The protective effect of astrocyte-derived 14,15-epoxyeicosatrienoic acid on hydrogen peroxide-induced cell injury in astrocyte-dopaminergic neuronal cell line co-culture.

Astrocytes perform several functions that are essential for normal neuronal activity. They play a critical role in neuronal survival during ischemia and other degenerative injuries and also modulate neuronal recovery by influencing neurite outgrowth. In this study, we investigated the neuroprotective effects of astrocyte-derived 14,15-epoxyeicosatrienoic acid (14,15-EET), metabolite of arachidonic acid by cytochrome P450 epoxygenases (CYP), against oxidative stress induced by hydrogen peroxide (H(2)O(2)). We found that dopaminergic neuronal cells (N27 cell line) stimulated with two different doses of H(2)O(2) (0.1 and 1mM) for 1h showed decreased cell viability compared to the control group, while astrocytes showed less cell death after stimulation with the same doses of H(2)O(2) for 1h. Dopaminergic neuronal cells (N27 cell line) pretreated with different doses of 14,15-EET (0.1-30 µM, 30 min) before H(2)O(2) stimulation also showed increased cell viability. Furthermore, pre-treatment of the co-cultured cells with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid, an inhibitor of the EET metabolizing enzyme, soluble epoxide hydrolase (sEH), before H(2)O(2) stimulation (1mM, for 1h) increased cell viability. It also increased the endogenous level of 14,15-EET in the media compared to control group. However, pretreatment with the CYP epoxygenase inhibitor miconazole (1-20 µM, 1h) before H(2)O(2) (1mM, 1h) stimulation showed decreased cell viability. Our data suggest that 14,15-EET which is released from astrocytes, enhances cell viability against oxidant-induced injury. Further understanding of the mechanism of 14,15-EET-mediated protection in dopaminergic neurons is imperative, as it could lead to novel therapeutic approaches for treating CNS neuropathologies, such as Parkinson's disease.

Differential effect of amyloid ß on the cytochrome P450 epoxygenase activity in rat brain.

One of the prominent features of Alzheimer's disease is the excessive accumulation of the protein amyloid beta (Aß) in certain areas of the brain leading to neurodegeneration. Aß is cytotoxic and disrupts several cytoprotective pathways. Recent literature has demonstrated that certain cytochrome P450 (CYP) products are neuroprotective, including epoxide metabolites of arachidonic acid (AA), epoxyeicosatrienoic acids (EETs). The action of Aß with respect to regionally produced EETs in the brain has yet to be defined. Epoxygenases metabolize AA into four regioisomers of EETs (14,15-, 11,12-, 8,9- and 5,6-EET). EETs are rapidly degraded into dihydroxyeicosatrienoic acids (DiHETEs) by soluble epoxide hydrolase (sEH). To determine the effect of Aß on the epoxygenase activity in different regions of the brain, microsomes were prepared from the cerebrum and cerebellum of adult Sprague-Dawley rats and incubated with 1 and 10 µM Aß for 30 min after which epoxygenase activity assay was performed. Mass spectrometry indicated that incubation with Aß reduced 14,15-EET production by 30% as compared to vehicle in the cerebrum, but not in the cerebellum. When we separated the cerebrum into cortex and hippocampus, significant decrease in the production of total EETs and DiHETEs were seen in presence of Aß (81% and 74%) in the cortex. Moreover, 11,12-EET production was decreased to ∼70% of vehicle in both cortex and hippocampus. Epoxygenase activity in the cultured astrocytes and neurons also showed reduction in total EET and DiHETE production (to 80% and ∼70% of vehicle respectively) in presence of Aß. Altogether, our data suggest that Aß reduces epoxygenase activity differentially in a region-specific and cell-specific manner. The reduction of cytoprotective EETs by Aß in the cerebrum may make it more prone to degeneration than the cerebellum. Further understanding of these interactions will improve our ability to protect against the pathology of Alzheimer's disease.

Contribution of epoxyeicosatrienoic acids to the hypoxia-induced activation of Ca2+ -activated K+ channel current in cultured rat hippocampal astrocytes.

