Ouabain, a specific inhibitor of the Na+/K+-pump, has previously been shown to interfere with intercellular communication. We have demonstrated a mechanism of this action of ouabain (1). We have showed that gap junctions between vascular smooth muscle cells (SMCs) are regulated through an interaction between the Na+/K+-pump and the Na+/Ca2+-exchanger leading to an increase in the intracellular calcium concentration ([Ca2+]i) in discrete areas near the plasma membrane. This regulation suggests a close association of the proteins in microdomains. We have also suggested that this Na+/K+-pump-containing microdomain is functionally linked to KATP channels via the local ion homeostasis and that this interaction can be bidirectional (1;2). [Ca2+]i in individual SMCs was imaged simultaneously with isometric force in rat mesenteric small arteries. Paired cultured rat aortic smooth muscle cells (A7r5) were used as a model for electrical coupling of SMC by measuring membrane capacitance (Cm). Using PCR, Western blotting and immunohistochemistry we aimed to identify the isoforms of membrane transporters involved in the suggested interaction in SMCs. SMCs were uncoupled (evaluated by inhibition of vasomotion and desynchronization of [Ca2+]i transients in vascular wall, or by reduction of Cm measured in paired A7r5 cells) when the Na+/K+-pump was inhibited either by a low concentration of ouabain (1-10 µM) or by ATP depletion. Reduction of Na+/K+-pump activity by removal of extracellular K+ also uncoupled cells, but only after inhibition of KATP channels. Inhibition of the Na+/Ca2+-exchange activity by SEA0400 or by lowering the extracellular Na+ concentration also uncoupled the cells. Depletion of [Na+]i and clamping low [Ca2+]i prevented the uncoupling. Two isoforms of the Na+/K+-ATPase α subunit (α1 and α2) were found in SMCs. Since rodent α1 Na+/K+-ATPase subunits are ouabain-resistant, we conclude that α2 Na+/K+-ATPase subunits is involved in regulation of the intercellular communications via interaction with the Na+/Ca2+-exchanger in spatially restricted spaces. The experiments suggest that the α2 Na+/K+-pump isoform may affect gap junction conductivity via localized changes in [Ca2+]i through modulation of Na+/Ca2+-exchanger activity.
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40th Sandbjerg meeting on membrane transport, 2008