Identification of a new P450s gene (AccCYP4AV1) and its roles in abiotic stress resistance in the Apis cerana cerana Fabricius.

Cytochrome P450 monooxygenases (P450s) play significant roles in protecting organisms from abiotic stress damage. Here, we report the sequence and characterization of a P450s gene (AccCYP4AV1), isolated from Apis cerana cerana Fabricius. The open reading frame of AccCYP4AV1 is 1506 base pairs long and encodes a predicted protein of 501 amino acids and 57.84 kDa, with an isoelectric point of 8.67. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis indicated that AccCYP4AV1 is more highly expressed in the midgut than in other tissues. In addition, the highest expression occurs in newly emerged adult workers, followed by the first instar of the larval stage. In addition, the expression of the AccCYP4AV1 was upregulated by low temperature (4 °C), ultraviolet radiation, hydrogen peroxide, paraquat, and dichlorvos treatments. In contrast, AccCYP4AV1 transcription was downregulated by other abiotic stress conditions: exposure to increased temperature (44 °C), deltamethrin, cadmium chloride, and mercury (II) chloride. Moreover, when AccCYP4AV1 was knocked-down by RNA interference, the results suggested that multiple antioxidant genes (AccsHSP22.6, AccSOD2, AccTpx1, and AccTpx4) were downregulated and antioxidant genes AccGSTO1 and AccTrx1 were upregulated. The activity levels of peroxidase and catalase were upregulated in the AccCYP4AV1-knocked-down samples, compared with those in the control groups. These findings suggest that the AccCYP4AV1 protein might be involved in the defense against abiotic stress damage.

A Role for the Orphan Human Cytochrome P450 2S1 in Polyunsaturated Fatty Acid ω-1 Hydroxylation Using an Untargeted Metabolomic Approach.

Cytochrome P450 (P450) 2S1 is one of the orphan P450s, known to be expressed but not having a defined function with an endogenous substrate or in drug oxidations. Although it has been clearly demonstrated to catalyze reductive reactions, its role in NADPH-dependent oxidations has been ambiguous. In our efforts to characterize orphan human P450 enzymes, we used an untargeted liquid chromatography-mass spectromterymetabolomic approach with recombinant human P450 2S1 and extracts of rat stomach and intestine, sites of P450 2S1 localization in humans and animals. The search yielded several candidates, including the product 19-hydroxyarachidonic acid. Subsequent 18O analysis and in vitro studies with commercial arachidonic acid and 19-hydroxyarachidonic acid were used to validate ω-1 hydroxylation of the former molecule as a NADPH- and O2-dependent reaction. Steady-state kinetic assays were done for ω-1 hydroxylation reactions of P450 2S1 with several other long-chain fatty acids, including arachidonic, linoleic, α-linolenic, eicosapentaenoic, and docosapentaenoic acids. Rates of hydroxylation were slow, but no detectable activity was seen with either medium-chain length or saturated fatty acids. P450 2S1 is known to be expressed, at least at the mRNA level, to the extent of some other non-3A subfamily P450s in the human gastrointestinal tract, and the activity may be relevant. We conclude that P450 2S1 is a fatty acid ω-1 hydroxylase, although the physiologic relevance of these oxidations remains to be established. The metabolomic approaches we employed in this study are feasible for orphan P450s and other enzymes, in regard to annotation of function, in mammals and other organisms. SIGNIFICANCE STATEMENT: An untargeted mass spectrometry approach was utilized to identify ω-1 hydroxylation of arachidonic acid as an oxidative reaction catalyzed by human cytochrome P450 2S1. The enzyme also catalyzes the relatively slow ω-1 hydroxylation of several other unsaturated long-chain fatty acids.

ß-adrenergic Receptor-stimulated Cardiac Myocyte Apoptosis: Role of Cytochrome P450 ω-hydroxylase.

