Overexpresion of 20-Hydroxyeicosatetraenoic Acid Synthesis Increases Vascular Tone
The cytochrome P450 (CYP)4A family catalyzes 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis which promotes vasoconstriction. We transfected gracilis arterioles (GA) with an expression plasmid (pcDNA3.1) containing the cDNA of CYP4A1, the low Km arachidonic acid ω-hydroxylase, and examined the consequences of increasing intracellular levels of 20-HETE on vascular diameter. GA were isolated from SD rats and incubated with a mixture of pcDNA3.1/CYP4A1 (20μg)or pcDNA3.1 (control plasmid, 20μg) and liposomes (200μg) in Dubecco’s modified Eagle medium with 10% Nu-serum for 18h. Immunoreactive CYP4A protein levels were markedly increased in GA transfected with the pcDNA3.1/CYP4A1 compared to those transfected with empty plasmid. CYP4A1-transfected GA demonstrated a 2-fold increase in 20-HETE synthesis (0.019 ± 0.005 vs 0.009 ± 0.0017 ng/mg/h in vessels transfected with the CYP4A1 and empty plasmid, respectively). Treated GA were mounted on a pressure-myograph and pressurized to 100 mmHg for functional studies. The internal diameter (ID) of GA transfected with pcDNA3.1/CYP4A1 (55 ± 2μm) was exceeded by that of GA transfected with empty plasmid DNA (99 ± 5μm). Acute treatment with an inhibitor of 20-HETE synthesis (DDMS, 30 μM) increased the ID of CYP4A1-transfected GA to 83 ± 7μm but not of GA transfected with empty plasmid DNA (105 ± 5μm). Likewise, acute treatment with 20-HEDE (10μM), a 20-HETE antagonist, increased the ID of CYP4A1-transfected GA to 90 ± 4μm but not of GA transfected with empty plasmid DNA (101 ± 8μm). These results, together with the observation that CYP4A1 expression and 20-HETE synthesis are detected in untreated arterioles, support the view that 20-HETE of vascular origin contributes to vascular tone.