Endothelial cells release several compounds, including prostacyclin, NO, and endothelium-derived hyperpolarizing factor (EDHF), that mediate the vascular effects of vasoactive hormones. The identity of EDHF remains unknown. Since arachidonic acid causes endothelium-dependent relaxations of coronary arteries through its metabolism to epoxyeicosatrienoic acids (EETs) by cytochrome P450, we wondered if the EETs represent EDHFs. Precontracted bovine coronary arteries relaxed in an endothelium-dependent manner to methacholine. The cytochrome P450 inhibitors, SKF 525A and miconazole, significantly attenuated these relaxations. They were also inhibited by tetraethylammonium (TEA),an inhibitor of Ca2+-activated K+ channels, and by high [K+]0 (20 mmol/L). Methacholine also caused hyperpolarization of coronary smooth muscle (-27 +/- 3.9 versus -40 +/- 5.1 mV), which was completely blocked by SKF 525A and miconazole. In vessels prelabeled with [3H] arachidonic acid, methacholine stimulated the release of 6-ketoprostaglandin F1alpha, 12-HETE, and the EETs. Arachidonic acid relaxed precontracted coronary arteries, which were also blocked by TEA, charybdotoxin, another Ca2+-activated K+ channel inhibitor, and high [K+]0. 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET relaxed precontracted coronary vessels (EC50, 1 X 10(-6) mol/L). The four regioisomers were equally active. TEA, charybdotoxin, and high [K+]0 attenuated the EET relaxations. 11,12-EET hyperpolarized coronary smooth muscle cells from -37 +/- 0.2 to -59 +/- 0.3 mV. In the cell-attached mode of patch clamp, both 14,15-EET and 11,12-EET increased the open-state probability of a Ca2+-activated K+ channel in coronary smooth muscle cells. This effect was blocked by TEA and charybdotoxin. These data support the hypothesis that the EETs are EDHFs.

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.

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

The metabolism of arachidonic acid (AA) into vasoactive products by cyclooxygenase and lipoxygenase enzymes has been well described, as has their biological relevance. Recently, a number of studies have demonstrated the ability of cytochrome P-450 (P450) enzymes to metabolize AA into biologically important regulators of vascular tone. There are two categories of vasoactive P450 metabolites, namely those catalyzed by epoxygenase enzymes which generate epoxyeicosatrienoic acids (EETs) and those enzymes which generate hydroxyeicosatetraenoic acids (HETEs). Except for 20-HETE, P450 metabolites of AA occur as stereo- and regioisomers which determine, to some extent, their biological activity. 5, 6-, 8, 9-, 11, 12- and 14, 15-EETs are generally potent dilators in a number of vascular beds with a sensitivity which appears to increase as the vasculature decreases in size toward capillaries. HETEs, such as 12R- and 20-HETE, can be potent activators of vascular tissue with 20-HETE contracting cerebral and renal microvessels at concentrations of < 10^-10M. Both EETs and HETEs can be made by vascular and extravascular tissue. Available data suggests that EETs are formed by endothelial and parenchymal tissue while HETEs can be endogenously formed in arterial muscle where they appear to act as second messengers. This review will discuss the molecular biology, stereochemistry, biological activity and importance of P450 metabolites of AA as para- and autocrine controllers of organ blood flow. We will also discuss the large diversity of P450 enzyme isoforms and how such diversity can provide for precise physiological control of vascular tone.

This study was undertaken to examine the effect of increasing transmural pressure on membrane electrical properties of cat middle cerebral arterial muscle. Middle cerebral arteries were removed from the cat brain, cannulated, and prepared so that transmural pressure within a segment could be manipulated. Intracellular membrane potential was recorded with glass microelectrodes at various transmural pressures. There was a positive slope relating changes in intracellular membrane potential as a function of transmural pressure with a correlation coefficient of 0.79. Blockade of nerve excitation with tetrodotoxin and inhibition of alpha-adrenergic receptors with phentolamine not only did not block the pressure-induced depolarization, but increased the slope of the intracellular membrane potential vs. pressure relationship. This slope was increased upon elevation of extracellular calcium concentration from 2.5 to 4.0 mM and was significantly reduced upon reduction of extracellular calcium concentration to 0.5 mM. When arterial preparations were equilibrated at 0 mm Hg prior to pressurization, action potentials were recorded only when pressure was initially elevated, while a sustained depolarization was recorded during the pressure plateau. However, when arteries were equilibrated at a transmural pressure of 100 mm Hg for 90 minutes, spontaneous action potentials were recorded which increased in frequency as a function of pressure until they were inactivated when intracellular membrane potential approached -30 mV at high transmural pressures. Photomicrographs demonstrated that these vessels either maintained or decreased diameter upon pressurization. These findings provide a cellular mechanism for myogenic regulation of cerebral arterial diameter.

The present study examined whether preglomerular arterioles of the rat produce 20-hydroxyeicosatetraenoic acid (20-HETE) and whether 20-HETE is vasoactive on these vessels. Raf preglomerular arterioles produced 20-HETE (4.8 +/- 1.0 pmol.min-1.mg-1, n = 7) and, to a lesser extent, 14-, 15-, 11-, and 12-dihydroxyeicosatetraenoic acid, 6-ketoprostaglandin F/alpha and prostaglandin E2 when incubated with [14C]larachidonic acid. The results of immunoblotting and reverse-transcription polymerase chain reaction experiments indicate that these vessels express mRNA and protein for a P-450 4A2 enzyme. With the use of a rat juxtamedullary nephron microvascular preparation perfused in vitro with a cell-free media, addition of 20-HETE (1 nM-1 microM) to the bath reduced the diameter of proximal and distal portions of the efferent arterioles. At a concentration of 1 microM, the diameter of the proximal and distal portions of the afferent arteriole fell by 14 +/- 1 and 16 +/- 3% after 20-HETE. The response to 20-HETE (1 microM) was not altered by blockade of cyclooxygenase, lipoxygenase, and p-450 pathways. Blockade of the large-conductance Ca(2+)-activated K+ channel with tetraethylammonium (1 mM) reduced the diameter of afferent arterioles by 10% and blocked the vasoconstrictor response to 20-HETE (1 microM). These results indicate that 20-HETE is an endogenous constrictor of preglomerular arterioles and suggest a role for the P-450 4A2 enzyme in the regulation of renal vascular tone.

The CB1 subtype of the cannabinoid receptor is present on neurons in the brain and mediates the perceptual effects of Δ9-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.