Insulin Stimulates the Activity of Na+/K+-Atpase in Human Peritoneal Mesothelial Cells

1997 ◽  
Vol 17 (2) ◽  
pp. 186-193 ◽  
Author(s):  
Janusz Witowski ◽  
Andrzej Breborowicz ◽  
Nicholas Topley ◽  
Leo Martis ◽  
Jan Knapowski ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Mesothelial Cells ◽  
Pcr Analysis ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Sodium Influx ◽  
Atpase Subunit ◽  
Peritoneal Mesothelial Cells ◽  
Human Peritoneal Mesothelial Cells ◽  
Dose Dependent

Objective To assess the effect of insulin on the Na+/ K+-ATPase expression and activity in human peritoneal mesothelial cells (HPMC). Methods HPMC were isolated from the omental tissue of non-uremic patients, grown to confluence and rendered quiescent by serum deprivation for 24 hours. The activity of Na+/K+-ATPase was determined by measuring the ouabain-sensitive86Rb uptake. To assess whether the effect of insulin was related to changes in [Na+]i the sodium influx was measured with 22Na and the activity of Na+/K+ -A TPase was assessed in the presence of amiloride. Expression of Na+/K+ -A TPaseα1’ α2 and β1-subunit mRNAs was determined by RT/PCR. Results Exposure of HPMC to insulin resulted in a time and dose-dependent increase in the Na+/K+-ATPase activity. After 60 minutes the ouabain-sensitive 86Rb up take (cpm/104 cells) was increased from 6650±796 in control cells to 9763±1212 in HPMC exposed to 100 mU/ mL insulin (1.5-fold increase; n=4, P<0.05). In addition, incubation of HPMC with 100 mU/mL insulin resulted in a time-dependent increase in the 22Na influx. Pre-exposure of HPMC to 1 mM amiloride reduced the activity of Na+/K+-A TPase but did not block the stimulatory effect of insulin. RT/PCR analysis revealed that HPMC constitutively expressed α1 and β1-subunit mRNAs while the α2-subunit mRNA was barely detectable. Exposure of HPMC to insulin for up to 24 hours was not associated with any changes in the expression of either α1’ α2 or B1-subunit. Conclusion Insulin stimulates the Na+/K+-ATPase activity in HPMC in a time and dose-dependent manner. This effect appears to mediated by an increase in [Na+]i and is not related to alterations in Na+/K+-ATPase subunit mRNAs expression.

Glycerol Toxicity for Human Peritoneal Mesothelial Cells in Culture: Comparison with Glucose

1994 ◽  
Vol 17 (5) ◽  
pp. 252-260 ◽  
Author(s):  
J. Witowski ◽  
J. Knapowski
Keyword(s):  
Mesothelial Cell ◽  
Cellular Protein ◽  
Mesothelial Cells ◽  
Dependent Manner ◽  
Peritoneal Mesothelial Cells ◽  
Proline Incorporation ◽  
Human Peritoneal Mesothelial Cells ◽  
Dose Dependent Decrease ◽  
Dose Dependent

Glycerol has been proposed as a substitute osmotic agent for glucose in peritoneal dialysis fluids. We have compared the effect of glycerol and glucose on the function of human peritoneal mesothelial cells (HPMC) in vitro. The viability of HPMC was not affected by glycerol (up to 250 mM), whereas it was reduced by glucose in a time- and dose-dependent manner, as assessed by the LDH release. Although the incubation of HPMC with glycerol induced a dose-dependent decrease in HPMC proliferation, the effect was significantly less inhibitory than that produced by glucose. In HPMC treated with 90 mM of glycerol or glucose the incorporation of [3H]-thymidine had reached 79.0±19.3% and 55.3+4.0% of the control (p<0.05 and p<0.01), respectively. As measured by the [methyl-14C]-choline incorporation, the intracellular amount of newly synthesized phospholipids was reduced from (cpm/μg cellular protein) 147±58 in control HPMC to 59+15 in cells exposed to 90 mM of glucose (p<0.01), but not affected by glycerol (163±65). On the other hand, both glycerol and glucose (90 mM) decreased the synthesis of proteins (as assessed by the [3H]-proline incorporation) and interfered with potassium (86Rb) transport mechanisms in HPMC. Our data suggest that there exist some possibly advantageous aspects of glycerol as far as mesothelial cell biocompatibility profile is concerned.

