1 Department of Internal Medicine, University of Tübingen, D-72076 Tübingen, Germany; 2 In Situ Hybridization Core Facility, Beth Israel Deaconess Medical Center, Boston, MA 02115; 3 Department of Physiology, University of Cambridge, Cambridge CB2 3EG; and 4 Cardiff School of Biosciences, Cardiff CF10 3U5, United Kingdom
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ABSTRACT |
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Basolateral
Na+-HCO/
crypts (in which no forskolin-induced
cell shrinkage occurs). We found 30% reduced
Na+-HCO
/
compared with CFTR +/+ crypts but similar
Na+-HCO
bicarbonate secretion; colon; ion transport; chloride secretion; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
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INTRODUCTION |
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ALL SEGMENTS OF
THE INTESTINAL tract secrete HCO
After the cloning of the renal
Na+-HCO/HCO
In the meantime, we and others found that two NBC1 subtypes with
different NH2-terminal ends exist. In the kidney, only the renal NBC1 (kNBC1) is expressed, whereas the intestinal/pancreatic subtype (pNBC1) is expressed more widely (2, 18). In the mouse colon, small amounts of kNBC1 are expressed in addition to pNBC1
(32). Interestingly, the cytoplasmic NH2
terminus of the pNBC1 subtype contains a highly conserved PKA consensus
site, which is lacking in the kNBC1 subtype, (2, 18). Thus
the structural basis for a direct cAMP-dependent regulation of
transport activity exists. However, whether the observed effect of cAMP on Na+-HCO
In intact intestinal epithelium, basolateral
Na+-HCO/
mice, which display no
cell shrinkage indicative of secretion (6), and in normal
crypts in the presence of K+-channel inhibition, which also
inhibits anion secretion (22). The results suggest that
cAMP activates colonic NBC independent of cAMP-induced changes in
intracellular pH (pHi), anion secretion, or cell volume.
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MATERIALS AND METHODS |
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Materials. S0859, an NBC1 inhibitor, was from Aventis (Frankfurt, Germany). This substance inhibits the heart/pancreatic NBC1 (at low sodium concentration) with an IC50 of 5.9 µM and does not affect NBC2/3 when expressed in Chinese hamster ovary cells (Willi Jansen, Aventis laboratories, unpublished observations). BSA (cell culture grade) was obtained from Paesel und Lorei (Frankfurt, Germany) and BCECF was obtained from Molecular Probes (Leiden, the Netherlands). All other chemicals were either obtained from Sigma (Deisenhofen, Germany) or from Merck (Darmstadt, Germany) at tissue culture grade or the highest grade available.
Animal breeding and genotyping.
For the experiments with control crypts, we used the CFTR +/+ mice that
were generated during the breeding of a CFTR-deficient mice strain. Due
to the increased postnatal death rate of the CFTR /
mice, the CFTR
+/+ littermates are much more abundant. For the experiments in which
CFTR
/
and +/+ crypts were compared, the crypts were derived from
littermates. The cystic fibrosis null mice (CFTRtm1Cam) had
been established in the laboratories of R. Ratcliff, W. H. Colledge, and M. Evans and previously characterized (27). They were raised in the animal facility of the Department of
Biochemistry in Tübingen and genotyped as previously described
(33). The protocol for the animal study was approved by
the local authorities for animal welfare (university veterinarian and
Regierungspräsidium Tübingen).
Preparation of colonic crypts.
After CO2 narcosis and cervical dislocation, a 3-cm segment
of proximal colon was excised, washed through with ice-cold buffer A
gassed with 100% O2 (in mM: 120 Na+, 7 K+, 1.2 Mg2+, 1.2 Ca2+, 120 Cl, 14 HEPES, 7 Tris, 3 H2PO
, 1 Mg2+, 5 pyruvate, 10 HEPES, 5 EDTA, 1% BSA, pH 7.4). The
segment was placed into a tube filled with solution B and vibrated at
10 Hz at room temperature. At defined intervals, the solution was
replaced and epithelial fragments were harvested by low-speed
centrifugation. Early fractions contained surface cells, later
fractions contained crypts, both easily identified under a microscope,
and their viability was tested by trypan blue exclusion.
