Role of P-450 Arachidonic Acid Epoxygenase in the Response of Cerebral Blood Flow to Glutamate in Rats
Background and Purpose Glutamate, a major excitatory neurotransmitter in the brain, has been implicated in the hyperemic response to increases in the activity of neurons, but the mechanism of glutamate-induced dilation of cerebral blood vessels is unknown. Glutamate has been shown to enhance the release of arachidonic acid (AA) in brain tissue and cultured astrocytes. We have previously shown that astrocytes metabolize AA to vasodilator products, epoxyeicosatrienoic acids (EETs), and express a P-450 AA epoxygenase, P-450 2C11. We tested the hypothesis that glutamate-induced dilation of cerebral arterioles is mediated in part by changes in the formation and release of EETs by perivascular astrocytes.
Methods Primary astrocyte cultures were prepared from 3-day-old rat pups. The cells were labeled with [14C]AA, and the effect of glutamate on the formation of EETs from [14C]AA by cultured astrocytes was studied. The expression of P-450 2C11 protein in the microsomal fractions of cultured astrocytes was assessed by Western blot. In vivo cerebral blood flow measurements were made in adult rats by laser-Doppler flowmetry after administration of glutamate into the subdural space of the rat before and after treatment with miconazole.
Results Glutamate treatment (100 μmol/L for 30 minutes) induced a threefold increase in the formation of EETs from [14C]AA by cultured astrocytes, and the increase was inhibited by miconazole (20 μmol/L), an inhibitor of P-450 AA epoxygenase. Treatment with glutamate (100 μmol/L) for 12 hours increased the expression of P-450 2C11 protein in the microsomal fraction of cultured astrocytes. The response of laser-Doppler cerebral blood flow to administration of glutamate (500 μmol/L) into the subdural space of the rat was significantly attenuated after treatment with miconazole (20 μmol/L for 30 minutes).
Conclusions These findings suggest a role for a P-450 AA epoxygenase in astrocytes in the coupling between the metabolic activity of neurons and regional blood flow in the brain.
The regulation of the cerebral circulation is extraordinarily dynamic, maintaining CBF over a wide range of arterial pressures while at the same time providing adequate substrate to meet the metabolic demands of neural tissue.1 2 Mechanisms responsible for providing nutritive blood flow to areas of neuronal activation remain poorly understood. Metabolic regulation of CBF is thought to involve release of vasoactive metabolites when neuronal activity increases; candidates include adenosine, prostacyclin, potassium, changing pH,1 and, more recently, NO.3
Glutamate is the most extensively distributed excitatory amino acid neurotransmitter in the cerebral cortex.4 While glutamate has no apparent action on cerebral arterial muscle or other cell types in the vascular wall,5 6 when applied to the surface of the brain in vivo it induces dilation of cerebral vessels.7 Such findings have led to the hypothesis that “spillover” of glutamate during neuronal activation initiates release of vasodilator metabolites, leading to a regional increase in CBF.
Astrocytes possess receptors for glutamate8 and constitute the most numerous cell type in the central nervous system.4 Previous reports have shown that glutamate induces release of AA from membrane-bound pools in astrocytes.9 We have recently cloned and sequenced a cytochrome P-450 (P-450) cDNA from cortical astrocytes, which is homologous to a previously sequenced P-450 2C11 cDNA from rat liver.10 This P-450 2C11 codes for an epoxygenase, which metabolizes AA to 5,6-, 8,9-, 11,12-, and 14,15-EETs.10 11,12- and 14,15-EETs dilate cerebral arteries by increasing K+ channel activity and hyperpolarizing the vascular muscle membrane.10 11 12 Inhibition of EET formation results in a marked reduction in baseline CBF13 and therefore must be considered a primary candidate involved in the physiological regulation of nutritive blood flow. The purpose of the present study was to test the hypothesis that glutamate-induced dilation of cerebral arterioles is mediated in part by the formation and release of EETs from perivascular astrocytes. We describe a combined in vitro and in vivo approach to demonstrate the action of glutamate on the formation and release of EETs and on the expression of P-450 2C11 epoxygenase by rat cortical astrocytes.
