Abstracts to Presentations for “HCO3- and CF”

listed in the order of the program schedule

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Soluble Adenylyl Cyclase as a Bicarbonate Sensor

Jochen Buck and Lonny R. Levin
Department of Pharmacology, Weill Medical College of Cornell University, New York, NY, USA

We recently described the purification and cloning of a novel form of mammalian adenylyl cyclase, the soluble adenylyl cyclase (sAC), which is structurally, molecularly, and biochemically distinct from the G protein-regulated hormone responsive transmembrane adenylyl cyclases (tmACs). sAC possesses no transmembrane domains and is insensitive to heterotrimeric G proteins and P site ligands, classic modulators of tmACs. Thus, sAC defines an independently regulated cAMP signaling system within mammalian cells.

sAC is directly stimulated by bicarbonate ion both in vivo in heterologously expressing cells and in vitro using purified protein. We found sAC to be the predominant form of AC in mammalian sperm which suggests it plays a specific role in germ cell physiology. sAC's direct activation by bicarbonate provides a mechanism for generating the cAMP required to complete the bicarbonate-induced processes necessary for fertilization, including hyperactivated motility, capacitation, and the acrosome reaction. Immunolocalization studies reveal sAC is also abundantly expressed in other tissues which respond to bicarbonate or carbon dioxide levels suggesting it may function as a general bicarbonate/CO2 sensor throughout the body. 

The Cellular Physiology of Carbonic Anhydrases

Sylvie Breton
Massachusetts General Hospital Program in Membrane Biology /Renal Unit,MGH, Boston, MA, USA

Carbonic anhydrases are zinc metalloenzymes that catalyze the reversible hydration of CO2 to form HCO3- and protons according to the following reaction: CO2 +H2O = H2CO3 = HCO3- + H+. The first reaction is catalyzed by carbonic anhydrase and the second reaction occurs instantaneously. The carbonic anhydrase (CA) gene family includes ten enzymatically active members, which are major players in many physiological processes, including renal and male reproductive tract acidification, bone resorption, respiration, gluconeogenesis, signal transduction, and formation of gastric acid 1. The newly identified CAIX (previously called MN) and CAXII are related to cell proliferation and oncogenesis 2, 3, 4. Carbonic anhydrase isozymes have different kinetic properties and they are present in various tissues 1 and in various cell compartments. CA I, II, III and VII are cytoplasmic, CA V is mitochondrial, and CA VI is present in salivary secretions. CA IV, IX, XII and XIV are membrane proteins: CA IV is a glycosyl-phosphatidylinositol-anchored protein, and CA IX, XII and XIV are transmembrane proteins.

The present work will focus on the roles of CAII and CAIV in transepithelial proton secretion and bicarbonate reabsorption processes. The localization of these isoforms in selected epithelia that are involved in net acid/base transport, such as kidney proximal tubules and collecting ducts, and tubules from the male reproductive tract will be reviewed.

Carbonic Anhydrase: In the Driver’s Seat for Bicarbonate Transport

Deborah Sterling, Reinhart A. F. Reithmeier and Joseph R. Casey
Department of Physiology University of Alberta, Edmonton, Alberta, Canada 

Carbonic anhydrases are a widely expressed family of enzymes that catalyze the reversible reaction: CO2 + H2O « HCO3- + H+ . These enzymes therefore both produce HCO3- for transport across membranes and consume HCO3- that has been transported across membranes. Thus these enzymes could be expected to have a key role in driving the transport of HCO3- across cells and epithelial layers. Plasma membrane anion exchange proteins (AE) transport chloride and bicarbonate across most mammalian membranes in a one-for-one exchange reaction and act as a model for our understanding of HCO3- transport processes. Recently it was shown that AE1, found in erythrocytes and kidney, binds carbonic anhydrase II (CAII) via the cytosolic C-terminal tail of AE1. To examine the physiological consequences of the interaction between CAII and AE1, we characterized Cl-/HCO3- exchange activity in transfected HEK293 cells. Treatment of AE1-transfected cells with acetazolamide, a CAII inhibitor, almost fully inhibited anion exchange activity, indicating that endogenous CAII activity is essential for transport. Further experiments to examine the role of the AE1/CAII interaction will include measurements of the transport activity of AE1 following mutation of the CAII binding site. In a second approach a functionally inactive CA mutant, V143Y, will be co-expressed with AE1 in HEK293 cells. Since over expression of V143Y CAII would displace endogenous wild-type CAII from AE1, a loss of transport activity would be observed if binding to the AE1 C-terminus is required for transport.