In situ hybridization of NBC1 in murine proximal colon. Nonradioactive in situ hybridization was performed as described (31), using two digoxigenin (DIG)-labeled cRNA probes that comprised nucleotides 143-2113 and 2234-3495 of rkNBC. Frozen sections (10 µm) were cut in a cryostat and captured onto Superfrost plus microscope slides (Fisher Scientific, Pittsburgh, PA). Sections were fixed and acetylated and hybridized at 68°C over three nights to the NBC probes (approximate concentration 100 ng/ml). Hybridized probe was visualized using alkaline phosphatase-conjugated anti-DIG Fab fragments (Roche, Indianapolis, IN) and 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium substrate (Kierkegard and Perry Laboratories, Gaithersburg, MD). Sections were rinsed several times in 100 mM Tris, 150 mM NaCl, 20 mM EDTA, pH 9.5, and placed on a coverslip with glycerol gelatin (Sigma, St. Louis, MO). Control sections were incubated in an identical concentration of the sense probe transcript.
Cloning of murine NBCn1 cDNA fragments.
RNA was isolated from mouse tissue, and first-strand cDNA was
synthesized as described previously (30). One described
forward primer [5'-GGAACAGTCATGCTGGATA-3' (24)] and two
heterologous reverse primers (5'-TGTTAGAGGTTGCCCAGCAAAC-3',
5'-AGGAGACATACAGGCACAGTATAGG-3', deduced from published sequence
information) were chosen for cloning NBCn1 cDNA fragments from murine
colon. Amplification by touchdown PCR, cloning of a 1104- and a 991-bp
PCR product, and sequencing was performed as described
(30). Both PCR products were ~350 nucleotides smaller
than expected, and sequencing revealed the lack of a 369-bp fragment
(compared with human and rat sequence). A homologous primer pair
(forward primer: 5'-AAGGGAGAACGTCAGAGAAGC-3', reverse primer:
5'-GATTACAGGAGACATACAGGC ACA-3') was deduced from the mouse sequence.
PCR resulted in one major band at the expected size of 890 bp. Its
molecular identity (NBCn1) was confirmed by restriction digest. A
weaker band at 1295 bp, which was most abundant in aorta, was
characterized by cloning and sequencing and represented NBCn1,
including the mentioned 368-bp fragment, confirming the existence of
two splice variants, of which only one was expressed in any significant
amount in the intestine (Fig. 1).
|
Semiquantitative RT-PCR. Homologous primers for murine NBC1 and NBCn1 were deduced from obtained (NBCn1, see Cloning of murine NBCn1 cDNA fragments) or published sequence information (NBC1: forward primer 5'-TAAACCAGAGAAGGACCAGC-3', reverse primer 5'-CGTCAGACATCAAGGTGGC-3'). An 18s rRNA fragment was amplified as an internal control with the use of primers supplied by Ambion (Austin, TX). Semiquantitative RT-PCR was performed as described previously (3, 30), only using different annealing temperatures (52°C for NBC1, 58°C for NBCn1, and 58°C for 18s rRNA). The relationship of the studied gene vs. 18s rRNA was calculated and regarded as the relative expression level.
pHi measurements.
Fluorescence microscopy for determination of pHi was
performed as described (5, 30), similar to previously
applied techniques (1, 20). After crypts were loaded for
30 min at room temperature with 5 µM BCECF in buffer A, crypts were
fixed between a glass coverslip and a 0.3-µm polycarbonate membrane
(Osmonics, Minnetonka, MN) on the stage of an inverted microscope
(Nikon Diaphot TMD, Nikon, Düsseldorf, Germany) and superfused
following the appropriate protocol with buffer A, buffer B (40 mM NaCl
replaced by NH4Cl), and buffer C [containing 120 mM
tetramethyl ammonium chloride (TMA) instead of NaCl]. For
CO2/HCO
Determination of the intrinsic intracellular buffering power.
