H(+)-K(+)-ATPase in rabbit cortical collecting duct B-type intercalated cell

1996 ◽  
Vol 270 (3) ◽  
pp. F518-F530 ◽  
Author(s):  
I. D. Weiner ◽  
A. E. Milton
Keyword(s):  
B Cell ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Cortical Collecting Duct ◽  
Intercalated Cell ◽  
Acid Load ◽  
Phi Regulation

The role of H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) in the cortical collecting duct (CCD) B-type intercalated cell (B cell) is unclear. This study examined whether H(+)-K(+)-ATPase contributes to B cell intracellular pH (pHi) regulation and, if so, whether it is present at the apical or basolateral membrane. B cell Na(+)-independent pHi recovery from an acid load was only partially inhibited by peritubular N-ethylmaleimide (NEM). Complete inhibition required combining peritubular NEM either with luminal Sch-28080 or with luminal K+ removal. In contrast, neither peritubular Sch-28080 nor peritubular K+ removal altered pHi regulation. Tomato lectin, which binds to the gastric H(+)-K(+)-ATPase beta-subunit, labeled the B cell apical membrane. We conclude that the rabbit CCD B cell possesses an apical H(+)-K(+)-ATPase that plays an important role in pHi recovery from an in vitro acid load.

Apical proton secretion by the inner stripe of the outer medullary collecting duct

1999 ◽  
Vol 276 (4) ◽  
pp. F606-F613 ◽  
Author(s):  
I. David Weiner ◽  
Amy E. Frank ◽  
Charles S. Wingo ◽  
Keyword(s):  
Collecting Duct ◽  
Principal Cell ◽  
Cortical Collecting Duct ◽  
Proton Secretion ◽  
Intercalated Cell ◽  
Acid Load ◽  
Medullary Collecting Duct ◽  
The One ◽  
Proton Transporters

The inner stripe of outer medullary collecting duct (OMCDis) is unique among collecting duct segments because both intercalated cells and principal cells secrete protons and reabsorb luminal bicarbonate. The current study characterized the mechanisms of OMCDis proton secretion. We used in vitro microperfusion, and we separately studied the principal cell and intercalated cell using differential uptake of the fluorescent, pH-sensitive dye, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Both the principal cell and intercalated cell secreted protons, as identified as Na+/H+exchange-independent intracellular pH (pHi) recovery from an intracellular acid load. Two proton transport activities were identified in the principal cell; one was luminal potassium dependent and Sch-28080 sensitive and the other was luminal potassium independent and luminal bafilomycin A1sensitive. Thus the OMCDisprincipal cell expresses both apical H+-K+-ATPase and H+-ATPase activity. Intercalated cell Na+/H+exchange-independent pHi recovery was approximately twice that of the principal cell and was mediated by pharmacologically similar mechanisms. We conclude 1) the OMCDis principal cell may contribute to both luminal potassium reabsorption and urinary acidification, roles fundamentally different from those of the principal cell in the cortical collecting duct; and 2) the OMCDis intercalated cell proton transporters are functionally similar to those in the principal cell, raising the possibility that an H+-K+-ATPase similar to the one present in the principal cell may contribute to intercalated cell proton secretion.

scholarly journals TRPV4 as a flow sensor in flow-dependent K+ secretion from the cortical collecting duct

2007 ◽  
Vol 292 (2) ◽  
pp. F667-F673 ◽  
Author(s):  
Junichi Taniguchi ◽  
Shuichi Tsuruoka ◽  
Atsuko Mizuno ◽  
Jun-ichi Sato ◽  
Akio Fujimura ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Basolateral Membrane ◽  
Cortical Collecting Duct ◽  
Urine Production ◽  
K Secretion ◽  
Distal Tubules ◽  
Transient Receptor

