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.

H-K-ATPase activity in PNA-binding intercalated cells of newborn rabbit cortical collecting duct

1997 ◽  
Vol 272 (2) ◽  
pp. F167-F177 ◽  
Author(s):  
A. Constantinescu ◽  
R. B. Silver ◽  
L. M. Satlin
Keyword(s):  
Atpase Activity ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Adult Rabbit ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Load ◽  
Ethanesulfonic Acid

Functional and immunocytochemical studies indicate that intercalated cells in the adult rabbit cortical collecting duct (CCD) possess an H-K-adenosinetriphosphatase (H-K-ATPase). Because growing subjects must retain K+ and excrete H+, we sought to determine whether H-K-ATPase is present in the CCD early in life and, if so, to assess its activity and polarity. H-K-ATPase activity was defined as the initial rate of Sch-28080-inhibitable K+-dependent cell pH (pHi) recovery observed, in the absence of Na+, in response to an in vitro acid load. Transporter activity was assayed in intercalated cells labeled with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and apical cell surface marker rhodamine peanut lectin (PNA) in split-open CCDs isolated from neonatal and adult New Zealand White rabbits. In Na+-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solutions (nominal absence of CO2/HCO3-), the rate of K+-dependent pH(i) recovery from a NH4Cl-induced acid load was similar in newborn (0.056 +/- 0.015 pH U/min, n = 9) and adult (0.060 +/- 0.019 pH U/min; n = 9, P = not significant) cells. This rate of K+-dependent pH(i) recovery was significantly reduced by 10-20 pM Sch-28080, an inhibitor of gastric H-K-ATPase, in both newborns (0.009 +/- 0.003 pH U/min, n = 7) and adults (0.013 +/- 0.007 pH U/min, n = 9) (P < 0.05 compared with rates in absence of inhibitor). To determine whether the location of the transporter is consistent with a role in K+ absorption and H+ secretion, pH(i) recovery of acutely acid-loaded intercalated cells in neonatal CCDs (n = 7) microperfused and bathed in the absence of Na+ and K+ was monitored after selective addition of K+ to either the luminal or basolateral membrane. Addition of 5 mM K+ led to a significantly greater rate of pH(i) recovery when it was added to the luminal rather than the peritubular solution (0.049 +/- 0.005 vs. 0.018 +/- 0.005 pH U/min, P < 0.05). We conclude that PNA-binding intercalated cells of the neonatal CCD possess H-K-ATPase activity, predominantly located in the apical membrane. This provides a mechanism for H secretion and K+ retention, processes required for growth.

Depletion of intercalated cells from collecting ducts of carbonic anhydrase II-deficient (CAR2 null) mice

1995 ◽  
Vol 269 (6) ◽  
pp. F761-F774 ◽  
Author(s):  
S. Breton ◽  
S. L. Alper ◽  
S. L. Gluck ◽  
W. S. Sly ◽  
J. E. Barker ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Collecting Duct ◽  
Water Channel ◽  
Immunofluorescent Staining ◽  
Specific Marker ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Principal Cells ◽  
Collecting Tubules ◽  
Deficient Mice

The kidneys of mice (CAR2-null mice) that are genetically devoid of carbonic anhydrase type II (CAII) were screened by immunocytochemistry with antibodies that distinguish intercalated and principal cells. Immunofluorescent localization of the anion exchanger AE1 and of the 56-kDa subunit of the vacuolar H(+)-adenosinetriphosphatase (H(+)-ATPase) was used to identify intercalated cells, while the AQP2 water channel was used as a specific marker for principal cells of the collecting duct. The CAII deficiency of the CAR2-null mice was first confirmed by the absence of immunofluorescent staining of kidney sections exposed to an anti-CAII antibody. Cells positive for AE1 and H(+)-ATPase were common in all collecting duct regions in normal mice but were virtually absent from the inner stripe of the outer medulla and the inner medulla of CAR2-null mice. The number of positive cells was also reduced threefold in the cortical collecting duct of CAR2-null animals compared with normal mice. In parallel, the percentage of AQP2-positive cells was correspondingly increased in the collecting tubules of CAII-deficient mice, whereas the total number of cells per tubule remained unchanged. These results suggest that intercalated cells are severely depleted and are replaced by principal cells in CAII-deficient mice. Quantitative analysis and double staining showed that, in the cortex, both type A and type B intercalated cells are equally affected. Elucidation of the mechanism(s) responsible for this phenotype will be of importance in understanding the origin and development of intercalated cells in the kidney.

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

1994 ◽  
Vol 267 (4) ◽  
pp. F509-F515 ◽  
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)

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

2012 ◽  
Vol 302 (1) ◽  
pp. F205-F215 ◽  
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.