3,4-Dideoxyglucosone-3-Ene Induces Apoptosis in Human Peritoneal Mesothelial Cells

2009 ◽  
Vol 29 (1) ◽  
pp. 44-51 ◽  
Author(s):  
Duk-Hyun Lee ◽  
Soon-Youn Choi ◽  
Hye-Myung Ryu ◽  
Chan-Duck Kim ◽  
Sun-Hee Park ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Caspase 3 ◽  
Degradation Products ◽  
Treated Group ◽  
Mesothelial Cells ◽  
Tunel Assay ◽  
Dependent Manner ◽  
Caspase Inhibitor ◽  
Peritoneal Mesothelial Cells ◽  
Human Peritoneal Mesothelial Cells

Objective Glucose degradation products (GDPs) are formed during heat sterilization and storage of peritoneal dialysis (PD) fluids. 3,4-dideoxyglucosone-3-ene (3,4-DGE) has been identified as the most bioreactive GDP. 3,4-DGE induces apoptosis in leukocytes and renal tubular epithelial cells. Our aim was to evaluate the apoptotic effects of 3,4-DGE on human peritoneal mesothelial cells (HPMCs). Methods Primary cultured HPMCs were treated with 25 or 50 μmol/L 3,4-DGE. MTT assay was used to determine cell viability. Apoptosis was measured using TUNEL assay and flow cytometry. Expressions of procaspase-3, Bax, and Bcl-2 were estimated by Western blot. Activity of caspase-3 was measured and the effect of the caspase inhibitor zVAD-fmk (Z-Val-Ala-DL-Asp-fluoromethylketone) was evaluated by TUNEL assay. Results 3,4-DGE treatment accelerated cell death in HPMCs in a dose- and time-dependent manner. Treatment with 3,4-DGE (25 and 50 μmol/L) significantly increased apoptosis compared to control ( p < 0.05 and p < 0.01 respectively) by TUNEL assay. Flow cytometry showed treatment with 50 μmol/L 3,4-DGE significantly increased apoptosis compared to control ( p < 0.05). Decreased expression of procaspase-3 and increased activity of caspase-3 were observed in the presence of 50 μmol/L 3,4-DGE compared to control and 25 μmol/L 3,4-DGE ( p < 0.05). 3,4-DGE-induced HPMC apoptosis was decreased after pretreatment with the pan-caspase inhibitor zVAD-fmk in the 50 μmol/L 3,4-DGE-treated group ( p < 0.001). The ratio of Bcl-2 to Bax expression was decreased in the 25 μmol/L and the 50 μmol/L 3,4-DGE-treated groups compared to control ( p < 0.05). Conclusions 3,4-DGE promotes apoptosis in HPMCs by a caspase-related mechanism.

scholarly journals Human Peritoneal Mesothelial Cells Display Phagocytic and Antigen-Presenting Functions to Contribute to Intraperitoneal Immunity

2016 ◽  
Vol 26 (5) ◽  
pp. 833-838 ◽  
Author(s):  
Tanya J. Shaw ◽  
Xiang Y. Zhang ◽  
Zhiming Huo ◽  
David Robertson ◽  
Patricia A. Lovell ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mhc Class Ii ◽  
Constitutive Expression ◽  
Class Ii ◽  
Mesothelial Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Peritoneal Mesothelial Cells ◽  
Human Peritoneal Mesothelial Cells ◽  
Antigen Presenting

