Sulfate and chloride transport in Caco-2 cells: differential regulation by thyroxine and the possible role of DRAgene

2001 ◽  
Vol 280 (4) ◽  
pp. G603-G613 ◽  
Author(s):  
W. A. Alrefai ◽  
S. Tyagi ◽  
F. Mansour ◽  
S. Saksena ◽  
I. Syed ◽  
...  
Keyword(s):  
Cell Line ◽  
Hormonal Regulation ◽  
Competitive Inhibition ◽  
Chloride Transport ◽  
Exchange Process ◽  
Rnase Protection Assay ◽  
Maximum Velocity ◽  
Rnase Protection ◽  
Exchange Processes ◽  
Exchange Activity

The current studies were undertaken to establish an in vitro cellular model to study the transport of SO[Formula: see text] and Cl− and hormonal regulation and to define the possible function of the downregulated in adenoma ( DRA) gene. Utilizing a postconfluent Caco-2 cell line, we studied the OH− gradient-driven35SO[Formula: see text] and 36Cl−uptake. Our findings consistent with the presence of an apical carrier-mediated 35SO[Formula: see text]/OH−exchange process in Caco-2 cells include: 1) demonstration of saturation kinetics [Michaelis-Menten constant ( K m) of 0.2 ± 0.08 mM for SO[Formula: see text] and maximum velocity of 1.1 ± 0.2 pmol · mg protein−1 · 2 min−1]; 2) sensitivity to inhibition by DIDS ( K i = 0.9 ± 0.3 μM); and 3) competitive inhibition by oxalate and Cl−but not by nitrate and short chain fatty acids, with a higher K i (5.95 ± 1 mM) for Cl−compared with oxalate ( K i = 0.2 ± 0.03 mM). Our results also suggested that the SO[Formula: see text]/OH− and Cl−/OH− exchange processes in Caco-2 cells are distinct based on the following: 1) the SO[Formula: see text]/OH− exchange was highly sensitive to inhibition by DIDS compared with Cl−/OH−exchange activity ( K i for DIDS of 0.3 ± 0.1 mM); 2) Cl− competitively inhibited the SO[Formula: see text]/OH− exchange activity with a high K i compared with the K mfor SO[Formula: see text], indicating a lower affinity for Cl−; 3) DIDS competitively inhibited the Cl−/OH− exchange process, whereas it inhibited the SO[Formula: see text]/OH− exchange activity in a mixed-type manner; and 4) utilizing the RNase protection assay, our results showed that 24-h incubation with 100 nM of thyroxine significantly decreased the relative abundance of DRA mRNA along with the SO[Formula: see text]/OH− exchange activity but without any change in Cl−/OH− exchange process. In summary, these studies demonstrated the feasibility of utilizing Caco-2 cell line as a model to study the apical SO[Formula: see text]/OH− and Cl−/OH− exchange processes in the human intestine and indicated that the two transporters are distinct and that DRA may be predominantly a SO[Formula: see text]transporter with a capacity to transport Cl− as well.

Silica-induced chemokine expression in alveolar type II cells is mediated by TNF-α

1998 ◽  
Vol 275 (6) ◽  
pp. L1110-L1119 ◽  
Author(s):  
Edward G. Barrett ◽  
Carl Johnston ◽  
Günter Oberdörster ◽  
Jacob N. Finkelstein
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Rnase Protection Assay ◽  
Alveolar Type ◽  
Mrna Levels ◽  
Type Ii ◽  
Rnase Protection ◽  
Tnf Α ◽  
Type Ii Cell ◽  
Ifn Γ

Recent evidence has suggested that epithelial cells may contribute to the inflammatory response in the lung after exposure to crystalline silica through the production of and response to specific growth factors, chemokines, and cytokines. However, the exact cellular and molecular responses of epithelial cells to silica exposure remains unclear. Using a murine alveolar type II cell line [murine lung epithelial (MLE)-15 cell line], we measured the early changes in various cytokine and chemokine mRNA species after exposure of the cells to 4–35 μg/cm2 of silica (cristobalite), interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and lipopolysaccharide (LPS) alone or in combination. Total mRNA was isolated and assayed with an RNase protection assay after 6 and 24 h of exposure. Cristobalite exposure alone led to an increase in monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-2, and regulated on activation normal T cells expressed and secreted (RANTES) mRNAs. Treatment with IFN-γ alone increased MCP-1 mRNA levels. Treatment with TNF-α or LPS alone led to an increase in MCP-1 and MIP-2 mRNA. The combination of cristobalite plus TNF-α led to an additive increase in MCP-1 and MIP-2, whereas cristobalite plus IFN-γ or LPS had a synergistic effect. We also found with a TNF-α-neutralizing antibody that TNF-α plays a major role in mediating the type II cell chemokine response to cristobalite exposure. The results indicate that the cristobalite-induced chemokine response in the lung epithelium is mediated in part by TNF-α and can be enhanced by macrophage- and lymphocyte-derived inflammatory mediators in an additive and synergistic fashion.

