Anion-dependent Mg2+ influx and a role for a vacuolar H+-ATPase in sheep ruminal epithelial cells

2003 ◽  
Vol 285 (1) ◽  
pp. G45-G53 ◽  
Author(s):  
Monika Schweigel ◽  
Holger Martens
Keyword(s):  
Epithelial Cells ◽  
Atpase Activity ◽  
Primary Culture ◽  
Intracellular Ph ◽  
Uptake Rate ◽  
Ruminal Fermentation ◽  
Fermentation Products ◽  
Fluorescence Techniques ◽  
Transport Inhibitors ◽  
Stimulation Of

The K+-insensitive component of Mg2+ influx in primary culture of ruminal epithelial cells (REC) was examined by means of fluorescence techniques. The effects of extracellular anions, ruminal fermentation products, and transport inhibitors on the intracellular free Mg2+ concentration ([Mg2+]i), Mg2+ uptake, and intracellular pH were determined. Under control conditions (HEPES-buffered high-NaCl medium), the [Mg2+]i of REC increased from 0.56 ± 0.14 to 0.76 ± 0.06 mM, corresponding to a Mg2+ uptake rate of 15 μM/min. Exposure to butyrate did not affect Mg2+ uptake, but it was stimulated (by 84 ± 19%) in the presence of [Formula: see text]. In contrast, Mg2+ uptake was strongly diminished if REC were suspended in [Formula: see text]-buffered high-KCl medium (22.3 ± 4 μM/min) rather than in HEPES-buffered KCl medium (37.5 ± 6 μM/min). After switching from high- to low-Cl– solution, [Mg2+]i was reduced from 0.64 ± 0.09 to 0.32 ± 0.16 mM and the [Formula: see text]-stimulated Mg2+ uptake was completely inhibited. Bumetanide and furosemide blocked the rate of Mg2+ uptake by 64 and 40%, respectively. Specific blockers of vacuolar H+-ATPase reduced the [Mg2+]i (36%) and Mg2+ influx (38%) into REC. We interpret this data to mean that the K+-insensitive Mg2+ influx into REC is mediated by a cotransport of Mg2+ and Cl– and is energized by an H+-ATPase. The stimulation of Mg2+ transport by ruminal fermentation products may result from a modulation of the H+-ATPase activity.

Hormonal regulation of Na+-K+-ATPase in cultured epithelial cells

1986 ◽  
Vol 251 (2) ◽  
pp. C186-C190 ◽  
Author(s):  
J. P. Johnson ◽  
D. Jones ◽  
W. P. Wiesmann
Keyword(s):  
Enzyme Activity ◽  
Epithelial Cells ◽  
Atpase Activity ◽  
Hormonal Regulation ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Toad Urinary Bladder ◽  
A6 Cells ◽  
Cultured Epithelial Cells ◽  
Stimulation Of

Aldosterone and insulin stimulate Na+ transport through mechanisms involving protein synthesis. Na+-K+-ATPase has been implicated in the action of both hormones. We examined the effect of aldosterone and insulin on Na+-K+-ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (ISC) in TB6C cells. Aldosterone increases Na+-K+-ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in ISC, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase ISC in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na+-K+-ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na+ entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na+-K+-ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on ISC.

scholarly journals Molecular Mechanism of Stimulation of Na-K-ATPase by Leukotriene D4 in Intestinal Epithelial Cells

2021 ◽  
Vol 22 (14) ◽  
pp. 7569
Author(s):  
Niraj Nepal ◽  
Subha Arthur ◽  
Molly R. Butts ◽  
Soudamani Singh ◽  
Balasubramanian Palaniappan ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Atpase Activity ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Leukotriene D4 ◽  
Intestinal Crypt ◽  
Calphostin C ◽  
Chronic Intestinal Inflammation ◽  
Crypt Cells ◽  
Stimulation Of

