Micromethodology for measuring ATPase activity in renal tubules: mineralocorticoid influence

The ATPase activity of rabbit isolated renal tubule segments was measured using a microtechnique in which the hydrolysis of ATP was enzymatically coupled to the appearance of an alkali-converted, highly fluorescent form of nicotinamide adenine dinucleotide. The methods are simple, reproducible, and have a high sensitivity in which picomole quantities of hydrolyzed ATP can readily be measured. Several methods for permeabilizing the cell membranes for measurement of Na+-K+-ATPase activity were evaluated, including osmotic (distilled water or 300 mM imidazole) and temperature (freezing) shock and addition of the nonionic detergent octylglucoside. An octylglucoside concentration of 0.5% was found to cause a maximum activation of the Na+-K+-ATPase and was comparable with that observed when tubules were permeabilized by exposure to distilled water and freezing. Incubation of tubules in 300 mM imidazole was less effective in permeabilizing the cell membranes. In all subsequent studies, the cells were permeabilized by exposure to distilled water and freezing as done by others. The methods were used to assay for the basal levels of Na+-K+-ATPase in the superficial proximal convoluted tubule, the superficial proximal straight tubule, and the cortical collecting tubule and were found to average 44.9 +/- 6.3, 26.4 +/- 2.4, and 11.8 +/- 2.2 pmol ADP X mm-1 X min-1, respectively. Furthermore, elevation of plasma mineralocorticoids by daily injections of deoxycorticosterone acetate (2 mg X kg-1 X day-1) for 4-15 days caused a doubling in the Na+-K+-ATPase activity of the cortical collecting duct, confirming the results of others. The methods presented can easily be adapted for microanalysis of other ATPases.

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Effect of vasopressin on Na+-K+-ATPase activity in rat cortical collecting duct

AJP Renal Physiology ◽

10.1152/ajprenal.1987.253.5.f874 ◽

1987 ◽

Vol 253 (5) ◽

pp. F874-F879 ◽

Cited By ~ 4

Author(s):

K. Tomita ◽

A. Owada ◽

Y. Iino ◽

N. Yoshiyama ◽

T. Shiigai

Keyword(s):

Atpase Activity ◽

Time Course ◽

Collecting Duct ◽

Cortical Collecting Duct ◽

Henle's Loop ◽

K Secretion ◽

Medullary Collecting Duct ◽

Hydrolysis Of

Vasopressin (V) causes a sustained increase in Na reabsorption and K secretion in isolated cortical collecting ducts (CCD) from rats. Because increased Na reabsorption may be associated with increased Na+-K+-ATPase activity, we investigated effects of V, given either in vivo or in vitro, on Na+-K+-ATPase activity in isolated nephron segments of rats. Na+-K+-ATPase activities were measured by coupling the hydrolysis of ATP to the production of a fluorescent nucleotide. In addition to CCD, other microdissected structures were medullary thick ascending limbs of Henle's loop, cortical thick ascending limbs of Henle's loop, and outer medullary collecting duct. To determine the time course of the response, Na+-K+-ATPase activities were measured in CCD 1 h, 3 h, 1 day, 3 days, and 7 days after intramuscular administrations of V. There was a significant increase in Na+-K+-ATPase activity in CCD after in vivo V administration for 7 days but not in any other segment. The activities increased after 3 days of administration of V. For in vitro experiments, CCD were incubated with 10(-6) M V for 1-3 h. Na+-K+-ATPase activities did not change after 1- or 3-h exposure of V in CCD in vitro. We conclude that prolonged V administration in vivo increases Na+-K+-ATPase activity in CCD. Because, in vitro exposure to V does not increase Na+-K+-ATPase activity, we conclude that rapid V-dependent increases in Na and K transport previously demonstrated in isolated perfused tubules are not dependent on a change in maximal Na+-K+-ATPase activity.

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

AJP Renal Physiology ◽

10.1152/ajprenal.2000.279.1.f195 ◽

2000 ◽

Vol 279 (1) ◽

pp. F195-F202 ◽

Cited By ~ 21

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.

