Traumatic injury of cultured astrocytes alters inositol (1,4,5)-trisphosphate-mediated signaling.

Our previous studies using an in vitro model of traumatic injury have shown that stretch injury of astrocytes causes a rapid elevation in intracellular free calcium ([Ca2+]i), which returns to near normal by 15 min postinjury. We have also shown that after injury astrocyte intracellular calcium stores are no longer able to release Ca2+ in response to signal transduction events mediated by the second messenger inositol (1,4,5)-trisphosphate (IP3, Rzigalinski et al., 1998). Therefore, we tested the hypothesis that in vitro injury perturbs astrocyte IP3 levels. Astrocytes grown on Silastic membranes were labeled with [3H]-myo-inositol and stretch-injured. Cells and media were acid-extracted and inositol phosphates isolated using anion-exchange columns. After injury, inositol polyphosphate (IPx) levels increased up to 10-fold over uninjured controls. Significant injury-induced increases were seen at 5, 15, and 30 min and at 24 and 48 h postinjury. Injury-induced increases in IPx were equivalent to the maximal glutamate and trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid-stimulated IPx production, however injury-induced increases in IPx were sustained through 24 and 48 h postinjury. Injury-induced increases in IPx were attenuated by pretreatment with the phospholipase C inhibitors neomycin (100 microM) or U73122 (1.0 microM). Since we have previously shown that astrocyte [Ca2+]i returns to near basal levels by 15 min postinjury, the current results suggest that IP3-mediated signaling is uncoupled from its target, the intracellular Ca2+ store. Uncoupling of IP3-mediated signaling may contribute to the pathological alterations seen after traumatic brain injury.

Influence of phenylmethylsulfonyl fluoride on anandamide brain levels and pharmacological effects.

The endogenous cannabinoid anandamide produces cannabimimetic effects similar to those produced by delta9-tetrahydrocannabinol (delta9-THC), but has a much shorter duration of action due to its rapid metabolism to arachidonic acid and polar metabolites via action of fatty acid amide hydrolase (FAAH). Our earlier observations that anandamide's effects persisted after brain levels of anandamide itself had substantially dropped prompted us to examine the influence of the irreversible amidase inhibitor, phenylmethyl sulfonyl fluoride (PMSF), on the brain levels and pharmacological effects of anandamide. As shown previously, pretreatment with PMSF resulted in a leftward shift of the anandamide dose effect curves for antinociception and hypothermia in male mice. Brain and plasma levels of anandamide, arachidonic acid and polar metabolites peaked at 1 min after i.v. injection with 3H-anandamide and remained high at 5 min post-injection, with levels falling sharply thereafter. Pretreatment with PMSF (30 mg/kg, i.p.) prior to an injection of 1 or 10 mg/kg 3H-anandamide resulted 5 min later in enhanced brain levels of anandamide compared to those obtained with 3H-anandamide plus vehicle injection. Levels of arachidonic acid and polar metabolites in brain were not significantly increased. The clear correspondence between brain levels of anandamide following pretreatment with PMSF and pharmacological activity suggests that this parent compound is responsible for the antinociception and hypothermia that occurred 5 min after injection. These results further suggest that metabolite contribution to anandamide's effects, if any, would occur primarily at later times.

Astrocytes generate isoprostanes in response to trauma or oxygen radicals.

Previous studies have shown that oxygen radical scavengers prevent the reduced cerebral blood flow that occurs following experimental traumatic brain injury. The exact chemical species responsible for the posttraumatic reduction in flow is unknown. We tested whether isoprostanes, which are formed by non-cyclooxygenase-dependent free radical attack of arachidonic acid and are vasoconstrictors of the cerebral circulation, are increased in astrocytes following stretch-induced trauma or injury with a free radical generating system. Isoprostane (8-epi-prostaglandin F2alpha) was analyzed in cells and in media by immunoassay. Confluent rat cortical astrocytes in culture were injured by a hydroxyl radical generating system consisting of hydrogen peroxide and ferrous sulfate or by rapid stretch of astrocytes grown on a deformable silastic membrane. Some cells were treated with the iron chelator deferoxamine for 1 h before injury. The hydroxyl generating system caused free and cell-bound isoprostanes to increase to more than 400% of control. After trauma, free and membrane bound isoprostanes increased to 321 +/- 34% and 229 +/- 23% of control, respectively, and posttraumatic increases were prevented by deferoxamine. Since astrocytes are in close proximity to cerebral vessels, posttraumatic free radical formation may increase the formation of isoprostanes, which in turn produce vasoconstriction and decrease cerebral blood flow.

