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.

Upregulation of 20-HETE Synthetic Cytochrome P450 Isoforms by Oxygen-Glucose Deprivation in Cortical Neurons.

20-Hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor, is a cytochrome P450 (CYP) 4A/4F-derived metabolite of arachidonic acid. Inhibition of 20-HETE synthesis protects brain from ischemic injury. However, that protection is not associated with changes in cerebral blood flow. The present study examined whether CYP4A isoforms are expressed in neurons, whether they produce 20-HETE in neurons, and whether neuronally derived 20-HETE exerts direct neurotoxicity after oxygen-glucose deprivation (OGD). The expression of Cyp4a10 and Cyp4a12a mRNA in cultured mouse cortical neurons increased significantly at 1 and 3 h after exposure to 1 h of OGD. Reoxygenation also markedly augmented the expression of CYP4A protein in neurons and increased 20-HETE levels in the culture medium. Cell viability after OGD increased after treatment with a 20-HETE synthesis inhibitor or an antagonist. That effect was reversed by co-administration of a 20-HETE agonist. These results indicate that neurons express Cyp4a10 and 4a12a, that expression of these isoforms is upregulated by OGD stress, and that neuronally derived 20-HETE directly contributes to neuronal death after reoxygenation.

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.

Rhythmic expression of cytochrome P450 epoxygenases CYP4x1 and CYP2c11 in the rat brain and vasculature.

Mammals have circadian variation in blood pressure, heart rate, vascular tone, thrombotic tendency, and cerebral blood flow (CBF). These changes may be in part orchestrated by circadian variation in clock gene expression within cells comprising the vasculature that modulate blood flow (e.g., fibroblasts, cerebral vascular smooth muscle cells, astrocytes, and endothelial cells). However, the downstream mechanisms that underlie circadian changes in blood flow are unknown. Cytochrome P450 epoxygenases (Cyp4x1 and Cyp2c11) are expressed in the brain and vasculature and metabolize arachidonic acid (AA) to form epoxyeicosatrienoic acids (EETs). EETs are released from astrocytes, neurons, and vascular endothelial cells and act as potent vasodilators, increasing blood flow. EETs released in response to increases in neural activity evoke a corresponding increase in blood flow known as the functional hyperemic response. We examine the hypothesis that Cyp2c11 and Cyp4x1 expression and EETs production vary in a circadian manner in the rat brain and cerebral vasculature. RT-PCR revealed circadian/diurnal expression of clock and clock-controlled genes as well as Cyp4x1 and Cyp2c11, within the rat hippocampus, middle cerebral artery, inferior vena cava, hippocampal astrocytes and rat brain microvascular endothelial cells. Astrocyte and endothelial cell culture experiments revealed rhythmic variation in Cyp4x1 and Cyp2c11 gene and protein expression with a 12-h period and parallel rhythmic production of EETs. Our data suggest there is circadian regulation of Cyp4x1 and Cyp2c11 gene expression. Such rhythmic EETs production may contribute to circadian changes in blood flow and alter risk of adverse cardiovascular events throughout the day.

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.

Epoxyeicosatrienoic acids pretreatment improves amyloid ß-induced mitochondrial dysfunction in cultured rat hippocampal astrocytes.

Amyloid-ß (Aß) has long been implicated as a causative protein in Alzheimer's disease. Cellular Aß accumulation is toxic and causes mitochondrial dysfunction, which precedes clinical symptoms of Alzheimer's disease pathology. In the present study, we explored the possible use of epoxyeicosatrienoic acids (EETs), epoxide metabolites of arachidonic acid, as therapeutic target against Aß-induced mitochondrial impairment using cultured neonatal hippocampal astrocytes. Inhibition of endogenous EET production by a selective epoxygenase inhibitor, MS-PPOH, caused a greater reduction in mitochondrial membrane potential in the presence of Aß (1, 10 µM) exposure versus absence of Aß. MS-PPOH preincubation also aggravated Aß-induced mitochondrial fragmentation. Preincubation of the cells with either 14,15- or 11,12-EET prevented this mitochondrial depolarization and fragmentation. EET pretreatment also further improved the reduction observed in mitochondrial oxygen consumption in the presence of Aß. Preincubation of the cells with EETs significantly improved cellular respiration under basal condition and in the presence of the protonophore, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). The uncoupling of ATP synthase from the electron transfer chain that occurred in Aß-treated cells was also prevented by preincubation with EETs. Lastly, cellular reactive oxygen species production, a hallmark of Aß toxicity, also showed significant reduction in the presence of EETs. We have previously shown that Aß reduces EET synthesis in rat brain homogenates and cultured hippocampal astrocytes and neurons (Sarkar P, Narayanan J, Harder DR. Differential effect of amyloid beta on the cytochrome P450 epoxygenase activity in rat brain. Neuroscience 194: 241-249, 2011). We conclude that reduction of endogenous EETs may be one of the mechanisms through which Aß inflicts toxicity and thus supplementing the cells with exogenous EETs improves mitochondrial dynamics and prevents metabolic impairment.

