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1.
We have examined the distribution and extent of phosphorylation of the tight junction-associated protein ZO-1 in the epithelial MDCK cell line, and in three cell types that do not form tight junctions: S180 (sarcoma) cells, S180 cells transfected with E-cadherin (S180L), and primary cultures of astrocytes. In shortterm calcium chelation experiments on MDCK cells, removal of extracellular calcium caused cells to pull apart. However, ZO-1 remained concentrated at the plasma membrane and no change in ZO-1 phosphorylation was observed. Maintenance of MDCK cells in low calcium medium, conditions where no tight junctions are found, resulted in altered ZO-1 distribution and lower total phosphorylation of the protein. In S180 cells, ZO-1 was diffusely distributed along the entire cell surface, with concentration of the antigen in motile regions of the cell. Cell-cell contact was not a prerequisite for ZO-1 localization at the plasma membrane in this cell type, and the phosphate content of ZO-1 was found to be lower in S180 cells relative to MDCK cells. Expression of Ecadherin in S180L cells did not alter either the distribution or phosphorylation of ZO-1. In contrast to S180 cells, ZO-1 in primary cultures of astrocytes was concentrated at sites of cell-cell contact, and the phosphorylation state was the same as that in control MDCK cells. Comparison of one-dimensional proteolytic digests of 32P-labeled ZO-1 revealed the phosphorylation of two peptides in control MDCK cells that was absent in both MDCK cells grown in low calcium and in S180 cells.We would like to thank Cheryl Richards for her help with the cell culture and immunohistochemistry; David Begg, Gary Firestone, Vik Maraj, Manijeh Pasdar and Colin Rasmussen for helpful discussions; Jaclyn Peebles and Greg Morrison for help with graphics and photography; and Grace Martin and Bob Campenot for rat tail collagen. We are grateful to all the members of our laboratories for their friendship, advice and support. This work was supported by an Establishment Award to B.R.S. from the Alberta Heritage Foundation for Medical Research and grants to B.R.S. from the Kidney Foundation of Canada and the Medical Research Council of Canada. A.H. is funded by a Studentship from the AHFMR. K.L.S. was supported by a grant from the National Institutes of Health (DK-42799) to Gary L. Firestone. B.R.S. is a Medical Research Council of Canada and AHFMR Scholar. 相似文献
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Bitte Aspenstrm-Fagerlund Birgitta Sundstrm Jonas Tallkvist Nils-Gunnar Ilbck Anders W. Glynn 《Chemico-biological interactions》2009,181(2):272-278
Passive paracellular absorption, regulated by tight junctions (TJs), is the main route for absorption of poorly absorbed hydrophilic substances. Surface active substances, such as fatty acids, may enhance absorption of these substances by affecting the integrity of TJ and increasing the permeability. It has been suggested that aluminium (Al) absorption occurs mainly by the paracellular route. Herein, we investigated if physiologically relevant exposures of fully differentiated Caco-2 cell monolayers to oleic acid and docosahexaenoic acid (DHA), which are fatty acids common in food, increase absorption of Al and the paracellular marker mannitol. In an Al toxicity test, mannitol and Al absorption through Caco-2 cell monolayers were similarly modulated by Al concentrations between 1 and 30 mM, suggesting that absorption of the two compounds occurred via the same pathways. Exposure of Caco-2 cell monolayers to non-toxic concentrations of Al (2 mM) and 14C-mannitol in fatty acid emulsions (15 and 30 mM oleic acid, 5 and 10 mM DHA) caused a decreased transepithelial electrical resistance (TEER). Concomitantly, fractional absorption of Al and mannitol, expressed as percentage of apical Al and mannitol retrieved at the basolateral side, increased with increasing dose of fatty acids. Transmission electron microscopy was applied to assess the effect of oleic acid on the morphology of TJ. It was shown that oleic acid caused a less structured morphology of TJ in Caco-2 cell monolayers. Taken together our findings indicate that fatty acids common in food increase the paracellular intestinal absorption of Al. These findings may influence future risk assessment of human Al exposure. 相似文献
4.