Brief hypoxia differentially regulates the activities of Ca(2+)-activated K(+) channels (K(Ca)) in a variety of cell types. We investigated the effects of hypoxia (<2% O(2)) on K(Ca) channel currents and on the activities of cytochrome P450 2C11 epoxygenase (CYP epoxygenase) in cultured rat hippocampal astrocytes. Exposure of astrocytes to hypoxia enhanced macroscopic outward K(Ca) current, increased the open state probability (NPo) of 71 pS and 161 pS single-channel K(Ca) currents in cell-attached patches, but failed to increase the NPo of both the 71 pS and 161 pS K(Ca) channel currents recorded from excised inside-out patches. The hypoxia-induced enhancement of macroscopic K(Ca) current was attenuated by pretreatment with tetraethylammonium (TEA, 1 mM) or during recording using low-Ca(2+) external bath solution. Exposure of astrocytes to hypoxia was associated with generation of superoxide as detected by staining of cells with the intracellular superoxide detection probe hydroethidine (HE), attenuation of the hypoxia-induced activation of unitary K(Ca) channel currents by superoxide dismutation with tempol, and as quantitated by high-pressure liquid chromatography/fluorescence assay using HE as a probe. In cultured astrocytes in which endogenous CYP epoxygenase activity has been inhibited with either miconazole or N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MSPPOH) hypoxia failed to increase the NPo of both the 71 pS and 161 pS K(Ca) currents and generation of superoxide. Hypoxia increased the level of P450 epoxygenase protein and production of epoxyeicosatrienoic acids (EETs) from cultured astrocytes, as determined by immunohistochemical staining and LC/MS analysis, respectively. Exogenous 11,12-EET increased the NPo of both the 71 pS and 161 pS K(Ca) single-channel currents only in cell-attached but not in excised inside-out patches of cultured astrocytes. These findings indicate that hypoxia enhances the activities of two types of unitary K(Ca) currents in astrocytes by a mechanism that appears to involve CYP epoxygenase-dependent generation of superoxide and increased production or release of EETs.

Reversal of delayed vasospasm by TS-011 in the dual hemorrhage dog model of subarachnoid hemorrhage.

PURPOSE: Arachidonic acid is avidly metabolized to a potent vasoconstrictor, 20-hydroxyeicosatetraenoic acid (20-HETE), in the cerebral circulation. 20-HETE has been reported to contribute to the acute fall in cerebral blood flow following subarachnoid hemorrhage (SAH), but its role in the development of delayed vasospasm is unknown. The present study examined whether delayed vasospasm is associated with elevations in 20-HETE in CSF in the dual hemorrhage model of SAH in dogs and if blockade of the synthesis of 20-HETE with N-(3-chloro-4-morpholin-4-yl)phenyl-N'-hydroxyimido formamide (TS-011) can reverse delayed vasospasm in this model. MATERIALS AND METHODS: Delayed vasospasm was induced in 22 adult beagle dogs by dual injection of blood (0.5 mL/kg) into the cisterna magna on days 1 and 4. Sequential samples of CSF were collected before intracisternal injections of blood on days 1 and 4 and after the development of delayed vasospasm on day 7. Sequential angiograms were obtained before and after intracisternal injection of blood on days 1 and 4 and before and 1 hour after administration of TS-011 (1 mg/kg IV) on day 7. RESULTS: The dogs consistently developed delayed vasospasm, and the diameter of the basilar artery fell to 68 +/- 3% (n = 15), 3 days after the second intracisternal injection of blood. The levels of 20-HETE in CSF increased from 4 +/- 2 to 39 +/- 16 pg/mL. In 9 dogs with delayed vasospasm, acute blockade of the synthesis of 20-HETE with TS011 (1 mg/kg IV) significantly increased the diameter of the basilar artery by 39%. Chronic administration of TS-011 (1 mg/kg per day) attenuated the development of delayed vasospasm, and the diameter of the basilar artery fell by 17 +/- 1% versus the 33 +/- 3% decrease in diameter seen in control animals 3 days following the second injection of blood into the cisterna magna. CONCLUSIONS: These results indicate that the development of delayed vasospasm in dogs is associated with an increase in 20-HETE levels in CSF, and acute blockade of the synthesis of 20-HETE with TS-011 reverses delayed vasospasm in this model.

Liquid chromatographic-electrospray ionization-mass spectrometric analysis of cytochrome P450 metabolites of arachidonic acid.

