Insulin-Induced Electrophysiology Changes in Human Pleura Are Mediated via Its Receptor

Background. Insulin directly changes the sheep pleural electrophysiology. The aim of this study was to investigate whether insulin induces similar effects in human pleura, to clarify insulin receptor's involvement, and to demonstrate if glibenclamide (hypoglycemic agent) reverses this effect. Methods. Human parietal pleural specimens were mounted in Ussing chambers. Solutions containing insulin or glibenclamide and insulin with anti-insulin antibody, anti-insulin receptor antibody, and glibenclamide were used. The transmesothelial resistance (RTM) was determined. Immunohistochemistry for the presence of Insulin Receptors (IRa, IRb) was also performed. Results. Insulin increased RTM within 1st min (P = .016), when added mesothelially which was inhibited by the anti-insulin and anti-insulin receptor antibodies. Glibenclamide also eliminated the insulin-induced changes. Immunohistochemistry verified the presence of IRa and IRb. Conclusion. Insulin induces electrochemical changes in humans as in sheep via interaction with its receptor. This effect is abolished by glibenclamide

Androgen-Induced Cell Migration: Role of Androgen Receptor/Filamin A Association

Background: Androgen receptor (AR) controls male morphogenesis, gametogenesis and prostate growth as well as development of prostate cancer. These findings support a role for AR in cell migration and invasiveness. However, the molecular mechanism involved in AR-mediated cell migration still remains elusive. Methodology/Principal Findings: Mouse embryo NIH3T3 fibroblasts and highly metastatic human fibrosarcoma HT1080 cells harbor low levels of transcriptionally incompetent AR. We now report that, through extra nuclear action, AR triggers migration of both cell types upon stimulation with physiological concentrations of the androgen R1881. We analyzed the initial events leading to androgen-induced cell migration and observed that challenging NIH3T3 cells with 10 nM R1881 rapidly induces interaction of AR with filamin A (FlnA) at cytoskeleton. AR/FlnA complex recruits integrin beta 1, thus activating its dependent cascade. Silencing of AR, FlnA and integrin beta 1 shows that this ternary complex controls focal adhesion kinase (FAK), paxillin and Rac, thereby driving cell migration. FAK-null fibroblasts migrate poorly and Rac inhibition by EHT impairs motility of androgen-treated NIH3T3 cells. Interestingly, FAK and Rac activation by androgens are independent of each other. Findings in human fibrosarcoma HT1080 cells strengthen the role of Rac in androgen signaling. The Rac inhibitor significantly impairs androgen-induced migration in these cells. A mutant AR, deleted of the sequence interacting with FlnA, fails to mediate FAK activation and paxillin tyrosine phosphorylation in androgen-stimulated cells, further reinforcing the role of AR/FlnA interaction in androgen-mediated motility. Conclusions/Significance: The present report, for the first time, indicates that the extra nuclear AR/FlnA/integrin beta 1 complex is the key by which androgen activates signaling leading to cell migration. Assembly of this ternary complex may control organ development and prostate cancer metastasis

Effects of varenicline therapy in combination with advanced behavioral support on smoking cessation and quality of life in inpatients with acute exacerbation of COPD, bronchial asthma, or community-acquired pneumonia: A prospective, open-label, preference-based, 52-week, follow-up trial

Quitting smoking is the most important element in the therapeutic management of chronic respiratory diseases. Combining pharmacotherapy with behavioral support increases smoking cessation success rates. In addition, hospitalized smokers have increased motivation to quit. We investigated the efficacy on smoking cessation, of varenicline in combination with behavioral support, in smokers hospitalized due to (a) acute exacerbation of chronic obstructive pulmonary disease (COPD), or (b) bronchial asthma attack, or (c) community-acquired pneumonia (CAP). The method used is prospective, open-label, preference-based, parallel group, 52-week trial. Patients chose the smoking cessation intervention they preferred: a standard regimen of varenicline combined with post-discharge advanced behavioral support (group A) or one private consultation session during hospitalization (group B). Follow-up phone calls were scheduled in weeks 1, 2, and 4 and months 3, 6, and 9. The final hospital visit was performed in week 52. Primary outcome was success rate defined as the percentage (%) of smoking abstinence at week 52 and secondary outcomes were (a) changes in quality of life (QoL) indicated by the scores on the Short Form 36 (SF36) questionnaire and (b) predictors of smoking abstinence investigated with multiple binary logistic regression. One hundred one patients were enrolled, 44 (43.6%) in group A and 57 (56.4%) in group B. Respective abstinence rates were 54.5% and 15.8% at week 12 and 52.3% and 14.0% at week 52. Scores on SF36 were statistically significantly increased in both groups. Predictors of smoking abstinence were varenicline (odds ratio (OR) 7.29; 95% confidence interval (CI) 2.15, 24.77; p = 0.001), age (OR 1.07; 95%CI 1.00, 1.15; p = 0.042), Fagerstrom score (OR 0.37; 95%CI 0.20, 0.68; p = 0.001), SF36 domains “vitality” (OR 1.12; 95%CI 1.04, 1.21; p = 0.003), and “social functioning” (OR 0.95; 95%CI 0.90, 1.00; p = 0.041). Varenicline in combination with behavioral support resulted in high abstinence rates inpatients hospitalized for exacerbation of COPD, asthma attack, or CAP, and improved QoL. © 2017, © The Author(s) 2017

In-depth bioinformatic study of the CLDN16 gene and protein: Prediction of subcellular localization to mitochondria

