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Objective: This study sought to identify any specific cytochrome P450 (CYP450) -4A enzyme isoforms expressed in arterioles and/or the surrounding parenchymal tissue of the rat cremaster muscle., Methods: RT-PCR was used to detect the presence of specific CYP450-4A isoforms in isolated muscle fibers and arterioles from the cremaster muscle of Sprague-Dawley rats; CYP450-4A protein expression was determined by Western blotting., Results: CYP450-4A3 mRNA was expressed in isolated muscle fibers and in cremasteric arterioles, while CYP450-4A8 mRNA was expressed only in cremasteric arterioles. CYP450-4A1 and CYP450-4A2 mRNA were not expressed in arterioles and skeletal muscle cells, although all four isoforms were strongly expressed in the liver. CYP450-4A protein was detected in both the isolated muscle fibers and in the isolated arterioles., Conclusions: The present study identifies the specific pattern of cytochrome P450-4A isoform expression in arterioles and parenchymal cells of the skeletal muscle microcirculation, and supports the hypothesis that the cytochrome P-450 enzymes may play a role in the regulation of microvascular function in the skeletal muscle microcirculation.

Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis induced by electrical stimulation in skeletal muscle. Less is known about the role of arachidonic acid metabolites in the control of growth of blood vessels in vivo. The present study examined the role of 20-hydroxyeicosatetraenoic acid (20-HETE) on the angiogenesis induced by electrical stimulation in skeletal muscle. The tibialis anterior and extensor digitorum longus muscles of rats were stimulated for 7 days. Electrical stimulation significantly increased the 20-HETE formation and angiogenesis in the muscles, which was blocked by chronic treatment with N-hydroxy-N'-(4-butyl-2-methylphenol)formamidine (HET0016) or 1-aminobenzotriazole (ABT). Chronic treatment with either HET0016 or ABT did not block the increases in VEGF protein expression in both muscles. To analyze the role of VEGF on 20-HETE formation, additional rats were treated with VEGF-neutralizing antibody (VEGF Ab). VEGF Ab blocked the increases of 20-HETE formation induced by stimulation. These results place 20-HETE in the downstream signaling pathway for angiogenesis and show that both VEGF and 20-HETE are involved in the angiogenesis induced by electrical stimulation in skeletal muscle.

Leukotriene B(4) (LTB(4)) is a lipid mediator that plays an important role in inflammation. Metabolism of LTB(4) by cytochrome P450 (CYP) enzymes belonging to the CYP4F subfamily is considered to be of importance for the regulation of inflammation. This study investigates LTB(4) metabolism by recombinant rat CYP4F5 and CYP4F6 expressed in a yeast system and by microsomes isolated from rat organs expressing CYP4F mRNA. CYP4F6 was found to convert LTB(4) into 19-hydoxy- and 18-hydroxy-LTB(4) with an apparent K(m) of 26 microM, and CYP4F5 was found to convert LTB(4) primarily into 18-hydroxy-LTB(4) with an apparent K(m) of 9.7 microM. The rate of formation of 18-hydroxy-LTB(4) by CYP4F5 was surprisingly high. At a substrate concentration of 30 microM, the rate of formation was about 15 nmol/min/mg microsomal protein, approximately 30 times faster than the reaction catalyzed by CYP4F6. Analysis of LTB(4) metabolism by microsomes isolated from various tissues from the rat suggests that CYP4F5 and CYP4F6 are active in the lung and to some extent in the brain, kidney, and testis. CYP4F5 and CYP4F6, due to their capacities to metabolize LTB(4), may play important roles in modulating inflammatory response in these organs.

