Departments of 1 Internal Medicine, 2 Hemodialysis and Apheresis, and 5 Pediatrics, Faculty of Medicine, Tokyo University Tokyo 113-0033; and Departments of 3 Pharmacology and Toxicology, and 4 Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mutations in
Na+-HCO
Na+-HCO
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE
NA+-HCO
The classic view of HCO/HCO
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunohistochemistry. Affinity-purified anti-kNBC-1 and anti-pNBC-1 antibodies were used to determine the localization of NBC-1 variants in pancreas. They are polyclonal antibodies against the NH2-terminal regions of human kNBC-1 (amino acids 4-16) or pNBC-1 (amino acids 2-12). The generation and specificity of these antibodies have been described elsewhere (40). Male Wistar rats were anesthetized with pentobarbital, and the pancreas was quickly removed and immersed in 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4. The specimens were then permeated by sequential incubation in PBS containing 10, 20, and 30% sucrose, embedded into Tissue-Tek OCT (Miles, Naperville, IL), and quickly frozen in liquid nitrogen. Sections 5 µm thick were prepared, air-dried, and immersed in PBS. Paraffin-embedded human pancreatic tissues were obtained with full informed consent from three patients who underwent the total pancreatectomy for the pancreatic carcinoma. The specimens had been immediately immersed in 10% formalin solution and then embedded in paraffin according to the standard method. Only areas that were confirmed to be histologically normal were used for the study. Sections 5 µm thick were cut from paraffin-embedded tissue blocks, rehydrated with xylene and graded alcohols, and immersed in PBS.
The immunofluorescence detection method was used for the immunohistological analysis. In brief, the rat pancreatic specimens were treated with normal goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA) to block nonspecific protein binding, followed by incubation with anti-kNBC-1 or anti-pNBC-1 antibodies (1:200 dilution in PBS) overnight at 4°C. Antigen deterioration in formalin-fixed, paraffin-embedded preparations is known to result in false negative findings. Therefore, for the paraffin-embedded human specimens, antigen retrieval by autoclave (121°C, 10 min in 0.01 mol/l citrate buffer, pH 6.0), which had been shown to be extremely useful in retrieving the masked antigenicity of proteins in paraffin sections (24), was adopted. After this pretreatment, the specimens were processed similarly as described for the rat pancreas. The rat and human specimens were subsequently incubated with the mixture of Alexa Fluora 488 goat anti-rabbit IgG (H + L), Alexa Fluora 568 phalloidin for labeling of actin, and TO-PROR-3 iodide for labeling of nuclei (all from Molecular Probes, Eugene, OR) for 60 min at room temperature. After being washed with PBS, the specimens were observed with a confocal laser scanning microscope (MRC-1024K, Japan Bio-Rad Laboratories, Tokyo, Japan).Transient expression of pNBC-1 and mutants. The wild-type human kNBC-1 and the kidney-type mutants, R298S and R510H, were cloned into a eukaryotic expression vector pcDNA3.1 (Invitrogen, San Diego, CA) (19) and designated as pkNBC, pR298S, and pR510H, respectively. The pcDNA3.1 containing the wild-type human pNBC-1 was constructed as follows (18). The DNA fragment spanning the pNBC-1 specific region was amplified by PCR from the cDNA of human corneal endothelial cells (41). This fragment was subcloned into the EcoRI sites of pcDNA3.1 in an appropriate orientation and designated as ppNBC-1-pre. The DNA fragment containing the remaining common region of NBC-1 was purified after digestion of pkNBC with AflII and subcloned into the AflII sites of ppNBC-1-pre. pcDNA3.1 containing the pancreatic-type mutants, R342S and R554H, was similarly constructed using pR298S and pR510H, respectively.
ECV304 cells were maintained in Medium199 (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Life Technologies). Cells were seeded onto 6-mm round coverslips and transfected with wild-type pNBC-1, R342S, or R554H using LipofectAMINE 2000 (Life Technologies) according to the manufacturer's instruction. In brief, cells were transfected with an equal amount of DNA in a 1:1 mixture of medium 199 and Opti-MEM I (Life Technologies) containing LipofectAMINE 2000 for 5 h. After the cells were incubated in medium199 supplemented with 10% FBS for 48 h, cell pH (pHi) measurement and Western blot analysis were performed.pHi measurement.
