"Tis Better to Give Than to Receive?" Health-related Benefits of Delivering Peer Support in Type 2 Diabetes: An Explanatory Sequential Mixed-methods Study.

OBJECTIVES: Existing peer support literature in diabetes has focussed predominantly on the health impact it has on the beneficiaries rather than the benefactors. In this mixed-methods study, we examined the effect of delivering peer support (vs receiving) on glycated hemoglobin (A1C) and diabetes distress (DD) at 3 and 12 months as part of a larger diabetes self-management support randomized controlled trial. Maintenance or improvement of outcomes was expected. We also assessed peer leaders' experiences with the program. METHODS: We utilized a sequential explanatory mixed-methods research design that included 58 adults with diabetes (i.e. peer leaders) who completed a 30-hour training program. Peer leaders (n=52) were matched with participants (adults with type 2 diabetes) and invited to undergo assessments at baseline, 3 months and 12 months. Primary clinical and psychosocial outcomes included A1C and DD, respectively. Secondary outcomes were cardiovascular risk factors and depressive symptoms. After the intervention, 17 peer leaders participated in semistructured interviews about their experience. RESULTS: Peer leaders had a mean age of 57.5±11 years and a long history of diabetes (13.9±11 years); over half were male (53.8%) and married/partnered (55.8%). At baseline, peer leaders were at target for A1C (7.0±0.9% [53±10 mmol/mol]) and reported a low level of DD (1.67±0.52). Of the 43 (82.7%) peer leaders who completed the 12-month study, A1C and DD remained stable over 12 months. Secondary outcomes also remained within the normal range from the start to the end of the intervention. CONCLUSION: Delivering peer support may help maintain glycemic control and DD over the long term.

Predicting the role of coping factors on pandemic-related anxiety.

The year 2020 saw the emergence of a worldwide pandemic caused by the novel coronavirus COVID-19. Measures against further spread of the virus were taken nearly everywhere in the world. Many countries also imposed social distancing rules and lockdowns on their population. This situation has caused a lot of fear and insecurity, along with reactance and even unrest in some countries. In this study, we measured the psychological concepts of resilience, reactance, positive schemas, social solidarity, and anxiety among psychiatric patients and in how far these factors influence their psychopathological anxiety during the pandemic. The aim was to better understand in what ways these factors influence pandemic anxiety to be able to reduce its negative psychological effects. Findings show a significant effect of positive schemas and social solidarity on the level of pandemic anxiety in a sample of psychiatric patients, but no correlation between resilience or reactance and pandemic anxiety. Based on these insights, the inclusion of positive schemas and social solidarity for therapy should be considered. Looking deeper into the relation between positive schemas and pandemic anxiety could provide insight into the different ways that schemas influence people's anxiety and determine whether some of them are particularly important.

Profiling "Success": Demographic and Personality Predictors of Effective Peer Leaders in a Diabetes Self-management Intervention.

BACKGROUND: The sociodemographic and personality profiles of effective peer leaders in the context of diabetes self-management interventions are poorly understood. In this study, we explored the demographic and personality characteristics of peer leaders participating in a 12-month, telephone-based type 2 diabetes self-management intervention. METHODS: We used a sequential explanatory mixed-methods research design and recruited 52 peer leaders. Thirty-seven peer leaders had at least 1 participant complete both the baseline and the 12-month assessments. Eligible peer leader candidates were English-speaking adults (≥21 years of age) with diabetes and a self-reported glycated hemoglobin (A1C) level of ≤8% who had access to a phone and transportation and were willing to attend a 30-hour training program. Peer leaders completed a self-report survey assessing sociodemographic characteristics and a Mini-International Personality Item Pool scale measuring the "Big 5" personality traits. After the intervention, 17 peer leaders participated in semistructured interviews on their program experience. We categorized peer leaders as effective if their participants sustained or improved their A1C and diabetes distress (DD) scores from baseline to 12 months, and as ineffective if their participants worsened on any of these parameters. RESULTS: Our cohort scored highest on agreeableness and lowest on neuroticism. Twenty peer leaders were considered effective, most of whom were male, married, employed and educated. They also had significantly lower mean DD levels (p=0.02) and a higher extroversion score (p=0.03) at baseline. CONCLUSIONS: Extroversion emerged as the best personality predictor of peer leader effectiveness. These results, in combination with interview responses, were used to produce a peer leader selection model.