AbstractMesothelial cells lining the peritoneal cavity are strategically positioned to respond to and counter intraperitoneal infections, cancer cells, and other challenges. We have investigated human peritoneal mesothelial cells (HPMCs) for phagocytic activity, expression of surface Major Histocompatibility Complex (MHC) class II and accessory molecules involved in antigen presentation, and the ability to present recall antigens to T cells. Phagocytosis of dextran, latex beads, andEscherichia coliwas observed by flow cytometry, and internalization was visualized using confocal and electron microscopy. Flow cytometry and/or cellular enzyme-linked immunosorbent assay showed constitutive expression of ICAM-1, LFA-3, and B7-1, but not B7-2 or MHC class II. Interferon-gamma induced MHC II and ICAM-1 expression in a dose- and time-dependent manner. Importantly, HPMCs induced autologous CD3+T-lymphocyte proliferation (3H incorporation) after pulse with recall antigen. Human peritoneal mesothelial cells equipped with phagocytic and antigen-presenting machinery are anticipated to have an integral role in intraperitoneal immune surveillance.

Interleukin-1β Stimulates Glucose Uptake of Human Peritoneal Mesothelial Cells In Vitro

1996 ◽  
Vol 16 (1_suppl) ◽  
pp. 58-60 ◽  
Author(s):  
Michael Kruse ◽  
Arezki Mahiout ◽  
Volker Kliem ◽  
Peter Kurz ◽  
Karl-Martin Koch ◽  
...  
Keyword(s):  
Glucose Uptake ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Interleukin 1 ◽  
Glucose Transporters ◽  
Mesothelial Cells ◽  
Dependent Manner ◽  
Peritoneal Mesothelial Cells ◽  
Human Peritoneal Mesothelial Cells

To investigate whether the glucose uptake (GU) of human peritoneal mesothelial cells (HPMC) is mediated by glucose transporters and whether this uptake is influenced by interleukin 1–β (IL-1β), we measured 2-deoxy-(3H)-GU of HPMC in vitro, after exposing the cells for different times (two and 12 hours) to increasing concentrations (0.1, 1.0, and 2.0 ng/mL) of IL-1 β. To exclude a noncarrier-mediated transport, GU was also tested in the presence of cytochalasin B. All experiments were performed in triplicate in the cells of two donors. Cytochalasin B inhibits GU of HPMC almost completely. GU of HPMC is not stimulated by insulin. GU is stimulated by IL-1 β in a dose-dependent manner. These data indicate a GU of HPMC, which is mediated by a glucose transporter and stimulated by IL-1 β. The increased uptake of glucose from the dialysate In patients with peritonitis may be mediated by a (cytokineinduced) increased activity of HPMC glucose transporters.

scholarly journals MiR-29b may suppresses peritoneal metastases through inhibition of the mesothelial–mesenchymal transition (MMT) of human peritoneal mesothelial cells

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Yuki Kimura ◽  
Hideyuki Ohzawa ◽  
Hideyo Miyato ◽  
Yuki Kaneko ◽  
Akira Saito ◽  
...  
Keyword(s):  
Morphological Changes ◽  
Negative Control ◽  
Mesothelial Cells ◽  
Peritoneal Metastases ◽  
Dependent Manner ◽  
Mesenchymal Transition ◽  
Peritoneal Mesothelial Cells ◽  
Human Peritoneal Mesothelial Cells ◽  
Tgf Β1

AbstractPeritoneal dissemination is a major metastatic pathway for gastrointestinal and ovarian malignancies. The miR-29b family is downregulated in peritoneal fluids in patients with peritoneal metastases (PM). We examined the effect of miR-29b on mesothelial cells (MC) which play critical a role in the development of PM through mesothelial-mesenchymal transition (MMT). Human peritoneal mesothelial cells (HPMCs) were isolated from surgically resected omental tissue and MMT induced by stimulation with 10 ng/ml TGF-β1. MiR-29b mimics and negative control miR were transfected by lipofection using RNAiMAX and the effects on the MMT evaluated in vitro. HPMC produced substantial amounts of miR-29b which was markedly inhibited by TGF-β1. TGF-β1 stimulation of HPMC induced morphological changes with decreased expression of E-cadherin and calretinin, and increased expression of vimentin and fibronectin. TGF-β1 also enhanced proliferation and migration of HPMC as well as adhesion of tumor cells in a fibronectin dependent manner. However, all events were strongly abrogated by simultaneous transfection of miR-29b. MiR-29b inhibits TGF-β1 induced MMT and replacement of miR-29b in the peritoneal cavity might be effective to prevent development of PM partly through the effects on MC.

