that resolve inflammation. These paradoxical findings require further studies to determine whether alcohol stimulates different CYP450 enzymes and whether the findings can be replicated in females.

Effect of Cytochrome P450 Metabolites of Arachidonic Acid in Nephrology.

Thirty-five years ago, a third pathway for the metabolism of arachidonic acid by cytochrome P450 enzymes emerged. Subsequent work revealed that 20-hydroxyeicosatetraenoic and epoxyeicosatrienoic acids formed by these pathways have essential roles in the regulation of renal tubular and vascular function. Sequence variants in the genes that produce 20-hydroxyeicosatetraenoic acid are associated with hypertension in humans, whereas the evidence supporting a role for variants in the genes that alter levels of epoxyeicosatrienoic acids is less convincing. Studies in animal models suggest that changes in the production of cytochrome P450 eicosanoids alter BP. However, the mechanisms involved remain controversial, especially for 20-hydroxyeicosatetraenoic acid, which has both vasoconstrictive and natriuretic actions. Epoxyeicosatrienoic acids are vasodilators with anti-inflammatory properties that oppose the development of hypertension and CKD; 20-hydroxyeicosatetraenoic acid levels are elevated after renal ischemia and may protect against injury. Levels of this eicosanoid are also elevated in polycystic kidney disease and may contribute to cyst formation. Our review summarizes the emerging evidence that cytochrome P450 eicosanoids have a role in the pathogenesis of hypertension, polycystic kidney disease, AKI, and CKD.

Cytochrome P450 eicosanoids in cerebrovascular function and disease.

Cytochrome P450 eicosanoids play important roles in brain function and disease through their complementary actions on cell-cell communications within the neurovascular unit (NVU) and mechanisms of brain injury. Epoxy- and hydroxyeicosanoids, respectively formed by cytochrome P450 epoxygenases and ω-hydroxylases, play opposing roles in cerebrovascular function and in pathological processes underlying neural injury, including ischemia, neuroinflammation and oxidative injury. P450 eicosanoids also contribute to cerebrovascular disease risk factors, including hypertension and diabetes. We summarize studies investigating the roles P450 eicosanoids in cerebrovascular physiology and disease to highlight the existing balance between these important lipid signaling molecules, as well as their roles in maintaining neurovascular homeostasis and in acute and chronic neurovascular and neurodegenerative disorders.

20-HETE Signals Through G-Protein-Coupled Receptor GPR75 (Gq) to Affect Vascular Function and Trigger Hypertension.

20-Hydroxyeicosatetraenoic acid (20-HETE), one of the principle cytochrome P450 eicosanoids, is a potent vasoactive lipid whose vascular effects include stimulation of smooth muscle contractility, migration, and proliferation, as well as endothelial cell dysfunction and inflammation. Increased levels of 20-HETE in experimental animals and in humans are associated with hypertension, stroke, myocardial infarction, and vascular diseases. To date, a receptor/binding site for 20-HETE has been implicated based on the use of specific agonists and antagonists. The present study was undertaken to identify a receptor to which 20-HETE binds and through which it activates a signaling cascade that culminates in many of the functional outcomes attributed to 20-HETE in vitro and in vivo. Using crosslinking analogs, click chemistry, binding assays, and functional assays, we identified G-protein receptor 75 (GPR75), currently an orphan G-protein-coupled receptor (GPCR), as a specific target of 20-HETE. In cultured human endothelial cells, 20-HETE binding to GPR75 stimulated Gαq/11 protein dissociation and increased inositol phosphate accumulation and GPCR-kinase interacting protein-1-GPR75 binding, which further facilitated the c-Src-mediated transactivation of epidermal growth factor receptor. This results in downstream signaling pathways that induce angiotensin-converting enzyme expression and endothelial dysfunction. Knockdown of GPR75 or GPCR-kinase interacting protein-1 prevented 20-HETE-mediated endothelial growth factor receptor phosphorylation and angiotensin-converting enzyme induction. In vascular smooth muscle cells, GPR75-20-HETE pairing is associated with Gαq/11- and GPCR-kinase interacting protein-1-mediated protein kinase C-stimulated phosphorylation of MaxiKβ, linking GPR75 activation to 20-HETE-mediated vasoconstriction. GPR75 knockdown in a mouse model of 20-HETE-dependent hypertension prevented blood pressure elevation and 20-HETE-mediated increases in angiotensin-converting enzyme expression, endothelial dysfunction, smooth muscle contractility, and vascular remodeling. This is the first report to identify a GPCR target for an eicosanoid of this class. The discovery of 20-HETE-GPR75 pairing presented here provides the molecular basis for the signaling and pathophysiological functions mediated by 20-HETE in hypertension and cardiovascular diseases.

