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.

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.

Interleukin-1 inhibition of Na(+)-K(+)-ATPase in inner medullary collecting duct cells: role of PGE2

1991 ◽  
Vol 261 (6) ◽  
pp. F1013-F1016 ◽  
Author(s):  
M. L. Zeidel ◽  
H. R. Brady ◽  
D. E. Kohan
Keyword(s):  
Atpase Activity ◽  
Collecting Duct ◽  
Interleukin 1 ◽  
Inhibitory Effect ◽  
Inner Medullary Collecting Duct ◽  
Duct Cells ◽  
Natriuretic Effect ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct ◽  
Stimulation Of

Interleukin-1 (IL-1), a cytokine produced by macrophages, causes an increase in Na+ excretion in experimental animals. Micropuncture studies have determined that the natriuretic effect of IL-1 is largely due to inhibition of Na+ reabsorption in the collecting duct. The current studies made use of suspensions of rabbit inner medullary collecting duct (IMCD) cells to examine the mechanism by which IL-1 regulates Na+ transport. IL-1 reduced ouabain-sensitive 86Rb+ uptake by 48% at 10 s, 36% at 30 s, and 29% at 60 s, suggesting an inhibitory effect on Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity. IL-1 inhibition of 86Rb+ uptake occurred in a dose-dependent manner. This effect appears to be mediated by prostaglandin E2 (PGE2) because 1) ibuprofen blocks the inhibitory effect of IL-1 on IMCD Na(+)-K(+)-ATPase activity, 2) IL-1 and PGE2 cause equivalent and nonadditive inhibition of 86Rb+ uptake, 3) IL-1 causes a two- to threefold increase in PGE2 content in IMCD cells, and 4) dose-response curves were similar for IL-1 stimulation of PGE2 content and inhibition of 86Rb+ uptake in IMCD cells. Thus the natriuretic effect of IL-1 is due, at least in part, to stimulation of PGE2 production by collecting duct cells with resultant inhibition of Na(+)-K(+)-ATPase activity.

Prostaglandin E2 inhibits Na+-K+-ATPase activity in the inner medullary collecting duct

1989 ◽  
Vol 257 (3) ◽  
pp. F424-F430 ◽  
Author(s):  
K. Jabs ◽  
M. L. Zeidel ◽  
P. Silva
Keyword(s):  
Atpase Activity ◽  
Prostaglandin E2 ◽  
Collecting Duct ◽  
Inner Medullary Collecting Duct ◽  
Activity Inhibition ◽  
Duct Cells ◽  
Pump Activity ◽  
Natriuretic Effect ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct

Prostaglandin E2 (PGE2) is natriuretic and inhibits collecting duct sodium transport by poorly defined mechanisms. To determine the mechanism of this inhibition, we have studied the effect of PGE2 on ouabain-sensitive (transport-dependent) oxygen consumption (QO2), ouabain-sensitive 86Rb+ uptake and ouabain-sensitive ATPase activity in fresh suspensions of rabbit inner medullary collecting duct cells, as well as Na+-K+-ATPase activity in inner medullary membranes. PGE2 (10(-5) M) reduced total QO2 by 21.6 +/- 2.3% (mean +/- SE) and reduced the ouabain-sensitive component of QO2 in IMCD cells. PGE2 failed to inhibit QO2 in the absence of sodium or in the presence of ouabain and blunted the increase in QO2 in response to amphotericin B. These results suggested that PGE2 inhibited Na+-K+-ATPase activity. Inhibition of pump activity was confirmed by measurements of 86Rb+ uptake: PGE2 (10(-5) M) reduced ouabain-sensitive 86Rb+ uptake by 57% at 10 s without altering equilibrium uptake. Furthermore, PGE2 (10(-6) M) reduced ouabain-sensitive ATPase activity by 46% in permeabilized inner medullary collecting duct cells. PGF2 alpha (10(-5) M) did not significantly alter QO2, 86Rb+ uptake, or Na+-K+-ATPase activity. These results demonstrate that PGE2 inhibits inner medullary collecting duct Na+-K+-ATPase activity and suggest a role for this inhibition in the natriuretic effect of PGE2.

