Molecular Cloning, Chromosomal Localization, Tissue Distribution,
and Functional Expression of the Human Pancreatic Sodium Bicarbonate
Cotransporter*
Natalia
Abuladze
,
Ivan
Lee,
Debra
Newman,
James
Hwang,
Kathryn
Boorer§,
Alexander
Pushkin, and
Ira
Kurtz¶
From the Division of Nephrology, Center for Health Sciences, and
§ Department of Physiology, UCLA School of Medicine,
Los Angeles, California 90095-1698
 |
ABSTRACT |
We report the cloning, sequence analysis, tissue
distribution, functional expression, and chromosomal localization of
the human pancreatic sodium bicarbonate cotransport protein (pancreatic NBC (pNBC)). The transporter was identified by searching the human expressed sequence tag data base. An I.M.A.G.E. clone W39298 was
identified, and a polymerase chain reaction probe was generated to
screen a human pancreas cDNA library. pNBC encodes a 1079-residue polypeptide that differs at the N terminus from the recently cloned human sodium bicarbonate cotransporter isolated from kidney (kNBC) (Burnham, C. E., Amlal, H., Wang, Z., Shull, G. E., and
Soleimani, M. (1997) J. Biol. Chem. 272, 19111-19114). Northern blot analysis using a probe specific for the N
terminus of pNBC revealed an ~7.7-kilobase transcript expressed
predominantly in pancreas, with less expression in kidney, brain,
liver, prostate, colon, stomach, thyroid, and spinal chord. In
contrast, a probe to the unique 5' region of kNBC detected an
~7.6-kilobase transcript only in the kidney. In situ
hybridization studies in pancreas revealed expression in the acini and
ductal cells. The gene was mapped to chromosome 4q21 using fluorescent
in situ hybridization. Expression of pNBC in Xenopus
laevis oocytes induced sodium bicarbonate cotransport. These data
demonstrate that pNBC encodes the sodium bicarbonate cotransporter in
the mammalian pancreas. pNBC is also expressed at a lower level in
several other organs, whereas kNBC is expressed uniquely in kidney.
 |
INTRODUCTION |
Sodium bicarbonate cotransport mediates the coupled movement of
Na+ and HCO3
ions across
the plasma membrane of many cells (1). This transport process is
involved in bicarbonate secretion/absorption and intracellular pH
(pHi)1 regulation.
Functional Na(HCO3)n cotransport was first identified in the salamander Ambystoma tigritum kidney (2) and has since been documented functionally in numerous other cell types
including pancreas (3-8), colon (9), liver (10-12), heart (13, 14),
retinal Müeller cells (15, 16), glial cells (17, 18), parietal
cells (19),and type II alveolar cells (20). Depending on the cell type,
the stoichiometry of Na+ to
HCO3
flux is 3:1, 2:1, or 1:1. As
characterized in many cell types, several features distinguish the
Na(HCO3)n cotransporter from other
bicarbonate-dependant transporters.1) Na(HCO3)n cotransport is not dependent on the presence of Cl
, 2)
transport is inhibited by stilbenes, and 3) transport is stimulated in
the presence of HCO3
.
In the kidney, Na(HCO3)n cotransport was initially
localized by functional studies to the basolateral membrane of the
proximal tubule where it plays an important role in mediating electrogenic basolateral bicarbonate efflux (2, 21, 22). Although
Na+-dependent and -independent
Cl
/base exchangers also contribute to basolateral
bicarbonate transport in the proximal tubule (23-27), current evidence
suggests that electrogenic Na(HCO3)n cotransport
mediates the majority of bicarbonate efflux in this nephron segment
(1). Romero et al. have recently cloned a renal electrogenic
sodium bicarbonate cotransporter (NBC) from rat (28) and salamander
kidney (29). Burnham et al. have reported the cloning of a
sodium bicarbonate cotransporter from human kidney (30). The NBC clone
isolated from salamander kidney encoded a 4.2-kb mRNA transcript
that was expressed predominantly in kidney, with less expression in
small intestine, large intestine, brain, eye, and bladder (29). Human NBC isolated from kidney encoded a ~7.6-kb mRNA and was
reportedly also expressed in pancreas and brain by Northern analysis
using a probe to the 3' region of kNBC (30). NBC expression in the kidney has recently been shown to be highest in the S1 proximal tubule,
with less expression in the proximal straight tubule (31). The high
level of expression in S1 proximal tubules is in keeping with the high
rate of transepithelial bicarbonate transport in this segment (32,
33).
The pancreas secretes digestive enzymes dissolved in a
HCO3
-rich fluid (34). Pancreatic
bicarbonate secretion is mediated by principal cells lining the
pancreatic ducts (34, 35). Previous functional studies have led to a
cell model that can account for transcellular bicarbonate secretion.
Apical bicarbonate secretion is thought to be mediated by an apical
Cl
/base exchanger acting in parallel with a small
conductance cystic fibrosis transmembrane conductance regulator
Cl
channel on the apical membrane (5, 36). Influx of
H+ equivalents during the process of apical bicarbonate
secretion requires the efflux of H+ or the influx of
bicarbonate in the steady state. Studies of pig, rat, and guinea
pig pancreatic ducts have demonstrated the presence of a basolateral
Na+/H+ antiporter, which serves an important
housekeeping role (5, 8, 37, 38). A basolateral vacuolar-type
H+/ATPase and Na(HCO3)n
cotransporter are thought to play an important role in
agonist-mediated bicarbonate secretion (5-8, 39). The relative
contribution of these transporters to basolateral H+/base
transport and their respective stimulation by secretogues in different
species is controversial. The importance of basolateral Na(HCO3)n cotransport has recently been documented
in studies of isolated rat and guinea pig pancreatic ducts (5-8, 39).