Cell-coated coverslips were incubated with HCO/HCO
-free HCO
, 2 H2PO
. Our preliminary experiments confirmed that this
treatment was sufficient to minimize the influence of
Na+-dependent Cl
/HCO
-free solution (18, 40). We have confirmed
that Na+/H+ exchange activity assayed by the
nigericin acidification was identical to that assayed by the
NH4Cl-pulse technique (18, 40). Therefore, the
problem of proton leak that might be caused by residual nigericin has
been shown to be minimal, if any, in our system. After cell
acidification, the coverslip was superfused for 3-4 min with
Na+-free, Cl
-free HCO
-free
HCO
-free HCO
Western blot analysis. Pancreas and kidneys were removed from Wistar rats and immediately homogenized in ice-cold buffer containing 280 mmol/l sucrose and 0.2 mmol/l Pefabloc SC (Boehringer Mannheim, Mannheim, Germany). The plasma membrane fraction was obtained by differential and discontinuous sucrose gradient procedures as described (18). Protein samples were boiled for 5 min in sample buffer, separated by SDS-PAGE on 7% acrylamide minigels, and blotted onto a nitrocellulose membrane. After incubation in blocking buffer, the membrane was treated with the diluted anti-NBC-1 antibodies (1:200) and then with horseradish peroxidase-conjugated anti-rabbit IgG (Bio-Rad, Richmond, CA) as the secondary antibody. The signal was detected by an ECL Plus system (Amersham, Aylesbury, UK).
Two days after transfection, ECV304 cells were lysed and the plasma membrane fraction was obtained. An equal amount of protein samples (50 µg/lane) was processed for Western blot analysis, as described above, using the antibody against NBC-1 COOH-terminal region (18) as the primary antibody.Reverse transcription-polymerase chain reaction. For reverse transcription-polymerase chain reaction (RT-PCR) analysis, total RNA was isolated from freshly isolated rat kidney and pancreas using the guanidinium isothiocyanate and phenol-chloroform extraction method described by Chomczynski and Sacchi (12). Primers were designed from the rat kNBC-1 sequence (9) as follows: 5'-GAT GTC CAC TGA AAA TGT GGA-3' (sense primer) and 5'-AGC ATG ACA GCC CTG CTC TGA-3' (antisense primer). These primers were set to amplify the kNBC-1 specific region and a part of the NBC-1 common region. The conditions for PCR were as follows: 30 cycles of 30 s at 94°C, 30 s at 60°C, and 45 s at 72°C, with an initial 9-min denaturing step and a final 5-min elongation step.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Western blot analysis.
To determine the dominant NBC-1 variant in pancreas, we first performed
Western blot analysis. For human pancreas, we could obtain only
paraffin-embedded specimens, which were not suitable for Western blot
analysis. Therefore, Western blot was performed only on rat pancreas.
As shown in Fig. 1, the anti-pNBC-1
antibody recognized a ~145-kDa band on immunoblots of rat pancreas.
On the other hand, the anti-kNBC-1 antibody did not recognize a
significant band in rat pancreas, although this antibody did recognize
a prominent band at ~130 kDa in rat kidney. These results suggest
that pNBC-1 is the dominant variant in pancreas. A recent study also
reported that the apparent molecular weight of pNBC-1 expressed in
pancreas is slightly larger than that of kNBC-1 expressed in kidney
(6). The molecular weight of pNBC-1 predicted from its
cDNA is ~121 kDa (1), whereas that of kNBC-1 is ~116
kDa (8), suggesting that NBC-1 proteins are
posttranslationally modified in slightly different ways in pancreas and
kidney. Consistent with this view, the antibody against the NBC-1
COOH-terminal region that recognizes both kNBC-1 and pNBC-1
(18) yielded a ~130-kDa band in rat kidney but a
~145-kDa band in rat pancreas (data not shown).
|
Localization of NBC-1 variants in rat and human pancreas. We next examined localization of NBC-1 variants in pancreas with the immunofluorescence detection method using confocal microscopy. Consistent with the results of Western blot analysis, the expression of pNBC-1 appeared dominant in both rat and human pancreas. However, there was a significant species difference in the patterns of pNBC-1 expression. The anti-pNBC-1 and anti-kNBC-1 antibodies did not label the islet cells in both rat and human pancreas.
