Sandrine V. Pierre

Identification of a Pool of Non-pumping Na/K-ATPase *

Recent studies have ascribed many non-pumping functions to the Na/K-ATPase. Here, we present experimental evidence demonstrating that over half of the plasma membrane Na/K-ATPase in LLC-PK1 cells is performing cellular functions other than ion pumping. This “non-pumping” pool of Na/K-ATPase, like the pumping pump, binds ouabain. Depletion of either cholesterol or caveolin-1 moves some of the “non-pumping” Na/K-ATPase into the pumping pool. Graded knock-down of the α1 subunit of the Na/K-ATPase eventually results in loss of this “non-pumping” pool while preserving the pumping pool. Our prior studies indicate that a loss of the non-pumping pool is associated with a loss of receptor function as evidenced by the failure of ouabain administration to induce the activation of Src and/or ERK. Therefore, our new findings suggest that a substantial amount of surface-expressed Na/K-ATPase, at least in some types of cells, may function as non-canonical ouabain-binding receptors.

Cardioprotective signaling to mitochondria

Mitochondria are central players in the pathophysiology of ischemia-reperfusion. Activation of plasma membrane G-coupled receptors or the Na,K-ATPase triggers cytosolic signaling pathways that result in cardioprotection. Our working hypothesis is that the occupied receptors migrate to caveolae, where signaling enzymes are scaffolded into signalosomes that bud off the plasma membrane and migrate to mitochondria. The signalosome-mitochondria interaction then initiates intramitochondrial signaling by opening the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)). MitoK(ATP) opening causes an increase in ROS production, which activates mitochondrial protein kinase C epsilon (PKCvarepsilon), which inhibits the mitochondrial permeability transition (MPT), thus decreasing cell death. We review the experimental findings that bear on these hypotheses and other modes of protection involving mitochondria.

The Na,K-ATPase Receptor Complex Its Organization and Membership

A major difference between the Na,K-ATPase ion pump and other P-type ATPases is its ability to bind cardiotonic steroids such as ouabain. Na,K-ATPase also interacts with many membrane and cytosolic proteins. In addition to their role in Na,K-ATPase regulation, it became apparent that some of the newly identified interactions are capable of organizing the Na,K-ATPase into various signaling complexes. This new function confers a ligand-like effect to cardiotonic steroids on cellular signal transduction. This article reviews these new developments and provides a comparison of Na,K-ATPase-mediated signal transduction with other receptors and ion transporters.

Conditioning the heart induces formation of signalosomes that interact with mitochondria to open mitoKATP channels

Perfusion of the heart with bradykinin triggers cellular signaling events that ultimately cause opening of mitochondrial ATP-sensitive K+ (mitoKATP) channels, increased H2O2 production, inhibition of the mitochondrial permeability transition (MPT), and cardioprotection. We hypothesized that the interaction of bradykinin with its receptor induces the assembly of a caveolar signaling platform (signalosome) that contains the enzymes of the signaling pathway and that migrates to mitochondria to induce mitoKATP channel opening. We developed a novel method for isolating and purifying signalosomes from Langendorff-perfused rat hearts treated with bradykinin. Fractions containing the signalosomes were found to open mitoKATP channels in mitochondria isolated from untreated hearts via the activation of mitochondrial PKC-epsilon. mitoKATP channel opening required signalosome-dependent phosphorylation of an outer membrane protein. Immunodetection analysis revealed the presence of the bradykinin B2 receptor only in the fraction isolated from bradykinin-treated hearts. Immunodetection and immunogold labeling of caveolin-3, as well as sensitivity to cholesterol depletion and resistance to Triton X-100, attested to the caveolar nature of the signalosomes. Ischemic preconditioning, ischemic postconditioning, and perfusion with ouabain also led to active signalosome fractions that opened mitoKATP channels in mitochondria from untreated hearts. These results provide initial support for a novel mechanism for signal transmission from a plasma membrane receptor to mitoKATP channels.

Alterations of Na+/K+-ATPase function in caveolin-1 knockout cardiac fibroblasts

Recent studies have demonstrated that the Na(+)/K(+)-ATPase is not only an ion pump, but also a membrane receptor that confers the ligand-like effects of cardiotonic steroids (CTS) such as ouabain on protein kinases and cell growth. Because CTS have been implicated in cardiac fibrosis, this study examined the role of caveolae in the regulation of Na(+)/K(+)-ATPase function and CTS signaling in cardiac fibroblasts. In cardiac fibroblasts prepared from wild-type and caveolin-1 knockout [Cav-1(-/-)] mice, we found that the absence of caveolin-1 did not affect total cellular amount or surface expression of Na(+)/K(+)-ATPase alpha1 subunit. However, it did increase ouabain-sensitive (86)Rb(+) uptake. While knockout of caveolin-1 increased basal activities of Src and ERK1/2, it abolished the activation of these kinases induced by ouabain but not angiotensin II. Finally, ouabain stimulated collagen synthesis and cell proliferation in wild type but not Cav-1(-/-) cardiac fibroblasts. Thus, we conclude that caveolae are important for regulating both pumping and signal transducing functions of Na(+)/K(+)-ATPase. While depletion of caveolae increases the pumping function of Na(+)/K(+)-ATPase, it suppresses CTS-induced signal transduction, growth, and collagen production in cardiac fibroblasts.

