Effect of Nearby Construction Activity on Endothelial Function, Sensitivity to Nitric Oxide, and Potassium Channel Activity in the Middle Cerebral Arteries of Rats.

The present study assessed the effect of nearby construction activity on the responses of rat middle cerebral arteries (MCA)to the endothelium-dependent vasodilator acetylcholine and the NO donor sodium nitroprusside (SNP) and the activity of MaxiK potassium channels in MCA smooth muscle cells from male Sprague-Dawley rats. Two monitoring systems were used to assess vibrations in the animal rooms during and immediately after construction activities near the research building where the animal facility is located. One was a commercially available system; the other was a Raspberry-Pi (RPi)-based vibration monitoring system designed in our laboratory that included a small computing unit attached to a rolling sensor (low sensitivity) and a piezoelectric film sensor (high sensitivity). Both systems recorded increased levels of vibration during construction activity outside the building. During the construction period, vasodilator responses to acetylcholine and SNP were abolished, and MaxiK single-channel current opening frequency and open-state probability in cell-attached patches of isolated MCA myocytes were dramatically decreased. Recovery of acetylcholine- and SNP-induced dilation was minimal in MCA from rats studied after completion of construction but housed in the animal facility during construction, whereas responses to acetylcholine and SNP were intact in rats purchased, housed, and studied after construction. Baseline levels of vibration returned after the completion of construction, concomitant with the recovery of normal endothelium-dependent vasodilation to acetylcholine and of NO sensitivity assessed by using SNP in MCA from animals obtained after construction. The results of this study indicate that the vibration associated with nearby construction can have highly disruptive effects on crucial physiologic phenotypes.

Regulation of Cerebral Blood Flow: Response to Cytochrome P450 Lipid Metabolites.

There have been numerous reviews related to the cerebral circulation. Most of these reviews are similar in many ways. In the present review, we thought it important to provide an overview of function with specific attention to details of cerebral arterial control related to brain homeostasis, maintenance of neuronal energy demands, and a unique perspective related to the role of astrocytes. A coming review in this series will discuss cerebral vascular development and unique properties of the neonatal circulation and developing brain, thus, many aspects of development are missing here. Similarly, a review of the response of the brain and cerebral circulation to heat stress has recently appeared in this series (8). By trying to make this review unique, some obvious topics were not discussed in lieu of others, which are from recent and provocative research such as endothelium-derived hyperpolarizing factor, circadian regulation of proteins effecting cerebral blood flow, and unique properties of the neurovascular unit. © 2018 American Physiological Society. Compr Physiol 8:801-821, 2018.

Detection of TRPV4 channel current-like activity in Fawn Hooded hypertensive (FHH) rat cerebral arterial muscle cells.

The transient receptor potential vallinoid type 4 (TRPV4) is a calcium entry channel known to modulate vascular function by mediating endothelium-dependent vasodilation. The present study investigated if isolated cerebral arterial myocytes of the Fawn Hooded hypertensive (FHH) rat, known to display exaggerated KCa channel current activity and impaired myogenic tone, express TRPV4 channels at the transcript and protein level and exhibit TRPV4-like single-channel cationic current activity. Reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining analysis detected the expression of mRNA transcript and translated protein of TRPV4 channel in FHH rat cerebral arterial myocytes. Patch clamp recording of single-channel current activity identified the presence of a single-channel cationic current with unitary conductance of ~85 pS and ~96 pS at hyperpolarizing and depolarizing potentials, respectively, that was inhibited by the TRPV4 channel antagonist RN 1734 or HC 067074 and activated by the potent TRPV4 channel agonist GSK1016790A. Application of negative pressure via the interior of the patch pipette increased the NPo of the TRPV4-like single-channel cationic current recorded in cell-attached patches at a patch potential of 60 mV that was inhibited by prior application of the TRPV4 channel antagonist RN 1734 or HC 067047. Treatment with the TRPV4 channel agonist GSK1016790A caused concentration-dependent increase in the NPo of KCa single-channel current recorded in cell-attached patches of cerebral arterial myocytes at a patch potential of 40 mV, which was not influenced by pretreatment with the voltage-gated L-type Ca2+ channel blocker nifedipine or the T-type Ca2+ channel blocker Ni2+. These findings demonstrate that FHH rat cerebral arterial myocytes express mRNA transcript and translated protein for TRPV4 channel and display TRPV4-like single-channel cationic current activity that was stretch-sensitive and activation of which increased the open state probability of KCa single-channel current in these arterial myocytes.

