Recent studies have indicated that arachidonic acid is primarily metabolized by cytochrome P-450 (CYP) enzymes in the brain, lung, kidney, and peripheral vasculature to 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) and that these compounds play critical roles in the regulation of renal, pulmonary, and cardiac function and vascular tone. EETs are endothelium-derived vasodilators that hyperpolarize vascular smooth muscle (VSM) cells by activating K(+) channels. 20-HETE is a vasoconstrictor produced in VSM cells that reduces the open-state probability of Ca(2+)-activated K(+) channels. Inhibitors of the formation of 20-HETE block the myogenic response of renal, cerebral, and skeletal muscle arterioles in vitro and autoregulation of renal and cerebral blood flow in vivo. They also block tubuloglomerular feedback responses in vivo and the vasoconstrictor response to elevations in tissue PO(2) both in vivo and in vitro. The formation of 20-HETE in VSM is stimulated by angiotensin II and endothelin and is inhibited by nitric oxide (NO) and carbon monoxide (CO). Blockade of the formation of 20-HETE attenuates the vascular responses to angiotensin II, endothelin, norepinephrine, NO, and CO. In the kidney, EETs and 20-HETE are produced in the proximal tubule and the thick ascending loop of Henle. They regulate Na(+) transport in these nephron segments. 20-HETE also contributes to the mitogenic effects of a variety of growth factors in VSM, renal epithelial, and mesangial cells. The production of EETs and 20-HETE is altered in experimental and genetic models of hypertension, diabetes, uremia, toxemia of pregnancy, and hepatorenal syndrome. Given the importance of this pathway in the control of cardiovascular function, it is likely that CYP metabolites of arachidonic acid contribute to the changes in renal function and vascular tone associated with some of these conditions and that drugs that modify the formation and/or actions of EETs and 20-HETE may have therapeutic benefits.

No abstract

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.

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 present study examined the effects of 20-hydroxyeicosatetraenoic acid (20-HETE) and 17-octadecynoic acid (17-ODYA), an inhibitor of the metabolism of arachidonic acid by P-450, on K(+)-channel activity in vascular smooth muscle cells (VSM) isolated from renal arterioles of the rat. Two types of K+ channels were characterized using inside-out excised membrane patches. One channel exhibited a large conductance (250.3 +/- 5 pS), was activated by membrane depolarization and elevations in cytoplasmic Ca2+ concentration, and was blocked by low concentrations (< 1 mM) of tetraethylammonium (TEA). The other K+ channel exhibited an intermediate conductance (46.3 +/- pS), was activated by membrane depolarization but not by changes in intracellular Ca2+ concentration, and was blocked by 4-aminopyridine (5 mM). Addition of 20-HETE to the bath (1-100 nM), reduced the frequency of opening of the large-conductance Ca(2+)-activated K+ channel recorded using cell-attached patches on VSM. It had no effect on the intermediate-conductance K+ channel: 17-ODYA (1 microM) increased the activity of the large-conductance Ca(2+)-activated K+ channel, and this effect was reversed by 20-HETE (10 nM). 20-HETE (1-1000 nM) reduced the diameter of isolated perfused small renal arteries of the rat by approximately 15% TEA (1 mM) blocked the vasoconstrictor response to 20-HETE (100 nM). These studies suggest that 20-HETE is an endogenously formed vasoconstrictor that acts in part by inhibiting the opening of the large-conductance Ca(2+)-activated K+ channel in renal arteriolar VSM.

Microsomal preparations of cat brain incubated with [14C]arachidonic acid produced epoxyeicosatrienoic acids (EETs) that eluted with the same retention times as synthetically prepared 5,6-, 8,9-, and 11,12-EETs. These compounds dilated serotonin-preconstricted, pressurized cat cerebral arteries in a dose-dependent fashion. Epoxide formation was not found in mitochondrial fractions and was dependent on the presence of NADPH. The maximum effects of 8,9-EET and 11,12-EET were greater than those of 5,6-EET. The cellular basis of this vasodilation was further investigated by examining the effects of 8,9-EET and 11,12-EET on K+ channel activity in vascular muscle cells freshly isolated from cat cerebral arteries. Both 8,9-EET and 11,12-EET increased the frequency of opening, mean open time, and open-state probability of a 98-pS K+ channel recorded in the cell-attached mode with 145 mM KCl in the pipette and 4.7 mM KCl in the bath. Blockade of K+ channel activity with tetraethylammonium attenuated the vasodilatory effects of 11,12-EET on serotonin-preconstricted cat cerebral arteries. These results suggest that endogenously formed EETs may participate in local regulation of cerebral blood flow by dilating cerebral arteries through a mechanism that involves activation of K+ channels.

Abstract-Consomic rats (SS.BN13), in which chromosome 13 from normotensive inbred Brown Norway rats from a colony maintained at the Medical College of Wisconsin (BN/Mcw) was introgressed into the background of Dahl salt-sensitive (SS/Mcw) rats, also maintained in a colony at the Medical College of Wisconsin, were bred. The present studies determined the mean arterial pressure (MAP) responses to salt and renal and peripheral vascular responses to norepinephrine and angiotensin II; 24-hour protein excretion and histological analyses were used to assess renal pathology in rats that received a high salt (4% NaCl) diet for 4 weeks. MAP of rats measured daily during the fourth week averaged 170Ϯ3. Key Words: hypertension, sodium dependent Ⅲ rats Ⅲ sodium Ⅲ chromosome 13 Ⅲ blood pressure Ⅲ consomic D ahl salt-sensitive (SS) rats exhibit many of the abnormalities that occur with hypertension in African Americans, 1,2 including blood pressure salt sensitivity, 3,4 insulin resistance, 5 and hyperlipidemia. 6 They have a low renin form of hypertension 3 that is refractory to treatment with converting enzyme inhibitors 7,8 and is effectively treated with diuretics. 7,9 Moreover, these rats rapidly develop severe progressive hypertensive glomerulosclerosis that leads to end-stage renal disease, as is commonly also seen in African Americans with hypertension. 9,10 For these reasons, insights and findings of the studies in SS rats may provide valuable clues to the genetic basis of hypertension and related traits in African Americans.The explosion of genomic resources in the rat has led to remarkable advances in identifying the regions of the rat genome that contain blood pressure quantitative trait loci (QTLs), as reviewed by Hamet et al, 11 Zicha and Kunes,12 and Rapp. 13 We have recently completed a linkage analysis based on an intercross of SS and Brown Norway (BN) salt-insensitive rats in which total genome scans using 238 polymorphic markers, evenly distributed throughout the genome, were scored. All F2 rats (113 males and 99 females) were extensively phenotyped for 239 measured or derived traits. This linkage analysis indicated the existence of a broad range of traits related to pathways of functional importance in hypertension that mapped to 19 chromosomes. 14,15 The development of congenic strains has been used by a number of laboratories, including our own, to confirm and narrow QTL regions of interest. 16,17 Despite the usefulness of congenic rat models in the deconstruction of complex traits and the identification of candidate genes, this work has been hampered by the time and expense involved in producing these informative recombinant rats. Even with the use of markerassisted selection to identify the rats best suited for backcrossing in generations, 18 we have found that the process of developing an inbred congenic strain requires nearly 2 years and 5 to 7 generations of backcrosses to achieve rats that are sufficiently isogenic to make meaningful comparisons.To overcome these limitations, we have been developing ...