Expression of Aquaporin-1 in Human Peritoneal Mesothelial Cells and Its Upregulation by GlucoseIn Vitro

2001 ◽  
Vol 12 (5) ◽  
pp. 1036-1045 ◽  
Author(s):  
KAR NENG LAI ◽  
FU KEUNG LI ◽  
HAO YUI LAN ◽  
SYDNEY TANG ◽  
ANITA W. L. TSANG ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Peritoneal Dialysis ◽  
Mesothelial Cells ◽  
Dependent Manner ◽  
Peritoneal Membrane ◽  
Dialysis Adequacy ◽  
Peritoneal Mesothelial Cells ◽  
Aqp1 Expression ◽  
Human Peritoneal Mesothelial Cells ◽  
Osmotic Agents

Abstract. Aquaporin (AQP) is a family of water channels that are highly selective for the passage of water and occasionally glycerol. In previous studies, only AQP1 was found in human peritoneal endothelial cells in both control subjects and patients on peritoneal dialysis. As human peritoneal mesothelial cells (HPMC) play an important role in dialysis adequacy and fluid balance in continuous ambulatory peritoneal dialysis patients, this study examined whether AQP1 is present in HPMC. It was found that AQP1 mRNA and protein are present in HPMC constitutively. The localization of AQP1 protein in peritoneal mesothelial cells was confirmed by double immunohistochemical staining of the mesothelial lining of human peritoneal membrane. More important, the expression of AQP1 in HPMC is not constitutive and the transcription and biosynthesis of AQP1 in HPMC is inducible by osmotic agents such as glucose and mannitol. There was significant enhancement of AQP1 biosynthesis upon exposure to glucose in a time- and dose-dependent manner (P< 0.0001). Similar findings were observed in the AQP1 biosynthesis by an endothelial cell line, EA.hy 926. Of particular interest, the upregulation in AQP1 mRNA or biosynthesis in mesothelial cells was always significantly higher than that of endothelial cells when the experiments were conducted under identical settings (P< 0.001). AQP1 expression in HPMC was demonstrated for the first time. Osmotic agents upregulate both mRNA and protein expression of this aquaporin. The role of AQP1 in HPMC in maintaining the ultrafiltration of the peritoneal membrane is potentially of clinical interest.

Effects of Peritoneal Dialysis Solutions on the Secretion of Growth Factors and Extracellular Matrix Proteins by Human Peritoneal Mesothelial Cells

2002 ◽  
Vol 22 (2) ◽  
pp. 171-177 ◽  
Author(s):  
Hunjoo Ha ◽  
Mi Kyung Cha ◽  
Hoo Nam Choi ◽  
Hi Bahl Lee
Keyword(s):  
Peritoneal Dialysis ◽  
Growth Factor ◽  
High Glucose ◽  
Transforming Growth Factor ◽  
Mesothelial Cells ◽  
Dependent Manner ◽  
Peritoneal Mesothelial Cells ◽  
Human Peritoneal Mesothelial Cells ◽  
Dialysis Solutions ◽  
Peritoneal Dialysis Solutions