Materials and Methods
Protocol 1: Effect of Glutamate on Formation of EETs by Cultured Astrocytes
Astrocytes were cultured from cerebral cortices and hippocampi of 2- to 3-day-old Sprague-Dawley rat brains under aseptic conditions. Primary cultures were prepared as previously described14 with slight modifications. Briefly, brain tissue was dissected free of meninges, cut into small pieces, and transferred to a sterile dish containing 20 U/mL papain (Worthington Biochemical Corp) and cysteine (1.5×10−4 g/mL; Sigma) dissolved in Earle’s balanced salt solution (Gibco BRL). Tissue pieces were incubated at 37°C for 40 minutes with gentle agitation and then washed three times in the feeding medium, which contains DMEM (Gibco BRL) with 10% fetal bovine serum (ICN Biomedicals) and 1% penicillin-streptomycin solution (Sigma). The tissue was then dissociated by trituration with a flame-narrowed Pasteur pipette. The cell suspension was diluted with feeding medium and seeded into 75-cm2 culture flasks (Costar) at an initial density of 2×105 cells per square centimeter. Cells were incubated at 37°C in a 95%/5% mixture of atmospheric air and CO2. The medium was changed after 2 days and subsequently twice a week.
Astrocytes were identified by indirect immunocytochemistry with the use of a monoclonal anti–glial fibrillary acidic protein mouse IgG (Boehringer Mannheim) as the primary antibody and fluorescein isothiocyanate–conjugated goat anti-mouse IgG (Boehringer Mannheim) as the secondary antibody. Approximately 3×104 cells in 1.5 mL medium were seeded on 22×22-mm coverslips (VWR) in a 35×10-mm Petri dish (Costar) and incubated at 37°C in 95% air/5% CO2 for 24 to 48 hours. Coverslips were then rinsed with DPBS containing Ca2+ and Mg2+ (Gibco BRL), and cells were fixed in cold methanol for 5 minutes at −20°C. Cells were permeabilized in 0.25% Triton X-100 in DPBS (Sigma) for 2 minutes at room temperature, blocked with 3% normal goat serum in DPBS for 30 minutes at room temperature, and incubated with 100 μL of 5 μg/mL (1:4 dilution in 0.1% normal goat serum) primary antibody for 1 hour at room temperature and finally with 100 μL of 2.68 μg/mL (1:500 dilution in 0.5% BSA/DPBS) of secondary antibody for 1 hour at room temperature in the dark. Coverslips were then washed with DPBS and mounted on a 25×75-mm slide (VWR) with a drop of histological mounting media. Slides were visualized with the aid of a Nikon Diaphot inverted microscope equipped for epifluorescence. The excitation wavelength was selected by narrow band filters (10 nm) centered at 495 nm, and images were acquired at a 525-nm emission frequency with a silicon-intensified CCD camera (Hamamatsu Photonics).
Assay of AA Metabolism
Confluent 10- to 14-day-old primary cultures of astrocytes were prepared as described above, washed three times in DMEM, and incubated overnight with 0.45 μCi of [14C]AA (57.0 mCi/mmol; Du Pont) added to 14 mL of 0.05% fatty acid–free BSA in DMEM. The medium was decanted, and cells were washed three times with DMEM and incubated further for 30 minutes. The conditioned DMEM was collected, and cells were scraped off the flask and homogenized in ice-cold PBS. In the experimental cultures, cells were washed three times in DMEM and incubated with glutamate in DMEM (100 μmol/L) for 30 minutes at 37°C before homogenization. In some experiments, miconazole (20 μmol/L) was added to the media 30 minutes before glutamate treatment. Control cultures were incubated with an equal volume of vehicle in DMEM. Acid lipids from both the cell homogenate and the medium were extracted twice with 3 mL of ethyl acetate, back extracted with 1 mL of distilled water, evaporated to dryness under nitrogen, and reconstituted in 0.5 mL of ethanol. Cell- and medium-associated products were separated by rpHPLC, as described previously.10 Briefly, AA products were separated with the use of a 2.1-mm×25-cm C-18 rpHPLC column (Supelco LC-18) and a linear solvent gradient ranging from 30:70:1 acetonitrile/water/acetic acid (vol/vol/vol) to 100:1 acetonitrile/acetic acid (vol/vol) over 50 minutes at a flow rate of 0.5 mL/min. AA metabolites were monitored with an on-line radioactive flow detector (model 171, Beckman System Gold). Products were identified by coelution with authentic standards.