Ishiguro et al. (7) reported that basolateral
Na(HCO3)n cotransport contributed up to 75% of
basolateral bicarbonate uptake during stimulation of transepithelial
bicarbonate transport by secretin. Furthermore, in isolated pancreatic
acini, Na(HCO3)n cotransport has been shown to
participate in the regulation of pHi after acid loads (3). On
the basis of HCO3
flux measurements and
thermodynamic considerations, it was concluded that this transporter
contributes to HCO3
efflux under
unstimulated conditions (3, 7) with a stoichiometry of 3:1 (3),
although direct measurements of the stoichiometry have thus far not
been performed. After depolarization of the basolateral membrane by
secretin (40), the electrochemical driving forces would favor
basolateral bicarbonate influx (7).
Although the functional characteristics of pancreatic
Na(HCO3)n cotransport have begun to be
investigated, the protein responsible for this function has not been
identified. We report here the cloning the human pancreatic
Na(HCO3)n cotransporter (pNBC). The predicted pNBC
polypeptide is 1079 amino acids in length, whereas the NBC variant
expressed in kidney (kNBC) consists of 1035 amino acids. The C-terminal
994 amino acids of pNBC and kNBC are identical. pNBC has a unique N
terminus of 85 amino acids that replaces the initial 41 amino acids in
kNBC. Expression of the cDNA encoding pNBC in Xenopus
oocytes results in sodium-dependent and
chloride-independent HCO3
transport,
which is inhibited by DIDS.
 |
EXPERIMENTAL PROCEDURES |
Cloning and Sequencing of pNBC--
A 159-bp PCR product
(2795-2954 bp in human pNBC) was generated using the human pancreas
NBC EST clone W39298 (I.M.A.G.E clone) as a template, random
primer-labeled with 32P and used to screen a human pancreas
gt10 cDNA library (CLONTECH, Palo Alto, CA).
A similar approach was utilized by Burnham et al. (30) while
the present studies were in progress to obtain an NBC clone from human
pancreas. Standard hybridization conditions were employed (42 °C,
50% formamide, 5× standard saline phosphate EDTA (SSPE), 5×
Denhardt's solution, 0.5% SDS, 0.2 mg/ml prehybridization herring
sperm DNA). The filters were washed three times with 1× SSC/0.1%SDS
(42 °C) and once with 0.1× SSC/0.1%SDS (25 °C) (1×SSC = 0.15 M NaCl and 0.015 M sodium citrate).
Positive clones were verified by sequencing. Two overlapping clones
(7.1) and (9.2.1) were obtained that contained the entire coding
region. To obtain a full-length clone containing the complete open
reading frame, these two clones were spliced together using a common
SpeI restriction site and subcloned into pPCR-Script SK(+) (Stratagene,
La Jolla, CA). To increase the stability of the capped RNA transcribed
from this clone, a poly(A) (70-mer) oligonucleotide was added to the 3'
end of the clone between a SalI and KpnI
restriction site. The 5' end of the coding sequence for pNBC was
confirmed by 5' rapid amplification of cDNA ends PCR amplification
and primer extension analysis. Furthermore, to confirm that the pNBC
amino acid sequence was derived from a bona fide transcript,
we amplified the entire open reading frame of human pNBC by reverse
transcription-PCR using Marathon Ready cDNA prepared from human
pancreas (CLONTECH, Palo Alto, CA) as a template.
Nucleotide sequences were determined bidirectionally by automated
sequencing (ABI 310 Perkin-Elmer) using Taq polymerase
(Ampli-Taq FS, Perkin-Elmer). Sequence assembly and analysis
was carried out using Geneworks software (Oxford, UK).
Northern Analysis and Tissue Distribution--
Northern blots
with various human tissues were obtained from
CLONTECH. The various probes were random
prime-labeled with [32P]dCTP to a specific activity of
about 1.5 × 109 dpm/µg. The filters were
prehybridized at 42 °C for 2 h using 50% formamide, 6× SSPE,
0.5% SDS, Denhardt's solution, and 0.1 mg/ml of sheared herring
testes denatured DNA. After the prehybridization, the filters were
incubated with the 32P probe using 25 ml of hybridization
buffer. The probes were denatured and added to the hybridization
solution at 107 dpm/ml. The filters were probed at 42 °C
for 18 h and washed in 1× SSC, 0.1% SDS at 45 °C for 60 min
(3 changes, 350 ml/wash); after exposure for 9.5 h, the filters
were rewashed in 1× SSC, 0.1%SDS at 65 °C for 30 min and 0.1×
SSC, 0.1%SDS at 65 °C for 1 h. The glyceraldehyde-3-phosphate
dehydrogenase DNA was T4 polynucleotide kinase (New England Biolabs,
Beverly, MA) labeled with [32P]
ATP to a specific
activity of 2.5 µCi/pmol. The filters were prehybridized at 42 °C
for 2 h using 50% formamide, 6x SSPE, 0.5% SDS, Denhardt's
solution, and 0.1 mg/ml sheared herring testes denatured DNA. After the
prehybridization, the filters were probed with the 32P
probe using 25 ml of hybridization buffer. The probe was denatured and
added to the hybridization solution at 0.5 pmol/ml. The filters were
probed at 42 °C for 18 h and then washed 3 times in 1x SSC, 0.1%SDS at 45 °C for 30 min. The following probes were used in the
Northern blot studies: 1) a 159-bp PCR product with a sequence common
to both pNBC and kNBC (nucleotides 2795-2954 pNBC sequence and 2695 to
2854 in the kNBC sequence); 2) synthetic oligonucleotide specific for
pNBC (nucleotides 118 to 212 in the pNBC sequence); 3) synthetic
oligonucleotide specific for kNBC (nucleotides 175 to 268 in the kNBC
sequence).