Inhibition of soluble epoxide hydrolase augments astrocyte release of vascular endothelial growth factor and neuronal recovery after oxygen-glucose deprivation.

Epoxyeicosatrienoic acids (EETs) are synthesized in astrocytes, and inhibitors of soluble epoxide hydrolase (sEH), which hydrolyzes EETs, reduce infarct volume in ischemic stroke. Astrocytes can release protective neurotrophic factors, such as vascular endothelial growth factor (VEGF). We found that addition of sEH inhibitors to rat cultured astrocytes immediately after oxygen-glucose deprivation (OGD) markedly increased VEGF concentration in the medium 48 h later and the effect was blocked by an EET antagonist. The sEH inhibitors increased EET concentrations to levels capable of increasing VEGF. When the sEH inhibitors were removed from the medium at 48 h, the increase in VEGF persisted for an additional 48 h. Neurons exposed to OGD and subsequently to astrocyte medium previously conditioned with OGD plus sEH inhibitors showed increased phosphorylation of their VEGF receptor-2, less TUNEL staining, and increased phosphorylation of Akt, which was blocked by a VEGF receptor-2 antagonist. Our findings indicate that sEH inhibitors, applied to cultured astrocytes after an ischemia-like insult, can increase VEGF secretion. The released VEGF then enhances Akt-enabled cell survival signaling in neurons through activation of VEGF receptor-2 leading to less neuronal cell death. These results suggest a new strategy by which astrocytes can be leveraged to support neuroprotection.

Expression of CYP 4A ω-hydroxylase and formation of 20-hydroxyeicosatetreanoic acid (20-HETE) in cultured rat brain astrocytes.

Astrocytes secrete vasodilator and vasoconstrictor factors via end feet processes, altering blood flow to meet neuronal metabolic demand. Compared to what is known about the ability of astrocytes to release factors that dilate local cerebral vasculature, very little is known regarding the source and identity of astrocyte derived constricting factors. The present study investigated if astrocytes express CYP 4A ω-hydroxylase and metabolize arachidonic acid (AA) to 20-hydroxyeicotetraenoic acid (20-HETE) that regulates KCa channel activity in astrocytes and cerebral arterial myocyte contractility. Here we report that cultured astrocytes express CYP 4A2/3 ω-hydroxylase mRNA and CYP 4A protein and produce 20-HETE and the CYP epoxygenase metabolites epoxyeicosatrienoic acids (EETs) when incubated with AA. The production of 20-HETE and EETs was enhanced following stimulation of metabotropic glutamate receptors (mGluR) on the astrocytes. Exogenous application of 20-HETE attenuated, whereas inhibition of 20-HETE production with HET-0016 increased the open state probabilities (NPo) of 71pS and 161pS KCa single-channel currents recorded from astrocytes. Exposure of isolated cerebral arterial myocytes to conditioned media from cultured astrocytes caused shortening of the length of freshly isolated cerebral arterial myocytes that was not evident following inhibition of astrocyte 20-HETE synthesis and action. These findings suggest that astrocytes not only release vasodilator EETs in response to mGluR stimulation but also synthetize and release the cerebral arterial myocyte constrictor 20-HETE that also functions as an endogenous inhibitor of the activity of two types of KCa channel currents found in astrocytes.

Contribution of epoxyeicosatrienoic acids to the cerebral blood flow response to hypoxemia.