Determination of the intracellular buffering power (i)
was performed as described by Boyarsky et al. (8) using
increasing or decreasing concentrations of NH4Cl
(0.5-80 mM TMA Cl replaced by NH4Cl in buffer C) and
measuring the resultant pHi changes.
i was
calculated over a wide pHi range (pH 6.6-7.8),
resulting in buffering curves that were not different between CFTR +/+
and CFTR
/
crypts (Fig. 2).
|
Statistics and calculation of proton fluxes.
Results are given as means ± SE. Proton fluxes were calculated by
multiplying the initial steep pHi slope after readdition of
sodium or removal of NH4Cl, respectively, which was
determined by regression analysis, with the total buffering capacity at
the initial pHi, (5) including the intrinsic
i and, in addition, the CO2-dependent
buffering capacity for
CO2/HCO
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RESULTS |
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In situ hybridization of NBC1 in mouse proximal colon.
We had previously observed rather high NBC1 expression levels in rabbit
proximal colon (18) and wondered whether colonic crypts,
in which the assessment of Na+-HCO
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NBC1 and NBCn1 expression levels in the murine intestine.
To have an estimate of the relative
Na+-HCO cotransporter
NKCC1 (not shown), whereas the expression of NBCn1 in the whole
proximal colon was higher than in crypts, suggesting that this isoform
is predominantly expressed in subepithelial layers, possibly muscular tissue.
Assessment of
Na+-HCO
|
Effect of forskolin of
Na+-HCO/
mice.
One possible mechanism for an indirect activation of
Na+-HCO
/
mice do not show forskolin-induced shrinkage (6, 37) and were
therefore used to address this question.
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Effect of K+-channel inhibition on
Na+-HCO
|
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DISCUSSION |
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This study investigates colonic
Na+-HCO
Apparent Na+-HCO secretion has
been obtained (19), suggesting an inward flux of
Na+/HCO
The first cloning of an Na+-HCO
These findings raised two interesting questions. First, is the
intestinal/pancreatic NBC1 stimulated by a rise in intracellular cAMP,
as suggested by indirect evidence? Second, could it be that the
difference in the NH2 terminus between the two NBC1
subtypes, far away from the actual membrane-spanning part of the
molecule, was the reason for the presumably different coupling ratios
between Na+ and HCO
Functional data demonstrate an inhibition of
Na+-HCO and HCO
We, therefore, searched for a model in which we could investigate
whether an increase in intracellular cAMP stimulates the intestinal/pancreatic NBC1 subtype in a native cellular system and
whether this stimulation was secondary to cAMP-stimulated secretion or
a direct effect on the cotransporter(s). Isolated colonic crypts are a
model in which vectorial anion secretion is still intact, as
demonstrated by marked cAMP-induced shrinkage and loss of intracellular
Cl after forskolin stimulation (6). They
also allow direct measurement of acid-activated
Na+-HCO
/
mice, which exhibit no electrogenic anion secretory
response in the colon. NBC1 in situ hybridization demonstrated that
NBC1 expression was located almost exclusively in the cryptal region,
which made isolated colonic crypts an ideal model to study the above
questions. Colonic NBCn1 expression levels were approximately one-third
those for NBC1 in total colonic tissue, but although we observed much higher NBC1 expression levels in colonic crypts than total colon, NBCn1
levels were markedly lower in crypts than total colon. This indicates
that NBCn1 is most likely predominantly nonepithelial in the colon. We
know from previous experiments that expression of NBC3
(Cl
-dependent NBC) is very low throughout the intestinal
tract (18) and that although some kNBC1 is expressed in
the colon, the pNBC1 subtype is expressed at far higher levels
(32).
Forskolin increased the acid-activated
Na+-HCO via a basolateral AE (19). Associated
pHi shifts, if they occur at all, may be so local that
single-cell microfluorometry does not pick them up.