In rat pancreas, the anti-pNBC-1 antibody labeled the acinar cells and the duct cells (Fig. 2, a and b). The nonimmunized rabbit IgG gave no fluorescence signals. Furthermore, the labeling by the anti-pNBC-1 antibody was diminished in the presence of antigen peptide (Fig. 2c), confirming the specificity of immunoreactions. In the acinar cells, the labeling appeared basolaterally dominant (Fig. 2a). In the medium and large ducts, such as the interlobular and the main ducts, the labeling was observed on both apical and basolateral membranes (Fig. 2, a and b), although the apical staining was less intense than the basolateral staining. In the intralobular ducts, the labeling was not so prominent and most of the intercalated ducts were not labeled. The anti-kNBC-1 antibody did not label the acinar cells, although it did label a limited number of the medium and large ducts (Fig. 2d). This occasional ductal labeling by anti-kNBC-1 antibody appeared rather apically dominant and was diminished in the presence of antigen peptide.
|
|
Detection of kNBC-1 mRNA in rat pancreas.
The anti-kNBC-1 antibody did not yield a clear band in Western blot
analysis, although it did label a limited number of ducts in rat
pancreas. This suggests that a small amount of kNBC-1 mRNA is expressed
in rat pancreas. To test for this view, we performed RT-PCR analysis.
As shown in Fig. 4, RT-PCR on the kNBC-1
specific region yielded a band of the expected size (800 bp) from rat
pancreas as well as from rat kidney.
|
Functional analysis of pNBC-1 mutants.
These immunohistological findings indicate that pNBC-1 is the dominant
variant in both rat and human pancreas. Because one patient with a
NBC-1 missense mutation presented high serum amylase levels, we decided
to examine whether disease-causing mutations (19) affect
the transport activity of pNBC-1. To accomplish this task, we
transiently expressed the wild-type pNBC-1 or the R342S or R554H
mutants in ECV304 cells and compared the rates of DIDS-sensitive
pHi recovery from acid-load in Cl-free
HCO
-free HCO
-free solution. In this case, readdition of
Na+ induced little pHi recovery in both cells
transfected with the wild-type pNBC-1 and vector alone. Thus the rates
of pHi recovery were 0.002 ± 0.002 pH/min for pNBC-1
(n = 8) and 0.001 ± 0.002 pH/min for vector
(n = 8), and both values were not significantly different from zero. In Cl
-free HCO
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To clarify the relative importance of NBC-1 variants in pancreatic
HCO
It should be pointed out that another NBC-1-related isoform (rb2NBC) has been cloned from rat brain (5). This isoform has a unique COOH terminus, but its NH2 terminus is identical to that of pNBC-1. It could be possible, therefore, that the anti-pNBC-1 antibody used in the present study also recognizes this isoform. However, our preliminary Western blot analysis on rat pancreas using the antibody against the unique COOH-terminal region of rb2NBC failed to yield a clear band, suggesting that pNBC-1 is really the dominant variant in pancreas.
In rat pancreas, the anti-pNBC-1 antibody labeled both acinar cells and duct cells. The labeling in acinar cells was quite intense and appeared rather basolateral dominant. Whereas the antibody labeled both apical and basolateral membranes in the medium and large ducts, most of the small, intercalated ducts were not labeled. In human pancreas, on the other hand, the anti-pNBC-1 antibody did not label the acinar cells, although it did intensively label the basolateral membranes of entire duct system, including the smallest intercalated cells. In view of the high proteolytic activity of acinar cells, we cannot exclude the possibility that a small amount of pNBC-1 is also expressed in human acinar cells but is not detected by the methods employed in the present study. However, the previous immunohistological studies using the antibodies against the common NBC-1 epitopes have also reported a very similar species difference in NBC-1 expression in pancreas. Thus, in rat pancreas, Thévenod et al. (38) have reported that NBC-1 is expressed in the basolateral membranes of acinar cells and in both apical and basolateral membranes of duct cells. In human pancreas, by contrast, Marino et al. (27) have reported that NBC-1 is expressed in the basolateral membranes of duct cells but not in the acinar cells. These results and observations may suggest different fluid and electrolyte secretion mechanisms in rat and human pancreas.