Preparation of Riboprobes for in Situ Hybridization--
To
prepare the riboprobes, the insert (9.2.1) was subcloned into
pPCR-script SK(+) (Stratagene) Riboprobes were synthesized by in
vitro transcription and labeled with 35S-CTP. For
generation of the antisense riboprobe, the plasmid was linearized with
SstI and transcribed by T7 RNA polymerase. For generation of
the sense riboprobe, the plasmid was linearized with KpnI
and transcribed with T3 RNA polymerase. The RNA transcripts were
purified by phenol-chloroform extractions and Sephadex G-50 spin
columns (Sigma). The final products were suspended in Tris-EDTA buffer
with 0.1 M dithiothreitol. The RNA transcripts were then sheared by alkaline hydrolysis at 68 °C for 5 min. After the
shearing, the reaction was neutralized by adding 3 M sodium
acetate, pH 5, to make a final acetate concentration of 0.3 M. Slices of mouse pancreas (1 mm) were fixed in 4%
formalin, and 5-µm sections were attached to glass slides (Fisher).
The slides were prewashed and digested for 15 min at 37 °C with
proteinase K. To reduce nonspecific background staining, the slides
were succinylated with succinic anhydride and acetylated with acetic
anhydride. The riboprobes were hybridized for 18 h at 45 °C.
The slides were then washed for 15 min in 2× SSC at room temperature,
followed by a wash (15 min) in 1 × SSC/50% formamide at
45 °C, then three washes in 2 × SSC/0.1% Triton X-100 at
60 °C for 15 min each, followed by two washes in 0.1 SSC at 60 °C
for 15 min each. The slides were then digested by RNase A (25 µg/ml;
Sigma) and RNase T1(25 units/ml; Sigma) for 40 min at 37 °C. The
slides were washed twice in 2× SSC at 60 °C for 15 min each and
then dehydrated in 0.3 M ammonium acetate, 70% ethanol for
5 min followed by a further 5 min of dehydration in 0.3 M
ammonium acetate, 95% ethanol. The slides were dipped into NTB2
emulsion solution (Eastman Kodak Co.) and exposed for 3 days at 4 °C
followed by hematoxylin/eosin staining. The sections were imaged using
a Zeiss Axiophot microscope (Max Erb, Los Angeles, CA) and digitized
using a Sony 3CCD color video camera (model DXC-960MD, Compix Imaging
Systems, Tuscon, AZ) with C Imaging software (Compix Imaging Systems,
Tuscon, AZ).
Fluorescent In Situ Hybridization--
The PCR probe used to
screen the pancreatic cDNA library was also used to screen an
arrayed PAC human genomic library (Genome Systems, St. Louis, MO). DNA
from clone F335 was identified by sequencing and was then labeled with
digoxigenin dUTP by nick translation. Labeled probe was combined with
sheared human DNA and hybridized to normal metaphase chromosomes
derived from phytohemagglutinin-stimulated peripheral blood lymphocytes
in a solution containing 50% formamide, 10% dextran sulfate, and 2×
SSC. Specific hybridization signals were detected by incubating the
hybridized slides in fluoresceinated antidigoxigenin antibodies
followed by counterstaining with 4',6-diamidino-2-phenylindole, dihydrochloride for one color experiments. Probe detection for two
color experiments was accomplished by incubating the slides in
fluoresceinated antidigoxigenin antibodies and Texas red avidin followed by counterstaining with 4',6-diamidino-2-phenylindole, dihydrochloride.
Xenopus Oocyte Expression--
The plasmid containing the
complete coding sequence of pNBC was linearized by digestion with
KpnI, and capped cRNA was prepared with T3 RNA polymerase
using a T3 mMessage mMachine kit RNA capping kit (Ambion, Austin, TX)
as recommended by the manufacturer. This cRNA was used for the
Xenopus oocyte expression studies. An aliquot of the
synthesized cRNA was run on a denaturing gel to verify the expected
size before oocyte injection. Defolliculated oocytes were injected with
50 nl of sterile water or a solution containing 1 ng/nl capped pNBC
cRNA (prepared as described above). They were then bathed in Barth's
medium at 18 °C.
