Ammonium transport by the colonic H+-K+-ATPase expressed in Xenopus oocytes

Marc Cougnon1, Patrice Bouyer1, Frédéric Jaisser2, Aleksander Edelman1, and Gabrielle Planelles1

1 Faculté de Médecine Necker, Institut National de la Santé et de la Recherche Médicale U. 467, Université Paris V, F-75015 Paris; and 2 Faculté de Médecine Xavier Bichat, Institut National de la Santé et de la Recherche Médicale U. 478, Université Paris VII, F-75018 Paris, France


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Functional expression of the rat colonic H+-K+-ATPase was obtained by coexpressing its catalytic alpha -subunit and the beta 1-subunit of the Na+-K+-ATPase in Xenopus laevis oocytes. We observed that, in oocytes expressing the rat colonic H+-K+-ATPase but not in control oocytes (expressing beta 1 alone), NH4Cl induced a decrease in 86Rb uptake and the initial rate of intracellular acidification induced by extracellular NH4Cl was enhanced, consistent with NH+4 influx via the colonic H+-K+-ATPase. In the absence of extracellular K+, only oocytes expressing the colonic H+-K+-ATPase were able to acidify an extracellular medium supplemented with NH4Cl. In the absence of extracellular K+ and in the presence of extracellular NH+4, intracellular Na+ activity in oocytes expressing the colonic H+-K+-ATPase was lower than that in control oocytes. A kinetic analysis of 86Rb uptake suggests that NH+4 acts as a competitive inhibitor of the pump. Taken together, these results are consistent with NH+4 competition for K+ on the external site of the colonic H+-K+-ATPase and with NH+4 transport mediated by this pump.

86Rb influx; intracellular sodium and pH measurements


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THE COMPLEMENTARY DNA cloned from rat distal colon by Crowson and Shull (11) belongs to the cDNAs for the phosphorylating class of ion-motive ATPases (P-ATPase family) and shares a high level of molecular homology with cDNAs for two other members of the P-type, K+-dependent ATPase family, the Na+-K+-ATPase and the gastric H+-K+-ATPase. The product of the colonic P-ATPase alpha -subunit gene, when expressed in the Xenopus oocyte heterologous expression system, exhibits functional and pharmacological properties similar to those of the gastric H+-K+-ATPase or of the Na+-K+-ATPase. Like the gastric H+-K+-ATPase, it mediates H+/K+ exchange, but unlike the gastric isoform, it is insensitive to Sch-28080 and is inhibited by millimolar concentrations of ouabain (10). Thus the pharmacological profile of the colonic H+-K+-ATPase is similar to that of the "ouabain-resistant" Na+-K+-ATPase, which is inhibited by high concentrations of the cardiac glycoside (28). By using the oocyte expression system, we have also recently shown that, like the Na+-K+-ATPase, the colonic H+-K+-ATPase can extrude Na+ from the cytosol, acting as both a Na+-K+-ATPase and a H+-K+-ATPase (9). More recently, it has been demonstrated that another of the nongastric H+-K+-ATPases, the human ATP1AL1-encoded protein, also acts as a Na+-K+- and H+-K+-transporting ATPase mediating primarily Na+/K+ exchange rather than H+/K+ exchange when expressed in HEK-293 cells (17).

As with the ATP1AL1 (17), the physiological role of ionic transport mediated by the colonic H+-K+-ATPase is difficult to delineate. Colonic H+-K+-ATPase is found mainly in the distal colon (11) and the distal part of the nephron (1). Its overexpression in kidney cells during a low-K+ diet is consistent with its involvement in renal K+ reabsorption (12, 18); in the distal colon, where it is constitutively expressed, it is not upregulated by a low-K+ diet (18), but dietary K+ depletion increases colonic K+ absorption (14, 25). The physiological relevance of Na+ transport by the pump has not been established. The presence of the colonic H+-K+-ATPase in the distal nephron and its mediation of H+ extrusion from the cell suggest that it could be involved in regulating the acid-base balance. However, this putative role is not firmly supported by the findings of previous reports, in particular by the lack of effect of metabolic acidosis on the colonic H+-K+-ATPase mRNA levels (12). Aldosterone status, which is often linked to K+ balance, does not change the expression of the colonic H+-K+-ATPase in the kidney (18). Functional studies of the role of the colonic H+-K+-ATPase in acid-base homeostasis of renal tubular cells are difficult to interpret, because of the segmental and cellular heterogeneities of the distal nephron, because of the lack of a specific inhibitor of the colonic H+-K+-ATPase, and especially because gastric H+-K+-ATPase, vacuolar H+-ATPase, and Na+-K+-ATPase are also present in this part of the nephron. The powerful tool of genetically modified animals has not yet clarified the roles of the colonic H+-K+-ATPase in renal ionic transport and physiology: when fed either a normal or a low-K+ diet, transgenic mice with an inactivated colonic H+-K+ alpha -subunit gene have the same Na+ and K+ urinary excretion as control mice (25). Thus functional expression of the colonic H+-K+-ATPase promises more help in understanding its mechanistic, functional, and pharmacological properties and may also stimulate investigations with other models.

