HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gross, E. Articles by Cotton, C. U. Search for Related Content PubMed PubMed Citation Articles by Gross, E. Articles by Cotton, C. U.

The stoichiometry of the electrogenic sodium bicarbonate cotransporter pNBC1 in mouse pancreatic duct cells is 2 HCO₃^-:1 Na⁺

E. Gross *† §, N. Abuladze, A. Pushkin, I. Kurtz and C. U. Cotton †‡

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

1. The electrogenic sodium bicarbonate cotransporter pNBC1 is believed to play a major role in the secretion of bicarbonate by pancreatic duct cells, by transporting bicarbonate into the cell across the basolateral membrane. Thermodynamics predict that this function can be achieved only if the reversal potential of the cotransporter is negative to the cell's membrane potential, or equivalently that the HCO₃^-:Na⁺ stoichiometry is not larger then 2:1. However, there are no data available on either the reversal potential or the HCO₃^-:Na⁺ stoichiometry of pNBC1 in pancreatic cells.

2. We studied pNBC1 function in mouse pancreatic duct cells. RT-PCR analysis of total RNA revealed that these cells contain the message for pNBC1, but not for kNBC1, NBC2 or NBC3.

3. To measure cotransporter activity, mouse pancreatic duct cells were grown to confluence on a porous substrate, mounted in an Ussing chamber, and the apical plasma membrane permeabilized with amphotericin B. Ion flux through pNBC1 was achieved by applying Na⁺ concentration gradients across the basolateral plasma membrane. The current through the cotransporter was isolated as the difference current due to the reversible inhibitor dinitrostilbene disulfonate (DNDS).

4. Current-voltage relationships for the cotransporter, measured at three different Na⁺ concentration gradients, were linear over a range of about 100 mV. The reversal potential data, obtained from these current-voltage relationships, all corresponded to a 2 HCO₃^-:1 Na⁺ stoichiometry.

5. The data indicate that pNBC1 is functionally expressed in mouse pancreatic duct cells. The cotransporter operates with a 2 HCO₃^-:1 Na⁺ stoichiometry in these cells, and mediates the transport of bicarbonate into the cell across the basolateral membrane.

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

One of the major functions of the exocrine pancreas is the secretion of large amounts of bicarbonate into the pancreatic juice. The high concentration of bicarbonate is required to dissolve digestive enzymes, secreted by the acinar glands, and to protect the duodenum and small intestine from the gastric acid. In humans, pigs and guinea-pigs, the bicarbonate concentration in the pancreatic duct can increase to 150 mM (Padfield et al. 1989) - about 5-fold greater than the bicarbonate concentration in the blood. This implies that bicarbonate accumulates in the pancreatic duct cells against its concentration gradient across the basolateral membrane. One mechanism for bicarbonate accumulation in these cells is by diffusion of CO₂ across the basolateral membrane, and its subsequent conversion into HCO₃^- and H⁺ by carbonic anhydrase. However, experiments in rabbits (Swanson & Solomon, 1975; Kuijpers et al. 1984), cats (Case et al. 1969, 1970), dogs (Banks & Sum, 1971), pigs (Raeder & Mathisen, 1982) and humans (Dyck et al. 1972; Anand et al. 1994) have indicated that inhibition of carbonic anhydrase with acetazolamide can only decrease bicarbonate secretion by ~50 %. These observations have led to the suggestion that the concentrative uptake of bicarbonate across the basolateral membrane in these cells is coupled to the transport of sodium down its concentration gradient.

Micropuncture experiments in the cat provided the first evidence that bicarbonate secretion in the pancreatic duct is coupled to chloride uptake across the apical membrane of duct cells (Case et al. 1969; Lightwood & Reber, 1977). This transport mode is consistent with the existence of a Cl^--HCO₃^- exchanger (AE) in the apical membrane of these cells, although the exact isoform(s) of AE has not been identified. Chloride exits the cell via an apical cystic fibrosis transmembrane conductance regulator (CFTR) (Gray et al. 1989; Crawford et al. 1991; Marino et al. 1991; Trezise & Buchwald, 1991). More recent studies with guinea-pig pancreatic ducts have demonstrated Cl^- independence of apical bicarbonate exit (Ishiguro et al. 1996).

Functional evidence for the existence of basolateral sodium-dependent bicarbonate transport in these cells comes from functional studies which have demonstrated that intracellular pH recovery is dependent on basolateral Na⁺ and HCO₃^- and can be inhibited by DIDS (Zhao et al. 1994; Villanger et al. 1995; Ishiguro et al. 1996; de Ondarza & Hootman, 1997). These results are consistent with either the presence of a Na⁺-dependent Cl^--HCO₃^- exchanger or a sodium bicarbonate cotransporter. Additional support for the existence of a sodium bicarbonate cotransporter in the pancreas has recently been provided by the cloning and characterization of the human pancreatic variant of the NBC1 gene, pNBC1 (Abuladze et al. 1998, 2000). pNBC1 has been localized to both ductal and acinar cells using in situ hybridization (Abuladze et al. 1998). More recent immunocytochemistry experiments have localized NBC1 to the basolateral surface of human duct cells (Marino et al. 1999), although the antibody used in the latter study cannot distinguish between kNBC1 and pNBC1.

