The role of the Na pump in regulating vascular smooth muscle (VSM) contractile function status has been studied for many years. Based on a variety of kinetic differences between VSM and other tissues and Na- independent pump activation by numerous agonists in VSM we have identified at least 2 ways the NaK pump in this tissue can be modulated; 1) ion level activation by intracellular Na+ and 2) protein level activation by phosphorylation of the pump complex itself or adjacent membrane proteins which can modulate pump function. Our hypotheses are that the unique Na+ K+ transport properties of VSM listed above are due to 1) the presence of different alpha and or beta isoforms of the Na+ pump. 2) specific effects of PKA and or PKC mediated phosphorylation of the pump complex. We have identified a truncated form of the alpha1 subunit of the Na+ pump (alpha1T) of molecular weight 66KD (compared to 100 KD) and have expressed it in Sf-9 insect cells. The expressed alpha1T complexes with beta subunit and support ouabain inhibited ATPase activity.
Specific Aims 1 -2 will determine whether alpha 1T can support pump function alone or whether it modulates transport functions of another isoform: 1) express alpha1T alone or with other isoforms in two mammalian expression systems (HeLa cells and COS-1 cells) and measure Na pump function by Na+ K+ ATPase activity. 86Rb uptake ouabain binding and inhibition and Na by SBFI and 2) utilizing the unique sequence of the alpha1T mRNA, and the 3' untranslated region resulting from alternative splicing, determine if alpha1T can function as a pump by antisense inhibition experiments in vascular smooth muscle cells.
Specific aims 3 -5 will address the specific kinase- phosphatase control mechanisms of the Na pump. 3) Correlate PKA and PKC supported phosphorylation of the alpha1 and alpha1T subunit with the various activities of the Na pump as above by kinase and phosphatase regulation. 4) Identify the phosphorylation sites on the alpha1 or alpha1T subunit by phosphopeptide analysis. 5) Prevent the effects of PKA and PKC supported phosphorylation of alpha1T by antisense inhibition and substrate competition of PKA and PKC sites and the identified alpha1T site. Competitive peptides will be introduced into the cells under study to be used as false substrates for phosphorylation. These data will define control of ion transport present in VSM and will help delineate the Na pump role in contractile regulation and will identify if the alpha 1T functions are specific for vascular smooth muscle.
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