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J Biol Chem, Vol. 273, Issue 28, 17689-17695, July 10, 1998

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

Top Abstract Introduction Procedures Results & Discussion References

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

Top Abstract Introduction Procedures Results & Discussion References

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 (pH_i)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 pH_i 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

Top Abstract Introduction Procedures Results & Discussion References

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 (pH_i)-- 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 pH_i 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 GenBank^TM have been scanned against the data base, and the following related human sequences have been identified (AF007216, P02730, P48751, U62531).

	RESULTS AND DISCUSSION

Top Abstract Introduction Procedures Results & Discussion References

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 GenBank^TM 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 GenBank^TM (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 Lys^46, 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.

View larger version (57K): [in this window] [in a new window]   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.

View larger version (90K): [in this window] [in a new window]   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 GenBank^TM (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.

View larger version (144K): [in this window] [in a new window]   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).

View larger version (182K): [in this window] [in a new window]   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).

Functional Expression of pNBC in Xenopus Oocytes-- We examined the functional properties of pNBC using measurements of pH_i, 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.

View larger version (12K): [in this window] [in a new window]   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 pH_i transients were measured in control oocytes and those injected with pNBC cRNA (Fig. 6). Resting pH_i was similar in both groups of oocytes, ~7.1 After extracellular Na⁺ removal, in control oocytes, pH_i increased by +0.008 ± 0.001 pH/min (n = 7). In contrast, in the cRNA-injected oocytes, pH_i decreased at a rate of 0.008 ± 0.001 pH/min (n = 5), p < 0.001. Na⁺ removal caused a similar decrease in pH_i 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 pH_i transient in cRNA-injected oocytes to 0.0025 ± 0.0003 pH/min (n = 3), p < 0.05. 

View larger version (11K): [in this window] [in a new window]   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 GenBank^TM/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: pH_i, 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.

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