Role of CYP eicosanoids in the regulation of pharyngeal pumping and food uptake in Caenorhabditis elegans

Cytochrome P450 (CYP)-dependent eicosanoids comprise epoxy- and hydroxy-metabolites of long-chain PUFAs (LC-PUFAs). In mammals, CYP eicosanoids contribute to the regulation of cardiovascular and renal function. Caenorhabditis elegans produces a large set of CYP eicosanoids; however, their role in worm's physiology is widely unknown. Mutant strains deficient in LC-PUFA/eicosanoid biosynthesis displayed reduced pharyngeal pumping frequencies. This impairment was rescued by long-term eicosapentaenoic and/or arachidonic acid supplementation, but not with a nonmetabolizable LC-PUFA analog. Short-term treatment with 17,18-epoxyeicosatetraenoic acid (17,18-EEQ), the most abundant CYP eicosanoid in C. elegans, was as effective as long-term LC-PUFA supplementation in the mutant strains. In contrast, 20-HETE caused decreased pumping frequencies. The opposite effects of 17,18-EEQ and 20-HETE were mirrored by the actions of neurohormones. 17,18-EEQ mimicked the stimulating effect of serotonin when added to starved worms, whereas 20-HETE shared the inhibitory effect of octopamine in the presence of abundant food. In wild-type worms, serotonin increased free 17,18-EEQ levels, whereas octopamine selectively induced the synthesis of hydroxy-metabolites. These results suggest that CYP eicosanoids may serve as second messengers in the regulation of pharyngeal pumping and food uptake in C. elegans.

20-Hydroxyeicosatetraenoic Acid Inhibition by HET0016 Offers Neuroprotection, Decreases Edema, and Increases Cortical Cerebral Blood Flow in a Pediatric Asphyxial Cardiac Arrest Model in Rats.

Vasoconstrictive and vasodilatory eicosanoids generated after cardiac arrest (CA) may contribute to cerebral vasomotor disturbances and neurodegeneration. We evaluated the balance of vasodilator/vasoconstrictor eicosanoids produced by cytochrome P450 (CYP) metabolism, and determined their role on cortical perfusion, functional outcome, and neurodegeneration after pediatric asphyxial CA. Cardiac arrest of 9 and 12 minutes was induced in 16- to 18-day-old rats. At 5 and 120 minutes after CA, we quantified the concentration of CYP eicosanoids in the cortex and subcortical areas. In separate rats, we inhibited 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis after CA and assessed cortical cerebral blood flow (CBF), neurologic deficit score, neurodegeneration, and edema. After 9 minutes of CA, vasodilator eicosanoids markedly increased versus sham. Conversely, after 12 minutes of CA, vasoconstrictor eicosanoid 20-HETE increased versus sham, without compensatory increases in vasodilator eicosanoids. Inhibition of 20-HETE synthesis after 12 minutes of CA decreased cortical 20-HETE levels, increased CBF, reduced neurologic deficits at 3 hours, and reduced neurodegeneration and edema at 48 hours versus vehicle-treated rats. In conclusion, cerebral vasoconstrictor eicosanoids increased after a pediatric CA of 12 minutes. Inhibition of 20-HETE synthesis improved cortical perfusion and short-term neurologic outcome. These results suggest that alterations in CYP eicosanoids have a role in cerebral hypoperfusion and neurodegeneration after CA and may represent important therapeutic targets.

Cytochrome p450 enzymes in the bioactivation of polyunsaturated Fatty acids and their role in cardiovascular disease.