Protocol 2: Effect of Glutamate on Expression of P-450 2C11 Protein in Astrocytes
Western blots of microsomes prepared from astrocytes cultured from the hippocampus of the rat brain were performed with the use of a polyclonal antibody raised in our laboratory against peptide sequences derived from P-450 2C11, as described previously.10 Confluent monolayers (10 to 14 days) of primary cultures of astrocytes were incubated with 100 μmol/L glutamate or the same volume of vehicle in the feeding medium for 24 hours at 37°C. Cells were washed three times with DPBS and scraped for preparation of microsomes by differential centrifugation, as previously described.10 Microsomal proteins (20 μg) were separated on a 7.5% sodium dodecyl sulfate–polyacrylamide gel and transferred electrophoretically to a 0.2-μm supported nitrocellulose membrane (Bio-Rad) in a transfer buffer (25 mmol/L Tris, 192 mmol/L glycine, pH 8.3) containing 20% vol/vol methanol at room temperature for 1 hour at 100 V. The membrane was blocked in a TBS-T buffer (50 mmol/L Tris, 0.2 mol/L NaCl, 0.08% Tween-20, pH 7.5) containing 5% nonfat dry milk at 4°C overnight. Then the membrane was incubated with P-450 2C11 antibody (1:3000 dilution in TBS-T containing 2% nonfat dry milk) for 2 hours at room temperature. The membrane was washed with TBS-T three times and incubated with the secondary antibody. Goat anti-rabbit IgG conjugated with horseradish peroxidase (Bio-Rad) was used as the secondary antibody. Incubations were performed at room temperature for 30 minutes at a 1:1000 dilution of the secondary antibody in TBS-T containing 2% nonfat dry milk. Avidin–horseradish peroxidase IgG (Bio-Rad) was included at 1:3000 dilution to visualize biotinylated marker proteins by enhanced chemiluminescence. Membranes were washed three times with TBS-T and detected by enhanced chemiluminescence (Amersham).
Protocol 3: Effect of Miconazole on Response of CBF to Glutamate
Adult (8- to 12-week-old) male Sprague-Dawley rats (Harlan, Indianapolis, Ind) were anesthetized with 60 mg/kg body wt IP of sodium pentobarbital (Anpro Pharmaceutical). A polyethylene cannula (PE-240, Intramedic, Fisher Scientific) was placed into the trachea to facilitate breathing. A femoral artery was cannulated for measurement of systemic arterial blood pressure and withdrawal of blood samples for monitoring arterial blood gases. Another cannula was inserted into the femoral vein for infusion of drugs and replacement of body fluids. Rats were administered 80 mg IP of gallamine (Sigma) and ventilated (model 683, Harvard Apparatus) with 30% oxygen in nitrogen. End-tidal carbon dioxide (model Lifespan 100, Biochem International) and airway pressure were continuously monitored. Anesthesia was supplemented at hourly intervals or when a toe pinch resulted in an increased systemic arterial blood pressure, with injections of 2.5 mg/kg body wt IP of sodium pentobarbital. Rats were placed on a heating pad, and the temperature was maintained at 37°C. After instrumentation, rats were mounted in a Kopf stereotaxic apparatus (model 900, David Kopf), and the scalp and connective tissue were removed over the parietal cranial bone. An area 2 to 3 mm in diameter was thinned in the center of the right parietal bone with a low-speed (<8000 rpm) air drill (model Rhino XP, Midwest Dental). Drilling was performed with a stereomicroscope until epidural and pial vessels were visible through the closed cranial window without penetrating the skull. Two small burr holes were drilled on opposite sides of the window for insertion of the inflow tubing and for drainage of excess fluid. The thin cranial plate in these two holes was carefully removed under the microscope, and an incision was made in the dura with care taken not to injure epidural or pial vessels. The tip of a PE-10 polyethylene catheter pulled to a tip diameter of 200 μm was inserted through the incision in the dura and advanced to the border of the cranial window. The other end of the tubing was attached to a syringe filled with aCSF by a three-way connection to allow for simultaneous infusion of drugs into the subdural space and measurement of inflow pressure. The distance between the tip of the inflow tubing and the branching site of the inflow pressure tubing was minimized to give a close estimate of local intracranial pressure. The incision in the second hole was left open for drainage of fluids.
