Mong Heng Wang

Kinetic profile of the rat CYP4A isoforms: arachidonic acid metabolism and isoform-specific inhibitors

20-Hydroxyeicosatetraenoic acid (HETE), the cytochrome P-450 (CYP) 4A ω-hydroxylation product of arachidonic acid, has potent biological effects on renal tubular and vascular functions and on the control of arterial pressure. We have expressed high levels of the rat CYP4A1, -4A2, -4A3, and -4A8 cDNAs, using baculovirus and Sf 9 insect cells. Arachidonic acid ω- and ω-1-hydroxylations were catalyzed by three of the CYP4A isoforms; the highest catalytic efficiency of 947 nM-1 ⋅ min-1for CYP4A1 was followed by 72 and 22 nM-1 ⋅ min-1for CYP4A2 and CYP4A3, respectively. CYP4A2 and CYP4A3 exhibited an additional arachidonate 11,12-epoxidation activity, whereas CYP4A1 operated solely as an ω-hydroxylase. CYP4A8 did not catalyze arachidonic or linoleic acid but did have a detectable lauric acid ω-hydroxylation activity. The inhibitory activity of various acetylenic and olefinic fatty acid analogs revealed differences and indicated isoform-specific inhibition. These studies suggest that CYP4A1, despite its low expression in extrahepatic tissues, may constitute the major source of 20-HETE synthesis. Moreover, the ability of CYP4A2 and -4A3 to catalyze the formation of two opposing biologically active metabolites, 20-HETE and 11,12-epoxyeicosatrienoic acid, may be of great significance to the regulation of vascular tone.20-Hydroxyeicosatetraenoic acid (HETE), the cytochrome P-450 (CYP) 4A omega-hydroxylation product of arachidonic acid, has potent biological effects on renal tubular and vascular functions and on the control of arterial pressure. We have expressed high levels of the rat CYP4A1, -4A2, -4A3, and -4A8 cDNAs, using baculovirus and Sf 9 insect cells. Arachidonic acid omega- and omega-1-hydroxylations were catalyzed by three of the CYP4A isoforms; the highest catalytic efficiency of 947 nM-1. min-1 for CYP4A1 was followed by 72 and 22 nM-1. min-1 for CYP4A2 and CYP4A3, respectively. CYP4A2 and CYP4A3 exhibited an additional arachidonate 11,12-epoxidation activity, whereas CYP4A1 operated solely as an omega-hydroxylase. CYP4A8 did not catalyze arachidonic or linoleic acid but did have a detectable lauric acid omega-hydroxylation activity. The inhibitory activity of various acetylenic and olefinic fatty acid analogs revealed differences and indicated isoform-specific inhibition. These studies suggest that CYP4A1, despite its low expression in extrahepatic tissues, may constitute the major source of 20-HETE synthesis. Moreover, the ability of CYP4A2 and -4A3 to catalyze the formation of two opposing biologically active metabolites, 20-HETE and 11, 12-epoxyeicosatrienoic acid, may be of great significance to the regulation of vascular tone.

Differential Regulation of Natriuresis by 20-Hydroxyeicosatetraenoic Acid in Human Salt-Sensitive Versus Salt-Resistant Hypertension

Background—Twenty-hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450 metabolite of arachidonic acid that produces vasoconstriction and inhibition of renal tubular sodium transport. In Dahl rats, a 20-HETE deficiency plays a role in salt-sensitive (SS) hypertension. In humans, there are no data on regulation of 20-HETE by salt intake or on a role for this compound in SS hypertension. Methods and Results—Thirteen salt-resistant (SR) and 13 SS hypertensive subjects had urine 20-HETE excretion measured during salt-loading and depletion. In all patients, 20-HETE was 66.6% higher in the salt-replete (1.75±0.25 &mgr;g/h) than in the salt-depleted state (1.05±0.16, P <0.003). There was no difference in 20-HETE excretion between SR and SS patients in either state of salt balance. In SR patients, sodium excretion during salt-loading correlated with 20-HETE (r =0.61, P <0.03) but not with blood pressure. In contrast, in SS patients, sodium excretion did not correlate with 20-HETE but did correlate with blood pressure (r =0.66, P <0.02). Finally, in the SS group only, there was a negative correlation between body mass index and 20-HETE excretion (r =−0.79, P <0.002) that was present during both salt-loading and depletion. Conclusions—We demonstrate for the first time that 20-HETE excretion is regulated by salt intake in hypertension. We find a disrupted relationship between sodium excretion and 20-HETE in SS patients, which results in dependence of their salt excretion on blood pressure and may be related to the magnitude of their obesity. We conclude that salt-sensitivity of blood pressure in essential hypertension may result from impairment of a natriuretic mechanism dependent on 20-HETE.

