In the brain, pressure-induced myogenic constriction of cerebral arteriolar muscle contributes to autoregulation of cerebral blood flow (CBF). This study examined the role of 20-HETE in autoregulation of CBF in anesthetized rats. The expression of P-450 4A protein and mRNA was localized in isolated cerebral arteriolar muscle of rat by immunocytochemistry and in situ hybridization. The results of reverse transcriptase-polymerase chain reaction studies revealed that rat cerebral microvessels express cytochrome P-450 4A1, 4A2, 4A3, and 4A8 isoforms, some of which catalyze the formation of 20-HETE from arachidonic acid. Cerebral arterial microsomes incubated with [(14)C]arachidonic acid produced 20-HETE. An elevation in transmural pressure from 20 to 140 mm Hg increased 20-HETE concentration by 6-fold in cerebral arteries as measured by gas chromatography/mass spectrometry. In vivo, inhibition of vascular 20-HETE formation with N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS), or its vasoconstrictor actions using 15-HETE or 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE), attenuated autoregulation of CBF to elevations of arterial pressure. In vitro application of DDMS, 15-HETE, or 20-HEDE eliminated pressure-induced constriction of rat middle cerebral arteries, and 20-HEDE and 15-HETE blocked the vasoconstriction action of 20-HETE. Taken together, these data suggest an important role for 20-HETE in the autoregulation of CBF.

The purpose of this study was to determine whether arachidonic acid can be converted to 20-hydroxyeicosatetraenoic acid (HETE) by P-450 enzymes in cat cerebral microvasculature, to identify the P-450 isoforms responsible for the formation of this metabolite, and to characterize the vasoactive effects of 20-HETE on these vessels. Cerebral microvessels were isolated by filling them with a suspension of magnetized iron oxide (particle size = 10 microns) and separated from minced cerebral cortical tissue using a magnet. Cat cerebral microvessels were homogenized and incubated with [14C]arachidonic acid (AA), and cytochrome P-450-dependent metabolites of AA were separated by reverse-phase high-pressure liquid chromatography. A major metabolite that coeluted with synthetic 20-HETE was identified. The formation of this metabolite was dependent on NADPH and was inhibited by 17-octadecynoic acid (ODYA), a specific suicide-substrate inhibitor of the omega-hydroxylation of AA by P-450 enzymes. Western blot analysis confirmed the presence of a P-450 enzyme of the 4A gene family in cat cerebral microvessels. Gas chromatography/mass spectrometry analysis revealed that this metabolite has an identical mass-to-charge ratio (391 m/z) as that of standard 20-HETE. Exogenous 20-HETE constricted pressurized cat pial arteries in a concentration-dependent manner with a threshold concentration of < 1.0 nM. 20-HETE (1 nM) inhibited the activity of a 217-pS K+ channel recorded in cell-attached patches of isolated cat cerebral microvascular muscle cells. Blockade of endogenous P-450 activity with 17-ODYA markedly increased the activity of the 217 pS K+ channel in these cells, an action that was completely reversed by a nanomolar concentration of 20-HETE, suggesting that 20-HETE might be an endogenous modulator of the 217 pS K+ channel in cerebral arterial muscle cells. These results demonstrate the presence of P-450 4A enzyme activity in the cerebral microvasculature of the cat that converts AA to 20-HETE. The potent vasoconstrictor effects of 20-HETE on cerebral vessels suggests that metabolites of P-450 enzymes of the 4A gene family could play an important role in regulating cerebral microvascular tone.

Our results demonstrate that a P450 2C11 mRNA is expressed in astrocytes and may be responsible for astrocyte epoxygenase activity. Given the vasodilatory effect of EETs, our findings suggest a role for astrocytes in the control of cerebral microcirculation mediated by P450 2C11-catalyzed conversion of AA to EETs. The mechanism of EET-induced dilation of rat cerebral microvessels may involve activation of K+ channels.