However, colonic crypts may shrink during agonist-activated secretion,
and it is feasible that this shrinkage, rather than the increase in
cAMP itself, increases Na+-HCO/
crypts. Interestingly, activation by forskolin was by 54% and,
therefore, similar than in normal littermates. These finding
demonstrate that Na+/HCO
Thus, in the absence of CFTR expression,
Na+-HCO
We next performed experiments with the chromanol HOE-293b. This
substance inhibits a certain type of K+ channels, and
Bleich and Warth (7) recently demonstrated that the
inhibition of these KCNQ channels virtually abolishes rat colonic
electrogenic anion secretion. We have tested the effect of HOE-293b on
the forskolin-induced Isc response in murine
proximal colon, and we also found a strong reduction (albeit no
abolition) of the secretory response by 100 µM HOE-293b. The
rationale of measuring the effect of forskolin on
Na+-HCO/
crypts. Second, and more importantly, the prevention of
forskolin-induced hyperpolarization of the basolateral membrane
potential (via KCNQ-channel activation) allows insight into the
electrogenicity of the Na+-HCO
Gross et al. (12, 13) found a coupling ratio for
Na+ to HCO
It would be highly interesting to study the stoichiometriy of the pNBC1
and a potential stimulation-associated change. Unfortunately, our
experimental model is not suitable to definitely determine the coupling
ratio for Na+ and HCO
In summary, this study demonstrates that the intestinal/pancreatic
subtype of NBC1 is strongly expressed in the colon and located
predominantly in the crypt region. NBCn1 is also expressed, with far
lower expression levels. A rise in intracellular cAMP increases
acid-activated Na+-HCO
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ACKNOWLEDGEMENTS |
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We thank C. Neff for expert technical assistance; Drs. A. Weichert and W. Kleemann from Aventis Pharma, Frankfurt, for kindly providing the S0859 compound; Prof. D. Mecke for the use of the animal facilities in the Dept. of Biochemistry of Tübingen University; and Prof. Dr. Michael Sessler, Dept. of Surgery of the Univ. of Tübingen, for constructive criticism.
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FOOTNOTES |
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This work was supported by Deutsche Forschungsgemeinschaft Grants Se-460/13-1 and Se-460/13-2, by the Mukoviszidose e.V., and by a grant from the Interdisziplinäres Zentrum für Klinische Forschung Tübingen (Project IIIC1).
Address for reprint requests and other correspondence: U. Seidler, Dept. of Gastroenterology and Hepatology, Hannover Medical School, Carl-Neuberg-Str. 1, 30623 Hannover, Germany (E-mail: seidler.ursula{at}mh-hannover.de).
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. Section 1734 solely to indicate this fact.
September 18, 2002;10.1152/ajpgi.00209.2002
Received 3 June 2002; accepted in final form 10 September 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abrahamse, SL,
Vis A,
Bindels RJ,
and
van Os CH.
Regulation of intracellular pH in crypt cells from rabbit distal colon.
Am J Physiol Gastrointest Liver Physiol
267:
G409-G415,
1994
2.
Abuladze, N,
Lee I,
Newman D,
Hwang J,
Boorer K,
Pushkin A,
and
Kurtz I.
Molecular cloning, chromosomal localization, tissue distribution, and functional expression of the human pancreatic sodium bicarbonate cotransporter.
J Biol Chem
273:
17689-17695,
1998
3.
Alper, SL,
Rossmann H,
Wilhelm S,
Stuart-Tilley AK,
Shmukler BE,
and
Seidler U.
Expression of AE2 anion exchanger in mouse intestine.
Am J Physiol Gastrointest Liver Physiol
277:
G321-G332,
1999
4.
Amlal, H,
Burnham CE,
and
Soleimani M.
Characterization of Na+/HCO
5.
Bachmann, O,
Sonnentag T,
Siegel WK,
Lamprecht G,
Weichert A,
Gregor M,
and
Seidler U.
Different acid secretagogues activate different Na+/H+ exchanger isoforms in rabbit parietal cells.
Am J Physiol Gastrointest Liver Physiol
275:
G1085-G1093,
1998
6.
Bachmann, O,
Wüchner K,
Rossmann H,
Leipziger J,
Gregor M,
and
Seidler U.
cAMP-mediated activation of NBC (Na+/HCO cotransporter) in murine colonic crypts (Abstract).
Gastroenterology
120:
A-701,
2002.
7.
Bleich, M,
and
Warth R.
The very small-conductance K+ channel KvLQT1 and epithelial function.