Because the colonic H+-K+-ATPase is mainly expressed in the renal medullary collecting duct (1, 23, 29) and in the colon, which are both involved in the net transport of ammonium, we looked for the involvement of this pump in NH+4 transmembrane transport after its functional expression in Xenopus oocytes. Measurements of 86Rb influx, intracellular pH (pHi) and extracellular pH (pHo), and intracellular Na+ activity ([Na+]i) are consistent with the colonic H+-K+-ATPase-mediated transport of NH+4.


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cRNA synthesis and expression in Xenopus laevis oocytes. cRNAs of the rat Na+-K+-ATPase beta 1-subunit (34) and of the catalytic alpha -subunit of the colonic H+-K+-ATPase, alpha c (11), were synthesized with the SP6 RNA polymerase (Promega).

Oocytes were injected with 2 ng of the Na+-K+-ATPase beta 1-subunit cRNAs to be used as control oocytes or were coinjected with 2 ng of beta 1 cRNAs plus 10 ng of alpha c cRNAs to express the colonic H+-K+-ATPase (9, 10). Experiments were performed 2 days after cRNA injection (9, 10). The oocytes were incubated at 18°C in an amphibian Ringer solution containing (in mM) 85 NaCl, 1 KCl, 1 CaCl2, and 1 MgCl2, buffered at pH 7.4 with TES-NaOH and supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin. As justified below, 10 µM ouabain, 5 mM barium, 10-5 M bumetanide, and 1 mM diphenylamine-2-carboxylic acid (DPC) were added to the Ringer during the 2-day incubation period ([Na+]i measurements) or during the experiments (86Rb uptake measurements and pHi and pHo measurements).

86Rb uptake. The effect of extracellular NH4Cl on K+ uptake by oocytes was tested by using 86Rb as a K+ surrogate. Batches of 8-10 oocytes were incubated before the experiment for 15 min at room temperature in a K+-free assay solution containing (in mM) 90 NaCl, 1 MgCl2, and 0.41 Ca(NO3)2, buffered at pH 7.4 with TES-NaOH. The assay solution contained 10 µM ouabain to inhibit the endogenous Na+-K+-ATPase, which is very sensitive to this inhibitor (6), and 5 mM barium to inhibit the oocyte membrane K+ conductance (8), because these transport systems mediate K+ influx and may also trigger NH+4 influx into the oocyte (8). Because Na+-K+-2Cl- symport and nonselective cationic conductance also allow K+ and NH+4 entry into the oocyte (4, 8, 20), they were inhibited by adding 10-5 M bumetanide and 1 mM DPC to the assay solution. Preliminary experiments showed that cH+-K+-ATPase mediates 80 or 95% of the total 86Rb uptake in the absence or presence of these inhibitors, respectively.

Preliminary experiments also showed that 86Rb uptake is linear for at least 12 min. The oocytes were therefore transferred to assay solutions (composition given above) containing 5 µCi/ml 86Rb and various concentrations of NH4Cl (at the expense of NaCl) for 12 min before counting. The assay solutions were supplemented with 200 µM KCl to only partially activate the colonic H+-K+-ATPase [Km for K+ is reported to be 730 µM (10)], except in the experiments to determine the apparent pump affinity for NH+4 (in which various concentrations of external KCl were added). The colonic H+-K+-ATPase was inhibited by adding 2 mM ouabain to the assay solution when necessary [whereas the endogenous Na+-K+-ATPase is sensitive to micromolar concentrations of ouabain (6), the colonic H+-K+-ATPase is only moderately sensitive to the cardiac glycoside (10)]. Individual oocytes were lysed with 100 µl of 5% SDS, mixed with 2 ml of scintillation medium, and counted.