Prolonged or excessive ß-adrenergic activation leads to cardiac myocyte loss and heart dysfunction; however, the underlying cellular mechanisms are still unclear. Therefore, we first confirmed the effect of isoproterenol (ISO), a ß-adrenergic receptor agonist, on cardiac toxicity using TUNEL and caspase activity assays in cultured rat cardiomyocytes. ISO treatment significantly increased cardiomyocyte apoptosis. Persistent ISO stimulation of cardiomyocytes also increased the expression of CYP4A3, a major CYP450 ω-hydroxylase that produces 20-hydroxyeicosatetraenoic acid (20-HETE) in a time-dependent manner. Next, we examined the effect of ISO and 20-HETE on cardiomyocyte apoptosis using annexin V and propidium iodide staining. Treatment with either 20-HETE or ISO significantly increased cardiomyocyte apoptosis, and inhibition of 20-HETE production using 17-ODYA, a CYP450 ω-hydroxylase inhibitor, dramatically attenuated ISO-induced cardiomyocyte apoptosis. To determine the apoptotic pathway involved, the mitochondrial membrane potential (ΔΨm) was measured by detecting the ratio of JC-1 green/red emission intensity. The results demonstrated that 17-ODYA significantly abolished ISO-induced disruption of ΔΨm and that 20-HETE alone induced a marked disruptive effect on ΔΨm in cardiomyocytes. In addition, 20-HETE-induced disruption of ΔΨm and apoptosis was significantly attenuated by KN93, a CaMKII inhibitor. Taken together, these results demonstrate that 20-HETE treatment induces significant apoptosis via mitochondrial-dependent pathways, and that inhibition of 20-HETE production using 17-ODYA attenuates ISO-induced cardiomyocyte apoptosis.

Genetics of Bietti Crystalline Dystrophy.

Bietti crystalline dystrophy (BCD) is an inherited retinal degenerative disease characterized by crystalline deposits in the retina, followed by progressive atrophy of the retinal pigment epithelium (RPE), choriocapillaris, and photoreceptors. CYP4V2 has been identified as the causative gene for BCD. The CYP4V2 gene belongs to the cytochrome P450 superfamily and encodes for fatty acid ω-hydroxylase of both saturated and unsaturated fatty acids. The CYP4V2 protein is localized most abundantly within the endoplasmic reticulum in the RPE and is postulated to play a role in the physiological lipid recycling system between the RPE and photoreceptors to maintain visual function. Electroretinographic assessments have revealed progressive dysfunction of rod and cone photoreceptors in patients with BCD. Several genotypes have been associated with more severe phenotypes based on clinical and electrophysiological findings. With the advent of multimodal imaging with spectral domain optical coherence tomography, fundus autofluorescence, and adaptive optics scanning laser ophthalmoscopy, more precise delineation of BCD severity and progression is now possible, allowing for the potential future development of targets for gene therapy.

Angiotensin II stimulation alters vasomotor response to adenosine in mouse mesenteric artery: role for A1 and A2B adenosine receptors.

BACKGROUND AND PURPOSE: Stimulation of the A1 adenosine receptor and angiotensin II receptor type-1 (AT1 receptor) causes vasoconstriction through activation of cytochrome P450 4A (CYP4A) and ERK1/2. Thus, we hypothesized that acute angiotensin II activation alters the vasomotor response induced by the non-selective adenosine receptor agonist, NECA, in mouse mesenteric arteries (MAs). EXPERIMENTAL APPROACH: We used a Danish Myo Technology wire myograph to measure muscle tension in isolated MAs from wild type (WT), A1 receptor and A2B receptor knockout (KO) mice. Western blots were performed to determine the expression of AT1 receptors and CYP4A. KEY RESULTS: Acute exposure (15 min) to angiotensin II attenuated the NECA-dependent vasodilatation and enhanced vasoconstriction. This vasoconstrictor effect of angiotensin II in NECA-treated MAs was abolished in A1 receptor KO mice and in WT mice treated with the A1 receptor antagonist DPCPX, CYP4A inhibitor HET0016 and ERK1/2 inhibitor PD98059. In MAs from A2B receptor KO mice, the vasoconstrictor effect of angiotensin II on the NECA-induced response was shown to be dependent on A1 receptors. Furthermore, in A2B receptor KO mice, the expression of AT1 receptors and CYP4A was increased and the angiotensin II-induced vasoconstriction enhanced. In addition, inhibition of KATP channels with glibenclamide significantly reduced NECA-induced vasodilatation in WT mice. CONCLUSIONS AND IMPLICATIONS: Acute angiotensin II stimulation enhanced A1 receptor-dependent vasoconstriction and inhibited A2B receptor-dependent vasodilatation, leading to a net vasoconstriction and altered vasomotor response to NECA in MAs. This interaction may be important in the regulation of BP.

Involvement of renal cytochromes P450 and arachidonic acid metabolites in diabetic nephropathy.