Adenosine A2A receptors and ATP-activated K(+) (KATP) channels contribute to part of the cerebral vasodilatory response to systemic hypoxia, but other mediators are likely involved. Epoxyeicosatrienoic acids (EETs) are cerebral vasodilators and are released from astrocytes exposed to hypoxia. Moreover, stimulation of metabotropic glutamate receptors (mGluR) produces vasodilation by an EET-dependent mechanism. Here, we tested the hypothesis that EET signaling and mGluR activation contribute to hypoxic vasodilation. Laser-Doppler flow was measured over cerebral cortex of anesthetized rats subjected to stepwise reductions in arterial oxygen saturation to 50-70%. Hypoxic reactivity was calculated as the slope of the change in laser-Doppler flow vs. the reciprocal of arterial oxygen content. Hypoxic reactivity significantly decreased from 9.2 ± 1.9 (±95% confidence interval) in controls with vehicle treatment to 2.6 ± 1.4 with the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid, to 3.0 ± 1.5 with the EET synthesis inhibitor MS-PPOH, to 1.9 ± 2.3 with the combined mGluR subtype 1 and 5 antagonists 2-methyl-6-(phenylethynyl)pyridine and LY367385, to 5.6 ± 1.2 with the KATP channel inhibitor glibenclamide, and to 5.8 ± 2.3 with the A2A receptor antagonist SCH58261. However, reactivity was not significantly altered by the A2B receptor antagonist MRS1754 (6.7 ± 1.8; P = 0.28 Dunnett's test) or by the 20-hydroxyeicosatetraenoic acid synthesis inhibitor HET0016 (7.5 ± 2.3; P = 0.6). These data indicate that, in addition to the known contributions of A2A receptors and KATP channels to the increase in cerebral blood flow during hypoxia, EETs and mGluRs make a major contribution, possibly by mGluR stimulation and hypoxia-induced release of EETs. In contrast, A2B receptors do not make a major contribution, and 20-hydroxyeicosatetraenoic acid does not significantly limit hypoxic vasodilation.

Enhanced large conductance K+ channel activity contributes to the impaired myogenic response in the cerebral vasculature of Fawn Hooded Hypertensive rats.

Recent studies have indicated that the myogenic response (MR) in cerebral arteries is impaired in Fawn Hooded Hypertensive (FHH) rats and that transfer of a 2.4 megabase pair region of chromosome 1 (RNO1) containing 15 genes from the Brown Norway rat into the FHH genetic background restores MR in a FHH.1(BN) congenic strain. However, the mechanisms involved remain to be determined. The present study examined the role of the large conductance calcium-activated potassium (BK) channel in impairing the MR in FHH rats. Whole-cell patch-clamp studies of cerebral vascular smooth muscle cells (VSMCs) revealed that iberiotoxin (IBTX; BK inhibitor)-sensitive outward potassium (K+) channel current densities are four- to fivefold greater in FHH than in FHH.1(BN) congenic strain. Inside-out patches indicated that the BK channel open probability (NPo) is 10-fold higher and IBTX reduced NPo to a greater extent in VSMCs isolated from FHH than in FHH.1(BN) rats. Voltage sensitivity of the BK channel is enhanced in FHH as compared with FHH.1(BN) rats. The frequency and amplitude of spontaneous transient outward currents are significantly greater in VSMCs isolated from FHH than in FHH.1(BN) rats. However, the expression of the BK-α and -ß-subunit proteins in cerebral vessels as determined by Western blot is similar between the two groups. Middle cerebral arteries (MCAs) isolated from FHH rats exhibited an impaired MR, and administration of IBTX restored this response. These results indicate that there is a gene on RNO1 that impairs MR in the MCAs of FHH rats by enhancing BK channel activity.

Endogenous events modulating myogenic regulation of cerebrovascular function.

The existence of arterial myogenic tone was first described by Bayliss in 1902, however, its association with pressure-dependent membrane depolarization was not observed until 1984. The factors that mediate myogenic arterial constriction remain unknown. One possible clue was a finding by our laboratory that cerebral arterial muscle cells express CYP 4A ω-hydroxylase enzyme that catalyzes the formation of the potent vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid (AA), the production of which increased by elevations of intravascular pressure. 20-HETE activates protein kinase C (PKC), inhibits Ca(2+)-activated K(+) (KCa) channels, depolarizes arterial muscle cell membrane, activates L-type Ca(2+) channels, increases intracellular Ca(2+) ([Ca(2+)]i) and mediates autoregulation of cerebral blood flow. Emerging evidence indicates that 20-HETE level increases in ischemia/reperfusion injury and stimulates production of reactive oxygen species (ROS) resulting in oxidative stress induced ischemic stroke injury, which can be prevented by inhibition of 20-HETE synthesis or action. The brain also expresses CYP epoxygenases that convert AA to the vasodilator epoxyeicosatrienoic acids (EETs), the production of which increases in ischemia and provide protection against ischemia-induced tissue damage. Basal or stimulus released ROS act to modify cerebral myogenic tone. Similar to other enzymes CYP epoxygenase and ω-hydroxylase also generate ROS that modify myogenic cerebral reactivity. Hypoxia per se or adenosine released during hypoxia induces increased production of ROS that alter cerebrovascular function. The capacity of the brain to express CYP enzymes that produce bioactive EETs and 20-HETE and generate ROS has a significant bearing in regulating the dynamics of cerebral blood flow and serve as potential therapeutic targets for the management of pathologic disorders of the cerebral circulation.