Arachidonic acid (AA) can be metabolized by cytochrome P450 (CYP) enzymes to many biologically active compounds including 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), their corresponding dihydroxyeicosatrienoic acids (DHETs), and 20-hydroxyeicosatetraenoic acid (20-HETE). These eicosanoids are potent regulators of vascular tone. We developed a liquid chromatography-electrospray ionization-mass spectrometry method to simultaneously determine 5,6-, 8,9-, 11,12-, and 14,15-EETs; 5,6-, 8,9-, 11,12-, and 14,15-DHETs; and 20-HETE. [2H8]EETs, [2H8]DHETs, and [2H2]20-HETE were used as internal standards. These compounds are readily separated on a C18 reverse-phase column using water:acetonitrile with 0.005% acetic acid as a mobile phase. The internal standards, [2H8]EETs, [2H8]DHETs, and [2H2]20-HETE, eluted slightly faster than the natural eicosanoids. The samples were ionized by electrospray with fragmentor voltage of 120 V and detected in a negative mode. The negative ion detection gave a lower background than the positive ion detection for these compounds. These eicosanoids exhibited high abundance of the ions corresponding to [M - 1]-. The m/z = 319, 337, and 319 ions were used for quantitation of EETs, DHETs, and 20-HETE, respectively. The detection limits using selected ion monitoring of these compounds are about 1 pg per injection. The position of functional groups and water content of mobile phase had a significant effect on the sensitivity of detection. Water content of 40% was found to give maximal sensitivity. The method was used to determine EETs, DHETs, and 20-HETE in bovine coronary artery endothelial cells, dog plasma, rat astrocytes, and rat kidney microsome samples.

Renal and cardiovascular actions of 20-hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acids.

1. Arachidonic acid (AA) is metabolized by cytochrome P450 (CYP)-dependent pathways to epoxyeicosatrienoic acids (EET) and 20-hydroxyeicosatetraenoic acid (20-HETE) in the kidney and the peripheral vasculature. 2. The present short review summarizes the renal and cardiovascular actions of these important mediators. 3. Epoxyeicosatrienoic acids are vasodilators produced by the endothelium that hyperpolarize vascular smooth muscle (VSM) cells by opening Ca2+-activated K+ (KCa) channels. 20-Hydroxyeicosatetraenoic acid is a vasoconstrictor that inhibits the opening of KCa channels in VSM cells. Cytochrome P450 4A inhibitors block the myogenic response of small arterioles to elevations in transmural pressure and autoregulation of renal and cerebral blood flow in vivo. Cytochrome P450 4A blockers also attenuate the vasoconstrictor response to elevations in tissue PO2, suggesting that this system may serve as a vascular oxygen sensor. Nitric oxide and carbon monoxide inhibit the formation of 20-HETE and a fall in 20-HETE levels contributes to the activation of KCa channels in VSM cells and the vasodilator response to these gaseous mediators. 20-Hydroxyeicosatetraenoic acid also mediates the inhibitory actions of peptide hormones on sodium transport in the kidney and the mitogenic effects of growth factors in VSM and mesangial cells. A deficiency in the renal production of 20-HETE is associated with the development of hypertension in Dahl salt-sensitive rats. 4. In summary, the available evidence indicates that CYP metabolites of AA play a central role in the regulation of renal, pulmonary and vascular function and that abnormalities in this system may contribute to the pathogenesis of cardiovascular diseases.

P-450 epoxygenase and NO synthase inhibitors reduce cerebral blood flow response to N-methyl-D-aspartate.

Epoxyeicosatrienoic acids are cerebral vasodilators produced in astrocytes by cytochrome P-450 epoxygenase activity. The P-450 inhibitor miconazole attenuates the increase in cerebral blood flow (CBF) elicited by glutamate. We evaluated whether epoxygenase activity is involved in the CBF response to activation of the N-methyl-D-aspartate (NMDA) receptor subtype by using two structurally distinct inhibitors, miconazole and N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH), a selective epoxygenase substrate inhibitor. Drugs were delivered locally through microdialysis probes in striata of anesthetized rats. Local CBF was measured by hydrogen clearance and compared with CBF in contralateral striatum receiving vehicle. Microdialysis perfusion of NMDA doubled CBF and increased nitric oxide (NO) production estimated by recovery of labeled citrulline in the dialysate during labeled arginine infusion. Perfusion of miconazole or MS-PPOH blocked the increase in CBF without decreasing citrulline recovery. Perfusion of N(omega)-nitro-L-arginine decreased baseline CBF and inhibited the CBF response to NMDA. Perfusion of MS-PPOH did not inhibit the CBF response to sodium nitroprusside. We conclude that both the P-450 epoxygenase and NO synthase pathways are involved in the local CBF response to NMDA receptor activation, and that the signaling pathway may be more complex than simply NO diffusion from neurons to vascular smooth muscle.