Cortical microvascular perfusion was monitored with a laser-Doppler flowmeter (PF3, Perimed) and a flow probe (PF316, “dental probe,” Perimed) with a tip diameter of 1 mm. The probe was lowered into the bottom of the cranial window with a micromanipulator into an area devoid of visible large blood vessels and was not moved for the duration of the experiment. A drop of mineral oil was applied at the probe tip to provide optical coupling between the probe and the tissue.
After surgery, ventilation with oxygen-enriched gas mixture (Fio2=30%) was instituted. MAP, inflow pressure, and airway pressure were continuously monitored, and the subdural space was perfused with aCSF at a rate that maintained inflow pressure value between 5 and 10 mm Hg. The composition of the aCSF mixed fresh daily was as follows (mmol/L): KCl 2.9, MgCl2 38, CaCl2 1.99, NaCl 131.9, NaHCO3 19, urea 6.63, glucose 3.69, with pH adjusted daily to 7.4. The solution was prewarmed to 37°C in a water bath and bubbled with 8% CO2 in atmospheric air for the duration of the experiment. The laser-Doppler flow probe was positioned over the cranial window, and laser-Doppler flow was monitored during a 30-minute stabilization period to allow for hemodynamic stability before the experiment was started. Twice during the experimental protocol, 0.25 mL of blood was withdrawn from the arterial line for measurement of arterial blood gases, arterial pH, and hemoglobin concentration (ABL-2, Radiometer). Volume withdrawn was replaced with an equal volume of 10% dextran 40 (Baxter) infused intravenously. After a 30-minute stabilization period, glutamate (5×10−4 mol/L) was perfused into the subdural space for 10 minutes at a rate of 2 μL/min. After a washout period with aCSF for 10 minutes, the subdural space was perfused with miconazole (20 μmol/L in aCSF) for 30 minutes, and glutamate treatment was repeated with miconazole present at the same time.
Data Acquisition and Analysis
MAP, laser-Doppler flow signal, inflow pressure, and airway pressure were displayed and recorded on an eight-channel analog-to-digital computer acquisition system (Codas, Dataq Instruments). Control laser-Doppler flow value was calculated by averaging laser-Doppler flow recordings over a 5-minute period at the end of the stabilization period. Peak laser-Doppler flow response after glutamate administration was expressed as percent change from control, with control laser-Doppler flow taken as 100%. Laser-Doppler flow response to glutamate after miconazole treatment was expressed as percent change of the peak laser-Doppler flow response from a 5-minute average of steady state recordings at the end of the 30-minute period of miconazole treatment.
Protocol 1: Effect of Glutamate on Formation of EETs by Cultured Astrocytes
Fig 1⇓ is a representative rpHPLC chromatogram demonstrating the effect of glutamate on formation of the peak that has an elution time similar to authentic EET standards in cultured astrocytes. Fig 2⇓ is a representative rpHPLC chromatogram demonstrating the effect of miconazole (panel B) on glutamate-induced formation of the product with an elution time similar to authentic EET standards. Figs 3⇓ and 4⇓ summarize the effects of a 30-minute incubation with 100 μmol/L glutamate on the metabolism of AA by cultured astrocytes. Glutamate treatment increased the percentage of EETs formed in astrocytes by approximately threefold, from 1.31±0.74% (n=5) to 4.29±0.91% (n=7) of the total counts extracted from astrocytes after an overnight incubation with [14C]AA (Fig 3⇓); the percentage of AA released into the medium (Fig 4⇓) was also increased by approximately threefold, from 9.44±3.69% (n=4) to 29.68±4.38% (n=7) compared with time-control, age-matched plates with the same initial seeding density. Based on the specific activity of AA substrate, the final concentration of EETs produced by astrocytes increased from 2.2±1.1 to 8.73±2.79 nmol/L with glutamate treatment.