Contribution of cytochrome P-450 4A1 and 4A2 to vascular 20-hydroxyeicosatetraenoic acid synthesis in rat kidneys

20-Hydroxyeicosatetraenoic acids (20-HETE), a biologically active cytochrome P-450 (CYP) metabolite of arachidonic acid in the rat kidney, can be catalyzed by CYP4A isoforms including CYP4A1, CYP4A2, and CYP4A3. To determine the contribution of CYP4A isoforms to renal 20-HETE synthesis, specific antisense oligonucleotides (ODNs) were developed, and their specificity was examined in vitro in Sf9 cells expressing CYP4A isoforms and in vivo in Sprague-Dawley rats. Administration of CYP4A2 antisense ODNs (167 nmol ⋅ kg body wt-1 ⋅ day-1iv for 5 days) decreased vascular 20-HETE synthesis by 48% with no effect on tubular synthesis, whereas administration of CYP4A1 antisense ODNs inhibited vascular and tubular 20-HETE synthesis by 52 and 40%, respectively. RT-PCR of microdissected renal microvessel RNA indicated the presence of CYP4A1, CYP4A2, and CYP4A3 mRNAs, and a CYP4A1-immunoreactive protein was detected by Western analysis of microvessel homogenates. Blood pressure measurements revealed a reduction of 17 ± 6 and 16 ± 4 mmHg in groups receiving CYP4A1 and CYP4A2 antisense ODNs, respectively. These studies implicate CYP4A1 as a major 20-HETE synthesizing activity in the rat kidney and further document the feasibility of using antisense ODNs to specifically inhibit 20-HETE synthesis and thereby investigate its role in the regulation of renal function and blood pressure.

20-HETE and Furosemide-Induced Natriuresis in Salt-Sensitive Essential Hypertension

Abstract—Cyclooxygenase metabolites of arachidonic acid modulate the natriuretic effect of furosemide. It is not known whether 20-HETE, a monooxygenase metabolite of arachidonic acid that also inhibits sodium transport, participates in the action of furosemide. We measured urine sodium (UNaV) and 20-HETE during furosemide diuresis (40 mg three times over 12 hours) in 12 salt-sensitive (SS) and 11 salt-resistant (SR), salt-replete hypertensive subjects (126±24 mmol/24 hours positive sodium balance produced by 160-mmol-sodium diet and 2 L saline infusion). Individual systolic blood pressure decreases from the salt-replete to the salt-depleted state were the index of salt-sensitivity. SS had low plasma renin with blunted responses to changes in salt balance, inappropriate plasma aldosterone, and an increased aldosterone/renin ratio. UNaV by furosemide was less in SS (263±25 mmol/12 hours) than in SR (351±25 mmol/12 hours, P <0.02) patients. 20-HETE was not different between SS and SR patients before (1.92±0.38 versus 1.37±0.34 &mgr;g/h) or after furosemide (1.52±0.27 versus 2.01±0.40 &mgr;g/h), but furosemide changed 20-HETE excretion in opposite direction in SR (0.63±0.26) versus SS (−0.40±0.17, P <0.005) patients. In all patients together, %&Dgr;20-HETE by furosemide correlated with %&Dgr;UNaV (r =0.56, P <0.01) and negatively with salt-sensitivity of blood pressure (r =−0.55, P <0.01). In SS, &Dgr;20-HETE by furosemide correlated with &Dgr;aldosterone/renin ratio (r =0.60, P <0.05), whereas 20-HETE during furosemide had a negative correlation with body mass index (r =−0.73, P <0.01). Our data suggest that 20-HETE modulates the natriuretic response to furosemide, and impaired natriuresis of SS involves a mechanism that alters the 20-HETE response to furosemide and is linked to salt-sensitivity of blood pressure.