The transient receptor vanilloid-4 (TRPV4) is a mechanosensitive, swell-activated cation channel that is abundant in the renal distal tubules. Immunolocalization studies, however, present conflicting data as to whether TRPV4 is expressed along the apical and/or basolateral membranes. To disclose the role of TRPV4 in flow-dependent K+ secretion in distal tubules in vivo, urinary K+ excretion and net transports of K+ and Na+ in the cortical collecting duct (CCD) were measured with an in vitro microperfusion technique in TRPV4 +/+ and TRPV4 −/− mice. Both net K+ secretion and Na+ reabsorption were flow dependently increased in the CCDs isolated from TRPV4 +/+mice, which were significantly enhanced by a luminal application of 50 μM 4α-phorbol-12,13-didecanoate (4αPDD), an agonist of TRPV4. No flow dependence of net K+ and Na+ transports or effects of 4αPDD on CCDs were observed in TRPV4 −/− mice. A basolateral application of 4αPDD had little effect on these ion transports in the TRPV4 +/+ CCDs, while the luminal application did. Urinary K+ excretion was significantly smaller in TRPV4 −/− than in TRPV4 +/+ mice when urine production was stimulated by a venous application of furosemide. These observations suggested an essential role of the TRPV4 channels in the luminal or basolateral membrane as flow sensors in the mechanism underlying the flow-dependent K+ secretion in mouse CCDs.

Impaired acid secretion in cortical collecting duct intercalated cells from H-K-ATPase-deficient mice: role of HKα isoforms

2008 ◽  
Vol 294 (3) ◽  
pp. F621-F627 ◽  
Author(s):  
I. Jeanette Lynch ◽  
Alicia Rudin ◽  
Shen-Ling Xia ◽  
Lisa R. Stow ◽  
Gary E. Shull ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Normal Diet ◽  
Cortical Collecting Duct ◽  
Initial Ph ◽  
Acid Load ◽  
Acid Extrusion ◽  
Deficient Mice ◽  
Α Subunits

Two classes of H pumps, H-K-ATPase and H-ATPase, contribute to luminal acidification and HCO3 transport in the collecting duct (CD). At least two H-K-ATPase α-subunits are expressed in the CD: HKα1 and HKα2. Both exhibit K dependence but have different inhibitor sensitivities. The HKα1 H-K-ATPase is Sch-28080 sensitive, whereas the pharmacological profile of the HKα2 H-K-ATPase is not completely understood. The present study used a nonpharmacological, genetic approach to determine the contribution of HKα1 and HKα2 to cortical CD (CCD) intercalated cell (IC) proton transport in mice fed a normal diet. Intracellular pH (pHi) recovery was determined in ICs using in vitro microperfusion of CCD after an acute intracellular acid load in wild-type mice and mice of the same strain lacking expression of HKα1, HKα2, or both H-K-ATPases (HKα1,2). A-type and B-type ICs were differentiated by luminal loading with BCECF-AM and peritubular chloride removal from CO2/HCO3-buffered solutions to identify the membrane locations of Cl/HCO3 exchange activity. H-ATPase- and Na/H exchange-mediated H transport were inhibited with bafilomycin A1 (100 nM) and EIPA (10 μM), respectively. Here, we report 1) initial pHi and buffering capacity were not significantly altered in the ICs of HKα-deficient mice, 2) either HKα1 or HKα2 deficiency resulted in slower acid extrusion, and 3) A-type ICs from HKα1,2-deficient mice had significantly slower acid extrusion compared with A-type ICs from HKα1-deficient mice alone. These studies are the first nonpharmacological demonstration that both HKα1 and HKα2 contribute to H secretion in both A-type and B-type ICs in animals fed a normal diet.

