NH+4 augments net acid secretion by a ouabain-sensitive mechanism in isolated perfused inner medullary collecting ducts

1996 ◽  
Vol 270 (3) ◽  
pp. F432-F439 ◽  
Author(s):  
S. M. Wall
Keyword(s):  
Acid Secretion ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Physiological Saline ◽  
Ammonia Concentration ◽  
Co2 Absorption ◽  
Proton Source ◽  
Total Ammonia ◽  
Saline Solutions

We have shown that NH4+ and K+ compete for extracellular binding on the Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) in the rat terminal inner medullary collecting duct (tIMCD). The present study explored whether the Na(+)-K(+)-ATPase modulates transepithelial net acid flux [JH+ = total CO2 absorption (JtCO2) + total ammonia secretion (JtAM)]. Tubules from the tIMCD were dissected from deoxycorticosterone (DOC)-treated rats and perfused in vitro. Perfusate and bath were identical physiological saline solutions containing 25 mM NaHCO3 + 6 mM NH4Cl or were NH4Cl or were NH4Cl free. With NH4+ present, the fall in total CO2 from perfusate to collected fluid (delta tCO2, 2.5 +/- 0.4 mM; n = 6) was accompanied by an increase in collected total ammonia concentration (0.2 +/- 0.1 mM). However, in the absence of NH4Cl, delta tCO2 was only 0.9 +/- 0.2 mM (P < 0.05, n = 5). To determine the mechanism of this NH4Cl-induced increase in net acid secretion, the effect of Na+ pump inhibition on net acid secretion was explored. With NH4Cl present, JCO2 was 3.8 +/- 0.5 pmol.mm-1.min-1 (ouabain absent) but declined to 1.6 +/- 0.3 pmol.mm-1.min-1 with ouabain addition to the bath (n = 7, P < 0.05). Furthermore, in the presence of NH4Cl, intracellular pH (pHi) increased from 7.05 +/- 0.02 to 7.15 +/- 0.02 (P < 0.05, n = 5) with ouabain addition and returned to 7.06 +/- 0.03 (P < 0.05) with ouabain removal. However, in the absence of NH4Cl, ouabain failed to reduce JtCO2 (P = NS, n = 5), and an increase in pHi was not observed (n = 4, P = NS). In conclusion, NH4+ augments net acid secretion likely by serving as a proton source for bicarbonate absorption and titration of other luminal buffers. This ammonium pathway is dependent on the basolateral membrane Na(+)-K(+)-ATPase.

Ouabain reduces net acid secretion and increases pHi by inhibiting NH 4 + uptake on rat tIMCD Na+-K+-ATPase

1997 ◽  
Vol 273 (6) ◽  
pp. F857-F868 ◽  
Author(s):  
Susan M. Wall
Keyword(s):  
Acid Secretion ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Physiological Saline ◽  
Weak Base ◽  
Pump Activity ◽  
Total Ammonia ◽  
Medullary Collecting Duct ◽  
Saline Solutions

In the rat terminal inner medullary collecting duct (tIMCD), Na+ pump inhibition reduces transepithelial net acid secretion ( J tAMM) [ J H = total CO2 absorption ( J tCO2) + total ammonia secretion] and increases resting intracellular pH (pHi). The increase in pHi and reduction in J H that follow ouabain addition do not occur in the absence of[Formula: see text] nor when [Formula: see text]is substituted with another weak base. The purpose of this study was to explore the mechanism of the [Formula: see text]-dependent reduction in J tCO2 and increase in pHi that follow ouabain addition. We hypothesized that [Formula: see text]enters the tIMCD cell through the Na+-K+-ATPase with proton release in the cytosol. To test this hypothesis, tIMCDs were dissected from deoxycorticosterone-treated rats and perfused in vitro with symmetrical physiological saline solutions containing 6 mM NH4Cl. Since K+ and[Formula: see text] compete for a common binding site on the Na+ pump, increasing extracellular K+ should limit[Formula: see text] (and hence net H+) uptake by the Na+ pump. Upon increasing extracellular K+ concentration from 3 to 12 mM, the [Formula: see text]-dependent, ouabain-induced increase in pHiand reduction in J tCO2 were attenuated. In the presence but not in the absence of[Formula: see text], reducing Na+ pump activity by limiting Na+ entry reduced J tCO2 and attenuated ouabain-induced alkalinization. Ouabain-induced alkalinization was not dependent on the presence of[Formula: see text]/CO2and was not reproduced with BaCl2or bumetanide addition. Therefore, ouabain-induced alkalinization is not mediated by the Na+-K+-2Cl−cotransporter or a [Formula: see text] transporter and is not mediated by changes in membrane potential. In conclusion, on the basolateral membrane of the tIMCD cell,[Formula: see text] uptake is mediated by the Na+-K+-ATPase. These data provide an explanation for the reduction in net acid secretion in the tIMCD observed following administration of amiloride or with dietary K+ loading.