Redox signaling via oxidative inactivation of PTEN modulates pressure-dependent myogenic tone in rat middle cerebral arteries.

The present study examined the level of generation of reactive oxygen species (ROS) and roles of inactivation of the phosphatase PTEN and the PI3K/Akt signaling pathway in response to an increase in intramural pressure-induced myogenic cerebral arterial constriction. Step increases in intraluminal pressure of cannulated cerebral arteries induced myogenic constriction and concomitant formation of superoxide (O2 (.-)) and its dismutation product hydrogen peroxide (H2O2) as determined by fluorescent HPLC analysis, microscopic analysis of intensity of dihydroethidium fluorescence and attenuation of pressure-induced myogenic constriction by pretreatment with the ROS scavenger 4,hydroxyl-2,2,6,6-tetramethylpiperidine1-oxyl (tempol) or Mito-tempol or MitoQ in the presence or absence of PEG-catalase. An increase in intraluminal pressure induced oxidation of PTEN and activation of Akt. Pharmacological inhibition of endogenous PTEN activity potentiated pressure-dependent myogenic constriction and caused a reduction in NPo of a 238 pS arterial KCa channel current and an increase in [Ca(2+)]i level in freshly isolated cerebral arterial muscle cells (CAMCs), responses that were attenuated by Inhibition of the PI3K/Akt pathway. These findings demonstrate an increase in intraluminal pressure induced increase in ROS production triggered redox-sensitive signaling mechanism emanating from the cross-talk between oxidative inactivation of PTEN and activation of the PI3K/Akt signaling pathway that involves in the regulation of pressure-dependent myogenic cerebral arterial constriction.

Arachidonic acid-induced dilation in human coronary arterioles: convergence of signaling mechanisms on endothelial TRPV4-mediated Ca2+ entry.

BACKGROUND: Arachidonic acid (AA) and/or its enzymatic metabolites are important lipid mediators contributing to endothelium-derived hyperpolarizing factor (EDHF)-mediated dilation in multiple vascular beds, including human coronary arterioles (HCAs). However, the mechanisms of action of these lipid mediators in endothelial cells (ECs) remain incompletely defined. In this study, we investigated the role of the transient receptor potential vanilloid 4 (TRPV4) channel in AA-induced endothelial Ca(2+) response and dilation of HCAs. METHODS AND RESULTS: AA induced concentration-dependent dilation in isolated HCAs. The dilation was largely abolished by the TRPV4 antagonist RN-1734 and by inhibition of endothelial Ca(2+)-activated K(+) channels. In native and TRPV4-overexpressing human coronary artery ECs (HCAECs), AA increased intracellular Ca(2+) concentration ([Ca(2+)]i), which was mediated by TRPV4-dependent Ca(2+) entry. The AA-induced [Ca(2+)]i increase was inhibited by cytochrome P450 (CYP) inhibitors. Surprisingly, the CYP metabolites of AA, epoxyeicosatrienoic acids (EETs), were much less potent activators of TRPV4, and CYP inhibitors did not affect EET production in HCAECs. Apart from its effect on [Ca(2+)]i, AA induced endothelial hyperpolarization, and this effect was required for Ca(2+) entry through TRPV4. AA-induced and TRPV4-mediated Ca(2+) entry was also inhibited by the protein kinase A inhibitor PKI. TRPV4 exhibited a basal level of phosphorylation, which was inhibited by PKI. Patch-clamp studies indicated that AA activated TRPV4 single-channel currents in cell-attached and inside-out patches of HCAECs. CONCLUSIONS: AA dilates HCAs through a novel mechanism involving endothelial TRPV4 channel-dependent Ca(2+) entry that requires endothelial hyperpolarization, PKA-mediated basal phosphorylation of TRPV4, and direct activation of TRPV4 channels by AA.

Role of dual-specificity protein phosphatase-5 in modulating the myogenic response in rat cerebral arteries.