Contribution of cytochrome P450 4A isoforms to renal functional response to inhibition of nitric oxide production in the rat

20‐Hydroxyeicosatetraenoic acid (20‐HETE), a major renal eicosanoid, regulates renal function and contributes to renal responses following withdrawal of nitric oxide (NO). However, the role of 20‐HETE‐synthesizing isoforms in renal function resulting from NO inhibition is unknown. The present study evaluated the role of cytochrome (CYP)4A1, ‐4A2 and ‐4A3 isoforms on renal function in the presence and absence of NO. Antisense oligonucleotides (ASODN) to CYP4A1, ‐4A2 and ‐4A3 reduced 20‐HETE synthesis and downregulated the expression of CYP4A isoforms in renal microsomes. Nω‐L‐nitromethyl arginine ester (L‐NAME, 25 mg kg−1), an inhibitor of NO production, increased mean arterial blood pressure (MABP, Δ=+18 to 26 mmHg), reduced renal blood flow (RBF, Δ= ‐1.8 to 2.9 ml min−1), increased renal vascular resistance (RVR, Δ=+47 to 54 mmHg ml−1 min−1), reduced glomerular filtration rate (GFR), but increased sodium excretion (UNaV). ASODN to CYP4A1 and ‐4A2 but not ‐4A3 reduced basal MABP and RVR and increased basal GFR, while ASODN to CYP4A2 significantly reduced basal UNaV suggesting a differential role for CYP4A isoforms in the regulation of renal function. ASODN to CYP4A2 but not ‐4A1 or ‐4A3 blunted the increase in MABP by L‐NAME (38 ± 9 %, P < 0.05). ASODN to CYP4A1, ‐4A2 and ‐4A3 attenuated the reduction in RBF and the consequent increase in RVR by L‐NAME with a potency order of CYP4A2 = CYP4A1 > CYP4A3. ASODN to CYP4A1 and ‐4A2 but not ‐4A3 attenuated L‐NAME‐induced reduction in GFR, but ASODN to all three CYP4A isoforms blunted the L‐NAME‐induced increase in UNaV (CYP4A3 > CYP4A1 >> CYP4A2). We conclude from these data that CYP4A isoforms contribute to different extents to basal renal function. Moreover, CYP4A2 contributes greatest to haemodynamic responses while CYP4A3 contributes greatest to tubular responses following NO inhibition. We therefore propose that NO differentially regulates the function of CYP4A1, ‐4A2, and ‐4A3 isoforms in the renal vasculature and the nephron.

Synthesis and Function of 20-Hydroxyeicosatetraenoic Acid in the Kidney

20-hydroxyeicosatetraenoic acid (20-HETE), the ω-hydroxylation product of arachidonic acid (AA), is the principal metabolite formed in tubular and vascular structures of the rat renal cortex and outer medulla. 20-HETE has potent biological activities and has been shown to contribute to the regulation of renal function and to the control of arterial pressure. In the renal tubules it inhibits sodium reabsorption, while in the renal microcirculation it is a vasoconstrictor and a regulator of the myogenic response. The ω-hydroxylation of fatty acids, including AA, is catalyzed by enzymes of the cytochrome P450 (CYP) 4A family. In the rat, four isoforms have been identified: CYP4A1, 4A2, 4A3, and 4A8. Our studies indicated that despite the high homology, these isoforms display distinct catalytic properties including differences in kinetic parameters, product profile and inhibitor sensitivity. While the constitutive level of expression of CYP4A1 is low, its recombinant form is the low Km AA-ω-hydroxylase and thus, by far, the most efficient 20-HETE synthesizing enzyme. Whereas CYP4A1 is solely an AA whydroxylase, CYP4A2 and CYP4A3 also catalyze AA 11,12-epoxidation. Systemic administration of CYP4A antisense oligodeoxynucleotides revealed that CYP4A1 contributes significantly to the renal tubular and vascular production of 20-HETE. Furthermore, these isoforms demonstrate unique intrarenal localization. Using molecular and pharmacological probes we demonstrated a unique CYP4A isoform-specific localization within the renal microvasculature. Transfection of CYP4A1 cDNA to renal microvessels resulted in enhanced 20-HETE synthesis and increased reactivity to phenylephrine. Thus, 20-HETE of vascular origin serves as a stimulatory regulator of vascular responses to constrictor agonists.