Glucose activates H+-ATPase in kidney epithelial cells

2004 ◽  
Vol 287 (1) ◽  
pp. C97-C105 ◽  
Author(s):  
Suguru Nakamura
Keyword(s):  
Epithelial Cells ◽  
Atpase Activity ◽  
Physiological Effect ◽  
Bicarbonate Transport ◽  
Epithelial Cell Line ◽  
Atpase Inhibitor ◽  
Concanamycin A ◽  
Vacuolar Acidification ◽  
Kidney Epithelial Cells ◽  
Effect Of Glucose

The vacuolar H+-ATPase (V-ATPase) acidifies compartments of the vacuolar system of eukaryotic cells. In renal epithelial cells, it resides on the plasma membrane and is essential for bicarbonate transport and acid-base homeostasis. The factors that regulate the H+-ATPase remain largely unknown. The present study examines the effect of glucose on H+-ATPase activity in the pig kidney epithelial cell line LLC-PK1. Cellular pH was measured by performing ratiometric fluorescence microscopy using the pH-sensitive indicator BCECF-AM. Intracellular acidification was induced with NH3/NH4+ prepulse, and rates of intracellular pH (pHi) recovery (after in situ calibration) were determined by the slopes of linear regression lines during the first 3 min of recovery. The solutions contained 1 μM ethylisopropylamiloride and were K+ free to eliminate Na+/H+ exchange and H+-K+-ATPase activity. After NH3/NH4+-induced acidification, LLC-PK1 cells had a significant pHi recovery rate that was inhibited entirely by 100 nM of the V-ATPase inhibitor concanamycin A. Acute removal of glucose from medium markedly reduced V-ATPase-dependent pHi recovery activity. Readdition of glucose induced concentration-dependent reactivation of V-ATPase pHi recovery activity within 2 min. Glucose replacement produced no significant change in cell ATP or ADP content. H+-ATPase activity was completely inhibited by the glycolytic inhibitor 2-deoxy-d-glucose (20 mM) but only partially inhibited by the mitochondrial electron transport inhibitor antimycin A (20 μM). The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (500 nM) abolished glucose activation of V-ATPase, and activity was restored after wortmannin removal. Glucose activates V-ATPase activity in kidney epithelial cells through the glycolytic pathway by a signaling pathway that requires PI3K activity. These findings represent an entirely new physiological effect of glucose, linking it to cellular proton secretion and vacuolar acidification.

scholarly journals Continuous growth of proximal tubular kidney epithelial cells in hormone-supplemented serum-free medium.

1982 ◽  
Vol 94 (3) ◽  
pp. 506-510 ◽  
Author(s):  
L Chuman ◽  
L G Fine ◽  
A H Cohen ◽  
M H Saier
Keyword(s):  
Epithelial Cells ◽  
Fluid Transport ◽  
Defined Medium ◽  
Epithelial Cell Line ◽  
Serum Free Medium ◽  
Free Medium ◽  
Continuous Growth ◽  
Serum Free ◽  
Proximal Tubular ◽  
Kidney Epithelial Cells

An epithelial cell line from pig kidney (LLC-PK1) with properties of proximal tubular cells can be maintained indefinitely in hormone-supplemented serum-free medium. Continuous growth requires the presence of seven factors: transferrin, insulin, selenium, hydrocortisone, triiodothyronine, vasopressin, and cholesterol. The hormone-defined medium (a) supports growth of LLC-PK1 cells at a rate of approaching that observed in serum-supplemented medium; (b) allows vectorial transepithelial salt and fluid transport as measured by hemicyst formation; and (c) influences cell morphology. The vasopressin dependency for growth and morphology can be partially replaced by isobutylmethylxanthine or dibutyryl cyclic AMP. The medium has been used to isolate rabbit proximal tubular kidney epithelial cells free of fibroblasts.

Ouabain potentiates the activation of ERK1/2 by carbachol in parotid gland epithelial cells; inhibition of ERK1/2 reduces Na+-K+-ATPase activity

2006 ◽  
Vol 290 (3) ◽  
pp. C702-C710 ◽  
Author(s):  
Deana Plourde ◽  
Stephen P. Soltoff
Keyword(s):  
Epithelial Cells ◽  
Atpase Activity ◽  
Egf Receptor ◽  
Fluid Secretion ◽  
Ros Generation ◽  
Atpase Inhibitor ◽  
Egfr Transactivation ◽  
Intracellular Ca ◽  
Single Cell Type ◽  
Atpase Inhibition