Identification of a region of rat chromosome 1 that impairs the myogenic response and autoregulation of cerebral blood flow in fawn-hooded hypertensive rats.

This study examined the effects of transfer of a 2.4-Mbp region of rat chromosome 1 (RNO1) from Brown Norway (BN) into fawn-hooded hypertensive (FHH) rats on autoregulation (AR) of cerebral blood flow (CBF) and the myogenic response of middle cerebral arteries (MCAs). AR of CBF was poor in FHH and FHH.1(BN) AR(-) congenic strains that excluded the critical 2.4-Mbp region. In contrast, AR was restored in FHH.1(BN) AR(+) congenic strains that included this region. The diameter of MCAs of FHH rats increased from 140 ± 14 to 157 ± 18 µm when transmural pressure was increased from 40 to 140 mmHg, but it decreased from 137 ± 5 to 94 ± 7 µm in FHH.1(BN) AR(+) congenic strains. Transient occlusion of MCAs reduced CBF by 80% in all strains. However, the hyperemic response following ischemia was significantly greater in FHH and AR(-) rats than that seen in AR(+) congenic strains (AR(-), 173 ± 11% vs. AR(+), 124 ± 5%). Infarct size and edema formation were also significantly greater in an AR(-) strain (38.6 ± 2.6 and 12.1 ± 2%) than in AR(+) congenic strains (27.6 ± 1.8 and 6.5 ± 0.9%). These results indicate that there is a gene in the 2.4-Mbp region of RNO1 that alters the development of myogenic tone in cerebral arteries. Transfer of this region from BN to FHH rats restores AR of CBF and vascular reactivity and reduces cerebral injury after transient occlusion and reperfusion of the MCA.

Attenuation of neonatal ischemic brain damage using a 20-HETE synthesis inhibitor.

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450 metabolite of arachidonic acid that that contributes to infarct size following focal cerebral ischemia. However, little is known about the role of 20-HETE in global cerebral ischemia or neonatal hypoxia-ischemia (H-I). The present study examined the effects of blockade of the synthesis of 20-HETE with N-hydroxy-N'-(4-n-butyl-2-methylphenyl) formamidine (HET0016) in neonatal piglets after H-I to determine if it protects highly vulnerable striatal neurons. Administration of HET0016 after H-I improved early neurological recovery and protected neurons in putamen after 4 days of recovery. HET0016 had no significant effect on cerebral blood flow. cytochrome P450 4A immunoreactivity was detected in putamen neurons, and direct infusion of 20-HETE in the putamen increased phosphorylation of Na(+), K(+) -ATPase and NMDA receptor NR1 subunit selectively at protein kinase C-sensitive sites but not at protein kinase A-sensitive sites. HET0016 selectively inhibited the H-I induced phosphorylation at these same sites at 3 h of recovery and improved Na(+), K(+) -ATPase activity. At 3 h, HET0016 also suppressed H-I induced extracellular signal-regulated kinase 1/2 activation and protein markers of nitrosative and oxidative stress. Thus, 20-HETE can exert direct effects on key proteins involved in neuronal excitotoxicity in vivo and contributes to neurodegeneration after global cerebral ischemia in immature brain.

Relative contribution of cyclooxygenases, epoxyeicosatrienoic acids, and pH to the cerebral blood flow response to vibrissal stimulation.