Protocol 2: Effect of Glutamate on Expression of P-450 2C11 Protein in Astrocytes
Fig 5⇓ shows a representative Western blot analysis of liver homogenates from male and female rats and of microsomes from glutamate-treated and untreated cultures of astrocytes using 2C11 antibody. The reactivity of the antibody with a 55-kD band in male but not in female rat liver demonstrates the specificity of the antibody to P-450 2C11, which is expressed in the liver of only male rats.10 P-450 2C11 was markedly increased in microsomes prepared from astrocytes treated with glutamate for 24 hours compared with untreated, time-control, and age-matched cultures with the same initial seeding density.
Protocol 3: Effect of Miconazole on Response of CBF to Glutamate
A summary of physiological variables before and after infusion of miconazole or vehicle is presented in the Table⇓. MAP, arterial Po2, Pco2, pH, and hemoglobin were well controlled and were not altered by administration of miconazole or vehicle.
Fig 6⇓ demonstrates original tracings of laser-Doppler flowmetry (bottom panel) and MAP (top panel) signals during subdural administration of glutamate (5×10−4 mol/L).
Fig 7⇓ summarizes the responses of CBF to perfusion of the subdural space with glutamate (5×10−4 mol/L) before and after miconazole treatment (20 μmol/L). CBF increased by 64.1±12.0% before and by 9.6±1.9% after miconazole treatment (n=5). CBF response to glutamate after perfusion with the vehicle was not different from the value before miconazole (n=5).
The purpose of the present study was to gain further insight into the hypothesis that the metabolites of P-450 AA epoxygenase function in the regulation of CBF. We found that glutamate enhanced the formation and release of EETs from rat brain astrocytes. We also found that the expression of a recently identified P-450 2C11 enzyme is upregulated after prolonged exposure to glutamate. Finally, the present study demonstrated that inhibition of P-450 enzyme activity markedly blunts the increase in laser-Doppler–measured CBF in response to glutamate in anesthetized rats.
Previous work from our laboratory demonstrated that cultured astrocytes metabolize AA by the P-450 pathway to EETs and that they express P-450 2C11 cDNA.10 P-450 2C11 enzymes catalyze formation of 5,6-, 8,9-, 11,12-, and 14,15-EETs from AA.10 A physiological role for EETs in the regulation of CBF is emerging. Ellis and coworkers15 found that mouse brain tissue and cultured rat astrocytes metabolize AA to EETs, which dilate cerebral arterioles. Furthermore, inhibition of P-450 enzymes results in a 30% reduction in baseline CBF.13
The present study is an attempt to define some of the physiological variables regulating EET production and release in brain parenchymal tissue. Glutamate is a major excitatory neurotransmitter in the brain and has been implicated in cerebral vasodilation accompanying elevated neuronal activity.16 When applied through a cranial window in vivo, glutamate dilates pial arterioles.7 However, glutamate has no effect on isolated cerebral blood vessels,5 6 demonstrating that glutamate-induced enhancement of CBF may be mediated by release of dilator substances from a nonvascular cell type in the brain. Except for the neuronal specific NMDA subtype, astrocytes contain all other glutamate receptor isoforms.4 Stimulation of metabotropic glutamate receptors induces release of AA from astrocytes.9 In agreement with the latter, we found that glutamate stimulated release of AA from cultured astrocytes. The mechanism through which glutamate enhances production and release of EETs from astrocytes cannot be directly ascertained from the results of the present study; however, given the above, it could be dependent on increasing availability of substrate for P-450 epoxygenase. Other mechanisms could involve release of preformed EETs from membrane-bound pools or enhancement of enzyme activity. It is difficult to speculate on the mechanism of the increase in 2C11 protein after 12-hour exposure to glutamate; however, the fact that it is increased indicates that at least some of the glutamate-induced generation of EETs is due to the 2C11 epoxygenase isoform. At this point, we do not know if 2C11 is the only or even major P-450 epoxygenase isoform present in astrocytes.