Out of the mouths of Peer Leaders: Perspectives on how to improve a telephone-based peer support intervention in type 2 diabetes.

OBJECTIVE: To explore the experiences of peer leaders with respect to delivering core components of a 12-month, telephone-based peer support intervention in type 2 diabetes within a tertiary-care setting. METHODS: Seventeen peer leaders were recruited and interviewed. Interviews lasted approximately 20 to 45 min, were audio-taped, and transcribed verbatim. The transcripts were analysed by two team members using the qualitative descriptive approach. FINDINGS: Peer leaders reported mutually beneficial and reciprocal relationships with participants. They encountered challenges in maintaining regular contact with participants and in motivating them to make lifestyle changes. To improve the programme, peer leaders suggested having more frequent - but shorter - training sessions and reducing the diabetes education component of the training programme. To enhance the intervention fidelity and retention rate, they recommended matching peer leaders to participants on more meaningful variables (e.g. diabetes-related commonalities, personality, life experiences, etc.) beyond just gender, geographic proximity and availability. They also requested more frequent face-to-face contacts with participants (Modality of Contact), and additional ongoing support from the research team. CONCLUSION: Peer leaders were satisfied with the intervention design. However, future studies may consider more comprehensive peer leader-matching algorithms and increased opportunities for in-person communication modalities. CLINICALTRIALS: gov Identifier: NCT02804620.

Masson's Tumor of the Hand: An Uncommon Histopathological Entity.

Masson's tumor or Masson's hemangioma, more precisely termed intravascular papillary endothelial hyperplasia (IPEH), is an uncommon benign vascular lesion of the skin and subcutaneous tissues which can be frequently confused with angiosarcoma. Although relatively rare, its accurate diagnosis is essential since it can clinically be similar to both benign and malignant lesions. We present a 39-year-old man with a round bulging arising from the left palm side of the hand with gradual growth in the last 5 months and on and off tenderness. The microscopic section demonstrated the papillary proliferation of endothelial cells in favor of Masson's hemangioma, which was sufficiently treated with excision alone.

Peer support interventions in type 2 diabetes: Review of components and process outcomes.

BACKGROUND: This review seeks to identify (a) the various components and process outcomes of type 2 diabetes peer support (PS) interventions and (b) the measures implemented to monitor intervention fidelity and evaluate outcomes in these studies. METHODS: The MEDLINE, PubMed, EMBASE (Excerpta Medica Database), CENTRAL (Cochrane Central Register of Controlled Trials), CINAHL (Cumulative Index to Nursing and Allied Health Literature), and PsycINFO databases were searched from inception to May 2019. Two reviewers independently screened and extracted data from eligible articles via the Template for Intervention Description and Replication (TIDieR) checklist (why, what, who provided, how, where, when and how much, tailoring, modifications, and how well). RESULTS: Twenty-three trials were included. The total number of participants was 7178. Most interventions were in primary care. Although face-to-face was the most common modality of contact, rates of contact were highest for telephone. Potential peer leaders (PLs) were identified primarily through recommendations from health professionals, based on their communication skills, glycosylated hemoglobin (HbA1c), and coaching interest. PLs were mostly female, university educated, and had a long history of diabetes (≥ 10 years). PL training varied significantly in length and content; the two most frequent topics were communication skills and diabetes knowledge. Although several studies implemented methods to evaluate "intervention fidelity," only few rigorously assessed the two key components of fidelity, "adherence" and "competence," through audio- and video-taping or direct observations. CONCLUSIONS: The impact of PS on participants' health outcomes is well investigated; however, the implementation and evaluation strategies vary significantly across these studies. In the present review, we define the various components of PS interventions and propose suggestions for enhancing the implementation and evaluation of future PS models.

The other functions of the sodium pump.