The present study examined the role of the dual-specificity protein phosphatase-5 (DUSP-5) in the pressure-induced myogenic responses of organ-cultured cerebral arterial segments. In these studies, we initially compared freshly isolated and organ-cultured cerebral arterial segments with respect to responses to step increases in intravascular pressure, vasodilator and vasoconstrictor stimuli, activities of the large-conductance arterial Ca(2+)-activated K(+) (K(Ca)) single-channel current, and stable protein expression of DUSP-5 enzyme. The results demonstrate maintained pressure-dependent myogenic vasoconstriction, DUSP-5 protein expression, endothelium-dependent and -independent dilations, agonist-induced constriction, and unitary K(Ca) channel conductance in organ-cultured cerebral arterial segments similar to that in freshly isolated cerebral arteries. Furthermore, using a permeabilization transfection technique in organ-cultured cerebral arterial segments, gene-specific small interfering RNA (siRNA) induced knockdown of DUSP-5 mRNA and protein, which were associated with enhanced pressure-dependent cerebral arterial myogenic constriction and increased phosphorylation of PKC-ßII. In addition, siRNA knockdown of DUSP-5 reduced levels of phosphorylated ROCK and ERK1 with no change in the level of phosphorylated ERK2. Pharmacological inhibition of ERK1/2 phosphorylation significantly attenuated pressure-induced myogenic constriction in cerebral arteries. The findings within the present studies illustrate that DUSP-5, native in cerebral arterial muscle cells, appears to regulate signaling of pressure-dependent myogenic cerebral arterial constriction, which is crucial for the maintenance of constant cerebral blood flow to the brain.

Protective effect of 20-HETE inhibition in a model of oxygen-glucose deprivation in hippocampal slice cultures.

Recent studies have indicated that inhibitors of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) may have direct neuroprotective actions since they reduce infarct volume after ischemia reperfusion in the brain without altering blood flow. To explore this possibility, the present study used organotypic hippocampal slice cultures subjected to oxygen-glucose deprivation (OGD) and reoxygenation to examine whether 20-HETE is released by organotypic hippocampal slices after OGD and whether it contributes to neuronal death through the generation of ROS and activation of caspase-3. The production of 20-HETE increased twofold after OGD and reoxygenation. Blockade of the synthesis of 20-HETE with N-hydroxy-N'-(4-butyl-2-methylphenol)formamidine (HET0016) or its actions with a 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, reduced cell death, as measured by the release of lactate dehydrogenase and propidium iodide uptake. Administration of a 20-HETE mimetic, 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid (5,14-20-HEDE), had the opposite effect and increased injury after OGD. The death of neurons after OGD was associated with an increase in the production of ROS and activation of caspase-3. These effects were attenuated by HET0016 and potentiated after the administration of 5,14-20-HEDE. These findings indicate that the production of 20-HETE by hippocampal slices is increased after OGD and that inhibitors of the synthesis or actions of 20-HETE protect neurons from ischemic cell death. The protective effect of 20-HETE inhibitors is associated with a decrease in superoxide production and activation of caspase-3.

H2O2-induced dilation in human coronary arterioles: role of protein kinase G dimerization and large-conductance Ca2+-activated K+ channel activation.

RATIONALE: Hydrogen peroxide (H(2)O(2)) serves as a key endothelium-derived hyperpolarizing factor mediating flow-induced dilation in human coronary arterioles (HCAs). The precise mechanisms by which H(2)O(2) elicits smooth muscle hyperpolarization are not well understood. An important mode of action of H(2)O(2) involves the oxidation of cysteine residues in its target proteins, including protein kinase G (PKG)-Iα, thereby modulating their activities. OBJECTIVE: Here we hypothesize that H(2)O(2) dilates HCAs through direct oxidation and activation of PKG-Iα leading to the opening of the large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel and subsequent smooth muscle hyperpolarization. METHODS AND RESULTS: Flow and H(2)O(2) induced pressure gradient/concentration-dependent vasodilation in isolated endothelium-intact and -denuded HCAs, respectively. The dilation was largely abolished by iberiotoxin, a BK(Ca) channel blocker. The PKG inhibitor Rp-8-Br-PET-cGMP also markedly inhibited flow- and H(2)O(2)-induced dilation, whereas the soluble guanylate cyclase inhibitor ODQ had no effect. Treatment of coronary smooth muscle cells (SMCs) with H(2)O(2) elicited dose-dependent, reversible dimerization of PKG-Iα, and induced its translocation to the plasma membrane. Patch-clamp analysis identified a paxilline-sensitive single-channel K(+) current with a unitary conductance of 246-pS in freshly isolated coronary SMCs. Addition of H(2)O(2) into the bath solution significantly increased the probability of BK(Ca) single-channel openings recorded from cell-attached patches, an effect that was blocked by the PKG-Iα inhibitor DT-2. H(2)O(2) exhibited an attenuated stimulatory effect on BK(Ca) channel open probability in inside-out membrane patches. CONCLUSIONS: H(2)O(2) dilates HCAs through a novel mechanism involving protein dimerization and activation of PKG-Iα and subsequent opening of smooth muscle BK(Ca) channels.