The increase in cerebral blood flow (CBF) during neuronal activation can be only partially attenuated by individual inhibitors of epoxyeicosatrienoic acids (EETs), cyclooxgenase-2, group I metabotropic glutamate receptors (mGluR), neuronal nitric oxide synthase (nNOS), N-methyl-D-aspartate receptors, or adenosine receptors. Some studies that used a high concentration (500 µM) of the cyclooxygenase-1 inhibitor SC-560 have implicated cyclooxygenase-1 in gliovascular coupling in certain model systems in the mouse. Here, we found that increasing the concentration of SC-560 from 25 µM to 500 µM over whisker barrel cortex in anesthetized rats attenuated the CBF response to whisker stimulation. However, exogenous prostaglandin E(2) restored the response in the presence of 500 µM SC-560 but not in the presence of a cyclooxygenase-2 inhibitor, thereby suggesting a limited permissive role for cyclooxygenase-1. Furthermore, inhibition of the CBF response to whisker stimulation by an EET antagonist persisted in the presence of SC-560 or a cyclooxygenase-2 inhibitor, thereby indicating that the EET-dependent component of vasodilation did not require cyclooxygenase-1 or -2 activity. With combined inhibition of cyclooxygenase-1 and -2, mGluR, nNOS, EETs, N-methyl-D-aspartate receptors, and adenosine 2B receptors, the CBF response was reduced by 60%. We postulated that the inability to completely block the CBF response was due to tissue acidosis resulting from impaired clearance of metabolically produced CO2. We tested this idea by increasing the concentration of superfused bicarbonate from 25 to 60 mM and found a markedly reduced CBF response to hypercapnia. However, increasing bicarbonate had no effect on the initial or steady-state CBF response to whisker stimulation with or without combined inhibition. We conclude that the residual response after inhibition of several known vasodilatory mechanisms is not due to acidosis arising from impaired CO2 clearance when the CBF response is reduced. An unidentified mechanism apparently is responsible for the rapid, residual cortical vasodilation during vibrissal stimulation.

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.

Astrocytes and the regulation of cerebral blood flow.

Moment-to-moment changes in local neuronal activity lead to dynamic changes in cerebral blood flow. Emerging evidence implicates astrocytes as one of the key players in coordinating this neurovascular coupling. Astrocytes are poised to sense glutamatergic synaptic activity over a large spatial domain via activation of metabotropic glutamate receptors and subsequent calcium signaling and via energy-dependent glutamate transport. Astrocyte foot processes can signal vascular smooth muscle by arachidonic acid pathways involving astrocytic cytochrome P450 epoxygenase, astrocytic cyclooxygenase-1 and smooth muscle cytochrome P450 omega-hydroxylase activities, and by astrocytic and smooth muscle potassium channels. Non-glutamatergic transmitters released from neurons, such as nitric oxide, cyclooxygenase-2 metabolites and vasoactive intestinal peptide, might modulate neurovascular signaling at the level of the astrocyte or smooth muscle. Thus, astrocytes have a pivotal role in dynamic signaling within the neurovascular unit. Important questions remain on how this signaling is integrated with other pathways in health and disease.

The intermediate-conductance calcium-activated potassium channel KCa3.1 contributes to atherogenesis in mice and humans.

Atherosclerosis remains a major cause of death in the developed world despite the success of therapies that lower cholesterol and BP. The intermediate-conductance calcium-activated potassium channel KCa3.1 is expressed in multiple cell types implicated in atherogenesis, and pharmacological blockade of this channel inhibits VSMC and lymphocyte activation in rats and mice. We found that coronary vessels from patients with coronary artery disease expressed elevated levels of KCa3.1. In Apoe(-/-) mice, a genetic model of atherosclerosis, KCa3.1 expression was elevated in the VSMCs, macrophages, and T lymphocytes that infiltrated atherosclerotic lesions. Selective pharmacological blockade and gene silencing of KCa3.1 suppressed proliferation, migration, and oxidative stress of human VSMCs. Furthermore, VSMC proliferation and macrophage activation were reduced in KCa3.1(-/-) mice. In vivo therapy with 2 KCa3.1 blockers, TRAM-34 and clotrimazole, significantly reduced the development of atherosclerosis in aortas of Apoe(-/-) mice by suppressing VSMC proliferation and migration into plaques, decreasing infiltration of plaques by macrophages and T lymphocytes, and reducing oxidative stress. Therapeutic concentrations of TRAM-34 in mice caused no discernible toxicity after repeated dosing and did not compromise the immune response to influenza virus. These data suggest that KCa3.1 blockers represent a promising therapeutic strategy for atherosclerosis.

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.

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.

Cytochrome P450 eicosanoids and cerebral vascular function.

