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.

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.

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.

NF-κB inhibits transcription of the H+-K+-ATPase α2-subunit gene: role of histone deacetylases

2002 ◽  
Vol 283 (5) ◽  
pp. F904-F911 ◽  
Author(s):  
Wenzheng Zhang ◽  
Bruce C. Kone
Keyword(s):  
Histone Deacetylases ◽  
Transcriptional Control ◽  
Protein Complexes ◽  
Trichostatin A ◽  
Collecting Duct ◽  
Proximal Promoter ◽  
Inner Medullary Collecting Duct ◽  
Nuclear Extracts ◽  
Medullary Collecting Duct

The H+-K+-ATPase α2 (HKα2) gene plays a central role in potassium homeostasis, yet little is known about its transcriptional control. We recently demonstrated that the proximal promoter confers basal transcriptional activity in mouse inner medullary collecting duct 3 cells. We sought to determine whether the κB DNA binding element at −104 to −94 influences basal HKα2 gene transcription in these cells. Recombinant NF-κB p50 footprinted the region −116/−94 in vitro. Gel shift and supershift analysis revealed NF-κB p50- and p65-containing DNA-protein complexes in nuclear extracts of mouse inner medullary collecting duct 3 cells. A promoter-luciferase construct with a mutated −104/−94 NF-κB element exhibited higher activity than the wild-type promoter in transfection assays. Overexpression of NF-κB p50, p65, or their combination trans-repressed the HKα2 promoter. The histone deacetylase (HDAC) inhibitor trichostatin A partially reversed NF-κB-mediated trans-repression of the HKα2 promoter. HDAC6 overexpression inhibited HKα2 promoter activity, and HDAC6 coimmunoprecipitated with NF-κB p50 and p65. These results suggest that HDAC6, recruited to the DNA protein complex, acts with NF-κB to suppress HKα2 transcription and identify NF-κB p50 and p65 as novel binding partners for HDAC6.

Suppression of acidification along inner medullary collecting duct

1988 ◽  
Vol 255 (2) ◽  
pp. F307-F312 ◽  
Author(s):  
H. H. Bengele ◽  
E. R. McNamara ◽  
J. H. Schwartz ◽  
E. A. Alexander
Keyword(s):  
Acid Secretion ◽  
Collecting Duct ◽  
Respiratory Alkalosis ◽  
Inner Medullary Collecting Duct ◽  
Titratable Acid ◽  
Arterial Ph ◽  
Medullary Collecting Duct ◽  
Collection Sites

The purpose of these experiments was to evaluate the effect of acute respiratory alkalosis (ARA) and chronic bicarbonate drinking (CBD) on inner medullary collecting duct (IMCD) acidification. Microcatheterization was used to measure pH and PCO2, and samples were simultaneously obtained for measurement of bicarbonate, titratable acid (TA), and ammonium. In ten ARA rats (arterial pH was 7.54 +/- 0.02; PCO2 was 20 +/- 1 mmHg), IMCD equilibrium pH was not different (deep pH was 5.65 +/- 0.06 and PCO2 was 20 +/- 1 mmHg; tip pH was 5.54 +/- 0.07 and PCO2 was 22 +/- 1 mmHg). Delivery of bicarbonate, TA, and ammonium also did not differ between collection sites. Thus net acidification along the IMCD was negligible. Nine rats drank NaHCO3 for 5-8 days (pH = 7.48 +/- 0.02) but did not receive NaHCO3 during the experiment so that arterial pH fell to 7.40 +/- 0.01. IMCD equilibrium pH was different at deep (pH was 5.68 +/- 0.06; PCO2 was 32 +/- 1 mmHg) and tip (pH was 5.57 +/- 0.04; PCO2 was 27 +/- 1 mmHg; P less than or equal to 0.05) collection sites. However, only minimal changes in the delivery of bicarbonate, TA, and ammonium were noted, and net acidification along the IMCD was negligible. In ten control rats, net acidification was 219 nmol/min between collection sites (P less than 0.001). We conclude that ARA and CBD abolish acidification along the IMCD. In addition, CBD produces an intrinsic modification along the IMCD, which suppresses acid secretion and persists after acute recovery from alkalemia.

