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.

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 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.

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)

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.

scholarly journals Contribution of the Na+-K+-2Cl− Cotransporter (NKCC1) to Transepithelial Transport of H+, NH4+, K+, and Na+ in Rat Outer Medullary Collecting Duct

2002 ◽  
Vol 13 (4) ◽  
pp. 827-835
Author(s):  
Susan M. Wall ◽  
Michael P. Fischer
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Transepithelial Transport ◽  
In Series ◽  
Acid Secreting ◽  
Medullary Collecting Duct ◽  
Nacl Secretion

ABSTRACT. In rat kidney, the “secretory” isoform of the Na-K-Cl cotransporter, NKCC1 (BSC-2), localizes to the basolateral membrane of the α intercalated cell, the acid secreting cell of the outer medullary collecting duct (OMCD). This laboratory has reported that NKCC1 mediates Cl− uptake across the basolateral membrane in series with Cl− secretion across the apical membrane in rat OMCD. NKCC1 transports NH4+, K+, and Na+ as well as Cl−; therefore, a role for the cotransporter in the process of HCl, NH4Cl, KCl, and NaCl secretion has been suggested. Thus, it was determined if bumetanide, an inhibitor of NKCC1, alters transepithelial cation transport in rat OMCD. OMCD tubules from deoxycorticosterone pivalate (DOCP)–treated rats were perfused in vitro. Hydration of CO2, rather than NH4+, provides the principle source of H+ for net acid secretion. In HCO3−/CO2-buffered solutions, no effect of bumetanide on net K+ flux was detected. Under some conditions, bumetanide addition resulted in a small reduction in secretion of net H+ equivalents. Transepithelial Na+ flux, JNa, was −1.5 ± 1.7 pmol/mm per min, values not different from zero. However, with the application of bumetanide to the bath, JNa was +5.2 ± 1.3 pmol/mm per min (P < 0.05), which indicates net Na+ absorption. In conclusion, inhibition of NKCC1 in rat OMCD changes transepithelial movement of Na+ and Cl−. The role of NKCC1 in the secretion of net H+ equivalents is small.

Electrophysiology of collecting duct H+ secretion: effect of inhibitors

1989 ◽  
Vol 256 (1) ◽  
pp. F79-F84 ◽  
Author(s):  
B. M. Koeppen
Keyword(s):  
Collecting Duct ◽  
Basolateral Membrane ◽  
Rabbit Kidney ◽  
Disulfonic Acid ◽  
Coupled Transport ◽  
Ionic Selectivity ◽  
Disulfonic Stilbene ◽  
Transepithelial Voltage ◽  
Medullary Collecting Duct

Segments of the outer medullary collecting duct were isolated from the inner stripe of the rabbit kidney (OMCDi), perfused in vitro, and impaled across their basolateral membranes with voltage-recording microelectrodes. The disulfonic stilbene 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) (10(-4) M) and the carbonic anhydrase inhibitor acetazolamide (10(-4) M) depolarized the lumen-positive transepithelial voltage (VT) toward 0 mV when added to the bath solution. Concurrently, the basolateral membrane voltage (Vbl) hyperpolarized. The hyperpolarization of Vbl, which averaged 19.3 +/- 2.9 mV (n = 11) for SITS and 22.7 +/- 3.5 mV (n = 11) for acetazolamide, was not due to an alteration in the ionic selectivity of the basolateral membrane, which was highly Cl- selective. The hyperpolarization of Vbl could best be explained by a decrease in the intracellular [Cl-], and the associated shift in the emf for Cl- (ECl) across the basolateral membrane. The decrease in intracellular [Cl-] could be attributed to inhibition of a Cl-HCO3 antiporter in the basolateral membrane. SITS appeared to inhibit this antiporter directly, whereas the effect of acetazolamide was indirect, probably secondary to a decrease in the intracellular [HCO3-]. Finally, both SITS and acetazolamide induced or unmasked an electroneutral K+-coupled transport system in the basolateral membrane.

Ammonium and bicarbonate transport in rat outer medullary collecting ducts

1992 ◽  
Vol 262 (1) ◽  
pp. F1-F7 ◽  
Author(s):  
M. F. Flessner ◽  
S. M. Wall ◽  
M. A. Knepper
Keyword(s):  
Collecting Duct ◽  
Co2 Concentration ◽  
Bicarbonate Transport ◽  
Concentration Gradients ◽  
Entry Rate ◽  
Collecting Ducts ◽  
Total Ammonia ◽  
Outer Stripe ◽  
Medullary Collecting Duct

