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.

scholarly journals Human ADPKD primary cyst epithelial cells with a novel, single codon deletion in the PKD1 gene exhibit defective ciliary polycystin localization and loss of flow-induced Ca2+ signaling

2007 ◽  
Vol 292 (3) ◽  
pp. F930-F945 ◽  
Author(s):  
Chang Xu ◽  
Sandro Rossetti ◽  
Lianwei Jiang ◽  
Peter C. Harris ◽  
Ursa Brown-Glaberman ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Nk Cells ◽  
Coiled Coil ◽  
Human Kidney ◽  
Coiled Coil Domain ◽  
Normal Human Kidney ◽  
Cyst Cells ◽  
Localization Signal ◽  
Normal Human ◽  
Autosomal Dominant Polycystic Kidney

Autosomal dominant polycystic kidney disease (ADPKD) gene products polycystin-1 (PC1) and polycystin-2 (PC2) colocalize in the apical monocilia of renal epithelial cells. Mouse and human renal cells without PC1 protein show impaired ciliary mechanosensation, and this impairment has been proposed to promote cystogenesis. However, most cyst epithelia of human ADPKD kidneys appear to express full-length PC1 and PC2 in normal or increased abundance. We show that confluent primary ADPKD cyst cells with the novel PC1 mutation ΔL2433 and with normal abundance of PC1 and PC2 polypeptides lack ciliary PC1 and often lack ciliary PC2, whereas PC1 and PC2 are both present in cilia of confluent normal human kidney (NK) epithelial cells in primary culture. Confluent NK cells respond to shear stress with transient increases in cytoplasmic Ca2+ concentration ([Ca2+]i), dependent on both extracellular Ca2+ and release from intracellular stores. In contrast, ADPKD cyst cells lack flow-sensitive [Ca2+]i signaling and exhibit reduced endoplasmic reticulum Ca2+ stores and store-depletion-operated Ca2+ entry but retain near-normal [Ca2+]i responses to ANG II and to vasopressin. Expression of wild-type and mutant CD16.7-PKD1(115–226) fusion proteins reveals within the COOH-terminal 112 amino acids of PC1 a coiled-coil domain-independent ciliary localization signal. However, the coiled-coil domain is required for CD16.7-PKD1(115–226) expression to accelerate decay of the flow-induced Ca2+ signal in NK cells. These data provide evidence for ciliary dysfunction and polycystin mislocalization in human ADPKD cells with normal levels of PC1.

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.

Mcl-1 is downregulated in cisplatin-induced apoptosis, and proteasome inhibitors restore Mcl-1 and promote survival in renal tubular epithelial cells

2007 ◽  
Vol 292 (6) ◽  
pp. F1710-F1717 ◽  
Author(s):  
Cheng Yang ◽  
Varsha Kaushal ◽  
Sudhir V. Shah ◽  
Gur P. Kaushal
Keyword(s):  
Epithelial Cells ◽  
Cytochrome C ◽  
Caspase 3 ◽  
Proteasome Inhibitors ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Tubular Epithelial Cells ◽  
Renal Tubular Epithelial Cells ◽  
Renal Tubular ◽  
Induced Apoptosis

Mcl-1 is an antiapoptotic member of the Bcl-2 family that plays an important role in cell survival. We demonstrate that proteasome-dependent regulation of Mcl-1 plays a critical role in renal tubular epithelial cell injury from cisplatin. Protein levels of Mcl-1 rapidly declined in a time-dependent manner following cisplatin treatment of LLC-PK1cells. However, mRNA levels of Mcl-1 were not altered following cisplatin treatment. Expression of other antiapoptotic members of the Bcl-2 family such as Bcl-2 and BclxL was not affected by cisplatin treatment. Cisplatin-induced loss of Mcl-1 occurs at the same time as the mitochondrial release of cytochrome c, activation of caspase-3, and initiation of apoptosis. Treatment of cells with cycloheximide, a protein synthesis inhibitor, revealed rapid turnover of Mcl-1. In addition, treatment with cycloheximide in the presence or absence of cisplatin demonstrated that cisplatin-induced loss of Mcl-1 results from posttranslational degradation rather than transcriptional inhibition. Overexpression of Mcl-1 protected cells from cisplatin-induced caspase-3 activation and apoptosis. Preincubating cells with the proteasome inhibitor MG-132 or lactacystin not only restored cisplatin-induced loss of Mcl-1 but also resulted in an accumulation of Mcl-1 that exceeded basal levels; however, Bcl-2 and BclxL levels did not change in response to MG-132 or lactacystin. The proteasome inhibitors effectively blocked cisplatin-induced mitochondrial release of cytochrome c, caspase-3 activation, and apoptosis. These studies suggest that proteasome regulation of Mcl-1 is crucial in the cisplatin-induced apoptosis via the mitochondrial apoptotic pathway and that Mcl-1 is an important therapeutic target in cisplatin injury to renal tubular epithelial cells.

