Expression of nucleotide-regulated Cl−currents in CF and normal mouse tracheal epithelial cell lines

2000 ◽  
Vol 279 (5) ◽  
pp. C1578-C1586 ◽  
Author(s):  
E. J. Thomas ◽  
S. E. Gabriel ◽  
M. Makhlina ◽  
S. P. Hardy ◽  
M. I. Lethem
Keyword(s):  
Epithelial Cell ◽  
Cell Lines ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
T Antigen ◽  
Epithelial Cell Lines ◽  
Chamber Studies ◽  
Cf Transmembrane Conductance Regulator

The dominant route for Cl− secretion in mouse tracheal epithelium is via Cl− channels different from the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the channel that is defective in CF. It has been proposed that the use of purinergic agonists to activate these alternative channels in human airways may be beneficial in CF. In the present study, two conditionally immortal epithelial cell lines were established from the tracheae of mice possessing the tsA58 T antigen gene, one of which [MTE18-(−/−)] was homozygous for a knockout of CFTR and the other [MTE7b-(+/−)] heterozygous for CFTR expression. In Ussing chamber studies, amiloride (10−4 M) and a cocktail of cAMP-activating agents (forskolin, IBMX, and dibutyryl cAMP) resulted in small changes in the short-circuit current ( I sc) and resistance of both cell lines, with larger increases in I scbeing elicited by ionomycin (10−6 M). Both cell lines expressed P2Y2 receptors and responded to the purinergic agonists ATP, UTP, and 5′-adenylylimidodiphosphate (10−4 M) with an increase in I sc. This response could be inhibited by DIDS and was abolished in the presence of Cl−-free Ringer solution. Reducing the mucosal Cl− concentration increased the response to UTP of both cell lines, with a significantly greater increase in MTE18-(−/−) cells. Pretreatment of these cells with thapsigargin caused a direct increase in I sc and inhibited the response to UTP. These data suggest that both cell lines express purinergic-regulated Cl− currents and may prove valuable tools in studying the properties of this pathway.

Extracellular UTP stimulates electrogenic bicarbonate secretion across CFTR knockout gallbladder epithelium

2000 ◽  
Vol 279 (1) ◽  
pp. G132-G138 ◽  
Author(s):  
Lane L. Clarke ◽  
Matthew C. Harline ◽  
Lara R. Gawenis ◽  
Nancy M. Walker ◽  
John T. Turner ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Receptor Activation ◽  
Biliary Disease ◽  
Anion Conductance ◽  
Intracellular Ca ◽  
Ph Stat ◽  
Chamber Studies

The loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial HCO3 − secretion contributes to the pathogenesis of pancreatic and biliary disease in cystic fibrosis (CF) patients. Recent studies have investigated P2Y2 nucleotide receptor agonists, e.g., UTP, as a means to bypass the CFTR defect by stimulating Ca2+-activated Cl− secretion. However, the value of this treatment in facilitating transepithelial HCO3 − secretion is unknown. Gallbladder mucosae from CFTR knockout mice were used to isolate the Ca2+-dependent anion conductance during activation of luminal P2Y2receptors. In Ussing chamber studies, UTP stimulated a transient peak in short-circuit current ( I sc) that declined to a stable plateau phase lasting 30–60 min. The plateau I sc after UTP was Cl− independent, HCO3 − dependent, insensitive to bumetanide, and blocked by luminal DIDS. In pH stat studies, luminal UTP increased both I sc and serosal-to-mucosal HCO3 − flux ( J s→m) during a 30-min period. Substitution of Cl− with gluconate in the luminal bath to inhibit Cl−/HCO3 −exchange did not prevent the increase in J s→mand I sc during UTP. In contrast, luminal DIDS completely inhibited UTP-stimulated increases in J s→m and I sc. We conclude that P2Y2 receptor activation results in a sustained (30–60 min) increase in electrogenic HCO3 − secretion that is mediated via an intracellular Ca2+-dependent anion conductance in CF gallbladder.

scholarly journals Synergy between Apc min and an activated ras mutation is sufficient to induce colon carcinomas.

1996 ◽  
Vol 16 (3) ◽  
pp. 884-891 ◽  
Author(s):  
G M D'Abaco ◽  
R H Whitehead ◽  
A W Burgess
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Soft Agar ◽  
T Antigen ◽  
Colonic Epithelial Cell ◽  
Large T Antigen ◽  
Ras Gene ◽  
Sv40 Large T Antigen ◽  
Epithelial Cell Lines