Different roles of the cardiac Na+/Ca2+-exchanger in ouabain-induced inotropy, cell signaling, and hypertrophy

Previous studies have shown that digitalis drugs, acting as specific inhibitors of cardiac Na(+)/K(+)-ATPase, not only cause positive inotropic effects, but also activate cell signaling pathways that lead to cardiac myocyte hypertrophy. A major aim of this work was to assess the role of Na(+)/Ca(2+)-exchanger, NCX1, in the above two seemingly related drug effects. Using a mouse with ventricular-specific knockout (KO) of NCX1, ouabain-induced positive inotropy that was evident in isolated wild-type (Wt) hearts was clearly reduced in KO hearts. Ouabain also increased Ca(2+) transient amplitudes in Wt myocytes, but not in KO myocytes. Ouabain-induced activations of ERK 1/2 were noted in Wt myocytes, but not in KO myocytes; however, ouabain activated PI3K1A and Akt in both Wt and KO myocytes. Protein synthesis rate, as a measure of hypertrophy, was increased by ouabain in Wt and KO myocytes; these drug effects were prevented by a PI3K inhibitor but not by a MEK/ERK inhibitor. Hypertrophy caused by ET-1, but not that induced by ouabain, was accompanied by upregulation of BNP gene in Wt and KO myocytes. The findings indicate 1) the necessity of NCX1 for positive inotropic action of ouabain; 2) the irrelevance of NCX1 and ERK 1/2 activation to ouabain-induced hypertrophy; and 3) that hypertrophy caused by ouabain through the activation of PI3K1A/Akt pathway is likely to be beneficial to the heart.

Preconditioning by subinotropic doses of ouabain in the Langendorff perfused rabbit heart.

Short exposure to low concentrations of digitalis drugs like ouabain protects the rat heart against ischemia/reperfusion injury through the activation of the Na/K-adenosine triphosphatase (ATPase)/Src receptor complex and subsequent stimulation of key intracellular cardioprotective signals. Rat Na/K-ATPase, however, is relatively insensitive to digitalis, and it is not known if similar results could be obtained in species with higher sensitivity. Thus, to determine whether ouabain pretreatment protects against ischemic injury and activates the Na/K-ATPase signaling cascade in a species with cardiac glycoside sensitivity comparable to humans, the present study was conducted in the rabbit model. In Langendorff perfused rabbit hearts, 20-minute exposure to 500-nM ouabain resulted in positive inotropy as evidenced by a significant increase in +dP/dt, and this increase was accompanied by the activation of several well-characterized downstream mediators of the cardiac Na/K-ATPase receptor pathway, including Src, Akt, ERK1/2, and protein kinase Cϵ. A short (4 minutes) administration of a subinotropic dose of ouabain (100 nM) followed by an 8-minute washout before 30 minutes of global ischemia and 120 minutes of reperfusion resulted in protection against cell death, as evidenced by a significant decrease in infarct size. These data indicate that ouabain administration activates the Na/K-ATPase signaling cascade and protects against ischemic injury in a species with high cardiac Na/K-ATPase sensitivity.

Modulation of cardiac Na+,K+-ATPase cell surface abundance by simulated ischemia-reperfusion and ouabain preconditioning.

