Differential regulation of H+-ATPases in MDCK-C11 cells by aldosterone and vasopressin

2009 ◽  
Vol 87 (9) ◽  
pp. 653-665 ◽  
Author(s):  
Priscilla M.C. Dos Santos ◽  
Fabio P. Freitas ◽  
Jeane Mendes ◽  
Ana Lucia Tararthuch ◽  
Ricardo Fernandez
Keyword(s):  
Atpase Activity ◽  
Mdck Cells ◽  
Collecting Duct ◽  
Colorimetric Method ◽  
Proton Pumps ◽  
Ph Optimum ◽  
Cell Monolayers ◽  
Dose Dependent Fashion ◽  
Specific Antagonist ◽  
Dose Dependent

The objective of the present work was to characterize the biochemical activity of the proton pumps present in the C11 clone of Madin–Darby canine kidney (MDCK) cells, akin to intercalated cells of the collecting duct, as well as to study their regulation by hormones like aldosterone and vasopressin. MDCK-C11 cells from passages 78 to 86 were utilized. The reaction to determine H+-ATPase activity was started by addition of cell homogenates to tubes contained the assay medium. The inorganic phosphate (Pi) released was determined by a colorimetric method modified from that described by Fiske and Subbarow. Changes in intracellular calcium concentration in the cells was determined using the Ca2+-sensing dye fluo-4 AM. Homogenates of MDCK-C11 cells present a bafilomycin-sensitive activity (vacuolar H+-ATPase), and a vanadate-sensitive activity (H+/K+-ATPase). The bafilomycin-sensitive activity showed a pH optimum of 6.12. ATPase activity was also stimulated in a dose-dependent fashion as K+ concentration was increased between 0 and 50 mmol·L–1, with an apparent Km for the release of Pi of 0.13 mmol·L–1 and Vmax of 22.01 nmol·mg–1·min–1. Incubation of cell monolayers with 10−8 mol·L–1 aldosterone for 24 h significantly increased vacuolar H+-ATPase activity, an effect prevented by 10−5 mol·L–1 spironolactone. Vacuolar H+-ATPase activity was also stimulated by 10−11 mol·L–1 vasopressin, an effect prevented by a V1 receptor-specific antagonist. This dose of vasopressin determined a sustained rise of cytosolic ionized calcium. We conclude that (i) homogenates of MDCK-C11 cells present a bafilomycin-sensitive (H+-ATPase) activity and a vanadate-sensitive (H+/K+-ATPase) activity, and (ii) vacuolar H+-ATPase activity is activated by aldosterone through a genomic pathway and by vasopressin through V1 receptors.

scholarly journals Potassium depletion increases proton pump (H+-ATPase) activity in intercalated cells of cortical collecting duct

2000 ◽  
Vol 279 (1) ◽  
pp. F195-F202 ◽  
Author(s):  
Randi B. Silver ◽  
Sylvie Breton ◽  
Dennis Brown
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Collecting Duct ◽  
Proton Pumps ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Collecting Ducts ◽  
Acid Load ◽  
Collecting Tubules ◽  
Atpase Staining

Intercalated cells (ICs) from kidney collecting ducts contain proton-transporting ATPases (H+-ATPases) whose plasma membrane expression is regulated under a variety of conditions. It has been shown that net proton secretion occurs in the distal nephron from chronically K+-depleted rats and that upregulation of tubular H+- ATPase is involved in this process. However, regulation of this protein at the level of individual cells has not so far been examined. In the present study, H+-ATPase activity was determined in individually identified ICs from control and chronically K+-depleted rats (9–14 days on a low-K+ diet) by monitoring K+- and Na+-independent H+ extrusion rates after an acute acid load. Split-open rat cortical collecting tubules were loaded with the intracellular pH (pHi) indicator 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and pHiwas determined by using ratiometric fluorescence imaging. The rate of pHi recovery in ICs in response to an acute acid load, a measure of plasma membrane H+-ATPase activity, was increased after K+ depletion to almost three times that of controls. Furthermore, the lag time before the start of pHirecovery after the cells were maximally acidified fell from 93.5 ± 13.7 s in controls to 24.5 ± 2.1 s in K+-depleted rats. In all ICs tested, Na+- and K+-independent pHi recovery was abolished in the presence of bafilomycin (100 nM), an inhibitor of the H+-ATPase. Analysis of the cell-to-cell variability in the rate of pHi recovery reveals a change in the distribution of membrane-bound proton pumps in the IC population of cortical collecting duct from K+-depleted rats. Immunocytochemical analysis of collecting ducts from control and K+-depleted rats showed that K+-depletion increased the number of ICs with tight apical H+ATPase staining and decreased the number of cells with diffuse or basolateral H+-ATPase staining. Taken together, these data indicate that chronic K+ depletion induces a marked increase in plasma membrane H+ATPase activity in individual ICs.

