1 Department of Physiology and Biophysics, Faculty of Health Sciences, Aarhus University, Aarhus University2 Department of Biomedicine - Forskning og uddannelse, Vest, Department of Biomedicine, Health, Aarhus University3 Institute of Neuroscience and Physiology, University of Gothenburg4 Department of Biomedicine - Forskning og uddannelse, Vest, Department of Biomedicine, Health, Aarhus University
The Na+/K+-ATPase is known to interact with many membrane and cytosolic proteins by organizing various signaling complexes. These interactions were suggested to be important in regulation of various cellular responses. Pumping activity of the Na+/K+-ATPase is suggested to be essential for some of these interactions, while other responses may be independent of pumping activity. The Na+/K+-pump differs from other P-type ATPases by its sensitivity to cardiotonic steroids such as ouabain. However, rodent tissues express both ouabain-insensitive (α1) and ouabain-sensitive (α2 and α3) isoforms of Na+/K+-ATPases. Based on our previous findings we hypothesized here that the ouabain-sensitive isoform of the Na+/K+-ATPase can regulate vascular tone by interaction with Na+/Ca2+-exchanger, gap junctions and KATP channels. [Ca2+]i in individual smooth muscle cells (SMCs) was imaged simultaneously with isometric force in rat mesenteric small arteries. Paired cultured rat smooth muscle cells (A7r5) were used as a model for electrical coupling of SMC by measuring membrane capacitance (Cm). PCR, Western blotting and immunohistochemistry were used to identify the membrane transporters. SMCs were uncoupled (evaluated by inhibition of vasomotion and desynchronization of [Ca2+]i transients in the vascular wall, or by reduction of Cm measured in paired A7r5 cells) when the Na+/K+-ATPase was inhibited either by a low concentration of ouabain (1-10 µM) or by ATP depletion. Inhibition of the Na+/Ca2+-exchange activity by SEA0400 or by lowering the extracellular Na+ concentration also uncoupled the cells. Reduction of Na+/K+-ATPase activity by removal of extracellular K+ uncoupled cells, but only after inhibition of KATP channels. This interaction was bidirectional. Depletion of [Na+]i and clamping [Ca2+]i at low levels prevented the uncoupling. Low ouabain concentration evoked spatially restricted [Ca2+]i transients along the cell periphery. All three isoforms of the Na+/K+-ATPase α subunit were found in SMCs but based on ouabain-sensitivity and co-precipitation experiments we suggest that α2 Na+/K+-ATPase subunit is involved in regulation of the intercellular communication. We have here shown that gap junctions between SMCs are regulated through an interaction between the Na+/K+-ATPase and the Na+/Ca2+-exchanger leading to an increase in [Ca2+]i in discrete areas near the plasma membrane. We have also suggested that this Na+/K+-pump-containing microdomain is functionally linked to KATP channels via the local ion homeostasis.
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12<sup>th</sup> International ATPase Conference - Na,K-ATPase and Related Transport ATPases of P-type: Structures, Mechanisms, and Roles in Health and Disease, 2008