Regulation of Na-independent Cl/HCO3 Exchangers 

Seth L. Alper, Marina N. Chernova, and Andrew K. Stewart
Molecular Medicine and Renal Units, Harvard Medical School, Beth Israel Deaconess Medical Center East Campus, Boston, MA, USA

Among human bicarbonate transporters, two major gene families encode Na-independent Cl/HCO3 exchangers: the SLC4 AE anion exchanger family, and the SLC26 “sulfate permease” anion transporter family. The SLC4 AE family contains at least three genes. Mutations in the AE1 gene cause autosomal dominant spherocytic anemia and distal renal tubular acidosis of both dominant and recessive forms. The SLC26 family consists of at least 10 members, 6 of which have been characterized. Mutations in three of these cause hereditary disease, including chondrodysplasia (SLC26A2), diarrhea (A3), and goiter/ deafness syndrome (A4). Little is known about the acute regulation of these modulators of intracellular and compartmental pH and volume. We have studied the structural elements required for short-term regulation of the SLC4 Cl/HCO3 exchangers AE1, AE2, and AE3, and more recently have initiated similar studies of the Down-regulated in Adenoma / Chloride-losing Diarrhea protein (DRA/CLD), SLC26A3. We have detected regulation of some but not all of these proteins by pH, tonicity, and NH4+. Small regions or single amino acid residues crucial to these regulations have been delineated. Whereas DRA and AE1 show little sensitivity to pH over the physiological range, intracellular and extracellular acidification both inhibit AE2 and AE3. The structural requirements of AE2 inhibition by protons depend on whether only pHi is varied, or pHi change is caused by imposed change in pHo. Additional forms of regulation will also be described. 

Detection of HCO3--Cl- and Na+-H+ Exchangers in Human Airways Epithelium

Faiq J. Al-Bazzaz, (with contributions by P. K. Dudeja, N. Hafez, S. Tyagi, C. A. Gailey, M. Toofanfard, W. A. Alrefai, T. M. Nazir and K. Ramaswamy)
Medical Service (MP111), VACHCS-Westside Division, University of Illinois, Chicago, IL, USA

Various mechanisms involving translocation of Na+ and Cl- across cell membranes have been identified. These include electrogenic processes, such as Na+ and Cl- channels, and electroneutral processes, such as Na+-H+ exchange. These mechanisms play a vital role in the regulation of intracellular pH and volume, vectorial transport of these ions, and proton or HCO3- secretion in various fluids, such as gastric, intestinal, exocrine pancreatic, and renal tubular secretions. Gene families for both the Na+-H+ exchanger (NHE) and Cl--HCO3- exchanges or anion exchangers (AEs) have been identified. 

The NHE gene family has been shown to include different isoforms (NHE1 to NHE6), and NHE1, NHE2, and NHE3 isoforms are the most characterized members of this gene family. NHE1 is considered to be the ubiquitous isoform localized to the basolateral membranes of the polarized epithelial cells and involved in housekeeping functions, whereas NHE2 and NHE3 isoforms have been considered to be the epithelial isoforms localized to the apical membranes of the polarized epithelial cells. NHE3 has been shown to be an important apical isoform involved in the vectorial Na+ transport in the kidney and intestinal epithelium. 

In the respiratory system, several studies have indicated the presence of Na+-H+ and Cl--HCO3- exchange activities in lung alveolar and tracheal tissues of various species. To date, the identity of the Na+-H+ (NHE) and Cl--HCO3- exchange (AE) isoforms and their regional distribution in human airways is not known. Molecular species of the NHE gene families and their relative abundance in the human airway regions was assessed utilizing the reverse transcription polymerase chain reaction (RT-PCR) and the RNase protection assay, respectively. Organ donor lung epithelia from various bronchial regions (small, medium, large, trachea) were harvested for RNA extraction. Gene specific primers for the human NHE isoforms were utilized for RT-PCR. Our results demonstrated that the NHE1 isoform was expressed in all the regions of the human airways, whereas NHE2 and NHE3 were not detected. RNase protection studies for NHE1 utilizing glyceraldehyde 3-phosphate dehydrogenase as an internal standard, demonstrated that there were regional differences in the NHE1 mRNA levels in human airways. NHE1 mRNA levels were significantly higher in the trachea compared with those in the distal bronchial regions (P < 0.05). NHE1 mRNA levels were the highest in the trachea followed by large > medium > small bronchi. The functional significance of this gradient in expression is unclear. The NHE1 isoform, which is usually expressed in the basolateral membrane domain has previously been suggested to be involved in a number of ‘housekeeping’ functions, including maintenance of intracellular pH and regulation of cell volume and cell proliferation. We did not detect NHE2 and NHE3 isoforms which are usually localized to the apical membrane of polarized epithelia. The absence of both of these putative apical membrane isoforms, NHE2 and NHE3, in the human proximal and distal airways suggests that the neutral NaCl absorptive process (involving dual ion exchange of Na+-H+ and Cl--HCO3-) may be absent in the luminal membranes of the human airway epithelial cells. Further studies are required to confirm this conclusion because other NHE isoforms such as NHE4 and NHE5 may be localized to luminal membranes of airways. 