Na-K-ATPase provides a favorable transcellular Na gradient required for the functioning of Na-dependent nutrient transporters in intestinal epithelial cells. The primary metabolite for enterocytes is glutamine, which is absorbed via Na-glutamine co-transporter (SN2; SLC38A5) in intestinal crypt cells. SN2 activity is stimulated during chronic intestinal inflammation, at least in part, secondarily to the stimulation of Na-K-ATPase activity. Leukotriene D4 (LTD4) is known to be elevated in the mucosa during chronic enteritis, but the way in which it may regulate Na-K-ATPase is not known. In an in vitro model of rat intestinal epithelial cells (IEC-18), Na-K-ATPase activity was significantly stimulated by LTD4. As LTD4 mediates its action via Ca-dependent protein kinase C (PKC), Ca levels were measured and were found to be increased. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, also mediated stimulation of Na-K-ATPase like LTD4, while BAPTA-AM (Ca chelator) and calphostin-C (Cal-C; PKC inhibitor) prevented the stimulation of Na-K-ATPase activity. LTD4 caused a significant increase in mRNA and plasma membrane protein expression of Na-K-ATPase α1 and β1 subunits, which was prevented by calphostin-C. These data demonstrate that LTD4 stimulates Na-K-ATPase in intestinal crypt cells secondarily to the transcriptional increase of Na-K-ATPase α1 and β1 subunits, mediated via the Ca-activated PKC pathway.

Beta 2-adrenergic function in cultured rat proximal tubule epithelial cells

1996 ◽  
Vol 271 (1) ◽  
pp. F71-F77 ◽  
Author(s):  
H. Singh ◽  
S. Linas
Keyword(s):  
Epithelial Cells ◽  
Atpase Activity ◽  
Proximal Tubule ◽  
Sodium Transport ◽  
Cultured Cells ◽  
Proximal Tubules ◽  
Beta 2 ◽  
Acid Loading ◽  
Rat Proximal Tubule ◽  
Stimulation Of

We conducted studies to determine whether functional beta 2-adrenoceptors are present in cultured rat proximal tubule epithelial cells. To determine whether cultured cells maintain polarity with respect to sodium transport, cells were acid loaded. Acid loading resulted in stimulation of sodium transport. Exposure of acid-loaded cells to alkaline extracellular pH further enhanced sodium transport (22Na flux at pH 7.50 was 68.1 +/- 44% above pH 7.00, P < 0.05). Cultured proximal tubules also exhibited basolateral 86Rb uptake, 65% of which was ouabain sensitive. Thus cultured cells maintain apical Na/H antiport and basolateral Na-K-adenosinetriphosphatase (Na-K-ATPase). Metaproterenol (10(-6) M), a selective beta 2-agonist, stimulated Na-K-ATPase activity by 36 +/- 6% above control (P < 0.05). The stimulatory effect was blocked by ICI-118551, a selective beta 2-antagonist. To determine whether metaproterenol-dependent increases in Na-K-ATPase were dependent on apical sodium entry, apical entry was blocked with dimethylamiloride or maximized with monensin. Both dimethylamiloride and monensin prevented metaproterenol activation of Na-K-ATPase. Metaproterenol-mediated increases in Na-K-ATPase activity were associated with increases in sodium transport (27 +/- 10% above control, P < 0.05), which was prevented by dimethylamiloride. In contrast to isoproterenol, metaproterenol did not stimulate cAMP production. In summary, we have shown that functional beta 2-adrenoceptors are present on cultured rat proximal tubules. beta 2-Adrenoceptor activation results in increases in Na-K-ATPase and Na transport as a consequence of increased apical sodium entry.