Production of 20-HETE and its role in autoregulation of cerebral blood flow.

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.

Role of cGMP versus 20-HETE in the vasodilator response to nitric oxide in rat cerebral arteries.

This study examined the response to nitric oxide (NO) in rat middle cerebral arteries (MCA). NO donors increased the activity of a 205-pS K(+) channel recorded from vascular smooth muscle (VSM) cells isolated from MCA 10-fold. Blockade of guanylyl cyclase activity with 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 10(-5) M) did not alter the effect of NO on this channel. In contrast, adding 20-hydroxyeicosatetraenoic acid (20-HETE) to the bath (10(-7) M) abolished the response to NO. NO donors also increased the diameter of serotonin-preconstricted MCA to 85% of control. Blockade of K(+) channels with iberiotoxin or a high-K(+) medium reduced this response by 50%. ODQ (10(-5) M) reduced this response by 47 +/- 3%, whereas preventing the fall of 20-HETE levels reduced the response by 59 +/- 2% (n = 5). Blockade of both pathways eliminated the response to NO donors. These results indicate that activation of K(+) channels contributes 50% to vasodilator response to NO in rat MCA. This is mediated by a fall in 20-HETE levels rather than a rise in cGMP levels or a direct effect of NO.

Molecular mechanisms controlling nutritive blood flow: role of cytochrome P450 enzymes.

This short review summarizes the potential role of cytochrome P450 (P450) in regulating blood flow in the brain tissue and in the skeletal muscle. We provide data showing that pressure-induced myogenic activity in the brain is largely responsible for autoregulation of CBF. This myogenic response to pressure is maintained, in part, by 20-HETE formation in arterial muscle cells through a P450 omega-hydroxylase coded for by a P450 4A cDNA. Autoregulation of CBF is a hallmark of the cerebral circulation and provides adequate nutritive blood flow despite large fluctuations in arterial pressure. Given the importance of oxidative metabolism in the brain, support of neuronal activity is mediated by functional hyperaemia to active neurones providing adequate delivery of oxidative substrate. We provide data demonstrating that this functional hyperaemia in the brain is regulated by astrocytes which sense neural activity and release dilator metabolites which shunt blood flow to active neurones. One of the metabolites released by astrocytes in this regard are epoxygenated products of arachidonic acid (AA) formed by P450 enzymes. These AA metabolites of P450 enzymes are epoxyeicosatrienoic acid (EETs). One of these P450 enzymes is coded by a 2C11 cDNA present in astrocytes. Furthermore, astrocytes are capable of inducing capillary angiogenesis which appears to be mediated, in part, by P450-derived EETs.

Cerebral microvascular endothelial cell tube formation: role of astrocytic epoxyeicosatrienoic acid release.

Cerebral microvascular endothelial cells (CMVEC) form tubes when cocultured with astrocytes (AS). Therefore, it appears that AS may be important in mediating angiogenesis in the brain. We hypothesized that AS modulate CMVEC tube formation by releasing a soluble factor. Thymidine incorporation in cultured CMVEC increased 305% when incubated with 50% conditioned AS medium for 24 h [control: 52,755 +/- 4,838 counts per minute (cpm) per well, conditioned 161,082 +/- 12,099 cpm/well, n = 8]. Because our laboratory has previously shown that AS can produce epoxyeicosatrienoic acids (EETs), which are known mitogens, we investigated whether release of EETs by AS is responsible for tube formation in the CMVEC-AS coculture. AS were seeded on Lab-Tek slides, CMVEC were seeded on the AS the next day, and cultures were allowed to progress for another 5 days with and without cytochrome P-450 epoxygenase blockade by 17-octadecynoic acid (17-ODYA). Tube formation in cocultures receiving 17-ODYA was significantly inhibited compared with control (93.8%). These data suggest that tube formation requires the release of EETs by AS.

Epoxyeicosatrienoic acids constrict isolated pressurized rabbit pulmonary arteries.