♦ Objective To compare the effects of different peritoneal dialysis solutions (PDS) on secretion of vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGFβ1), procollagen I C-terminal peptide (PICP), procollagen III N-terminal peptide (PIIINP), and fibronectin by cultured human peritoneal mesothelial cells (HPMC). ♦ Design Using M199 culture medium as control, commercial PDS containing 1.5% or 4.25% glucose and 40 mmol/L lactate [Dianeal 1.5 (D 1.5) and Dianeal 4.25 (D 4.25), respectively; Baxter Healthcare, Deerfield, Illinois, USA]; PDS containing 1.5% or 4.25% glucose with 25 mmol/L bicarbonate and 15 mmol/L lactate [Physioneal 1.5 (P 1.5) and Physioneal 4.25 (P 4.25), respectively; Baxter]; and PDS containing 7.5% icodextrin [Extraneal (E); Baxter] were tested. Growth-arrested and synchronized HPMC were continuously stimulated for 48 hours by test PDS diluted twofold with M199, TGFβ1 1 ng/mL, or different concentrations of icodextrin. VEGF, TGFβ1, and fibronectin secreted into the media were analyzed by ELISA, and PICP and PIIINP by radioimmunoassay. ♦ Results Dianeal 1.5, D 4.25, and P 4.25, but not P 1.5 and E, significantly increased VEGF secretion compared with control M199. D 4.25- and P 4.25-induced VEGF secretion was significantly higher than induction by D 1.5 and P 1.5, respectively, suggesting that high glucose may be involved in the induction of VEGF. Physioneal 1.5- and P 4.25-induced VEGF secretion was significantly lower than induction by D 1.5 and D 4.25, respectively, suggesting a role for glucose degradation products (GDP) in VEGF production. TGFβ1 secretion was significantly increased by D 4.25 and E. Icodextrin increased TGFβ1 secretion in a dose-dependent manner. All PDS tested significantly increased secretion of PIIINP compared with control. D 1.5- and D 4.25-induced PIIINP secretion was significantly higher than P 1.5, P 4.25, and E. Physioneal 4.25-induced PIIINP secretion was significantly higher than P 1.5, again implicating high glucose and GDP in PIIINP secretion by HPMC. There was no significant increase in PICP or fibronectin secretion using any of the PDS tested. Addition of TGFβ1 1 ng/mL into M199 control significantly increased VEGF, PICP, PIIINP, and fibronectin secretion by HPMC. ♦ Conclusions The present study provides direct evidence that HPMC can secrete VEGF, TGFβ1, and PIIINP in response to PDS, and that HPMC may be actively involved in the development and progression of the peritoneal membrane hyperpermeability and fibrosis observed in long-term PD patients. This study also suggests that both high glucose and GDP in PDS may play important roles in inducing VEGF and PIIINP production/secretion by HPMC.

scholarly journals MiR-29b suppresses peritoneal metastases through inhibition of the mesothelial-mesenchymal transition (MMT) of human peritoneal mesothelial cells

2021 ◽  
Author(s):  
Yuki Kimura ◽  
Hideyuki Ohzawa ◽  
Hideyo Miyato ◽  
Yuki Kaneko ◽  
Kazuya Takahashi ◽  
...  
Keyword(s):  
Morphological Changes ◽  
Negative Control ◽  
Mesothelial Cells ◽  
Peritoneal Metastases ◽  
Dependent Manner ◽  
Mesenchymal Transition ◽  
Peritoneal Mesothelial Cells ◽  
Human Peritoneal Mesothelial Cells ◽  
And Migration

Abstract Background: Peritoneal dissemination is a major metastatic pathway for gastrointestinal and ovarian malignancies. The miR-29b family is downregulated in peritoneal fluids in patients with peritoneal metastases (PM). We examined the effect of miR-29b on mesothelial cells (MC) which play critical a role in the development of PM through mesothelial-mesenchymal transition (MMT). Methods: Human peritoneal mesothelial cells (HPMCs) were isolated from surgically resected omental tissue and MMT induced by stimulation with 10 ng/ml TGF-b1. MiR-29b mimics and negative control miR were transfected by lipofection using RNAiMAX and the effects on the MMT evaluated in vitro. Results: HPMC produced substantial amounts of miR-29b which was markedly inhibited by TGF-b1. TGF-b1 stimulation of HPMC induced morphological changes with decreased expression of E-cadherin and calretinin, and increased expression of vimentin and fibronectin. TGF-b1 also enhanced proliferation and migration of HPMC as well as adhesion of tumor cells in a fibronectin dependent manner. However, all events were strongly abrogated by simultaneous transfection of miR-29b. Conclusion: MiR-29b inhibits TGF-b1 induced MMT and replacement of miR-29b in the peritoneal cavity might be effective to prevent development of PM partly through the effects on MC.