Contribution of the Na+-K+-2Cl−cotransporter NKCC1 to Cl− secretion in rat OMCD

2001 ◽  
Vol 280 (5) ◽  
pp. F913-F921 ◽  
Author(s):  
Susan M. Wall ◽  
Michael P. Fischer ◽  
Pramod Mehta ◽  
Kathryn A. Hassell ◽  
Stanley J. Park
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Physiological Role ◽  
Fluid Secretion ◽  
Transepithelial Potential ◽  
In Series

In rat kidney the “secretory” isoform of the Na+-K+-2Cl− cotransporter (NKCC1) localizes to the basolateral membrane of the α-intercalated cell. The purpose of this study was to determine whether rat outer medullary collecting duct (OMCD) secretes Cl− and whether transepithelial Cl− transport occurs, in part, through Cl− uptake across the basolateral membrane mediated by NKCC1 in series with Cl− efflux across the apical membrane. OMCD tubules from rats treated with deoxycorticosterone pivalate were perfused in vitro in symmetrical HCO[Formula: see text]/CO2-buffered solutions. Cl− secretion was observed in this segment, accompanied by a lumen positive transepithelial potential. Bumetanide (100 μM), when added to the bath, reduced Cl− secretion by 78%, although the lumen positive transepithelial potential and fluid flux were unchanged. Bumetanide-sensitive Cl− secretion was dependent on extracellular Na+ and either K+ or NH[Formula: see text], consistent with the ion dependency of NKCC1-mediated Cl− transport. In conclusion, OMCD tubules from deoxycorticosterone pivalate-treated rats secrete Cl−into the luminal fluid through NKCC1-mediated Cl− uptake across the basolateral membrane in series with Cl− efflux across the apical membrane. The physiological role of NKCC1-mediated Cl− uptake remains to be determined. However, the role of NKCC1 in the process of fluid secretion could not be demonstrated.

scholarly journals Inhibition of Na(+)-independent H+ pump by Na(+)-induced changes in cell Ca2+.

1991 ◽  
Vol 98 (4) ◽  
pp. 791-813 ◽  
Author(s):  
S R Hays ◽  
R J Alpern
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Chelating Agent ◽  
Cell Ph ◽  
Pump Activity ◽  
Acid Load ◽  
Medullary Collecting Duct ◽  
Induced Changes

Apical membrane H+ extrusion in the renal outer medullary collecting duct, inner stripe, is mediated by a Na(+)-independent H+ pump. To examine the regulation of this transporter, cell pH and cell Ca2+ were measured microfluorometrically in in vitro perfused tubules using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and fura-2, respectively. Apical membrane H+ pump activity, assayed as cell pH recovery from a series of acid loads (NH3/NH+4 prepulse) in the total absence of ambient Na+, initially occurred at a slow rate (0.06 +/- 0.02 pH units/min), which was not sufficient to account for physiologic rates of H+ extrusion. Over 15-20 min after the initial acid load, the rate of Na(+)-independent cell pH recovery increased to 0.63 +/- 0.09 pH units/min, associated with a steady-state cell pH greater than the initial pre-acid load cell pH. This pattern suggested an initial suppression followed by a delayed activation of the apical membrane H+ pump. Replacement of peritubular Na+ with choline or N-methyl-D-glucosamine resulted in an initial spike increase in cell Ca2+ followed by a sustained increase in cell Ca2+. The initial rate of Na(+)-independent cell pH recovery could be increased by elimination of the Na+ removal-induced sustained cell Ca2+ elevation by: (a) performing studies in the presence of 135 mM peritubular Na+ (1 mM peritubular amiloride used to inhibit basolateral membrane Na+/H+ antiport); (b) clamping cell Ca2+ low with dimethyl-BAPTA, an intracellular Ca2+ chelating agent; or (c) removal of extracellular Ca2+. Cell acidification induced a spike increase in cell Ca2+. The late acceleration of Na(+)-independent cell pH recovery was independent of Na+ removal and of the method used to acidify the cell, but was eliminated by prevention of the cell Ca2+ spike and markedly delayed by the microfilament-disrupting agent, cytochalasin B. This study demonstrates that peritubular Na+ removal results in a sustained elevation in cell Ca2+, which inhibits the apical membrane H+ pump. In addition, rapid cell acidification associated with a spike increase in cell Ca2+ leads to a delayed activation of the H+ pump. Thus, cell Ca2+ per se, or a Ca(2+)-activated pathway, can modulate H+ pump activity.

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.