The potential physiological significance of the above findings is highlighted by the finding that inhibition of P-450 epoxygenase activity largely inhibits the response to exogenous glutamate. Assuming for the moment that the in vivo source of epoxygenase activity lies within astrocytes, it could be speculated that spillover of glutamate during neuronal activation could stimulate release of EETs to dilate cerebral arterioles and increase blood flow to that area. More functional work will be needed to further support this hypothesis.
Activation of the neuronal NMDA receptor leads to the formation of NO by a Ca2+/calmodulin–dependent neuronal NO synthase pathway, leading to the hypothesis that glutamate-induced dilation is mediated by NO release through NMDA receptor activation.16 17 Several lines of evidence argue against this hypothesis. First, NMDA receptors do not appear to be involved in synaptic transmission, since selective blockade of these receptors does not affect synaptic transmission in the hippocampus despite a high abundance of NMDA receptors.18 Second, selective blockade of NMDA receptors has no effect on resting vascular tone,16 and mutant mice that do not express the gene for neuronal NO synthase demonstrate no apparent cerebrovascular pathology and exhibit an autoregulatory capacity similar to that of the wild-type animals.19 If glutamate released in the brain during synaptic activity contributes to cerebrovascular tone by activation of NMDA receptors and NO production, one would expect pharmacological blockade or genetic “knockout” of these receptors to affect resting vascular tone and blood flow.20 We have previously reported that inhibition of P-450 AA epoxygenase decreases baseline CBF without affecting responsiveness to sodium nitroprusside.13 Third, it has been demonstrated that NO released from neurons acts to enhance transmitter release from presynaptic neurons.21 Thus, NMDA-induced dilation of cerebral arteries and its attenuation by NO synthase inhibition may be related to the availability of neurotransmitters in the extracellular fluid22 rather than to the action of NMDA-evoked release of NO. In support of the latter notion is the finding that dilation of cerebral arterioles in response to nitroglycerin and nitroprusside is mediated by activation of trigeminal fibers that innervate cerebral vessels and release of calcitonin gene–related peptide.23 Finally, activation of the quisqualate (AMPA) receptor subtype also leads to the release of NO from neurons24 25 26 but, in contrast to NMDA and kainate glutamate receptors subtypes, fails to dilate cerebral vessels.
In summary, we have presented evidence to support the hypothesis that glutamate-induced dilation of cerebral arterioles is mediated at least in part by changes in the formation and release of EETs by astrocytes. The close proximity of astrocytes to both synaptic clefts and cerebral microvessels makes a neurotransmitter-sensitive release of a vasodilator substance a plausible mechanism to couple neuronal activity to regional increases in CBF.
Selected Abbreviations and Acronyms
|aCSF||=||artificial cerebrospinal fluid|
|AMPA||=||α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid|
|CBF||=||cerebral blood flow|
|DMEM||=||Dulbecco’s modified Eagle’s medium|
|DPBS||=||Dulbecco’s phosphate-buffered saline|
|MAP||=||mean systemic arterial blood pressure|
|rpHPLC||=||reverse-phase high-performance liquid chromatography|
This study was supported in part by grants HL-33833 and NS-32321 from the National Institutes of Health and by grant 3440-02P from the Department of Veterans Affairs Administration. The authors would like to thank Nathan Heeray for his technical assistance.
- Received October 29, 1996.
- Revision received January 29, 1997.
- Accepted January 30, 1997.
- Copyright © 1997 by American Heart Association
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