NH3 permeation through the apical membrane of MDCK cells is via a lipid pathway

1988 ◽  
Vol 255 (1) ◽  
pp. F135-F141
Author(s):  
K. Golchini ◽  
I. Kurtz
Keyword(s):  
Activation Energy ◽  
Acetic Acid ◽  
Plasma Membrane ◽  
Time Course ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Cell Monolayers ◽  
Dose Dependent

The pathway for NH3 permeation across the apical membrane of MDCK cells was determined by measuring the effect of membrane fluidizing agents, protein reactive agents, and temperature on cellular NH3 influx. The rate of NH3 influx was calculated from the time course of increase in intracellular pH (pHi), measured with 2,7-biscarboxyethyl-5(6)-carboxyfluorescein, when MDCK cell monolayers were exposed to NH4Cl. The apical membrane NH3 permeability was 7.13 +/- 0.37 x 10(-3) cm/s (n = 12) at 37 degrees C and 1.23 +/- 0.07 x 10(-3) cm/s (n = 7) at 18 degrees C. In comparison, apical membrane permeability at 37 degrees C to the weak acids, valeric acid and acetic acid, were 1.39 +/- 0.11 x 10(-2) cm/s (n = 4) and 6.93 +/- 0.11 x 10(-3) cm/s (n = 4), respectively. The activation energy for NH3 permeation was 15.0 +/- 1.0 kcal/mol (17.5 degrees C-37.5 degrees C). In the presence of the membrane fluidizing agents, heptanol or chloroform, NH3 permeability increased in a dose-dependent manner. Heptanol (15 mM) significantly decreased the activation energy for NH3 permeation to 4.4 +/- 0.6 kcal/mol, P less than 0.001. The carboxyl reactive agent (1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluensulfonic acid 1 mM), aminoreactive agents (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid 50 microM; picrylsulphonic acid 1 mM), the sulphydryl reactive agent (p-chloromercuriphenylsulfonic acid 1 mM), and the nonspecific membrane protein cleaving agent pronase (1 mg/ml) had no effect on the NH3 influx. The results suggest that NH3 permeates the plasma membrane of MDCK cells via a lipid pathway.

Acute and early effects of aldosterone on Na-K-ATPase activity in Madin-Darby canine kidney epithelial cells

1993 ◽  
Vol 264 (6) ◽  
pp. F1021-F1026 ◽  
Author(s):  
M. Shahedi ◽  
K. Laborde ◽  
L. Bussieres ◽  
C. Sachs
Keyword(s):  
Atpase Activity ◽  
Time Course ◽  
De Novo ◽  
Mdck Cells ◽  
Early Effect ◽  
Catalytic Sites ◽  
Early Effects ◽  
Dose Dependent ◽  
De Novo Protein Synthesis ◽  
Darby Canine Kidney

The time course and mechanism of early effects of aldosterone on renal Na-K-adenosinetriphosphatase (Na-K-ATPase) activity and number of units were studied in MDCK cells. Aldosterone induced a time- and dose-dependent stimulation of Na-K-ATPase activity. The stimulatory effect of aldosterone on activity and number of pump units increased progressively and was inhibited by spironolactone. In presence of cycloheximide, the stimulatory effect of aldosterone on activity and number of catalytic sites persisted to the same extent until 30 min and decreased by 20% after 60 min. In these cells, dimethylamiloride addition during preincubation abolished the aldosterone-induced stimulation in Na-K-ATPase activity up to 60 min. In contrast, furosemide addition did not alter the effect of aldosterone on Na-K-ATPase activity. The present study demonstrates an early effect of aldosterone on Na-K-ATPase activity that can be separated into the following two successive periods: 1) increase in pump number due to insertion of presynthetized units secondary to Na entry through an amiloride-sensitive apical pathway; and 2) an increase in pump number by de novo protein synthesis.