Pflügers Arch
440:
202-206,
2000[ISI][Medline].
8.
Boyarsky, G,
Ganz MB,
Sterzel RB,
and
Boron WF.
pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO
9.
Choi, I,
Romero MF,
Khandoudi N,
Bril A,
and
Boron WF.
Cloning and characterization of a human electrogenic Na+-HCO
10.
Curci, S,
Debellis L,
and
Fromter E.
Evidence for rheogenic sodium bicarbonate cotransport in the basolateral membrane of oxyntic cells of frog gastric fundus.
Pflügers Arch
408:
497-504,
1987[ISI][Medline].
11.
Goddard, PJ,
Takahashi S,
Milbank AJ,
Silen W,
and
Soybel DI.
HCO
12.
Gross, E,
Abuladze N,
Pushkin A,
Kurtz I,
and
Cotton CU.
The stoichiometry of the electrogenic sodium bicarbonate cotransporter pNBC1 in mouse pancreatic duct cells is 2 HCO
13.
Gross, E,
Hawkins K,
Pushkin A,
Sassani P,
Dukkipati R,
Abuladze N,
Hopfer U,
and
Kurtz I.
Phosphorylation of Ser(982) in the sodium bicarbonate cotransporter kNBC1 shifts the HCO
14.
Isenberg, JI,
Ljungstrom M,
Safsten B,
and
Flemstrom G.
Proximal duodenal enterocyte transport: evidence for Na+-H+ and Cl-HCO
15.
Ishibashi, K,
Sasaki S,
and
Marumo F.
Molecular cloning of a new sodium bicarbonate cotransporter cDNA from human retina.
Biochem Biophys Res Commun
246:
535-538,
1998[ISI][Medline].
16.
Ishiguro, H,
Naruse S,
Steward MC,
Kitagawa M,
Ko SB,
Hayakawa T,
and
Case RM.
Fluid secretion in interlobular ducts isolated from guinea-pig pancreas.
J Physiol
511:
407-422,
1998
17.
Ishiguro, H,
Steward MC,
Wilson RW,
and
Case RM.
Bicarbonate secretion in interlobular ducts from guinea-pig pancreas.
J Physiol
495:
179-191,
1996[Abstract].
18.
Jacob, P,
Christiani S,
Rossmann H,
Lamprecht G,
Vieillard-Baron D,
Muller R,
Gregor M,
and
Seidler U.
Role of Na+/HCO
19.
Jacob, P,
Rossmann H,
Neff C,
Weichert A,
Gregor M,
and
Seidler U.
Multiple transport pathways are involved in cellular Cl uptake during colonic anion secretion (Abstract).
Gastroenterology
120:
A-699,
2002.
20.
Kaunitz, JD.
Preparation and characterization of viable epithelial cells from rabbit distal colon.
Am J Physiol Gastrointest Liver Physiol
254:
G502-G512,
1988
21.
Kurashima, K,
Yu FH,
Cabado AG,
Szabo EZ,
Grinstein S,
and
Orlowski J.
Identification of sites required for down-regulation of Na+/H+ exchanger NHE3 activity by cAMP-dependent protein kinase. Phosphorylation-dependent and -independent mechanisms.
J Biol Chem
272:
28672-28679,
1997
22.
Lohrmann, E,
Burhoff I,
Nitschke RB,
Lang HJ,
Mania D,
Englert HC,
Hropot M,
Warth R,
Rohm W,
and
Bleich M.
A new class of inhibitors of cAMP-mediated Cl secretion in rabbit colon, acting by the reduction of cAMP-activated K+ conductance.
Pflügers Arch
429:
517-530,
1995[ISI][Medline].
23.
Müller-Berger, S,
Ducoudret O,
Diakov A,
and
Frömter E.
The renal Na+-HCO
24.
Praetorius, J,
Hager H,
Nielsen S,
Aalkjaer C,
Friis UG,
Ainsworth MA,
and
Johansen T.
Molecular and functional evidence for electrogenic and electroneutral Na+-HCO
25.
Pushkin, A,
Abuladze N,
Newman D,
Lee I,
Xu G,
and
Kurtz I.