Oxygen modulates Na+absorption in middle ear epithelium

1999 ◽  
Vol 276 (2) ◽  
pp. C312-C317 ◽  
Author(s):  
F. Portier ◽  
T. van den Abbeele ◽  
E. Lecain ◽  
E. Sauvaget ◽  
B. Escoubet ◽  
...  
Keyword(s):  
Cell Line ◽  
Otitis Media ◽  
Middle Ear ◽  
Chronic Hypoxia ◽  
Short Circuit ◽  
Rnase Protection Assay ◽  
Secretory Otitis Media ◽  
Healthy Human ◽  
Rnase Protection ◽  
Α Subunit

The physiology of the middle ear is primarily concerned with keeping the cavities air filled and fluid free to allow transmission of the sound vibrations from the eardrum to the inner ear. Middle ear epithelial cells are thought to play a key role in this process, since they actively transport Na+ and water. The [Formula: see text] of the middle ear cavities varies from 44 to 54 mmHg in healthy human ears but may be lower in the course of secretory otitis media. The effect of chronic hypoxia on ion transport was investigated on a middle ear cell line using the short-circuit current technique. Chronic hypoxia reversibly decreased the rate of Na+absorption across the MESV cell line. Although a decrease in cellular ATP content was observed, the decrease of Na+ absorption seemed related to a primary modulation of apical Na+entry. As revealed by RNase protection assay, the decrease in the rate of apical Na+ entry strictly paralleled the decrease in the expression of transcripts encoding the α-subunit of the epithelial Na+channel. This effect of oxygen on Na+ absorption might account for 1) the presence of fluid in the middle ear in the course of secretory otitis media and 2) the beneficial effect of the ventilation tube in treating otitis media that allows the[Formula: see text] to rise and restores the fluid clearance.

scholarly journals Glutathione depletion is associated with decreased Bcl-2 expression and increased apoptosis in cholangiocytes

1998 ◽  
Vol 275 (4) ◽  
pp. G749-G757 ◽  
Author(s):  
Adriane Celli ◽  
Florencia G. Que ◽  
Gregory J. Gores ◽  
Nicholas F. LaRusso
Keyword(s):  
Oxidative Stress ◽  
Protein Synthesis ◽  
Cell Line ◽  
Half Life ◽  
Cultured Cells ◽  
Morphological Changes ◽  
Rnase Protection Assay ◽  
Glutathione Depletion ◽  
Rnase Protection ◽  
Human Bile

Cholangiocytes are the target of a group of liver diseases termed the cholangiopathies that include conditions characterized by periductal inflammation and cholangiocyte apoptosis. Because inflammation is associated with oxidative stress, we developed the hypothesis that cholangiocytes exposed to oxidative stress will be depleted of endogenous cytoprotective molecules, leading to cholangiocyte apoptosis. To begin to test this hypothesis, we explored the relationships among glutathione (GSH) depletion, expression of Bcl-2 (a protooncogene that inhibits apoptosis), and apoptosis in a nonmalignant human cholangiocyte cell line. Monolayers of human bile duct epithelial cells, derived from normal liver and immortalized by SV40 transformation, were depleted of GSH using buthionine sulfoximine (BSO). Bcl-2 expression was assessed by quantitative immunoblot analysis, and apoptosis quantified by fluorescence microscopy using the DNA binding dye 4′,6′-diamidino-2-phenylindole. Bcl-2 message was assessed by RNase protection assay, and Bcl-2 protein synthesis and half-life by pulse-chase analysis. Exposure of human cholangiocytes in culture to BSO reduced GSH levels by 93 ± 3% ( P < 0.01). In addition, treatment of cholangiocytes with BSO reduced Bcl-2 levels by 87 ± 2% ( P < 0.01) and was associated with a time-dependent increase in the number of cells undergoing apoptosis; ∼11 ± 1% of cultured cells demonstrated morphological changes of apoptosis by 72 h compared with 1.5 ± 0.1% in untreated cholangiocytes ( P < 0.01). Maintenance of GSH levels by addition of glutathione ethyl ester in the presence of BSO blocked the BSO-associated increase in apoptosis in BSO-treated cholangiocytes and also prevented the decrease in Bcl-2 protein. BSO treatment of cholangiocytes did not change steady-state levels of bcl-2 mRNA or Bcl-2 protein synthesis. However, Bcl-2 protein half-life decreased 57% in BSO-treated vs. untreated cells. Our results using a human cholangiocyte cell line demonstrate that reduction in the cellular levels of an antioxidant such as GSH results in increased degradation of Bcl-2 protein and an increase in apoptosis. These data provide a mechanistic link between the consequences of oxidative stress and cholangiocyte apoptosis, an observation that may be important in the pathogenesis of the inflammatory cholangiopathies.