As a secondary active, electrogenic, transport system, thermodynamic considerations suggest that the sodium concentration gradient could drive the transport of bicarbonate into the cell via an electrogenic sodium bicarbonate cotransporter only if the reversal potential of the cotransporter is more negative then the resting membrane potential. The reversal potential of the cotransporter is determined by the HCO₃^-:Na⁺ stoichiometry. Although pNBC1 has been shown to transport Na⁺ and HCO₃^- (Abuladze et al. 1998), and is electrogenic (Choi et al. 1999), no data are available on the stoichiometry or reversal potential of pNBC1 in pancreatic duct cells.

In the present study we demonstrate for the first time that pNBC1 in mammalian pancreatic duct cells is electrogenic with a stoichiometry of 2 HCO₃^-:1 Na⁺. The current generated by the cotransporter in these cells can be blocked by DNDS, and is Na⁺ dependent and Cl^- independent. These results suggest that pNBC1 mediates cellular bicarbonate uptake in these cells.

	METHODS

Top Abstract Introduction Methods Results Discussion References

Cell culture

Experiments were carried out with the cell line mPEC1, derived from ductal fragments of an ImmortaMouse (Takacs-Jarrett et al. 2001). Passages 30-35 were used for the present study. Cells were grown on Ethicon-coated (20 % Ethicon in 60 % ethanol) Millicell-CM filters (diameter, 30 mm) in a 1:1 mixture of Dulbecco's Modified Eagle's Medium and Ham's F12, supplemented with 10 ng ml^-1 epithelial growth factor, 0.5 mM isobutyl methyl xanthine (IBMX), 30 g ml^-1 penicillin G, 50 g ml^-1 streptomycin sulfate, 50 units ml^-1 nystatin, 10 units ml^-1 interferon , 2 mM glutamine and 2.5 % fetal bovine serum (exocrine medium). Usually, about 6 X 10⁵ cells were seeded and grown to confluence in 5 days.

Pancreatic duct isolation

Six mice were killed by exposure to a rising concentration of CO₂ and their pancreases removed and placed in ice-cold Hepes-buffered Ringer solution (HBR). The tissue was cut into small fragments and digested at 37°C in a solution of HBR that contained 0.25 mg ml^-1 collagenase I, 0.25 mg ml^-1 collagenase IV, 0.1 mg ml^-1 DNase and 0.025 mg ml^-1 soybean trypsin inhibitor. At 15 min intervals the digest was dispersed with a 10 ml pipette. After 60 min the digest was filtered through 149 m nylon mesh. The pancreatic fragments retained on the filter were collected and resuspended in a solution of rat tail collagen. After the collagen solution had formed a gel, tissue culture medium (exocrine medium supplemented with 10 % fetal bovine serum) was added and the gels were placed in a tissue culture incubator. During the next 24-48 h the ends of the ductal fragments sealed and the ducts became inflated. At this point the gel was digested with collagenase (1 mg ml^-1 each of collagenase I and IV) and the inflated ducts were collected with a glass micropipette.

Electrophysiology

Filter inserts were mounted vertically in a thermostatically controlled Ussing chamber equipped with gas inlets for CO₂ bubbling, and separate reservoirs for the perfusion of apical and basolateral compartments. Electrophysiological measurements were carried out with an epithelial voltage-clamp amplifier (EC825, Warner Instruments, Hamden, CT, USA). Data were digitized at 100 kHz and recorded through an A/D converter (PowerLab/400, AD Instruments, Castle Hill, Australia) on a Pentium PC for further analysis. Data were filtered at 0.5 Hz. Current-voltage relationships were measured by stepping the voltage command from -100 mV to +100 mV in 10 mV steps, using the stimulator utility of the Chart program (AD Instruments). Cells were continuously perfused with modified Ringer solution (for composition of solutions see Table 1), at a rate of about 3 ml min^-1. All solutions were kept under constant CO₂ pressure throughout the experiment. To measure the cotransporter-related current, cell monolayers were permeabilized with 20 M apical amphotericin B, from a 100 mM stock solution in DMSO, as described previously (Gross & Hopfer, 1996). Given that H⁺ readily equilibrates across the permeabilized membrane and assuming the cell membrane is permeable to CO₂, the concentration of HCO₃^-, at equilibrium, can be calculated from the Henderson-Hasselbalch relation: [HCO₃^-] = 3 X 10^-5 X 760 X f X 10^{(pH -6.1)}, where f is the partial pressure of CO₂ (P_CO2).