Na(+),K(+)-ATPase and cell survival were investigated in a cellular model of ischemia-reperfusion (I/R)-induced injury and protection by ouabain-induced preconditioning (OPC). Rat neonatal cardiac myocytes were subjected to 30 min of substrate and coverslip-induced ischemia followed by 30 min of simulated reperfusion. This significantly compromised cell viability as documented by lactate dehydrogenase release and Annexin V/propidium iodide staining. Total Na(+),K(+)-ATPase α(1)- and α(3)-polypeptide expression remained unchanged, but cell surface biotinylation and immunostaining studies revealed that α(1)-cell surface abundance was significantly decreased. Na(+),K(+)-ATPase-activity in crude homogenates and (86)Rb(+) transport in live cells were both significantly decreased by about 30% after I/R. OPC, induced by a 4-min exposure to 10 μM ouabain that ended 8 min before the beginning of ischemia, increased cell viability in a PKCε-dependent manner. This was comparable with the protective effect of OPC previously reported in intact heart preparations. OPC prevented I/R-induced decrease of Na(+),K(+)-ATPase activity and surface expression. This model also revealed that Na(+),K(+)-ATPase-mediated (86)Rb(+) uptake was not restored to control levels in the OPC group, suggesting that the increased viability was not conferred by an increased Na(+),K(+)-ATPase-mediated ion transport capacity at the cell membrane. Consistent with this observation, transient expression of an internalization-resistant mutant form of Na(+),K(+)-ATPase α(1) known to have increased surface abundance without increased ion transport activity successfully reduced I/R-induced cell death. These results suggest that maintenance of Na(+),K(+)-ATPase cell surface abundance is critical to myocyte survival after an ischemic attack and plays a role in OPC-induced protection. They further suggest that the protection conferred by increased surface expression of Na(+),K(+)-ATPase may be independent of ion transport.

Ceacam1 deletion causes vascular alterations in large vessels

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes hepatic insulin clearance and endothelial survival. However, its role in the morphology of macrovessels remains unknown. Mice lacking Ceacam1 (Cc1-/-) exhibit hyperinsulinemia, which causes insulin resistance and fatty liver. With increasing evidence of an association among hyperinsulinemia, fatty liver disease, and atherosclerosis, we investigated whether Cc1-/- exhibited vascular lesions in atherogenic-prone aortae. Histological analysis revealed impaired endothelial integrity with restricted fat deposition and aortic plaque-like lesions in Cc1-/- aortae, likely owing to their limited lipidemia. Immunohistochemical analysis indicated macrophage deposition, and in vitro studies showed increased leukocyte adhesion to aortic wall, mediated in part by elevation in vascular cell adhesion molecule 1 levels. Basal aortic eNOS protein and NO content were reduced, in parallel with reduced Akt/eNOS and Akt/Foxo1 phosphorylation. Ligand-induced vasorelaxation was compromised in aortic rings. Increased NADPH oxidase activity and plasma 8-isoprostane levels revealed oxidative stress and lipid peroxidation in Cc1-/- aortae. siRNA-mediated CEACAM1 knockdown in bovine aortic endothelial cells adversely affected insulins stimulation of IRS-1/PI 3-kinase/Akt/eNOS activation by increasing IRS-1 binding to SHP2 phosphatase. This demonstrates that CEACAM1 regulates both endothelial cell autonomous and nonautonomous mechanisms involved in vascular morphology and NO production in aortae. Systemic factors such as hyperinsulinemia could contribute to the pathogenesis of these vascular abnormalities. Cc1-/- mice provide a first in vivo demonstration of distinct CEACAM1-dependent hepatic insulin clearance linking hepatic to macrovascular abnormalities.

Modulation of Na+-K+-ATPase cell surface abundance through structural determinants on the α1-subunit

Through their ion-pumping and non-ion-pumping functions, Na(+)-K(+)-ATPase protein complexes at the plasma membrane are critical to intracellular homeostasis and to the physiological and pharmacological actions of cardiotonic steroids. Alteration of the abundance of Na(+)-K(+)-ATPase units at the cell surface is one of the mechanisms for Na(+)-K(+)-ATPase regulation in health and diseases that has been closely examined over the past few decades. We here summarize these findings, with emphasis on studies that explicitly tested the involvement of defined regions or residues on the Na(+)-K(+)-ATPase α1 polypeptide. We also report new findings on the effect of manipulating Na(+)-K(+)-ATPase membrane abundance by targeting one of these defined regions: a dileucine motif of the form [D/E]XXXL[L/I]. In this study, opossum kidney cells stably expressing rat α1 Na(+)-K(+)-ATPase or a mutant where the motif was disrupted (α1-L499V) were exposed to 30 min of substrate/coverslip-induced-ischemia followed by reperfusion (I-R). Biotinylation studies suggested that I-R itself acted as an inducer of Na(+)-K(+)-ATPase internalization and that surface expression of the mutant was higher than the native Na(+)-K(+)-ATPase before and after ischemia. Annexin V/propidium iodide staining and lactate dehydrogenase release suggested that I-R injury was reduced in α1-L499V-expressing cells compared with α1-expressing cells. Hence, modulation of Na(+)-K(+)-ATPase cell surface abundance through structural determinants on the α-subunit is an important mechanism of regulation of cellular Na(+)-K(+)-ATPase in various physiological and pathophysiological conditions, with a significant impact on cell survival in face of an ischemic stress.