Regulation by calcitonin of Na(+)-K(+)-Cl- cotransport in a rabbit thick ascending limb cell line

1992 ◽  
Vol 263 (3) ◽  
pp. C563-C572 ◽  
Author(s):  
T. Vuillemin ◽  
J. Teulon ◽  
M. Geniteau-Legendre ◽  
B. Baudouin ◽  
S. Estrade ◽  
...  
Keyword(s):  
Cell Line ◽  
Hormonal Regulation ◽  
Channel Blocker ◽  
Maximum Velocity ◽  
Simian Virus 40 ◽  
Kinetic Studies ◽  
Simian Virus ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Camp Production

The hormonal regulation of a Na(+)-K(+)-Cl- cotransport was investigated in a renal tubule cell line (RC.SV2 cells) transformed by the simian virus 40. This cell line has the main characteristics of cells from the thick ascending limb of Henle, including the presence of Tamm-Horsfall protein and stimulation of adenosine 3',5'-cyclic monophosphate (cAMP) production by calcitonin (CT). Kinetic studies with 22Na+, 36Cl-, and 86Rb+ indicated the existence of a Na(+)-K(+)-Cl- cotransport with a stoichiometry of 1Na+:1K+: 2Cl-. All compounds stimulating cAMP production enhanced the ouabain-resistant bumetanide-sensitive (Or-Bs) Rb+ influx mediated by Na(+)-K(+)-Cl- cotransport. CT (100 ng/ml) increased the Or-Bs influx twofold by enhancing maximum velocity without changing the apparent Michaelis constant. The K(+)-channel blocker barium blunted the CT-stimulated Or-Bs influx by 64-74%, whereas the Cl(-)-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate reduced the CT-stimulated influx by 28-40%. These results suggest that CT stimulates the Na(+)-K(+)-Cl- cotransport by a cAMP-dependent mechanism and that K+ recycling through K+ membrane channels is an important modulator of cotransporter-mediated ion fluxes.

Differential expression and hormonal regulation of alternatively spliced IGF-I mRNA transcripts in salmon

1994 ◽  
Vol 12 (1) ◽  
pp. 25-37 ◽  
Author(s):  
S J Duguay ◽  
P Swanson ◽  
W W Dickhoff
Keyword(s):  
Hormonal Regulation ◽  
Coho Salmon ◽  
Juvenile Fish ◽  
Rnase Protection Assay ◽  
Mrna Levels ◽  
Gh Treatment ◽  
Rnase Protection ◽  
Mrna Transcripts ◽  
Igf I ◽  
Alternatively Spliced