Bernhard Hoffmann Isolde Nagel Wolfgang Clauss 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1990,160(4):381-388
Summary Regulation of the paracellular pathway in rabbit distal colon by the hormone aldosterone was investigated in vitro in Ussing chambers by means of transepithelial and microelectrode techniques. To evaluate the cellular and paracellular resistances an equivalent circuit analysis was used. For the analysis the apical membrane resistance was altered using the antibiotic nystatin. Under control conditions two groups of epithelia were found, each clearly dependent on the light: dark regime. Low-transporting epithelia (LT) were observed in the morning and high-transporting epithelia (HT) in the afternoon. Na+ transport was about 3-fold higher in HT than in LT epithelia. Incubating epithelia of both groups with 0.1 mol·1-1 aldosterone on the serosal side nearly doubled in LT epithelia the short circuit current and transepithelial voltage but the transepithelial resistance was not influenced. Maximal values were reached after 4–5 h of aldosterone treatment. In HT epithelia due to the effect of aldosterone all three transepithelial parameters remained constant over time. Evaluation of the paracellular resistance revealed a significant increase after aldosterone stimulation in both epithelial groups. This increase suggests that tight junctions might have been regulated by aldosterone. The hormonal effect on electrolyte transport was also dependent on the physiological state of the rabbit colon. Since net Na+ absorption in distal colon is, in addition to transcellular absorption capacity, also dependent on the permeability of the paracellular pathway, the regulation of tight junctions by aldosterone may be a potent mechanism for improving Na+ absorption during hormone-stimulated ion transport.Abbreviations
V
t
transepithelial potential difference (mV)
-
R
t
transepithelial resistance (·cm2)
-
G
t
transepithelial conductance (mS·cm-2)
- Isc
calculated short circuit current (A·cm-2)
-
V
a
apical membrane potential difference (mV)
-
V
bl
basolateral membrane potential difference (mV)
-
voltage divider ratio
-
R
a
apical membrane resistance (·cm2)
-
R
bl
basolateral membrane resistance (·cm2)
-
R
c
cellular resistance ( of apical and basolateral resistance) (·cm2)
-
R
p
resistance of the paracellular pathway (·cm2)
-
G
a
apical membrane conductance (mS·cm-2)
-
G
bl
basolateral membrane conductance (mS·cm-2)
-
G
p
paracellular conductance (mS·cm-2)
-
G
t
transepithelial conductance (mS·cm-2)
-
HT
contr
high transporting control epithelia
-
LT
contr
low transporting control epithelia
-
HT
aldo
aldosterone incubated high transporting epithelia
-
LT
aldo
aldosterone incubated low transporting epithelia 相似文献
5.
Ascorbic acid prevents increased endothelial permeability caused by oxidized low density lipoprotein
《Free radical research》2013,47(11):1359-1368
AbstractMildly oxidized low density lipoprotein (mLDL) acutely increases the permeability of the vascular endothelium to molecules that would not otherwise cross the barrier. This study has shown that ascorbic acid tightens the permeability barrier in the endothelial barrier in cells, so this work tested whether it might prevent the increase in endothelial permeability due to mLDL. Treatment of EA.hy926 endothelial cells with mLDL decreased intracellular GSH and activated the cells to further oxidize the mLDL. mLDL also increased endothelial permeability over 2 h to both inulin and ascorbate in cells cultured on semi-permeable filters. This effect was blocked by microtubule and microfilament inhibitors, but not by chelation of intracellular calcium. Intracellular ascorbate both prevented and reversed the mLDL-induced increase in endothelial permeability, an effect mimicked by other cell-penetrant antioxidants. These results suggest a role for endothelial cell ascorbate in ameliorating an important facet of endothelial dysfunction caused by mLDL. 相似文献
6.
Michela Buccioni Diego Dal Ben Catia Lambertucci Aleix Martí Navia Massimo Ricciutelli Andrea Spinaci Rosaria Volpini Gabriella Marucci 《Bioorganic & medicinal chemistry》2019,27(15):3328-3333
In recent years, special attention has been paid to the A3 adenosine receptor (A3AR) as a possible pharmacological target to treat intestinal inflammation. In this work, it was set up a novel method to quantify the concentration of a promising anti-inflammatory agent inside and outside of intestinal barrier using the everted gut sac technique. The compound chosen for the present study is one of the most potent and selective A3AR agonist reported so far, named AR 170 (N6-methyl-2-phenylethynyl-5′-N-methylcarboxamidoadenosine). In order to evaluate the intestinal absorption of AR 170 the radioligand binding assay in comparison with HPLC-DAD was used. Results showed that the compound is absorbed via passive diffusion by paracellular pathway. The concentrations determined in the serosal (inside the sac) fluid by radioligand binding assay are in good agreement with those obtained through the widely used HPLC/MS protocol, demonstrating the reliability of the method. It is worthwhile to note that the radioligand binding assay allows detecting very low concentrations of analyte, thus offering an excellent tool to measure the intestinal absorption of receptor ligands. Moreover, the AR 170 quantity outside the gut sac and the interaction with A3AR could presuppose good topical anti-inflammatory effects of this compound. 相似文献
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Y.-H. Tai J. Flick S.A. Levine J.L. Madara G.W.G. Sharp M. Donowitz 《The Journal of membrane biology》1996,149(1):71-79