The AE gene family includes four structurally and functionally related anion exchangers, AE1, AE2, AE3 and AE4. The AE1 has been suggested to be erythroid whereas the AE2 isoform has been suggested to be the epithelial isoform. AE3 has been suggested to be a neuronal homologue and AE4 is apically located in b-intercalated cells of the kidney. The presence of AE2 polypeptide has been detected in rabbit lung alveoli and medium airway epithelial cells. The AE2 polypeptide has been reported to be localized to the basolateral membranes of the alveolar epithelial cell monolayers, choroid plexus epithelium, and gastric parietal cells in the human and rat kidney and not in the apical membranes in both the rabbit ileum and stomach. Recent studies have suggested the role of anion exchangers in the transport of sulfate and Cl- in bovine trachea and human bronchial cells. There is also evidence for the presence of a Cl--HCO3- exchange activity in equine trachea and a Cl--HCO3- exchange process (Na+-independent) in the rat type II alveolar epithelial cells. This activity was shown to be localized to the basolateral side of the alveolar epithelial cells grown as monolayers. To detect AE isoforms in the human tracheobronchial tree, we used gene specific primers for human AE isoforms. We detected AE2 and brain-AE3 isoforms, but we did not detect AE1 or cardiac-AE3 isoforms. The levels of AE2 mRNA were similar in large as well as small bronchi. Immunohistochemistry indicates staining for AE2 in the bronchial epithelium only, no staining was detected in non-epithelial cells. Membrane localization and detailed functional roles of the AE2 and brain-AE3 isoforms expressed in the human airways remain to be defined. 

Differential expression of the NHE1 isoform and the uniform distribution of AE2 and brain-AE3 isoforms in the human airways may have functional significance related to the airway absorption and secretion of electrolytes. 

Electroneutral Na+-Driven HCO–3 Transporters

Walter F. Boron
Department of Cell and Molecular Physiology, Yale University, New Haven, CT, USA

The known members of the HCO–3 transporter superfamily include the Cl-HCO3 exchangers (AEs) and three major groups of Na+-coupled HCO–3 transporters: (i) electrogenic Na/HCO3 cotransporters (NBCe), (ii) electroneutral Na/HCO3 cotransporters (NBCn) and (iii) Na+-driven Cl-HCO3 exchangers (NDCBE). The NBCe family plays an important role in transepithelial HCO–3 transport in the kidney and pancreas, as well as in intracellular-pH (pHi) regulation in many cells (e.g., astrocytes). The NBCn and NDCBE families are thought mainly to be important for pHi regulation in a wide variety of cells.

NBCn1, cloned from vascular smooth muscle, is expressed most highly in spleen and testis. At the amino-acid level, it is ~55% identical to NBCe. NBCn1 requires Na+ and HCO–3, but is independent of Cl–. It is unusual among family members in being poorly sensitive to DIDS. Although cotransport is electroneutral, expression of NBCn1 in oocytes is associated with a substantial Na+ current that is slowly stimulated by DIDS. This Na+ current could reflect slippage of the cotransporter.

NDCBE appears to be the major pHi regulator in many neurons. It is highly expressed in brain and testis, and is ~50% identical to NBCe1 and ~75% identical to NBCn1. NDCBE requires Na+, HCO–3 and Cl–. It is electroneutral, not associated with any channel activity, and is highly sensitive to DIDS. 36Cl-flux measurements show that the unidirectional Cl– efflux requires external Na+ and HCO–3, and blocked by DIDS. Using Na+ electrodes to measure the DIDS-sensitive net Na+ efflux, we found that the HCO–3 to Na+ stoichiometry is 2:1, as expected for a Na+-driven Cl-HCO3 exchanger.