ABSTRACT Salmon have been shown to express alternatively spliced IGF-I mRNA transcripts coding for four different IGF-I prohormones. These transcripts, now designated Ea-1, Ea-2, Ea-3 and Ea-4, differ in size due to the inclusion of additional sequences in the E domain-coding region of the molecule. In this study, the tissue distribution and hormonal regulation of expression of alternatively spliced IGF-I mRNA transcripts were investigated in coho salmon. IGF-I mRNAs were detected by solution hybridization/RNase protection assay in all tissues examined. GH treatment significantly increased hepatic IGF-I mRNA content. Hepatic IGF-I mRNA levels were not influenced by prolactin or somatolactin. Heart, fat, brain, kidney, spleen and ovary IGF-I mRNA levels were not affected by GH, prolactin or somatolactin. Ea-1, Ea-3 and Ea-4 mRNA transcripts were detectable in the liver, and Ea-1 and Ea-3 levels increased dramatically in response to GH treatment, whereas the amount of Ea-4 mRNA was unchanged. Most non-hepatic tissues expressed only the Ea-4 transcript, and expression was not influenced by GH, prolactin or somatolactin. Ea-1 and Ea-3 transcripts were visible in gill samples from fish treated with GH. The ovaries of juvenile fish expressed Ea-1, Ea-2 and Ea-4. The amounts of these transcripts were not changed by gonadotrophin treatment. During smoltification of juvenile coho salmon, liver and gill IGF-I mRNA levels increased with increasing plasma GH and thyroxine concentrations. Muscle, brain and ovary IGF-I mRNA levels were unchanged during this period. These data suggest that the liver is a major site of IGF-I production in response to GH. Heart, fat, brain, kidney, spleen and ovary did not show increased IGF-I mRNA levels in response to GH treatment. GH and prolactin had inconsistent effects on muscle IGF-I mRNA levels. Somatolactin and a gonadotrophin preparation did not stimulate IGF-I expression in tissues of juvenile fish. Differences in tissue GH responsiveness can be partially explained by the expression of alternatively spliced IGF-I mRNAs. Of the four hepatic IGF-I mRNA transcripts, Ea-1 and Ea-3 are GH-responsive, while Ea-2 and Ea-4 are not. Most non-hepatic tissues express only the Ea-4 transcript, and IGF-I mRNA levels do not increase after GH treatment. The increased IGF-I mRNA levels observed in gill tissue during smoltification suggest that other factors, in addition to GH, may regulate IGF-I expression. These data are also consistent with the hypothesis that IGF-I may mediate the osmoregulatory functions of GH during sea water adaptation.

A new quantitative solution hybridisation-RNase protection assay for APP and APLP2 mRNA

1996 ◽  
Vol 43 (1-2) ◽  
pp. 77-84 ◽  
Author(s):  
Janet A Johnston ◽  
Svante Norgren ◽  
Göran Annerén ◽  
Richard F Cowburn ◽  
Lars Lannfelt
Keyword(s):  
Rnase Protection Assay ◽  
Protection Assay ◽  
Rnase Protection

scholarly journals A cell cycle analysis of growth-related genes expressed during T lymphocyte maturation.

1990 ◽  
Vol 111 (6) ◽  
pp. 2693-2701 ◽  
Author(s):  
J N Feder ◽  
C J Guidos ◽  
B Kusler ◽  
C Carswell ◽  
D Lewis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcriptional Regulation ◽  
Steady State ◽  
T Lymphocyte ◽  
Ribonucleotide Reductase ◽  
Cellular Proliferation ◽  
Rnase Protection Assay ◽  
Minor Component ◽  
Mrna Levels ◽  
Rnase Protection

Fetal liver or bone marrow-derived T lymphocyte precursors undergo extensive, developmentally regulated proliferation in response to inductive signals from the thymic microenvironment. We have used neonatal mouse thymocytes size-separated by centrifugal elutriation to study the cell cycle stage-specific expression of several genes associated with cell proliferation. These include genes involved in the biosynthesis of deoxyribonucleotide precursors, such as dihydrofolate reductase (DHFR), thymidylate synthase (TS), and the M1 and M2 subunits of ribonucleotide reductase, as well as c-myc, a cellular oncogene of unknown function. Using nuclear run-on assays, we observed that the transcription rates for these genes, with the exception of TS, are essentially invariant not only throughout the cell cycle in proliferating cells, but also in noncycling (G0) cells. The TS gene showed a transient increase in transcription rate in cells which bordered between a proliferating and nonproliferating status. Studies of an elutriated T cell line, S49.1, yielded similar results, indicating that the process of immortalization has not affected the transcriptional regulation of these genes. Analysis of steady-state mRNA levels using an RNase protection assay demonstrated that the levels of DHFR and TS mRNA accumulate as thymocytes progress through the cell cycle. In contrast, only the M2 subunit of ribonucleotide reductase showed cyclic regulation. Finally, in contrast to cultured cell models, we observed an abrupt fivefold increase in the steady-state level of c-myc mRNA in the transition from G1 to S-phase. We conclude from these studies that the transcriptional regulation of specific genes necessary for cellular proliferation is a minor component of the developmental modulation of the thymocyte cell cycle.

