Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase
Lijun Liu1, Kamiar Mohammadi, Behrouz Aynafshar
1Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43614, USA.
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 Ca(2+) concentration ([Ca(2+)](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 alpha(1)- and alpha(2)-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 alpha-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 alpha(1)-isoform and caveolin-3; and 3) 50-60% increases in caveolar Src and alpha(2)-isoform. These findings, in conjunction with previous observations, show that components of the pathways that link Na(+)/K(+)-ATPase to ERK1/2 and [Ca(2+)](i) are organized within cardiac caveolae microdomains. They also suggest that ouabain-induced recruitments of Src and alpha(2)-isoform to caveolae are involved in the manifestation of the positive inotropic effect of ouabain.
Insights
Cardiac caveolae organize signaling pathways involving Na(+)/K(+)-ATPase and ERK1/2, crucial for heart contractility. Ouabain treatment recruits Src and Na(+)/K(+)-ATPase alpha(2)-isoform to these caveolae, influencing heart muscle function.
Area of Science:
- Cardiovascular Physiology
- Cellular Signaling
- Membrane Biology
Background:
- Ouabain binding to Na(+)/K(+)-ATPase triggers signaling pathways regulating cell growth and cardiac function.
- Early ouabain effects in cardiac cells involve ERK1/2 activation, impacting intracellular calcium and contractility.
- The role of caveolae, specialized membrane microdomains, in these rapid signaling events remains to be fully elucidated.
Purpose of the Study:
- To investigate the involvement of caveolae in early ouabain-induced signaling pathways in cardiac myocytes.
- To determine the localization of Na(+)/K(+)-ATPase subunits and signaling proteins within cardiac caveolae.
- To examine the effect of ouabain on the composition and activation status of proteins within isolated cardiac caveolae.
Main Methods:
- Immunoblot analysis to detect Na(+)/K(+)-ATPase subunits, Src, EGFR, ERK1/2, and caveolin-3 in isolated caveolae.
- Detergent-free procedures for caveolae isolation from cardiac myocytes, ventricles, and kidney tissues.
- Immunofluorescence microscopy to visualize protein localization in adult cardiac myocytes.
- Treatment of isolated contracting rat hearts with ouabain and subsequent analysis of caveolar content and protein activation.
Main Results:
- Na(+)/K(+)-ATPase alpha(1) and alpha(2) isoforms, Src, EGFR, and ERK1/2 were found in isolated cardiac caveolae.
- Caveolin-3 and Na(+)/K(+)-ATPase alpha-isoforms showed partial colocalization in cardiac myocyte sarcolemma and T-tubules.
- Ouabain increased caveolar phosphorylated/activated ERK1/2, Src, and the Na(+)/K(+)-ATPase alpha(2)-isoform, without altering total ERK1/2, caveolar alpha(1)-isoform, or caveolin-3.
Conclusions:
- Cardiac caveolae serve as microdomains organizing signaling components linking Na(+)/K(+)-ATPase to ERK1/2 and intracellular calcium.
- Ouabain-induced recruitment of Src and the Na(+)/K(+)-ATPase alpha(2)-isoform to caveolae is implicated in the positive inotropic effect.
- Caveolae play a critical role in mediating rapid signaling events initiated by Na(+)/K(+)-ATPase activation in the heart.
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