Autophagy is associated with apoptosis in cisplatin injury to renal tubular epithelial cells

2008 ◽  
Vol 294 (4) ◽  
pp. F777-F787 ◽  
Author(s):  
Cheng Yang ◽  
Varsha Kaushal ◽  
Sudhir V. Shah ◽  
Gur P. Kaushal
Keyword(s):  
Cell Death ◽  
Epithelial Cells ◽  
Beclin 1 ◽  
Lag Phase ◽  
Tubular Epithelial Cells ◽  
Renal Tubular Epithelial Cells ◽  
Normal Cells ◽  
Life And Death ◽  
Renal Tubular ◽  
Induced Apoptosis

Autophagy has emerged as another major “programmed” mechanism to control life and death much like “programmed cell death” is for apoptosis in eukaryotes. We examined the expression of autophagic proteins and formation of autophagosomes during progression of cisplatin injury to renal tubular epithelial cells (RTEC). Autophagy was detected as early as 2–4 h after cisplatin exposure as indicated by induction of LC3-I, conversion of LC3-I to LC3-II protein, and upregulation of Beclin 1 and Atg5, essential markers of autophagy. The appearance of cisplatin-induced punctated staining of autophagosome-associated LC3-II upon GFP-LC3 transfection in RTEC provided further evidence for autophagy. The autophagy inhibitor 3-methyladenine blocked punctated staining of autophagosomes. The staining of normal cells with acridine orange displayed green fluorescence with cytoplasmic and nuclear components in normal cells but displayed considerable red fluorescence in cisplatin-treated cells, suggesting formation of numerous acidic autophagolysosomal vacuoles. Autophagy inhibitors LY294002 or 3-methyladenine or wortmannin inhibited the formation of autophagosomes but induced apoptosis after 2–4 h of cisplatin treatment as indicated by caspase-3/7 and -6 activation, nuclear fragmentation, and cell death. This switch from autophagy to apoptosis by autophagic inhibitors further suggests that the preapoptotic lag phase after treatment with cisplatin is mediated by autophagy. At later stages of cisplatin injury, apoptosis was also found to be associated with autophagy, as autophagic inhibitors and inactivation of autophagy proteins Beclin 1 and Atg5 enhanced activation of caspases and apoptosis. Our results demonstrate that induction of autophagy mounts an adaptive response, suppresses cisplatin-induced apoptosis, and prolongs survival of RTEC.

scholarly journals House Dust Mite Induces IL-33 Release From Human Nasal Epithelial Cells via P2Y Purinergic Signals and ERK/P38 MAPK Pathways.

