Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase

Abstract

Ouabain binding to Na+/K+-ATPase activates Src/epidermal growth factor receptor (EGFR) to initiate multiple signal pathways that regulate growth. In cardiac myocytes and the intact heart, the early ouabain-induced pathways that cause rapid activations of ERK1/2 also regulate intracellular Ca2+ concentration ([Ca2+]i) and contractility. The goal of this study was to explore the role of caveolae in these early signaling events. Subunits of Na+/K+-ATPase were detected by immunoblot analysis in caveolae isolated from cardiac myocytes, cardiac ventricles, kidney cell lines, and kidney outer medulla by established detergent-free procedures. Isolated rat cardiac caveolae contained Src, EGFR, ERK1/2, and 20–30% of cellular contents of α1- and α2-isoforms of Na+/K+-ATPase, along with nearly all of cellular caveolin-3. Immunofluorescence microscopy of adult cardiac myocytes showed the presence of caveolin-3 and α-isoforms in peripheral sarcolemma and T tubules and suggested their partial colocalization. Exposure of contracting isolated rat hearts to a positive inotropic dose of ouabain and analysis of isolated cardiac caveolae showed that ouabain caused 1) no change in total caveolar ERK1/2, but a two- to threefold increase in caveolar phosphorylated/activated ERK1/2; 2) no change in caveolar α1-isoform and caveolin-3; and 3) 50–60% increases in caveolar Src and α2-isoform. These findings, in conjunction with previous observations, show that components of the pathways that link Na+/K+-ATPase to ERK1/2 and [Ca2+]i are organized within cardiac caveolae microdomains. They also suggest that ouabain-induced recruitments of Src and α2-isoform to caveolae are involved in the manifestation of the positive inotropic effect of ouabain.