	RESULTS

Top Abstract Introduction Methods Results Discussion References

In addition to the pancreatic (pNBC1) and kidney (kNBC1) variants of the NBC1 gene, two other isoforms have recently been cloned: NBC2 from retina (Ishibashi et al. 1998), and a non-electrogenic NBC3 from skeletal muscle (Pushkin et al. 1999a,b). To check which of these clones are present in mPEC1 cells, total RNA was extracted, reverse transcribed and amplified with primers specific for pNBC1, kNBC1, NBC2 or NBC3. As illustrated in Fig. 1, mPEC1 cells express pNBC1, but not any of the other three proteins.

		View larger version [in this window] [in a new window]
Figure 1 RT-PCR amplification of mPEC1 cells reveals the message for pNBC1 Total RNA from mPEC1 cells or mouse pancreatic duct fragments was reversed transcribed using AMV reverse transcriptase (RT). Specific regions of pNBC1, kNBC1, NBC2 or NBC3 were amplified using primers as described in the Methods section. M, 1 kb ladder; lane 1 (mPEC1), pNBC1 primers, +RT; lane 2 (mPEC1), pNBC1 primers, -RT; lane 3 (mPEC1), kNBC1 primers, +RT; lane 4 (mouse kidney), kNBC1 primers, +RT; lane 5 (mouse kidney), kNBC1 primers, -RT; lane 6 (mPEC1), NBC2 primers, +RT; lane 7 (mouse testes), NBC2 primers, +RT; lane 8 (mouse testes), NBC2 primers, -RT; lane 9 (mPEC1), NBC3 primers, +RT; lane 10 (mouse heart), NBC3 primers, +RT; lane 11 (mouse heart), NBC3 primers, -RT; lane 12 (mouse pancreas), pNBC1 primers, +RT; lane 13 (mouse pancreas), pNBC1 primers, -RT.

Current-voltage relationships of pNBC1

The ionic permeability of the apical membrane of mPEC1 cells was increased with 20 M amphotericin B. This manipulation effectively removes the electrical resistance of the apical plasma membrane and renders the membrane permeable to small monovalent ions, but not to those with higher valencies, and remains restricted for several hours to the plasma membrane to which it was applied (Kirk & Dawson, 1983; Backman et al. 1992; Illek et al. 1993; Acevedo, 1994; Gross & Hopfer, 1996). Under these conditions, the electrical properties of the basolateral membrane can be monitored with extracellular electrodes.

Figure 2 shows a typical protocol for collecting current- voltage relationships. After permeabilization of the apical membrane with amphotericin B, a 5-fold sodium concentration gradient (high on the basolateral side) was applied across the basolateral membrane, while the concentration of HCO₃^- across that membrane was symmetrical. Amphotericin B permeabilizes the membrane to H⁺, and CO₂ equilibrates across the plasma membrane (both apical and basolateral) by free diffusion. Under these circumstances the concentration of bicarbonate in the cytosolic face of the basolateral membrane is clamped to that of the apical compartment. Transepithelial voltage was then stepped from -100 to +100 mV in 10 mV steps and the resulting current at each voltage measured (continuous line in Fig. 2A). The NBC inhibitor DNDS (Coppola & Fromter, 1994; Gross & Hopfer, 1996; Devor et al. 1999) was then added to the basolateral solution for 10 min before a second current- voltage relationship was collected (dotted line in Fig. 2A). The DNDS-sensitive current, i.e. the net current through pNBC1, is the difference between the current measured in the presence of DNDS and that measured in the absence of DNDS.

		View larger version [in this window] [in a new window]
Figure 2 Bicarbonate dependence of pNBC1 current A 5-fold sodium concentration gradient was applied across the cell monolayer, which had been apically permeabilized with 20 M amphotericin B, in the presence of CO2/bicarbonate (A and B) by perfusing the basolateral compartment with solution 50Na and the apical compartment with solution 10Na. A, voltage pulse protocol used to collect I-V relationships in the absence (continuous line) and presence (dotted line) of 2 mM DNDS. Voltage was stepped from -100 to +100 mV in 10 mV steps. The current at each voltage was recorded for 5 s. B, I-V relationships in the absence () and presence () of DNDS obtained by averaging the current at each voltage over 5 s. C, I-V relationships in the absence () and presence () of DNDS for a 5-fold sodium concentration gradient but in the nominal absence of CO2/bicarbonate.

Figure 2B shows the current-voltage relationships in the absence and presence of DNDS. The current at each voltage was obtained from the voltage pulse protocol shown in Fig. 2A, by averaging the current at each voltage step for 5 s.

To test whether the DNDS-sensitive current depends on the presence of bicarbonate, we repeated the experiment, this time perfusing the cells with the same solutions as in Fig. 2B but in the nominal absence of CO₂/HCO₃^-. As can be seen in Fig. 2C, withdrawal of HCO₃^- from the solutions abolished the DNDS-sensitive component of the current.