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.

Effect of peritubular hypertonicity on water and urea transport of inner medullary collecting duct

1992 ◽  
Vol 262 (3) ◽  
pp. F338-F347 ◽  
Author(s):  
L. H. Kudo ◽  
K. R. Cesar ◽  
W. C. Ping ◽  
A. S. Rocha
Keyword(s):  
Hydraulic Conductivity ◽  
Collecting Duct ◽  
Osmotic Gradient ◽  
Urea Transport ◽  
Perfusion Fluid ◽  
Cyclic Monophosphate ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct ◽  
Series Of Experiments

The effect of bath fluid hypertonicity on hydraulic conductivity (Lp) and [14C]urea permeability (Pu) of the distal inner medullary collecting duct (IMCD) was studied in the absence and in the presence of vasopressin (VP) using the in vitro microperfusion technique of rat IMCD. In the first three groups of IMCD, we observed that in the absence of VP the Lp was not different from zero when the osmotic gradient was created by hypotonic perfusate and isotonic bath fluid, but it was significantly greater than 1.0 x 10(-6) cm.atm-1.s-1 when the osmotic gradient was created by hypertonic bath and isotonic perfusion fluid. The increase in Lp was observed when the hypertonicity of the bath fluid was produced by the addition of NaCl or raffinose, but no such effect was observed with urea. The stimulated effect of bath fluid hypertonicity on Lp was also observed in the IMCD obtained from Brattleboro homozygous rats in which VP is absent. The NaCl hypertonic bath increased the Pu in the absence of VP. In another series of experiments with VP (10(-10) M) we observed that the hypertonic bath fluid increased in a reversible manner the VP-stimulated Lp of distal IMCD. However, the NaCl hypertonicity of the bath fluid was not able to increase dibutyryladenosine 3',5'-cyclic monophosphate-stimulated Lp. The Pu stimulated by VP (10(-10) M) increased twofold when the bath fluid was hypertonic. Therefore hypertonicity of the peritubular fluid produced by the addition of NaCl or raffinose increases the Lp and Pu in the absence and in the presence of VP. No such effect was noted with the addition of urea.

Rubidium absorption and proton secretion by rabbit outer medullary collecting duct via H-K-ATPase

1992 ◽  
Vol 263 (5) ◽  
pp. F849-F857 ◽  
Author(s):  
C. S. Wingo ◽  
F. E. Armitage
Keyword(s):  
Collecting Duct ◽  
Restricted Diet ◽  
Atpase Inhibitor ◽  
Marked Contrast ◽  
Proton Secretion ◽  
Subsequent Effect ◽  
Transepithelial Voltage ◽  
Medullary Collecting Duct ◽  
Atpase Inhibition ◽  
Lines Of Evidence

Multiple lines of evidence support the hypothesis that the outer medullary collecting duct from the inner stripe (OMCDi) possesses a functional proton-potassium-activated adenosinetriphosphatase (H-K-ATPase). To examine the effect of inhibition of H-K-ATPase on Rb efflux, we measured the Rb tracer rate efflux coefficient (KRb) across the OMCDi of animals adapted to a K-restricted diet using the selective K-competitive H-K-ATPase inhibitor, Sch 28080. Sch 28080 (10 microM) did not significantly alter transepithelial voltage (VT) but significantly decreased KRb by 41%. We further examined the effect of 10% peritubular CO2 on KRb and the subsequent effect of Sch 28080 (10 microM) on KRb. After exposure to 10% CO2 for 120 min, vehicle-treated tubules exhibited a small but significant increase in KRb without a significant change in VT. In contrast, 10 microM Sch 28080 significantly decreased KRb by 44% without affecting VT. The lack of an effect of H-K-ATPase inhibition on VT in the presence of either 5% or 10% CO2 was in marked contrast to the effect of carbonic anhydrase inhibition (CAI). CAI consistently and significantly decreased VT either in the presence of 5% or 10% CO2. To address whether H-K-ATPase also participates in proton secretion we examined the effect of Sch 28080 (10 microM) on net bicarbonate absorption by the OMCDi of rabbits fed a normal rabbit ration and rabbits adapted to a K-restricted diet.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals A mathematical model of the inner medullary collecting duct of the rat: acid/base transport