The vascular response to changes in oxygen levels in the blood and tissue is a highly adaptive physiological response that functions to match tissue oxygen supply to metabolic demand. Defining the cellular mechanisms that can sense physiologically relevant changes in P o 2 and adjust vascular diameter are vital to our understanding of this process. A cytochrome P450 (P450) enzyme of the 4A family of ω-hydroxylases was localized in renal microvessels, renal cortex, and a striated muscle microvascular bed (cremaster) of the rat. In the presence of molecular oxygen, this P450 enzyme catalyzes formation of 20-HETE from arachidonic acid (AA). Prior studies have shown that 20-HETE potently contracts renal and cerebral arteries and arterioles. The present study demonstrates that 20-HETE constricts striated muscle arterioles as well. In both intact renal microvessels and enriched renal cortical microsomal enzyme preparations, the formation of 20-HETE was linearly dependent on P o 2 between 20 and 140 mm Hg. Homogenates of cremaster tissue produced 20-HETE when incubated with AA. They also expressed message for P450 4A enzyme, as determined by Southern and Western blots. Administration of 17-octadecynoic acid (17-ODYA), which is a P450 4A inhibitor, attenuated the constriction of third-order cremasteric arterioles in response to elevation of superfusion solution P o 2 from ≈3 to 5 mm Hg to ≈35 mm Hg. 17-ODYA had no effect on basal vascular tone or response of cremaster arterioles to vasoactive compounds. These results demonstrate the existence of P450 ω-hydroxylase activity and 20-HETE formation in the vasculature and parenchyma of at least two microvascular beds. Our data suggest that a P450 enzyme of the 4A family has the potential to function as an oxygen sensor in mammalian microcirculatory beds and to regulate arteriolar caliber by generating 20-HETE in an oxygen-dependent manner.

Cerebral arteries express cytochrome P450 4A enzymes (P450 4A) and produce 20‐ hydroxyeicosatetraenoic acid (20‐HETE), a potent constrictor of pial arterioles. It is not known which cell type in the vessel wall is responsible for the formation of 20‐HETE. We examined whether freshly isolated cerebral arterial muscle cells (VSMCs) express P450 4A and produce 20‐HETE. We also studied the effect of 20‐HETE on pressurized cerebral arteries and on whole‐cell L‐type Ca2+current (ICa) recorded in cat cerebral VSMCs. Cat cerebral VSMCs incubated with [14C]arachidonic acid ([14C]AA) produced 20‐HETE (3.9 ± 1.1 pmol min−1 (mg protein)−1). Reverse transcription‐polymerase chain reaction studies revealed that cat cerebral VSMCs express mRNA for P450 4A which metabolizes AA to 20‐HETE. Cloning and sequencing of the cDNA amplified from mRNA isolated from VSMCs showed > 96 % amino acid homology to the rat and human P450 4A2 and 4A3. 20‐HETE (1–300 nM) induced a concentration‐dependent constriction of cat cerebral arteries, which was inhibited by nifedipine. Addition of 10 and 100 nM 20‐HETE to the bath increased peak ICa by 50 ± 3 and 100 ± 10 %, respectively. This effect was not influenced by altering the frequency of depolarization. 20‐HETE (100 nM) failed to increase ICa in the presence of nifedipine. These results demonstrate that cat cerebral VSMCs express P450 4A enzyme, and produce 20‐HETE which activates L‐type Ca2+ channel current to promote cerebral vasoconstriction.