Epoxyeicosatrienoic acid-dependent cerebral vasodilation evoked by metabotropic glutamate receptor activation in vivo.

Group I metabotropic glutamate receptors (mGluR) on astrocytes have been shown to participate in cerebral vasodilation to neuronal activation in brain slices. Pharmacological stimulation of mGluR in brain slices can produce arteriolar constriction or dilation depending on the initial degree of vascular tone. Here, we examined whether pharmacological stimulation of mGluR in vivo increases cerebral blood flow. A 1-mM solution of the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) superfused at 5 µl/min over the cortical surface of anesthetized rats produced a 30 ± 2% (±SE) increase in blood flow measured by laser-Doppler flowmetry after 15-20 min. The response was completely blocked by superfusion of group I mGluR antagonists and attenuated by superfusion of an epoxyeicosatrienoic acid (EET) antagonist (5 ± 4%), an EET synthesis inhibitor (11 ± 3%), and a cyclooxygenase-2 inhibitor (15 ± 3%). The peak blood flow response was not significantly affected by administration of inhibitors of cyclooxygenase-1, neuronal nitric oxide synthase, heme oxygenase, adenosine A(2B) receptors, or an inhibitor of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE). The blood flow response gradually waned following 30-60 min of DHPG superfusion. This loss of the flow response was attenuated by a 20-HETE synthesis inhibitor and was prevented by superfusion of an inhibitor of epoxide hydrolase, which hydrolyzes EETs. These results indicate that pharmacological stimulation of mGluR in vivo increases cerebral blood flow and that the response depends on the release of EETs and a metabolite of cyclooxygenase-2. Epoxide hydrolase activity and 20-HETE synthesis limit the duration of the response to prolonged mGluR activation.

Pressure-induced myogenic tone and role of 20-HETE in mediating autoregulation of cerebral blood flow.

While myogenic force in response to a changing arterial pressure has been described early in the 20th century, it was not until 1984 that the effect of a sequential increase in intraluminal pressure on cannulated cerebral arterial preparations was found to result in pressure-dependent membrane depolarization associated with spike generation and reduction in lumen diameter. Despite a great deal of effort by different laboratories and investigators, the identification of the existence of a mediator of the pressure-induced myogenic constriction in arterial muscle remained a challenge. It was the original finding by our laboratory that demonstrated the capacity of cerebral arterial muscle cells to express the cytochrome P-450 4A enzyme that catalyzes the formation of the potent vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid, the production of which in cerebral arterial muscle cells increases with the elevation in intravascular pressure. 20-HETE activates protein kinase C and causes the inhibition of Ca(²+)-activated K(+) channels, depolarizes arterial muscle cell membrane, and activates L-type Ca(²+) channel to increase intracellular Ca(²+) levels and evoke vasoconstriction. The inhibition of 20-HETE formation attenuates pressure-induced arterial myogenic constriction in vitro and blunts the autoregulation of cerebral blood flow in vivo. We suggest that the formation and action of cytochrome P-450-derived 20-HETE in cerebral arterial muscle could play a critically important role in the control of cerebral arterial tone and the autoregulation of cerebral blood flow under physiological conditions.

Adenosine can mediate its actions through generation of reactive oxygen species.