There was some concern as to whether addition of amphotericin B to the cell monolayers might destroy the polarity of the epithelial monolayer by disrupting the tight junction integrity. We thus repeated the experiment by perfusing the cells with Na⁺- and K⁺- containing solution (solution 4K in Table 1) and measured the activity of the Na⁺,K⁺-ATPase, a functional marker of the basolateral membrane, following permeabilization with amphotericin B. The Na⁺,K⁺-ATPase is a widely accepted basolateral marker in epithelial transport studies, and should thus co-localize with pNBC1 on the basolateral membrane. Figure 3 shows the effect of apical application of 20 M amphotericin B. The permeabilization process can be conveniently followed by the short-circuit current (I_SC).

After permeabilization, I_SC (equivalent to cation flux from the apical to basal compartment) increased steeply and then levelled off. Based on several criteria, the steady-state current largely represents Na⁺,K⁺-ATPase turnover. (1) The current could be reversibly inhibited by replacement of K⁺ with NMDG⁺ in both apical and basal solution (not shown). (2) It could also be reversibly inhibited by 1 mM basal ouabain (Fig. 3). (3) The current also depended on the presence of Na⁺ in the perfusion solutions (data not shown). These results demonstrate that the amphotericin-treated monolayers retained a substantial degree of cellular and monolayer integrity; however, the apical membrane became permeable to monovalent ions and thus electrically conductive.

		View larger version [in this window] [in a new window]
Figure 3 Polarized expression of the basolateral marker Na+,K+-ATPase in apically permeabilized pancreatic duct cells Cells were mounted in an Ussing chamber and the transepithelial voltage was clamped to zero. Cells were perfused initially with a solution containing K+ and Na+ (solution 4K in Table 1). At the indicated time the apical surface was permeabilized by adding 20 M amphotericin B, which resulted in an increase in short-circuit current, ISC. The increased ISC following apical application of amphotericin B reflects the activity of the Na+,K+-ATPase, as demonstrated by a decrease in ISC upon basolateral application of 1 mM of the enzyme's inhibitor ouabain, or upon removal of K+ (not shown).

To check whether any primary active transporter systems (i.e. ATPases) might contribute to the measured currents in our experiments, we also measured the DNDS-sensitive current-voltage relationship under symmetrical substrate concentrations, by perfusing both sides with solution 20Na (Table 1) containing 20 mM Na⁺ and 9 mM HCO₃. The results of this experiment are depicted in Fig. 4 (). As can be seen from the figure, the current- voltage relationship of pNBC1 under these symmetrical conditions goes through the origin (0,0), arguing against the contribution of any active transport system to the measured currents.

The stoichiometry of the Na⁺-HCO₃^- cotransporter

The HCO₃^-:Na⁺ stoichiometry of pNBC1 was calculated from the reversal potential (V_rev) and eqn (1), as described previously (Gross & Hopfer, 1996):

(1)

where n is the number of bicarbonate anions cotransported with each sodium cation, and the subscripts i and o represent intra- and extracellular concentrations of the indicated ion. R, T and F have their usual meanings.

The reversal potential

For a symmetrical HCO₃^- concentration, the reversal potential depends logarithmically on the magnitude and direction of the Na⁺ concentration gradient. We, thus, measured V_rev for pNBC1 in pancreatic duct cells from its current-voltage relationship for three different sodium concentration gradients (Fig. 4 and Table 2). Figure 4 shows the change in current (I) versus voltage for apically permeabilized monolayers, with Na⁺ concentration gradients of 20/10 (solutions 20Na/10Na in Table 1), 50/10 (solutions 50Na/10Na) and 80/10 (solutions 80Na/10Na), applied in either the APÚBL or the BLÚAP direction. I is the difference in current in the absence and presence of 2 mM DNDS and V is the transepithelial voltage. The reversal potential was determined from the intercept of the plot with the voltage axis.

		View larger version [in this window] [in a new window]
Figure 4 I-V relationships for pNBC1 in apically permeabilized monolayers Cell membranes were permeabilized with 20 M amphotericin B. I is the DNDS-sensitive current. The potential of the basal compartment was taken as zero. The ratio of apical to basolateral Na+ concentration (AP/BL) was as follows: , 10 mM/80 mM (solutions 10Na/80Na); , 10/50 (10Na/50Na); , 10/20 (10Na/20Na); , 20/20 (20Na/20Na); , 20/10; , 50/10; and , 80/10. The reversal potentials for the different gradients were evaluated graphically from the intersection of the lines with the X-axis and are tabulated in Table 2.