H(+)-K(+)-ATPase mediates net acid secretion in rat terminal inner medullary collecting duct

1996 ◽  
Vol 271 (5) ◽  
pp. F1037-F1044 ◽  
Author(s):  
S. M. Wall ◽  
A. V. Truong ◽  
T. D. DuBose
Keyword(s):  
Acid Secretion ◽  
Collecting Duct ◽  
Specific Inhibitor ◽  
Co2 Absorption ◽  
Atpase Inhibitor ◽  
Inner Medullary Collecting Duct ◽  
Total Ammonia ◽  
Medullary Collecting Duct ◽  
Atpase Inhibition

Studies in our laboratory have demonstrated total CO2 absorption (JtCO2) and total ammonia secretion in the terminal inner medullary collecting duct (tIMCD) perfused in vitro. The purpose of the present study was to determine whether the H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) participates in proton secretion or JtCO2 in this segment. Tubules from the middle third of the tIMCD were dissected from rats with chronic metabolic acidosis (300 mM NH4Cl, 3-4 days in drinking water) and perfused in vitro. Perfusate and bath were symmetrical solutions containing 5 mM KCl, 6 mM NH4Cl, and 25 mM NaHCO3. Bafilomycin A1 (5 nM), a specific inhibitor of the H(+)-ATPase, did not affect JtCO2 compared with baseline (JtCO2, 3.0 +/- 1.0 and 3.0 +/- 0.8; n = 6, P = not significant) or with time controls (n = 4). With removal of luminal K+, JtCO2 fell from 2.8 +/- 0.6 to 1.6 +/- 0.4 pmol.mm-1.min-1 (n = 5, P < 0.05). To further evaluate K(+)-sensitive JtCO2, the effect of H(+)-K(+)-ATPase inhibition on JtCO2 was explored using the specific H(+)-K(+)-ATPase inhibitor, Sch-28080. Addition of 10 microM Sch-28080 to the luminal perfusate decreased JtCO2 (2.7 +/- 0.4 to 1.4 +/- 0.5 pmol.mm-1. min-1; n = 5, P < 0.05) but did not alter transepithelial membrane potential. Thus luminal Sch-28080 addition, as well as luminal K+ removal, limits apical H+ exit or OH-/HCO3- entry. These results demonstrate that net acid secretion is mediated by the H(+)-K(+)-ATPase in the tIMCD.

scholarly journals Intercalated cell-specific Rh B glycoprotein deletion diminishes renal ammonia excretion response to hypokalemia

2013 ◽  
Vol 304 (4) ◽  
pp. F422-F431 ◽  
Author(s):  
Jesse M. Bishop ◽  
Hyun-Wook Lee ◽  
Mary E. Handlogten ◽  
Ki-Hwan Han ◽  
Jill W. Verlander ◽  
...  
Keyword(s):  
Metabolic Acidosis ◽  
Collecting Duct ◽  
Ammonia Excretion ◽  
Ammonia Concentration ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Intercalated Cell ◽  
Transporter Family ◽  
Total Ammonia ◽  
Normal Increase

The ammonia transporter family member, Rh B Glycoprotein (Rhbg), is an ammonia-specific transporter heavily expressed in the kidney and is necessary for the normal increase in ammonia excretion in response to metabolic acidosis. Hypokalemia is a common clinical condition in which there is increased renal ammonia excretion despite the absence of metabolic acidosis. The purpose of this study was to examine Rhbg's role in this response through the use of mice with intercalated cell-specific Rhbg deletion (IC-Rhbg-KO). Hypokalemia induced by feeding a K+-free diet increased urinary ammonia excretion significantly. In mice with intact Rhbg expression, hypokalemia increased Rhbg protein expression in intercalated cells in the cortical collecting duct (CCD) and in the outer medullary collecting duct (OMCD). Deletion of Rhbg from intercalated cells inhibited hypokalemia-induced changes in urinary total ammonia excretion significantly and completely prevented hypokalemia-induced increases in urinary ammonia concentration, but did not alter urinary pH. We conclude that hypokalemia increases Rhbg expression in intercalated cells in the cortex and outer medulla and that intercalated cell Rhbg expression is necessary for the normal increase in renal ammonia excretion in response to hypokalemia.