Adenosine is an important cerebral vasodilator, but mediating mechanisms are not understood. We investigated the expression of adenosine receptor subtypes in isolated cerebral arterial muscle cells (CAMCs), and their role in adenosine-induced superoxide (O(2)(-)) generation and reduction in cerebral arterial tone. Reverse transcriptase-PCR, western blotting, and immunofluorescence studies have shown that CAMCs express transcript and protein for A1, A(2A), A(2B), and A(3) adenosine receptors. Stimulation of CAMCs with adenosine or the A(2A) agonist CGS-21680 increased the generation of O(2)(-) that was attenuated by the inhibition of A(2A) and A(2B) adenosine receptor subtypes, or by the peptide inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase gp91ds-tat, or by the mitochondria uncoupler 2,4-dinitrophenol. Application of adenosine or CGS-21680 dilated pressure-constricted cerebral arterial segments that were prevented by the antioxidants superoxide dismutase (SOD) conjugated to polyethylene glycol (PEG) and PEG-catalase or by the A(2B) adenosine receptor antagonist MRS-1754, or by the mixed A(2A) and A(2B) antagonist ZM-241385. Antagonism of the A(2A) and A(2B) adenosine receptors had no effect on cerebral vasodilatation induced by nifedipine. These findings indicate that adenosine reduces pressure-induced cerebral arterial tone through stimulation of A(2A) and A(2B) adenosine receptors and generation of O(2)(-) from NADPH oxidase and mitochondrial sources. This signaling pathway could be one of the mediators of the cerebral vasodilatory actions of adenosine.

Onset of pulmonary ventilation in fetal sheep produces pial arteriolar constriction dependent on cytochrome p450 omega-hydroxylase activity.

With the onset of ventilation at birth, cerebral blood flow decreases as oxygenation increases, but the mechanism of cerebral vasoconstriction is unknown. Cytochrome P-450 omega-hydroxylase activity metabolizes arachidonic acid to 20-HETE, a potent vasoconstrictor, in a physiologically relevant O(2)-dependent manner. We tested the hypothesis that the omega-hydroxylase inhibitor, 17-octadecynoic acid (17-ODYA), reduces cerebral vasoconstriction during in utero ventilation with O(2) in fetal sheep. In anesthetized pregnant sheep near term, the fetal head was exposed with the rest of the body remaining in utero. Pial arteriolar diameter was measured by intravital microscopy through a closed cranial window superfused with vehicle or 17-ODYA. Mechanical ventilation of the fetal lungs with a high O(2) mixture to increase arterial Po(2) from approximately 20 to approximately 90 Torr markedly decreased pial arteriolar diameter by 24 + or - 3% (+ or - SE) without a change in arterial pressure. In contrast, superfusion of 17-ODYA completely blocked the decrease in diameter (2 + or - 3%) with increased oxygenation. Vasoconstriction to hypocapnia was intact after returning to the baseline intrauterine oxygenation state, thereby indicating that the effect of 17-ODYA was selective for increased oxygenation. In cerebral arteries isolated from fetal sheep, increasing oxygenation increased 20-HETE production. We conclude that cytochrome P-450 omega-hydroxylase activity makes an important contribution to cerebral vasoconstriction associated with the onset of ventilation at birth.

Antiangiogenic effect of inhibitors of cytochrome P450 on rats with glioblastoma multiforme.

Cytochrome P450 epoxygenase catalyzes 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) from arachidonic acid (AA). In 1996, our group identified the expression of the cytochrome P450 2C11 epoxygenase (CYP epoxygenase) gene in astrocytes. Because of our finding an array of physiological functions have been attributed to EETs in the brain, one of the actions of EETs involves a predominant role in brain angiogenesis. Blockade of EETs formation with different epoxygenase inhibitors decreases endothelial tube formation in cocultures of astrocytes and capillary endothelial cells. The intent of this investigation was to determine if pharmacologic inhibition of formation of EETs is effective in reducing capillary formation in glioblastoma multiforme with a concomitant reduction in tumor volume and increase in animal survival time. Two mechanistically different inhibitors of CYP epoxygenase, 17-octadecynoic acid (17-ODYA) and miconazole, significantly reduced capillary formation and tumor size in glial tumors formed by injection of rat glioma 2 (RG2) cells, also resulting in an increased animal survival time. However, we observed that 17-ODYA and miconazole did not inhibit the formation of EETs in tumor tissue. This implies that 17-ODYA and miconazole appear to exert their antitumorogenic function by a different mechanism that needs to be explored.