scholarly journals EVALUATION OF PRO-APOPTOTIC EFFECTS OF β-MONOLINOLEIN ON METASTATIC BREAST CANCER CELL LINE MDA-MB-231

2019 ◽  
pp. 235-240 ◽  
Author(s):  
MOHAMMED SHAFI SOFI
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Western Blot ◽  
Trypan Blue ◽  
Metastatic Breast ◽  
Pcr Analysis ◽  
Dependent Manner ◽  
Cell Cycle Regulators ◽  
Rt Pcr ◽  
Dose Dependent

Objectives: The objective of this study was to evaluate the β-monolinolein as a potential therapy for breast cancer treatment. Methods: The cytotoxic activity of β-monolinolein was evaluated by 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trypan blue exclusion assay. The cellular cytotoxicity and levels of cytosolic enzyme, lactate dehydrogenase (LDH), were measured by assessing μmoles of nicotinamide adenine dinucleotide/well/min. To confirm whether β-monolinolein induces apoptosis in 3,4-methylenedioxyamphetamine (MDA)- MB-231 cells, western blot and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis were performed. Results: For the 1st time, it was demonstrated that β-monolinolein strongly inhibits the growth of MDA-MB-231 cells, with an half maximal inhibitory concentration value of 12.5 μg/ml. <90% of cell death was achieved at higher concentrations after 48 h of treatment. Trypan blue assay showed that the cell viability was significantly decreased in a dose-dependent manner in MDA-MB-231 cells after 48 h of treatment. On the other hand, LDH activities in the cultured media were significantly elevated in a dose-dependent manner as compared to the control. Further, the western blot analysis showed that β-monolinolein leads to change in expression levels of important cell cycle regulators such as p21, Bax, Bcl-Xl, and Bcl-2 in MDA-MB-231 cells. The semiquantitative RT-PCR results indicated a significant upregulation of proapoptotic genes such as p53, p21, and Bax and downregulation of antiapoptotic gene Bcl-2. Conclusion: These results indicate that β-monolinolein leads to change in expression of various cell cycle/apoptotic regulators and hence induces death in MDA-MB-231 cells.

Parathyroid Ca2+-conducting currents are modulated by muscarinic receptor agonists and antagonists

1997 ◽  
Vol 273 (5) ◽  
pp. E880-E890 ◽  
Author(s):  
Wenhan Chang ◽  
Tsui-Hua Chen ◽  
Stacy A. Pratt ◽  
Benedict Yen ◽  
Michael Fu ◽  
...  
Keyword(s):  
Muscarinic Receptors ◽  
Inositol Phosphate ◽  
Receptor Protein ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Pipette Solution ◽  
Pcr Products ◽  
Inositol Phosphate Accumulation ◽  
Receptor Cdna ◽  
Dose Dependent

Parathyroid cells express Ca2+-conducting cation currents, which are activated by raising the extracellular Ca2+ concentration ([Ca2+]o) and blocked by dihydropyridines. We found that acetylcholine (ACh) inhibited these currents in a reversible, dose-dependent manner (50% inhibitory concentration ≈10−8 M). The inhibitory effects could be mimicked by the agonist (+)-muscarine. The effects of ACh were blunted by the antagonist atropine and reversed by removing ATP from the pipette solution. (+)-Muscarine enhanced the adenosine 3′,5′-cyclic monophosphate (cAMP) production by 30% but had no effect on inositol phosphate accumulation in parathyroid cells. Oligonucleotide primers, based on sequences of known muscarinic receptors (M1-M5), were used in reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify receptor cDNA from parathyroid poly (A)+ RNA. RT-PCR products displayed >90% nucleotide sequence identity to human M2- and M4-receptor cDNAs. Expression of M2-receptor protein was further confirmed by immunoblotting and immunocytochemistry. Thus parathyroid cells express muscarinic receptors of M2 and possibly M4 subtypes. These receptors may couple to dihydropyridine-sensitive, cation-selective currents through the activation of adenylate cyclase and ATP-dependent pathways in these cells.

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