Measurement of Oocyte Intracellular pH
(pHi)--
3-6 days post-injection, optical
recordings were made at 22-24 °C. Intracellular pH was monitored
using the fluorescent probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and a
microfluorometer coupled to the microscope (41). Individual
defolliculated oocytes were held in place with pipettes attached to low
suction with vegetal pole surface closest to the 40× objective. Before
loading with BCECF, the background intensity from each oocyte was
digitized at 500 nm and 440 nm (530-nm emission). The oocytes were
loaded with 32 µM BCECF-acetoxymethyl ester for 1 h
before experimentation in the following solution: NaCl (108 mM), KCl (2 mM), CaCl2 (1 mM), MgCl2 (1 mM), and Hepes (8 mM), pH 7.4. Calibration of intracellular BCECF in the
oocytes was performed at the end of each experiment by monitoring the
500/440-nm fluorescence excitation ratio at various values of
pHi in the presence of high K+ nigericin standards
as described previously (42). Three experimental protocols were
performed: 1) Na+ removal/addition. The oocytes were bathed
in the following Na+-containing solution for 30 min: NaCl
(100 mM); KCl (2 mM); CaCl2 (1 mM); MgCl2 (1 mM);
NaHCO3 (8 mM) and bubbled with 1.5%
CO2, pH 7.4. After a steady state was reached,
Na+ was removed by bathing the oocytes in the following
Na+-free solution: TMA-Cl (100 mM), KCl (2 mM), CaCl2 (1 mM),
MgCl2 (1 mM), TMA-HCO3 (8 mM) bubbled with 1.5% CO2, pH 7.4; 2)
Na+ removal/addition with DIDS (0.3 mM); and 3)
Na+ removal/addition in Cl
-free solutions
with EIPA (10 µM). For the latter experiments, the
oocytes were loaded with BCECF in the following Cl
-free
solution: sodium gluconate (108 mM), potassium gluconate (2 mM), calcium gluconate (7 mM), magnesium
gluconate (2 mM), Hepes (8 mM), pH 7.4. The
oocytes were bathed in the following Na+-containing
Cl
-free solution for ~30 min with EIPA (10 µM): sodium gluconate (100 mM), potassium
gluconate (2 mM), calcium gluconate (7 mM), magnesium gluconate (2 mM), NaHCO3 (8 mM), pH 7.4. bubbled with 1.5% CO2, pH 7.4. After a steady state was reached, Na+ was removed by
bathing the oocytes in the following Na+-free,
Cl
-free solution with EIPA (10 µM): TMA-OH
(100 mM), D-gluconic acid lactone (100 mM), potassium gluconate (2 mM), calcium
gluconate (7 mM), magnesium gluconate (2 mM),
TMA-HCO3 (8 mM) bubbled with 1.5%
CO2, pH 7.4.
22Na+ Influx
Measurements--
Defolliculated oocytes were injected with pNBC cRNA
(50 nl, 1 µg/ul) or water and incubated in Barth's medium for 3-6
days at 18 °C before study. The oocytes were preincubated for 1 h in 1 ml of a Na+-free solution containing: TMA-Cl (108 mM), KCl (2 mM), CaCl2 (1 mM), MgCl2 (1 mM), and Hepes (8 mM), pH 7.4. The oocytes were then transferred into 1.4 ml
of the following Na+-containing solution: NaCl (100 mM), KCl (2 mM), CaCl2 (1 mM), MgCl2 (1 mM),
NaHCO3 (8 mM) bubbled with 1.5%
CO2, pH 7.4 with 2 µCi of 22Na+.
A 10-µl aliquot was removed from the influx solution for later determination of 22Na+-specific activity.
22Na+ influx was measured after 15 min and
terminated with three washes of ice-cold Na+-free stop
solution. The influx experiments were repeated in the presence of DIDS
(0.3 mM). In the DIDS-containing experiments, the oocytes
were exposed to 0.3 mM DIDS for 30 min before and throughout the influx period.
36Cl
Influx Measurements--
The
oocytes were preincubated in 1 ml for 1 h in a
Cl
-free solution containing sodium gluconate (108 mM), potassium gluconate (2 mM), calcium
gluconate (7 mM), magnesium gluconate (2 mM), and Hepes (8 mM), pH 7.4. The oocytes were then transferred
into 1.4 ml of the following Cl
-containing solution: NaCl
(100 mM), KCl (2 mM), CaCl2 (1 mM), MgCl2 (1 mM),
NaHCO3 (8 mM) bubbled with 1.5%
CO2, pH 7.4. with 3.3 µCi of
36Cl
. A 10-µl aliquot was removed from the
influx solution for later determination of
36Cl
-specific activity.
36Cl
influx was measured after 15 min and
terminated with three washes of ice-cold stop solution.
The sequences submitted to GenBankTM have been scanned
against the data base, and the following related human sequences have been identified (AF007216, P02730, P48751, U62531).
 |
RESULTS AND DISCUSSION |
Isolation of cDNA Clones and Characterization of Multiple Human
NBC Transcripts--
The cloning of pNBC was based on the
identification of human pancreatic cDNA clone in the
GenBankTM data base. To obtain full-length pNBC, we
screened a human pancreas
gt10 cDNA library using a probe
generated by PCR amplification of the human EST sequence. Two
overlapping clones were obtained that were fused at a shared SpeI
restriction site to generate a full-length clone containing the entire
open reading frame. To confirm that the pNBC sequence was derived from
a bona fide transcript, reverse transcription-PCR was used
to generate a full-length PCR product containing the complete open
reading frame. Analysis of the full-length clone revealed a 1079-amino
acid open reading frame beginning with the initial methionine as well
as 117 bp of 5'-untranslated region. Additional overlapping
3'-untranslated rapid amplification of cDNA ends sequences were
almost identical to the sequence recently published by Burnham et
al. (30) except for minor changes likely due to polymorphisms. The
nucleotide sequence of human pNBC has been submitted to the
GenBankTM (accession number AF011390).
Structure of pNBC--
The overall structure of pNBC is similar to
kNBC (30) and other members of the anion exchange gene family (43).
Specifically, pNBC has 12 predicted transmembrane regions and
hydrophilic intracellular N- and C-terminal regions. As shown in Fig.
1, the sequences of pNBC and kNBC are
identical after the Ser residue at position 42 of kNBC and position
86 of the pancreatic sequence. Unlike the kidney sequence, the N
terminus of pNBC before the region common to both polypeptides contains
blocks of charged amino acids. Further distinctive features of the N
terminus of pNBC are 1) the consensus phosphorylation site for protein
kinase A beginning at Lys46, 2) the consensus
phosphorylation sites for protein kinase C beginning at
Ser38 and Ser65, and 3) the casein kinase II
phosphorylation site beginning at Ser68. In contrast, amino
acids 1-41 of kNBC lack consensus phosphorylation sites. pNBC and kNBC
polypeptides are predicted to have identical transmembrane and
C-terminal regions.