Stretch-induced injury alters mitochondrial membrane potential and cellular ATP in cultured astrocytes and neurons.

Energy deficit after traumatic brain injury (TBI) may alter ionic homeostasis, neurotransmission, biosynthesis, and cellular transport. Using an in vitro model for TBI, we tested the hypothesis that stretch-induced injury alters mitochondrial membrane potential (delta(psi)m) and ATP in astrocytes and neurons. Astrocytes, pure neuronal cultures, and mixed neuronal plus glial cultures grown on Silastic membranes were subjected to mild, moderate, and severe stretch. After injury, delta(psi)m was measured using rhodamine-123, and ATP was quantified with a luciferin-luciferase assay. In astrocytes, delta(psi)m dropped significantly, and ATP content declined 43-52% 15 min after mild or moderate stretch but recovered by 24 h. In pure neurons, delta(psi)m declined at 15 min only in the severely stretched group. At 48 h postinjury, delta(psi)m remained decreased in severely stretched neurons and dropped in moderately stretched neurons. Intracellular ATP content did not change in any group of injured pure neurons. We also found that astrocytes and neurons release ATP extracellularly following injury. In contrast to pure neurons, delta(psi)m in neurons of mixed neuronal plus glial cultures declined 15 min after mild, moderate, or severe stretch and recovered by 24-48 h. ATP content in mixed cultures declined 22-28% after mild to severe stretch with recovery by 24 h. Our findings demonstrate that injury causes mitochondrial dysfunction in astrocytes and suggest that astrocyte injury alters mitochondrial function in local neurons.

Calcium influx factor, further evidence it is 5, 6-epoxyeicosatrienoic acid.

We present evidence in astrocytes that 5,6-epoxyeicosatrienoic acid, a cytochrome P450 epoxygenase metabolite of arachidonic acid, may be a component of calcium influx factor, the elusive link between release of Ca2+ from intracellular stores and capacitative Ca2+ influx. Capacitative influx of extracellular Ca2+ was inhibited by blockade of the two critical steps in epoxyeicosatrienoic acid synthesis: release of arachidonic acid from phospholipid stores by cytosolic phospholipase A2 and cytochrome P450 metabolism of arachidonic acid. AAOCF3, which inhibits cytosolic phospholipase A2, blocked thapsigargin-stimulated release of arachidonic acid as well as thapsigargin-stimulated elevation of intracellular free calcium. Inhibition of P450 arachidonic acid metabolism with SKF525A, econazole, or N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide, a substrate inhibitor of P450 arachidonic acid metabolism, also blocked thapsigargin-stimulated Ca2+ influx. Nano- to picomolar 5, 6-epoxyeicosatrienoic acid induced [Ca2+]i elevation consistent with capacitative Ca2+ influx. We have previously shown that 5, 6-epoxyeicosatrienoic acid is synthesized and released by astrocytes. When 5,6-epoxyeicosatrienoic acid was applied to the rat brain surface, it induced vasodilation, suggesting that calcium influx factor may also serve a paracrine function. In summary, our results suggest that 5,6-epoxyeicosatrienoic acid may be a component of calcium influx factor and may participate in regulation of cerebral vascular tone.

Alterations in calcium-mediated signal transduction after traumatic injury of cortical neurons.

Calcium influx and elevation of intracellular free calcium ([Ca2+]i), with subsequent activation of degradative enzymes, is hypothesized to cause cell injury and death after traumatic brain injury. We examined the effects of mild-to-severe stretch-induced traumatic injury on [Ca2+]i dynamics in cortical neurons cultured on silastic membranes. [Ca2+]i was rapidly elevated after injury, however, the increase was transient with neuronal [Ca2+]i returning to basal levels by 3 h after injury, except in the most severely injured cells. Despite a return of [Ca2+]i to basal levels, there were persistent alterations in calcium-mediated signal transduction through 24 h after injury. [Ca2+]i elevation in response to glutamate or NMDA was enhanced after injury. We also found novel alterations in intracellular calcium store-mediated signaling. Neuronal calcium stores failed to respond to a stimulus 15 min after injury and exhibited potentiated responses to stimuli at 3 and 24 h post-injury. Thus, changes in calcium-mediated cellular signaling may contribute to the pathology that is observed after traumatic brain injury.