pHi and [Na+]i measurements. Simultaneous measurements of membrane potential (Vm) and of intracellular proton activity expressed as pHi or of [Na+]i were performed with double-barreled (ion selective and conventional) microelectrodes. The fabrication of ion-selective microelectrodes and the compositions of their filling solutions have been described elsewhere (2). The H+ ionophore 95291 (Fluka) and the Na+ ionophore 71176 (Fluka) were used for pHi and [Na+]i measurements, respectively. Before use, the microelectrodes were beveled on a microgrinder (De Marco Engineering). Their slopes (S) were determined before intracellular measurement by measuring the change in potential caused by a 10-fold change in Na+ or H+ concentration in the extracellular fluid; S was checked again after each puncture. The intracellular activity of ion i (Ai) was calculated from the relation Ai = Aref · 10(Vsel-Vm)/S where Aref is the activity (in mM) of ion i in the reference solution and Vsel is the measured electrochemical potential difference (in mV) for ion i.

For pH-selective microelectrodes, S was 54-57 mV (pH of the testing solutions: 7.4-8.4); no interference with NH+4 was detected. For Na+-selective microelectrodes, S was 50-56 mV when changing extracellular Na+ concentration ([Na+]o) from 100 to 10 mM. In this solution, 90 mM KCl was added to maintain the osmolarity and to take into account the slight interference of the high intracellular K+ activity with [Na+]i measurements (2, 9).

Single oocytes were placed in a perfusion microchamber and were punctured by selective microelectrodes under stereomicroscopic control. The electrical circuit has been described elsewhere (2). Superfusate solutions (containing 10 µM ouabain, 5 mM BaCl2, 10-5 M bumetanide, and 1 mM DPC) were delivered by a gravimetric system equipped with an electronic rapid-switch device.

pHo measurements. pHo was measured as previously described (10, 19). Briefly, individual oocytes (beta 1- or alpha cbeta 1-expressing oocytes) were placed in an oil-surrounded droplet (1 µl) of a weakly buffered (0.5 mM MOPS; pH 7.6) solution containing (in mM) 110 NaCl, 0.5 MgCl2, and 0.5 CaCl2, sometimes supplemented with 10 mM NH4Cl; 10 µM ouabain, 5 mM BaCl2, 10-5 M bumetanide, and 1 mM DPC were always present. A double-barreled pH-selective microelectrode (the conventional barrel was used as the reference) was introduced into the droplet to record pHo evolution. In this experimental series, S was 51-58 mV (pH of the testing solutions was 6.8-7.8). Reported pHo values were obtained after 20 min.

Data analysis. Results are given as means ± SE. The significance of the results was assessed by Student's unpaired t-test. P < 0.05 was considered significant.


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Effect of NH+4 on 86Rb uptake. In this experimental series, 86Rb uptake was measured in beta 1- and in alpha cbeta 1-expressing oocytes in the presence of 200 µM KCl and of inhibitors of endogenous membrane K+ transport systems (as detailed in METHODS). Functional expression of the colonic H+-K+-ATPase dramatically increased ouabain-sensitive 86Rb uptake compared with the expression of beta 1 alone (Fig. 1, A and B). The addition of 1-85 mM NH4Cl to assay solutions significantly reduced 86Rb uptake by alpha cbeta 1-expressing oocytes (Fig. 1A). NH4Cl did not affect 86Rb uptake by beta 1-expressing oocytes (Fig. 1B). These results are consistent with an NH4Cl-induced effect on the ionic transport mediated by the colonic H+-K+-ATPase.


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Fig. 1.   86Rb uptake by beta 1- and alpha cbeta 1-expressing oocytes. 86Rb uptake was measured in presence of 200 µM KCl and inhibitors of K+ transport pathways [10 µM ouabain, 5 mM barium, 10-5 M bumetanide, and 1 mM diphenylamine-2-carboxylic acid (DPC)]. A: effect of increasing NH4Cl concentration ([NH4Cl]) on 86Rb uptake by alpha cbeta 1-expressing oocytes (n = 9-24 oocytes from 3 independent experiments). Inset: 86Rb uptake by alpha cbeta 1-expressing oocytes exposed to 2 mM ouabain; presence of 10 mM NH4Cl is indicated below bar. B: 86Rb uptake by beta 1-expressing oocytes (n = 8-18 oocytes from 2 independent experiments). Solid bars, exposure to 2 mM ouabain. Presence of 10 mM NH4Cl is indicated below bars. Data are means ± SE. Significance of results was assessed by Student's unpaired t-test. NS, not significant. * P < 0.05.