The eicosanoids 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs), which are generated from the metabolism of arachidonic acid by cytochrome P450 (CYP) enzymes, possess a wide array of biological actions, including the regulation of blood flow to organs. 20-HETE and EETs are generated in various cell types in the brain and cerebral blood vessels, and contribute significantly to cerebral blood flow autoregulation and the coupling of regional brain blood flow to neuronal activity (neurovascular coupling). Investigations are beginning to unravel the molecular and cellular mechanisms by which these CYP eicosanoids regulate cerebral vascular function and the changes that occur in pathological states. Intriguingly, 20-HETE and the soluble epoxide hydrolase (sEH) enzyme that regulates EET levels have been explored as molecular therapeutic targets for cerebral vascular diseases. Inhibition of 20-HETE, or increasing EET levels by inhibiting the sEH enzyme, decreases cerebral damage following stroke. The improved outcome following cerebral ischaemia is a consequence of improving cerebral vascular structure or function and protecting neurons from cell death. Thus, the CYP eicosanoids are key regulators of cerebral vascular function and novel therapeutic targets for cardiovascular diseases and neurological disorders.

Electromagnetic energy (670 nm) stimulates vasodilation through activation of the large conductance potassium channel (BKCa).

INTRODUCTION: Peripheral artery disease (PAD) is a highly morbid condition in which impaired blood flow to the limbs leads to pain and tissue loss. Previously we identified 670 nm electromagnetic energy (R/NIR) to increase nitric oxide levels in cells and tissue. NO elicits relaxation of smooth muscle (SMC) by stimulating potassium efflux and membrane hyperpolarization. The actions of energy on ion channel activity have yet to be explored. Here we hypothesized R/NIR stimulates vasodilation through activation of potassium channels in SMC. METHODS: Femoral arteries or facial arteries from C57Bl/6 and Slo1-/- mice were isolated, pressurized to 60 mmHg, pre-constricted with U46619, and irradiated twice with energy R/NIR (10 mW/cm2 for 5 min) with a 10 min dark period between irradiations. Single-channel K+ currents were recorded at room temperature from cell-attached and excised inside-out membrane patches of freshly isolated mouse femoral arterial muscle cells using the patch-clamp technique. RESULTS: R/NIR stimulated vasodilation requires functional activation of the large conductance potassium channels. There is a voltage dependent outward current in SMC with light stimulation, which is due to increases in the open state probability of channel opening. R/NIR modulation of channel opening is eliminated pharmacologically (paxilline) and genetically (BKca α subunit knockout). There is no direct action of light to modulate channel activity as excised patches did not increase the open state probability of channel opening. CONCLUSION: R/NIR vasodilation requires indirect activation of the BKca channel.

Increased blood pressure in mice lacking cytochrome P450 2J5.

The cytochrome P450 (CYP) enzymes participate in a wide range of biochemical functions, including metabolism of arachidonic acid and steroid hormones. Mouse CYP2J5 is abundant in the kidney where its products, the cis-epoxyeicosatrienoic acids (EETs), modulate sodium transport and vascular tone. To define the physiological role of CYP2J5 in the kidney, knockout mice were generated using a conventional gene targeting approach. Cyp2j5 (-/-) mice develop normally and exhibit no overt renal pathology. While renal EET biosynthesis was apparently unaffected by the absence of CYP2J5, deficiency of this CYP in female mice was associated with increased blood pressure, enhanced proximal tubular transport rates, and exaggerated afferent arteriolar responses to angiotensin II and endothelin I. Interestingly, plasma 17beta-estradiol levels were reduced in female Cyp2j5 (-/-) mice and estrogen replacement restored blood pressure and vascular responsiveness to normal levels. There was no evidence of enhanced estrogen metabolism, or altered expression or activities of steroidogenic enzymes in female Cyp2j5 (-/-) mice, but their plasma levels of luteinizing hormone and follicle stimulating hormone were inappropriately low. Together, our findings illustrate a sex-specific role for CYP2J5 in regulation of blood pressure, proximal tubular transport, and afferent arteriolar responsiveness via an estrogen-dependent mechanism.

Interaction of tumor cells and astrocytes promotes breast cancer brain metastases through TGF-ß2/ANGPTL4 axes.