Diabetic nephropathy (DN) is one of the most serious complications of type I and type II diabetes. DN is characterized by hyperfiltration, hypertrophy, extracellular matrix accumulation, and proteinuria. This advances into renal fibrosis and loss of renal function. Reactive oxygen species (ROS) and TGF-beta have been implicated in the pathogenesis of diabetic nephropathy. Early stages of diabetic nephropathy are also associated with alterations in renal sodium handling as well as hypertension; both are processes linked by involvement of the arachidonic acid (AA) metabolites, 20-hydroxyeicosatetraenoic acid (20-HETE, produced by cytochrome P450-4a, (CYP4A) and epoxyeicosatrienoic acids (EETs). Indeed, metabolism of AA is increased in a rat model of diabetes. In this study, we demonstrate that rats with streptozotocin-induced diabetes of 1 month duration develop renal hypertrophy and increased fibronectin and TGF-beta1 expression/cortical levels concomitant with an increase in CYP4A expression and 20 HETE production. These results were also paralleled by an increase in reactive oxygen species (ROS) production and NADPH oxidase activity. Treatment of diabetic rats with HET0016, selective inhibitor of CYP 4A, prevented all these changes. Our results suggest that diabetes-induced induction of CYP4A and 20-HETE production could be a major pathophysiological mechanism leading to activation of ROS through an NADPH dependent pathway and TGF-beta1 thus resulting in major renal pathology. Inhibitors of 20-HETE production could thus have an important therapeutic potential in the treatment of diabetic nephropathy.

Adenosine A1 receptors link to smooth muscle contraction via CYP4a, protein kinase C-α, and ERK1/2.

Adenosine A1 receptor (A1AR) activation contracts smooth muscle, although signaling mechanisms are not thoroughly understood. Activation of A1AR leads to metabolism of arachidonic acid, including the production of 20-hydroxyeicosatetraenoic acid (20-HETE) by cytochrome P4504a (CYP4a). The 20-HETE can activate protein kinase C-α (PKC-α), which crosstalks with extracellular signal-regulated kinase (ERK1/2) pathway. Both these pathways can regulate smooth muscle contraction, we tested the hypothesis that A1AR contracts smooth muscle through a pathway involving CYP4a, PKC-α, and ERK1/2. Experiments included isometric tension recordings of aortic contraction and Western blots of signaling molecules in wild type (WT) and A1AR knockout (A1KO) mice. Contraction to the A1-selective agonist 2-chloro-N cyclopentyladenosine (CCPA) was absent in A1KO mice aortae, indicating the contractile role of A1AR. Inhibition of CYP4a (HET0016) abolished 2-chloro-N cyclopentyladenosine-induced contraction in WT aortae, indicating a critical role for 20-HETE. Both WT and A1KO mice aortae contracted in response to exogenous 20-HETE. Inhibition of PKC-α (Gö6976) or ERK1/2 (PD98059) attenuated 20-HETE-induced contraction equally, suggesting that ERK1/2 is downstream of PKC-α. Contractions to exogenous 20-HETE were significantly less in A1KO mice; reduced protein levels of PKC-α, p-ERK1/2, and total ERK1/2 supported this observation. Our data indicate that A1AR mediates smooth muscle contraction via CYP4a and a PKC-α-ERK1/2 pathway.

The cytochrome P450 4A/F-20-hydroxyeicosatetraenoic acid system: a regulator of endothelial precursor cells derived from human umbilical cord blood.

Endothelial progenitor cells (EPCs) contribute to physiological and pathological neovascularization. Previous data have suggested that the cytochrome P450 4A/F (CYP4A/F)-20-hydroxyeicosatetraenoic acid (20-HETE) system regulates neovascularization. Therefore, we studied whether the angiogenic effects of the CYP4A/F-20-HETE system involve regulation of EPC function. We extracted human umbilical cord blood and isolated EPCs, which express AC133(+)CD34(+) and kinase insert domain receptor (KDR) surface markers and contain mRNA and protein for CYP4A11 and CYP4A22 enzymes, as opposed to mesenchymal stem cells, which only express negligible amounts of CYP4A11/22. When EPCs were incubated with arachidonic acid, they produced 20-HETE, which stimulated the cells to proliferate and migrate, as did vascular endothelial growth factor. Incubation with 1 µM N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine (HET0016), a selective inhibitor of 20-HETE synthesis, reduced the proliferative and migratory effects of vascular endothelial growth factor and also significantly abolished EPC migration mediated by stroma-derived factor-1α, as did (6,15) 20-hydroxyeicosadienoic acid. Coculturing EPCs and endothelial cells on a Matrigel matrix led to tube formation, which in turn was inhibited by both HET0016 and 20-hydroxyeicosadienoic acid. We concluded that the CYP4A/F-20-HETE system is expressed in EPCs and can act as both an autocrine and a paracrine regulatory factor.

