1 Faculté de
Médecine Necker, Functional expression of the rat colonic
H+-K+-ATPase
was obtained by coexpressing its catalytic
86Rb influx; intracellular
sodium and pH measurements
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 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+
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.
cRNA synthesis and expression in Xenopus laevis oocytes.
cRNAs of the rat
Na+-K+-ATPase
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
-subunit and the
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
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
-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).
-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.
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
1-subunit (34) and of the
catalytic
-subunit of the colonic
H+-K+-ATPase,
c (11), were synthesized with
the SP6 RNA polymerase (Promega).
1-subunit cRNAs to be used as
control oocytes or were coinjected with 2 ng of
1 cRNAs plus 10 ng of
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.
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, 10pHo measurements.
pHo was measured as previously
described (10, 19). Briefly, individual oocytes
(1- or
c
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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RESULTS |
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Effect of NH+4 on
86Rb uptake.
In this experimental series, 86Rb
uptake was measured in 1- and
in
c
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
1 alone (Fig.
1, A and
B). The addition of 1-85 mM
NH4Cl to assay solutions
significantly reduced 86Rb uptake
by
c
1-expressing
oocytes (Fig. 1A).
NH4Cl did not affect
86Rb uptake by
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.
|
|
|
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
|
|
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
c
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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DISCUSSION |
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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
c
association, i.e., the catalytic
c subunit together with the
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
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
c
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
c
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,
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
c
1-expressing
oocytes was unaffected by NH4Cl
(Fig. 1A,
inset). Finally, a major effect of
Vm and
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
c
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,
, the proton-equivalent flux is only partly reflected by
dpHi/dt.
Nonetheless, even if
were lower in
c
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
c
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
c
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
c
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
c
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
c
1-
and
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.
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ACKNOWLEDGEMENTS |
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We thank M. S. Crowson and G. E. Shull for donating the full-length
cDNA coding for the -subunit of the colonic P-ATPase and J. B. Lingrel for kindly providing the rat
Na+-K+-ATPase
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.
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FOOTNOTES |
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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.
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