Membrane recycling and epithelial cell function

1989 ◽  
Vol 256 (1) ◽  
pp. F1-F12 ◽  
Author(s):  
D. Brown
Keyword(s):  
Plasma Membrane ◽  
Cell Function ◽  
Collecting Duct ◽  
Water Channel ◽  
Cell Types ◽  
Apical Plasma Membrane ◽  
Proton Pumps ◽  
Intercalated Cells ◽  
Membrane Recycling ◽  
Membrane Composition

The plasma membrane composition of virtually all eucaryotic cells is established, maintained, and modified by the process of membrane recycling. Specific plasma membrane components are inserted by exocytosis of transport vesicles, and are removed by endocytosis of segments of the membrane in which particular proteins are concentrated. In the kidney collecting duct, vasopressin induces the cycling of vesicles that are thought to carry water channels to and from the apical plasma membrane of principal cells, thus modulating the water permeability of this membrane. In the intercalated cells of the collecting duct, hydrogen ion secretion is controlled by the recycling of vesicles carrying proton pumps to and from the plasma membrane. In both cell types, "coated" carrier vesicles are involved, but whereas clathrin-coated vesicles participate in water channel recycling, the vesicles in intercalated cells are coated with the cytoplasmic domains of proton pumps. Following a brief outline of membrane recycling in general, this review summarizes previous data on membrane recycling in the collecting duct and related transporting epithelia and discusses some selected points relating to the role of membrane recycling and cell-specific function in the collecting duct.

scholarly journals Intercalated Cell Subtypes in Connecting Tubule and Cortical Collecting Duct of Rat and Mouse

1999 ◽  
Vol 10 (1) ◽  
pp. 1-12 ◽  
Author(s):  
JIN KIM ◽  
YOUNG-HEE KIM ◽  
JUNG-HO CHA ◽  
C. CRAIG TISHER ◽  
KIRSTEN M. MADSEN
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
Collecting Duct ◽  
Type A ◽  
Band 3 ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Type B ◽  
Intercalated Cell ◽  
Basolateral Plasma Membrane

Abstract. At least two populations of intercalated cells, type A and type B, exist in the connecting tubule (CNT), initial collecting tubule (ICT), and cortical collecting duct (CCD). Type A intercalated cells secrete protons via an apical H+ - ATPase and reabsorb bicarbonate by a band 3-like Cl-/HCO3- exchanger, AE1, located in the basolateral plasma membrane. Type B intercalated cells secrete bicarbonate by an apical Cl-/HCO3- exchanger that is distinct from AE1 and remains to be identified. They express H+ -ATPase in the basolateral plasma membrane and in vesicles throughout the cytoplasm. A third type of intercalated cell with apical H+ -ATPase, but no AE1, has been described in the CNT and CCD of both rat and mouse. The prevalence of the third cell type is not known. The aim of this study was to characterize and quantify intercalated cell subtypes, including the newly described third non A-non B cell, in the CNT, ICT, and CCD of the rat and mouse. A triple immunolabeling procedure was developed in which antibodies to H+ -ATPase and band 3 protein were used to identify subpopulations of intercalated cells, and segment-specific antibodies were used to identify distal tubule and collecting duct segments. In both rat and mouse, intercalated cells constituted approximately 40% of the cells in the CNT, ICT, and CCD. Type A, type B, and non A-non B intercalated cells were observed in all of the three segments, with type A cells being the most prevalent in both species. In the mouse, however, non A-non B cells constituted more than half of the intercalated cells in the CNT, 39% in the ICT, and 22% in the CCD, compared with 14, 7, and 5%, respectively, in the rat. In contrast, type B intercalated cells accounted for only 8 to 16% of the intercalated cells in the three segments in the mouse compared with 26 to 39% in the rat. It is concluded that striking differences exist in the prevalence and distribution of the different types of intercalated cells in the CNT, ICT, and CCD of rat and mouse. In the rat, the non A-non B cells are fairly rare, whereas in the mouse, they constitute a major fraction of the intercalated cells, primarily at the expense of the type B intercalated cells.

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

1992 ◽  
Vol 262 (4) ◽  
pp. F692-F695 ◽  
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.

Low-NaCl diet increases H-K-ATPase in intercalated cells from rat cortical collecting duct

1998 ◽  
Vol 275 (1) ◽  
pp. F94-F102 ◽  
Author(s):  
Randi B. Silver ◽  
Han Choe ◽  
Gustavo Frindt
Keyword(s):  
Plasma Levels ◽  
Collecting Duct ◽  
Plasma Aldosterone ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Elevated Plasma ◽  
Ratiometric Fluorescence ◽  
Collecting Tubule ◽  
Acid Load ◽  
Cortical Collecting Tubule