Intracellular free calcium dynamics in stretch-injured astrocytes.

We have previously developed an in vitro model for traumatic brain injury that simulates a major component of in vivo trauma, that being tissue strain or stretch. We have validated our model by demonstrating that it produces many of the posttraumatic responses observed in vivo. Sustained elevation of the intracellular free calcium concentration ([Ca2+]i) has been hypothesized to be a primary biochemical mechanism inducing cell dysfunction after trauma. In the present report, we have examined this hypothesis in astrocytes using our in vitro injury model and fura-2 microphotometry. Our results indicate that astrocyte [Ca2+]i is rapidly elevated after stretch injury, the magnitude of which is proportional to the degree of injury. However, the injury-induced [Ca2+]i elevation is not sustained and returns to near-basal levels by 15 min postinjury and to basal levels between 3 and 24 h after injury. Although basal [Ca2+]i returns to normal after injury, we have identified persistent injury-induced alterations in calcium-mediated signal transduction pathways. We report here, for the first time, that traumatic stretch injury causes release of calcium from inositol trisphosphate-sensitive intracellular calcium stores and may uncouple the stores from participation in metabotropic glutamate receptor-mediated signal transduction events. We found that for a prolonged period after trauma astrocytes no longer respond to thapsigargin, glutamate, or the inositol trisphosphate-linked metabotropic glutamate receptor agonist trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid with an elevation in [Ca2+]i. We hypothesize that changes in calcium-mediated signaling pathways, rather than an absolute elevation in [Ca2+]i, is responsible for some of the pathological consequences of traumatic brain injury.

The biodisposition and metabolism of anandamide in mice.

The endogenous cannabinoid anandamide (AN) has been reported to produce pharmacological effects similar to those of delta9-tetrahydrocannabinol but with a shorter duration of action. Also, AN is known to be metabolized to arachidonic acid. The purpose of this study was to examine the time course of distribution and metabolism of AN. Male mice were each administered 20 microCi 3H-AN and 50 mg/kg AN (i.v.). At 1, 5, 15 and 30 min after administration, the animals were sacrificed, and various tissues were removed, solubilized and counted to determine the distribution of 3H. Also, samples from brain, adrenal gland and plasma were extracted with ethyl acetate and analyzed by HPLC to separate 3H-labeled AN, arachidonic acid and other metabolites. AN was detectable in brain by 1 min after injection. At 1 min after injection, the rank order of radioactivity per milligram or microliter of tissue was adrenal > lung > kidney > plasma > heart > liver > diaphragm > brain > fat. Although the 1 and 5 min metabolic profiles of brain 3H showed that AN was clearly present, most AN had already been transformed to arachidonic acid and other polar metabolites, and there were almost no detectable brain levels of AN at 15 and 30 min. In plasma and adrenal gland, AN was the predominant form at 1 and 5 min. Our experiments confirm that AN quickly reaches the brain and suggest that rapid metabolism of AN plays a key role in the time course of the pharmacological activity of this naturally occurring cannabinoid receptor ligand.

Effect of Ca2+ on in vitro astrocyte injury.

Current literature suggests that a massive influx of Ca2+ into the cells of the CNS induces cell damage associated with traumatic brain injury (TBI). Using an in vitro model for stretch-induced cell injury developed by our laboratory, we have investigated the role of extracellular Ca2+ in astrocyte injury. The degree of injury was assessed by measurement of propidium iodide uptake and release of lactate dehydrogenase. Based on results of in vivo models of TBI developed by others, our initial hypothesis was that decreasing extracellular Ca2+ would result in a reduction in astrocyte injury. Quite unexpectedly, our results indicate that decreasing extracellular Ca2+ to levels observed after in vivo TBI increased astrocyte injury. Elevating the extracellular Ca2+ content to twofold above physiological levels (2 mM) produced a reduction in cell injury. The reduction in injury afforded by Ca2+ could not be mimicked with Ba2+, Mn2+, Zn2+, or Mg2+, suggesting that a Ca(2+)-specific mechanism is involved. Using 45Ca2+, we demonstrate that injury induces a rapid influx of extracellular Ca2+ into the astrocyte, achieving an elevation in total cell-associated Ca2+ content two- to threefold above basal levels. Pharmacological elevation of intracellular Ca2+ levels with the Ca2+ ionophore A23187 or thapsigargin before injury dramatically reduced astrocyte injury. Our data suggest that, contrary to popular assumptions, an elevation of total cell-associated Ca2+ reduces astrocyte injury produced by a traumatic insult.