scholarly journals Characterization of the receptor-destroying enzyme activity from infectious salmon anaemia virus

2002 ◽  
Vol 83 (11) ◽  
pp. 2693-2697 ◽  
Author(s):  
Marianne Kristiansen ◽  
Marianne K. Frøystad ◽  
Anne Lise Rishovd ◽  
Tor Gjøen
Keyword(s):  
Endocytic Pathway ◽  
Infectious Salmon Anaemia Virus ◽  
Serine Residue ◽  
Ph Optimum ◽  
Dose Dependent Fashion ◽  
Infected Cells ◽  
Bona Fide ◽  
Dose Dependent ◽  
Infectious Salmon Anaemia

Infectious salmon anaemia virus (ISAV) infects cells via the endocytic pathway and, like many other enveloped viruses, ISAV contains a receptor-destroying enzyme. We have analysed this acetylesterase activity with respect to substrate specificity, enzyme kinetics, inhibitors, temperature and pH stability. The ISAV acetylesterase was inhibited by di-isopropyl fluorophosphate (DFP) in a dose-dependent fashion but not by other known hydrolase inhibitors, suggesting that a serine residue is part of the active site. The pH optimum of the enzyme was in the range 7·5–8·0 and the enzymatic activity was lessened at temperatures above 40 °C. The effect of DFP on agglutination/elution of erythrocytes by ISAV demonstrated that the acetylesterase activity is the bona fide receptor-destroying enzyme. A haemadsorption assay was used to analyse whether the esterase was active on the surface of infected cells or not.

PGE2 inhibits Na-K-ATPase activity and ouabain binding in MDCK cells

1993 ◽  
Vol 264 (1) ◽  
pp. F61-F65 ◽  
Author(s):  
R. Cohen-Luria ◽  
G. Rimon ◽  
A. Moran
Keyword(s):  
Atpase Activity ◽  
Mdck Cells ◽  
Collecting Duct ◽  
Direct Reduction ◽  
Specific Effect ◽  
Scatchard Analysis ◽  
Ouabain Binding ◽  
Intact Cells ◽  
Concomitant Reduction ◽  
Natriuretic Effect

In the present study we report on a direct effect of prostaglandin E2 (PGE2) on ouabain binding and Na-K-adenosinetriphosphatase (Na-K-ATPase) activity in a clone of Madin-Darby canine kidney cells, a renal cell line with collecting duct properties. Incubation of the cells with low concentrations (pM) of PGE2 produced a concomitant reduction of approximately 50% in the activity of Na-K-ATPase in the cell homogenate and in ouabain binding to the intact cells (half-maximal inhibition of approximately 0.1 pM). The inhibition was apparent within 10 min of preincubation of the cells with PGE2. Scatchard analysis of the binding demonstrated that the treatment with PGE2 reduced the number of ouabain binding sites without a change in the dissociation constant. PGE1 and PGF2 alpha (10 nM) did not affect ouabain binding or Na-K-ATPase activity. The fast, potent, and specific effect of PGE2 suggests that the diuretic/natriuretic effect of prostaglandins of the E series in the collecting tubule, in addition to the interference with the activity of arginine vasopressin, may result from a direct reduction in the number of the Na-K-ATPase active units, via a prostaglandin receptor.