Metastatic outcomes depend on the interactions of metastatic cells with a specific organ microenvironment. Our previous studies have shown that triple-negative breast cancer (TNBC) MDA-MB-231 cells passaged in astrocyte-conditioned medium (ACM) show proclivity to form brain metastases, but the underlying mechanism is unknown. The combination of microarray analysis, qPCR, and ELISA assay were carried out to demonstrate the ACM-induced expression of angiopoietin-like 4 (ANGPTL4) in TNBC cells. A stable ANGPTL4-knockdown MDA-MB-231 cell line was generated by ANGPTL4 short-hairpin RNA (shRNA) and inoculated into mice via left ventricular injection to evaluate the role of ANGPTL4 in brain metastasis formation. The approaches of siRNA, neutralizing antibodies, inhibitors, and immunoprecipitation were used to demonstrate the involved signaling molecules. We first found that ACM-conditioned TNBC cells upregulated the expression of ANGPTL4, a secreted glycoprotein whose effect on tumor progression is known to be tumor microenvironment- and tumor-type dependent. Knockdown of ANGPTL4 in TNBC MDA-MB-231 cells with shRNA decreased ACM-induced tumor cell metastatic growth in the brain and attributed to survival in a mouse model. Furthermore, we identified that astrocytes produced transforming growth factor-beta 2 (TGF-ß2), which in part is responsible for upregulation of ANGPTL4 expression in TNBC through induction of SMAD signaling. Moreover, we identified that tumor cells communicate with astrocytes, where tumor cell-derived interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) increased the expression of TGF-ß2 in astrocytes. Collectively, these findings indicate that the invading TNBC cells interact with astrocytes in the brain microenvironment that facilitates brain metastases of TNBC cells through a TGF-ß2/ANGPTL4 axis. This provides groundwork to target ANGPTL4 as a treatment for breast cancer brain metastases.

Interaction of mechanisms involving epoxyeicosatrienoic acids, adenosine receptors, and metabotropic glutamate receptors in neurovascular coupling in rat whisker barrel cortex.

Adenosine, astrocyte metabotropic glutamate receptors (mGluRs), and epoxyeicosatrienoic acids (EETs) have been implicated in neurovascular coupling. Although A(2A) and A(2B) receptors mediate cerebral vasodilation to adenosine, the role of each receptor in the cerebral blood flow (CBF) response to neural activation remains to be fully elucidated. In addition, adenosine can amplify astrocyte calcium, which may increase arachidonic acid metabolites such as EETs. The interaction of these pathways was investigated by determining if combined treatment with antagonists exerted an additive inhibitory effect on the CBF response. During whisker stimulation of anesthetized rats, the increase in cortical CBF was reduced by approximately half after individual administration of A(2B), mGluR and EET antagonists and EET synthesis inhibitors. Combining treatment of either a mGluR antagonist, an EET antagonist, or an EET synthesis inhibitor with an A(2B) receptor antagonist did not produce an additional decrement in the CBF response. Likewise, the CBF response also remained reduced by approximately 50% when an EET antagonist was combined with an mGluR antagonist or an mGluR antagonist plus an A(2B) receptor antagonist. In contrast, A(2A) and A(3) receptor antagonists had no effect on the CBF response to whisker stimulation. We conclude that (1) adenosine A(2B) receptors, rather than A(2A) or A(3) receptors, play a significant role in coupling cortical CBF to neuronal activity, and (2) the adenosine A(2B) receptor, mGluR, and EETs signaling pathways are not functionally additive, consistent with the possibility of astrocytic mGluR and adenosine A(2B) receptor linkage to the synthesis and release of vasodilatory EETs.

Antinociception produced by 14,15-epoxyeicosatrienoic acid is mediated by the activation of beta-endorphin and met-enkephalin in the rat ventrolateral periaqueductal gray.

Cytochrome P450 genes catalyze formation of epoxyeicosatrienoic acids (EETs) from arachidonic acid. The effects of 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET microinjected into the ventrolateral periaqueductal gray (vlPAG) on the thermally produced tail-flick response were studied in male Sprague-Dawley rats. 14,15-EET microinjected into vlPAG (3-156 pmol) dose-dependently inhibited the tail-flick response (ED50 = 32.5 pmol). In contrast, 5,6-EET, 8,9-EET, and 11,12-EET at a dose of 156 pmol were not active when injected into the vlPAG. 14,15-EET failed to displace the radiobinding of [3H][D-Ala2,NHPe4, Gly-ol5]enkephalin (mu-opioid receptor ligand) or [3H]naltrindole (delta-opioid receptor ligand) in crude membrane fractions of rat brain. Tail-flick inhibition produced by 14,15-EET from vlPAG was blocked by intra-vlPAG pretreatment with antiserum against beta-endorphin or Met-enkephalin or the mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) or the delta-opioid receptor antagonist naltrindole but not with dynorphin A[1-17] antiserum or the kappa-opioid receptor antagonist nor-binaltorphimine. In addition, tail-flick inhibition produced by 14,15-EET treatment was blocked by intrathecal pretreatment with Met-enkephalin antiserum, naltrindole, or CTOP but not with beta-endorphin antiserum. It is concluded that 1) 14,15-EET itself does not have any affinity for mu- or delta-opioid receptors and 2) 14,15-EET activates beta-endorphin and Met-enkephalin, which subsequently act on mu- and delta-opioid receptors to produce antinociception.