Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase.
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.
Liu
Lijun
L
Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43614, USA.
Mohammadi
Kamiar
K
Aynafshar
Behrouz
B
Wang
Haojie
H
Li
Daxiang
D
Liu
Jiang
J
Ivanov
Alexander V
AV
Xie
Zijian
Z
Askari
Amir
A
D000818:Animals; D000831:Animals, Newborn; D021941:Caveolae / Q000737:chemistry / Q000378:metabolism*; D051242:Caveolin 1; D051244:Caveolin 3; D022461:Caveolins / Q000378:metabolism; D002458:Cell Fractionation; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D010042:Ouabain / Q000378:metabolism; D021122:Protein Subunits / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine; D019061:src-Family Kinases / Q000378:metabolism
C495540:CAV1 protein, human; C495537:Cav1 protein, rat; C495549:Cav3 protein, rat; D051242:Caveolin 1; D051244:Caveolin 3; D022461:Caveolins; D004791:Enzyme Inhibitors; D021122:Protein Subunits; D010042:Ouabain; D066246:ErbB Receptors; D019061:src-Family Kinases; D020928:Mitogen-Activated Protein Kinases; D000254:Sodium-Potassium-Exchanging ATPase