The above results do not indicate whether NH4Cl acts directly on the pump or if the pump is affected by other effects of NH4Cl on the oocyte. In oocytes, the extracellular addition of NH4Cl is known to induce intracellular acidification (due to NH+4 influx into the cell and its subsequent partial dissociation into NH3 and H+) and membrane depolarization (4, 8); both phenomena may persist despite the inhibition of the oocyte Na+-K+-ATPase, K+ conductances, Na+-K+-2Cl- symport, and cationic nonselective conductance (8). To determine whether a change in Vm (Delta Vm) and/or a change in pHi (Delta pHi) could reduce 86Rb uptake by alpha cbeta 1-expressing oocytes, we compared the effects of NH4Cl and of trimethylamine-HCl on Vm and pHi and on 86Rb uptake. It was reported that trimethylamine-HCl qualitatively affects Vm and pHi, as does NH4Cl (5, 33). In experiments using double-barreled pH-selective microelectrodes, Vm and pHi were monitored in oocytes exposed to a 200 µM KCl Ringer solution (plus 10 µM ouabain, 5 mM BaCl2, 10-5 M bumetanide, and 1 mM DPC), then to the same solution containing 10 mM NH4Cl or 10 mM trimethylamine-HCl. Results from this experimental series are summarized in Table 1. In alpha cbeta 1-expressing oocytes, which, as previously reported (10), had a higher pHi than control oocytes [7.71 ± 0.03 (n = 12) vs. 7.28 ± 0.02 (n = 12); P < 0.001], as in beta 1-expressing oocytes, both amines caused membrane depolarization and cell acidification. In a separate experimental series, oocyte 86Rb uptake in medium containing NH4Cl or trimethylamine-HCl was measured. As shown in Fig. 2, 10 mM trimethylamine-HCl did not affect 86Rb uptake by alpha cbeta 1-expressing oocytes, but 10 mM NH4Cl did. This suggests that neither Delta Vm nor Delta pHi could account for the NH4Cl-induced 86Rb uptake decrease. However, Table 1 reveals that, in alpha cbeta 1-expressing oocytes, 10 mM trimethylamine-HCl caused less cell acidification than did 10 mM NH4Cl (P < 0.05). The possibility that a threshold Delta pHi was required to reduce 86Rb uptake was eliminated because 5 mM NH4Cl caused cell acidification similar to that induced by 10 mM trimethylamine-HCl [Delta pHi = 0.35 ± 0.01 (n = 4) vs. 0.36 ± 0.02 (n = 3); P = 0.61] but significantly decreased 86Rb uptake (Fig. 1). The lack of effect of trimethylamine-HCl on 86Rb uptake is consistent with the noninvolvement of Delta Vm and Delta pHi in the NH4Cl-induced decrease in 86Rb uptake observed in alpha cbeta 1-expressing oocytes, and thus is consistent with a direct effect of NH4Cl on the ionic transport mediated by colonic H+-K+-ATPase.

                              
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Table 1.   Effects of NH4Cl and trimethylamine-HCl on membrane potential and intracellular pH in beta 1- and alpha cbeta 1-expressing oocytes



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Fig. 2.   Effects of 10 mM trimethylamine-HCl and 10 mM NH4Cl on 86Rb uptake by alpha cbeta 1-expressing oocytes. 86Rb uptake was measured in presence of 200 µM KCl and inhibitors of K+ transport pathways (10 µM ouabain, 5 mM barium, 10-5 M bumetanide, and 1 mM DPC). Presence of 10 mM trimethylamine-HCl (TMACl) or of NH4Cl is indicated below bars. Results are means ± SE; n = 12-14 oocytes from 2 independent experiments. NS, not significant. * P < 0.05.

NH+4 may act as a functional substrate of the colonic H+-K+-ATPase. The reduction in 86Rb uptake observed in the presence of 200 µM KCl [i.e., a concentration below the potassium Km of the colonic H+-K+-ATPase (10)] and of NH4Cl (from the 1 mM concentration) suggests that NH+4 may be either a competitive substrate for the colonic H+-K+-ATPase, or a noncompetitive inhibitor of the pump. We addressed this question with the following experiments.