Repeated cocaine administration reduces bradykinin-induced dilation of pial arterioles.

Using the acute cranial window technique in rabbits under surgical anesthesia, we tested the vasoactivity of acetylcholine (ACh, 10(-8)-10(-5) M), bradykinin (BK, 10(-8)-10(-5) M), and asphyxia (10% O2, 9% CO2, balance N2) after subchronic pretreatment with cocaine. After repeated administration of cocaine (20 mg.kg-1.day-1 sc x 7 days), the BK-induced dilation of pial arterioles was reduced by 51%. Previous work showed that BK produces dilation of pial arterioles by a cyclooxygenase-dependent oxygen radical-mediated mechanism and that in rabbits the BK-induced dilation is dependent on both vascular and nonvascular cyclooxygenase. Selective blockade of vascular cyclooxygenase, in addition to cocaine treatment, did not produce any greater inhibition of the BK-induced dilation. The dilation in response to ACh and asphyxia was unaltered by cocaine. Levels of cerebrospinal fluid prostaglandins suggest cocaine pretreatment may inhibit cerebral vascular prostaglandin production. Together, cerebrospinal fluid prostaglandin and vasoreactivity data indicate cocaine pretreatment selectively inhibits the vascular cyclooxygenase-dependent mechanism mediating the BK-induced dilation. This decreased response to BK in cocaine-treated rabbits may result from decreased oxygen radical production concomitant with decreased vascular prostaglandin production. Alternatively, oxygen radical scavenging may be increased after cocaine treatment. We speculate that cocaine-induced alterations in cerebrovascular function and metabolism may be related to the increased incidence of stroke reported to occur in human cocaine users.

Stretch-induced injury of cultured neuronal, glial, and endothelial cells. Effect of polyethylene glycol-conjugated superoxide dismutase.

BACKGROUND AND PURPOSE: There is abundant evidence that after in vivo traumatic brain injury, oxygen radicals contribute to changes in cerebrovascular structure and function; however, the cellular source of these oxygen radicals is not clear. The purpose of these experiments was to use a newly developed in vitro tissue culture model to elucidate the effect of strain, or stretch, on neuronal, glial, and endothelial cells and to determine the effect of the free radical scavenger polyethylene glycol-conjugated superoxide dismutase (PEG-SOD; pegorgotein, Dismutec) on the response of each cell type to trauma. METHODS: Rat brain astrocytes, neuronal plus glial cells, and aortic endothelial cells were grown in cell culture wells with 2-mm-thick silastic membrane bottoms. A controllable, 50-millisecond pressure pulse was used to transiently deform the silastic membrane and thus stretch the cells. Injury was assessed by quantifying the number of cells that took up the normally cell-impermeable dye propidium iodide. Some cultures were pretreated with 100 to 300 U/mL PEG-SOD. RESULTS: Increasing degrees of deformation produced increased cell injury in astrocytes, neuronal plus glial cultures, and aortic endothelial cells. By 24 hours after injury, all cultures showed evidence of repair as demonstrated by cells regaining their capacity to exclude propidium iodide. Compared with astrocytes or neuronal plus glial cultures, endothelial cells were much more resistant to stretch-induced injury and more quickly regained their capacity to exclude propidium iodide. PEG-SOD had no effect on the neuronal or glial response to injury but reduced immediate posttraumatic endothelial cell dye uptake by 51%. CONCLUSIONS: These studies further document the utility of the model for studying cell injury and repair and further support the vascular endothelial cell as a site of free radical generation and radical-mediated injury. On the assumption that, like aortic endothelial cells, stretch-injured cerebral endothelial cells also produce oxygen radicals, our results further suggest the endothelial cell as a site of therapeutic action of free radical scavengers after traumatic brain injury.

Anandamide and delta 9-THC dilation of cerebral arterioles is blocked by indomethacin.