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Mal protein stabilizes luminal membrane PLC-β3 and negatively regulates ENaC in mouse cortical collecting duct cells

AJP Renal Physiology ◽

10.1152/ajprenal.00446.2018 ◽

2019 ◽

Vol 317 (4) ◽

pp. F986-F995

Author(s):

Kubra M. Tuna ◽

Bing-Chen Liu ◽

Qiang Yue ◽

Zinah M. Ghazi ◽

He-Ping Ma ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Expression ◽

Collecting Duct ◽

Renal Tubules ◽

Cortical Collecting Duct ◽

Open Probability ◽

Anionic Phospholipid ◽

Kinase Substrate ◽

Luminal Membrane ◽

Collecting Duct Cells

Abnormally high epithelial Na+ channel (ENaC) activity in the aldosterone-sensitive distal nephron and collecting duct leads to hypertension. Myelin and lymphocyte (Mal) is a lipid raft-associated protein that has been previously shown to regulate Na+-K-2Cl− cotransporter and aquaporin-2 in the kidney, but it is not known whether it regulates renal ENaC. ENaC activity is positively regulated by the anionic phospholipid phosphate phosphatidylinositol 4,5-bisphosphate (PIP2). Members of the myristoylated alanine-rich C-kinase substrate (MARCKS) family increase PIP2 concentrations at the plasma membrane, whereas hydrolysis of PIP2 by phospholipase C (PLC) reduces PIP2 abundance. Our hypothesis was that Mal protein negatively regulates renal ENaC activity by stabilizing PLC protein expression at the luminal plasma membrane. We investigated the association between Mal, MARCKS-like protein, and ENaC. We showed Mal colocalizes with PLC-β3 in lipid rafts and positively regulates its protein expression, thereby reducing PIP2 availability at the plasma membrane. Kidneys of 129Sv mice injected with MAL shRNA lentivirus resulted in increased ENaC open probability in split-open renal tubules. Overexpression of Mal protein in mouse cortical collecting duct (mpkCCD) cells resulted in an increase in PLC-β3 protein expression at the plasma membrane. siRNA-mediated knockdown of MAL in mpkCCD cells resulted in a decrease in PLC-β3 protein expression and an increase in PIP2 abundance. Moreover, kidneys from salt-loaded mice showed less Mal membrane protein expression compared with non-salt-loaded mice. Taken together, Mal protein may play an essential role in the negative feedback of ENaC gating in principal cells of the collecting duct.

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Intracellular Na+ Controls Cell Surface Expression of Na,K-ATPase via a cAMP-independent PKA Pathway in Mammalian Kidney Collecting Duct Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e02-11-0720 ◽

2003 ◽

Vol 14 (7) ◽

pp. 2677-2688 ◽

Cited By ~ 49

Author(s):

Manlio Vinciguerra ◽

Georges Deschênes ◽

Udo Hasler ◽

David Mordasini ◽

Martine Rousselot ◽

...

Keyword(s):

Cell Membrane ◽

Cell Surface ◽

Atpase Activity ◽

Collecting Duct ◽

Surface Expression ◽

Cell Surface Expression ◽

Cortical Collecting Duct ◽

Mammalian Kidney ◽

Duct Cells ◽

Collecting Duct Cells

In the mammalian kidney the fine control of Na+ reabsorption takes place in collecting duct principal cells where basolateral Na,K-ATPase provides the driving force for vectorial Na+ transport. In the cortical collecting duct (CCD), a rise in intracellular Na+ concentration ([Na+]i) was shown to increase Na,K-ATPase activity and the number of ouabain binding sites, but the mechanism responsible for this event has not yet been elucidated. A rise in [Na+]i caused by incubation with the Na+ ionophore nystatin, increased Na,K-ATPase activity and cell surface expression to the same extent in isolated rat CCD. In cultured mouse mpkCCDcl4 collecting duct cells, increasing [Na+]i either by cell membrane permeabilization with amphotericin B or nystatin, or by incubating cells in a K+-free medium, also increased Na,K-ATPase cell surface expression. The [Na+]i-dependent increase in Na,K-ATPase cell-surface expression was prevented by PKA inhibitors H89 and PKI. Moreover, the effects of [Na+]i and cAMP were not additive. However, [Na+]i-dependent activation of PKA was not associated with an increase in cellular cAMP but was prevented by inhibiting the proteasome. These findings suggest that Na,K-ATPase may be recruited to the cell membrane following an increase in [Na+]i through cAMP-independent PKA activation that is itself dependent on proteasomal activity.