Na/K-ATPase (the sodium pump) was discovered in the 1950s as the plasma membrane enzyme that carries out the coupled active transports of Na+ and K+ across the membranes of nearly all eukaryotic cells. It was not until the 1990s when it was shown that besides pumping ions, Na/K-ATPase is also capable of stimulus-induced interactions with neighboring proteins that lead to activations of signal transduction pathways causing cell growth. This article is an attempt to review the progress of the research on these signaling functions of sodium pump during the past 2-3 decades. The covered topics include (a) the controversial digitalis-induced growth activations through the epidermal growth factor receptor and Src kinase in cardiac myocytes and several other cell types; (b) the extensive findings on digitalis-induced growth activations in cardiac myocytes and other cell types through phosphatidylinositol 3-kinases; and (c) a number of interesting but insufficiently studied signaling functions of the sodium pump.

The sodium pump and digitalis drugs: Dogmas and fallacies.

The sodium pump (Na/K-ATPase) is a plasma membrane enzyme that transports Na+ and K+ against their physiological gradients in most eukaryotic cells. Besides pumping ions, the enzyme may also interact with neighboring proteins to activate cell signaling pathways that regulate cell growth. Digitalis drugs, useful for the treatment of heart failure and atrial arrhythmias, inhibit the pumping function of Na/K-ATPase and stimulate its signaling function. In the current field of research on the sodium pump and digitalis drugs, some issues that are commonly accepted to be well established are not so, and this may impede progress. Here, several such issues are identified, their histories are discussed, and their open discussions are urged. The covered unsettled questions consist of (a) the suggested hormonal role of endogenous digitalis compounds; (b) the specificity of Na/K-ATPase as the receptor for digitalis compounds; (c) the relevance of the positive inotropic action of digitalis to its use for the treatment of heart failure; (d) the conflicting findings on digitalis-induced signaling function of Na/K-ATPase; and (e) the uncertainties about the structure of Na/K-ATPase in the native cell membrane.

Comparison of Digitalis Sensitivities of Na+/K+-ATPases from Human and Pig Kidneys.

Digitalis drugs are selective inhibitors of the plasma membrane Na+/K+-ATPase. There are many studies on molecular mechanisms of digitalis interaction with purified pig kidney enzyme, with the tacit assumption that it is a good model of human kidney enzyme. However, previous studies on crude or recombinant human kidney enzymes are limited, and have not resulted in consistent findings on their digitalis sensitivities. Hence, we prepared comparably purified enzymes from human and pig kidneys and determined inhibitory constants of digoxin, ouabain, ouabagenin, bufalin, and marinobufagenin (MBG) on enzyme activity under optimal turnover conditions. We found that each compound had the same potency against the two enzymes, indicating that (i) the pig enzyme is an appropriate model of the human enzyme, and (ii) prior discrepant findings on human kidney enzymes were either due to structural differences between the natural and recombinant enzymes or because potencies were determined using binding constants of digitalis for enzymes under nonphysiological conditions. In conjunction with previous findings, our newly determined inhibitory constants of digitalis compounds for human kidney enzymes indicate that (i) of the compounds that have long been advocated to be endogenous hormones, only bufalin and MBG may act as such at kidney tubules, and (ii) beneficial effects of digoxin, the only digitalis with extensive clinical use, does not involve its inhibitory effect on renal tubular Na+/K+-ATPase.

Evolution of the α-Subunit of Na/K-ATPase from Paramecium to Homo sapiens: Invariance of Transmembrane Helix Topology.