Abnormal myocyte Ca2+homeostasis in rabbits with pacing-induced heart failure

1998 ◽  
Vol 275 (4) ◽  
pp. H1441-H1448 ◽  
Author(s):  
Atsushi Yao ◽  
Zhi Su ◽  
Akihiko Nonaka ◽  
Iram Zubair ◽  
Kenneth W. Spitzer ◽  
...  
Keyword(s):  
Heart Failure ◽  
Ventricular Myocytes ◽  
Rnase Protection Assay ◽  
Protection Assay ◽  
Rnase Protection ◽  
Contraction Coupling ◽  
Intracellular Ca ◽  
Rapid Pacing ◽  
Sodium Binding ◽  
The Times

To determine whether there are abnormalities in myocyte excitation-contraction coupling and intracellular Ca2+concentration ([Ca2+]i) homeostasis in pacing-induced heart failure (PF), we measured L-type Ca2+ current ( I Ca,L) and Na+/Ca2+exchanger current ( I Na/Ca) with voltage clamp and measured intracellular Na+ concentration ([Na+]i) and [Ca2+]iwith the use of sodium-binding benzofuran isophthalate (SBFI) and fluo 3 in ventricular myocytes isolated from control and paced rabbits. The peak systolic and diastolic levels and the amplitude of electrically stimulated [Ca2+]itransients (0.25 Hz, extracellular Ca2+ concentration = 1.08 mM) were significantly less in PF myocytes. Also, there was prolongation of the times to peak and decline of [Ca2+]itransients. I Ca,Ldensity was markedly decreased in PF myocytes. I Na/Ca at −40 mV elicited by rapid exposure to 0 Na+ solution with a rapid solution switcher was significantly reduced in PF myocytes, suggesting that the function of the Na+/Ca2+exchanger is impaired in these myocytes. In PF myocytes the decline of the [Ca2+]itransient when the Na+/Ca2+exchanger was abruptly disabled was markedly prolonged compared with the decline in control myocytes, consistent with depressed sarcoplasmic reticulum (SR) Ca2+-ATPase function. RNase protection assay showed decreased levels of Na+/Ca2+exchanger and SR Ca2+-ATPase mRNA in PF hearts, consistent with the function studies. We conclude that the functions of L-type Ca2+channels, Na+/Ca2+exchanger, and SR Ca2+-ATPase are impaired in myocytes from rabbit hearts with failure induced by rapid pacing. These abnormalities result in reduced [Ca2+]itransients and systolic and diastolic dysfunction and appear to account for the abnormal ventricular function observed.

RNase Protection Assay

2019 ◽  
pp. 109-116
Author(s):  
Jianzhu Zhao ◽  
Jun Tang ◽  
Justin Elfman ◽  
Hui Li
Keyword(s):  
Rnase Protection Assay ◽  
Protection Assay ◽  
Rnase Protection

Chloride transport by the cortical and outer medullary collecting duct

1987 ◽  
Vol 253 (2) ◽  
pp. F203-F212 ◽  
Author(s):  
V. L. Schuster ◽  
J. B. Stokes
Keyword(s):  
Chloride Transport ◽  
Collecting Duct ◽  
Exchange Process ◽  
Basolateral Membrane ◽  
Fine Tuning ◽  
Transport Systems ◽  
Acid Base ◽  
Intercalated Cell ◽  
Cl Channel ◽  
Collecting Tubule

The processes by which chloride is transported by the cortical and outer medullary collecting tubule have been most extensively studied using in vitro microperfusion of rabbit tubules. Chloride appears to be transported by three major mechanisms. First, Cl can be actively reabsorbed by an electroneutral Cl-HCO3 exchanger localized to the apical membrane of the HCO3-secreting (beta-type) intercalated cell. Cl exits this cell via a basolateral Cl channel. This anion exchange process can also operate in a Cl self-exchange mode, is stimulated acutely by beta-adrenergic agonists and cAMP, and is regulated chronically by in vivo acid-base status. Second, Cl can diffuse passively down electrochemical gradients via the paracellular pathway. Although this pathway does not appear to be selectively permeable to Cl, it is large enough to allow for significant passive reabsorption. Third, Cl undergoes recycling across the basolateral membrane of the H+-secreting (alpha-type) intercalated cell. HCO3 exit from this cell brings Cl into the cell via electroneutral Cl-HCO3 exchange; Cl then exits the cell via a Cl channel. Cl transport is thus required for acidification and alkalinization of the urine. Both of these processes exist in the cortical collecting tubule. Their simultaneous operation allows fine tuning of acid-base excretion. In addition, these transport systems, when functioning at equal rates, effect apparent electrogenic net Cl absorption without changing net HCO3 transport. These systems may play an important role in regulating Cl balance.

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