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.

Cyclooxygenase inhibitors suppress inhibitory effect of PGE2 on Na-K-ATPase in MDCK cells

1994 ◽  
Vol 267 (1) ◽  
pp. F94-F98 ◽  
Author(s):  
R. Cohen-Luria ◽  
A. Moran ◽  
G. Rimon
Keyword(s):  
Arachidonic Acid ◽  
Atpase Activity ◽  
Mdck Cells ◽  
Inhibitory Effect ◽  
Cyclooxygenase Inhibitors ◽  
Inhibitory Effects ◽  
Ouabain Binding ◽  
Madin Darby Canine Kidney ◽  
Inflammatory Drugs ◽  
Darby Canine Kidney

In a previous work, we showed that picomolar concentrations of prostaglandin E2 (PGE2) inhibit Na-K-ATPase activity and ouabain binding in a clone of Madin-Darby canine kidney (MDCK) cells. In the present study, we demonstrate that the inhibitory effects of PGE2 on Na-K-ATPase activity, ouabain-sensitive Rb+ uptake, and ouabain binding in MDCK cells were diminished by treatment of the cells with nonsteroidal anti-inflammatory drugs. These results suggested that products of arachidonic acid synthesized through the cyclooxygenase pathway are involved in the inhibitory mechanism of PGE2. Treatment of the cells with arachidonic acid resulted in inhibition of ouabain binding, and the inhibition was eliminated by cyclooxygenase inhibitors. These observations further support the involvement of cyclooxygenase products in the PGE2-induced inhibitory process. Finally, we demonstrated that dopamine inhibits Rb+ influx and ouabain binding in MDCK cells similarly to PGE2. Cyclooxygenase inhibitors suppressed the inhibition of ouabain binding by dopamine, thus also suggesting the involvement of cyclooxygenase products in the inhibitory effect of dopamine.

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.

scholarly journals Synthesis of plasmalemmal glycoproteins in intestinal epithelial cells. Separation of Golgi membranes from villus and crypt cell surface membranes; glycosyltransferase activity of surface membrane

1978 ◽  
Vol 77 (3) ◽  
pp. 722-734 ◽  
Author(s):  
MM Weiser ◽  
MM Neumeier ◽  
A Quaroni ◽  
K Kirsch
Keyword(s):  
Cell Surface ◽  
Atpase Activity ◽  
Surface Membrane ◽  
Basal Membrane ◽  
Crypt Cell ◽  
Velocity Sedimentation ◽  
Intact Cells ◽  
Microvillus Membrane ◽  
Villus Cell ◽  
Crypt Cells

The relationship between Golgi and cell surface membranes of intestinal cells was studied. These membranes were isolated from intestinal crypt cells and villus cells. The villus cell membranes consisted of microvillus membrane, a Golgi-rich fraction, and two membrane fractions interpreted as representing lateral-basal membranes. The villus cell microvillus membrane was purified by previously published techniques while the other membranes were obtained from isolated cells by differential centrifugation and density gradient velocity sedimentation. The two membrane fractions obtained from villus cells and considered to be lateral-basal membranes were enriched for Na+,K+-ATPase activity, but one also showed enrichment in glycosyltransferase activity. The Golgi membrane fraction was enriched for glycosyltransferase activity and had low to absent Na+,K+-ATPase activity. Adenylate cyclase activity was present in all membrane fractions except the microvillus membrane but co-purified with Golgi rather than lateral-basal membranes. Electron microscopy showed that the Golgi fraction consisted of variably sized vesicles and cisternalike structures. The two lateral-basal membrane fractions showed only vesicles of smaller, more uniform size. After 125I labeling of isolated intact cells, radioactivity was found associated with the lateral-basal and microvillus membrane fractions and not with the Golgi fraction. Antibody prepared against lateral-basal membrane fractions reacted with the surface membrane of isolated villus cells. The membrane fractions from isolated crypt cells demonstrated that all had high glycosyltransferase activity. The data show that glycosyltransferase activity, in addition to its Golgi location, may be a significant property of the lateral-basal portion of the intestinal villus cell plasma membrane. Data obtained with crypt cells support earlier data and show that the crypt cell surface membrane possesses glycosyltransferase activity.