Little information is available regarding the vasoactive effects of epoxyeicosatrienoic acids (EETs) in the lung. We demonstrate that 5, 6-, 8,9-, 11,12-, and 14,15-EETs contract pressurized rabbit pulmonary arteries in a concentration-dependent manner. Constriction to 5,6-EET methyl ester or 14,15-EET is blocked by indomethacin or ibuprofen (10(-5) M), SQ-29548, endothelial denuding, or submaximal preconstriction with the thromboxane mimetic U-46619. Constriction of pulmonary artery rings to phenylephrine is blunted by treatment with the epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide. Pulmonary arteries and peripheral lung microsomes metabolize arachidonate to products that comigrate on reverse-phrase HPLC with authentic regioisomers of 5,6-, 8,9-, 11,12-, and 14,15-EETs, but no cyclooxygenase products of EETs could be demonstrated. Proteins of the CYP2B, CYP2E, CYP2J, CYP1A, and CYP2C subfamilies are present in pulmonary artery and peripheral lung microsomes. Constriction of isolated rabbit pulmonary arteries to EETs is nonregioselective and depends on intact endothelium and cyclooxygenase, consistent with the formation of a pressor prostanoid compound. These data raise the possibility that EETs may contribute to regulation of pulmonary vascular tone.

Contribution of 20-HETE to vasodilator actions of nitric oxide in the cerebral microcirculation.

BACKGROUND AND PURPOSE: The present study examined the contributions of a rise in cGMP versus a fall in 20-HETE levels to the vasodilator response to nitric oxide (NO) in the cerebral circulation of the rat. METHODS: Intact rat middle cerebral and basilar arteries were bathed in physiological saline solution containing indomethacin (5 micromol/L) and baicalein (0.5 micromol/L) and pressurized at 90 mm Hg. Relaxations to sodium nitroprusside (SNP) were studied before and after addition of [1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one] (ODQ, a guanylyl cyclase blocker), 8R,9S, 11S-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cycloocta-(c, d, e)-trinden-1-one (KT5823, a protein kinase G blocker), and 20-hydroxyeicosatetraenoic acid (20-HETE). Cerebral blood flow was measured by using a laser Doppler flow probe over a thin cranial window in anesthetized rats, and the effects of intracerebroventricular infusion of 1-hexamine, 6-(2-hydroxy-1-methyl-2-nitrosohydrazino)N-methyl (MAHMA nonoate) and dibromododecenyl methylsulfimide (DDMS) were determined. RESULTS: SNP-induced dilation of serotonin-preconstricted (0.2 micromol/L) middle cerebral arteries (10(-7) to 10(-3) mol/L) was attenuated in arteries treated with ODQ (10 micromol/L) or KT5823 (1 micromol/L) by 52% and 27%, respectively. Preventing the NO-induced fall in intracellular 20-HETE, by adding 20-HETE (100 nmol/L) to the bath, reduced the dilation to SNP by 62%. Simultaneous administration of ODQ and 20-HETE markedly attenuated the SNP-induced dilation by 90%. In basilar arteries, ODQ (10 micromol/L) alone completely blocked the response to SNP. Infusion of MAHMA nonoate (10 nmol/min ICV) in anesthetized rats increased cerebral blood flow by 52% before and 8% after blockade of the endogenous production of 20-HETE with DDMS (50 pmol/min). CONCLUSIONS: These results suggest that NO dilates cerebral arteries through both cGMP-dependent and cGMP-independent pathways and that inhibition of 20-HETE formation contributes to the cerebral vasodilator response to NO both in vitro and in vivo.

Cytochrome P450 2C is an EDHF synthase in coronary arteries.

In most arterial beds a significant endothelium-dependent dilation to various stimuli persists even after inhibition of nitric oxide synthase and cyclo-oxygenase. This dilator response is preceded by an endothelium-dependent hyperpolarization of vascular smooth muscle cells, which is sensitive to a combination of the calcium-dependent potassium-channel inhibitors charybdotoxin and apamin, and is assumed to be mediated by an unidentified endothelium-derived hyperpolarizing factor (EDHF). Here we show that the induction of cytochrome P450 (CYP) 2C8/34 in native porcine coronary artery endothelial cells by beta-naphthoflavone enhances the formation of 11,12-epoxyeicosatrienoic acid, as well as EDHF-mediated hyperpolarization and relaxation. Transfection of coronary arteries with CYP 2C8/34 antisense oligonucleotides results in decreased levels of CYP 2C and attenuates EDHF-mediated vascular responses. Thus, a CYP-epoxygenase product is an essential component of EDHF-mediated relaxation in the porcine coronary artery, and CYP 2C8/34 fulfils the criteria for the coronary EDHF synthase.

