scholarly journals ATP Release Mechanisms in Primary Cultures of Epithelia Derived from the Cysts of Polycystic Kidneys

1999 ◽  
Vol 10 (2) ◽  
pp. 218-229
Author(s):  
PATRICIA D. WILSON ◽  
JEFFREY S. HOVATER ◽  
CASH C. CASEY ◽  
JAMES A. FORTENBERRY ◽  
ERIK M. SCHWIEBERT
Keyword(s):  
Epithelial Cells ◽  
Primary Cultures ◽  
Atp Release ◽  
Cyst Fluid ◽  
Extracellular Nucleotides ◽  
Channel Blockers ◽  
Ion Channel Blockers ◽  
Autosomal Dominant Polycystic Kidney ◽  
Cyst Fluids

Abstract. Autosomal dominant polycystic kidney disease (ADPKD) cyst enlargement is exacerbated by accumulation of fluid within the lumen of the cyst. Extracellular nucleotides and nucleosides stimulate fluid and chloride (Cl-) secretion across epithelia and are potent autocrine and paracrine agonists within tissues. This study tests the hypothesis that ATP may be released by ADPKD epithelial cells. Once released, extracellular nucleotides and their metabolites may become “trapped” in the cyst lumen. As a consequence, extracellular ATP may augment ADPKD cyst enlargement through stimulation of salt and water secretion across ADPKD epithelia that encapsulate ADPKD cysts. To test this hypothesis, bioluminescence detection assays of ATP released from primary cultures of human ADPKD epithelial cells were compared with non-ADPKD human epithelial primary cultures. ADPKD cultures release comparable or greater amounts of ATP than non-ADPKD cultures derived from proximal tubule or cortex. ATP release in both ADPKD and non-ADPKD primary epithelial monolayers was directed largely into the apical medium; however, basolateral-directed ATP release under basal and stimulated conditions was also observed. Hypotonicity potentiated ATP release into the apical and basolateral medium in a reversible manner. Reconstitution of isotonic conditions with specific osmoles or inhibition with mechanosensitive ion channel blockers dampened hypotonicity-induced ATP release. “Flashfrozen” cyst fluids from ADPKD cysts, harvested from multiple donor kidneys, were screened by luminometry. A subset of cyst fluids contained as much as 0.5 to 10 μM ATP, doses sufficient to stimulate purinergic receptors. Taken together, these results show that ADPKD and non-ADPKD human epithelial primary cultures release ATP under basal and stimulated conditions and that ATP is released in vitro and into the cyst fluid by cystic epithelial cells in concentrations sufficient to stimulate ATP receptors. It is hypothesized that extracellular nucleotide release and signaling may contribute detrimentally to the gradual expansion of cyst fluid volume that is a hallmark of ADPKD.

VEGF receptor inhibition blocks liver cyst growth in pkd2(WS25/−) mice

2007 ◽  
Vol 293 (1) ◽  
pp. C419-C428 ◽  
Author(s):  
Claudia R. Amura ◽  
Kelley S. Brodsky ◽  
Rachel Groff ◽  
Vincent H. Gattone ◽  
Norbert F. Voelkel ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epithelial Cells ◽  
Liver Cyst ◽  
Cyst Fluid ◽  
Vegf Receptor ◽  
In Vitro Proliferation ◽  
Vegf Signaling ◽  
Cyst Growth ◽  
Cyst Fluids

