Endothelial cells release nitric oxide, prostaglandin I2 and endothelium-derived hyperpolarizing factor (EDHF) that reduce vascular tone and counteract vasoconstriction. Ischemic heart disease, heart failure and hyperten- sion are associated with vasoconstriction that is attributed, in part, to reductions in endothelium-dependent di- lation. Therapeutic approaches restoring endothelial function represent promising new treatments for cardio- vascular diseases with a major impact on health. We have identified a 15-lipoxygenase (LO) metabolite of ara- chidonic acid, 11(R),12(S),15(S)-trihydroxy-eicosatrienoic acid (11,12,15-THETA), that acts as an EDHF. It relax arteries, in part, by activating apamin-sensitive, small conductance, calcium-activated potassium (SKCa) channels and hyperpolarizing smooth muscle cells (SMCs). It also acts by inhibition of RhoA activation. 11,12,15-THETA mediates a portion of the endothelium-dependent relaxations to acetylcholine (ACh), in- creases in flow and AA and opposes the action of vasoconstrictors. We will test the hypothesis that 11,12,15- THETA mediates relaxations to ACh and AA by a unique combination of mechanisms: (1) activating a hetero- trimeric guanine nucleotide binding protein (G protein) that opens SKCa channels resulting in hyperpolarization of SMCs and (2) inhibiting the activation of the small GTPase RhoA reducing the calcium sensitivity of contrac- tile proteins. These studies will indicate that 11,12,15-THETA is an EDHF and an endothelium-derived calcium (Ca) desensitizer. 11(R),12(S),15(S)-THETA increases GTP335S binding to rabbit arterial membranes implicat- ing a G protein-coupled binding site/receptor. Using biochemical and molecular methods, we will identify the G protein(s) that is coupled to 11,12,15-THETA action. Using patch clamp and bioassay methods, we will char- acterize the role of the G protein in the regulation of SKCa channel activity, hyperpolarization and dilation by 11,12,15-THETA. These studies will provide new insights into the molecular mechanism of action of 11,12,15- THETA and the regulation of vascular SKCa channel activity. Agonists such as norepinephrine and angiotensin promote constriction by activating the RhoA-Rho kinase cascade that sensitizes contractile proteins to Ca. 11,12,15-THETA decreases the formation of the active form of RhoA, RhoA-GTP. These studies will test the hypothesis that 11,12,15-THETA causes relaxation by inhibiting RhoA activation resulting in decreased Rho kinase activity increasing myosin light chain phosphatase activity and decreasing phosphorylated myosin light chain. These studies will access the effect of 11,12,15-THETA on RhoA activity in SMCs and arteries and de- termine the contribution of RhoA inhibition to the relaxations to 11,12,15-THETA. The role of a G protein in RhoA inhibition by 11,12,15-THETA will be determined. These studies will identify two new mechanisms of action for the endothelium-derived relaxing factor 11,12,15-THETA and provide a new therapeutic approach to enhancing endothelial function.
The local regulation of the dilation and constriction of blood vessels is important in normal blood pressure control and contributes to pathological conditions as hypertension, diabetes and angina. These studies have defined a new class of vasodilators that are formed by the blood vessel wall. Knowledge of the mechanisms of action of these new endogenous vasodilators will help our understanding of the regulation of vascular tone and blood pressure.
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