Role of caveolae in ouabain-induced proliferation of cultured vascular smooth muscle cells of the synthetic phenotype

Abstract

Role of caveolae in ouabain-induced proliferation of cultured vascular smooth muscle cells of the synthetic phenotype. Am J Physiol Heart Circ Physiol 287: H2173-H2182, 2004. First published July 15, 2004 doi:10.1152/ajpheart.00352.2004.-We have shown earlier that low concentrations of ouabain that do not perturb the ionic milieu can initiate proliferation of vascular smooth muscle cells (VSMCs) in the synthetic phenotype from three different species: canine, rodent, and human. This effect occurs by activation of Src and the epidermal growth factor receptor (EGFR), and thus supports the concept of an additional, nonionic, transducing function of the Na pump. The present study presents data suggesting that such activation occurs through specific Na pump sites localized to the caveolae, and subsequent interactions with selected signaling proteins resident within the same membrane microdomain. Our data show that at rest, 30% of the total number of Na pumps are concentrated within the caveolae. When the various VSMCs were treated with proliferating concentrations of ouabain, the key protein content in isolated caveolae was increased. However, the recruited proteins were different between the different tissues. Thus ouabain activated the recruitment of both the Na pump ␣ 1-subunit and EGFR in the caveolae from rat A7r5 cells, whereas in both human and canine cells, ouabain activated the recruitment of Src, with the caveolar content of the other proteins remaining constant. These data demonstrate that ouabain interacts with the ␣1-subunit of the Na pump that resides within the caveolar domain, and such interaction selectively recruits signal transducing proteins to this microdomain resulting in their activation, which is necessary for the initiation of the proliferative cascade. Na pump; ouabain signaling; vascular smooth muscle proliferation THE NA PUMP maintains normal ion gradients across the cell membrane of virtually all mammalian cells, and the mechanism of ion transport has been studied extensively for many years (7,21). Recently, however, it has become apparent that pump function can be modulated by a variety of interacting proteins with mechanisms other than enzymatic phosphorylation of PKA and PKC. Such protein modulators are the FXYD family of proteins, i.e., phospholemman (15, 16), the ␥-subunit (28), and corticosteroid hormone-induced factor (16), which have been known for some time but whose functions have only recently been delineated. Their interaction with the Na pump can alter affinity for both ATP and ions (42).However, these are not the only protein moieties that can interact with the pump. Recently, a number of studies have appeared indicating that the pump can also interact with proteins generally associated with the well-known growth-related transducing cascades. The functional manifestation of these pump-protein interactions are quite tissue specific (see below), and a common bond between all of these observations is that they are initiated by the binding of the cardiac glycoside inhibitor oua...

Examination of the Cellular Mechanisms by Which Marinobufagenin Inhibits Cytotrophoblast Function

“…Xie (60) reported that in addition to promoting ion transport, Na ϩ /K ϩ -ATPase acts as a signal transducer in a cell-specific manner. Allen and co-workers (61,62) demonstrated that the cardiotonic steroid ouabain stimulates vascular smooth muscle cell proliferation, which correlates with increased epidermal growth factor transactivation and Src phosphorylation. Moreover, binding of ouabain to the sodium pump induces the phosphorylation of various MAPKs and subsequently activates the transcription factors AP-1 (activator protein-1) and NFB (63).…”

Reduction of Na/K-ATPase Potentiates Marinobufagenin-induced Cardiac Dysfunction and Myocyte Apoptosis

“…Reduction of cardiac Na/K-ATPase is also related with decreased cardiac contractile function in humans and in animals (1,10,11). Previous reports showed that exposure of cells to low concentrations of cardiotonic steroids (CTS) 2 stimulates cell growth (12)(13)(14). Our recent work also shows that binding of ouabain to Na/K-ATPase in pig renal proximal tubule cells stimulates Src/Akt/mTOR signaling pathways and results in a stimulation of cell growth.…”

β-Subunit of cardiac Na+-K+-ATPase dictates the concentration of the functional enzyme in caveolae

“…Protein A/G plus agarose was purchased from Santa Cruz Biotechnology. 22 Na ϩ was purchased from Perkin-Elmer Life Sciences (Boston, MA). Kidney medullas were prepared by dissection from kidneys.…”