Ouabain Interaction with Cardiac Na+/K+-ATPase Initiates Signal Cascades Independent of Changes in Intracellular Na+ and Ca2+

We have shown previously that partial inhibition of the cardiac myocyte Na+/K+-ATPase activates signal pathways that regulate myocyte growth and growth-related genes and that increases in intracellular Ca2+ concentration ([Ca2+]i) and reactive oxygen species (ROS) are two essential second messengers within these pathways. The aim of this work was to explore the relation between [Ca2+]i and ROS. When myocytes were in a Ca2+-free medium, ouabain caused no change in [Ca2+]i, but it increased ROS as it did when the cells were in a Ca2+-containing medium. Ouabain-induced increase in ROS also occurred under conditions where there was little or no change in [Na+]i. Exposure of myocytes in Ca2+-free medium to monensin did not increase ROS. Increase in protein tyrosine phosphorylation, an early event induced by ouabain, was also independent of changes in [Ca2+]i and [Na+]i. Ouabain-induced generation of ROS in myocytes was antagonized by genistein, a dominant negative Ras, and myxothiazol/diphenyleneiodonium, indicating a mitochondrial origin for the Ras-dependent ROS generation. These findings, along with our previous data, indicate that increases in [Ca2+]i and ROS in cardiac myocytes are induced by two parallel pathways initiated at the plasma membrane: One being the ouabain-altered transient interactions of a fraction of the Na+/K+-ATPase with neighboring proteins (Src, growth factor receptors, adaptor proteins, and Ras) leading to ROS generation, and the other, inhibition of the transport function of another fraction of the Na+/K+-ATPase leading to rise in [Ca2+]i. Evidently, the gene regulatory effects of ouabain in cardiac myocytes require the downstream collaborations of ROS and [Ca2+]i

Self-Normalized Importance Sampling for Neural Language Modeling

To mitigate the problem of having to traverse over the full vocabulary in the softmax normalization of a neural language model, sampling-based training criteria are proposed and investigated in the context of large vocabulary word-based neural language models. These training criteria typically enjoy the benefit of faster training and testing, at a cost of slightly degraded performance in terms of perplexity and almost no visible drop in word error rate. While noise contrastive estimation is one of the most popular choices, recently we show that other sampling-based criteria can also perform well, as long as an extra correction step is done, where the intended class posterior probability is recovered from the raw model outputs. In this work, we propose self-normalized importance sampling. Compared to our previous work, the criteria considered in this work are self-normalized and there is no need to further conduct a correction step. Through self-normalized language model training as well as lattice rescoring experiments, we show that our proposed self-normalized importance sampling is competitive in both research-oriented and production-oriented automatic speech recognition tasks.Comment: Accepted at INTERSPEECH 202

SDT: A Low-cost and Topology-reconfigurable Testbed for Network Research

Network experiments are essential to network-related scientific research (e.g., congestion control, QoS, network topology design, and traffic engineering). However, (re)configuring various topologies on a real testbed is expensive, time-consuming, and error-prone. In this paper, we propose \emph{Software Defined Topology Testbed (SDT)}, a method for constructing a user-defined network topology using a few commodity switches. SDT is low-cost, deployment-friendly, and reconfigurable, which can run multiple sets of experiments under different topologies by simply using different topology configuration files at the controller we designed. We implement a prototype of SDT and conduct numerous experiments. Evaluations show that SDT only introduces at most 2\% extra overhead than full testbeds on multi-hop latency and is far more efficient than software simulators (reducing the evaluation time by up to 2899x). SDT is more cost-effective and scalable than existing Topology Projection (TP) solutions. Further experiments show that SDT can support various network research experiments at a low cost on topics including but not limited to topology design, congestion control, and traffic engineering.Comment: This paper will be published in IEEE CLUSTER 2023. Preview version onl

Intracellular Reactive Oxygen Species Mediate the Linkage of Na+/K+-ATPase to Hypertrophy and its Marker Genes in Cardiac Myocytes