In the first approach, we analyzed the rate of initial Delta pHi induced by 10 mM NH4Cl in the presence of 200 µM KCl; as discussed in DISCUSSION, the slope of pHi as a function of time (dpHi/dt) can be considered to be a reflection of the initial NH+4 influx into the oocyte. If NH4Cl inhibited the colonic H+-K+-ATPase, dpHi/dt values in both alpha cbeta 1-expressing oocytes and beta 1-expressing oocytes should be similar because they would reflect NH+4 influx via residual endogenous NH+4 pathways (uninhibited by 10 µM ouabain, 5 mM BaCl2, 10-5 M bumetanide, and 1 mM DPC). If NH+4 entered the cell via the colonic H+-K+-ATPase, dpHi/dt should be enhanced in alpha cbeta 1-expressing oocytes, reflecting NH+4 influx by residual endogenous NH+4 pathways plus NH+4 influx via the colonic H+-K+-ATPase. In beta 1-expressing oocytes, dpHi/dt measured in the presence of NH4Cl was 0.054 ± 0.002 pH units (UpH)/min (n = 9). In alpha cbeta 1-expressing oocytes, NH4Cl-induced dpHi/dt was significantly enhanced (0.097 ± 0.004 UpH/min; n = 9; P < 0.001). When alpha cbeta 1-expressing oocytes were exposed to 2 mM ouabain, NH4Cl-induced dpHi/dt was 0.051 ± 0.004 UpH/min (n = 7), similar to that observed in beta 1-expressing oocytes (P > 0.4). These results are consistent with NH+4 influx into the oocyte via the colonic H+-K+-ATPase. As a control test, it was observed that trimethylamine-HCl (10 mM) induced similar dpHi/dt values in beta 1- and in alpha cbeta 1-expressing oocytes [0.051 ± 0.002 UpH/min (n = 6) vs. 0.047 ± 0.001 UpH/min (n = 3); P = 0.1], consistent with the absence of effect of this amine 86Rb uptake.

Next, we tested the capacity of alpha cbeta 1-expressing oocytes to acidify their extracellular medium in the absence of extracellular KCl. As shown in Table 2, the lack of this extracellular substrate prevented the extracellular acidification previously reported (10). However, when 10 mM NH4Cl was added, alpha cbeta 1-expressing oocytes acidified the KCl-free medium, which is opposite to the effect observed for beta 1-expressing oocytes. This last observation excluded the possibility that NH4Cl may cause extracellular acidification by a mechanism unrelated to colonic H+-K+-ATPase function. Consistent with this conclusion was the finding that adding 2 mM ouabain to the NH4Cl-containing KCl-free medium prevented the pHo decrease caused by alpha cbeta 1-expressing oocytes. These results are consistent with NH+4 being able to replace K+ on colonic H+-K+-ATPase.

                              
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Table 2.   Effect of NH4Cl on the extracellular pH of beta 1- and alpha cbeta 1-expressing oocytes

To further support this conclusion, oocytes were incubated for 2 days in KCl-free Ringer solution (supplemented with 10 µM ouabain, 10-5 M bumetanide, 5 mM BaCl2, and 1 mM DPC) before [Na+]i was measured. The absence of K+ should lead to colonic H+-K+-ATPase inhibition. Consequently [Na+]i values should be the same in alpha cbeta 1- and beta 1-expressing oocytes, because colonic H+-K+-ATPase-mediated Na+ extrusion from cytosol should be blocked (9). [Na+]i was measured for oocytes superfused with the KCl-free solution (composition as above) or with a similar solution containing 1 or 10 mM NH4Cl. Results are summarized in Fig. 3. In the absence of NH4Cl, [Na+]i values were indeed similar in beta 1-expressing and in alpha cbeta 1-expressing oocytes. The presence of NH4Cl in the superfusate did not alter [Na+]i in beta 1-expressing oocytes, whereas it significantly decreased [Na+]i in alpha cbeta 1-expressing oocytes, except when 2 mM ouabain was added. This confirms that NH+4 can replace K+ on the colonic H+-K+-ATPase. Figure 3, inset, shows that, with a 1 or 10 mM concentration of external KCl, [Na+]i is slightly lower in alpha cbeta 1-expressing oocytes than with 1 or 10 mM NH4Cl, a finding that is consistent with a higher pump affinity for K+ than for NH+4.