Table 2 summarizes the results for the reversal potentials and the calculated stoichiometries (n). The data in Table 2 are consistent with a 2 HCO₃^-:1 Na⁺ stoichiometry for each of the three concentration gradients tested.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

The exocrine pancreas secretes a bicarbonate-rich fluid into the pancreatic duct. The large concentrations of bicarbonate (up to 150 mM in humans), necessary to dissolve digestive enzymes secreted by the acinar glands, is also thought to protect the small intestine from the gastric acid that enters the duodenum. The current model for bicarbonate secretion across these cells calls for bicarbonate entry into the cell across the basolateral membrane via pNBC1. RT-PCR analysis of total RNA revealed that our cells contain the message for pNBC1 but not for kNBC1, NBC2 or NBC3. This result establishes the fact that our cell line is a suitable model for studying the biophysical and physiological properties of pNBC1 in its native, endogenous, environment. For pNBC1 to transport bicarbonate into the cell, its reversal potential should be more negative than the basolateral membrane potential. With [Na⁺]_i of 14 mM, [Na⁺]_o of 140 mM, [HCO₃^-]_o of 25 mM and [HCO₃^-]_i of 15 mM, eqn (1) predicts a reversal potential of -86 mV, for a 2 HCO₃^-:1 Na⁺ stoichiometry and -50 mV for a 3:1 stoichiometry. Thus, at a membrane potential range of -30 to -65 mV (Novak & Pahl, 1993), a cotransporter with a 2:1 stoichiometry would transport bicarbonate into the cell, while a 3:1 cotransporter (e.g. in the renal proximal tubule) would mainly transport bicarbonate out of the cell. Our results, showing a 2:1 stoichiometry for pNBC1, are consistent with the transport of bicarbonate into pancreatic duct cells by pNBC1.

In a more recent study, Shumaker et al. (1999) have suggested that impaired bicarbonate secretion in the pancreas of cystic fibrosis patients results from the inability of the duct cells to depolarize the membrane potential sufficiently in order to drive enough bicarbonate influx through pNBC1, due to the lack of active apical CFTR Cl^- channels. This proposition is based on the assumption that pNBC1 in pancreatic duct cells is electrogenic. Our results support this assumption.

In the present study, we studied the stoichiometry of pNBC1 in mammalian pancreatic epithelial cells. In a previous study we employed a similar approach to measure the stoichiometry of the renal electrogenic sodium bicarbonate cotransporter (Gross & Hopfer, 1996) and found that the cotransporter exhibited a 3 HCO₃:1 Na⁺ stoichiometry. The results of the present study suggest that pNBC1 in pancreatic cells operates with a different stoichiometry from that of kNBC1 in kidney cells. It is possible that the different stoichiometries exhibited by the two NBC1 variants are sequence related. Human pNBC1 is a 1079 amino acid polypeptide (Abuladze et al. 1998), while human kNBC1 is a 1035 amino acid polypeptide (Burnham et al. 1997). The primary structure of the two variants is almost identical except for a small region in their amino-termini. Two recent studies (Heyer et al. 1999; Sciortino & Romero, 1999) have reported that the stoichiometry of kNBC1 from rat kidney expressed in Xenopus oocytes is 2 HCO₃:1 Na⁺. As discussed above, a 2:1 stoichiometry would not be able to support bicarbonate reabsorption from the glomerular filtrate. Furthermore, this finding is in contrast to the 3:1 stoichiometry reported previously for kNBC1 in rat (Gross & Hopfer, 1996), rabbit (Yoshitomi & Fromter, 1984, 1985) and Necturus (Lopes et al. 1987) proximal tubule. It seems likely that the oocyte expression system does not mirror the environment in the mammalian proximal tubule.

Alternatively, the different stoichiometries could result from interaction(s) of the cotransporter proteins with some, as yet unknown, cell-specific regulatory factors that might be present in one organ but absent in the other. Studies are currently in progress to distinguish and test these possibilities.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