Effects of respiratory acidosis on HCO3- transport by rabbit collecting tubules

1988 ◽  
Vol 255 (4) ◽  
pp. F656-F665 ◽  
Author(s):  
T. D. McKinney ◽  
K. K. Davidson
Keyword(s):  
Calcium Concentration ◽  
Basolateral Membrane ◽  
Respiratory Acidosis ◽  
Bicarbonate Transport ◽  
Co2 Absorption ◽  
Distal Nephron ◽  
Outer Medulla ◽  
Collecting Tubules ◽  
Stimulation Of

These studies were performed to determine whether in vitro elevation of bath PCO2 with associated reduction in pH (acute respiratory acidosis) affected bicarbonate transport by isolated perfused rabbit cortical collecting tubules (CCT) and collecting tubules from the outer (OMCTos) and inner (OMCTis) stripes of the outer medulla. When the PCO2 was elevated and pH reduced from approximately 7.4 to 7.0 the rate of total CO2 absorption increased to 252% of that observed at pH 7.4 in CCT, 146% in OMCTos, and 150% in OMCTis. In OMCTis, pretreatment with colchicine inhibited the stimulation of total CO2 absorption associated with respiratory acidosis, whereas lumicolchicine did not. Similar inhibition was observed in the presence of maptam and a low calcium concentration and in the presence of a calmodulin inhibitor. No differences were observed in apical or basolateral membrane morphometry of principal or intercalated cells between control tubules and those subjected to respiratory acidosis. The results indicate that acute respiratory acidosis stimulates acidification by the rabbit distal nephron in vitro through a process(es) that, at least in OMCTis, evidently involves the cell cytoskeleton and changes in cell calcium and calmodulin activities.

Conductive properties of the rabbit outer medullary collecting duct: inner stripe

1985 ◽  
Vol 248 (4) ◽  
pp. F500-F506 ◽  
Author(s):  
B. M. Koeppen
Keyword(s):  
Cell Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Rabbit Kidney ◽  
Apical Cell Membrane ◽  
Basolateral Cell Membrane ◽  
Medullary Collecting Duct ◽  
Conductive Properties

Segments of outer medullary collecting duct were dissected from the inner stripe of the rabbit kidney (OMCDi) and perfused in vitro. The conductive properties of the tubule epithelium and individual cell membranes were determined by means of cable analysis and intracellular voltage-recording microelectrodes. In 35 tubules the transepithelial voltage (VT) and resistance (RT) averaged 17.2 +/- 1.4 mV, lumen positive, and 58.6 +/- 5.3 k omega X cm, respectively. The basolateral membrane voltage, (Vbl) was -29.2 +/- 2.1 mV (n = 23). The apical cell membrane did not contain appreciable ion conductances, as evidenced by the high values of apical cell membrane fractional resistance (fRa = Ra/Ra + Rb), which approached unity (0.99 +/- 0.01; n = 23). Moreover, addition of amiloride or BaCl2 to the tubule lumen was without effect on the electrical characteristics of the cell, as was a twofold reduction in luminal [Cl-]. The conductive properties of the basolateral cell membrane were assessed with bath ion substitutions. A twofold reduction in bath [Cl-] depolarized Vbl by 14.7 +/- 0.4 mV (theoretical, 17 mV), while a 10-fold increase in bath [K+] resulted in only a 0.9 +/- 0.4 mV depolarization (theoretical, 61 mV). Substituting bath Na+ with tetramethylammonium (from 150 to 75 mM) was without effect. Reducing bath [HCO-3] from 25 to 5 mM (constant PCO2) resulted in a steady-state depolarization of Vbl of 8.4 +/- 0.4 mV that could not be attributed to conductive HCO-3 movement. Thus, the basolateral cell membrane is predominantly Cl- selective.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Functional characterization of alpha- and beta-intercalated cell types in rabbit cortical collecting duct