We showed before that in cardiac myocytes partial inhibition of Na+/K+-ATPase by nontoxic concentrations of ouabain causes hypertrophy and transcriptional regulations of growth-related marker genes through multiple Ca2+-dependent signal pathways many of which involve Ras and p42/44 mitogen-activated protein kinases. The aim of this work was to explore the roles of intracellular reactive oxygen species (ROS) in these ouabain-initiated pathways. Ouabain caused a rapid generation of ROS within the myocytes that was prevented by preexposure of cells to N-acetylcysteine (NAC) or vitamin E. These antioxidants also blocked or attenuated the following actions of ouabain: inductions of the genes of skeletal α-actin and atrial natriuretic factor, repression of the gene of the α3-subunit of Na+/K+-ATPase, activation of mitogen-activated protein kinases, activation of Ras-dependent protein synthesis, and activation of transcription factor NF-κB. Induction of c-fos and activation of AP-1 by ouabain were not sensitive to NAC. Ouabain-induced inhibition of active Rb+ uptake through Na+/K+-ATPase and the resulting rise in intracellular Ca2+ were also not prevented by NAC. A phorbol ester that also causes myocyte hypertrophy did not increase ROS generation, and its effects on marker genes and protein synthesis were not affected by NAC. We conclude the following: (a) ROS are essential second messengers within some but not all signal pathways that are activated by the effect of ouabain on Na+/K+-ATPase; (b) the ROS-dependent pathways are involved in ouabain-induced hypertrophy; (c) increased ROS generation is not a common response of the myocyte to all hypertrophic stimuli; and (d) it may be possible to dissociate the positive inotropic effect of ouabain from its growth-related effects by alteration of the redox state of the cardiac myocyte

Regulation of Apical NHE3 Trafficking by Ouabain-Induced Activation of Basolateral Na/K-ATPase Receptor Complex

The long-term effects of ouabain on transepithelial Na+ transport involve transcriptional downregulation of apical Na+/H+ exchanger isoform 3 (NHE3). The aim of this study was to determine whether ouabain could acutely regulate NHE3 via a posttranscriptional mechanism in LLC-PK1 cells. We observed that the basolateral, but not apical, application of ouabain for 1 h significantly reduced transepithelial Na+ transport. This effect was not due to changes in the integrity of tight junctions or increases in the intracellular Na+ concentration. Ouabain regulated the trafficking of NHE3 and subsequently inhibited its activity, a process independent of intracellular Na+ concentration. Ouabain-induced NHE3 trafficking was abolished by either cholesterol depletion or Src inhibition. Moreover, ouabain increased the intracellular Ca2+concentration. Pretreatment of cells with the intracellular Ca2+ chelator BAPTA-AM blocked ouabain-induced trafficking of NHE3. Also, blockade of Na+-K+-ATPase endocytosis by a phosphatidylinositol 3-kinase inhibitor was equally effective in attenuating ouabain-induced NHE3 trafficking. These data indicate that ouabain acutely stimulates NHE3 trafficking by activating the basolateral Na+-K+-ATPase signaling complex. Taken together with our previous observations, we propose that ouabain can simultaneously regulate basolateral Na+-K+-ATPase and apical NHE3, leading to inhibition of transepithelial Na+ transport. This mechanism may be relevant to proximal tubular Na+ handling during conditions associated with increases in circulating endogenous cardiotonic steroids

Gender differences in the development of uremic cardiomyopathy following partial nephrectomy: Role of progesterone

Gender difference has been suggested as a risk factor for developing cardiovascular and renal diseases in humans and experimental animals. As a major sex hormone, progesterone was reported to compete with cardiotonic steroid binding to Na/K-ATPase. Our previous publication demonstrated that cardiotonic steroids (e.g., marinobufagenin) play an important role in the development of experimental uremic cardiomyopathy. We also observed that the putative mineralocorticoid antagonists, spironolactone and its major metabolite canrenone, antagonize binding of cardiotonic steroids to Na/K-ATPase in a competitive manner and also ameliorate experimental uremic cardiomyopathy induced by partial nephrectomy. In the following studies, we noted that progesterone displayed competitive inhibition of cardiotonic steroid binding to Na/K-ATPase and partially inhibited collagen synthesis induced by marinobufagenin in cultured cardiac fibroblasts. Therefore, we sought to examine whether female rats displayed less uremic cardiomyopathy than male rats when subjected to partial nephrectomy. Although partial nephrectomy caused the induction of smaller increases in blood pressure of female rats, they appeared to be similarly susceptible to cardiac remodeling induced by partial nephrectomy in terms of hypertrophy and fibrosis as age-matched male rats. The possible explanations for our findings are therefore discussed