ABULADZE N., LEE, I., NEWMAN, D., HWANG, J., BOORER, K., PUSHKIN, A. & KURTZ, I. (1998). Molecular cloning, chromosomal localization, tissue distribution, and functional expression of the human pancreatic sodium bicarbonate cotransporter. Journal of Biological Chemistry 273, 17689-17695	[Abstract/Full Text]
ABULADZE N., SONG, M., PUSHKIN, A., NEWMAN, D., LEE, I., NICHOLAS, S. & KURTZ, I. (2000). Structural organization of human NBC1 gene kNBC1 is transcribed from alternative promoter in intron 3. Gene 251, 109-122	[Medline]
ACEVEDO M. (1994). Effect of acetyl choline on ion transport in sheep tracheal epithelium. Pflügers Archiv 427, 543-546	[Medline]
ANAND B. S., GOODGAME, R. & GRAHAM, D. Y. (1994). Pancreatic secretion in man: effect of fasting, drugs, pancreatic enzymes, and somatostatin. American Journal of Gastroenterology 89, 267-270	[Medline]
BACKMAN K., HARRISON, B., MEYSENBERG, M., SCHWARTZ, C. & GERMANN, W. (1992). Inactivation of a volume-sensitive basolateral potassium conductance in turtle colon: effect of metabolic inhibitors. Biochimica et Biophysica Acta 1105, 89-96	[Medline]
BANKS P. A. & SUM, P. T. (1971). Mode of action of acetazolamide on pancreatic exocrine secretion. Archives of Surgery 102, 505-508	[Medline]
BURNHAM C. E., AMLAL, H., WANG, Z., SHULL, G. E. & SOLEIMANI, M. (1997). Cloning and functional expression of a human kidney Na+:HCO3- cotransporter. Journal of Biological Chemistry 272, 19111-19114	[Abstract/Full Text]
CASE R. M., HARPER, A. A. & SCRATCHERD, T. (1969). The secretion of electrolytes and enzymes by the pancreas of the anaesthetized cat. Journal of Physiology 201, 335-348	[Medline]
CASE R. M., SCRATCHERD, T. & WYNNE, R. D. (1970). The origin and secretion of pancreatic juice bicarbonate. Journal of Physiology 210, 1-15	[Medline]
CHOI I., ROMERO, M. F., KHANDOUDI, N., BRIL, A. & BORON, W. F. (1999). Cloning and characterization of a human electrogenic Na+-HCO3- cotransporter isoform (hhNBC). American Journal of Physiology 276, C576-584	[Medline]
COPPOLA S. & FROMTER, E. (1994). An electrophysiological study of angiotensin II regulation of Na-HCO3 cotransport and K conductance in renal proximal tubule. I. Effect of picomolar concentrations. Pflügers Archiv 427, 143-150	[Medline]
CRAWFORD I., MALONEY, P. C., ZEITLIN, P. L., GUGGINO, W. B., HYDE, S. C., TURLEY, H., GATTER, K. C., HARRIS, A. & HIGGINS, C. F. (1991). Immunocytochemical localization of the cystic fibrosis gene product CFTR. Proceedings of the National Academy of Sciences of the USA 88, 9262-9266	[Abstract]
DE ONDARZA J. & HOOTMAN, S. R. (1997). Confocal microscopic analysis of intracellular pH regulation in isolated guinea pig pancreatic ducts. American Journal of Physiology 272, G124-134	[Medline]
DEVOR D. C., SINGH, A. K., LAMBERT, L. C., DELUCA, A., FRIZZELL, R. A. & BRIDGES, R. J. (1999). Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells. Journal of General Physiology 113, 743-760	[Abstract/Full Text]
DYCK W. P., HIGHTOWER, N. C. & JANOWITZ, H. D. (1972). Effect of acetazolamide on human pancreatic secretion. Gastroenterology 62, 547-552	[Medline]
GRAY M. A., HARRIS, A., COLEMAN, L., GREENWELL, J. R. & ARGENT, B. E. (1989). Two types of chloride channel on duct cells cultured from human fetal pancreas. American Journal of Physiology 257, C240-251	[Medline]
GROSS E. & HOPFER, U. (1996). Activity and stoichiometry of Na:HCO3 cotransport in immortalized renal proximal tubule cells. Journal of Membrane Biology 152, 245-252	[Medline]
HEYER M., MULLER-BERGER, S., ROMERO, M. F., BORON, W. F. & FROMTER, E. (1999). Stoichiometry of the rat kidney Na-HCO3- cotransporter expressed in Xenopus laevis oocytes. Pflügers Archiv 438, 322-329	[Medline]
ILLEK B., FISCHER, H. & CLAUSS, W. (19930). Quinidine sensitive K+ channels in the basolateral membrane of embryonic coprodeum epithelium: regulation by aldosterone and thyroxine. Journal of Comparative Physiology B 163. 556-562,
ISHIBASHI K., SASAKI, S. & MARUMO, F. (1998). Molecular cloning of new sodium bicarbonate cotransporter cDNA from human retina. Biochemical and Biophysical Research Communications 246, 535-538	[Medline]
ISHIGURO H., STEWARD, M. C., LINDSAY, A. R. & CASE, R. M. (1996). Accumulation of intracellular HCO3- by Na+-HCO3- cotransport in interlobular ducts from guinea-pig pancreas. Journal of Physiology 495, 169-178	[Abstract]