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Fig. 1.
Comparison of the N terminus of pNBC and
kNBC. a, ATG translation start sites are
underlined. The nucleotide sequence common to pNBC and kNBC
is highlighted in gray. b, predicted
pNBC and kNBC polypeptide sequences. The sequences of pNBC and kNBC
differ before the Ser residue at position 42 of kNBC and position 86 of
the pancreatic sequence. In pNBC, several consensus phosphorylation
sites are depicted: 1) protein kinase A beginning at Lys46
(dotted line; 2) protein kinase C beginning at
Ser38 and Ser65 (thick line); and 3)
casein kinase II at Ser68 (thin line). Amino
acids 1-41 of kNBC lack consensus phosphorylation sites. The amino
acid sequence common to pNBC and kNBC is highlighted in
gray.
|
|
Tissue Expression of pNBC and kNBC--
The expression of pNBC and
kNBC was examined in various human tissues by Northern blot analysis
(Fig. 2). Specific probes were prepared
that contained the unique N-terminal region of each isoform. A probe to
the common 3'-coding region of pNBC and kNBC recognized transcripts in
RNA samples from the pancreas, with less expression in kidney, brain,
liver, prostate, colon, stomach, thyroid, and spinal chord. Burnham
et al. (30), using a probe to nucleotides 2737 to 2973 of
kNBC (2837 to 3073 in pNBC), detected transcripts in kidney, pancreas
and brain. Importantly, the results of the present study demonstrate
that the 5'-coding region of pNBC differs from the 5' terminus of the
human NBC sequence reported by Burnham et al. (30). The
C-terminal 994 amino acids of pNBC and kNBC are identical. pNBC has a
unique N terminus of 85 amino acids that replaces the first 41 amino
acids in kNBC. This observation is consistent with the failure of a
probe derived from the 5' terminus of kNBC to detect the ~7.7-kb pNBC
transcript in the Northern blot experiments. As shown in Fig. 2, the
expression of the ~7.6-kb kNBC transcript was restricted exclusively
to kidney. The kNBC probe failed to detect a transcript in any tissue
other than kidney (despite longer exposure times, and lower
stringency), suggesting that the kNBC N terminus is unique. The
mechanism responsible for the tissue-specific expression of kNBC and
pNBC is unknown but may involve activation of a downstream promoter
and/or differential splicing of the primary transcripts in kidney.

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Fig. 2.
Northern blot analysis of expression of pNBC
and kNBC in human tissues: Lanes 1-23: 1,
heart; 2, brain; 3, placenta; 4, lung;
5, liver; 6, skeletal muscle; 7,
kidney; 8, pancreas; 9, spleen; 10,
thymus; 11, prostate; 12, testis; 13,
ovary; 14, small intestine; 15, colon;
16, peripheral blood leukocytes; 17, stomach;
18, thyroid; 19, spinal chord; 20,
lymph node; 21, trachea; 22, adrenal gland;
23, bone marrow. Each lane was loaded with ~2
µg of poly(A)+ human RNA. The multiple tissue Northern
blots were from CLONTECH. The following
32P-labeled NBC probes were used: a, a 159-bp
PCR product with sequence common to both pNBC and kNBC (nucleotides
2795-2954 pNBC sequence and 2695 to 2854 in the kNBC sequence);
b, synthetic oligonucleotide specific for pNBC (nucleotides
118 to 212 in the pNBC sequence); c, synthetic
oligonucleotide specific for kNBC (nucleotides 175 to 268 in the kNBC
sequence). The blots were also probed for glyceraldehyde-3-phosphate as
shown in d.
|
|
In separate experiments, a Northern blot from a variety of human
tissues was screened with a probe derived from the unique 5' portion of
the pancreas cDNA to confirm that the sequence is represented in
the ~7.7-kb mRNA transcript. As shown in Fig. 2, this probe
detected a ~7.7-kb mRNA abundant in pancreas and present at lower
levels in kidney, brain, liver, prostate, colon, stomach, thyroid, and
spinal chord. The size of the transcripts in most tissues was identical
to that of the pancreatic NBC transcript identified with a probe common
to the 3'-coding region of pNBC and kNBC. However, in thyroid, a
transcript of higher mobility was detected by both the specific pNBC
probe and the C-terminal probe common to kNBC and pNBC. This transcript
may represent a variant of pNBC.
In Situ Hybridization of pNBC--
To determine in greater detail
the distribution of pNBC within the pancreas, we used in
situ hybridization with a pNBC 35S-labeled riboprobe.
Mouse pancreas was used because of tissue availability. Mouse pNBC was
cloned using a PCR-based strategy. The sequence of mouse pNBC was found
to be 93% identical to the human sequence and has been deposited in
GenBankTM (accession number AF020195). Microautoradigraph
analysis of frozen pancreas sections hybridized with the pNBC riboprobe
showed strong expression in the pancreatic ducts and pancreatic acini (Fig. 3). A signal was not detected in
the islets.

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Fig. 3.
