NA-PUMP ISOFORM-SPECIFIC REGULATION OF VASCULAR FUNCTION

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DESCRIPTION (Adapted from the application): The expression of the Na+-K+ ATPase (NKA) a subunit isoforms (a-isoforms) is tissue specific, developmentally regulated, and under hormonal and neurogenic control, but the physiological basis for NXA a-isoforms is largely unknown. Our overall hypothesis is that the relative distribution and subcellular location of NKA isoforms as well as the total NKA activity are critical to regulation of vascular smooth muscle function. We propose to elucidate the role of NIKA a-isoforms in the regulation of vascular smooth muscle contractility and metabolism. Our long-term goal is to extend this knowledge of their physiological function to disease states, such as hypertension and diabetes, in which the relative expression of NKA a-isoforms is altered. Our overall strategy employs recently developed a 1 & a2 isoform knockout (KO) mice. We propose to develop new mouse models, using the SMP-8 smooth muscle specific promoter. These mice can be crossbred and bred to the KOs to provide additional important models with wide ranges of total NKA activity and a-isoform distribution. We will correlate a-isoform with vascular function at cellular, vessel and whole animal levels to quantify the relations between NKA activity, a-isoform expression and vascular function. Specific Aim 1. To determine the efftcts of NKA a-isoforms and their relative expression on vascular contractility and cardiovascular function. Our hypothesis is that contractility is strongly affected by the a2 isoform. Contractility in aorta, portal vein and resistance vessels will be compared to cardiovascular parameters in the whole animal. Specific Aim 2. To quantify the subcellular localization of NKA a-isoforms and correlate the relative expression with Ca2t and Na+-ion handling in vascular smooth muscle cells. We will use, immunohistological techniques coupled with 3D-microscopic imaging for the subcellular localization of NKA a-isoforms compared to specific compartmental markers. Cytosolic and subcellular [Ca2+]i and [Na+]i will be assessed using fluorescent dye technology. The applicant will test the hypothesis that altered ion homeostasis and contractility are due to specific NKA isoform expression and localization. Specific Aim 3. To determine the effect of NKA alpha-isoforms and their relative expression on vascular metabolism and isoform-specific energetics. Measurements of oxygen consumption and lactate production will be used to access the in vivo ATP utilization of each alpha-isoform based on their differential sensitivity to inhibition by ouabain. The applicant will test the hypotheses that these isoforms are independently regulated and that the observed glucose-sensitivity of activation of vascular smooth muscle is a consequence of NKA alpha-isoform distribution.