Previous in vitro studies have demonstrated spontaneous bicarbonate absorption in the outer stripe portion of the rat outer medullary collecting duct (OMCD) and inner medullary collecting duct, but net acid transport has not been studied in the inner stripe of the rat OMCD (OMCDIS). When we perfused isolated OMCDIS segments with identical bath and perfusate solutions containing HCO-3 and NH4Cl, HCO-3 was spontaneously absorbed, and total ammonia was spontaneously secreted at rapid rates in tubules from both deoxycorticosterone (DOC)-treated and untreated rats. We next measured the NH3 flux due to imposed NH3 concentration gradients. Carbonic anhydrase (CA), when added to the lumen, enhanced the NH3 flux, implying an absence of endogenous CA. The NH3 permeability was 0.0042 +/- 0.0007 cm/s. By measuring the luminal pH in perfused OMCDIS segments with an imposed lumen-to-bath NH3 gradient, we determined the pH at the end of the lumen to be 0.23 units below the equilibrium pH calculated from the simultaneously measured total CO2 concentration in collected fluid, confirming the lack of luminal CA. These results are consistent with the view that ammonium secretion in the OMCDIS occurs predominantly by H+ secretion and parallel NH3 diffusion. A luminal disequilibrium pH due to H+ secretion in the absence of endogenous luminal CA enhances the NH3 entry rate. Spontaneous net acid secretion appears to occur more rapidly in the OMCD than in other parts of the rat collecting duct system.

Acidification adaptation in cultured inner medullary collecting duct cells

1993 ◽  
Vol 264 (5) ◽  
pp. F765-F769 ◽  
Author(s):  
R. Mankus ◽  
J. H. Schwartz ◽  
E. A. Alexander
Keyword(s):  
Lucifer Yellow ◽  
Collecting Duct ◽  
C Cells ◽  
Inner Medullary Collecting Duct ◽  
Pump Activity ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct ◽  
New Protein

Chronic acid feeding stimulates the rat inner medullary collecting duct (IMCD) to increase acid secretion in vivo (acidification adaptation), but the mechanism for this phenomenon is unknown. Our purpose was to determine whether IMCD cells undergo adaptation in vitro and to explore the mechanism of this response. Confluent cultured rat IMCD cells were exposed to incubation media supplemented with 10(-7) M deoxycorticosterone acetate, pH 7.0 [acid incubated (AI)] or 7.7 [control (C)], for 48 h, and cell pH (pHi) was determined using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Resting pHi was 7.46 +/- 0.05 for AI and 7.25 +/- 0.04 for C (P < 0.05). N-ethylmaleimide-sensitive pHi recovery after an acute acid pulse was 0.030 +/- 0.002 for AI and 0.020 +/- 0.002 pH U/min for C (P < 0.05). However, when AI and C cells were incubated with 7 x 10(-6) M cycloheximide, the increment in pHi and enhanced proton pump activity was abolished. In addition, exocytic function, as measured by Lucifer yellow release, was increased significantly in AI cells. In summary, incubation of IMCD cells in acid medium stimulates acidification adaptation by a mechanism dependent on new protein synthesis.

Adaptation of inner medullary collecting duct to dehydration involves a paracellular pathway

1995 ◽  
Vol 268 (1) ◽  
pp. F53-F63 ◽  
Author(s):  
B. Flamion ◽  
K. R. Spring ◽  
M. Abramow
Keyword(s):  
Water Permeability ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Osmotic Water Permeability ◽  
Inner Medullary Collecting Duct ◽  
Osmotic Water ◽  
Medullary Collecting Duct ◽  
Paracellular Pathways

Prolonged fluid restriction in rats is accompanied by functional modifications of the terminal part of the inner medullary collecting duct (IMCD) revealed by a sustained increase in arginine vasopressin (AVP)-independent transepithelial osmotic water permeability (PTE) in vitro. The cellular basis of this adaptation was explored in isolated and perfused terminal IMCDs of Sprague-Dawley rats using video and fluorescence microscopy. Basolateral membrane osmotic water permeability (Posm), transcellular Posm, and PTE were measured in quick sequence in every tubule. They were expressed per unit area of basolateral membrane corrected for infoldings, based on previous stereological studies and assuming no major change in membrane surface area between hydrated and dehydrated animals. Compared with IMCDs of rats with a high water intake, IMCDs of rats deprived of fluid for 36 h displayed a significantly higher basal PTE (24.9 +/- 5.1 vs. 6.1 +/- 0.6 microns/s), a similar basolateral Posm, and a higher transcellular Posm, implying a higher permeability of the apical membrane, despite the absence of exogenous AVP. However, when IMCDs of thirsted rats were exposed to AVP in vitro, their transcellular Posm (36.0 +/- 2.4 microns/s) was significantly smaller than their PTE determined simultaneously (51.8 +/- 7.1 microns/s), suggesting that part of the water flow may follow a paracellular route. A change in paracellular pathways was supported by higher apparent permeabilities to [14C]sucrose (0.85 +/- 0.27 vs. 0.28 +/- 0.04 x 10(-5) cm/s) and to [methoxy-3H]inulin (0.25 +/- 0.04 vs. 0.14 +/- 0.03 x 10(-5) cm/s) in IMCDs of thirsted rats. The nonelectrolyte permeabilities were affected neither by AVP nor by urea-rich bathing solutions. We conclude that in vivo factors related to dehydration produce a conditioning effect on terminal IMCD, which includes stabilization of the apical membrane in a state of high Posm and opening up of paracellular pathways revealed by a higher permeability to water and nonelectrolytes. The role of these adaptive phenomena remains unclear but may pertain to the sudden transitions between antidiuresis and diuresis.

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