Localization of pNBC mRNA in mouse
pancreas detected by in situ hybridization.
a and c, antisense probe; b and
d, sense probe. pNBC mRNA expression was detected in
pancreatic acini (A) and ductal cells (D). No
labeling was detected in the islets (I). Bright-field
microphotograph: a and b, magnification, 400×;
c and d, magnification, 200×.
|
|
Chromosomal Localization of Human NBC--
The initial experiment
resulted in the specific labeling of the long arm of a group B
chromosome, which was believed to be chromosome 4 on the basis of size,
morphology, and banding pattern. A second experiment was conducted in
which a biotin-labeled probe that is specific for the centromere of
chromosome 4 (D4ZI) was cohybridized with clone F335. This experiment
resulted in specific labeling of the centromere of chromosome 4. Measurements of 10 specifically labeled chromosomes 4 demonstrated that
clone F335 maps to a position that is 19% that of the distance from
the centromere to the telomere of chromosome arm 4q, an area that
corresponds to band 4q21. A total of 80 metaphase cells were examined,
with 71 exhibiting specific labeling (Fig.
4).

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Fig. 4.
Chromosomal localization of the NBC
gene. The NBC gene is localized to chromosome 4 at position 4q21
on metaphase spreads of human peripheral lymphocytes
(arrows).
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Functional Expression of pNBC in Xenopus Oocytes--
We examined
the functional properties of pNBC using measurements of pHi,
22Na, and 36Cl
uptake after
injecting the corresponding polyadenylated cRNA into Xenopus
oocytes. Polyadenylated cRNA prepared as described above was injected
into oocytes and allowed to express for 72 h. Augmented
22Na uptake was observed in oocytes injected with pNBC cRNA
(Fig. 5). The uptake was 16-fold greater
than control oocytes, p < 0.001. Uptake was
significantly inhibited in the presence of DIDS (0.3 mM).
Cl
uptake was not significantly affected by pNBC cRNA
injection: 0.16 ± 0.05 nmol/h/oocyte in controls
(n = 8) and 0.21 ± 0.02 (n = 9)
nmol/h/oocyte in cRNA injected oocytes, p = NS.

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Fig. 5.
Functional expression of pNBC Xenopus
laevis oocytes. 22Na uptake (expressed as
nmol/h/oocyte) in oocytes injected with water or with pNBC cRNA is
shown. Expression was assessed in the presence and absence of 0.3 mM DIDS. Each bar represents the mean ± S.E. of 10 to 14 oocytes. 22Na uptake was significantly
increased by pNBC cRNA versus H2O-injected
oocytes and inhibited in the presence of DIDS (0.3 mM); *
indicates p < 0.001 cRNA versus
H2O-injected and cRNA versus cRNA plus DIDS.
36Cl uptake (expressed as nmol/h/oocyte) was
also assessed in oocytes injected with water or with pNBC cRNA.
Cl uptake was not significantly affected by pNBC cRNA
injection.
|
|
Further studies were done in which pHi transients were measured
in control oocytes and those injected with pNBC cRNA (Fig.
6). Resting pHi was similar in
both groups of oocytes, ~7.1 After extracellular Na+
removal, in control oocytes, pHi increased by +0.008 ± 0.001 pH/min (n = 7). In contrast, in the cRNA-injected
oocytes, pHi decreased at a rate of
0.008 ± 0.001 pH/min (n = 5), p < 0.001. Na+ removal caused a similar decrease in pHi in
cRNA-injected oocytes in the absence of chloride with 10 µM EIPA;
0.010 ± 0.001 pH/min (n = 3), p = NS. DIDS (0.3 mM), significantly
decreased the Na+-induced pHi transient in
cRNA-injected oocytes to
0.0025 ± 0.0003 pH/min
(n = 3), p < 0.05.

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|
Fig. 6.
Intracellular pH measurements in
Xenopus laevis oocytes. a, removal and
readdition of Na+ in H20-injected oocytes;
b, removal and readdition of Na+ in
cRNA-injected oocytes; c, removal and readdition of
Na+ in cRNA-injected oocytes in the absence of
Cl in the presence of EIPA (10 µM); and
d, removal and readdition of Na+ in
cRNA-injected oocytes in the presence of DIDS (0.3 mM).
|
|
The Physiological Role of pNBC-mediated
Na(HCO3)n Cotransport--
The highest
level of pNBC expression was found in the pancreas, with lower levels
of expression in kidney, brain, liver, prostate, colon, stomach,
thyroid, and spinal chord. The results of the present study are
compatable with previous functional studies that have demonstrated
Na(HCO3)n cotransport in pancreatic ductal cells
and acini (3, 5-8, 39). It has been hypothesized that basolateral
Na(HCO3)n cotransport in pancreatic ductal cells
plays a modulatory role in ductal fluid secretion (5, 7). Under resting
conditions, the basolateral cotransporter would mediate cellular
bicarbonate efflux when the basolateral membrane potential is ~
70
mV (40). After stimulation of bicarbonate secretion by secretin, the
basolateral membrane voltage of rat duct cells depolarizes to ~
40
to
20 mV (40). Under these conditions, the cotransporter would
mediate bicarbonate influx (7). After stimulation by secretin,
basolateral bicarbonate uptake by guinea pig pancreatic ductal cells is
mediated in part by the basolateral Na(HCO3)n
cotransporter (5-8, 39), although a basolateral H+-ATPase
may also play a role (5, 6, 8). Two important physiological roles for
bicarbonate secretion by pancreatic centroacinar cells and ductal cells
have been proposed (44): 1) the solubilization of secreted proteins and
vesicular retrieval of secreted proteins from the acinar lumen and 2)
neutralization of the acidic chyme delivered into the upper intestine
from the stomach. In the absence of secretogogues, cellular bicarbonate
efflux via by the basolateral Na(HCO3)n
cotransporter coupled to apical Na+/H+ exchange
may mediate transepithelial H+ secretion in the main and
common pancreatic ducts (5).