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Fig. 3.   Effect of extracellular NH4Cl on intracellular Na+ activity ([Na+]i) in beta 1- and alpha cbeta 1-expressing oocytes. Intracellular Na+-selective microelectrodes were used to measure [Na+]i in beta 1-expressing (solid bars) and in alpha cbeta 1-expressing (open bars) oocytes. Oocytes were bathed by a K+-free medium (with 10 µM ouabain, 5 mM barium, 10-5 M bumetanide, and 1 mM DPC), sometimes supplemented with 1 or 10 mM NH4Cl, as indicated below bars. Hatched bar, [Na+]i in alpha cbeta 1-expressing oocytes exposed to 10 mM NH4Cl-2 mM ouabain. Results are means ± SE; n = 3-8 oocytes from 3 independent experiments. Significance of results is indicated with respect to [Na+]i measured in beta 1-expressing oocytes. NS, not significant. * P < 0.05. Inset: to compare effects of extracellular NH4Cl and extracellular KCl (1 or 10 mM, as indicated below bars) on [Na+]i, [Na+]i was also measured in oocytes bathed by a NH4Cl-free medium (in presence of 10 µM ouabain, 5 mM barium, 10-5 M bumetanide, and 1 mM DPC), sometimes supplemented with 1 or 10 mM KCl. Results are means ± SE; n = 3-6 oocytes. Significance of results is indicated with respect to [Na+]i measured in beta 1-expressing oocytes. * P < 0.05.

Apparent affinity of the colonic H+-K+-ATPase for NH+4. Lastly, we estimated the apparent affinity constant of the colonic H+-K+-ATPase for NH+4. The uptake of 86Rb by alpha cbeta 1-expressing oocytes was measured in the presence of 0.2, 1, or 5 mM KCl, with or without NH4Cl (1, 5, or 10 mM) in the medium. Results from one representative experiment are shown in Fig. 4. The double-reciprocal plot of velocity vs. K+ concentration (1/V vs. 1/[K+]) in the presence of different NH+4 concentrations ([NH+4]) suggested that maximal velocity (Vmax) is independent of [NH+4] (Fig. 4A), consistent with competitive inhibition between NH+4 and K+. A replot of the slopes of the reciprocal plot vs. [NH+4] (Fig. 4B) gave a straight line, consistent with pure competitive inhibition (30), and was used to determine the apparent inhibition constant for NH+4 (Ki). From this experiment and three others, Ki was found to be 6.3 ± 0.8 mM.


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Fig. 4.   Representative experiment of kinetics of 86Rb uptake by alpha cbeta 1-expressing oocytes in presence of increasing NH+4 concentrations. A: plot of 1/V vs. 1/[K+] (V = velocity; [K+] = K+ concn = 0.2, 1, and 5 mM) in absence of NH+4 (open circle ) or in presence of 1 , 5 , or 10 mM NH+4 (). Results are means from 5-8 oocytes. Km for K+ is 666 µM in absence of NH+4 and increases with increasing [NH+4], whereas maximal velocity (Vmax) is unchanged. B: plot of slopes of reciprocal plots (shown in A) against [NH4Cl], fitted by linear regression (r = 0.987). Intercept on abscissa gives -Ki = -7.66 mM where Ki is inhibitor constant.


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It is well known that NH+4 may substitute for K+ in numerous membrane transport systems of various cells, because of the similarity between NH+4 and K+ hydrated radii (21). Abundant literature concerns NH+4 transport by the renal Na+-K+-2Cl- cotransporter and Na+-K+-ATPase (for reviews, see Refs. 16, 21, and 32). However, very few studies have been devoted to possible NH+4 transport by the renal H+-K+-ATPases. It was reported that NH+4 stimulates Sch-28080-sensitive and ouabain-insensitive ATP hydrolysis in the rat outer medullary collecting duct (OMDC) (15). In that study, NH+4 transport was not specifically demonstrated, but the pharmacological profile of NH+4-supported ATP hydrolysis was consistent with that of the gastric H+-K+-ATPase (24) and not with that of the colonic H+-K+-ATPase [which is Sch-28080 insensitive and ouabain sensitive (10)]. The colon, from which colonic H+-K+-ATPase was cloned (11), is the major site of NH+4 reabsorption, but, to our knowledge, the involvement of this pump in this transport has not been investigated. Therefore, to determine whether the colonic H+-K+-ATPase could mediate NH+4 transport, we studied the effects of NH4Cl on 86Rb uptake, on pHi, and on [Na+]i after functional expression in Xenopus oocytes of the renal alpha cbeta association, i.e., the catalytic alpha c subunit together with the beta 1 subunit of the Na+-K+-ATPase (7). Our results are consistent with NH+4 transport mediated by the colonic H+-K+-ATPase.