Extracellular K+-dependent H+ extrusion after an acute acid load, an index of H/K exchange, was monitored in intercalated cells (ICs) from rat cortical collecting tubule (CCT) using ratiometric fluorescence imaging of the intracellular pH (pHi) indicator, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The hypothesis tested was that 12- to 14-day NaCl deprivation increases H-K-ATPase in rat ICs. The rate of H/K exchange in the low-NaCl ICs was double that of controls. In control ICs, H/K exchange was inhibited by Sch-28080 (10 μM). In the low-NaCl ICs, it was partially blocked by Sch-28080 or ouabain (1 mM). Simultaneous addition of both inhibitors abolished K-dependent pHirecovery. The induced H/K exchange observed with NaCl restriction was not due to elevated plasma aldosterone as evidenced by experiments on ICs from rats implanted with osmotic minipumps administering aldosterone such that plasma levels were comparable to those of NaCl-deficient rats. The results suggest that NaCl deficiency induces two isoforms of H-K-ATPase in ICs and that this effect is not mediated by elevated plasma aldosterone.

Intracellular band 3 immunostaining in type A intercalated cells of rabbit kidney

1992 ◽  
Vol 262 (6) ◽  
pp. F1015-F1022
Author(s):  
K. M. Madsen ◽  
J. Kim ◽  
C. C. Tisher
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Type A ◽  
Band 3 ◽  
Rabbit Kidney ◽  
Apical Plasma Membrane ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Electron Microscopic

Intercalated cells (ICs) in the collecting duct and the connecting tubule (CNT) are involved in H+ secretion and HCO3- reabsorption. H+ secretion is mediated by an H(+)-adenosinetriphosphatase in the apical plasma membrane, whereas a band 3-like Cl(-)-HCO3- exchanger in the basolateral membrane is responsible for HCO3- reabsorption. Recent studies have reported that a band 3-like protein is also present in mitochondria in rabbit ICs. The purpose of this study was to establish the subcellular location of the band 3-like Cl(-)-HCO3- exchanger in rabbit ICs by electron microscopic immunocytochemistry using a monoclonal antibody, IVF12, against erythrocyte band 3 protein. Rabbit kidneys were preserved by in vivo perfusion with a paraformaldehyde-lysine-periodate solution and processed for immunocytochemistry using a horseradish peroxidase preembedding technique. Band 3 immunostaining was observed on the basolateral plasma membrane of ICs in the outer medullary collecting duct and type A cells in the cortical collecting duct (CCD) and CNT. In addition, distinct staining for band 3 was present in numerous small vesicles and in multivesicular bodies in type A ICs in the CCD and CNT. However, there was no evidence of band 3 immunostaining of mitochondria or of the apical plasma membrane in any cells of the collecting duct. These observations suggest that basolateral Cl(-)-HCO3- exchangers in type A ICs in the rabbit kidney are stored in intracellular vesicles and possibly degraded in the vascular-lysosomal system when these cells are in a resting state. The previously reported band 3 immunolabeling of mitochondria could not be confirmed.

scholarly journals Ultrastructural localization of H+ATPase in rabbit cortical collecting duct.

1994 ◽  
Vol 4 (8) ◽  
pp. 1546-1557 ◽  
Author(s):  
J W Verlander ◽  
K M Madsen ◽  
D K Stone ◽  
C C Tisher
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Collecting Duct ◽  
Rabbit Kidney ◽  
Apical Plasma Membrane ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Thin Sections ◽  
Cytoplasmic Vesicles ◽  
Basolateral Plasma Membrane

In contrast to results obtained in the rat kidney, studies of H+ATPase localization in the rabbit kidney have failed to demonstrate basolateral plasma membrane H+ATPase immunoreactivity in intercalated cells in the cortical collecting duct (CCD). Previous studies have relied on light microscopic immunofluorescence techniques, which have limited resolution. Therefore, the immunogold procedure was used to localize H+ATPase in rabbit collecting ducts at the ultrastructural level. Rabbit kidneys were preserved in vivo with periodate-lysine-paraformaldehyde or glutaraldehyde solutions, and samples of cortex were embedded in Lowicryl K4M. Thin sections were labeled for H+ATPase by the immunogold procedure with a rabbit polyclonal antibody against the 70-kd subunit of bovine brain H+ATPase. Three patterns of localization of H+ATPase were observed. The majority of intercalated cells in the CCD exhibited label over cytoplasmic vesicles only. In these cells, no label was associated with either the apical or basolateral plasma membranes. In a second group of cells, label for H+ATPase was observed along the basolateral plasma membrane and over cytoplasmic vesicles throughout the cell. Rarely, intercalated cells with H+ATPase label along the apical plasma membrane and over the apical cytoplasmic vesicles were observed in the CCD. In the initial collecting tubule and connecting segment, intercalated cells with either pronounced apical or basolateral plasma membrane label prevailed, whereas few cells exhibited label restricted to the cytoplasmic vesicles. In summary, in the rabbit CCD, three patterns of H+ATPase distribution exist in intercalated cells, two of which conform to published models of type A and type B intercalated cells.

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