Na/K-ATPase is a key plasma membrane enzyme involved in cell signaling, volume regulation, and maintenance of electrochemical gradients. The α-subunit, central to these functions, belongs to a large family of P-type ATPases. Differences in transmembrane (TM) helix topology, sequence homology, helix-helix contacts, cell signaling, and protein domains of Na/K-ATPase α-subunit were compared in fungi (Beauveria), unicellular organisms (Paramecia), primitive multicellular organisms (Hydra), and vertebrates (Xenopus, Homo sapiens), and correlated with evolution of physiological functions in the α-subunit. All α-subunits are of similar length, with groupings of four and six helices in the N- and C-terminal regions, respectively. Minimal homology was seen for protein domain patterns in Paramecium and Hydra, with high correlation between Hydra and vertebrates. Paramecium α-subunits display extensive disorder, with minimal helix contacts. Increases in helix contacts in Hydra approached vertebrates. Protein motifs known to be associated with membrane lipid rafts and cell signaling reveal significant positional shifts between Paramecium and Hydra vulgaris, indicating that regional membrane fluidity changes occur during evolution. Putative steroid binding sites overlapping TM-3 occurred in all species. Sites associated with G-protein-receptor stimulation occur both in vertebrates and amphibia but not in Hydra or Paramecia. The C-terminus moiety "KETYY," necessary for the Na(+) activation of pump phosphorylation, is not present in unicellular species indicating the absence of classical Na(+)/K(+)-pumps. The basic protein topology evolved earliest, followed by increases in protein domains and ordered helical arrays, correlated with appearance of α-subunit regions known to involve cell signaling, membrane recycling, and ion channel formation.

Ouabain prevents pathological cardiac hypertrophy and heart failure through activation of phosphoinositide 3-kinase α in mouse.

BACKGROUND: Use of low doses of digitalis to prevent the development of heart failure was advocated decades ago, but conflicting results of early animal studies dissuaded further research on this issue. Recent discoveries of digitalis effects on cell signal pathways prompted us to reexamine the possibility of this prophylactic action of digitalis. The specific aim of the present study was to determine if subinotropic doses of ouabain would prevent pressure overload-induced cardiac remodeling in the mouse by activating phosphoinositide 3-kinase α (PI3Kα). RESULTS: Studies were done on an existing transgenic mouse deficient in cardiac PI3Kα (p85-KO) but with normal cardiac contractility, a control mouse (Con), and on cultured adult cardiomyocytes. In Con myocytes, but not in p85-KO myocytes, ouabain activated PI3Kα and Akt, and caused cell growth. This occurred at low ouabain concentrations that did not activate the EGFR-Src/Ras/Raf/ERK cascade. Con and p85-KO mice were subjected to transverse aortic constriction (TAC) for 8 weeks. A subinotropic dose of ouabain (50 µg/kg/day) was constantly administrated by osmotic mini-pumps for the first 4 weeks. All mice were monitored by echocardiography throughout. Ouabain early treatment attenuated TAC-induced cardiac hypertrophy and fibrosis, and improved cardiac function in TAC-operated Con mice but not in TAC-operated p85-KO mice. TAC downregulated α2-isoform of Na(+)/K(+)-ATPase but not its α1-isoform in Con hearts, and ouabain treatment prevented the downregulation of α2-isoform. TAC-induced reduction of α2-isoform did not occur in p85-KO hearts. CONCLUSIONS: Our results show that (a) safe doses of ouabain prevent or delay cardiac remodeling of pressure overloaded mouse heart; and (b) these prophylactic effects are due to ouabain binding to α2-isoform resulting in the selective activation of PI3Kα. Our findings also suggest that potential prophylactic use of digitalis for prevention of heart failure in man deserves serious consideration.

Digitalis-induced cell signaling by the sodium pump: on the relation of Src to Na(+)/K(+)-ATPase.

In addition to performing its essential transport function, the sodium pump also activates multiple cell signaling pathways in response to digitalis drugs such as ouabain. Based mainly on cell-free studies with mixtures of purified Src kinase and Na(+)/K(+)-ATPase, a well-advocated hypothesis on how ouabain initiates the activation of signaling pathways is that there is a preexisting physiological complex of inactive Src bound to the α-subunit of Na(+)/K(+)-ATPase, and that ouabain binding to this subunit disrupts the bound Src and activates it. Because of the published disagreements of the results of such cell-free experiments of two other laboratories, our aim was to attempt the resolution of these discrepancies. We reexamined the effects of ouabain, vanadate, and oligomycin on mixtures of Src, Na(+)/K(+)-ATPase, Mg(2+), and ATP as specified in prior studies; and assayed for Src-418 autophosphorylation as the measure of Src activation. In contrast to the findings of the proponents of the above hypothesis, our results showed similar effects of the three inhibitors of Na(+)/K(+)-ATPase; indicating that Src activation in such experiments is primarily due to the ATP-sparing effect of the ATPase inhibitor on the mixture of two enzymes competing for ATP. We conclude that there is no solid evidence for direct molecular interaction of Src with Na(+)/K(+)-ATPase under physiological conditions.