In our study, difficulties arising from the presence of endogenous NH+4 pathways were circumvented 1) by performing the experiments in the presence of inhibitors of the NH+4 transport systems so far identified in the oocyte (8), 2) by using beta 1-expressing oocytes as controls, and 3) by inhibiting the colonic H+-K+-ATPase with millimolar concentrations of ouabain (10). We conclude that the significant 86Rb uptake decrease by alpha cbeta 1-expressing oocytes caused by NH4Cl (Fig. 1A) did not reflect an effect of NH4Cl on the ionic transport mediated by endogenous K+ pathways and that alpha cbeta 1 expression did not induce the overexpression or activation of endogenous K+- and NH+4-stimulated transport systems because 1) in the presence of NH4Cl, beta 1-expressing oocytes did not show a change in 86Rb uptake (Fig. 1B) and 2) in the presence of 2 mM ouabain, the 86Rb uptake by alpha cbeta 1-expressing oocytes was unaffected by NH4Cl (Fig. 1A, inset). Finally, a major effect of Delta Vm and Delta pHi on the function of the expressed colonic H+-K+-ATPase was ruled out because trimethylamine-HCl did not alter 86Rb uptake; this lack of effect also indicates that amines have no general effect on the pump. Taken together, these results are consistent with a direct effect of NH+4 on the colonic H+-K+-ATPase.

Results of functional experiments performed in the presence of a low K+ concentration or in K+-free medium are consistent with the acceptance of NH+4 as a substrate for the colonic H+-K+-ATPase. The pHo decrease, observed with no external K+ and with NH+4, is consistent with H+ extrusion by the colonic H+-K+-ATPase: of the excreted protons, some combined with NH3 but enough remained free to decrease the droplet pH. The pHo experimental series indicated that NH+4 may substitute for K+ and also that expression of alpha cbeta 1 subunits leads to an NH+4/H+ exchange in the absence of external K+. The decrease in [Na+]i under K+-free conditions [with endogenous Na+-K+-ATPase inhibited by micromolar concentrations of ouabain (6)] suggests that the colonic H+-K+-ATPase can also extrude Na+, as it does in the presence of external K+ (9). We therefore conclude that the exchange of Na+ and H+ with either NH+4 or K+ is mediated by the colonic H+-K+-ATPase. The initial rate of change of pHi points to a colonic H+-K+-ATPase-mediated NH+4 influx. Because of the intracellular buffering power, omega , the proton-equivalent flux is only partly reflected by dpHi/dt. Nonetheless, even if omega  were lower in alpha cbeta 1-expressing oocytes than in control oocytes (because of a high pHi), several observations support the view that enhancement of dpHi/dt is a reliable index of an increased NH+4 flux. First, in the presence of 2 mM ouabain, the NH4Cl-induced dpHi/dt in alpha cbeta 1-expressing oocytes is similar to that in control oocytes, despite their different pHi values (7.28 ± 0.03, n = 3, vs. 7.66 ± 0.004, n = 7). Second, trimethylamine-HCl induced similar dpHi/dt values in alpha cbeta 1-expressing oocytes and in control oocytes (here again, pHi values were different). Third, this compound induced a significantly lower dpHi/dt than did NH4Cl in alpha cbeta 1-expressing oocytes (here, pHi values were the same). Thus these results support the interpretation that enhanced NH4Cl-induced dpHi/dt reflected an increased NH4Cl influx, unless the H+ extrusion rate was lower in alpha cbeta 1-expressing oocytes than in control oocytes. The latter possibility is unlikely, because the differences between the inward Na+ chemical gradients and the outward H+ chemical gradients (thus the driving force for Na+/H+ exchange) in alpha cbeta 1- and beta 1-expressing oocytes (47 vs. 42 mV; calculated from [Na+]i and pHi measurements) are not different; moreover, functional expression of H+-K+-ATPase would, if anything, enhance H+ extrusion. Taken together, these results therefore support the conclusion that NH+4 is transported into the cell by the colonic H+-K+-ATPase at a higher rate than that at which protons are extruded, resulting in dpHi/dt enhancement. The simplest explanation for a proton-equivalent influx overwhelming the proton efflux is the concomitant Na+ efflux by the colonic H+-K+-ATPase (9). It could also be that the colonic H+-K+-ATPase is also electrogenic, carrying net positive charge(s) into the cell: this condition would also lead to a proton-equivalent influx exceeding the proton efflux.