The results of the present study indicate that the pNBC is also
expressed at lower levels in kidney, brain, liver, prostate, colon,
stomach, thyroid, and spinal chord. The N terminus of pNBC has a unique
consensus phosphorylation site for protein kinase A beginning at
Lys46, consensus phosphorylation sites for protein kinase C
beginning at Ser38 and Ser65, and a casein
kinase II phosphorylation site beginning at Ser68, which
kNBC lacks. Of interest, cAMP stimulates transepithelial bicarbonate
secretion and basolateral Na(HCO3)n cotransport in
pancreatic ducts (7, 45), whereas in the renal proximal tubule, cAMP
inhibits basolateral Na(HCO3)n cotransport (46).
The unique N terminus of pNBC not shared by kNBC could have an
important regulatory role in functioning as a target for phosphorylation by protein kinase A.
Na(HCO3)n cotransport has been functionally
demonstrated in pancreas (3-8), kidney (1, 2, 21, 22, 25-27, 31),
leech glial cells (17, 18), retinal Müller cells (15, 16), type
II alveolar cells (20), hepatocytes (10-12), colon (9), gastric
parietal cells (19), cardiac Purkinje fibers (13) and ventricular
myocytes (14), although the stoichiometry of the transported species
appears to be tissue-dependent. The lack of detectable
transcripts in heart and lung with any of the three probes used in this
study suggests the possibility that cardiac and lung
Na(HCO3)n cotransport is mediated by an alternative
protein(s). The finding that prostate and thyroid have transcripts that
are labeled by the pNBC probe is of interest, given that these tissues
have not been previously investigated for the presence of functional
Na(HCO3)n cotransport. Furthermore the higher
mobility of the thyroid transcript suggests that this tissue expresses
a variant of pNBC not present in other organs.
 |
ACKNOWLEDGEMENT |
We thank Dr. E. M. Wright for providing the
oocytes.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant DK46976, the Iris and B. Gerald Cantor Foundation, the Max Factor
Family Foundation, the Verna Harrah Foundation, the Richard and Hinda
Rosenthal Foundation, and the Fredericka Taubitz Foundation.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF011390 and AF020195.
Supported by National Kidney Foundation of Southern California
Training Grant J891002.
¶
To whom correspondence should be addressed: UCLA Division of
Nephrology, 10833 Le Conte Ave., Rm. 7-155 Factor Bldg., Los Angeles,
CA 90095-1689. Tel.: 310-206-6741; Fax: 310-825-6309; E-mail:
IKurtz{at}med1.medsch.ucla.edu.
1
The abbreviations used are: pHi,
intracellular pH; NBC, sodium bicarbonate cotransport protein; kNBC,
kidney NBC; pNBC, pancreatic NBC; kb, kilobases; bp, base pair(s);
DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; PCR, polymerase
chain reaction; EST, Expressed Sequence Tag; BCECF,
2',7'-biscarboxyethyl-5,6-carboxyfluorescein; TMA, tetramethylammonium; EIPA,
5-(N-ethyl-N-isopropyl)-amiloride; NS, not
significant.
 |
REFERENCES |
-
Boron, W. F.,
and Boulpaep, E. L.
(1989)
Kidney Int.
36,
392-402[Medline]
[Order article via Infotrieve]
-
Boron, W. F.,
and Boulpaep, E. L.
(1983)
J. Gen. Physiol.
81,
53-94[Abstract]
-
Muallem, S.,
and Loessberg, P. A.
(1990)
J. Biol. Chem.
265,
12806-12812[Abstract/Free Full Text]
-
Zhao, H.,
Katzumoto, U.,
Star, R. A.,
and Muallem, S.
(1994)
Am. J. Physiol.
36,
C385-C396
-
Zhao, H.,
Star, R. A.,
and Muallem, S.
(1994)
J. Gen. Physiol.
104,
57-85[Abstract]
-
Villanger, O.,
Veel, T.,
and Raeder, M. G.
(1995)
Gastroenterology
108,
850-859[Medline]
[Order article via Infotrieve]
-
Ishiguro, H., M.,
Steward, C.,
Lindsay, A. R.,
and Case, R. M.
(1996)
J. Physiol. (Lond.)
495,
169-178[Abstract]
-
de Ondarza, J.,
and Hootman, S. R.
(1997)
Am. J. Physiol.
272,
G124-G134[Abstract/Free Full Text]
-
Vazhaikkurichi, M.,
Rajendran, M.,
Oesterlin, M.,
and Binder, H. J
(1991)
J. Clin. Invest.
88,
1379-1385[Medline]
[Order article via Infotrieve]
-
Fitz, J. G.,
Persico, M.,
and Scharschmidt, B. F.
(1989)
Am. J. Physiol.
256,
G491-G500[Abstract/Free Full Text]
-
Gleeson, D.,
Smith, N. D.,
and Boyer, J. L.
(1989)
J. Clin. Invest.
84,
312-321[Medline]
[Order article via Infotrieve]
-
Weintraub, W. H.,
and Machen, T. E.
(1989)
Am. J. Physiol.
257,
G317-G327[Abstract/Free Full Text]
-
Dart, C.,
and Vaughan-Jones, R. D.
(1992)
J. Physiol. (Lond.)
451,
365-385[Abstract]
-
Lagadic-Gossman, D,
Buckler, K. J.,
and Vaughan-Jones, R. D.