Mechanisms of Mg2+ transport in cultured ruminal epithelial cells

2000 ◽  
Vol 278 (3) ◽  
pp. G400-G408 ◽  
Author(s):  
Monika Schweigel ◽  
Jürgen Vormann ◽  
Holger Martens
Keyword(s):  
Epithelial Cells ◽  
Cell Membrane ◽  
Intracellular Ph ◽  
Control Solution ◽  
Fluorescent Indicators ◽  
Chemical Gradients ◽  
Transcellular Pathway ◽  
Sodium Binding ◽  
Free Media ◽  
Stimulation Of

Net Mg2+ absorption from the rumen is mainly mediated by a transcellular pathway, with the greater part (62%) being electrically silent. To investigate this component of Mg2+transport, experiments were performed with isolated ruminal epithelial cells (REC). Using the fluorescent indicators mag-fura 2, sodium-binding benzofuran isophthalate, and 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, we measured the intracellular free Mg2+ concentration ([Mg2+]i), the intracellular Na+ concentration ([Na+]i), and the intracellular pH (pHi) of REC under basal conditions, after stimulation with butyrate and [Formula: see text], and after changing the transmembrane chemical gradients for Mg2+, H+, and Na+. REC had a mean resting pHi of 6.83 ± 0.1, [Mg2+]i was 0.56 ± 0.14 mM, and [Na+]i was 18.95 ± 3.9 mM. Exposure to both [Formula: see text] and[Formula: see text]/butyrate led to a stimulation of Mg2+ influx that amounted to 27.7 ± 5 and 29 ± 10.6 μM/min, respectively, compared with 15 ± 1 μM/min in control solution. The increase of [Mg2+]iwas dependent on extracellular Mg2+ concentration ([Mg2+]e). Regulation of pHi has been demonstrated to be Na+ dependent and is performed, for the most part, by a Na+/H+ exchanger. The recovery of pHi was fully blocked in nominally Na+-free media, even if [Mg2+]e was stepwise increased from 0 to 7.5 mM. However, an increase of [Mg2+]i was observed after reversing the transmembrane Na+ gradient. This rise in [Mg2+]i was pH independent, K+ insensitive, dependent on [Mg2+]e, imipramine and quinidine sensitive, and accompanied by a decrease of [Na+]i. The results are consistent with the existence of a Na+/Mg2+ exchanger in the cell membrane of REC. The coupling between butyrate, CO2/[Formula: see text] and Mg2+ transport may be mediated by another mechanism, perhaps by cotransport of Mg2+ and[Formula: see text].

scholarly journals Rapid Effects of Dexamethasone on Intracellular pH and Na+/H+ Exchanger Activity in Human Bronchial Epithelial Cells

2005 ◽  
Vol 280 (43) ◽  
pp. 35807-35814 ◽  
Author(s):  
Valia A. Verrière ◽  
Darina Hynes ◽  
Sheila Faherty ◽  
James Devaney ◽  
Jean Bousquet ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Intracellular Ph ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Protein Kinase ◽  
Bronchial Epithelial ◽  
Camp Dependent Protein Kinase ◽  
Mitogen Activated Protein ◽  
Dependent Protein ◽  
Stimulation Of

Glucocorticoids have been shown to produce rapid nongenomic responses in airway epithelia. By using an intracellular pH (pHi) spectrofluorescence imaging system and the NH4 Cl acid-loading technique, we have shown that the synthetic glucocorticoid,dexamethasone, accelerated intracellular pH recovery after an acid load in a human bronchial epithelial cell line (16HBE14o– cells). Exposure to NH4Cl (20 mm) elicited an intracellular acidification, followed by a pHi recovery. Inhibition of the Na+/H+ exchanger decreased the steady-state pHi and antagonized the dexamethasone stimulation of pHi regulation. The rapid effect of dexamethasone on pHi was neither affected by the inhibitor of transcription, cycloheximide, nor by the classical glucocorticoid and mineralocorticoid receptors antagonists, RU486 and spironolactone, respectively. The dexamethasone effect on pHi regulation was reduced by inhibitors of adenylate cyclase, cAMP-dependent protein kinase and mitogenactivated protein kinase (ERK1/2). By using a PepTag assay system and Western blotting, we have shown that dexamethasone stimulated cAMP-dependent protein kinase and mitogen-activated protein kinase activities. Taken together our results provide evidence for the rapid stimulation of Na+/H+ exchange activity by glucocorticoids in bronchial epithelial cells via a nongenomic mechanism involving cAMP-dependent protein kinase and mitogen-activated protein kinase ERK1/2 pathways.