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Nitric oxide inhibits bafilomycin-sensitive H(+)-ATPase activity in rat cortical collecting duct

AJP Renal Physiology ◽

10.1152/ajprenal.1994.267.4.f509 ◽

1994 ◽

Vol 267 (4) ◽

pp. F509-F515 ◽

Cited By ~ 13

Author(s):

A. Tojo ◽

N. J. Guzman ◽

L. C. Garg ◽

C. C. Tisher ◽

K. M. Madsen

Keyword(s):

Nitric Oxide ◽

Atpase Activity ◽

Ph Regulation ◽

Collecting Duct ◽

Competitive Inhibitor ◽

No Production ◽

Intercalated Cells ◽

Cortical Collecting Duct ◽

Dose Dependent Decrease ◽

Nos Isoforms

Nitric oxide (NO) is a messenger molecule that is produced from L-arginine by NO synthase (NOS). Some NOS isoforms are present in cells constitutively, whereas others can be induced by cytokines. Recent evidence suggests that NO inhibits intracellular pH regulation by the vacuolar H(+)-adenosinetriphosphatase (ATPase) in macrophages, which contain an inducible form of NOS. The vacuolar H(+)-ATPase is involved in proton secretion in intercalated cells in the collecting duct. We have therefore examined the effect of NO on bafilomycin-sensitive H(+)-ATPase activity in individual cortical collecting ducts (CCD) microdissected from collagenase-treated kidneys of normal rats using a fluorometric microassay. Incubation of CCD with the NO donors, sodium nitroprusside (0.1 and 1 mM) or 3-morpholino-sydnonimine hydrochloride (SIN-1, 30 microM), caused a dose-dependent decrease in H(+)-ATPase activity. Incubation of CCD with lipopolysaccharide (LPS) and interferon-gamma, which induces NOS in macrophages, decreased H(+)-ATPase activity by 85%. This effect was prevented by simultaneous incubation with N omega-nitro-L-arginine, a competitive inhibitor of NOS, indicating that the decrease in H(+)-ATPase activity was caused by NO production. Incubation with 8-bromo-guanosine 3',5'-cyclic monophosphate (cGMP) also inhibited H(+)-ATPase activity, suggesting that NO may exert its effect in the CCD via activation of guanylyl cyclase and production of cGMP. Immunohistochemistry using antibodies to the macrophage-type NOS revealed strong labeling of intercalated cells in the CCD, confirming the presence of NOS in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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Time-dependent actions of aldosterone and amiloride on Na+-K+-ATPase of cortical collecting duct

AJP Renal Physiology ◽

10.1152/ajprenal.1988.254.5.f689 ◽

1988 ◽

Vol 254 (5) ◽

pp. F689-F696 ◽

Cited By ~ 7

Author(s):

R. A. Hayhurst ◽

R. G. O'Neil

Keyword(s):

Atpase Activity ◽

Latent Period ◽

Collecting Duct ◽

Constant Infusion ◽

Kinetic Properties ◽

Cortical Collecting Duct ◽

Initial Activity ◽

Simultaneous Treatment ◽

Single Mechanism ◽

Variable Modulation

The actions of aldosterone on Vmax Na+-K+-ATPase activity and length of latent period were assessed for the rabbit cortical collecting duct (CCD). Initially, animals were moderately aldosterone depleted and then treated with a constant infusion of physiological doses of aldosterone. Aldosterone administration had no influence after 3 h but caused a detectable increase with 6 (borderline significance) or more hours. An apparent plateau was reached between 24 and 48 h at twice the initial activity. This aldosterone-induced stimulation could be abolished by simultaneous treatment of the animals with amiloride, demonstrating a Na+-dependent modulation of the Vmax Na+-K+-ATPase activity. The aldosterone-stimulated enzyme had kinetic properties similar to those reported by others, but the latent period for aldosterone action on the Vmax Na+-K+-ATPase activity averaged near 6 h in the present study, as opposed to the highly variable period (from 1 h to several days) seen by others. This latent period variability was shown to be directly related to the initial Vmax Na+-K+-ATPase activity in the CCD and could be likened to an "end product dependent" latent period, i.e., the lower the initial end product (Vmax Na+-K+-ATPase activity) the shorter the latent period. Hence aldosterone's actions on the Na+-K+-ATPase of the CCD would be consistent with a single mechanism of action, i.e., increased synthesis, but with a variable modulation of this synthesis, which is dependent on the initial Vmax Na+-K+-ATPase activity of the CCD cells and/or the initial aldosterone status of the animal.

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Luminal flow modulates H+-ATPase activity in the cortical collecting duct (CCD)

AJP Renal Physiology ◽

10.1152/ajprenal.00179.2011 ◽

2012 ◽

Vol 302 (1) ◽

pp. F205-F215 ◽

Cited By ~ 13

Author(s):

Wen Liu ◽

Núria M. Pastor-Soler ◽

Carlos Schreck ◽

Beth Zavilowitz ◽

Thomas R. Kleyman ◽

...