Salt modulates vascular response through adenosine A(2A) receptor in eNOS-null mice: role of CYP450 epoxygenase and soluble epoxide hydrolase.

High salt (HS) intake can change the arterial tone in mice, and the nitric oxide (NO) acts as a mediator to some of the receptors mediated vascular response. The main aim of this study was to explore the mechanism behind adenosine-induced vascular response in HS-fed eNOS(+/+) and eNOS(-/-) mice The modulation of vascular response by HS was examined using aortas from mice (eNOS(+/+) and eNOS(-/-)) fed 4% (HS) or 0.45% (NS) NaCl-diet through acetylcholine (ACh), NECA (adenosine-analog), CGS 21680 (A(2A) AR-agonist), MS-PPOH (CYP epoxygenase-blocker; 10(-5) M), AUDA (sEH-blocker; 10(-5) M), and DDMS (CYP4A-blocker; 10(-5) M). ACh-response was greater in HS-eNOS(+/+) (+59.3 ± 6.3%) versus NS-eNOS(+/+) (+33.3 ± 8.0%; P < 0.05). However, there was no response in both HS-eNOS(-/-) and NS-eNOS(-/-). NECA-response was greater in HS-eNOS(-/-) (+37.4 ± 3.2%) versus NS-eNOS(-/-) (+7.4.0 ± 3.8%; P < 0.05). CGS 21680-response was also greater in HS-eNOS(-/-) (+45.4 ± 5.2%) versus NS-eNOS(-/-)(+5.1 ± 5.0%; P < 0.05). In HS-eNOS(-/-), the CGS 21680-response was reduced by MS-PPOH (+7.3 ± 3.2%; P < 0.05). In NS-eNOS(-/-), the CGS 21680-response was increased by AUDA (+38.2 ± 3.3%; P < 0.05) and DDMS (+30.1 ± 4.1%; P < 0.05). Compared to NS, HS increased CYP2J2 in eNOS(+/+) (35%; P < 0.05) and eNOS(-/-) (61%; P < 0.05), but decreased sEH in eNOS(+/+) (74%; P < 0.05) and eNOS(-/-) (40%; P < 0.05). Similarly, CYP4A decreased in HS-eNOS(+/+) (35%; P < 0.05) and HS-eNOS(-/-) (34%; P < 0.05). These data suggest that NS causes reduced-vasodilation in both eNOS(+/+) and eNOS(-/-) via sEH and CYP4A. However, HS triggers possible A(2A)AR-induced relaxation through CYP epoxygenase in both eNOS(+/+) and eNOS(-/-).

Renoprotective effects of soy protein hydrolysates in N(omega)-nitro-L-arginine methyl ester hydrochloride-induced hypertensive rats.

Pepsin-digested soy protein hydrolysate (SPH) has been reported to be responsible for many of the physiological benefits associated with soy protein consumption. In the present study, we investigated the effects of SPH with angiotensin-converting enzyme (ACE) inhibitory potential on blood pressure and renal injuries in rats with N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced hypertension. Rats were fed a diet containing L-NAME (50 mg/kg body weight) with or without SPH (at 1%, 3%, or 5%) for 6 weeks. We found that ingestion of SPH ameliorated the development of hypertension during the 6-week experimental period. SPH was also found to ameliorate renal function by decreasing urinary protein excretion and elevating the creatinine clearance rate. The levels of kidney ACE activity, malonaldehyde, tumor necrosis factor-a and plasminogen activator inhibitor-1, and the expression of CYP4A decreased in the 5% SPH group. Consumption of 5% SPH also ameliorated renal damage according to the histopathological analysis. These findings suggest that SPH might ameliorate the elevation of blood pressure and show renoprotective effects in nitric oxide (NO)-deficient rats, and one possible mechanism might be mediation via its ACE inhibitory activity.