Cloning, characterization and chromosomal assignment of NBC4, a new member of the sodium bicarbonate cotransporter family.
Biochim Biophys Acta
1493:
215-218,
2000[ISI][Medline].
26.
Rajendran, VM,
Oesterlin M,
and
Binder HJ.
Sodium uptake across basolateral membrane of rat distal colon. Evidence for Na-H exchange and Na-anion cotransport.
J Clin Invest
88:
1379-1385,
1991[ISI][Medline].
27.
Ratcliff, R,
Evans MJ,
Cuthbert AW,
MacVinish LJ,
Foster D,
Anderson JR,
and
Colledge WH.
Production of a severe cystic fibrosis mutation in mice by gene targeting.
Nat Genet
4:
35-41,
1993[ISI][Medline].
28.
Romero, MF,
Fong P,
Berger UV,
Hediger MA,
and
Boron WF.
Cloning and functional expression of rNBC, an electrogenic Na+-HCO
29.
Romero, MF,
Hediger MA,
Boulpaep EL,
and
Boron WF.
Expression cloning and characterization of a renal electrogenic Na+/HCO
30.
Rossmann, H,
Bachmann O,
Vieillard-Baron D,
Gregor M,
and
Seidler U.
Na+/HCO
31.
Schmitt, BM,
Berger UV,
Douglas RM,
Bevensee MO,
Hediger MA,
Haddad GG,
and
Boron WF.
Na/HCO3 cotransporters in rat brain: expression in glia, neurons, and choroid plexus.
J Neurosci
20:
6839-6848,
2000
32.
Seidler, U,
Bachmann O,
Jacob P,
Christiani S,
Blumenstein I,
and
Rossmann H.
Na+/HCO
33.
Seidler, U,
Blumenstein I,
Kretz A,
Viellard-Baron D,
Rossmann H,
Colledge WH,
Evans M,
Ratcliff R,
and
Gregor M.
A functional CFTR protein is required for mouse intestinal cAMP-, cGMP- and Ca2+-dependent.
J Physiol
505:
411-423,
1997[Abstract].
34.
Seidler, U,
Rossmann H,
Jacob P,
Bachmann O,
Christiani S,
Lamprecht G,
and
Gregor M.
Expression and function of Na+/HCO
35.
Shumaker, H,
Amlal H,
Frizzell R,
Ulrich CD,
and
Soleimani M.
CFTR drives Na+/HCO
36.
Townsley, MC,
and
Machen TE.
Na+/HCO
37.
Valverde, MA,
O'Brien JA,
Sepulveda FV,
Ratcliff R,
Evans MJ,
and
Colledge WH.
Inactivation of the murine cftr gene abolishes cAMP-mediated but not Ca2+-mediated secretagogue-induced volume decrease in small-intestinal crypts.
Pflügers Arch
425:
434-438,
1993[ISI][Medline].
38.
Wang, CZ,
Yano H,
Nagashima K,
and
Seino S.
The Na+-driven Cl/HCO
39.
Weinman, EJ,
Steplock D,
Donowitz M,
and
Shenolikar S.
NHERF associations with sodium-hydrogen exchanger isoform 3 (NHE3) and ezrin are essential for cAMP-mediated phosphorylation and inhibition of NHE3.
Biochemistry
39:
6123-6129,
2000[ISI][Medline].
40.
Yanaka, A,
Carter KJ,
Goddard PJ,
and
Silen W.
Effect of luminal acid on intracellular pH in oxynticopeptic cells in intact frog gastric mucosa.
Gastroenterology
100:
606-618,
1991[ISI][Medline].
41.
Yun, CH,
Oh S,
Zizak M,
Steplock D,
Tsao S,
Tse CM,
Weinman EJ,
and
Donowitz M.
cAMP-mediated inhibition of the epithelial brush border Na+/H+ exchanger, NHE3, requires an associated regulatory protein.
Proc Natl Acad Sci USA
94:
3010-3015,
1997
42.
Zhao, H,
Star RA,
and
Muallem S.
Membrane localization of H+ and HCO