Keyword(s):

Atpase Activity ◽

Collecting Duct ◽

Axial Flow ◽

Bk Channel ◽

Cortical Collecting Duct ◽

Dolichos Biflorus ◽

Intracellular Ca ◽

Acid Load ◽

Luminal Flow ◽

Principal And Intercalated Cells

Epithelial Na+ channel (ENaC)-mediated Na+ absorption and BK channel-mediated K+ secretion in the cortical collecting duct (CCD) are modulated by flow, the latter requiring an increase in intracellular Ca2+ concentration ([Ca2+]i), microtubule integrity, and exocytic insertion of preformed channels into the apical membrane. As axial flow modulates HCO3− reabsorption in the proximal tubule due to changes in both luminal Na+/H+ exchanger 3 and H+-ATPase activity (Du Z, Yan Q, Duan Y, Weinbaum S, Weinstein AM, Wang T. Am J Physiol Renal Physiol 290: F289–F296, 2006), we sought to test the hypothesis that flow also regulates H+-ATPase activity in the CCD. H+-ATPase activity was assayed in individually identified cells in microperfused CCDs isolated from New Zealand White rabbits, loaded with the pH-sensitive dye BCECF, and then subjected to an acute intracellular acid load (NH4Cl prepulse technique). H+-ATPase activity was defined as the initial rate of bafilomycin-inhibitable cell pH (pHi) recovery in the absence of luminal K+, bilateral Na+, and CO2/HCO3−, from a nadir pH of ∼6.2. We found that 1) an increase in luminal flow rate from ∼1 to 5 nl·min−1·mm−1 stimulated H+-ATPase activity, 2) flow-stimulated H+ pumping was Ca2+ dependent and required microtubule integrity, and 3) basal and flow-stimulated pHi recovery was detected in cells that labeled with the apical principal cell marker rhodamine Dolichos biflorus agglutinin as well as cells that did not. We conclude that luminal flow modulates H+-ATPase activity in the rabbit CCD and that H+-ATPases therein are present in both principal and intercalated cells.

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Mechanism of enhanced Na-K-ATPase activity in cortical collecting duct from rats with nephrotic syndrome.

Journal of Clinical Investigation ◽

10.1172/jci116328 ◽

1993 ◽

Vol 91 (4) ◽

pp. 1295-1300 ◽

Cited By ~ 32

Author(s):

E Féraille ◽

B Vogt ◽

M Rousselot ◽

C Barlet-Bas ◽

L Cheval ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Atpase Activity ◽

Collecting Duct ◽

Cortical Collecting Duct

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H(+)-K(+)-ATPase activity in rat collecting duct segments

AJP Renal Physiology ◽

10.1152/ajprenal.1992.262.4.f692 ◽

1992 ◽

Vol 262 (4) ◽

pp. F692-F695 ◽

Cited By ~ 13

Author(s):

J. D. Gifford ◽

L. Rome ◽

J. H. Galla

Keyword(s):

Atpase Activity ◽

Marked Decrease ◽

Collecting Duct ◽

Intercalated Cells ◽

Cortical Collecting Duct ◽

Acid Base ◽

Medullary Collecting Duct ◽

Gastric Proton Pump

Previous studies have suggested the presence of an H(+)-K(+)-ATPase in rat cortical and medullary intercalated cells with similar properties to the gastric proton pump. The purpose of this study was to determine the functional contribution of an H(+)-K(+)-adenosinetriphosphatase(ATPase) to total CO2 (tCO2) transport along the rat collecting duct. After baseline determination of tCO2 transport in isolated perfused collecting duct segments, Sch 28080 (10 microM) was added to either the perfusate or bath. When Sch 28080 was added to the perfusate, there was no effect in the cortical collecting duct (CCD, 20.8 +/- 6.7 vs. 25.3 + 3.0 pmol.mm-1.min-1), but a marked decrease in tCO2 absorption was effected in both the outer medullary (OMCD, 37.6 + 6.2 vs. 10.7 +/- 4.1 pmol.mm-1.min-1) and initial inner medullary collecting duct (IMCD1, 34.4 +/- 8.1 vs. 16.2 +/- 5.6 pmol.mm-1.min-1). In the CCD from rats with acute alkalosis in vivo, Sch 28080 added to the bath inhibited tCO2 secretion in the CCD (-17.1 +/- 4.4 vs 3.5 + 3.3 pmol.mm-1.min-1). These findings suggest that 1) H(+)-K(+)-ATPase is important in tCO2 absorption in the OMCD and IMCD1 and in tCO2 secretion in the CCD, 2) HCO3(-)-absorbing intercalated cells differ functionally in the cortex and medulla, 3) HCO3- secretion is not the reverse process of HCO3- absorption in the CCD, and 4) H(+)-K(+)-ATPase is important in distal acidification under normal and altered acid-base conditions.

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