Arachidonic acid metabolites of the cytochrome P450 monooxygenase pathway have recently been found to play a major role in modulating vascular tone in the renal and cerebral circulations (1-3). The major cytochrome P450 metabolite of arachidonic acid produced in the cerebral and renal vasculature is 20-hydroxyeicosatetraenoic acid (20-HETE) 1 (4 -6). 20-HETE is a potent vasoconstrictor in isolated cat cerebral and rat renal microvessels over the concentration range of 10 Ϫ11 to 10 Ϫ9 M (4, 5). The underlying cellular-ionic mechanism of this vasoconstrictor response appears to be depolarization-induced influx of calcium secondary to inhibition of large conductance calcium-activated potassium channels (K Ca ) (4, 6, 7). Independent of the depolarization induced by inhibitory effects on K Ca , recent data indicate that 20-HETE also activates L-type calcium channels in a concentration-dependent manner, an effect that is antagonized by nifedipine (8, 9).Several reports identify a role for 20-HETE in the regulation of renal tubular ion transport. In cells of the thick ascending limb of the rat kidney, 20-HETE decreases the open state probability of an apical 70 pS K ϩ channel (10), thus regulating K ϩ recycling across the membrane and Na ϩ resorption. In the medullary thick ascending limb of the loop of Henle, NaϪ transport activity is reduced by 20-HETE (11). In proximal tubular epithelial cells, the activity of the NaATPase is reduced by 20-HETE, an effect that is dependent upon activation of protein kinase C (PKC) (12-14). These observations implicate 20-HETE in a diverse array of effector functions. However, the exact signal transduction pathway by which 20-HETE exerts these effects is unknown. Most of the effects described above could be related to an increased activity of PKC (15-17). Because several cis-unsaturated fatty acids, including arachidonic acid and its metabolites, activate PKC (18 -20), and activation of PKC decreases the activity of K Ca (21-24) and promotes vasoconstriction (25), we hypothesize that the effects of 20-HETE on cerebral arterial tone and whole-cell K ϩ channel current involve activation of PKC. In this report, we provide functional evidence indicating that 20-HETE promotes cerebral vasoconstriction and inhibition of whole-cell K ϩ current via a pathway that involves PKC. We also provide biochemical evidence that 20-HETE increases the phosphorylation of myristoylated, alanine-rich PKC substrate (MARCKS) in cultured cat cerebral vascular smooth muscle cells (VSMCs) in a concentration-related and PKC-dependent manner. EXPERIMENTAL PROCEDURESIsolated Pressurized Vessel Studies-Isolated cat middle cerebral arteries (outside diameter, 200 -400 m; length, 10 -12 mm) were placed in a perfusion chamber, cannulated with glass micropipettes, and secured in place with 8-O polyethylene suture (Ethicon, Inc., Somerville, NJ), and side branches were tied off with 10-O polyethylene suture using a stereomicroscope (Carl Zeiss, Inc., Berlin, Germany). The arterial segments were bathed in physiological ...

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 NIAID Radiation and Nuclear Countermeasures Program is developing medical agents to mitigate the acute and delayed effects of radiation that may occur from a radionuclear attack or accident. To date, most such medical countermeasures have been developed for single organ injuries. Angiotensin converting enzyme (ACE) inhibitors have been used to mitigate radiation-induced lung, skin, brain and renal injuries in rats. ACE inhibitors have also been reported to decrease normal tissue complication in radiation oncology patients. In the current study we have developed a rat partial-body irradiation (leg-out PBI) model with minimal bone marrow sparing (one leg shielded) that results in acute and late injuries to multiple organs. In this model, the ACE inhibitor lisinopril (at ∼24 mg m-2 day-1 started orally in the drinking water at 7 days after irradiation and continued to ≥150 days) mitigated late effects in the lungs and kidneys after 12.5 Gy leg-out PBI. Also in this model, a short course of saline hydration and antibiotics mitigated acute radiation syndrome following doses as high as 13 Gy. Combining this supportive care with the lisinopril regimen mitigated overall morbidity for up to 150 days after 13 Gy leg-out PBI. Furthermore lisinopril was an effective mitigator in the presence of the growth factor G-CSF (100 μg kg-1 day-1 from days 1-14) which is FDA-approved for use in a radionuclear event. In summary, by combining lisinopril (FDA-approved for other indications) with hydration and antibiotics, we mitigated acute and delayed radiation injuries in multiple organs.