Various members of the cytochrome P450 (CYP) superfamily have the capacity of metabolizing omega-6 and omega-3 polyunsaturated fatty acids (n-6 and n-3 PUFAs). In most mammalian tissues, CYP2C and CYP2J enzymes are the major PUFA epoxygenases, whereas CYP4A and CYP4F subfamily members function as PUFA hydroxylases. The individual CYP enzymes differ in their substrate specificities as well as regio- and stereoselectivities and thus produce distinct sets of epoxy and/or hydroxy metabolites, collectively termed CYP eicosanoids. Nutrition has a major impact on the endogenous CYP-eicosanoid profile. "Western diets" rich in n-6 PUFAs result in a predominance of arachidonic acid-derived metabolites, whereas marine foodstuffs rich in n-3 PUFAs shift the profile to eicosapentaenoic and docosahexaenoic acid-derived metabolites. In general, CYP eicosanoids are formed as second messengers of numerous hormones, growth factors and cytokines regulating cardiovascular and renal function, and a variety of other physiological processes. Imbalances in the formation of individual CYP eicosanoids are linked to the development of hypertension, myocardial infarction, maladaptive cardiac hypertrophy, acute kidney injury, stroke and inflammatory disorders. The underlying mechanisms are increasingly understood and may provide novel targets for the prevention and treatment of these disease states. Suitable pharmacological agents are under development and first proofs of concept have been obtained in animal models.

Cytochrome P450 eicosanoids in hypertension and renal disease.

Purpose of reviewCytochrome (CYP) P450 metabolites of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) contribute to the regulation of renal tubular and vascular function. This review highlights the results of the recent genetic studies in humans and rodent models, indicating that these eicosanoids participate in the control of blood pressure (BP), chronic kidney disease (CKD), renal ischemia–reperfusion injury (IRI) and polycystic kidney disease (PKD).Recent findingsEndogenous 20-HETE has been reported to play an essential role in the myogenic and tubuloglomerular feedback responses in the afferent arteriole, and a deficiency of 20-HETE contributes to the development of hypertension and renal injury in Dahl S rats. Mutations in CYP4A11 and CYP4F2 have been linked to elevated BP in humans. EETs have been shown to regulate epithelial sodium channel in the collecting duct, lower BP and have renoprotective properties. 20-HETE also opposes the development of CKD and IRI, and may play a role in PKD.SummaryThese studies indicate that CYP P450 metabolites of arachidonic acid play an important role in the control of BP, CKD, AKI and PKD. Drugs targeting these pathways could be useful in the treatment of IRI and CKD.

Urinary CYP eicosanoid excretion correlates with glomerular filtration in African-Americans with chronic kidney disease

Previous studies have indicated that cytochrome P450 (CYP) metabolites of arachidonic acid (AA), i.e., 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs), play an important role in the regulation of renal tubular and vascular function. The present study for the first time profiled HETEs and epoxygenase derived dihydroxyeicosatetraenoic acid diHETEs levels in spot urines and plasma in 262 African American patients from the University of Mississippi Chronic Kidney Disease Clinic and 31 African American controls. Significant correlations in eGFR and urinary 20-HETE/creatinine and 19-HETE/creatinine levels were observed. The eGFR increased by 17.47 [p=0.001] and 60.68 [(p=0.005]ml/min/for each ng/mg increase in 20-HETE and 19-HETE levels, respectively. Similar significant positive associations were found between the other urinary eicosanoids and eGFR and also with 19-HETE/urine creatinine concentration and proteinuria. We found that approximately 80% of plasma HETEs and 30% diHETEs were glucuronidated and the fractional excretion of 20-HETE was less than 1%. These results suggest that there is a significant hepatic source of urinary 20-HETE glucuronide and EETs with extensive renal biotransformation to metabolites which may play a role in the pathogenesis of CKD.