2021 ◽  
Author(s):  
Bin Liu ◽  
Pei Zhou ◽  
Tingting Feng ◽  
Jiping Li
Keyword(s):  
Epithelial Cells ◽  
House Dust ◽  
House Dust Mite ◽  
Atp Release ◽  
Extracellular Atp ◽  
Nasal Epithelial Cells ◽  
Interleukin 33 ◽  
Atp Assay ◽  
Human Nasal Epithelial Cells ◽  
Dust Mite

Abstract BackgroundAllergic rhinitis (AR) is an inflammatory disease of the nasal mucosa, which is triggered by stimulations of environmental allergens such as house dust mite (HDM). Th2-type proinflammatory factor interleukin 33 (IL-33) plays an important role in the pathogenesis of AR, but it remains unknown how IL-33 products in human nasal epithelial cells (HNECs) mediated by HDM. MethodsWe investigated the effect of HDM allergens by analyzing the accumulation of Ca2+ levels and IL-33 release in HNECs. Involvements of Adenosine triphosphate (ATP)-dependent activation of P2Y-PLC-IP3 pathways, downstream of Ca2+ signaling and P38/ERK pathways were studied, using the P2Y-PLC-IP3 pathways agonists, the calcium chelators, and P38/ERK pathways inhibitors. ResultsDer p induced expression of IL-33 mRNA and protein in HNECs via ERK/P38 pathways. Average 69.4% of co-localization of quinacrine and Lyso-tracker fluorescent puncta revealed that ATP was mainly stored in the lysosomes of nasal epithelial cells. After stimulation with Der p, ATP released from lysosomes by P2Y-PLC-IP3-Ca2+pathways. The ATP assay of HNECs culture supernatants implied an acute accumulation of extracellular ATP immediately after the Der p stimulation. Using P2Y-PLC-IP3 signaling inhibitors, we found that the Der p-induced IL-33 release was dependent on ATP-P2Y-PLC-IP3 signaling, followed by abolishing the ERK/P38 pathways. ConclusionDer p induced an acute accumulation of extracellular ATP which activated PY2-PLC-IP3 pathways to induce Ca2+ releasing from endoplasmic reticulum (ER), and intracellular Ca2+ induced ATP release from lysosomes from HNECs. ATP activated PY2-PLC-IP3 pathways followed by transactivation of ERK/P38 pathways which induced the expression of IL-33 mRNA and protein.

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.

Kinetics of ATP release and cell volume regulation of hyposmotically challenged goldfish hepatocytes

2008 ◽  
Vol 294 (1) ◽  
pp. R220-R233 ◽  
Author(s):  
Diego E. Pafundo ◽  
Osvaldo Chara ◽  
María P. Faillace ◽  
Gerhard Krumschnabel ◽  
Pablo J. Schwarzbaum
Keyword(s):  
Atp Hydrolysis ◽  
Extracellular Volume ◽  
Regulatory Volume Decrease ◽  
Cell Volume Regulation ◽  
Atp Release ◽  
Extracellular Atp ◽  
P2 Receptor ◽  
Animal Cells ◽  
Receptor Antagonists ◽  
Volume Decrease

In most animal cells, hypotonic swelling is followed by a regulatory volume decrease (RVD) thought to prevent cell death. In contrast, goldfish hepatocytes challenged with hypotonic medium (180 mosM, HYPO) increase their volume 1.7 times but remain swollen and viable for at least 5 h. Incubation with ATPγS (an ATP analog) in HYPO triggers a 42% volume decrease. This effect is concentration dependent ( K1/2 = 760 nM) and partially abolished by P2 receptor antagonists (64% inhibition). A similar induction of RVD is observed with ATP, UTP, and UDP, whereas adenosine inhibits RVD. Goldfish hepatocytes release more than 500 nM ATP during the first minutes of HYPO with no induction of RVD. The fact that similar concentrations of ATPγS did trigger RVD could be explained by showing that ATPγS induced ATP release. Finally, we observed that in a very small extracellular volume, hepatocytes do show a 56% RVD. This response was diminished by P2 receptor antagonists (73%) and increased (73%) when the extracellular ATP hydrolysis was inhibited 72%. Using a mathematical model, we predict that during the first 2 min of HYPO exposure the extracellular [ATP] is mainly governed by ATP diffusion and by both nonlytic and lytic ATP release, with almost no contribution from ecto-ATPase activity. We show that goldfish hepatocytes under standard HYPO (large volume) do not display RVD unless this is triggered by the addition of micromolar concentrations of nucleotides. However, under very low assay volumes, sufficient endogenous extracellular [ATP] can build up to induce RVD.

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