Anandamide (AN, arachidonyl ethanolamide) has been isolated from the brain and shown to be an endogenous ligand for the delta 9-tetrahydrocannabinol (delta 9-THC) receptor. The purpose of these studies was to determine whether AN or delta 9-THC can affect the cerebral circulation. With the use of the closed cranial window AN and delta 9-THC (10(-13)-10(-3) M) were topically applied to rabbit cerebral arterioles and effects on diameter were measured with a microscope. AN and delta 9-THC similarly induced a dose-dependent dilation starting at concentrations as low as 10(-12) M. Maximum dilation for AN was 25% and that for delta 9-THC 22%. Topical coapplication of indomethacin, a cyclooxygenase inhibitor, completely blocked dilation, whereas the free radical scavengers superoxide dismutase and catalase or the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) had no effect on AN-induced dilation. The cerebrospinal fluid level of prostaglandin E2 increased only in response to 10(-7) M and greater AN and was not affected by delta 9-THC. [3H]AN superfused through the cranial window was 20% converted to arachidonic acid. These results show that AN and delta 9-THC can modulate cerebral arterioles, likely by stimulating release and metabolism of endogenous arachidonic acid. Whether dilation is due to vasodilator eicosanoids, or other vasoactive agents whose synthesis or release is cyclooxygenase dependent, is uncertain.

Effect of protein kinase C modulators on 14,15-epoxyeicosatrienoic acid incorporation into astroglial phospholipids.

Our previous studies have shown that 14,15-epoxyeicosatrienoic acid (14,15-EET) is a major product of arachidonic acid metabolism in astrocytes. The purpose of this study was to investigate cellular regulation of 14,15-EET incorporation, distribution, and metabolism in primary cultures of rat brain cortical astrocytes. Incorporation of 14,15-EET into astrocytes was lower (93,390 +/- 11,121 dpm/5 x 10(6) cells) than incorporation of 8,9-EET (226,500 +/- 5,567 dpm/5 x 10(6) cells) and arachidonic acid (321,600 +/- 1,200 dpm/5 x 10(6) cells). 14,15-EET was distributed in the order neutral lipids and free fatty acids (solvent front) >> phosphatidylcholine (PC) > phosphatidylinositol (PI) > phosphatidylethanolamine. In contrast, 8,9-EET and arachidonic acid were exclusively incorporated into PC. During incubation, astroglial epoxide hydrolase selectively metabolized 14,15-EET, but not 8,9-EET, to its vic-diol. Although 4-phenylchalcone oxide, a potent inhibitor of epoxide hydrolase, completely inhibited 14,15-EET metabolism, a large amount of cell-incorporated radioactivity remained as free 14,15-EET. Long-term exposure of astrocytes to 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA) resulted in a time-dependent incorporation of 14,15-EET into PI but not in control cells exposed to 4 alpha-phorbol 12,13-didecanoate. PKC down-regulation completely inhibited epoxide hydrolase metabolism of 14,15-EET. Following recovery of down-regulated PKC, 1 week after treatment with 4 beta-PMA, astrocytes regained their normal pattern of low incorporation of 14,15-EET. Protein kinase C (PKC) inhibition by staurosporine enhanced 14,15-EET incorporation without affecting its metabolism to 14,15-dihydroxyeicosatrienoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

A new model for rapid stretch-induced injury of cells in culture: characterization of the model using astrocytes.

The purpose of this study was to develop a simple, reproducible model for examining the morphologic, physiologic, and biochemical consequences of stretch-induced injury on tissue-cultured cells of brain origin. Rat cortical astrocytes from 1- to 2-day-old rats were cultured to confluency in commercially available 25-mm-diameter tissue culture wells with a 2-mm-thick flexible silastic bottom. A cell injury controller was used to produce a closed system and exert a rapid positive pressure of known amplitude (psi) and duration (msec). The deformation of the membrane, and thus the stretch of the cells growing on the membrane, was proportional to the amplitude and duration of the air pressure pulse. Extent of cell injury was qualitatively assessed by light and electron microscopy and quantitatively assessed by nuclear uptake of the fluorescent dye propidium iodide, which is excluded from cells with intact membranes. Lactate dehydrogenase (LDH) enzyme release was measured spectrophotometrically. Cell injury was found to be proportional to the extent of the silastic membrane deformation. Increasing cell stretch caused mitochondrial swelling and vacuolization as well as disruption of glial filaments. Stretching also caused increased dye uptake, with maximum dye uptake occurring with a 50 msec pressure pulse duration, whereas deformations produced over longer periods of time (seconds) caused little dye uptake. With increasing postinjury survival fewer cells took up dye, implying cell repair. LDH release was also proportional to the amplitude of cell stretch, with maximum release occurring within 2 h of injury. In summary we have developed a simple, reproducible model to produce graded, strain-related injuries in cultured cells. Our continuing experiments suggest that this model can be used to study the biochemistry and physiology of injury as well as serve as a tool to examine the efficacy of therapeutic agents.