Abstract 101: Tissue-specific Deletion of Collectrin in the Proximal Tubular Epithelium Increases Arterial Pressure and Augments Salt-sensitivity

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Sylvia Cechova ◽  
Pei-Lun Chu ◽  
Joseph C Gigliotti ◽  
Fan Chan ◽  
Thu H Le
Keyword(s):  
Blood Pressure ◽  
Blood Flow ◽  
Renal Blood Flow ◽  
Collecting Duct ◽  
Critical Role ◽  
Specific Effect ◽  
Salt Sensitivity ◽  
Kidney Cortex ◽  
Mrna Levels ◽  
Proximal Tubular

Background: Collectrin ( Tmem27 ) is a key regulator of blood pressure (BP) and modulator of the bioavailability of nitric oxide (NO) and superoxide. It is highly expressed in the kidney in the proximal tubule (PT), collecting duct, and throughout the vascular endothelium. We reported that collectrin plays a critical role as a chaperone for the reabsorption of all amino acids (AAs) in the PT, and for the uptake of the cationic AA L-arginine (L-Arg) in endothelial cells. Global collectrin knockout ( Tmem27 Y/- ) mice display baseline hypertension (HTN), augmented salt-sensitive hypertension (SSH), and decreased renal blood flow. Objective and Methods: To determine the PT-specific effect of collectrin on BP homeostasis and salt sensitivity, we used the Cre -loxP approach and PEPCK-Cre to generate a mouse line lacking collectrin specifically in the PT-- PEPCK-Cre + Tmem27 Y/Flox mice. PEPCK-Cre - Tmem27 Y/Flox mice were used as control. Radiotelemetry was used to measure BP for 2 weeks at baseline and 2 weeks on high salt diet (HSD). Renal blood flow at baseline and on HSD was measured using contrast enhanced ultrasound in the same mice. Results: Successful deletion of collectrin in the PT was confirmed by assessing mRNA levels using real-time RT-PCR, immunohistochemistry staining of renal tissues using anti-collectrin antibody, and quantitation of protein from kidney cortex by Western analysis. Compared to control PEPCK-Cre - Tmem27 Y/Flox mice (n=6), PEPCK-Cre + Tmem27 Y/Flox mice (n=6) displayed significantly higher systolic BP (SBP) at baseline (120.0 ± 2.5 vs 131.6 ± 2.9 mm Hg; p = 0.014) and after HSD (135.3 ± 2.6 vs 151.5 ± 5.2 mm Hg; p = 0.019). Renal blood flow was not different between groups, at baseline nor after HSD. Conclusion: Collectrin in the PT plays an important role in blood pressure homeostasis and response to sodium intake, independent of renal blood flow. Increasing proximal tubular collectrin activity may be a novel therapeutic strategy for the treatment of hypertension and salt-sensitivity.

