Ouabain-induced Signaling and Vascular Smooth Muscle Cell Proliferation

Abstract

The hypothesis of this study is that the sodium pump complex acts as an intracellular signal-transducing molecule in canine vascular smooth muscle cells through its interaction with other membrane and cytoskeletal proteins. We have demonstrated that 1 nM ouabain induced transactivation of the epidermal growth factor receptor (EGFR), resulting in increased proliferation and bromodeoxyuridine (BrdUrd) uptake. Immunoprecipitation and Western blotting showed that the EGFR and Src were phosphorylated within 5 min of 10 ؊9 M ouabain stimulation. Both ouabain-induced DNA synthesis (BrdUrd uptake) and MAPK42/44 phosphorylation were inhibited by the Src inhibitor PP2, the EGFR kinase inhibitor AG1478, the tyrosine kinase inhibitor genistein, and the MEK1 inhibitor PD98059. Ouabain concentrations higher than 1 nM had little or no stimulating effect on proliferation or BrdUrd uptake but did minimally activate ERK1/2. Thus, low concentrations of ouabain, which do not inhibit the sodium pump sufficiently to perturb the resting cellular ionic milieu, initiate a transactivational signaling cascade leading to vascular smooth muscle cell proliferation.The role of the sodium pump as a regulator of intracellular ionic balance has been well documented. However, research in this area has recently shown that this protein complex has the potential to function in ways that apparently do not involve these well documented ionic shifts. For example, Peng et al. (1) reported that the sodium pump complex can function as a molecular signal transducer in rat cardiac myocytes. Kometiani et al. (2) further showed that 10 Ϫ4 -10 Ϫ5 M (10 -100 M) ouabain activated cardiac myocyte hypertrophy and MAPK42/ 44 1 phosphorylation. Recently many growth factor signaling pathways have been shown to involve EGFR transactivation in VSMCs (3, 4) as was also shown earlier in cardiac myocytes (5). In this paper we describe the ability of low concentrations of ouabain (0.1-1.0 nM) to induce proliferation of cultured canine vascular smooth muscle cells via a signaling cascade involving Src, EGFR, and MAPK42/44. The isolation of ouabain-like substances from the plasma and urine samples of both healthy and hypertensive individuals (6), as well as a variety of animal tissues, has suggested potentially important paradigms for these agents in the modulation of cell function through interaction with the sodium pump. MATERIALS AND METHODSAll chemicals were obtained from Sigma. Ouabain (o-3125 and o-5754) from Sigma and ouabain (75640) from Fluka were used. All kinase inhibitors were from Calbiochem. Antibodies for Src, phosphotyrosine (4G10, monoclonal), and EGFR were from Upstate Biotechnology Inc. (Waltham, MA). The antibody for MAPK42/44 (E10, monoclonal) was from Cell Signaling Technology Inc. (New England BioLabs, Beverly, MA). Anti-active EGFR antibody was purchased from Transduction Laboratories.Vascular Smooth Muscle Cell Culture-Vascular smooth muscle cells were isolated from the saphenous veins of mongrel dogs by a two-step enzymatic digestion procedure as...

Selective association of endogenous ouabain with subclinical organ damage in treated hypertensive patients

“…EO take part in the development of hypertension through increased sympathetic tone, and in direct prohypertrophic effects on myocytes of heart and arteries, as well as causing collagen overproduction and cardiomyopathy in uraemic rat heart. 13,14 These processes can lead to cardiac hypertrophy in vivo. 17 In our study, we have found association between left ventricular diastolic dysfunction (E/A) and level of plasma EO in HT þ DM þ CKD group.…”

“…10 Beyond the classical effects of EO, the digitalis-like factors have genomic effects inducing hypertrophy in myocardial and vascular smooth muscle cells because of the activation of numerous known intracellular signaling pathways. [11][12][13] The digitalis-like factors also stimulate fibroblast causing collagen overproduction and fibrosis in rats, which is perhaps important in the development of cardiomyopathy. 14 Some human studies have shown that the elevated plasma EO positively correlates with systolic and diastolic blood pressure in untreated hypertensive individuals and further studies have found positive correlation with left ventricular mass index, and left ventricular end-diastolic volume and cardiac dysfunction.…”

Src-mediated Inter-receptor Cross-talk between the Na+/K+-ATPase and the Epidermal Growth Factor Receptor Relays the Signal from Ouabain to Mitogen-activated Protein Kinases

“…Furthermore, our previous work also demonstrated that ouabain-induced increases in reactive oxygen species production in HeLa cells and rat cardiac myocytes correlated well with the ouabain sensitivities of the ␣ 1 isoforms expressed in these cells (10). It was also reported that in canine smooth muscle cells, MAPKs were activated by 1 nM ouabain that would bind to about 5% of the enzyme in these cells (21). Clearly, the signal transducing effects of ouabain are indeed due to its interaction with Na ϩ /K ϩ -ATPase.…”

Na/K-ATPase Tethers Phospholipase C and IP3 Receptor into a Calcium-regulatory Complex

“…First, binding of ouabain to the signaling Na/KATPase activates multiple signaling pathways and regulates transcription and translation of many genes in cardiac myocytes and other cell types. Second, activation of several of these pathways is independent of changes in intracellular ion concentrations (Liu et al, 2000;Aydemir-Koksoy et al, 2001;Miyakawa-Naito et al, 2003). Third, Na/K-ATPase is found to interact directly with neighboring membrane proteins and organized cytosolic cascades of signaling complexes to transmit the ouabain signal to different intracellular compartments (Xie and Cai, 2003).…”

“…Recent work from several laboratories indicates that the enzyme also functions as a signaltransducing receptor for both endogenous and exogenous cardiotonic steroids such as ouabain (Kometiani et al, 1998;Xie et al, 1999;Haas et al, 2000Haas et al, , 2002Liu et al, 2000;Aizman et al, 2001;Aydemir-Koksoy et al, 2001;Miyakawa-Naito et al, 2003). First, binding of ouabain to the signaling Na/KATPase activates multiple signaling pathways and regulates transcription and translation of many genes in cardiac myocytes and other cell types.…”