Proliferation of cyst-lining epithelial cells is an integral part of autosomal dominant polycystic kidney disease (ADPKD) cyst growth. Cytokines and growth factors within cyst fluids are positioned to induce cyst growth. Vascular endothelial growth factor (VEGF) is a pleiotropic growth factor present in ADPKD liver cyst fluids (human 1,128 ± 78, mouse 2,787 ± 136 pg/ml) and, to a lesser extent, in ADPKD renal cyst fluids (human 294 ± 41, mouse 191 ± 90 pg/ml). Western blotting showed that receptors for VEGF (VEGFR1 and VEGFR2) were present in both normal mouse bile ducts and pkd2(WS25/−) liver cyst epithelial cells. Treatment of pkd2(WS25/−) liver cyst epithelial cells with VEGF (50–50,000 pg/ml) or liver cyst fluid induced a proliferative response. The effect on proliferation of liver cyst fluid was inhibited by SU-5416, a potent VEGF receptor inhibitor. Treatment of pkd2(WS25/−) mice between 4 and 8 mo of age with SU-5416 markedly reduced the cyst volume density of the liver (vehicle 9.9 ± 4.3%, SU-5416 1.8 ± 0.7% of liver). SU-5416 treatment between 4 and 12 mo of age markedly protected against increases in liver weight [pkd2(+/+) 4.8 ± 0.2%, pkd2(WS25/−)-vehicle 10.8 ± 1.9%, pkd2(WS25/−)-SU-5416 4.8 ± 0.4% body wt]. The capacity of VEGF signaling to induce in vitro proliferation of pkd2(WS25/−) liver cyst epithelial cells and inhibition of in vivo VEGF signaling to retard liver cyst growth in pkd2(WS25/−) mice indicates that the VEGF signaling pathway is a potentially important therapeutic target in the treatment of ADPKD liver cyst disease.

scholarly journals Attenuated, flow-induced ATP release contributes to absence of flow-sensitive, purinergic Cai2+ signaling in human ADPKD cyst epithelial cells

2009 ◽  
Vol 296 (6) ◽  
pp. F1464-F1476 ◽  
Author(s):  
Chang Xu ◽  
Boris E. Shmukler ◽  
Katherine Nishimura ◽  
Elzbieta Kaczmarek ◽  
Sandro Rossetti ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Atp Hydrolysis ◽  
Atp Release ◽  
Extracellular Atp ◽  
Mrna Levels ◽  
Normal Cells ◽  
Altered Expression ◽  
Cyst Cells ◽  
Autosomal Dominant Polycystic Kidney ◽  
Renal Tubular

Flow-induced cytosolic Ca2+ Cai2+ signaling in renal tubular epithelial cells is mediated in part through P2 receptor (P2R) activation by locally released ATP. The ability of P2R to regulate salt and water reabsorption has suggested a possible contribution of ATP release and paracrine P2R activation to cystogenesis and/or enlargement in autosomal dominant polycystic kidney disease (ADPKD). We and others have demonstrated in human ADPKD cyst cells the absence of flow-induced Cai2+ signaling exhibited by normal renal epithelial cells. We now extend these findings to primary and telomerase-immortalized normal and ADPKD epithelial cells of different genotype and of both proximal and distal origins. Flow-induced elevation of Cai2+ concentration ([Ca2+]i) was absent from ADPKD cyst cells, but in normal cells was mediated by flow-sensitive ATP release and paracrine P2R activation, modulated by ecto-nucleotidase activity, and abrogated by P2R inhibition or extracellular ATP hydrolysis. In contrast to the elevated ATP release from ADPKD cells in static isotonic conditions or in hypotonic conditions, flow-induced ATP release from cyst cells was lower than from normal cells. Extracellular ATP rapidly reduced thapsigargin-elevated [Ca2+]i in both ADPKD cyst and normal cells, but cyst cells lacked the subsequent, slow, oxidized ATP-sensitive [Ca2+]i recovery present in normal cells. Telomerase-immortalized cyst cells also exhibited altered CD39 and P2X7 mRNA levels. Thus the loss of flow-induced, P2R-mediated Cai2+ signaling in human ADPKD cyst epithelial cells was accompanied by reduced flow-sensitive ATP release, altered purinergic regulation of store-operated Ca2+ entry, and altered expression of gene products controlling extracellular nucleotide signaling.