EGF and PGE2 inhibit rabbit CCD Na+ transport by different mechanisms: PGE2 inhibits Na(+)-K+ pump

1993 ◽  
Vol 264 (4) ◽  
pp. F670-F677 ◽  
Author(s):  
D. H. Warden ◽  
J. B. Stokes
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Membrane Conductance ◽  
Membrane Voltage ◽  
Na Channel ◽  
Cortical Collecting Duct ◽  
Na Transport ◽  
Flux Measurements ◽  
Epidermal Growth

The rabbit cortical collecting duct absorbs Na+ by a transport system comprised of an apical membrane Na+ channel and a basolateral membrane Na(+)-K(+)-adenosinetriphosphatase. The rate of Na+ absorption across this epithelium is acutely inhibited by several hormones and autacoids including epidermal growth factor (EGF) and prostaglandin E2 (PGE2). We used electrophysiological analysis to determine which Na+ transport mechanism is primarily regulated in response to EGF and PGE2. We used concentrations of EGF and PGE2 that inhibited Na+ absorption to a comparable degree. We assessed the effects of these agents on Na+ transport primarily by the calculated equivalent current; the validity of this indicator was verified using simultaneous tracer flux measurements. EGF and PGE2 had different effects on the intracellular electrophysiological parameters. EGF (in the presence of a cyclooxygenase inhibitor) hyperpolarized the apical membrane voltage in a manner analogous to the Na(+)-channel blocker amiloride, reduced the transepithelial conductance, and increased the fractional resistance of the apical membrane. In comparison, PGE2 depolarized the apical membrane voltage in a manner analogous to the Na(+)-K+ pump inhibitor ouabain, and caused no significant changes in transepithelial conductance or apical membrane conductance. The finding that EGF hyperpolarized the apical membrane indicates that this agent attenuates Na+ absorption by reducing apical Na+ entry due to a decrease in the magnitude of the apical membrane Na+ conductance. In contrast, the electrophysiological changes produced by PGE2 indicate primary inhibition of the basolateral Na(+)-K+ pump following PGE2 treatment.

Phenotypic plasticity in the intercalated cell: the hensin pathway

1998 ◽  
Vol 275 (2) ◽  
pp. F183-F190 ◽  
Author(s):  
Qais Al-Awqati ◽  
S. Vijayakumar ◽  
C. Hikita ◽  
J. Chen ◽  
J. Takito
Keyword(s):  
Collecting Duct ◽  
Basolateral Membrane ◽  
Cell Types ◽  
Band 3 ◽  
Cytoskeletal Proteins ◽  
Β Cell ◽  
Biochemical Basis ◽  
Intercalated Cell ◽  
Immortalized Cell

The collecting duct of the renal tubule contains two cell types, one of which, the intercalated cell, is responsible for acidification and alkalinization of urine. These cells exist in a multiplicity of morphological forms, with two extreme types, α and β. The former acidifies the urine by an apical proton-translocating ATPase and a basolateral Cl/HCO3 exchanger, which is an alternately spliced form of band 3. This kidney form of band 3, kAE1, is present in the apical membrane of the β-cell, which has the H+-ATPase on the basolateral membrane. We had suggested previously that metabolic acidosis leads to conversion of β-types to α-types. To study the biochemical basis of this plasticity, we used an immortalized cell line of the β-cell and showed that these cells convert to the α-phenotype when plated at superconfluent density. At high density these cells localize a new protein, which we term “hensin,” to the extracellular matrix, and hensin acts as a molecular switch capable of changing the phenotype of these cells in vitro. Hensin induces new cytoskeletal proteins, makes the cells assume a more columnar shape and retargets kAE1 and the H+-ATPase. These recent studies suggest that the conversion of β- to α-cells, at least in vitro, bears many of the hallmarks of terminal differentiation.