(1992)
J. Physiol. (Lond.)
458,
361-384[Abstract]
-
Newman, E. A.,
and Astion, M. L.
(1991)
Glia
4,
424-428[Medline]
[Order article via Infotrieve]
-
Newman, E. A.
(1996)
J. Neurosci.
16,
159-168[Abstract]
-
Deitmer, J. W.,
and Schlue, W.-R.
(1987)
J. Physiol. (Lond.)
388,
261-283[Abstract]
-
Deitmer, J. W.
(1991)
J. Gen. Physiol.
98,
637-655[Abstract]
-
Machen, T. E.,
Townsley, T. E.,
Paradiso, A. M.,
Wenzl, E.,
and Negulescu, P. A.
(1989)
Ann. N. Y. Acad. Sci.
574,
447-462[Medline]
[Order article via Infotrieve]
-
Lubman, R. L.,
Chao, D. C.,
and Crandall, E. D.
(1995)
Respir. Physiol.
100,
15-24[CrossRef][Medline]
[Order article via Infotrieve]
-
Alpern, R. J.
(1985)
J. Gen. Physiol.
86,
613-636[Abstract]
-
Yoshitomi, K.,
Burckhardt, B.-C.,
and Frömter, E.
(1985)
Pfluegers Arch. Eur. J. Physiol.
405,
360-366[Medline]
[Order article via Infotrieve]
-
Alpern, R. J.,
and Chambers, M.
(1987)
J. Gen. Physiol.
89,
581-598[Abstract]
-
Guggino, W. B.,
London, E. L. Boulpaep,
and Giebisch, G.
(1983)
J. Membr. Biol.
71,
227-240[Medline]
[Order article via Infotrieve]
-
Kurtz, I.
(1989)
J. Clin. Invest.
83,
616-622[Medline]
[Order article via Infotrieve]
-
Nakhoul, N. L.,
Chen, L. K.,
and Boron, W. F.
(1990)
Am. J. Physiol.
258,
F371-F381[Abstract/Free Full Text]
-
Sasaki, S.,
and Yoshiyama, N.
(1988)
J. Clin. Invest.
81,
1004-1011[Medline]
[Order article via Infotrieve]
-
Romero, M. F.,
Hediger, M. A.,
Boulpaep, E. L.,
and Boron, W. F.
(1996)
J. Am. Soc. Nephrol.
7,
1259 (abstr.)
-
Romero, M. F.,
Hediger, M. A.,
Boulpaep, E. L.,
and Boron, W. F
(1997)
Nature
387,
409-413[CrossRef][Medline]
[Order article via Infotrieve]
-
Burnham, C. E.,
Amlal, H.,
Wang, Z.,
Shull, G. E.,
and Soleimani, M.
(1997)
J. Biol. Chem.
272,
19111-19114[Abstract/Free Full Text]
-
Abuladze, N.,
Lee, I,
Newman, D.,
Hwang, J,
Pushkin, A.,
and Kurtz, I.
(1998)
Am. J. Physiol.
43,
628-633
-
Jacobson, H. R.
(1981)
Am. J. Physiol.
240,
F54-F62[Medline]
[Order article via Infotrieve]
-
Schwartz, G. J.,
and Evan, A. P.
(1983)
Am. J. Physiol.
245,
F382-F390[Medline]
[Order article via Infotrieve]
-
Kuijpers, G. A. J.,
and Depont, J. J. H. H.
(1987)
Annu. Rev. Physiol.
49,
87-103[CrossRef][Medline]
[Order article via Infotrieve]
-
Case, M. R.
(1989)
Curr. Opin. Gastroenterol.
5,
665-681
-
Gray, M. A.,
Harris, A.,
Coleman, L.,
Greenwell, J. R.,
and Argent, B. E.
(1993)
Am. J. Physiol.
264,
C591-C602[Abstract/Free Full Text]
-
Stuenkel, E. L.,
Machen, T. E.,
and Williams, J. A.
(1988)
Am. J. Physiol.
254,
G925-G930[Abstract/Free Full Text]
-
Veel, T. O.,
Villanger, O.,
Holthe, M. S.,
Cragoe, E. J.,
and Raeder, M. G.
(1992)
Acta Physiol. Scand.
144,
239-246[Medline]
[Order article via Infotrieve]
-
Ishiguro, H.,
Steward, M. C.,
Wilson, R. W.,
and Case, R. M.
(1996)
J. Physiol. (Lond.)
495,
179-191[Abstract]
-
Novak, I.,
and Pahl, C.
(1993)
Pfluegers Arch. Eur. J. Physiol.
425,
272-279[Medline]
[Order article via Infotrieve]
-
Kurtz, I.
(1987)
J. Clin. Invest.
80,
928-935[Medline]
[Order article via Infotrieve]
-
Sasaki, S.,
Ishibashi, K.,
Nagai, T.,
and Marumo, F.
(1992)
Biochim. Biophys. Acta
1137,
45-51[Medline]
[Order article via Infotrieve]
-
Alper, S.
(1991)
Annu. Rev. Physiol.
53,
549-564[CrossRef][Medline]
[Order article via Infotrieve]
-
Freedman, S. D.,
and Scheele, G. A.
(1994)
Ann N. Y. Acad. Sci.
713,
199-206[Abstract]
-
Case, R. M.,
and Scratcherd, T.
(1972)
J. Physiol. (Lond.)
223,
649-667[Medline]
[Order article via Infotrieve]
-
Arruda, J. A. L.,
and Ruiz, O. S.
(1992)
Am. J. Physiol.
262,
F560-F565[Abstract/Free Full Text]
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