Cell signaling associated with Na(+)/K(+)-ATPase: activation of phosphatidylinositide 3-kinase IA/Akt by ouabain is independent of Src.

Exposure of intact cells to selective inhibitors of Na(+)/K(+)-ATPase such as ouabain activates several growth-related cell signaling pathways. It has been suggested that the initial event of these pathways is the binding of ouabain to a preexisting complex of Src with Na(+)/K(+)-ATPase of the plasma membrane. The aim of this work was to evaluate the role of Src in the ouabain-induced activation of phosphatidylinositide 3-kinase 1A (PI3K1A) and its downstream consequences. When fibroblasts devoid of Src (SYF cells) and controls (Src(++) cells) were exposed to ouabain, PI3K1A, Akt, and proliferative growth were similarly stimulated in both cell lines. Ouabain-induced activation of Akt was not prevented by the Src inhibitor PP2. In contrast, ERK1/2 were not activated by ouabain in SYF cells but were stimulated in Src(++) cells; this was prevented by PP2. In isolated adult mouse cardiac myocytes, where ouabain induces hypertrophic growth, PP2 also did not prevent ouabain-induced activation of Akt and the resulting hypertrophy. Ouabain-induced increases in the levels of co-immunoprecipitation of the α-subunit of Na(+)/K(+)-ATPase with the p85 subunit of PI3K1A were noted in SYF cells, Src(++) cells, and adult cardiac myocytes. In conjunction with previous findings, the results presented here indicate that (a) if there is a preformed complex of Src and Na(+)/K(+)-ATPase, it is irrelevant to ouabain-induced activation of the PI3K1A/Akt pathway through Na(+)/K(+)-ATPase and (b) a more likely, but not established, mechanism of linkage of Na(+)/K(+)-ATPase to PI3K1A is the ouabain-induced interaction of a proline-rich domain of the α-subunit of Na(+)/K(+)-ATPase with the SH3 domain of the p85 subunit of PI3K1A.

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.

Caveolin-Na/K-ATPase interactions: role of transmembrane topology in non-genomic steroid signal transduction.

Progesterone and its polar metabolite(s) trigger the meiotic divisions in the amphibian oocyte through a non-genomic signaling system at the plasma membrane. Published site-directed mutagenesis studies of ouabain binding and progesterone-ouabain competition studies indicate that progesterone binds to a 23 amino acid extracellular loop of the plasma membrane α-subunit of Na/K-ATPase. Integral membrane proteins such as caveolins are reported to form Na/K-ATPase-peptide complexes essential for signal transduction. We have characterized the progesterone-induced Na/K-ATPase-caveolin (CAV-1)-steroid 5α-reductase interactions initiating the meiotic divisions. Peptide sequence analysis algorithms indicate that CAV-1 contains two plasma membrane spanning helices, separated by as few as 1-2 amino acid residues at the cell surface. The CAV-1 scaffolding domain, reported to interact with CAV-1 binding (CB) motifs in signaling proteins, overlaps transmembrane (TM) helix 1. The α-subunit of Na/K-ATPase (10 TM helices) contains double CB motifs within TM-1 and TM-10. Steroid 5α-reductase (6 TM helices), an initial step in polar steroid formation, contains CB motifs overlapping TM-1 and TM-6. Computer analysis predicts that interaction between antipathic strands may bring CB motifs and scaffolding domains into close proximity, initiating allostearic changes. Progesterone binding to the α-subunit may thus facilitate CB motif:CAV-1 interaction, which in turn induces helix-helix interaction and generates both a signaling cascade and formation of polar steroids.

Comparative properties of caveolar and noncaveolar preparations of kidney Na+/K+-ATPase.