Endogenous ATP release regulates Cl− secretion in cultured human and rat biliary epithelial cells

1999 ◽  
Vol 276 (6) ◽  
pp. G1391-G1400 ◽  
Author(s):  
Richard M. Roman ◽  
Andrew P. Feranchak ◽  
Kelli D. Salter ◽  
Yu Wang ◽  
J. Gregory Fitz
Keyword(s):  
Epithelial Cells ◽  
Cell Volume ◽  
Purinergic Signaling ◽  
Cell Volume Regulation ◽  
Atp Release ◽  
Extracellular Atp ◽  
Extracellular Nucleotides ◽  
Cell Swelling ◽  
Biliary Epithelial Cells ◽  
Normal Rat

P2Y receptor stimulation increases membrane Cl− permeability in biliary epithelial cells, but the source of extracellular nucleotides and physiological relevance of purinergic signaling to biliary secretion are unknown. Our objectives were to determine whether biliary cells release ATP under physiological conditions and whether extracellular ATP contributes to cell volume regulation and transepithelial secretion. With the use of a sensitive bioluminescence assay, constitutive ATP release was detected from human Mz-ChA-1 cholangiocarcinoma cells and polarized normal rat cholangiocyte monolayers. ATP release increased rapidly during cell swelling induced by hypotonic exposure. In Mz-ChA-1 cells, removal of extracellular ATP (apyrase) and P2 receptor blockade (suramin) reversibly inhibited whole cell Cl− current activation and prevented cell volume recovery during hypotonic stress. Moreover, exposure to apyrase induced cell swelling under isotonic conditions. In intact normal rat cholangiocyte monolayers, hypotonic perfusion activated apical Cl−currents, which were inhibited by addition of apyrase and suramin to bathing media. These findings indicate that modulation of ATP release by the cellular hydration state represents a potential signal coordinating cell volume with membrane Cl− permeability and transepithelial Cl−secretion.

scholarly journals New Gene Markers Involved in Molecular Processes of Tissue Repair, Response to Wounding and Regeneration Are Differently Expressed in Fibroblasts from Porcine Oral Mucosa during Long-Term Primary Culture

Animals ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1938
Author(s):  
Artur Bryja ◽  
Patrycja Sujka-Kordowska ◽  
Aneta Konwerska ◽  
Sylwia Ciesiółka ◽  
Maria Wieczorkiewicz ◽  
...  
Keyword(s):  
Wound Healing ◽  
Epithelial Cells ◽  
Expression Profile ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Primary Cultures ◽  
Gene Markers ◽  
Morphological Process

The mechanisms of wound healing and vascularization are crucial steps of the complex morphological process of tissue reconstruction. In addition to epithelial cells, fibroblasts play an important role in this process. They are characterized by dynamic proliferation and they form the stroma for epithelial cells. In this study, we have used primary cultures of oral fibroblasts, obtained from porcine buccal mucosa. Cells were maintained long-term in in vitro conditions, in order to investigate the expression profile of the molecular markers involved in wound healing and vascularization. Based on the Affymetrix assays, we have observed three ontological groups of markers as wound healing group, response to wounding group and vascularization group, represented by different genes characterized by their expression profile during long-term primary in vitro culture (IVC) of porcine oral fibroblasts. Following the analysis of gene expression in three previously identified groups of genes, we have identified that transforming growth factor beta 1 (TGFB1), ITGB3, PDPN, and ETS1 are involved in all three processes, suggesting that these genes could be recognized as markers of repair specific for oral fibroblasts within the porcine mucosal tissue.

Abnormal polarization of EGF receptors and autocrine stimulation of cyst epithelial growth in human ADPKD

1995 ◽  
Vol 269 (2) ◽  
pp. C487-C495 ◽  
Author(s):  
J. Du ◽  
P. D. Wilson
Keyword(s):  
Epithelial Cell ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Scatchard Analysis ◽  
Egf Receptors ◽  
Renal Epithelial Cell ◽  
Western Blots ◽  
High Affinity ◽  
Cyst Fluids