A previous functional study has shown the presence of
Na+-HCO
We have previously shown that missense mutations identified in pRTA
patients reduce the transport activity of kNBC-1. The present study
confirmed that these mutations also impair pNBC-1 function, which could
potentially explain the pancreatic phenotype associated with pRTA
(19, 21). We cannot exclude the possibility, however, that
these mutations might also affect the targeting and/or recycling of the
NBC-1 protein in human tissues. Recently, Shumaker et al.
(37) have shown that both CF transmembrane conductance regulator (CFTR) and NBC-1 cooperatively function to accomplish HCO and HCO
channel activity itself, may actually be responsible
for the pancreatic phenotype in these cases. A study in CFTR knockout mice (16) also supports the view that acidic pH changes in
luminal secretions play quite an important role in the pathogenesis of pancreatic dysfunction observed in CF. Because only a few NBC-1 mutations have been identified so far (3, 19, 20), a
definite conclusion cannot be drawn at present as to a possible link
between NBC-1 inactivation and pancreatic dysfunction. In addition,
clear evidence of pancreatitis is not yet reported in another pRTA
patient with S427L mutation in kNBC-1 (3). Nevertheless,
it is tempting to speculate that the inactivation of pNBC-1 may, at
least in some patients, decreases ductal HCO
Nonalcoholic chronic pancreatitis is a potentially life-threatening disease (14). Although idiopathic pancreatitis is frequently associated with CFTR mutations, recent studies suggest that other genetic factors may be also involved (13, 30). To better understand the pathogenesis of idiopathic pancreatitis and to establish the more effective therapeutic strategy, future studies are required, including the genetic testing of pNBC-1 mutations.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Kumamoto Immunochemical Laboratory (Kumamoto, Japan) for providing anti-NBC-1 antibodies.
![]() |
FOOTNOTES |
---|
This study was in part supported by grants 12671024 and 14571013 from the Ministry of Education, Science, and Culture of Japan.
Address for reprint requests and other correspondence: G. Seki, Dept. of Internal Medicine, Faculty of Medicine, Tokyo Univ., 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (E-mail: georgeseki-tky{at}umin.ac.jp).
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.
First published November 20, 2002;10.1152/ajpcell.00166.2002
Received 11 April 2002; accepted in final form 14 November 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
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
2.
Abuladze, N,
Song M,
Pushkin A,
Newman D,
Lee I,
Nicholas S,
and
Kurtz I.
Structural organization of the human NBC1 gene: kNBC1 is transcribed from an alternative promoter in intron 3.
Gene
251:
109-122,
2000[ISI][Medline].
3.
Alper, SL.
Genetic diseases of acid-base transporters.
Annu Rev Physiol
64:
899-923,
2002[ISI][Medline].
4.
Argent, BE,
and
Case RM.
Pancreatic ducts: cellular mechanism and control of bicarbonate secretion.
In: Physiology of the Gasrointestinal Tract, , edited by Johnson LR.. New York: Raven, 1994, p. 1473-1497.
5.
Bevensee, MO,
Schmitt BM,
Choi I,
Romero MF,
and
Boron WF.
An electrogenic Na+-HCO
6.
Bok, D,
Schibler MJ,
Pushkin A,
Sassani P,
Abuladze N,
Naser Z,
and
Kurtz I.
Immunolocalization of electrogenic sodium-bicarbonate cotransporters pNBC1 and kNBC1 in the rat eye.
Am J Physiol Renal Physiol
281:
F920-F935,
2001
7.
Boron, WF,
and
Boulpaep EL.
Intracellular pH regulation in the renal proximal tubule of the salamander. Basolateral HCO
8.
Burnham, CE,
Amlal H,
Wang Z,
Shull GE,
and
Soleimani M.
Cloning and functional expression of a human kidney Na+:HCO
9.
