Transfection of CYP4A1 cDNA increases vascular reactivity in renal interlobar arteries

Abstract

HETE, a cytochrome P-450 4A (CYP4A1)-derived arachidonic acid metabolite, is a major eicosanoid formed in renal and extrarenal microcirculation. 20-HETE inhibits Ca 2ϩ -activated K ϩ channels in vascular smooth muscle cells and thereby may modulate vascular reactivity. We transfected renal interlobar arteries with an expression plasmid containing the cDNA of CYP4A1, the low-Km arachidonic acid -hydroxylase, and examined the consequences of increasing 20-HETE synthesis on constrictor responses to phenylephrine. CYP4A1-transfected interlobar arteries demonstrated a twofold increase in CYP4A protein levels and 20-HETE production compared with arteries transfected with the empty plasmid; they also showed increased sensitivity to phenylephrine, as evidenced by a decrease in EC50 from 0.37 Ϯ 0.04 M in plasmid-transfected arteries to 0.07 Ϯ 0.01 M in CYP4A1-transfected arteries. The increased sensitivity to phenylephrine was greatly attenuated by , a selective inhibitor of 20-HETE synthesis, and by 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, a specific 20-HETE antagonist. This effect of DDMS was reversed by addition of 20-HETE, further substantiating the notion that increased levels of 20-HETE contribute to the increased sensitivity to phenylephrine in vessels overexpressing CYP4A1. These data suggest that 20-HETE of vascular origin sensitizes renal vascular smooth muscle to phenylephrine. 20-hydroxyeicosatetraenoic acid; arachidonic acid; phenylephrine; cytochrome P-450 SMALL ARTERIAL VESSELS MANUFACTURE 20-HETE, a product of arachidonic acid metabolism by cytochrome P-450 enzymes of the 4A family (CYP4A1) (8,12,17,26). Exogenous 20-HETE was reported to contract or relax vascular smooth muscle, depending on the animal species, type of vessels, and experimental conditions. Cyclooxygenase-dependent and -independent mechanisms have been implicated in 20-HETE-induced vascular contraction (6, 16, 17) and vascular relaxation (2, 4).Renal preglomerular vessels express CYP4A proteins, produce 20-HETE, and are constricted by exogenous 20-HETE. The constrictor action of exogenous 20-HETE in pressurized canine arcuate arteries, rat renal interlobular arteries, and rat renal afferent arterioles is independent of cyclooxygenase activity and has been attributed to inhibition of the opening of Ca 2ϩ -activated K ϩ channels in vascular smooth muscle cells, leading to cellular depolarization and increased Ca 2ϩ entry (8,12,14,23,28). Recent studies support the notion that endogenous 20-HETE subserves vasconstrictor mechanisms in the rat kidney. For example, inhibitors of 20-HETE synthesis were shown to increase blood flow to the kidney (29), to increase the diameter of preconstricted interlobular arteries denuded of endothelium (11), and to attenuate vasconstrictor responses elicited by endothelin (10, 22), angiotensin II (5), or increases in transmural pressure (14). 20-HETE produced by the smooth muscle isolated from renal preglomerular vessels was also shown to exert a tonic inhibitory influence on the activity of large-conductance C...

Vascular CO Counterbalances the Sensitizing Influence of 20-HETE on Agonist-Induced Vasoconstriction

“…The level of 20-HETE in renal interlobar arteries was reduced by treatment with DDMS, implying that vascular 20-HETE production is dependent on the activity of 1 or more CYP4A oxygenases. 2,5 CO is known to interfere with the activity of cytochrome P450 enzymes and therefore is expected to decrease 20-HETE synthesis. 9 In keeping with this notion, we found that the level of 20-HETE falls in renal interlobar arteries incubated in buffer containing exogenous CO (1 mol/L).…”

“…In this regard, we confirmed previous reports that the sensitivity of small arteries to phenylephrine and vasopressin is decreased by exogenous CO 1 and increased by exogenous 20-HETE. 2,4 The reactivity of the vessels to the constrictor agonists also is decreased and increased, respectively, by CO and 20-HETE manufactured by the renal vessels themselves because their sensitivity to phenylephrine and vasopressin was found to increase in response to HO inhibition with CrMP 1 and to decrease after CYP4A inhibition with DDMS. 2,4 Notably, neither exogenous CO nor CrMP effected changes in responsiveness to the constrictor agonist in vascular preparations undergoing CYP4A inhibition with DDMS.…”

“…Renal interlobar artery specimens were transferred into glass vials (5 mL) containing 1 mL of Krebs buffer saturated with 95% O 2 -5% CO 2 and complemented with NADPH (1 mmol/L), N -nitro-L-arginine methyl ester (L-NAME; 1 mmol/L) and indomethacin (10 mol/L). The vials were capped tightly with rubberized Teflon liners, and the samples were incubated at 37°C for 60 minutes in the absence and presence of DDMS (30 mol/L), CrMP (30 mol/L), or CO (1 mol/L).…”

“…1,2 The rings were bathed in Krebs buffer (37°C) containing L-NAME (1 mmol/L) and gassed with 95% O 2 -5% CO 2 , unless indicated otherwise. After a 30-minute equilibration interval, concentration-response curves to phenylephrine (10 Ϫ9 to 5ϫ10 Ϫ5 mol/L) or vasopressin (10 Ϫ11 to 10 Ϫ7 mol/L) were constructed in the absence and the presence of test agents by cumulatively increasing the concentration of agonist every 2 to 3 minutes and recording the resulting changes in tension at the end of the period.…”

“…1,2 CO, a product of heme metabolism by heme oxygenase (HO) isoforms Ϫ1 and Ϫ2, 3 reduces the sensitivity of vascular smooth muscle to phenylephrine and vasopressin. 1 In contrast, 20-HETE, a product of arachidonic acid -hydroxylation by isoforms of the cytochrome P450 4A family (CYP4A), 4,5 increases the sensitivity of vascular smooth muscle to phenylephrine 2 and vasopressin. 4 The reciprocal modulatory actions of CO and 20-HETE on vascular reactivity to phenylephrine are linked, respectively, to stimulation and inhibition of large conductance calcium-activated potassium (K Ca ) channels in vascular smooth muscle.…”