Stimulation of total CO2 flux by 10% CO2 in rabbit CCD: role of an apical Sch-28080- and Ba-sensitive mechanism

1994 ◽  
Vol 267 (1) ◽  
pp. F114-F120 ◽  
Author(s):  
X. Zhou ◽  
C. S. Wingo
Keyword(s):  
Collecting Duct ◽  
Co2 Flux ◽  
K Channel ◽  
Co2 Absorption ◽  
Cortical Collecting Duct ◽  
Atpase Inhibitor ◽  
Contrast Exposure ◽  
Stimulation Of

These studies examine the effect of ambient PCO2 on net bicarbonate (total CO2) absorption by the in vitro perfused cortical collecting duct (CCD) from K-replete rabbits and the mechanism responsible for this effect. Exposure to 10% CO2 increased net bicarbonate flux (total CO2 flux, JtCO2) by 1.8-fold (P < 0.005), and this effect was inhibited by luminal 10 microM Sch-28080, an H-K-adenosinetriphosphatase (H-K-ATPase) inhibitor. In contrast, exposure to 10% CO2 significantly decreased Rb efflux, and this decrement in Rb efflux was blocked by luminal 2 mM Ba, a K channel blocker. Thus transepithelial tracer Rb flux did not increase upon exposure to 10% CO2 as we have observed in this segment under K-restricted conditions. The observation that 10% CO2 increased net bicarbonate absorption without a change in absorptive Rb flux suggested that 10% CO2 increased apical K recycling. To test this hypothesis, we examined whether luminal Ba inhibited the stimulation of luminal acidification induced by 10% CO2. If apical K exit were necessary for full activation of proton secretion, then inhibiting K exit should indirectly affect the stimulation of JtCO2 by 10% CO2. In fact, the effect of 10% CO2 on JtCO2 in the presence of 2 mM luminal Ba was quantitatively indistinguishable from the effect of 10% CO2 on JtCO2 in the presence of 10 microM luminal Sch-28080.(ABSTRACT TRUNCATED AT 250 WORDS)

Apical and basolateral membrane H+ extrusion mechanisms in inner stripe of rabbit outer medullary collecting duct

1990 ◽  
Vol 259 (4) ◽  
pp. F628-F635 ◽  
Author(s):  
S. R. Hays ◽  
R. J. Alpern
Keyword(s):  
Initial Rate ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Control Level ◽  
Atpase Inhibitor ◽  
Initial Control ◽  
Acid Load ◽  
Extrusion Mechanism ◽  
Medullary Collecting Duct

To examine mechanisms of H+ extrusion in the inner stripe of outer medullary collecting duct (OMCDIS), cell pH (pHi) was measured microfluorometrically in in vitro perfused tubules by use of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In total absence of luminal and peritubular Na+, pHi recovery from an acid load (NH3/NH+4 pulse) occurred at an initial rate of 0.13 +/- 0.02 pH units/min, whereas in the presence of 135 mM peritubular Na+, pHi recovered at 1.40 +/- 0.28 pH units/min. Na(+)-dependent pHi recovery was completely inhibited by 1.0 mM peritubular amiloride. Luminal Na+ (135 mM) addition had no effect on pHi recovery. Na(+)-independent pHi recovery from acid load was manifest by a triphasic response: 1) initial slow alkalinization; 2) slow cell acidification; and 3) a final phase that exhibited gradually increasing rates of alkalinization, returning pHi above the initial control level (pre-NH3/NH+4 pulse). Luminal N-ethylmaleimide (NEM, 500 microM), an H(+)-ATPase inhibitor, significantly inhibited initial rate of pHi recovery and total pHi recovery; whereas 500 microM peritubular NEM had no effect on initial rate of pHi recovery. Luminal SCH 28080 (100 microM), an H(+)-K(+)-ATPase inhibitor, had no effect on initial rate of pHi recovery or total pHi recovery. Thus rabbit OMCDIS possesses both an apical membrane NEM-sensitive, SCH 28080-insensitive, Na(+)-independent H+ extrusion mechanism (likely a simple H(+)-translocating ATPase) and a basolateral membrane amiloride-sensitive Na(+)-H+ antiporter.

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