Endothelial and nonendothelial cyclooxygenase mediate rabbit pial arteriole dilation by bradykinin.

Aspirin (acetylsalicylic acid, ASA) was administered to rabbits in an attempt to inhibit selectively endothelial cyclooxygenase activity and therefore to determine its role in bradykinin-induced radical-mediated dilation of cerebral arterioles. With the use of the cranial window technique in anesthetized rabbits, pial arteriolar diameters were recorded in response to topically applied bradykinin, acetylcholine, and ventilation with 10% O2-9% CO2 gas mixture. Prostaglandins were measured in isolated cerebral microvessels and cerebrospinal fluid (CSF) using radioimmunoassay. Microvessel prostaglandin production was reduced significantly by 90 mg/kg i.v. ASA, whereas acetylcholine-stimulated increases of CSF prostaglandins were not similarly affected. This treatment reduced bradykinin-induced dilation of pial arterioles by 47%. After concurrent 90 mg/kg i.v. ASA plus 300 microM ASA topically applied to the brain, stimulated increases of CSF prostaglandins were reduced by 79%, while bradykinin-induced dilation was reduced by 78%. ASA did not reduce the dilator activity of either acetylcholine or ventilation with 10% O2-9% CO2. Acetylcholine- but not bradykinin-induced dilation was reduced by NG-nitro-L-arginine methyl ester. These results indicate intravenous ASA produced a relatively selective inhibition of cerebral microvascular cyclooxygenase and partial inhibition of bradykinin-induced dilation. Further inhibition of dilation occurred following ASA administered both systemically and topically to the brain. This indicates two sources of cyclooxygenase, endothelial and nonendothelial, mediate the bradykinin-induced dilation of rabbit pial arterioles. Furthermore, systemic doses of ASA do not eliminate brain prostaglandin formation.

Metabolism of arachidonic acid to epoxyeicosatrienoic acids, hydroxyeicosatetraenoic acids, and prostaglandins in cultured rat hippocampal astrocytes.

We have recently shown that brain slices are capable of metabolizing arachidonic acid by the epoxygenase pathway. The purpose of this study was to begin to determine the ability of individual brain cell types to form epoxygenase metabolites. We have examined the astrocyte epoxygenase pathway and have also confirmed metabolism by the cyclooxygenase and lipoxygenase enzyme systems. Cultured rat hippocampal astrocyte homogenate, when incubated with radiolabeled [3H]arachidonic acid, formed products that eluted in four major groups designated as R17-30, R42-50, R51-82, and R83-90 based on their retention times in reverse-phase HPLC. These fractions were further segregated into as many as 13 peaks by normal-phase HPLC and a second reverse-phase HPLC system. The principal components in each peak were structurally characterized by gas chromatography/electron impact-mass spectrometry. Based on HPLC retention times and gas chromatography/electron impact-mass spectrometry analysis, the more polar fractions (R17-30) contained prostaglandin D2 as the major cyclooxygenase product. Minor products included 6-keto prostaglandin F1 alpha, prostaglandin E2, prostaglandin F2 alpha, and thromboxane B2. Fractions R42-50, R51-82, and R83-90 contained epoxygenase and lipoxygenase-like products. The major metabolite in fractions R83-90 was 5,6-epoxyeicosatrienoic acid (EET). Fractions R51-82 contained 14,15- and 8,9-EETs, 12- and 5-hydroxyeicosatetraenoic acids, and 8,9- and 5,6-dihydroxyeicosatrienoic acids (DHETs). In fractions R42-50, 14,15-DHET was the major product. When radiolabeled [3H]14,15-EET was incubated with astrocyte homogenate, it was rapidly metabolized to [3H]14,15-DHET. The metabolism was inhibited by submicromolar concentration of 4-phenylchalcone oxide, a potent inhibitor of epoxide hydrolase activity. Formation of other polar metabolites such as triols or epoxy alcohols from 14,15-DHET was not observed. In conclusion, astrocytes readily metabolize arachidonic acid to 14,15-EET, 5,6-EET, and their vicinal-diols. Previous studies suggest these products may affect neuronal function and cerebral blood flow.