Effect of milrinone on left ventricular relaxation and Ca2+ uptake function of cardiac sarcoplasmic reticulum

2000 ◽  
Vol 279 (4) ◽  
pp. H1898-H1905 ◽  
Author(s):  
Masafumi Yano ◽  
Michihiro Kohno ◽  
Tomoko Ohkusa ◽  
Mamoru Mochizuki ◽  
Jutaro Yamada ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Left Ventricular ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Phosphodiesterase 3 ◽  
Dose Dependent Fashion ◽  
Membrane Bound ◽  
Dose Dependent ◽  
Higher Sensitivity

Milrinone, a phosphodiesterase 3 (PDE3) inhibitor, is known to enhance left ventricular (LV) contractility by an inhibition of the breakdown of cAMP through the mechanism inhibiting PDE3. However, it is unclear whether milrinone also exerts positive lusitropy, like dobutamine. Here, we assessed the effects of milrinone on in vivo LV relaxation, as well as the Ca2+-ATPase activity and the Ca2+uptake function of the cardiac sarcoplasmic reticulum (SR), compared with the effect of dobutamine on those functions. After dobutamine (3 μg · kg−1 · min−1) was administered, the peak value of the first derivative of LV pressure (+dP/d t) increased by 46%, whereas the time constant (τ) of LV pressure decay decreased by 6.9%, respectively. After milrinone (10 μg/kg) was administered, the peak +dP/d tincreased to a similar extent as dobutamine (46%), whereas τ decreased much more than dobutamine (19.9%; P < 0.05). In LV crude homogenate, the thapsigargin-sensitive, Ca2+-ATPase activity-cAMP relationships was significantly less increased by milrinone compared with dobutamine ( P< 0.05), indicating the higher sensitivity of the SR Ca2+-ATPase activity on cAMP by milrinone than by dobutamine. In the SR vesicles purified from LV muscles, the addition of cAMP increased the SR Ca2+ uptake in a dose-dependent fashion, and the PDE3 inhibitors (milrinone and cGMP) significantly augmented this response ( P < 0.05). Hence, milrinone substantially improved LV relaxation in association with an acceleration of the SR Ca2+-ATPase activity and the SR Ca2+ uptake. This acceleration might be due to an inhibition of the membrane-bound PDE3 in the SR, leading to a local elevation of cAMP.

scholarly journals Recognition of intestinal epithelial HIF-1α activation by Pseudomonas aeruginosa

2007 ◽  
Vol 292 (1) ◽  
pp. G134-G142 ◽  
Author(s):  
Nachiket J. Patel ◽  
Olga Zaborina ◽  
Licheng Wu ◽  
Yingmin Wang ◽  
Donald J. Wolfgeher ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Hypoxia Inducible Factor ◽  
Opportunistic Pathogen ◽  
Intestinal Epithelial ◽  
Human Intestinal Epithelial Cell ◽  
Cell Monolayers ◽  
Dose Dependent Fashion ◽  
Epithelial Cell Monolayers ◽  
Dose Dependent

Human intestinal epithelial cell monolayers (Caco-2) subjected to hypoxia and reoxygenation release soluble factors into the apical medium that activate the virulence of the opportunistic pathogen Pseudomonas aeruginosa to express the potent barrier-dysregulating protein PA-I lectin/adhesin. In this study, we defined the role of hypoxia-inducible factor (HIF)-1α in this response. We tested the ability of medium from Caco-2 cells with forced expression of HIF-1α to increase PA-I expression in P. aeruginosa and found that medium from Caco-2 cells overexpressing HIF-1α increased PA-I expression compared with medium from control cells ( P < 0.001, ANOVA). To identify the components responsible for this response, medium was fractionated by molecular weight and subjected to mass spectroscopy, which identified adenosine as the possible mediator. Both adenosine and its immediate downstream metabolite inosine induced PA-I expression in P. aeruginosa in a dose-dependent fashion. Because inosine was not detectable in the medium of Caco-2 cells exposed to hypoxia or overexpressing HIF-1α, we hypothesized that P. aeruginosa itself might metabolize adenosine to inosine. Using mutant and parental strains of P. aeruginosa, we demonstrated that P. aeruginosa metabolized adenosine to inosine via adenosine deaminase and that the conditioned medium enhanced the extracellular accumulation of inosine. Together, these results provide evidence that P. aeruginosa can recognize and respond to extracellular end products of intestinal hypoxia that are released after activation of HIF-1α. The ability of P. aeruginosa to metabolize adenosine to inosine may represent a subversive microbial virulence strategy that deprives the epithelium of the cytoprotective actions of adenosine.

scholarly journals Cholinergic inhibition of Na-K-ATPase via activation of protein kinase C in Madin-Darby canine kidney cells.