ISHIGURO H., STEWARD, M. C., WILSON, R. W. & CASE, R. M. (1996). Bicarbonate secretion in interlobular ducts from guinea-pig pancreas. Journal of Physiology 495, 179-191	[Abstract]
KIRK K. L. & DAWSON, D. C. (1983). Basolateral potassium channel in turtle colon: Evidence for single file flow. Journal of General Physiology 82, 297-313	[Abstract]
KUIJPERS G. A., VAN NOOY, I. G., DE PONT, J. J. & BONTING, S. L. (1984). The mechanism of fluid secretion in the rabbit pancreas studied by means of various inhibitors. Biochimica et Biophysica Acta 778, 324-331	[Medline]
LIGHTWOOD R. & REBER, H. A. (1977). Micropuncture study of pancreatic secretion in the cat. Gastroenterology 72, 61-66	[Abstract]
LOPES A. G., SIEBENS, A. W., GIEBISCH, G. & BORON, W. F. (1987). Electrogenic Na/HCO3 cotransport across basolateral membrane of isolated perfused Necturus proximal tubule. American Journal of Physiology 253, F340-350	[Medline]
MARINO C. R., JEANES, V., BORON, W. F. & SCHMITT, B. M. (1999). Expression and distribution of the Na+-HCO3- cotransporter in human pancreas. American Journal of Physiology 277, G487-494	[Medline]
MARINO C. R., MATOVCIK, L. M., GORELICK, F. S. & COHN, J. A. (1991). Localization of the cystic fibrosis transmembrane conductance regulator in pancreas. Journal of Clinical Investigation 88, 712-716	[Medline]
NOVAK I. & PAHL, C. (1993). Effect of secretin and inhibitors of HCO3-/H+ transport on the membrane voltage of rat pancreatic duct cells. Pflügers Archiv 425, 272-279	[Medline]
PADFIELD P. J., GARNER, A. & CASE, R. M. (1989). Patterns of pancreatic secretion in the anaesthetised guinea pig following stimulation with secretin, cholecystokinin octapeptide, or bombesin. Pancreas 4, 204-209	[Medline]
PUSHKIN A., ABULADZE, N., LEE, I., NEWMAN, D., HWANG, J. & KURTZ, I. (1999a). Cloning, tissue distribution, genomic organization, and functional characterization of NBC3, a new member of the sodium bicarbonate cotransporter family Journal of Biological Chemistry 274, 16569-16575.	[Abstract/Full Text]
PUSHKIN A., ABULADZE, N., LEE, I., NEWMAN, D., HWANG, J. & KURTZ, I. (1999b). Mapping of the human NBC3 (SLC4A7) gene to chromosome 3p22. Genomics 57, 321-322	[Medline]
RAEDER M. & MATHISEN, O. (1982). Abolished relationship between pancreatic HCO3- secretion and arterial pH during carbonic anhydrase inhibition. Acta Physiologica Scandinavia 114, 97-102
SCIORTINO C. M. & ROMERO, M. F. (1999). Cation and voltage dependence of rat kidney electrogenic Na+-HCO3- cotransporter, rkNBC, expressed in oocytes. American Journal of Physiology 277, F611-623	[Medline]
SHUMAKER H., AMLAL, H., FRIZZELL, R., ULRICH, C. D. II & SOLEIMANI, M. (1999). CFTR drives Na+-nHCO3- cotransport in pancreatic duct cells: a basis for defective HCO3- secretion in CF. American Journal of Physiology 276, C16-25	[Medline]
SWANSON C. H. & SOLOMON, A. K. (1975). Micropuncture analysis of the cellular mechanisms of electrolyte secretion by the in vitro rabbit pancreas. Journal of General Physiology 65, 22-45	[Abstract]
TAKACS-JARRETT M., SWEENEY, W. E., AVNER, E. D. & COTTON, C. U. (2001). Generation and phenotype of cell line from CF and non-CF mice that carry the H-2-Kb-ts58 transgene. American Journal of Physiology, Cell Physiology 280, C228-236.
TREZISE A. E. & BUCHWALD, M. (1991). In vivo cell-specific expression of the cystic fibrosis transmembrane conductance regulator. Nature 353, 434-437	[Medline]
VILLANGER O., VEEL, T. & RAEDER, M. G. (1995). Secretin causes H+/HCO3- secretion from pig pancreatic ductules by vacuolar-type H(+)-adenosine triphosphatase. Gastroenterology 108, 850-859	[Abstract]
YOSHITOMI K. & FROMTER, E. (1984). Cell pH of rat renal proximal tubule in vivo and the conductive nature of peritubular HCO3 exit. Pflügers Archiv 402, 300-305	[Medline]
YOSHITOMI K. & FROMTER, E. (1985). How big is the electrochemical potential difference of Na across rat renal proximal tubular cell membranes in vivo? Pflügers Archiv 405, S121-126.	[Medline]
ZHAO H., STAR, R. A. & MUALLEM, S. (1994). Membrane localization of H+ and HCO3- transporters in the rat pancreatic duct. Journal of General Physiology 104, 57-85	[Abstract]

