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...

Background and purpose: Reactive oxygen and nitrogen species play an important role in the development of diabetic cardiomyopathy. They can activate matrix metalloproteinases (MMPs), and MMP-2 in particular is known to mediate early consequences of oxidative stress injury in the heart. Therefore, we investigated the role of MMP-2 and the effect of the MMP inhibitor doxycycline on the changes of heart function caused by diabetes. Experimental approach: Using streptozotocin-induced diabetic rats, we evaluated the effect of doxycycline on both mechanical and electrical function of isolated hearts, papillary muscle and cardiomyocytes. Key results: Doxycycline abolished the diabetes-induced depression in left ventricular developed pressure and the rates of changes in developed pressure in isolated hearts and normalized the prolongation of the action potential in papillary muscles. In cardiomyocytes isolated from doxycycline-treated diabetic rats, the altered kinetic parameters of Ca 2 þ transients, depressed Ca 2 þ loading of sarcoplasmic reticulum and basal intracellular Ca 2 þ level, and the spatio-temporal properties of Ca 2 þ sparks were significantly restored. Gelatin zymography and western blot data indicated that the diabetes-induced alterations in MMP-2 activity and protein level, level of tissue inhibitor of matrix metalloproteinase-4 and loss of troponin I were restored to control levels with doxycycline. Conclusions and implications: Our data suggest that these beneficial effects of doxycycline on the mechanical, electrical and biochemical properties of the diabetic rat heart appear, at least in part, to be related to inhibition of MMP activity, implying a role for MMPs in the development of diabetic cardiomyopathy.

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Reactive oxygen species (ROS) play important roles in the development of diabetic cardiomyopathy. Matrix metalloproteinases (MMPs) can get activated by ROS and contribute to loss of myocardial contractile function in oxidative stress injury. Previously we have shown that either a MMP-2 inhibitor doxycycline or an antioxidant selenium treatment in vivo prevented diabetes-induced cardiac dysfunction significantly. In addition, there is an evidence for impaired cardiac responsiveness to beta-adrenoceptor (beta AR) stimulation in experimental animals with diabetes. The exact nature of linkage between the functional depression in cardiac responses to catecholamines and the variations in uncoupling of beta AR in diabetes has not been clearly defined. Therefore, we aimed to evaluate the effect of in vivo administration of doxycycline on beta AR responses of isolated hearts from diabetic rats and compare these data with two well-known antioxidants; sodium selenate and (n-3) fatty acid-treated diabetic rats. We examined the changes in the basal cardiac function in response to the beta AR stimulation, adenylate cyclase activity, and beta AR affinity to its agonist, isoproterenol. These results showed that antioxidant treatment of diabetic rats could protect the hearts against diabetes-induced depression in beta AR responses, significantly while doxycycline did not have any significant beneficial action on these parameters. As a summary, present data, in part, demonstrate that antioxidants and MMP inhibitors could both regulate MMP function but may also utilize different mechanisms of action in cardiomyocytes, particularly related with beta AR signaling pathway.

The Na(+) pump and its regulation is important for maintaining membrane potential and transmembrane Na(+) gradient in all mammalian cells and thus is essential for cell survival and function. Vascular smooth muscle cells (VSMC) have a relatively low number of pump sites on their membrane compared with other cells. We wished to determine the mechanisms for regulating the number of pump sites in these cells. We used canine saphenous vein VSMC cultured in 10% serum and passaged one time. These cells were subcultured in 5% serum media with low K(+) (1 mM vs. control of 5 mM), and their pump expression was assessed. These VSMC upregulated their pump sites as early as 4 h after treatment (measured by [(3)H]ouabain binding). At this early time point, there was no detectable increase in protein expression of either alpha(1)- or beta(1)-subunits of the pump shown by Western blots. When the cells were treated with the phosphoinositide 3-kinase (PI-3-K) inhibitor LY-294002 (which is known to inhibit cytoplasmic transport processes) in low-K(+) media, the pump site upregulation was inhibited. These data suggest that the low-K(+)-induced upregulation of Na(+) pump number can occur by translocation of preformed pumps from intracellular stores.

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Diabetes is characterized with increased oxidant stress, vasculopathy, and neuropathy. In diabetic vasculopathy, the observed thickening of the media and intima is not only a result of vascular smooth muscle cell proliferation but also due to modification of the extracellular matrix by these cells. Also, there is hampered membrane function and a reduction in sodium pump expression in the vessels of the diabetic animals. Selenium, being a trace element, has both insulinomimetic and antioxidant effects. Thus, we hypothesized that selenium treatment will reduce proliferation, restore physiology, and correct increased proliferation signaling of diabetic aorta. Diabetes was induced by streptozotocin (50 mg/kg body weight), and rats were then treated with sodium selenate (15 mumol/kg body weight/day) for 4 weeks. Our data from diabetic rats showed an increase in proliferation rate and matrix metalloproteinase activity in aortic cell cultures. We observed marked increases in MAPK phosphorylation and caveolin 1 expression but a decrease in Na(+)/K(+) ATPase activity in diabetic rat aorta homogenates. Selenium treatment resulted in complete normalization of the above parameters to control level, while it increased Na(+)/K(+) pump activity by 40%. Our results suggest that selenium treatment of diabetics can play beneficial role in protecting vascular architecture and function against diabetes-induced pathology.