Background and Objectives: The defects in the CLDN16 gene are a cause of primary hypomagnesemia (FHHNC), which is characterized by massive renal magnesium wasting, resulting in nephrocalcinosis and renal failure. The mutations occur throughout the gene’s coding region and can impact on intracellular trafficking of the protein or its paracellular pore forming function. To gain more understanding about the mechanisms by which CLDN16 mutations can induce FHHNC, we performed an in-depth computational analysis of the CLDN16 gene and protein, focusing specifically on the prediction of the latter’s subcellular localization. Materials and Methods: The complete nucleotide or amino acid sequence of CLDN16 in FASTA format was entered and processed in 14 databases. Results: One CpG island was identified. Twenty five promoters/enhancers were predicted. The CLDN16 interactome was found to consist of 20 genes, mainly involved in kidney diseases. No signal peptide cleavage site was identified. A probability of export to mitochondria equal to 0.9740 and a cleavable mitochondrial localization signal in the N terminal of the CLDN16 protein were predicted. The secondary structure prediction was visualized. Νo phosphorylation sites were identified within the CLDN16 protein region by applying DISPHOS to the functional class of transport. The KnotProt database did not predict any knot or slipknot in the protein structure of CLDN16. Seven putative miRNA binding sites within the 3’-UTR region of CLDN16 were identified. Conclusions: This is the first study to identify mitochondria as a probable cytoplasmic compartment for CLDN16 localization, thus providing new insights into the protein’s intracellular transport. The results relative to the CLDN16 interactome underline its role in renal pathophysiology and highlight the functional dependence of CLDNs-10, 14, 16, 19. The predictions pertaining to the miRNAs, promoters/enhancers and CpG islands of the CLDN16 gene indicate a strict regulation of its expression both transcriptionally and post-transcriptionally. © 2019 by the authors. Licensee MDPI, Basel, Switzerland

Role of Electrolytes and Glucose in the Insulin-Induced Electrochemical Effect in Sheep Pleura

Aim: Insulin induces electrochemical alterations in sheep visceral and parietal pleura, an effect abolished by the Na(+)-channel blocker amiloride and the Na(+)-K(+) pump inhibitor ouabain. The aim of this study was to further investigate the role of different electrolytes and glucose in these electrochemical changes. Materials and methods: Sheep pleural specimens were mounted in Ussing chambers. Insulin (10(-7) M) was added mesothelially in Na(+), K(+), Ca(2+)-free, low H(+) and glucose solutions. In other experiments, specimens were pretreated with K(+) and Ca(2+)-free Krebs solutions. Trans-mesothelial Resistance was determined. Results: Insulin did not increase Trans-mesothelial Resistance of visceral and parietal pleura in K(+)-free (p = 0.008 and p = 0.028 respectively), Ca(2+)-free (p = 0.006 and p = 0.012 respectively) and low glucose (p = 0.009 and p = 0.03 respectively) solutions. This effect was totally inhibited in Na(+)-free solutions or in specimens pretreated with Ca(2+)-free Krebs solution and partially inhibited, when low H(+) solutions were used (p = 0.042 for visceral and p = 0.045 for parietal). Conclusion: Insulin-induced electrochemical changes in sheep pleura are mainly associated with alterations in Na(+) and Ca(2+) concentrations. Since amiloride and ouabain abolish these electrochemical changes, it may be suggested that insulin could influence the pleural fluid recycling, mainly via the Na(+) transportation system, irrespective of the glucose content

Human parietal pleura present electrophysiology variations according to location in pleural cavity

The aim of the study was to investigate if human pleura from different anatomical locations presents electrophysiology differences. Specimens were stripped over the 2nd-5th rib (cranial), 8th-10th rib (caudal), and mediastinum during open surgery and were mounted between Ussing chambers. Amiloride and ouabain were added towards mesothelial surface and trans-mesothelial potential difference (PDTM) was measured after 1, 5, 10 and 20 min. Trans-membrane resistance (RTM) was calculated from Ohm's law. R TM increased after amiloride addition, for cranial (net increase of 0.40 Ω·cm2) and caudal (1.16 Ω·cm 2) pleural pieces. Mediastinal pleura RTM remained unchanged (0.09 Ω·cm2). RTM increase was higher for caudal than cranial (P = 0.029) or mediastinal tissues (P = 0.002). RTM increased after ouabain addition for caudal (1.35 Ω·cm2) and cranial (0.56 Ω·cm2) pleural pieces. Mediastinal pleural tissue did not respond (0.20 Ω·cm2). Caudally located pleura responded greater than cranial (P = 0.043) or mediastinal (P = 0.003) pleural tissues. Human pleura shows electrophysiology differences according to the location within the pleural cavity. Surgeons may waste mediastinal pleura when needed but should leave intact caudal parietal pleura, which seems to be electrophysiologically the most important part of the pleural cavity

Insulin Alters the Permeability of Sheep Pleura

Aim: Insulin promotes ion transportation across epithelia, mainly kidneys, leading to water and electrolyte abnormalities, possibly causing 'insulin oedema syndrome', which rarely presents as pleural effusion. Direct stimulation of sheep pleura by insulin and the possible electrophysiology mechanisms involved were investigated. Material and Methods: Sheep visceral and parietal pleural specimens were mounted between Ussing chambers. Insulin solutions (10(-9) to 10(-5) M), L-NAME, Nitroprussid sodium, amiloride and ouabain were used. Trans-mesothelial Resistance was determined. Immunohistochemistry for presence of Insulin Receptors was performed. Results: Insulin increased Trans-mesothelial Resistance within 1st minute when added mesothelially of visceral (p = 0.008) and parietal pleura (p = 0.046) for concentrations higher than 10(-7) M. L-NAME or Nitroprussid sodium didn't but amiloride and ouabain inhibited insulin's effect. Immunohistochemistry revealed the presence of Insulin Receptors. Conclusion: Insulin changes the permeability of sheep pleura by altering its electrophysiology and may interfere in pleural effusion formation. Involvement of Insulin Receptors may be suggested