To evaluate previously proposed functions of renal caveolar Na(+)/K(+)-ATPase, we modified the standard procedures for the preparation of the purified membrane-bound kidney enzyme, separated the caveolar and noncaveolar pools, and compared their properties. While the subunits of Na(+)/K(+)-ATPase (α,ß,γ) constituted most of the protein content of the noncaveolar pool, the caveolar pool also contained caveolins and major caveolar proteins annexin-2 tetramer and E-cadherin. Ouabain-sensitive Na(+)/K(+)-ATPase activities of the two pools had similar properties and equal molar activities, indicating that the caveolar enzyme retains its ion transport function and does not contain nonpumping enzyme. As minor constituents, both caveolar and noncaveolar pools also contained Src, EGFR, PI3K, and several other proteins known to be involved in stimulous-induced signaling by Na(+)/K(+)-ATPase, indicating that signaling function is not limited to the caveolar pool. Endogenous Src was active in both pools but was not further activated by ouabain, calling into question direct interaction of Src with native Na(+)/K(+)-ATPase. Chemical cross-linking, co-immunoprecipitation, and immunodetection studies showed that in the caveolar pool, caveolin-1 oligomers, annexin-2 tetramers, and oligomers of the α,ß,γ-protomers of Na(+)/K(+)-ATPase form a large multiprotein complex. In conjunction with known roles of E-cadherin and the ß-subunit of Na(+)/K(+)-ATPase in cell adhesion and noted intercellular ß,ß-contacts within the structure of Na(+)/K(+)-ATPase, our findings suggest that interacting caveolar Na(+)/K(+)-ATPases located at renal adherens junctions maintain contact of two adjacent cells, conduct essential ion pumping, and are capable of locus-specific signaling in junctional cells.

Progesterone modulation of transmembrane helix-helix interactions between the alpha-subunit of Na/K-ATPase and phospholipid N-methyltransferase in the oocyte plasma membrane.

BACKGROUND: Progesterone binding to the surface of the amphibian oocyte initiates the meiotic divisions. Our previous studies with Rana pipiens oocytes indicate that progesterone binds to a plasma membrane site within the external loop between the M1 and M2 helices of the alpha-subunit of Na/K-ATPase, triggering a cascade of lipid second messengers and the release of the block at meiotic prophase. We have characterized this site, using a low affinity ouabain binding isoform of the alpha1-subunit. RESULTS: Preparations of isolated plasma membranes from Rana oocytes demonstrate that physiological levels of progesterone (or the non-metabolizable progestin R5020) successively activate phosphatidylethanolamine-N-methyltransferase (PE-NMT) and sphingomyelin synthase within seconds. Inhibition of PE-NMT blocks the progesterone induction of meiosis in intact oocytes, whereas its initial product, phosphatidylmonomethylethanolamine (PME), can itself initiate meiosis in the presence of the inhibitor. Published X-ray crystallographic data on Na/K-ATPase, computer-generated 3D projections, heptad repeat analysis and hydrophobic cluster analysis of the transmembrane helices predict that hydrophobic residues L, V, V, I, F and Y of helix M2 of the alpha1-subunit interact with F, L, G, L, L and F, respectively, of helix M3 of PE-NMT. CONCLUSION: We propose that progesterone binding to the first external loop of the alpha1-subunit facilitates specific helix-helix interactions between integral membrane proteins to up-regulate PE-NMT, and, that successive interactions between two or more integral plasma membrane proteins induce the signaling cascades which result in completion of the meiotic divisions.

Progesterone binding to the alpha1-subunit of the Na/K-ATPase on the cell surface: insights from computational modeling.