20-HETE increases superoxide production and activates NAPDH oxidase in pulmonary artery endothelial cells.

Reactive oxygen species (ROS) signal vital physiological processes including cell growth, angiogenesis, contraction, and relaxation of vascular smooth muscle. Because cytochrome P-450 family 4 (CYP4)/20-hydroxyeicosatetraenoic acid (20-HETE) has been reported to enhance angiogenesis, pulmonary vascular tone, and endothelial nitric oxide synthase function, we explored the potential of this system to stimulate bovine pulmonary artery endothelial cell (BPAEC) ROS production. Our data are the first to demonstrate that 20-HETE increases ROS in BPAECs in a time- and concentration-dependent manner as detected by enhanced fluorescence of oxidation products of dihydroethidium (DHE) and dichlorofluorescein diacetate. An analog of 20-HETE elicits no increase in ROS and blocks 20-HETE-evoked increments in DHE fluorescence, supporting its function as an antagonist. Endothelial cells derived from bovine aortas exhibit enhanced ROS production to 20-HETE quantitatively similar to that of BPAECs. 20-HETE-induced ROS production in BPAECs is blunted by pretreatment with polyethylene-glycolated SOD, apocynin, inhibition of Rac1, and a peptide-based inhibitor of NADPH oxidase subunit p47(phox) association with gp91. These data support 20-HETE-stimulated, NADPH oxidase-derived, and Rac1/2-dependent ROS production in BPAECs. 20-HETE promotes translocation of p47(phox) and tyrosine phosphorylation of p47(phox) in a time-dependent manner as well as increased activated Rac1/2, providing at least three mechanisms through which 20-HETE activates NADPH oxidase. These observations suggest that 20-HETE stimulates ROS production in BPAECs at least in part through activation of NADPH oxidase within minutes of application of the lipid.

Role of 20-HETE in the hypoxia-induced activation of Ca2+-activated K+ channel currents in rat cerebral arterial muscle cells.

The mechanism of sensing hypoxia and hypoxia-induced activation of cerebral arterial Ca(2+)-activated K(+) (K(Ca)) channel currents and vasodilation is not known. We investigated the roles of the cytochrome P-450 4A (CYP 4A) omega-hydroxylase metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE), and generation of superoxide in the hypoxia-evoked activation of the K(Ca) channel current in rat cerebral arterial muscle cells (CAMCs) and cerebral vasodilation. Patch-clamp analysis of K(+) channel current identified a voltage- and Ca(2+)-dependent 238 +/- 21-pS unitary K(+) currents that are inhibitable by tetraethylammonium (TEA, 1 mM) or iberiotoxin (100 nM). Hypoxia (<2% O(2)) reversibly enhanced the open-state probability (NP(o)) of the 238-pS unitary K(Ca) current in cell-attached patches. This effect of hypoxia was not observed on unitary K(Ca) currents recorded from either excised inside-out or outside-out membrane patches. Inhibition of CYP 4A omega-hydroxylase activity increased the NP(o) of K(Ca) single-channel current. Hypoxia reduced the basal endogenous level of 20-HETE by 47 +/- 3% as well as catalytic formation of 20-HETE in cerebral arterial muscle homogenates as determined by liquid chromatography-mass spectrometry analysis. The concentration of authentic 20-HETE was reduced when incubated with the superoxide donor KO(2). Exogenous 20-HETE (100 nM) attenuated the hypoxia-induced activation of the K(Ca) current in CAMCs. Hypoxia did not augment the increase in NP(o) of K(Ca) channel current induced by suicide inhibition of endogenous CYP 4A omega-hydroxylase activity with 17-octadecynoic acid. In pressure (80 mmHg)-constricted cerebral arterial segments, hypoxia induced dilation that was partly attenuated by 20-HETE or by the K(Ca) channel blocker TEA. Exposure to hypoxia caused the generation of intracellular superoxide as evidenced by intense staining of arterial muscle with the fluorescent probe hydroethidine, by quantitation using fluorescent HPLC analysis, and by attenuation of the hypoxia-induced activation of the K(Ca) channel current by superoxide dismutation. These results suggest that the exposure of CAMCs to hypoxia results in the generation of superoxide and reduction in endogenous level of 20-HETE that may account for the hypoxia-induced activation of arterial K(Ca) channel currents and cerebral vasodilation.