1998 ◽  
Vol 274 (5) ◽  
pp. F856-F867 ◽  
Author(s):  
Alan M. Weinstein
Keyword(s):  
Mathematical Model ◽  
Acid Secretion ◽  
Collecting Duct ◽  
Model Calculations ◽  
Principal Mode ◽  
Inner Medullary Collecting Duct ◽  
Tubule Lumen ◽  
Nephron Segment ◽  
The Face ◽  
Medullary Collecting Duct

A mathematical model of the inner medullary collecting duct (IMCD) of the rat has been developed that is suitable for simulating luminal buffer titration and ammonia secretion by this nephron segment. Luminal proton secretion has been assigned to an H-K-ATPase, which has been represented by adapting the kinetic model of the gastric enzyme by Brzezinski et al. (P. Brzezinski, B. G. Malmstrom, P. Lorentzon, and B. Wallmark. Biochim. Biophys. Acta 942: 215–219, 1988). In shifting to a 2 H+:1 ATP stoichiometry, the model enzyme can acidify the tubule lumen ∼3 pH units below that of the cytosol, when luminal K+ is in abundance. Peritubular base exit is a combination of ammonia recycling and[Formula: see text] flux (either via[Formula: see text] exchange or via a Cl− channel). Ammonia recycling involves[Formula: see text] uptake on the Na-K-ATPase followed by diffusive NH3 exit [S. M. Wall. Am. J. Physiol. 270 ( Renal Physiol. 39): F432–F439, 1996]; model calculations suggest that this is the principal mode of base exit. By virtue of this mechanism, the model also suggests that realistic elevations in peritubular K+ concentration will compromise IMCD acid secretion. Although ammonia recycling is insensitive to carbonic anhydrase (CA) inhibition, the base exit linked to [Formula: see text] flux provides a CA-sensitive component to acid secretion. In model simulations, it is observed that increased luminal NaCl entry increases ammonia cycling but decreases peritubular [Formula: see text]exchange (due to increased cell Cl−). This parallel system of peritubular base exit stabilizes acid secretion in the face of variable Na+ reabsorption.

Distribution of luminal carbonic anhydrase activity along rat inner medullary collecting duct

1991 ◽  
Vol 260 (5) ◽  
pp. F738-F748 ◽  
Author(s):  
S. M. Wall ◽  
M. F. Flessner ◽  
M. A. Knepper
Keyword(s):  
Carbonic Anhydrase ◽  
Collecting Duct ◽  
Co2 Concentration ◽  
Carbonic Anhydrase Activity ◽  
Inner Medullary Collecting Duct ◽  
Proton Source ◽  
Total Ammonia ◽  
Medullary Collecting Duct ◽  
Disequilibrium Ph ◽  
Ammonia Flux

The isolated perfused tubule technique was utilized to determine whether endogenous luminal carbonic anhydrase is present in the initial or terminal parts of the inner medullary collecting duct (IMCD) of the rat. This was accomplished by measuring the luminal disequilibrium pH in the presence of a large luminal proton source created by perfusing the lumen with a solution containing 10 mM NH4Cl. (NH3 efflux causes H+ to be released from NH+4 in the lumen). The disequilibrium pH was calculated by subtracting the equilibrium pH from the measured pH at the end of the tubule lumen. The end-luminal equilibrium pH was calculated from the total CO2 concentration in the collected fluid, as measured by microcalorimetry. The end-luminal pH was determined by measuring the fluorescent signal from the the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), which was added to the luminal perfusate in its nonesterified form. In the initial IMCD, there was no measurable disequilibrium pH. With the addition of the carbonic anhydrase inhibitor acetazolamide to the luminal fluid, a significant acidic pH disequilibrium was elicited. In the terminal IMCD under control conditions a statistically significant acidic disequilibrium pH was measured. The disequilibrium was obliterated when exogenous carbonic anhydrase was added to the luminal perfusate. These findings were verified by measuring total ammonia flux by ultramicrofluorometry. The results demonstrate endogenous luminal carbonic anhydrase activity in the initial IMCD but a lack of enzyme activity in the terminal IMCD.

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