Progesterone triggers the resumption of meiosis in the amphibian oocyte through a signaling system at the plasma membrane. Analysis of [(3)H]ouabain and [(3)H]progesterone binding to the plasma membrane of the Rana pipiens oocyte indicates that progesterone competes with ouabain for a low affinity ouabain binding site on a 112kDa alpha1-subunit of the membrane Na/K-ATPase. Published amino acid sequences from both low and high affinity ouabain binding alpha1-subunits are compared, together with published site-directed mutagenesis studies of ouabain binding. We propose that the progesterone binding site is located in the external loop (23 amino acids) between the M1-M2 transmembrane helices. Analysis of loop topology and the countercurrent hydrophobicity/polarity gradients within the M1-M2 loop further suggest that the polar beta and hydrophobic alpha surfaces of the planar progesterone molecule interact with opposite sides of the amino acid loop. The 19-angular methyl group of progesterone is essential for activity; it could bind to the C-terminal region of the M1-M2 loop. Maximum biological activity requires formation of hydrogen-bond networks between the 3-keto group of progesterone and Arg(118), Asp(129) and possibly Glu(122-124) in the C-terminal region of the loop. The 20-keto group hydrogen may in turn hydrogen bond to Cys(111) near the M1 helix. Peptide flexibility undergoes a maximal transition near the midway point in the M1-M2 loop, suggesting that folding occurs within the loop, which further stabilizes progesterone binding.

Association of PI3K-Akt signaling pathway with digitalis-induced hypertrophy of cardiac myocytes.

Our previous studies on cardiac myocytes showed that positive inotropic concentrations of the digitalis drug ouabain activated signaling pathways linked to Na(+)-K(+)-ATPase through Src and epidermal growth factor receptor (EGFR) and led to myocyte hypertrophy. In view of the known involvement of phosphatidylinositol 3-kinase (PI3K)-Akt pathways in cardiac hypertrophy, the aim of the present study was to determine whether these pathways are also linked to cardiac Na(+)-K(+)-ATPase and, if so, to assess their role in ouabain-induced myocyte growth. In a dose- and time-dependent manner, ouabain activated Akt and phosphorylation of its substrates mammalian target of rapamycin and glycogen synthase kinase in neonatal rat cardiac myocytes. Akt activation by ouabain was sensitive to PI3K inhibitors and was also noted in adult myocytes and isolated hearts. Ouabain caused a transient increase of phosphatidylinositol 3,4,5-trisphosphate content of neonatal myocytes, activated class IA, but not class IB, PI3K, and increased coimmunoprecipitation of the alpha-subunit of Na(+)-K(+)-ATPase with the p85 subunit of class IA PI3K. Ouabain-induced activation of ERK1/2 was prevented by Src, EGFR, and MEK inhibitors, but not by PI3K inhibitors. Activation of Akt by ouabain, however, was sensitive to inhibitors of PI3K and Src, but not to inhibitors of EGFR and MEK. Similarly, ouabain-induced myocyte hypertrophy was prevented by PI3K and Src inhibitors, but not by an EGFR inhibitor. These findings 1) establish the linkage of the class IA PI3K-Akt pathway to Na(+)-K(+)-ATPase and the essential role of this linkage to ouabain-induced myocyte hypertrophy and 2) suggest cross talk between these PI3K-Akt pathways and the signaling cascades previously identified to be associated with cardiac Na(+)-K(+)-ATPase.

Interactions of K+ATP channel blockers with Na+/K+-ATPase.

Two K(+) (ATP) channel blockers, 5-hydroxydecanoate (5-HD) and glyburide, are often used to study cross-talk between Na(+)/K(+)-ATPase and these channels. The aim of this work was to characterize the effects of these blockers on purified Na(+)/K(+)-ATPase as an aid to appropriate use of these drugs in studies on this cross-talk. In contrast to known dual effects (activating and inhibitory) of other fatty acids on Na(+)/K(+)-ATPase, 5-HD only inhibited the enzyme at concentrations exceeding those that block mitochondrial K(+) (ATP) channels. 5-HD did not affect the ouabain sensitivity of Na(+)/K(+)-ATPase. Glyburide had both activating and inhibitory effects on Na(+)/K(+)-ATPase at concentrations used to block plasma membrane K(+) (ATP) channels. The findings justify the use of 5-HD as specific mitochondrial channel blocker in studies on the relation of this channel to Na(+)/K(+)-ATPase, but question the use of glyburide as a specific blocker of plasma membrane K(+) (ATP) channels, when the relation of this channel to Na(+)/K(+)-ATPase is being studied.