Impaired Pancreatic Ductal Bicarbonate Secretion in Cystic Fibrosis

Manoocher Soleimani
Division of Nephrology and Hypertension Department of Medicine and Veterans Affairs Medical Center at Cincinnati, University of Cincinnati Medical Center, Cincinnati, OH, USA

Patients with cystic fibrosis demonstrate a defect in HCO3- secretion by their pancreatic duct cells. However, attempts toward understanding or correcting this defect have been hampered by a lack of knowledge regarding the cellular and molecular mechanisms mediating HCO3- transport in these cells. Recent functional and molecular studies indicate a major role for a basolateral electrogenically-driven Na+: HCO3- cotransporter (NBC1) in mediating the transport of HCO3- into the duct cells. The HCO3- exits at the lumen predominantly via two recently discovered apical HCO3- transporters. cAMP, which mediates the stimulatory effect of secretin on pancreatic ductal HCO3- secretion, potentiates the basolateral Na+: HCO3- cotransporter due to generation of a favorable electrogenic gradient as a result of membrane depolarization by Cl--secreting CFTR. Two apical HCO3- transporters drive the secretion of bicarbonate into the pancreatic duct lumen. Recent studies indicate that CFTR upregulates the expression of these two apical HCO3- transporters. In addition, CFTR may distinctly upregulate the expression of certain water channels and facilitate the secretion of fluid into the duct lumen. In brief, current research suggests that the defect in pancreatic HCO3- secretion in patients with cystic fibrosis is multifactorial and involves the alteration in the function/expression of transporters at the basolateral and luminal membrane domains of the duct cells. 

CFTR and Regulation of Golgi pH

Grischa Chandy, Minnie Wu, Michael Grabe, Hsiao-Ping Moore, Terry E. Machen
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA, USA

CFTR conducts both Cl and HCO3, and CFTR could regulate pH of both the airway surface liquid (pHASL) and also the Golgi (pHG. For example, by conducting HCO3 from airway cells into the ASL, CFTR may affect pHASL and the activities of antibacterial factors and bacterial-secreted products. CFTR may also affect pHG through its ability to conduct Cl and thereby control membrane potential and the ability of the H-ATPase to acidify the Golgi lumen. It has been proposed that pHG of CF cells is alkaline compared to normal, that this altered pH affects sialyltransferase and other enzymes controlling biochemical composition of the plasma membrane, and that altered surface biochemistry increases bacterial binding. We generated a number of plasmids encoding chimera proteins that were transfected into HeLa cells and also CF (JME, CFT1) and normal or CFTR-corrected (HBE, CFT1-CFTR) airway epithelial cells. Ratio imaging microscopy and the pH-sensitivity of the chimeras were used to measure pHG. A GFP-sialyltransferase chimera that localized to the Golgi showed that all the cells (in HCO3/CO2-buffered solutions) had pH = 6.4 – 6.7, and there was no correlation between the presence or absence of CFTR and pHG. Thus, CFTR seemed not to be involved in controlling pHG. These and other experiments using an avidin-galactosyltransferase chimera in combination with a membrane-permeant, pH-sensitive biotin indicated that pHG was controlled not by Cl permeability and membrane potential, but by the H-ATPase and a H leak. These data and those from other labs indicate that the CFTR likely plays a minor role in organelle pH regulation. 

Functional Interactions of HCO3- with CFTR

Mike Gray, Catherine O’Reilly#, John Winpenny* and Barry Argent
Department of Physiological Sciences, University Medical School, Newcastle upon Tyne, UK, #Biomedical Imaging Group, Department of Physiology, University of Massachusetts Medical Centre, Worcester, MA, USA, and *School of Health Sciences, University of Sunderland, Sunderland, UK 

Disruption of normal CFTR-mediated Cl- transport is associated with cystic fibrosis. CFTR is also required for HCO3- transport in many tissues such as the lungs, G.I tract, and pancreas, although the exact role CFTR plays is uncertain. Given the importance of CFTR in HCO3- transport by so many CF-affected organ systems, it is perhaps surprising that relatively little is known about the interactions of HCO3- ions with CFTR. We have used patch clamp recordings from native pancreatic duct cells to study HCO3- permeation and interaction with CFTR. Ion selectivity studies show that CFTR is between 3-5 times more selective for Cl- over HCO3-, making it unlikely that significant amounts of HCO3- permeate through CFTR. In addition, we also find that extracellular HCO3- has a novel inhibitory effect on cAMP-stimulated CFTR currents carried by Cl-. The block by HCO3- was rapid, relatively independent of voltage and occurred over a physiological range of HCO3- concentrations. These data show that luminal HCO3- acts as a potent regulator of CFTR, and suggests that inhibition involves an external anion-binding site on the channel. This work has implications not only for elucidating mechanisms of HCO3- transport in epithelia, but also for approaches used to treat CF.