Acknowledgements

The authors wish to thank Mrs Karen Hawkins and Drs Anoop A. Kumar and Michael B. Ganz for technical assistance and valuable comments on the manuscript. This work was supported by grants from the American Heart Association and Kidney Foundation of Ohio to Eitan Gross; NIH grants DK46976, DK58563 and DK07789, 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 to Ira Kurtz; and NIH grant DK53318 to Calvin U. Cotton. Natasha Abuladze is supported by a training grant from the National Kidney Foundation of Southern California (J891002).

Corresponding author

E. Gross: VA Medical Center, MS151(W), 10701 East Boulevard, Cleveland, OH 44106, USA.

Email: ezg{at}po.cwru.edu

This article has been cited by other articles:

				M.-H. Chang, J. DiPiero, F. D. Sonnichsen, and M. F. Romero Entry to "Formula Tunnel" Revealed by SLC4A4 Human Mutation and Structural Model J. Biol. Chem., June 27, 2008; 283(26): 18402 - 18410. [Abstract] [Full Text] [PDF]

				R. Pedrosa, N. Goncalves, U. Hopfer, P. A. Jose, and P. Soares-da-Silva Activity and Regulation of Na+-HCO3- Cotransporter in Immortalized Spontaneously Hypertensive Rat and Wistar-Kyoto Rat Proximal Tubular Epithelial Cells Hypertension, May 1, 2007; 49(5): 1186 - 1193. [Abstract] [Full Text] [PDF]

				S. Yamaguchi and T. Ishikawa Electrophysiological characterization of native Na+-HCO3- cotransporter current in bovine parotid acinar cells J. Physiol., October 1, 2005; 568(1): 181 - 197. [Abstract] [Full Text] [PDF]

				T. Yamamoto, P. Swietach, A. Rossini, S.-H. Loh, R. D. Vaughan-Jones, and K. W. Spitzer Functional diversity of electrogenic Na+-HCO3- cotransport in ventricular myocytes from rat, rabbit and guinea pig J. Physiol., January 15, 2005; 562(2): 455 - 475. [Abstract] [Full Text] [PDF]

				N. Abuladze, A. Pushkin, S. Tatishchev, D. Newman, P. Sassani, and I. Kurtz Expression and localization of rat NBC4c in liver and renal uroepithelium Am J Physiol Cell Physiol, September 1, 2004; 287(3): C781 - C789. [Abstract] [Full Text] [PDF]

				O. Bachmann, H. Rossmann, U. V. Berger, W. H. Colledge, R. Ratcliff, M. J. Evans, M. Gregor, and U. Seidler cAMP-mediated regulation of murine intestinal/pancreatic Na+/HCO3- cotransporter subtype pNBC1 Am J Physiol Gastrointest Liver Physiol, January 1, 2003; 284(1): G37 - G45. [Abstract] [Full Text] [PDF]

				E. Gross and I. Kurtz Structural determinants and significance of regulation of electrogenic Na+-HCO3- cotransporter stoichiometry Am J Physiol Renal Physiol, November 1, 2002; 283(5): F876 - F887. [Abstract] [Full Text] [PDF]

				H. Ishiguro, M.C. Steward, Y. Sohma, T. Kubota, M. Kitagawa, T. Kondo, R.M. Case, T. Hayakawa, and S. Naruse Membrane Potential and Bicarbonate Secretion in Isolated Interlobular Ducts from Guinea-pig Pancreas J. Gen. Physiol., October 29, 2002; 120(5): 617 - 628. [Abstract] [Full Text] [PDF]

				V. Gresz, T.-H. Kwon, H. Vorum, T. Zelles, I. Kurtz, M. C. Steward, C. Aalkjaer, and S. Nielsen Immunolocalization of electroneutral Na+-HCO3- cotransporters in human and rat salivary glands Am J Physiol Gastrointest Liver Physiol, August 1, 2002; 283(2): G473 - G480. [Abstract] [Full Text] [PDF]

				P. Sassani, A. Pushkin, E. Gross, A. Gomer, N. Abuladze, R. Dukkipati, G. Carpenito, and I. Kurtz Functional characterization of NBC4: a new electrogenic sodium-bicarbonate cotransporter Am J Physiol Cell Physiol, February 1, 2002; 282(2): C408 - C416. [Abstract] [Full Text] [PDF]

				D. Bok, M. J. Schibler, A. Pushkin, P. Sassani, N. Abuladze, Z. Naser, and I. Kurtz Immunolocalization of electrogenic sodium-bicarbonate cotransporters pNBC1 and kNBC1 in the rat eye Am J Physiol Renal Physiol, November 1, 2001; 281(5): F920 - F935. [Abstract] [Full Text] [PDF]

				E. Gross, K. Hawkins, A. Pushkin, P. Sassani, R. Dukkipati, N. Abuladze, U. Hopfer, and I. Kurtz Phosphorylation of Ser982 in the sodium bicarbonate cotransporter kNBC1 shifts the HCO3- : Na+ stoichiometry from 3 : 1 to 2 : 1 in murine proximal tubule cells J. Physiol., December 12, 2001; 537(3): 659 - 665. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gross, E. Articles by Cotton, C. U. Search for Related Content PubMed PubMed Citation Articles by Gross, E. Articles by Cotton, C. U.