Cannabinoid CB1 receptor of cat cerebral arterial muscle functions to inhibit L-type Ca2+ channel current.

The CB1 subtype of the cannabinoid receptor is present on neurons in the brain and mediates the perceptual effects of Delta9-tetrahydrocannabinol and other cannabinoids. We found that cat cerebral arterial smooth muscle cells (VSMC) contain the protein for the CB1 receptor and express a cDNA that has >98% amino acid homology to the CB1 cDNA expressed in rat and human neurons. Activation of the CB1 cannabinoid receptor has been shown to decrease the opening of N-type voltage-gated Ca2+ channels in neurons through a pertussis toxin-sensitive GTP-binding protein. In the present study we tested the hypothesis that activation of the cannabinoid CB1 receptor in cerebral VSMC inhibits voltage-gated Ca2+ channels and results in cerebral vasodilation. The predominant Ca2+ current identified in cat cerebral VSMC is a voltage-gated, dihydropyridine-sensitive, L-type Ca2+ current. The cannabimimetic drug WIN-55,212-2 (10-100 nM) induced concentration-dependent inhibition of peak L-type Ca2+ current, which reached a maximum of 82 +/- 4% at 100 nM (n = 14). This effect was mimicked by the putative endogenous CB1-receptor agonist anandamide, which produced a concentration-related reduction of peak L-type Ca2+ current with a maximum inhibition (at 300 nM) of 39 +/- 4% (n = 12). The inhibitory effects of both ligands on peak L-type Ca2+ currents were abolished by pertussis toxin pretreatment and application of the CB1-receptor antagonist SR-141716A (100 nM, n = 5). Both WIN-55,212-2 and anandamide produced concentration-dependent relaxation of preconstricted cerebral arterial segments that was abolished by SR-141716A. These results indicate that the CB1 receptor is expressed in cat cerebral VSMC and that the cerebral vasculature is one of the targets for endogenous cannabinoids. These findings suggest that the CB1 receptor and its endogenous ligand may play a fundamental role in the regulation of cerebral arterial tone and reactivity by modulating the influx of Ca2+ through L-type Ca2+ channels.

Cerebrovascular alterations in protein kinase C-mediated constriction in stroke-prone rats.

BACKGROUND AND PURPOSE: Cerebrovascular pressure-dependent constriction may involve the smooth muscle production of diacylglycerol, which could facilitate constriction by activating protein kinase C (PKC). A dysfunctional PKC system could promote the loss of pressure-dependent constriction. We attempted to determine whether the alterations in pressure-dependent constriction in the middle cerebral arteries (MCAs) observed in relation to stroke development in Wistar-Kyoto stroke-prone spontaneously hypertensive rats (SHRsp) were associated with defects in the ability of the arteries to constrict in response to PKC activation. METHODS: MCAs were sampled from SHRsp before and after stroke development and in stroke-resistant Wistar-Kyoto spontaneously hypertensive rats. A pressure myograph was used to test the ability of the arteries to constrict in response to a 100 mm Hg pressure step and subsequently to contract in response to phorbol 12,13-dibutyrate in the presence of nifedipine (3 micromol/L). RESULTS: Pressure-dependent constriction and constriction in response to phorbol dibutyrate in the MCAs were inhibited by PKC inhibitors (staurosporine [40 nmol/L], chelerythrine [12 micromol/L], bisindolylmaleimide [5 micromol/L]), declined with age before stroke development in SHRsp, and were absent after stroke. There was a significant relationship between pressure- and phorbol dibutyrate-induced constriction (r=0.815, P<0. 05). CONCLUSIONS: Phorbol esters interact with the same activation site as diacylglycerol to stimulate PKC. An inability to constrict in response to phorbol dibutyrate may reflect unresponsiveness to diacylglycerol and may contribute to the loss of pressure-dependent constriction associated with stroke in the MCAs of SHRsp. The loss of this autoregulatory function before stroke could increase the risk of cerebral hemorrhage.