Coordination of Pancreatic HCO3- Secretion by Protein-Protein Interactions Between Membrane Transporters 

Kyung Hwan Kim1, Wooin Ahn1, Jin Ah Lee1, Shmuel Muallem2, and Min Goo Lee1
1Department of Pharmacology and BK 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea, and 2the Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA

We studied the regulatory interactions between CFTR and the HCO3- salvage mechanism of NHE3 using molecular, biochemical and functional approaches. CFTR, NHE3, and EBP50, a scaffolding protein which has two PDZ domains, were co-immunoprecipitated from PS120 cells and mouse pancreas and were co-localized in the luminal area of the pancreatic duct cells. We found that CFTR regulates NHE3 activity by both acute and chronic mechanisms. Acutely, CFTR augmented the cAMP-dependent inhibition of NHE3 in both PS120 cells and pancreatic ducts. In a chronic mechanism, CFTR increases expression of NHE3 in the luminal membrane of pancreatic duct cells. These findings reveal that CFTR controls the overall HCO3- homeostasis by regulating both HCO3- secretory and salvage mechanisms. In addition, protein complexes on the plasma membrane of duct cells are highly organized for efficient HCO3- secretion. 

Microelectrode and Impedance Analysis of Anion Secretion in CALU-3 Cells

Tsutomu Tamada, Martin J. Hug1, Raymond A. Frizzell and Robert J. Bridges2
1Institut für Physiologie, Abt. Vegetative Physiologie, Westfälische Wilhelms Universiät Münster, Münster, Germany, and 2Cell Biology and Physiology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA


Calu-3 cells secrete bicarbonate in response to cAMP agonists but can be stimulated to secrete chloride with potassium channel activating agonists. Microelectrode and impedance analysis experiments were performed to obtain a better understanding of the conductances and driving forces involved in these different modes of anion secretion in Calu-3 cells. Microelectrode studies revealed an apical and basolateral membrane depolarization upon the addition of forskolin (Vap –51 mV vs. –22 mV; Vbl –59 mV vs. –43 mV) that paralleled the hyperpolarization of the mucosal negative transepithelial voltage (Vt –8 mV vs. –21 mV). These changes were accompanied by a decrease in the apical membrane fractional resistance (FRa) from approximately 0.50 to 0.06, consistent with the activation of an apical membrane conductance. The subsequent addition of 1-EBIO, a potassium channel activator, hyperpolarized Vap to –27 mV, Vbl to –58 mV and Vt to –31 mV. Impedance analysis revealed the apical membrane resistance (Rap) of the forskolin-stimulated cells was less than 20 ohms cm2, indeed in many monolayers Rap fell to less than 5 ohms cm2. The impedance derived estimate of the basolateral membrane resistance (Rbl) was approximately 170 ohms cm2 in forskolin treated cells and fell to 50 ohms cm2 with the addition of 1-EBIO. Using these values for the Rbl and the FRa value of 0.06 yields a Rap of approximately 10 ohms cm2. Thus, by two independent methods, forskolin-stimulated Calu-3 cells are seen to have a very high apical membrane conductance of greater than 100 mS/cm2. Therefore, we would assert that even at one-tenth the anion selectivity for chloride, this high conductance could support the conductive exit of bicarbonate across the apical membrane. We further propose that this high apical membrane conductance serves to clamp the apical membrane potential at the equilibrium potential for chloride and thereby provides the driving force for bicarbonate secretion in forskolin-stimulated Calu-3 cells. The hyperpolarization caused by 1-EBIO provides a driving force for chloride exit across the apical membrane, inhibits the influx of bicarbonate on the Na:HCO3 cotransporter across the basolateral membrane, activates the basolateral membrane Na:K:2Cl cotransporter and thereby provides the switch from bicarbonate secretion to chloride secretion. 