In agreement with functional studies, kinetic data presented in Fig. 4 indicate that NH+4 acts as a competitive inhibitor of K+. The apparent Ki for NH+4 was estimated to be ~6.5 mM. This value appears to be higher than the Km for K+, which is below 1 mM (Ref. 10 and the present study; see Fig. 4A), but both values were obtained by measuring the uptake of 86Rb, which is a K+ surrogate. As previously indicated, NH+4 was reported to stimulate an Sch-28080-sensitive K+-ATPase activity in rat OMDC (15) with a rather high affinity constant, 2 mM. However, from that study and the present one, it cannot be concluded that the gastric H+-K+-ATPase has a higher affinity for NH+4 than the colonic H+-K+-ATPase, because the two studies use different techniques and different cell systems. By measuring 86Rb fluxes mediated by a ouabain-sensitive K+ transport system in inner medullary cells of the rat, an apparent Ki for NH+4 of 11 mM was reported (33). In that study, it was proposed that the ouabain-sensitive system that mediates NH+4 entry into the cell was the Na+-K+-ATPase, NH+4 being transported from the interstitium into the cytosol. Whereas colonic H+-K+-ATPase was considered to be mostly expressed in OMDC (23, 29), a recent study reported its presence in the inner medullary collecting duct (IMDC) (26). Thus whether the ouabain-sensitive, NH+4-transporting system reported by Wall and Koger (33) represents only the Na+-K+-ATPase or might also represent the colonic H+-K+-ATPase is not yet clear.

Our results shed light on the possible transport of NH+4 by the colonic H+-K+-ATPase in the medullary collecting duct and in the colon. In the medullary collecting duct, luminal NH+4 is secreted mainly by means of H+ secretion in parallel with NH3 diffusion (see Ref. 13 for a review). The apical location of the colonic H+-K+-ATPase is supported by immunolocalization (using polyclonal antibodies) in OMCD (29) and by a recent functional study of IMDC (26). Apical reabsorption of NH+4 in place of K+ by the colonic H+-K+-ATPase would imply that this pump mediates NH+4 transport against a net NH+4 secretion. In the thick ascending limb of Henle's loop, in which net NH+4 reabsorption occurs, a negative feedback regulation of NH+4 absorption was proposed to account for the apical Na+/H+ (NH+4) exchanger (3). By analogy, it could be proposed that the entry of NH+4 into the cell from the lumen via an apically located colonic H+-K+-ATPase may be a feedback mechanism of net NH+4 excretion during hypokalemia, a condition that induces colonic H+-K+-ATPase overexpression (1, 12, 18, 23, 26). Hypokalemia increases NH+4 production by the proximal tubule and increases NH+4 reabsorption in the thick ascending limb of Henle's loop, finally resulting in increased urinary excretion of NH+4 (21). Thus hypokalemia-induced colonic H+-K+/NH+4-ATPase expression may limit metabolic alkalosis, which is partly the result of increased NH+4 excretion.

In the colon from which it was cloned (11), the colonic H+-K+-ATPase is localized on the luminal side (29). The lumen of the colon is bathed by a very high ammonia (NH3 plus NH+4) concentration; the small part absorbed accounts for almost all circulating ammonia. The mechanisms of ammonia absorption by the colon are of pathophysiological interest (because of the deleterious effects of a high ammonia concentration in blood), but have not been clearly identified (27). It was recently reported that the luminal side of colonic crypts acts as a permeation barrier for either NH3 or NH+4, a finding which is consistent with NH+4 absorption via the paracellular pathway or via cells other than the crypt cells (31); as a matter of fact, it was previously concluded that the apical membrane of surface cells of rat colon is endowed with large NH+4 permeation (22). Because the colonic H+-K+-ATPase is present in surface cells but not in crypt cells (18), we suggest that further work should focus on the involvement of colonic H+-K+-ATPase in colonic NH+4 absorption.

In conclusion, our findings indicate that NH+4 competes with K+ for an external site of colonic H+-K+-ATPase, when it is expressed in Xenopus oocytes. The transport of NH+4 by colonic H+-K+-ATPase in various cells and its physiological relevance remain to be established, and the ionic transport mode(s) of this cationic pump has to be further determined.


    ACKNOWLEDGEMENTS

We thank M. S. Crowson and G. E. Shull for donating the full-length cDNA coding for the alpha -subunit of the colonic P-ATPase and J. B. Lingrel for kindly providing the rat Na+-K+-ATPase beta 1-subunit cDNA. We thank T. Anagnostopoulos for continuous encouragement throughout this study, S. R. Thomas for helpful comments, and P. Hulin for technical assistance.


    FOOTNOTES

M. Cougnon was supported by a fellowship from the Fondation pour la Recherche Médicale, and P. Bouyer was supported by a fellowship from the Association pour la Recherche contre le Cancer.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. Planelles, Institut National de la Santé et de la Recherche Médicale, U. 467, Faculté de Médecine Necker, Université Paris V, F-75015 Paris, France (E-mail: planelle{at}necker.fr).

Received 1 February 1999; accepted in final form 18 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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