The underlying mechanism of the hyperproliferative response of human autosomal dominant polycystic kidney disease (ADPKD) epithelia was studied. Epidermal growth factor (EGF) protein is highly expressed in ADPKD cyst epithelia in vivo, and primary cultures are hyperesponsive to mitogenic stimulation by EGF in vitro. Doses of > 1 ng/ml EGF were highly mitogenic to ADPKD epithelia. 3H-labeled thymidine proliferation assays showed that cyst fluids and ADPKD epithelial cell-conditioned media also stimulated renal epithelial cell proliferation and contained EGF immunoreactivity (6, 30, and 37 kDa) as detected by Western blots. Radioimmunoassays detected mean levels of 2.87 and 1.4 ng/ml EGF in cyst fluids from early (proliferative) and end-stage ADPKD cysts, respectively. Scatchard analysis of 125I-labeled EGF binding to apical and basolateral membrane showed high-affinity binding to basolateral membranes of normal and ADPKD kidneys but additional unique high-affinity receptor binding to apical membranes of ADPKD but not normal kidneys. Cross-linking analysis and antiphosphotyrosine Western analysis demonstrated functionally active apical EGF receptors at 150-170 kDa. These results suggest mediation of cyst expansion via an autocrine loop involving EGF synthesis and processing by cyst epithelial cells, apical secretion into cyst lumens, and subsequent binding to and phosphorylation of apical membrane EGF receptors. These findings are consistent with a membrane protein polarization defect in ADPKD cyst epithelia.

Mechanical stimulation initiates cell-to-cell calcium signaling in ovine lens epithelial cells

1996 ◽  
Vol 109 (2) ◽  
pp. 355-365 ◽  
Author(s):  
G.C. Churchill ◽  
M.M. Atkinson ◽  
C.F. Louis
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Gap Junction ◽  
Mechanical Stimulation ◽  
Primary Cultures ◽  
Channel Blockers ◽  
Extracellular Medium ◽  
Gap Junction Channels ◽  
Cell Propagation ◽  
Cytosolic Factor

Although abnormalities in calcium regulation have been implicated in the development of most forms of cataract, the mechanisms by which Ca2+ is regulated in the cells of the ocular lens remain poorly defined. Cell-to-cell Ca2+ signaling was investigated in primary cultures of ovine epithelial cells using the Ca(2+)-reporter dye fura-2 and fluorescence microscopy. Mechanical stimulation of a single cell with a micropipette initiated a propagated increase in cytosolic free Ca2+ that spread from the stimulated cell through 2–8 tiers of surrounding cells. During this intercellular Ca2+ wave, cytosolic Ca2+ increased 2- to 12-fold from resting levels of approximately 100 nM. Nanomolar extracellular Ca2+ did not affect the cell-to-cell propagation of the Ca2+ wave, but reduced the magnitude of the cytosolic Ca2+ increases, which was most evident in the mechanically-stimulated cell. Depletion of intracellular Ca2+ stores with thapsigargin eliminated the propagated intercellular Ca2+ wave, but did not prevent the cytosolic Ca2+ increase in the mechanically-stimulated cell, which required extracellular Ca2+ and was attenuated by the addition of the Ca2+ channel blockers Ni2+, Gd3+ and La3+ to the medium. These results are most easily explained by a mechanically-activated channel in the plasma membrane of the stimulated cell. The propagated increase in cytosolic Ca2+ appeared to be communicated to adjacent cells by the passage of an intracellular messenger other than Ca2+ through gap junction channels. However, if the plasma membrane of the mechanically-stimulated cell was ruptured such that there was loss of cytosolic contents, the increase in cytosolic Ca2+ in the surrounding cells was elicited by both a messenger passing through gap junction channels and by a cytosolic factor(s) diffusing through the extracellular medium. These results demonstrate the existence of intercellular Ca2+ signaling in lens cells, which may play a role in regulating cytosolic Ca2+ in the intact lens.

Estrogen-dependent expression of the cystic fibrosis transmembrane regulator gene in a novel uterine epithelial cell line

1994 ◽  
Vol 107 (9) ◽  
pp. 2439-2448
Author(s):  
L. Rochwerger ◽  
S. Dho ◽  
L. Parker ◽  
J.K. Foskett ◽  
M. Buchwald
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Steroid Hormones ◽  
Primary Cultures ◽  
T Antigen ◽  
Epithelial Cell Line ◽  
Normal Female ◽  
Western Blots ◽  
Uterine Epithelial Cell