Studies of HCO3- Transport in Relation to Fluid Secretion from Submucosal Glands

Nam Soo Joo, Mauri Krouse & Jeffrey J. Wine
Cystic Fibrosis Research Laboratory, Stanford University, Stanford, CA, USA


The role of HCO3- transport in relation to fluid secretion by submucosal glands was studied in sheep by replacing HCO3- with HEPES and nominally eliminating CO2 in the gassing. In response to HCO3- removal, basal mucus secretion was reduced to 45 ± 14% of control (39 glands from 3 sheep, P < 0.01) and peak, carbachol-stimulated secretion was reduced to 33 ± 10% of control, (33 glands from 3 sheep, P < 0.01). The remaining secretory response to carbachol was essentially eliminated by bumetanide. To determine if part of the reduction might result from intracellular acidification, we replaced HCO3- with either 25 mM HEPES or 1 mM HEPES + 24 mM NaCl. These produced similar levels of inhibition (60-75%) respectively. Measurements of mucus pH indicate that HCO3- levels in mucus generated with 25 mM bath HCO3- are <25 mM. We hypothesize that HCO3- transport is important for mucus secretion, but that the mechanism is not simply HCO3--mediated fluid secretion.

pH Regulation and Bicarbonate Transport of Isolated Porcine Submucosal Glands

Martin J. Hug1 and Robert J. Bridges2
1Institut für Physiologie, Abt. Vegetative Physiologie, Westfälische Wilhelms Universiät Münster, Münster, Germany, and 2Cell Biology and Physiology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA 

We have previously demonstrated that the airway serous cell line Calu-3 employs a number of pH regulatory mechanisms required for bicarbonate secretion by these cells. The aim of the present study was to investigate the pH regulatory mechanisms of serous cells of freshly isolated submucosal glands (SMG). Pig SMG were dissected out of pig tracheas obtained from a local slaughterhouse. Single glands were transferred into the chamber of an inverted microscope, immobilized by two holding pipettes and the serous cells loaded with the fluorescent pH probe BCECF. Fluorescence was monitored from small areas consisting of up to 20 cells. The fluorescence ratio of the emission after excitation at 488nm and 436nm respectively was used to estimate cytosolic pH (pHi). Resting pHi of SMG cells in the absence of HCO3/CO2 was 7.1 ± 0.06 (n=24). Addition of a HCO3/CO2 buffered solution to the bath acidified the cells by 0.18 ± 0.03 (n=18). pHi rapidly recovered to a slightly more alkaline value than baseline pHi. Removal of the HCO3/CO2 buffer strongly alkalinized SMG cells by 0.2 ± 0.03 (n=18). To challenge pH regulatory mechanisms we exposed the cells to 20 mmol/l NH4+ in the absence and presence of HCO3/CO2. In both cases we observed a rapid increase in pHi followed by a slight recovery. Washout of NH4 strongly acidified the cells. Realkalinization of pHi could only be observed in the presence of Na. This effect was inhibited by the addition of the specific NHE1 blocker (HOE 694, 10 –100 micromol/l) with an IC50 of approximately 20 micromol/l. Full recovery of pHi in the presence of HOE 694 was observed when the cells were bathed in HCO3/CO2 solution. Addition of Forskolin (5 micromol/l) in the presence of HCO3/CO2 did not significantly alter pHi or change pHi recovery after acid loading. We conclude that SMG cells possess both HCO3 dependent and HCO3 independent pHi regulatory mechanisms that require the presence of extracellular Na. Further studies are required to understand whether the bicarbonate transported to regulate pHi is linked to the overall secretory capacity of SMG serous cells.

Pancreatic Ductal Bicarbonate Secretion: Past, Present and Future

Maynard Case1, Hiroshi Ishiguro2 & Martin Steward1
1University of Manchester, School of Biological Sciences, Manchester, UK 2Research Center of Health, Physical Fitness and Sports, Nagoya University, Japan