The pressure-natriuresis relationship is shifted to higher pressures in genetic and experimental models of hypertension; however, the factors responsible for altering kidney function remain to be determined. In spontaneously hypertensive (SHR) and Lyon hypertensive rats, the resetting of pressure-natriuresis results from increased preglomerular renal vascular tone, whereas sodium reabsorption is elevated in the thick ascending loop of Henle (TALH) of Dahl S rats. Recently, a new route for the renal metabolism of arachidonic acid (AA) has been described, and there is evidence that this pathway contributes to the resetting of renal function in hypertension. In the kidney, cytochrome P450 (CYP) enzymes metabolize AA primarily to 20-HETE and EETs. 20-HETE is a potent constrictor of renal arterioles that has an important role in autoregulation of renal blood flow and tubuloglomerular feedback. 20-HETE and EETS also inhibit sodium reabsorption in the proximal tubule and TALH. In the SHR, the renal production of 20-HETE is elevated and inhibitors of the formation of 20-HETE decrease arterial pressure. Blockade of 20-HETE formation also reduces blood pressure or improves renal function in deoxycorticosterone acetate (DOCA)-salt, angiotensin II--infused, and Lyon hypertensive rats. In contrast, 20-HETE formation is reduced in the TALH of Dahl S rats and this contributes to elevated sodium reabsorption. Induction of 20-HETE synthesis improves pressure-natriuresis and lowers blood pressure in Dahl S rats, whereas inhibitors of the synthesis of 20-HETE promote the development of hypertension in Lewis rats. These findings indicate that the renal production of CYP metabolites of AA is altered in genetic and experimental models of hypertension and that this system contributes to the resetting of pressure-natriuresis and the development of hypertension in some models.

Recent studies indicate that arachidonic acid is primarily metabolized by cytochrome P450 enzymes of the 4A and 2C families in the kidney to 20-hydroxyeicosatetraenoic acid (HETE), epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids. These compounds play central roles in the regulation of renal tubular and vascular function. 20-HETE is produced by renal vascular smooth muscle (VSM) cells and is a potent constrictor that depolarizes VSM cells by blocking the calcium-activated potassium channel. Inhibition of the formation of 20-HETE blocks the myogenic response of isolated renal arterioles in vitro, and autoregulation of renal blood flow and tubuloglomerular feedback responses in vivo. EETs are products formed in the endothelium and are potent dilators that activate the calcium-activated potassium channel in renal VSM. Endothelial-dependent vasodilators stimulate the release of EETs, and these compounds appear to serve as an endothelial-derived hyperpolarizing factor. EETs and 20-HETE are produced in the proximal tubule. There, they regulate sodium/potassium-ATPase activity and serve as second messengers for the natriuretic effects of dopamine, parathyroid hormone and angiotensin II. 20-HETE is also produced in the thick ascending loop of Henle. It regulates sodium-potassium-chloride transport in this nephron segment. The renal production of cytochrome P450 metabolites of arachidonic acid is altered in hypertension, diabetes, toxemia of pregnancy, and hepatorenal syndrome. Given the importance of cytochrome P450 metabolites of arachidonic acid in the control of renal function, it is likely that changes in this system contribute to the abnormalities in renal function that are associated with many of these conditions.

This study describes a fluorescent HPLC assay for measuring 20-hydroxyeicosatetraenoic acid (20-HETE) and other cytochrome P-450 metabolites of arachidonic acid in urine, tissue, and interstitial fluid. An internal standard, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, was added to samples, and the lipids were extracted and labeled with 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate. P-450 metabolites were separated on a C18 reverse-phase HPLC column. Coelution and gas chromatography-mass spectrometry studies confirmed the identity of the 20-HETE peak. The 20-HETE peak can be separated from those for dihydroxyeicosatrienoic acids, other HETEs, and epoxyeicosatrienoic acids. Known amounts of 20-HETE were used to generate a standard curve (range 1-10 ng, r(2) = 0.98). Recovery of 20-HETE from urine averaged 95%, and the intra-assay variation was <5%. Levels of 20-HETE were measured in 100 microliter of urine and renal interstitial fluid or 0.1 mg of renal tissue. The assay was evaluated by studying the effects of 1-aminobenzotriazole (ABT) on the excretion of 20-HETE in rats. ABT reduced excretion of 20-HETE by >65% and inhibited the formation of 20-HETE by renal microsomes. The availability of this assay should facilitate work in this field.