A male-specific fatty acid omega-hydroxylase, SXE1, is necessary for efficient male mating in Drosophila melanogaster.

In Drosophila, sexual differentiation, physiology, and behavior are thought to be mediated by numerous male- and female-specific effector genes whose expression is controlled by sex-specifically expressed transcriptional regulators. One such downstream effector gene, sex-specific enzyme 1 (sxe1, cyp4d21), has been identified in a screen for genes with sex-biased expression in the head. Sxe1 was also identified in another screen as a circadian regulated gene. Here, we analyzed the spatial and temporal regulation of sxe1 and identified a function for this gene in male courtship. We show that male-specific transcriptional regulator DSX(M) and the clock genes are necessary for cycling of sxe1 mRNA during the diurnal cycle. Similar to sxe1 mRNA, expression of SXE1 protein oscillates in a diurnal fashion, with highest protein levels occurring around midnight. SXE1 protein expression is restricted to nonneuronal cells associated with diverse sensory bristles of both the chemo- and mechanosensory systems. Suppression or knockout of sxe1 significantly reduces mating success throughout the diurnal cycle. Finally, the metabolomic profile of wild-type and sxe1 mutant males revealed that sxe1 likely functions as a fatty acid omega-hydroxylase, suggesting that male courtship and mating success is mediated by small compounds generated by this enzyme.

Expression of CYP4A1 in U251 human glioma cell induces hyperproliferative phenotype in vitro and rapidly growing tumors in vivo.

Exogenous 20-hydroxyeicosatetraenoic acid (20-HETE) increases the growth of human glioma cells in vitro. However, glioma cells in culture show negligible 20-HETE synthesis. We examined whether inducing the expression of a 20-HETE synthase in a human glioma U251 cell line would increase proliferation. U251 cells transfected with CYP4A1 cDNA (termed U251 O) increased the formation of 20-HETE from less than 1 to over 60 pmol/min/mg proteins and increased their proliferation rate by 2-fold (p < 0.01). Compared with control U251, U251 O cells were rounded, smaller, showed a disorganized cytoskeleton, exhibited reduced vinculin staining, and were easily detached from the growing surface. They showed a marked increase in dihydroethidium staining, suggesting increased oxidative stress. The expression of phosphorylated extracellular signal-regulated kinase 1/2, cyclin D1/2, and vascular endothelial growth factor was markedly elevated in U251 O. The hyperproliferative and signaling effects seen in U251 O cells are abolished by selective CYP4A inhibition of 20-HETE formation with HET0016 [N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine], by small interfering RNA against the enzyme, and by the putative 20-HETE antagonist, 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid. In vivo, implantation of U251O cells in the brain of nude rats resulted in a approximately 10-fold larger tumor volume (10 days postimplantation) compared with animals receiving mock-transfected U251 cells. These data show that elevations in 20-HETE synthesis in U251 cells lead to an increased growth both in vitro and in vivo. This suggests that 20-HETE may have proto-oncogenic properties in U251 human gliomas. Further studies are needed to determine whether 20-HETE plays a role promoting growth of some human gliomas.

Cytochrome P450 omega hydroxylase (CYP4) function in fatty acid metabolism and metabolic diseases.

The cytochrome P450 gene 4 family (CYP4) consists of a group of over 63 members that omega-hydroxylate the terminal carbon of fatty acids. In mammals, six subfamilies have been identified and three of these subfamily members show a preference in the metabolism of short (C7-C10)-CYP4B, medium (C10-C16)-CYP4A, and long (C16-C26)-CYP4F, saturated, unsaturated and branched chain fatty acids. These omega-hydroxylated fatty acids are converted to dicarboxylic acids, which are preferentially metabolized by the peroxisome beta-oxidation system to shorter chain fatty acids that are transported to the mitochondria for complete oxidation or used either to supply energy for peripheral tissues during starvation or in lipid synthesis. The differential regulation of the CYP4A and CYP4F genes during fasting, by peroxisome proliferators and in non-alcoholic fatty liver disease (NAFLD) suggests different roles in lipid metabolism. The omega-hydroxylation and inactivation of pro-inflammatory eicosanoids by members of the CYP4F subfamily and the association of the CYP4F2 and CYP4F3 genes with inflammatory celiac disease indicate an important role in the resolution of inflammation. Several human diseases have been genetically linked to the expression CYP4 gene polymorphic variants, which may link human susceptibility to diseases of lipid metabolism and the activation and resolution phases of inflammation. Understanding how the CYP4 genes are regulated during the fasting and feeding cycles and by endogenous lipids will provide therapeutic avenues in the treatment of metabolic disorders of lipid metabolism and inflammation.