1993 ◽  
Vol 4 (2) ◽  
pp. 195-205 ◽  
Author(s):  
L C Garg ◽  
P K Saha ◽  
D Mohuczy-Dominiak
Keyword(s):  
Atpase Activity ◽  
Mdck Cells ◽  
Collecting Duct ◽  
Madin Darby Canine Kidney ◽  
Cholinergic Agonists ◽  
Kinase C ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct ◽  
Darby Canine Kidney ◽  
Canine Kidney

Recently, it was reported that muscarinic-type cholinergic receptors coupled to the phosphoinositide messenger system are present in the rabbit inner medullary collecting duct and Madin-Darby canine kidney (MDCK) cells. The receptor density in MDCK cells is 50 times more than that in inner medullary collecting duct cells. To examine if muscarinic receptor activation influences Na-K-ATPase, the effects of a cholinergic agonist, carbachol, on Na-K-ATPase activity in MDCK cells were measured. Carbachol inhibited Na-K-ATPase activity in a time- and concentration-dependent manner. A maximum of approximately 80% of the enzyme activity was inhibited in 160 min with an EC50 of 5 microM carbachol. The inhibition of Na-K-ATPase activity was reversible; up to 80% of the enzyme activity was recovered within 4 h after carbachol was removed. The inhibitory effect of carbachol was blocked by a muscarinic antagonist atropine and by inhibitors of protein kinase C (PKC), 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine HCl, and N-(2-(methylamino)ethyl)-5-isoquinoline sulfonamide HCl. Direct activators of PKC, phorbol 12-myristate 13-acetate, N(n-heptyl)-5-chloro-1-naphthalene sulfonamide, and phosphatidyl serine, also inhibited Na-K-ATPase activity in MDCK cells, and their effect was also blocked by PKC inhibitors. These results indicate that cholinergic agonists inhibit Na-K-ATPase activity in MDCK cells by the activation of PKC. It is concluded that the inhibition of Na-K-ATPase by PKC may, in part, be responsible for the natriuretic action of cholinergic agonists, which have been shown to stimulate phosphoinositide hydrolysis in renal collecting duct cells.

Plasminogen Interactions with Immobilized Fibrinogen

1989 ◽  
Vol 62 (04) ◽  
pp. 1078-1082 ◽  
Author(s):  
Burt Adelman ◽  
Patricia Ouynn
Keyword(s):  
Immunosorbent Assay ◽  
Aminocaproic Acid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Binding Regions ◽  
Dose Dependent Fashion ◽  
Epsilon Aminocaproic Acid ◽  
Dose Dependent

SummaryThis report describes the binding of plasminogen to fibrinogen adsorbed onto polystyrene wells. Binding was determined by enzyme linked immunosorbent assay. Both glu- and lys-plasminogen bound to immobilized fibrinogen in a dose-dependent fashion. However, more lys- than glu-plasminogen bound when equal concentrations of either were added to immobilized fibrinogen. Plasminogen binding was inhibited by epsilon aminocaproic acid indicating that binding was mediated via lysine-binding regions of plasminogen. Soluble fibrinogen added in excess of immobilized fibrinogen did not compete for plasminogen binding but fibrinogen fragments produced by plasmin digestion of fibrinogen did. Treatment of immobilized fibrinogen with thrombin caused a small but significant (p <0.01) increase in plasminogen binding. These studies demonstrate that immobilized fibrinogen binds both glu- and lys-plasminogen and that binding is mediated via lysine-binding regions. These interactions may facilitate plasminogen binding to fibrinogen adsorbed on to surfaces and to cells such as platelets which bind fibrinogen.

Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

1995 ◽  
Vol 73 (05) ◽  
pp. 805-811 ◽  
Author(s):  
Yasuo Takahashi ◽  
Yoshitaka Hosaka ◽  
Hiromi Niina ◽  
Katsuaki Nagasawa ◽  
Masaaki Naotsuka ◽  
...  
Keyword(s):  
Human Urine ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Rabbit Lung ◽  
Soluble Thrombomodulin ◽  
Molecular Weights ◽  
Dose Dependent Fashion ◽  
Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

Sign in / Sign up

Export Citation Format

Share Document