ROLE OF 20-HETE IN REGULATING VASCULAR OXYGEN RESPONSE

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Oxygen dependent autoregulatory mechanisms are critical in regulating organ blood flow and vascular resistance. While these have been widely studied, the cellular and molecular "sensors" responsible for oxygen sensing in the peripheral circulation are unknown. This project will examine the hypotheses that 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 metabolite of arachidonic acid, is an important mediator of the vasoconstrictor response to increased oxygen availability in normotensive animals, and that increases either in the expression of the cytochrome P4504A alpha-hydroxylase enzymes that produce 20-HETE or in the sensitivity of resistance vessels to 2-HETE contribute to the enhanced constriction of resistance arteries in response to increased oxygen availability that has been demonstrated in various models of hypertension. The role of 20-HETE in mediating vascular response to increased oxygen availability will be assessed in the rat cremaster muscle using three different methods of inhibiting the formation or action of 20-HETE [17- octadecenoic acid (17-ODYA), dibromododeconylmethyl sulfinmide (DDMS), and 19-hydroxyeicosatetraenoic acid (19-HETE)]. Oxygen availability to the cremaster muscle will be increased by: 1) increasing the P0/2 of the superfusion solution flowing over the tissue, 2) infusion of the allosteric hemoglobin effector RSR-13, and 3) increasing the inspired fraction of 0/2 (FI0/2). Microvascular responses to increased 0/2 availability will be assessed by the direct measurement of arteriolar diameter and RBC velocity and the calculation of blood flow in individual arterioles. P0/2 in the tissue and on the surface of the microvessels will be measured with 0/2 microelectrodes to verify that changes in tissue and arteriolar P0/2 during increased 0/2 availability are in the range of P0/2 where 20-HETE formation is 0/2 sensitive. The location of the "sensor" for 20-HETE mediated oxygen responses in the microcirculation will be investigated by determining the location of the enzyme responsible for 20- HETE formation (P4504A alpha-hydroxylase) using Western blotting and by identifying the P4504A alpha-hydroxylase isoform using RT-PCR in isolated arterioles and skeletal muscle parenchymal tissue. The role of 20-HETE in mediating whole body vascular responses to increased O/2 availability and acute volume expansion will be evaluated in conscious animals by determining cardiac output, total peripheral resistance, regional blood flows and regional vascular resistance during increased FI0/2, infusion of the allosteric hemoglobin effector RSR-13, and acute volume expansion with and without blockade of 20-HETE formation with DDMS. The contribution of 20-HETE to the enhanced vasoconstrictor response to increased P0/2 in hypertension will be tested in two experimental models of hypertension previously reported to exhibit an enhanced response to increased oxygen availability: the reduced renal mass (RRM) hypertensive rat and the spontaneously hypertensive rat (SHR). The contribution of an increased expression of P4504A alpha-hydroxylase to the enhanced constriction of arterioles in response to elevated P0/2 in the hypertensive animals will be assessed by using Western blots and competitive RT-PCR to evaluate the expression of the P4504A alpha-hydroxylase enzyme in the whole cremaster muscle and in isolated arterioles and parenchymal tissue of hypertensive and normotensive animals. The role of an increased sensitivity of the resistance vessels to 20-HETE in mediating the enhanced response to elevated P0/2 in hypertensive animals will be assessed by evaluating the sensitivity of the in situ microcirculation and isolated arterioles and resistance arteries to exogenously added 20-HETE. Taken together, these experiments should provide an integrated understanding of the role of 20- HETE in mediating vascular responses to increased oxygen availability at the molecular level, at the level of the individual resistance vessel, and at the level of the peripheral circulation in the intact, conscious animal.