Ovariectomy, but not estrogen deficiency, increases CYP4A modulation of alpha(1)-adrenergic vasoconstriction in aging female rats.

BACKGROUND: Postmenopausal women are more likely to develop cardiovascular disease (CVD) than premenopausal women. Increased vasoconstriction in the peripheral vasculature may underlie this risk. In vascular smooth muscle, cytochrome P450 4A (CYP4A) enzymes form the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE). CYP4A modulation of alpha(1)-adrenergic vasoconstriction is increased in aging male rats; however, this pathway has not been investigated in aging females. To generate an appropriate model of menopause, we ovariectomized aged Sprague-Dawley rats to create an aged, ovarian-depleted phenotype. Because estrogen has profound effects on the peripheral vasculature, we also determined the effect of estrogen replacement on CYP4A modulation of vasoconstriction. METHODS: Aged (15-16 months) rats were assigned to be intact or ovariectomized. Ovariectomized rats received either placebo (OVX) or 17beta-estradiol (OVX-E) subcutaneously for 4 weeks. Mesenteric arteries were isolated and constricted with the alpha(1)-adrenergic agonist phenylephrine or intraluminal pressure in the absence or presence of the CYP4A inhibitor, DDMS. RESULTS: Ovariectomy increased CYP4A modulation of alpha(1)-adrenergic vasoconstriction. This was unaffected by estrogen replacement. Arteries from OVX-E animals exhibited increased phenylephrine sensitivity and forced dilation relative to arteries from intact and OVX animals. Myogenic tone was increased in both OVX and OVX-E animals relative to intact rats; however, CYP4A inhibition had no effect on myogenic tone in any group. CONCLUSIONS: In aged female rats, ovariectomy caused an increase in CYP4A modulation of alpha(1)-adrenergic vasoconstriction that was not prevented by estrogen replacement. Future study of these pathways may provide important targets for the prevention of CVD in aging women.

Emerging mechanisms for growth and protection of the vasculature by cytochrome P450-derived products of arachidonic acid and other eicosanoids.

Arachidonic acid (AA) is an essential fatty acid that is metabolized by cyclooxygenase (COX), lipoxygenase (LOX) or cytochrome P450 (CYP) enzymes to generate eicosanoids which in turn mediate a number of biological activities including regulation of angiogenesis. While much information on the effects of COX and LOX products is known, the physiological relevance of the CYP-derived products of AA are less well understood. CYP enzymes are highly expressed in the liver and kidney, but have also been detected at lower levels in the brain, heart and vasculature. A number of these enzymes, including members of the CYP 4 family, predominantly catalyze conversion of AA to 20-hydroxyeicosatetraenoic acid (20-HETE) while the CYP epoxygenases generate mainly epoxyeicosatrienoic acids (EETs). This review will focus on the emerging roles of inhibitors of eicosanoid production with emphasis on the CYP pathways, in the regulation of angiogenesis and tumor growth. We also discuss current observations describing the protective effects of EETs for survival of the endothelium.

Glial cells dilate and constrict blood vessels: a mechanism of neurovascular coupling.

Neuronal activity evokes localized changes in blood flow. Although this response, termed neurovascular coupling, is widely used to monitor human brain function and diagnose pathology, the cellular mechanisms that mediate the response remain unclear. We investigated the contribution of glial cells to neurovascular coupling in the acutely isolated mammalian retina. We found that light stimulation and glial cell stimulation can both evoke dilation or constriction of arterioles. Light-evoked and glial-evoked vasodilations were blocked by inhibitors of cytochrome P450 epoxygenase, the synthetic enzyme for epoxyeicosatrienoic acids. Vasoconstrictions, in contrast, were blocked by an inhibitor of omega-hydroxylase, which synthesizes 20-hydroxyeicosatetraenoic acid. Nitric oxide influenced whether vasodilations or vasoconstrictions were produced in response to light and glial stimulation. Light-evoked vasoactivity was blocked when neuron-to-glia signaling was interrupted by a purinergic antagonist. These results indicate that glial cells contribute to neurovascular coupling and suggest that regulation of blood flow may involve both vasodilating and vasoconstricting components.