1991 ◽  
Vol 261 (3) ◽  
pp. F377-F385 ◽  
Author(s):  
H. Furuya ◽  
M. D. Breyer ◽  
H. R. Jacobson
Keyword(s):  
Collecting Duct ◽  
Basolateral Membrane ◽  
Functional Characterization ◽  
Cell Types ◽  
Disulfonic Acid ◽  
Cortical Collecting Duct ◽  
Electrical Measurements ◽  
Collecting Ducts

Single-cell electrical measurements and spectrophotometric determinations of intracellular pH were used to determine unique features of alpha- and beta-intercalated cells (alpha-IC, beta-IC) in in vitro perfused rabbit cortical collecting ducts (CCD). pHi rose in alpha-IC and fell in beta-IC after bath Cl- removal. Luminal Cl- removal did not change pHi of alpha-IC, but pHi of beta-IC rose by 0.36 +/- 0.01 pH units. Cl- concentration-dependent recovery of beta-IC pHi revealed a Cl- Km of 18.7 mM for the luminal Cl(-) -HCO3- exchanger. Measurements of basolateral membrane voltage (Vbl) also showed two IC cell types. Removal of luminal Cl- did not change Vbl in alpha-IC, whereas Vbl hyperpolarized by a mean of 73.2 +/- 3.5 mV in beta-IC. Reducing bath Cl- depolarized both alpha- and beta-IC Vbl. In alpha-IC a large repolarization of 39.8 +/- 5.2 mV followed acute depolarization after bath Cl- removal. Reducing bath HCO3- (constant CO2) had little effect on beta-IC Vbl, whereas alpha-IC Vbl depolarized by 5.2 +/- 0.7 mV. Reducing luminal HCO3- in the absence of luminal Cl- produced a 17.6 +/- 1.8 mV depolarization in beta-IC. This change was independent of luminal Na+ and was not blocked by luminal 10(-4) M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In beta-IC, Vbl was not altered by either bath or lumen DIDS in the presence of luminal Cl-. However, when luminal Cl- was removed, luminal DIDS reversibly depolarized Vbl by 9.6 +/- 2.9 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular actions of cAMP on HCO3(-)-secreting cells of rabbit CCD: dependence on in vivo acid-base status

1994 ◽  
Vol 266 (4) ◽  
pp. F528-F535 ◽  
Author(s):  
C. Emmons ◽  
J. B. Stokes
Keyword(s):  
Anion Exchanger ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Disulfonic Acid ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Base ◽  
Acid Base Status

HCO3- secretion by cortical collecting duct (CCD) occurs via beta-intercalated cells. In vitro CCD HCO3- secretion is modulated by both the in vivo acid-base status of the animal and by adenosine 3',5'-cyclic monophosphate (cAMP). To investigate the mechanism of cAMP-induced HCO3- secretion, we measured intracellular pH (pHi) of individual beta-intercalated cells of CCDs dissected from alkali-loaded rabbits perfused in vitro. beta-Intercalated cells were identified by demonstrating the presence of an apical anion exchanger (cell alkalinization in response to removal of lumen Cl-). After 180 min of perfusion to permit decrease of endogenous cAMP, acute addition of 0.1 mM 8-bromo-cAMP or 1 microM isoproterenol to the bath caused a transient cellular alkalinization (> 0.20 pH units). In the symmetrical absence of either Na+, HCO3-, or Cl-, cAMP produced no change in pHi. Basolateral dihydrogen 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (0.1 mM) for 15 min before cAMP addition also prevented this alkalinization. In contrast to the response of cells from alkali-loaded rabbits, addition of basolateral cAMP to CCDs dissected from normal rabbits resulted in an acidification of beta-intercalated cells (approximately 0.20 pH units). The present studies demonstrate the importance of the in vivo acid-base status of the animal in the regulation of CCD HCO3- secretion by beta-intercalated cells. The results identify the possible existence of a previously unrecognized Na(+)-dependent Cl-/HCO3- exchanger on the basolateral membrane of beta-intercalated cells in alkali-loaded rabbits.

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