Burnham, CE,
Flagella M,
Wang Z,
Amlal H,
Shull GE,
and
Soleimani M.
Cloning, renal distribution, and regulation of the rat Na+-HCO
10.
Choi, I,
Romero MF,
Khandoudi N,
Bril A,
and
Boron WF.
Cloning and characterization of a human electrogenic Na+-HCO
11.
Choi, JY,
Muallem D,
Kiselyov K,
Lee MG,
Thomas PJ,
and
Muallem S.
Aberrant CFTR-dependent HCO
12.
Chomczynski, P,
and
Sacchi N.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:
156-159,
1987[ISI][Medline].
13.
Cohn, JA,
Friedman KJ,
Noone PG,
Knowles MR,
Silverman LM,
and
Jowell PS.
Relation between mutations of the cystic fibrosis gene and idiopathic pancreatitis.
N Engl J Med
339:
653-658,
1998
14.
Etemad, B,
and
Whitcomb DC.
Chronic pancreatitis: diagnosis, classification, and new genetic developments.
Gastroenterology
120:
682-707,
2001[ISI][Medline].
15.
Freedman, SD,
Kern HF,
and
Scheele GA.
Acinar lumen pH regulates endocytosis, but not exocytosis, at the apical plasma membrane of pancreatic acinar cells.
Eur J Cell Biol
75:
153-162,
1998[ISI][Medline].
16.
Freedman, SD,
Kern HF,
and
Scheele GA.
Pancreatic acinar cell dysfunction in CFTR(/
) mice is associated with impairments in luminal pH and endocytosis.
Gastroenterology
121:
950-957,
2001[ISI][Medline].
17.
Gaskin, KJ,
Durie PR,
Corey M,
Wei P,
and
Forstner GG.
Evidence for a primary defect of pancreatic HCO
18.
Hara, C,
Satoh H,
Usui T,
Kunimi M,
Noiri E,
Tsukamoto K,
Taniguchi S,
Uwatoko S,
Goto A,
Racusen LC,
Inatomi J,
Endou H,
Fujita T,
and
Seki G.
Intracellular pH regulatory mechanism in a human renal proximal cell line (HKC-8): evidence for Na+/H+ exchanger, Cl/HCO
19.
Igarashi, T,
Inatomi J,
Sekine T,
Cha SH,
Kanai Y,
Kunimi M,
Tsukamoto K,
Satoh H,
Shimadzu M,
Tozawa F,
Mori T,
Shiobara M,
Seki G,
and
Endou H.
Mutations in SLC4A4 cause permanent isolated proximal renal tubular acidosis with ocular abnormalities.
Nat Genet
23:
264-266,
1999[ISI][Medline].
20.
Igarashi, T,
Inatomi J,
Sekine T,
Seki G,
Shimadzu M,
Tozawa F,
Takeshima Y,
Takumi T,
Takahashi T,
Yoshikawa N,
Nakamura H,
and
Endou H.
Novel nonsense mutation in the Na+/HCO
21.
Igarashi, T,
Ishii T,
Watanabe K,
Hayakawa H,
Horio K,
Sone Y,
and
Ohga K.
Persistent isolated proximal renal tubular acidosis-a systemic disease with a distinct clinical entity.
Pediatr Nephrol
8:
70-71,
1994[ISI][Medline].
22.
Ishiguro, H,
Steward MC,
Lindsay AR,
and
Case RM.
Accumulation of intracellular HCO
23.
Jentsch, TJ,
Keller SK,
Koch M,
and
Wiederholt M.
Evidence for coupled transport of bicarbonate and sodium in cultured bovine corneal endothelial cells.
J Membr Biol
81:
189-204,
1984[ISI][Medline].
24.
Kawai, K,
Serizawa A,
Hamana T,
and
Tsutsumi Y.
Heat-induced antigen retrieval of proliferating cell nuclear antigen and p53 protein in formalin-fixed, paraffin-embedded sections.
Pathol Int
44:
759-764,
1994[ISI][Medline].
25.
Kopelman, H,
Corey M,
Gaskin K,
Durie P,
Weizman Z,
and
Forstner G.
Impaired chloride secretion, as well as bicarbonate secretion, underlies the fluid secretory defect in the cystic fibrosis pancreas.
