Hyperglycemia and diabetes are associated with impaired endothelium-dependent dilation of coronary conduit vessels, due in part to quenching of the vasodilator nitric oxide by production excess superoxide. In contrast, little is known regarding the effect of oxidative stress on hyperpolarization-mediated vasodilation. This is important since coronary arteriolar dilation, especially in humans, depends less upon nitric oxide and more upon hyperpolarization via opening of K + channels. Voltage-dependent (Kv) K + channels are one of several K + channels highly expressed in coronary vascular smooth muscle (VSM). They play an important role in regulating resting tone and participate in physiological dilation. Much of the vascular dysfunction associated with diabetes and hyperglycemia is due to excess production of reactive oxygen species (ROS). However the extent to which vasomotor responses mediated through K + channels and hyperpolarization are affected by elevations in glucose, and whether this influence involves the production of ROS is not known. The overall aim of this application is to determine the effect of high glucose (either in the tissue medium bathing isolated vessels or in the serum of diabetic rats) on hyperpolarization-mediated coronary dilation and coronary VSM Kv channel activity, and to establish a mechanistic role for superoxide in both the altered channel activity and hyperpolarization-induced dilation of coronary arterioles. Videomicroscopy, membrane potential, patch clamping, and Western blot analysis of isolated coronary arterioles will be used in both rat and dog models to test three specific aims.
In aim 1 we shall examine the hypothesis that high glucose reduces both Kv channel activity and the associated dilation to isoproterenol and forskolin in rat coronary arterioles.
In aim 2 we shall determine the contribution of superoxide to the reduced Kv channel activity following exposure to high glucose.
In aim 3 we shall identify the source of vascular superoxide generation following exposure to high glucose or hyperglycemia. Rat coronary arterioles incubated in normal or high glucose or isolated from diabetic and control rats or dogs are either mounted onto micropipettes for diameter measurements or dissociated into single VSM cells for K + current measurements. Vessels from the same preparations are also saved for Western blot analysis. This combination of pharmacological, electrophysiological and molecular assessment of vascular and cellular function provides a powerful mechanism for studying hyperpolarization-dependent vascular regulatory process. These studies will have important implications with regard to mechanisms of coronary vasomotor regulation in diabetes and provide novel mechanisms of altered coronary vasoregulation. The results may suggest new therapeutic approaches for the treatment of microvascular dysfunction in patients with diabetes. ? ?