14,15-Epoxyeicosa-5,8,11-trienoic Acid (14,15-EET) Surrogates: Carboxylate Modifications

The cytochrome P450 eicosanoid 14,15-epoxyeicosa-5,8,11-trienoic acid (14,15-EET) is a powerful endogenous autacoid that has been ascribed an impressive array of physiologic functions including regulation of blood pressure. Because 14,15-EET is chemically and metabolically labile, structurally related surrogates containing epoxide bioisosteres were introduced and have become useful in vitro pharmacologic tools but are not suitable for in vivo applications. A new generation of EET mimics incorporating modifications to the carboxylate were prepared and evaluated for vasorelaxation and inhibition of soluble epoxide hydrolase (sEH). Tetrazole 19 (ED50 0.18 μM) and oxadiazole-5-thione 25 (ED50 0.36 μM) were 12- and 6-fold more potent, respectively, than 14,15-EET as vasorelaxants; on the other hand, their ability to block sEH differed substantially, i.e., 11 vs >500 nM. These data will expedite the development of potent and specific in vivo drug candidates.

Acute effects of red wine on cytochrome P450 eicosanoids and blood pressure in men

The vasodilation accompanying acute alcohol ingestion is hard to reconcile with the strong evidence linking chronic alcohol consumption with hypertension. Cytochrome P450 (CYP450) eicosanoids derived from arachidonic acid include vasodilator epoxyeicosatrienoic acids (EETs) and the vasoconstrictor 20-hydroxyeicosatrienoic acid (20-HETE). This study aimed to examine the relationship between CYP450 eicosanoids and blood pressure (BP), and compared the effect of single session of drinking red wine with de-alcoholized red wine (DRW) or water over 24 h. Twenty-five normotensive men were randomly assigned to drink either 375 ml of red wine (41 g of alcohol) or the equivalent volume of DRW or water, with a light meal on 3 separate days. Ambulatory BP and heart rate were measured over 24 h. Blood samples were obtained before and 2, 4 and 24 h after beverage consumption. Blood pressure fell in the first 4 h after red wine consumption (P = 0.001), but was significantly higher after 20 h (P = 0.037). Plasma 20-HETE fell in the 2 h after consumption of all beverages, but over the 24-h period was relatively higher after red wine consumption (P = 0.025). The largest difference in 20-HETE was 2 h after consuming red wine and coincided with the highest blood alcohol level. There were no significant effects of red wine on plasma EETs. Acute consumption of alcohol as red wine results in a relative increase in plasma levels of the vasoconstrictor 20-HETE over 24 h without affecting EETs, and may contribute to the BP elevation that associates with a binge drinking pattern or be a homeostatic response to the acute fall in BP induced by alcohol.

Circulation: Clinical Summaries

<i>Circulation:</i> Clinical Summaries

Abstract 348: Urinary CYP Eicosanoid Excretion Correlates with Glomerular Filtration in African-Americans with Chronic Kidney Disease

Previous studies have indicated that cytochrome P450 (CYP) metabolites of arachidonic acid, ie, 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) play an important role in the regulation of renal tubular function and vascular tone. More recent studies suggest that variants in the CYP4A11 and CYP4F2 genes are linked to the development of hypertension in a variety of human population studies. However, little is known about the role of 20-HETE or EETs in the pathogenesis of hypertension or diabetic induced renal disease because CYP eicosanoids have not been measured in patients with CKD. The present study profiled HETEs and the dihydroxy metabolites of EETs (DHETs) levels, from spot urines at the time of their clinic visit using LC/MS/MS in 106 African-American patients from the University of Mississippi (UMC) Chronic Kidney Disease (CKD) Clinic with various etiologies of renal disease. Informed consent was obtained for this UMMC IRB approved protocol. Significant positive correlations were found between urinary 5,6- DHETE, 8,9-DHETE, 11,12-DIHETE, 14,15-DHETE, 20-HETE and 19-, 15- and 8-HETE levels and estimated GFR (eGFR) as derived from the MDRD. The magnitude of the influence of urinary eicosanoid levels on eGFR was relatively profound since the slopes of these relationships indicated that there is a 5-10% decrement eGFR associated with each ng/ml fall in urinary eicosanoid levels. These results suggest that a decline in the renal formation of 20-HETE and/or EETs may contribute to the pathogenesis of CKD. At very least, urinary CYP eicosanoids may serve as a useful biomarker for progressive disease.