Gastroenterology
95:
349-355,
1988[ISI][Medline].
26.
Kristidis, P,
Bozon D,
Corey M,
Markiewicz D,
Rommens J,
Tsui LC,
and
Durie P.
Genetic determination of exocrine pancreatic function in cystic fibrosis.
Am J Hum Genet
50:
1178-1184,
1992[ISI][Medline].
27.
Marino, CR,
Jeanes V,
Boron WF,
and
Schmitt BM.
Expression and distribution of the Na+-HCO
28.
Montrose, MH,
and
Murer H.
Regulation of intracellular pH by cultured opossum kidney cells.
Am J Physiol Cell Physiol
259:
C110-C120,
1990
29.
Muallem, S,
and
Loessberg PA.
Intracellular pH-regulatory mechanisms in pancreatic acinar cells. I. Characterization of H+ and HCO
30.
Noone, PG,
Zhou Z,
Silverman LM,
Jowell PS,
Knowles MR,
and
Cohn JA.
Cystic fibrosis gene mutations and pancreatitis risk: relation to epithelial ion transport and trypsin inhibitor gene mutations.
Gastroenterology
121:
1310-1319,
2001[ISI][Medline].
31.
Novak, I,
and
Greger R.
Electrophysiological study of transport systems in isolated perfused pancreatic ducts: properties of the basolateral membrane.
Pflügers Arch
411:
58-68,
1988[ISI][Medline].
32.
Romero, MF,
Hediger MA,
Boulpaep EL,
and
Boron WF.
Expression cloning and characterization of a renal electrogenic Na+/HCO
33.
Roussa, E,
Romero MF,
Schmitt BM,
Boron WF,
Alper SL,
and
Thevenod F.
Immunolocalization of anion exchanger AE2 and Na+-HCO
34.
Scheele, GA,
Fukuoka SI,
Kern HF,
and
Freedman SD.
Pancreatic dysfunction in cystic fibrosis occurs as a result of impairments in luminal pH, apical trafficking of zymogen granule membranes, and solubilization of secretory enzymes.
Pancreas
12:
1-9,
1996[ISI][Medline].
35.
Schmitt, BM,
Berger UV,
Douglas RM,
Bevensee MO,
Hediger MA,
Haddad GG,
and
Boron WF.
Na/HCO
36.
Seki, G,
Coppola S,
and
Frömter E.
The Na+-HCO
37.
Shumaker, H,
Amlal H,
Frizzell R,
Ulrich CD, 2nd,
and
Soleimani M.
CFTR drives Na+-HCO
38.
Thévenod, F,
Roussa E,
Schmitt BM,
and
Romero MF.
Cloning and immunolocalization of a rat pancreatic Na+ bicarbonate cotransporter.
Biochem Biophys Res Commun
264:
291-298,
1999[ISI][Medline].
39.
Thomas, JA,
Buchsbaum RN,
Zimniak A,
and
Racker E.
Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ.
Biochemistry
18:
2210-2218,
1979[ISI][Medline].
40.
Usui, T,
Hara M,
Satoh H,
Moriyama N,
Kagaya H,
Amano S,
Oshika T,
Ishii Y,
Ibaraki N,
Hara C,
Kunimi M,
Noiri E,
Tsukamoto K,
Inatomi J,
Kawakami H,
Endou H,
Igarashi T,
Goto A,
Fujita T,
Araie M,
and
Seki G.
Molecular basis of ocular abnormalities associated with proximal renal tubular acidosis.
J Clin Invest
108:
107-115,
2001
41.
Usui, T,
Seki G,
Amano S,
Oshika T,
Miyata K,
Kunimi M,
Taniguchi S,
Uwatoko S,
Fujita T,
and
Araie M.
Functional and molecular evidence for Na+-HCO
42.
Yoshitomi, K,
Burckhardt BC,
and
Frömter E.
Rheogenic sodium-bicarbonate cotransport in the peritubular cell membrane of rat renal proximal tubule.
Pflügers Arch
405:
360-366,
1985[ISI][Medline].
43.
Zhao, H,
Star RA,
and
Muallem S.
Membrane localization of H+ and HCO