Colon carcinomas appear to arise from the cumulative effect of mutations to several genes (APC, DCC, p53, ras, hMLH1, and hMSH2). By using novel colonic epithelial cell lines derived from the Immorto mouse, named the YAMC (young adult mouse colon) cell line, and an Immorto-Min mouse hybrid, named the IMCE (Immorto-Min colonic epithelial) cell line, carrying the Apc min mutation, we investigated the effect of an activated v-Ha-ras gene on tumor progression. The YAMC and IMCE cell lines are normal colonic epithelial cell lines which are conditionally immortalized by virtue of expression of a temperature-sensitive simian virus 40 (SV40) large T antigen. Under conditions which permit expression of a functional SV40 large T antigen (33 degrees C plus gamma interferon), neither the YAMC nor the IMCE cell line grows in soft agar or is tumorigenic in nude mice. In vitro, when the SV40 large T antigen is inactivated (39 degrees C without gamma interferon), the cells stop proliferating and die. By infecting the YAMC and IMCE cell lines with a replication-defective psi2-v-Ha-ras virus, we derived cell lines which overexpress the v-Ha-ras gene (YAMC-Ras and IMCE-Ras). In contrast to the parental cell lines, under conditions in which the SV40 large T antigen is inactive, both the YAMC-Ras and IMCE-Ras cell lines continue to proliferate. Initally YAMC-Ras cells do not form tumors; however, tumors are visible after 90 days of incubation. IMCE-Ras cells form colonies in soft agar under both permissive and nonpermissive culture conditions. Furthermore, IMCE-Ras cells form tumors in nude mice within 3 weeks. The phenotype of the IMCE-Ras cell line thus clearly demonstrates that a defective Apc allele and an activated ras gene are sufficient to transform normal colonic epithelial cells and render them tumorigenic.

Immortalised mouse submandibular epithelial cell lines retain polarised structural and functional properties

1996 ◽  
Vol 109 (12) ◽  
pp. 2789-2800 ◽  
Author(s):  
B.M. Laoide ◽  
Y. Courty ◽  
I. Gastinne ◽  
C. Thibaut ◽  
O. Kellermann ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cell Lines ◽  
T Antigen ◽  
Growth Potential ◽  
Specific Gene ◽  
Type I ◽  
Collagen Gels ◽  
Ductal Cell ◽  
Structural And Functional Properties ◽  
Epithelial Cell Lines

The mouse submandibular gland (SMG) is an excellent model for the study of many important biological phenomena such as hormonal regulation of differentiation, neurotransmitter control of secretion, epithelial transport, exocytosis and endocytosis as well as the regulation of mouse SMG specific gene expression, in particular, NGF, EGF and renin. The postnatal development and sexual dimorphism of the mouse gland permits the isolation of male SMGs of different ages, corresponding to different stages of differentiation, particularly with respect to the cytodifferentiation of ductal cell types. We have immortalized SMG epithelial cell lines using mice transgenic for the large T antigen of SV40 or polyoma viruses. Epithelial clusters from the dissected glands were placed in culture and cell lines were established from the immortalized population. Two cell lines, SIMS and SIMP, which retain structural and functional characteristics, are described here. The cell lines are immortalised but not transformed, as judged by the absence of anchorage independent growth potential and the lack of tumour formation in athymic nude mice. Confocal and electron microscopy examination demonstrate that SIMP and SIMS cells express E-cadherin and ZO-1 and have features of polarised epithelial cells. In addition, they form spherical cysts with a wide lumen when grown in type I collagen gels. When grown on a filter support SIMS cells form a tight monolayer, exhibit vectorial transport function and show exclusive Na+, K(+)-ATPase localisation to the basolateral domain. We determined the cell type restricted expression of cytokeratin markers in the mouse SMG in vivo and we demonstrate that SIMS and SIMP cell lines express duct-specific cytokeratins. Finally, the expression of a set of differentiation markers, including EGF, NGF and renin, was detected by RT-PCR and by indirect immunofluorescence staining in these lines. Thus, these polarised ductal cell lines, as well as having important intrinsic properties, represent well characterised mouse epithelial models which, until now, have not been readily available for cellular studies.

scholarly journals Novel human bronchial epithelial cell lines for cystic fibrosis research

2009 ◽  
Vol 296 (1) ◽  
pp. L82-L91 ◽  
Author(s):  
M. L. Fulcher ◽  
S. E. Gabriel ◽  
J. C. Olsen ◽  
J. R. Tatreau ◽  
M. Gentzsch ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cell ◽  
Cell Lines ◽  
Cell Structure ◽  
Short Circuit ◽  
Growth Potential ◽  
Secretory Cells ◽  
Primary Cells ◽  
Bronchial Epithelial ◽  
Epithelial Cell Lines