Eicosanoid formation by a cytochrome P450 isoform expressed in the pharynx of Caenorhabditis elegans

Caenorhabditis elegans harbours several CYP (cytochrome P450) genes that are homologous with mammalian CYP isoforms important to the production of physiologically active AA (arachidonic acid) metabolites. We tested the hypothesis that mammals and C. elegans may share similar basic mechanisms of CYP-dependent eicosanoid formation and action. We focused on CYP33E2, an isoform related to the human AA-epoxygenases CYP2C8 and CYP2J2. Co-expression of CYP33E2 with the human NADPH-CYP reductase in insect cells resulted in the reconstitution of an active microsomal mono-oxygenase system that metabolized EPA (eicosapentaenoic acid) and, with lower activity, also AA to specific sets of regioisomeric epoxy- and hydroxy-derivatives. The main products included 17,18-epoxyeicosatetraenoic acid from EPA and 19-hydroxyeicosatetraenoic acid from AA. Using nematode worms carrying a pCYP33E2::GFP reporter construct, we found that CYP33E2 is exclusively expressed in the pharynx, where it is predominantly localized in the marginal cells. RNAi (RNA interference)-mediated CYP33E2 expression silencing as well as treatments with inhibitors of mammalian AA-metabolizing CYP enzymes, significantly reduced the pharyngeal pumping frequency of adult C. elegans. These results demonstrate that EPA and AA are efficient CYP33E2 substrates and suggest that CYP-eicosanoids, influencing in mammals the contractility of cardiomyocytes and vascular smooth muscle cells, may function in C. elegans as regulators of the pharyngeal pumping activity.

Cytochrome P450 eicosanoids and cerebral vascular function

The eicosanoids 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs), which are generated from the metabolism of arachidonic acid by cytochrome P450 (CYP) enzymes, possess a wide array of biological actions, including the regulation of blood flow to organs. 20-HETE and EETs are generated in various cell types in the brain and cerebral blood vessels, and contribute significantly to cerebral blood flow autoregulation and the coupling of regional brain blood flow to neuronal activity (neurovascular coupling). Investigations are beginning to unravel the molecular and cellular mechanisms by which these CYP eicosanoids regulate cerebral vascular function and the changes that occur in pathological states. Intriguingly, 20-HETE and the soluble epoxide hydrolase (sEH) enzyme that regulates EET levels have been explored as molecular therapeutic targets for cerebral vascular diseases. Inhibition of 20-HETE, or increasing EET levels by inhibiting the sEH enzyme, decreases cerebral damage following stroke. The improved outcome following cerebral ischaemia is a consequence of improving cerebral vascular structure or function and protecting neurons from cell death. Thus, the CYP eicosanoids are key regulators of cerebral vascular function and novel therapeutic targets for cardiovascular diseases and neurological disorders.

Eicosanoid profiling in cerebral vessels and brain of WKY, SHR, and SHR‐SP rats

A previous F2 linkage analysis identified a QTL for sensitivity to cerebral ischemia on rat chromosome 5 in the region of the cytochrome P-450 (CYP) 4A genes. CYP4A is a potential candidate gene because it metabolizes arachidonic acid to form 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor of cerebral arteries. The goal of this study was to use liquid chromatography mass spectrometry (LC/MS) to characterize CYP eicosanoid formation in cerebral vessels and surrounding brain parenchyma of Wistar-Kyoto (WKY), spontaneously hypertensive (SHR), and spontaneously hypertensive stroke prone (SHR-SP) rats. Microdissected cerebral vessels and microsomes prepared from the remaining brain tissue were incubated in 0.1M potassium phosphate buffer in the presence of 1mM NADPH, 40μM arachidonic acid, 2μM indomethacin and oxygen at 37°C for 90 minutes. Samples were homogenized and extracted with ethyl acetate. Intact cerebral microvessels and brain microsomes generated 5-, 9-, 11-, 12-, 15-, and 20-HETE, as well as 14,15- and 11,12-EET when incubated with arachidonic acid. The formation of 20-HETE by cerebral vessels of SHR and SHR-SP rats averaged 0.2814±0.050 and 0.2719±0.033pmol/min/mg protein, respectively (n=5). This was significantly greater than that seen in WKY rats (0.1509±0.012 pmol/min/mg protein, n=5). These studies support the potential for differences in the formation of 20-HETE by cerebral arteries to contribute to genetic differences in sensitivity to ischemic stroke. This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-59996 and HL-29587.