The Na+-K+-ATPase and the ERK1/2 pathway appear to be linked in some fashion in a variety of cells. The Na+-K+-ATPase inhibitor ouabain can promote ERK1/2 activation. This activation involves Src, intracellular Ca2+ concentration ([Ca2+]i) elevation, reactive oxygen species (ROS) generation, and EGF receptor (EGFR) transactivation. In contrast, ERK1/2 can mediate changes in Na+-K+-ATPase activity and/or expression. Thus signaling between ERK1/2 and Na+-K+-ATPase can occur from either direction. Whether such bidirectionality can occur within the same cell has not been reported. In the present study, we have demonstrated that while ouabain (1 mM) produces only a small (∼50%) increase in ERK1/2 phosphorylation in freshly isolated rat salivary (parotid acinar) epithelial cells, it potentiates the phosphorylation of ERK1/2 by submaximal concentrations of carbachol, a muscarinic receptor ligand that initiates fluid secretion. Although ERK1/2 is only modestly phosphorylated when cells are exposed to 1 mM ouabain or 10−6 M carbachol, the combination of these agents promotes ERK1/2 phosphorylation to near-maximal levels achieved by a log order carbachol concentration. These effects of ouabain are distinct from Na+-K+-ATPase inhibition by lowering extracellular K+, which promotes a rapid and large increase in ERK1/2 phosphorylation. ERK1/2 potentiation by ouabain (EC50 ∼100 μM) involves PKC, Src, and alterations in [Ca2+]i but not ROS generation or EGFR transactivation. In addition, inhibition of ERK1/2 reduces Na+-K+-ATPase activity (measured as stimulation of Qo2 by carbachol and the cationophore nystatin). These results suggest that ERK1/2 and Na+-K+-ATPase may signal to each other in each direction under defined conditions in a single cell type.

scholarly journals The Vacuolar-TypeH+-ATPase in Ovine Rumen Epithelium is Regulated by Metabolic Signals

2010 ◽  
Vol 2010 ◽  
pp. 1-12 ◽  
Author(s):  
Judith Kuzinski ◽  
Rudolf Zitnan ◽  
Christina Warnke-Gurgel ◽  
Monika Schweigel
Keyword(s):  
Epithelial Cells ◽  
Atpase Activity ◽  
Metabolic Control ◽  
Energy Availability ◽  
Antimycin A ◽  
Atpase Inhibitor ◽  
B Subunit ◽  
Rumen Epithelium ◽  
Free Glucose ◽  
Rumen Epithelial Cells

In this study, the effect of metabolic inhibition (MI) by glucose substitution with 2-deoxyglucose (2-DOG) and/or application of antimycin A on ovine rumen epithelial cells (REC) vacuolar-typeH+-ATPase (vH+-ATPase) activity was investigated. Using fluorescent spectroscopy, basalpHiof REC was measured to be7.3±0.1inHCO3−-free, glucose-containing NaCl medium. MI induced a strongpHireduction (−0.44±0.04pH units) with a more pronounced effect of 2-DOG compared to antimycin A (−0.30±0.03versus−0.21±0.03pH units). Treatment with foliomycin, a specific vH+-ATPase inhibitor, decreased RECpHiby0.21±0.05pH units. After MI induction, this effect was nearly abolished (−0.03±0.02pH units). In addition, membrane-associated localization of vH+-ATPase B subunit disappeared. Metabolic control of vH+-ATPase involving regulation of its assembly state by elements of the glycolytic pathway could provide a means to adapt REC ATP consumption according to energy availability.

Three-dimensional Growth of Renal Epithelial Cells in Vitro: A Tool in Toxicity Testing

1993 ◽  
Vol 21 (2) ◽  
pp. 191-195 ◽  
Author(s):  
Knut-Jan Andersen ◽  
Erik Ilsø Christensen ◽  
Hogne Vik
Keyword(s):  
Epithelial Cells ◽  
Three Dimensional ◽  
Toxicity Testing ◽  
Renal Tissue ◽  
Epithelial Cell Line ◽  
Multicellular Spheroids ◽  
Renal Epithelial Cell ◽  
Renal Epithelial Cells

The tissue culture of multicellular spheroids from the renal epithelial cell line LLC-PK1 (proximal tubule) is described. This represents a biological system of intermediate complexity between renal tissue in vivo and simple monolayer cultures. The multicellular structures, which show many similarities to kidney tubules in vivo, including a vectorial water transport, should prove useful for studying the potential nephrotoxicity of drugs and chemicals in vitro. In addition, the propagation of renal epithelial cells as multicellular spheroids in serum-free culture may provide information on the release of specific biological parameters, which may be suppressed or masked in serum-supplemented media.

Stimulation of both human bronchial epithelium and neutrophils is needed for maximal interactive adhesion

1996 ◽  
Vol 270 (1) ◽  
pp. L80-L87 ◽  
Author(s):  
P. G. Bloemen ◽  
M. C. Van den Tweel ◽  
P. A. Henricks ◽  
F. Engels ◽  
M. J. Van de Velde ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cultured Cells ◽  
Bronchial Epithelium ◽  
Bronchial Epithelial Cells ◽  
Intercellular Adhesion Molecule ◽  
Tnf Alpha ◽  
Epithelial Cell Line ◽  
Ifn Gamma ◽  
Bronchial Epithelial ◽  
Stimulation Of