Immortalization of human bronchial epithelial (hBE) cells often entails loss of differentiation. Bmi-1 is a protooncogene that maintains stem cells, and its expression creates cell lines that recapitulate normal cell structure and function. We introduced Bmi-1 and the catalytic subunit of telomerase (hTERT) into three non-cystic fibrosis (CF) and three ΔF508 homozygous CF primary bronchial cell preparations. This treatment extended cell life span, although not as profoundly as viral oncogenes, and at passages 14 and 15, the new cell lines had a diploid karyotype. Ussing chamber analysis revealed variable transepithelial resistances, ranging from 200 to 1,200 Ω·cm2. In the non-CF cell lines, short-circuit currents were stimulated by forskolin and inhibited by CFTR(inh)-172 at levels mostly comparable to early passage primary cells. CF cell lines exhibited no forskolin-stimulated current and minimal CFTR(inh)-172 response. Amiloride-inhibitable and UTP-stimulated currents were present, but at lower and higher amplitudes than in primary cells, respectively. The cells exhibited a pseudostratified morphology, with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells, and occasional ciliated cells. CF and non-CF cell lines produced similar levels of IL-8 at baseline and equally increased IL-8 secretion in response to IL-1β, TNF-α, and the Toll-like receptor 2 agonist Pam3Cys. Although they have lower growth potential and more fastidious growth requirements than viral oncogene transformed cells, Bmi-1/hTERT airway epithelial cell lines will be useful for several avenues of investigation and will help fill gaps currently hindering CF research and therapeutic development.

Asymmetric effects of H2O2 on alveolar epithelial barrier properties

1993 ◽  
Vol 264 (3) ◽  
pp. L308-L315 ◽  
Author(s):  
K. J. Kim ◽  
D. J. Suh
Keyword(s):  
Alveolar Epithelial Cell ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Barrier Properties ◽  
Ringer Solution ◽  
Alveolar Epithelial Cells ◽  
Dependent Manner ◽  
Polycarbonate Membrane ◽  
Alveolar Epithelial

The effects of H2O2 on active ion transport and resistance to passive solute flow were studied utilizing rat alveolar epithelial cell monolayers cultured on permeable supports. Type II alveolar epithelial cells were plated onto tissue culture-treated polycarbonate membrane filters. The resulting confluent monolayers on days 3 and 4 were mounted in a modified Ussing chamber and bathed on both sides with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Ringer solution. These monolayers have a high transepithelial resistance (> 2,000 omega.cm2) and actively transport Na+ from apical fluid. H2O2 (0-100 mM) was then delivered to either apical or basolateral fluid. The changes in short-circuit current (Isc) and monolayer resistance (R) in response to the exogenous hydroperoxide were measured. To determine the degree of cellular catalase participation in protection against H2O2 injury to the barrier, experiments were repeated in the presence of 20 mM aminotriazole (ATAZ; an inhibitor of catalase) in the same bathing fluid as the hydroperoxide. Results indicated that H2O2 decreased Isc and R gradually in a dose-dependent manner. The effective concentration of apical H2O2 at which Isc (or R) was decreased by 50% at 1 h (ED50) was approximately 4 mM. However, basolateral H2O2 exposure led to ED50 for Isc (and R) of approximately 0.04 mM. Inhibition of cellular catalase yielded ED50 for Isc (and R) of approximately 0.4 mM when H2O2 was given apically, while ED50 for basolateral exposure to H2O2 did not change in the presence of ATAZ. The rate of H2O2 consumption in apical and basolateral bathing fluids was the same, while cellular catalase activity rose gradually with time in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of normal and cystic fibrosis phenotypes by continuous airway epithelial cell lines

1990 ◽  
Vol 259 (6) ◽  
pp. L496-L505 ◽  
Author(s):  
D. M. Jefferson ◽  
J. D. Valentich ◽  
F. C. Marini ◽  
S. A. Grubman ◽  
M. C. Iannuzzi ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cell ◽  
Cell Lines ◽  
T Antigen ◽  
Ionophore A23187 ◽  
Airway Epithelial ◽  
Sv40 Large T Antigen ◽  
Continuous Cell Lines ◽  
Epithelial Cell Lines ◽  
Normal Controls

Continuous epithelial cell lines from individuals with cystic fibrosis (CF) and normal controls are required to understand the genetic and cellular defects in CF. We used retroviruses to transduce SV40 large T antigen into nasal epithelial cells. Transformed continuous cell lines were isolated that expressed epithelial markers, cytokeratin, and tight junctions. Northern blot analysis shows that all of the cell lines express the putative CF gene mRNA. Studies of transepithelial electrolyte transport show that CF and normal cell lines develop a transepithelial electrical resistance. Normal but not CF cell lines secreted Cl- in response to agonists that increase cellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) (isoproterenol, forskolin, and a membrane-permeant analogue of cAMP) or in response to a tumor-promoting phorbol ester that activates protein kinase C. In contrast, the Ca2(+)-elevating agonist bradykinin and the Ca2+ ionophore A23187 stimulated secretion in both normal and CF cell lines. The continuous cell lines we have produced maintain their proper phenotypes and will serve as useful tools in understanding the pathophysiology of CF.

Sign in / Sign up

Export Citation Format

Share Document