Elevation in intracellular Ca2+ acting via protein kinase C (PKC) is shown to regulate tight junction resistance in T84 cells, a human colon cancer line and a model Cl− secretory epithelial cell. The Ca2+ ionophore A23187, which was used to increase the intracellular Ca2+ concentration, caused a decrease in tight junction resistance in a concentration- and time-dependent manner. Dual Na+/mannitol serosal-to-mucosal flux analysis performed across the T84 monolayers treated with 2 μm A23187 revealed that A23187 increased both fluxes and that in the presence of ionophore there was a linear relationship between
the Na+ and mannitol fluxes with a slope of 56.4, indicating that the decrease in transepithelial resistance was due to a decrease
in tight junction resistance. Whereas there was no effect of 0.1 μm A23187, 1 or 2 μm produced a 55% decrease in baseline resistance in 1 hr and 10 μm decreased resistance more than 80%. The A23187-induced decrease in tight junction resistance was partially reversible by
washing 3 times with a Ringer's-HCO3 solution containing 1% BSA. The A23187 effect on resistance was dependent on intracellular Ca2+; loading the T84 cells with the intracellular Ca2+ chelator BAPTA significantly reduced the decrease in tight junction resistance caused by A23187. This intracellular Ca2+ effect was mediated by protein kinase C and not calmodulin. While the protein kinase C antagonist H-7 totally prevented the
action of A23187 on tight junction resistance, the Ca2+/calmodulin inhibitor W13 did not have any effect. Sphingosine, another inhibitor of PKC, partially reduced the A23187-induced
decline in tight junction resistance. The PKC agonist PMA mimicked the A23187 effect on resistance, although the effect was
delayed up to 1 hr after exposure. In addition, however, PMA also caused an earlier increase in resistance, indicating it
had an additional effect in addition to mimicking the effect of elevating Ca2+. The effects of a phospholipase inhibitor (mepacrine) and of inhibitors of arachidonic acid metabolism (indomethacin for
the cyclooxygenase pathway, NDGA for the lipoxygenase pathway, and SKF 525A for the epoxygenase pathway) on the A23187 action
were also examined. None of these agents altered the A23187-induced decrease in resistance. Monolayers exposed to 2 μm A23187 for 1 hr were stained with fluorescein conjugated phalloidin, revealing that neighboring cells did not part one from
another and that A23187 did not have a detectable effect on distribution of F-actin in the perijunctional actomyosin ring.
The results indicate that elevation in intracellular Ca2+ decreases tight junction resistance in the T84 monolayer, acting through protein kinase C by a mechanism which does not involve visible changes in the perijunctional actomyosin
ring.
Received: 14 July 1995/Revised: 25 September 1995 相似文献
9.
Tokuda S Niisato N Nakajima K Marunaka Y 《Biochemical and biophysical research communications》2008,366(2):464-470
In the present study, we investigated the effect of osmolality on the paracellular ion conductance (Gp) composed of the Na+ conductance (GNa) and the Cl− conductance (GCl). An osmotic gradient generated by NaCl with relatively apical hypertonicity (NaCl-absorption-direction) induced a large increase in the GNa associated with a small increase in the GCl, whereas an osmotic gradient generated by NaCl with relatively basolateral hypertonicity (NaCl-secretion-direction) induced small increases in the GNa and the GCl. These increases in the Gp caused by NaCl-generated osmotic gradients were diminished by the application of sucrose canceling the NaCl-generated osmotic gradient. The osmotic gradient generated by basolateral application of sucrose without any NaCl gradients had little effects on the Gp. However, this basolateral application of sucrose produced a precondition drastically quickening the time course of the action of the NaCl-generated osmotic gradient on the Gp. Further, we found that application of the basolateral hypotonicity generated by reduction of NaCl concentration shifted the localization of claudin-1 to the apical from the basolateral side. These results indicate that the osmotic gradient regulates the paracellular ion conductive pathway of tight junctions via a mechanism dependent on the direction of NaCl gradients associated with a shift of claudin-1 localization to the apical side in renal A6 epithelial cells. 相似文献
10.
V. Calderón A. Lázaro R.G. Contreras L. Shoshani C. Flores-Maldonado L. González-Mariscal G. Zampighi M. Cereijido 《The Journal of membrane biology》1998,164(1):59-69
Tight junctions (TJs) are cell-to-cell contacts made of strands, which appear as ridges on P faces and complementary furrows
on E faces on freeze fracture replicas. Evidences and opinions on whether these strands are composed of either membrane-bound
proteins or lipid micelles are somewhat varied. In the present work we alter the lipid composition of Madin-Darby canine kidney
monolayers using a novel approach, while studying (i) their transepithelial electrical resistance, a parameter that depends
on the degree of sealing of the TJs; (ii) the apical-to-basolateral flux of 4 kD fluorescent dextran (JDEX), that reflects the permeability of the intercellular spaces; (iii) the ability of TJs to restrict apical-to-basolateral
diffusion of membrane lipids; and (iv) the pattern of distribution of endogenous and transfected occludin, the sole membrane
protein presently known to form part of the TJs. We show that changing the total composition of phospholipids, sphingolipids,
cholesterol and the content of fatty acids, does not alter TER nor the structure of the strands. Interestingly, enrichment
with linoleic acid increases the JDEX by 631%. The fact that this increase is not reflected in a decrease of TER, suggests that junctional strands do not act as
simple resistive elements but may contain mobile translocating mechanisms.
Received: 7 November 1997/Revised: 20 March 1998 相似文献