It has become clear that the bronchial epithelium is not just a passive barrier but plays an active role in inflammation. It can produce several inflammatory mediators and does express cell adhesion molecules of which intercellular adhesion molecule (ICAM)-1 can be upregulated by cytokines like interferon (IFN)-gamma. In the present study, we analyzed in detail the interaction of neutrophils with human bronchial epithelial cells, both primary cultured cells and the bronchial epithelial cell line BEAS-2B. Confluent monolayers of epithelial cells were incubated with freshly isolated 51Cr-labeled neutrophils for 30 min at 37 degrees C; then the nonadherent cells were removed by washing gently. Stimulation of the epithelial cells with IFN-gamma or the combination of IFN-gamma and tumor necrosis factor-alpha (TNF-alpha) (which doubles the ICAM-1 expression) increased neutrophil adhesion. Activation of the neutrophils themselves with N-formylmethionyl-leucyl-phenylalanine (fMLP), platelet-activating factor, or TNF-alpha also caused a profound enhancement of the adhesion. A significant additional increase was found when the epithelial cells had been exposed to IFN-gamma and the neutrophils were stimulated with fMLP simultaneously. This effect was even more pronounced with epithelium preincubated with IFN-gamma and TNF-alpha. With the use of monoclonal antibodies against CD18 and ICAM-1, it was demonstrated that the increased adhesion was mainly mediated by the ICAM-1/beta 2-integrin interaction. This study highlights that both the activation state of the bronchial epithelial cells and the activation state of the neutrophils are critical for their interactive adhesion.

Aberrant responses to growth-regulatory signals by variant kidney epithelial cells

1988 ◽  
Vol 254 (5) ◽  
pp. F747-F753
Author(s):  
M. M. Walsh-Reitz ◽  
R. I. Feldman ◽  
F. G. Toback
Keyword(s):  
Epithelial Cells ◽  
Growth Regulation ◽  
Growth Inhibitor ◽  
Soft Agar ◽  
Isoenzyme Analysis ◽  
Variant Cell ◽  
Kidney Epithelial Cells ◽  
Potent Growth ◽  
Potent Growth Inhibitor ◽  
Low K

Cultures that achieved a higher cell density than expected were noted during study of growth regulation in monkey kidney epithelial cells of the BSC-1 line. Multiplication of the variant cells was accelerated, compared with parental cells, as the cultures approached confluence. Cytogenetic analysis, immunofluorescence antibody reactions with specific monkey serum, isoenzyme analysis, microbiological studies, and lack of growth in soft agar indicated that the variant cells were not a contaminating cell type, lacked new isoenzymes, were free of microbial contamination, and were not transformed. Confluent variant cultures did not respond to a purified growth inhibitor protein produced by BSC-1 cells that inhibits multiplication and reduces cell Na content in subconfluent variant and parental cells. Vasopressin, which is a mitogen for parental cells, was a potent growth inhibitor for confluent cultures of variant cells. Low-K or high-Na media, which stimulate proliferation of parental cells, had no effect on growth of the variant cell line. These results suggest that enhanced multiplication of the variant cells is mediated by altered signal transduction pathways and/or receptors for growth-regulatory molecules.

Protective effects of astaxanthin on lipopolysaccharide-induced inflammation in bovine endometrial epithelial cells†

2019 ◽  
Vol 102 (2) ◽  
pp. 339-347 ◽  
Author(s):  
Fa-Chun Wan ◽  
Chen Zhang ◽  
Qing Jin ◽  
Chen Wei ◽  
Hong-Bo Zhao ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epithelial Cells ◽  
B Cell Lymphoma ◽  
Resistant Bacteria ◽  
Epithelial Cell Line ◽  
Protective Effects ◽  
Necrosis Factor Alpha ◽  
Antibiotic Resistant ◽  
Injury Treatment ◽  
Endometrial Epithelial Cells

Abstract Astaxanthin (AST), a natural antioxidant carotenoid, has been shown to exert anti-inflammatory effects. However, to our knowledge, no study has specifically addressed the potential protective effects of AST against bovine endometritis. The purpose of this study was to examine whether treatment with AST could protect endometrial epithelial cells against lipopolysaccharide (LPS)-induced inflammatory injury. Treatment of bovine endometrial (BEND) epithelial cell line with AST reduced LPS-induced production of interleukin-6 and tumor necrosis factor-alpha, increased the cellular activity of superoxide dismutase and catalase, decreased the proportion of apoptotic cells, and promoted the production of insulin-like growth factor and epithelial growth factor. The effects of AST were mediated through the downregulation of B-cell lymphoma 2 (Bcl-2) associated X, apoptosis regulator (Bax), and cleaved caspase-3 and through the upregulation of Bcl-2. Moreover, AST significantly increased the expression of the tight junction proteins (TJP) claudin, cadherin-1, and TJP1, which play an essential role in the maintenance of host endometrial defense barrier against pathogen infection. Collectively, these results demonstrated that treatment with AST protected against oxidative stress, prevented cell apoptosis, promoted BEND cells viability, and increased the production of growth factors, in addition to activating the endometrial defense barrier. Therefore, AST is a promising therapeutic agent for the prevention and treatment of endometritis. This finding is of utmost importance in the present times when the excessive use of antibiotics has resulted in the development of antibiotic-resistant bacteria.

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