Cadmium is more toxic to LLC-PK1cells than to MDCK cells acting on the cadherin-catenin complex

1998 ◽  
Vol 275 (1) ◽  
pp. F143-F153 ◽  
Author(s):  
L. B. Zimmerhackl ◽  
F. Momm ◽  
G. Wiegele ◽  
M. Brandis
Keyword(s):  
Mdck Cells ◽  
Morphological Changes ◽  
Collecting Duct ◽  
Cadmium Toxicity ◽  
Distal Tubule ◽  
Insoluble Fraction ◽  
Atp Depletion ◽  
Rhodamine Phalloidin ◽  
Apical Side ◽  
E Cadherin

Cadmium toxicity to renal cells was investigated in Madin-Darby canine kidney (MDCK) and LLC-PK1cells as models of the distal tubule/collecting duct and proximal tubule, respectively. Cells were grown on two-compartment filters and exposed to 0.1–50 μM Cd2+. In MDCK cells, Cd2+was more toxic from the basolateral than from the apical side and dependent on the extracellular Ca2+concentration. Toxicity was evident within 24 h, as shown by a decrease in transepithelial resistance (TER), reduced proliferation (bromodeoxyuridine incorporation), reduction in ATP concentration, and morphological changes. On confocal microscopy, E-cadherin and α-catenin staining patterns indicated interference with the cadherin-catenin complex. LLC-PK1cells showed a similar toxicity pattern, which was evident at lower Cd2+concentrations. An increase of E-cadherin and α-catenin molecules in the Triton X-100-insoluble fraction was detectable at high Cd2+concentrations in LLC-PK1cells but not in MDCK cells. Lactate dehydrogenase release indicated membrane leakage in LLC-PK1cells. Rhodamine-phalloidin staining, a probe for F-actin filaments, demonstrated alterations of the actin cytoskeleton in both cell lines. In conclusion, cadmium caused ATP depletion and interfered with the cadherin-catenin complex and probably the tight junctions changing renal cell morphology and function.

ANP-stimulated cGMP egression in renal principal cells: abrogation of polarity by SV40 large T

1996 ◽  
Vol 270 (4) ◽  
pp. C1051-C1060 ◽  
Author(s):  
V. Millul ◽  
D. Prie ◽  
M. Geniteau-Legendre ◽  
M. C. Verpont ◽  
B. Baudouin ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Collecting Duct ◽  
Physiological Role ◽  
Specific Effect ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Temperature Sensitive ◽  
Cyclic Monophosphate ◽  
First Order ◽  
Principal Cells

Egression of atrial natriuretic peptide (ANP)-stimulated guanosine 3', 5'-cyclic monophosphate (cGMP) was compared with that of isoproterenol-stimulated adenosine 3', 5'-cyclic monophosphate (cAMP) in a rabbit collecting duct cell line transformed with a temperature-sensitive strain of simian virus 40 (SV40). At 39.5 degrees C (inactivated large T), cells exhibit major features of principal cells, whereas at 33 degrees C (functional large T) they lose most of their specific properties. When cells were grown on plastic at 39.5 degrees C, both cyclic nucleotides were predominantly released extracellularly via probenecid-sensitive carriers. Probenecid (3mM) reduced the ratios of extracellular cGMP and cAMP by 84 and 70%, respectively. The amount of extracellular cGMP or cAMP ws linearly correlated with the time integral of the intracellular cyclic nucleotide, suggesting first-order kinetics. The apparent first-order rate constant (k) was sixfold greater for cGMP (0.139 +/- 0.037 min-1, n = 3 experiments) than for cAMP (0.022 +/- 0.003(-1), n = 3 experiments). 3-Isobutyl-1-methylxanthine markedly inhibited extrusion of cGMP (k = 0.022 +/- 0.003 min-1), whereas that of cAMP was unchanged. When cells were grown on filters at 39.5 degrees C, both nucleotides were predominantly released in the apical medium but with a greater polarity for cGMP (83 +/- 4%, n = 6 experiments) than for cAMP (60 +/- 6%, n = 3 experiments) and a prevailing apical localization of the probenecid-sensitive carrier. Activation of SV40 large T at 33 degrees C did not alter cyclic nucleotide transport characteristics but abolished the polarity of probenecid-sensitive cyclic nucleotide extrusion. These results suggest a physiological role for luminal cGMP in the rabbit collecting duct and a specific effect of large T on the probenecid-sensitive carrier polarity.

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