Stimulation of both human bronchial epithelium and neutrophils is needed for maximal interactive adhesion

1996 ◽  
Vol 270 (1) ◽  
pp. L80-L87 ◽  
Author(s):  
P. G. Bloemen ◽  
M. C. Van den Tweel ◽  
P. A. Henricks ◽  
F. Engels ◽  
M. J. Van de Velde ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cultured Cells ◽  
Bronchial Epithelium ◽  
Bronchial Epithelial Cells ◽  
Intercellular Adhesion Molecule ◽  
Tnf Alpha ◽  
Epithelial Cell Line ◽  
Ifn Gamma ◽  
Bronchial Epithelial ◽  
Stimulation Of

It has become clear that the bronchial epithelium is not just a passive barrier but plays an active role in inflammation. It can produce several inflammatory mediators and does express cell adhesion molecules of which intercellular adhesion molecule (ICAM)-1 can be upregulated by cytokines like interferon (IFN)-gamma. In the present study, we analyzed in detail the interaction of neutrophils with human bronchial epithelial cells, both primary cultured cells and the bronchial epithelial cell line BEAS-2B. Confluent monolayers of epithelial cells were incubated with freshly isolated 51Cr-labeled neutrophils for 30 min at 37 degrees C; then the nonadherent cells were removed by washing gently. Stimulation of the epithelial cells with IFN-gamma or the combination of IFN-gamma and tumor necrosis factor-alpha (TNF-alpha) (which doubles the ICAM-1 expression) increased neutrophil adhesion. Activation of the neutrophils themselves with N-formylmethionyl-leucyl-phenylalanine (fMLP), platelet-activating factor, or TNF-alpha also caused a profound enhancement of the adhesion. A significant additional increase was found when the epithelial cells had been exposed to IFN-gamma and the neutrophils were stimulated with fMLP simultaneously. This effect was even more pronounced with epithelium preincubated with IFN-gamma and TNF-alpha. With the use of monoclonal antibodies against CD18 and ICAM-1, it was demonstrated that the increased adhesion was mainly mediated by the ICAM-1/beta 2-integrin interaction. This study highlights that both the activation state of the bronchial epithelial cells and the activation state of the neutrophils are critical for their interactive adhesion.

ICAM-1-independent adhesion of neutrophils to phorbol ester-stimulated human airway epithelial cells

1999 ◽  
Vol 277 (3) ◽  
pp. L465-L471 ◽  
Author(s):  
Alessandro Celi ◽  
Silvana Cianchetti ◽  
Stefano Petruzzelli ◽  
Stefano Carnevali ◽  
Filomena Baliva ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Bronchial Epithelial Cells ◽  
Neutrophil Adhesion ◽  
Late Time ◽  
Airway Epithelial ◽  
Bronchial Epithelial ◽  
Human Airway ◽  
Stimulation Of ◽  
Human Airway Epithelial Cells

Intercellular adhesion molecule-1 (ICAM-1) is the only inducible adhesion receptor for neutrophils identified in bronchial epithelial cells. We stimulated human airway epithelial cells with various agonists to evaluate whether ICAM-1-independent adhesion mechanisms could be elicited. Phorbol 12-myristate 13-acetate (PMA) stimulation of cells of the alveolar cell line A549 caused a rapid, significant increase in neutrophil adhesion from 11 ± 3 to 49 ± 7% (SE). A significant increase from 17 ± 4 to 39 ± 6% was also observed for neutrophil adhesion to PMA-stimulated human bronchial epithelial cells in primary culture. Although ICAM-1 expression was upregulated by PMA at late time points, it was not affected at 10 min when neutrophil adhesion was already clearly enhanced. Antibodies to ICAM-1 had no effect on neutrophil adhesion. In contrast, antibodies to the leukocyte integrin β-chain CD18 totally inhibited the adhesion of neutrophils to PMA-stimulated epithelial cells. These results demonstrate that PMA stimulation of human airway epithelial cells causes an increase in neutrophil adhesion that is not dependent on ICAM-1 upregulation.

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