When stimulated with secretin, pancreatic ducts secrete an isotonic HCO3--rich fluid. It is widely believed that HCO3- is generated from CO2 in the epithelial cells by a basolateral Na+/H+ exchanger (NHE) working in conjunction with carbonic anhydrase (CA). HCO3- efflux across the luminal membrane is thought to occur by exchange for Cl- on an anion exchanger (AE) working in parallel with the CFTR Cl- channel. However, this model is based largely on studies of rat pancreatic ducts which secrete a mixture of Cl- and HCO3-, the latter achieving concentrations generally no higher than about 75 mM. In the guinea-pig and many other species, the ducts secrete HCO3- at a much higher concentration, often approaching 150 mM. This appears to be true even in the nominal absence of luminal Cl- and it can be argued on thermodynamic grounds that such HCO3- concentrations could not be achieved by the luminal transport mechanism described above. While basolateral HCO3- uptake undoubtedly involves the NHE and CA, in guinea-pig ducts it also involves a Na+-HCO3- cotransporter (NBC). Since the NBC is electrogenic, this could contribute significantly to an electrochemical gradient supporting passive HCO3- efflux across the luminal membrane. However, the identity of such an efflux pathway remains unclear. The involvement of the AE at the luminal membrane can probably be excluded for two reasons. Firstly, measurements of intracellular pH and Cl- concentration in microperfused guinea-pig ducts confirm that the Cl- and HCO3- concentration gradients across the luminal membrane would favour HCO3- reabsorption rather than secretion. Secondly, and perhaps fortunately, it seems that the luminal membrane AE becomes inactive when the luminal concentration of HCO3- reaches concentrations of 125 mM or above. In contrast to earlier studies on rat ducts, the membrane potential Em in guinea-pig duct cells does not depolarise appreciably upon stimulation and remains at –60 to –70 mV. This suggests that, even with 125 mM or more HCO3- in the lumen and an estimated 20 mM in the cytoplasm, there is still an electrochemical gradient favouring HCO3- secretion to the lumen. Under the same conditions, the intracellular Cl- concentration drops to very low levels (approximately 7 mM) presumably because, although Cl- may leave freely through the CFTR channels in the luminal membrane, there is no significant pathway for Cl- uptake across the basolateral membrane. Consequently the competition between HCO3- and Cl- for efflux via an anion conductance at the luminal membrane would tend to favour HCO3- secretion. Whether CFTR provides such a pathway for HCO3- remains to be seen.

CFTR-Mediated, Cl--Dependent HCO3- Transport 

Joo Young Choi, Shigeru B. H. Ko, Kenichi Ishibashi+, Daniella Muallem, Philip Thomas, Min Goo Lee# and Shmuel Muallem. 

Department of Physiology, UT Southwestern Medical Center, Dallas; +Department of Pharmacology, Jichi Medical School, Kawachi-gun, Tochigi-ken, Japan, and #Department of Pharmacology, Yonsei University College of Medicine, Seoul, Korea

That abnormal Cl- channel activity by CFTR leads to CF is now amply established. However, identification of CF-causing mutants with normal Cl- channel activity indicates that other CFTR-dependent processes contribute to the disease. Indeed, CFTR regulates other transporters, including Cl--coupled HCO3- transport. The alkaline fluids secreted by normal and acidic fluids by mutant CFTR-expressing tissues point to the importance of this activity. Therefore, we examined Cl--coupled HCO3- transport by CFTR mutants that retain substantial or normal Cl- channel activity. We found that mutants reported to cause CF with pancreatic insufficiency (PI) did not support HCO3- transport and those associated with pancreatic sufficiency (PS) showed reduced HCO3- transport. These findings demonstrate the importance of HCO3- transport in the function of secretory epithelia and in CF. 

The association of Cl--coupled HCO3- transport with CF prompted us to study the possible mechanism of this transport. When expressed in heterogonous systems, CFTR can function as a Cl- and HCO3- channel. However, when stimulated with protein kinase A (PKA), the CFTR-supported Cl--dependent HCO3- transport does not conform to ion flow through a conductive pathway. The mechanism of this activity and the protein that mediates the transport are not known. We expressed all known anion exchangers (AE1,2,3,4) with CFTR and used mutations in CFTR and transmembrane domains (TM) one and two constructs of CFTR to conclude that a) CFTR does not stimulate the activity of a known AE. b) CFTR itself mediates the Cl--coupled HCO3- transport. c) TM1 of CFTR mediates Cl- channel activity whereas TM2 mediates the Cl--dependent HCO3- transport. d) The CFTR-supported Cl--dependent HCO3- transport has properties of coupled Cl-/HCO3- exchange.

150 mM HCO3- - How Does the Pancreas Do it? Clues From Computer Modelling of the Duct Cell

Y. Sohma1, M.A. Gray2, Y. Imai1 & B.E. Argent2

1Department of Physiology, Osaka Medical College, Takatsuki, Osaka, Japan, and 2 Department of Physiological Sciences, University Medical School, Newcastle upon Tyne, UK

The pancreatic duct secretes near isotonic NaHCO3. Experimental data suggests that HCO3- secretion occurs via Cl-/HCO3- exchangers working in parallel with Cl- channels (CFTR and CaCC). As yet, no other HCO3- transporters have been discovered on the apical membrane of the duct cell. Programming the currently available experimental data into our computer model shows that while the anion exchanger/Cl- channel mechanism will produce a relatively large volume of a HCO3--rich fluid, it can only raise the luminal HCO3- concentration up to about 70 mM. To achieve secretion of ~150 mM NaHCO3 it is necessary to: (i) reduce the conductive Cl- permeability and increase the conductive HCO3- permeability of the apical membrane, and (ii) reduce the activity of the apical Cl-/HCO3- exchangers. Under these conditions most of the HCO3- is secreted via a conductive pathway. We propose that HCO3- secretion occurs mainly by the exchanger in duct segments near the acini (luminal HCO3- concentration up to ~70 mM), but mainly via channels further down the ductal tree (raising luminal HCO3- to ~150 mM). We speculate that the switch between these two secretory mechanisms is controlled by a series of luminal signals (e.g. pH, HCO3- concentration) acting on the apical membrane transporters. 