We have demonstrated previously the modulation of CFTR expression by estrogen in vivo in the rat uterine epithelium. The purpose of this study was to establish a suitable in vitro system to investigate the regulation of CFTR by steroid hormones. Primary cultures of rat uterine epithelial cells, which showed high levels of CFTR expression in vitro, were infected with an adeno/SV40 virus. One clone, UIT 1.16, which retained the morphology of the primary epithelial cells yet proliferated beyond the life span of the primary culture, was isolated and characterized. Successful immortalization of UIT 1.16 cells was verified by the presence of a band corresponding to the SV40 large T-antigen in western blots, as well as by their ability to proliferate continuously. Transmission electron microscopy studies revealed that these cells maintained the characteristics of a polarized epithelium with well-established membrane domains and specialized intercellular junctions. A high transepithelial electrical resistance was also observed when cells were assayed in modified Ussing chambers. When the basolateral cellular membrane of cells grown in vitrogen-coated filters was permeabilized with nystatin, a forskolin-stimulated Cl- permeability was observed in the apical membrane, similar to that present in other CFTR-expressing epithelial cells. UIT 1.16 cells showed high levels of CFTR expression on northern blots. The expression of CFTR was dependent on the presence of estrogen in the culture medium, since almost undetectable levels of CFTR mRNA were observed when the cells were cultured in medium containing serum depleted of steroid hormones. However, addition of estrogen to this medium prevented the disappearance of CFTR mRNA, confirming estrogen-regulated expression of CFTR in the UIT 1.16 cell line. The newly developed UIT 1.16 cell line provides a valuable model to analyze the regulation of CFTR expression by steroid hormones. Moreover, the cell line could also be used to investigate the role of CFTR in the uterus during the normal female cycle as well as for the study of other uterine epithelial functions and the agents that regulate them.

scholarly journals Pathways for ATP release by bovine ciliary epithelial cells, the initial step in purinergic regulation of aqueous humor inflow

2010 ◽  
Vol 299 (6) ◽  
pp. C1308-C1317 ◽  
Author(s):  
Ang Li ◽  
Chi Ting Leung ◽  
Kim Peterson-Yantorno ◽  
Claire H. Mitchell ◽  
Mortimer M. Civan
Keyword(s):  
Epithelial Cells ◽  
Cell Lines ◽  
Aqueous Humor ◽  
Primary Cultures ◽  
Atp Release ◽  
Initial Step ◽  
Bafilomycin A1 ◽  
Pannexin 1 ◽  
Quantitative Expression ◽  
Vesicular Release

ATP release by nonpigmented (NPE) and pigmented (PE) ciliary epithelial cells is the enabling step in purinergic regulation of aqueous humor formation, but the release pathways are unknown. We measured ATP release from primary cultures of bovine mixed NPE and PE (bCE) cells and transformed bovine NPE and PE cells, using the luciferin-luciferase reaction. Hypotonicity-triggered bCE ATP release was inhibited by the relatively selective blocker of pannexin-1 (PX1) hemichannels (probenecid, 1 mM, 47 ± 2%), by a connexin inhibitor (heptanol, 1 mM, 49 ± 4%), and by an inhibitor of vesicular release (bafilomycin A1, 25 ± 2%), but not by the P2X7 receptor (P2RX7) antagonist KN-62. Bafilomycin A1 acts by reducing the driving force for uptake of ATP from the cytosol into vesicles. The reducing agent dithiothreitol reduced probenecid-blockable ATP release. Similar results were obtained with NPE and PE cell lines. Pannexins PX1–3, connexins Cx43 and Cx40, and P2RX7 were identified in native cells and cell lines by RT-PCR. PX1 mRNA expression was confirmed by Northern blots; its quantitative expression was comparable to that of Cx43 by real-time PCR. Heterologous expression of bovine PX1 in HEK293T cells enhanced swelling-activated ATP release, inhibitable by probenecid. We conclude that P2RX7-independent PX1 hemichannels, Cx hemichannels, and vesicular release contribute comparably to swelling-triggered ATP release. The relatively large response to dithiothreitol raises the possibility that the oxidation-reduction state is a substantial regulator of PX1-mediated ATP release from bovine ciliary epithelial cells.

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