Na+HCO3- Cotransport in Normal and CF Intestine 

U. Seidler, O. Bachmann, P. Jacob, S. Christiani, H. Rossmann
Dept. of Medicine, University of Tubingen, Tubingen,Germany

All segments of the intestine secrete HCO3- ions, albeit at very different secretory rates. In a search for the HCO3- supply mechanisms to the enterocyte we cloned and sequenced an intestinal subtype of the Na+HCO3- cotransporter isoform I (dNBC1). Within the intestine, we found particularly high NBC1 expression levels in the duodenum and proximal colon. Experiments with stripped rabbit duodenum in Ussing-chambers revealed that Na+HCO3- cotransport (NBC) and CO2 hydration/Na+/H+exchange were equally important duodenal HCO3- supply pathways and were both upregulated during cAMP-mediated secretion. In the proximal colon, however, where HCO3- secretion was low but NBC1 expression even higher than in the duodenum, NBC, coupled to basolateral Cl-/HCO3- exchange, was an important alternative Cl- supply pathway to Na+K+2Cl- cotransport (NKCC) during cAMP-stimulated colonic Cl- secretion. In order to assess whether a cellular increase in cAMP stimulated NBC and NKCC rates directly or via a change in cellular volume and/or [Cl-]i,, we fluorometrically assessed NBC and NKCC transport rates, cell volume and [Cl-]i before and during forskolin-stimulation in isolated colonic crypts from normal and CFTR -/- mice. Although forskolin stimulation decreased cell volume and [Cl-]i only in normal, not in CFTR -/- crypts, it activated NBC and NKCC to a similar degree in both normal and CFTR - /- crypts. We conclude that, depending on the intestinal segment, NBC1 plays an important role in basolateral HCO3- or Cl- uptake, and is directly stimulated by cAMP.

Bicarbonate Secretion in the Murine Gallbladder – Lessons for the Treatment of Cystic Fibrosis

Alan W. Cuthbert
Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, UK

The epithelium lining the gallbladder of mammalian species has absorptive and secretory functions. An important function is the secretion of a bicarbonate rich fluid that helps neutralise stomach acid and provides an appropriate environment for intestinal enzymes. In cystic fibrosis (CF) this secretory function is lost. This study concerns the bicarbonate secreting activity of murine gallbladders in vitro using wild type and CF mice and four main questions are considered as follows: (a) Does the murine gallbladder secrete bicarbonate electrogenically and is this prevented in CF? (b) Can the secretory activity in CF gallbladders be restored by gene therapy or pharmacologically? (c) How is the cystic fibrosis transmembrane conductance regulator (CFTR) involved in bicarbonate secretion? (d) Does the data offer prospects for the treatment of CF? Work from both, the author's laboratory and the literature, will be reviewed. Consideration of the currently available data indicates that the wild type murine gallbladder does secrete bicarbonate electrogenically and that this is absent in CF mice. Further it has been demonstrated that bicarbonate secretory activity can be restored by both gene therapy and by the use of drugs. The role of CFTR in bicarbonate secretion remains equivocal. Much evidence suggests that CFTR can act as a channel for HCO3 ions as well as Cl ions, while others propose a parallel arrangement of CFTR with a Cl-/HCO3- exchanger is necessary. The matter is further complicated by the regulatory role of CFTR on other transporting activities. Opportunities for possible application to man are discussed.

Intestinal HCO3- Secretion in Cystic Fibrosis Mice

Lane L. Clarke, Xavier Stien and Nancy M. Walker
Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO, USA

Gene-targeted disruption of the cystic fibrosis transmembrane conductance regulator (CFTR) in mice results in an intestinal disease phenotype that is remarkably similar to bowel disease in cystic fibrosis (CF) patients. In the intestinal segment downstream from the stomach (i.e., the duodenum), CFTR plays an important role in bicarbonate secretion that protects the epithelium from acidic gastric effluent. In this report, we examine the role of CFTR in cAMP-stimulated bicarbonate secretion in the murine duodenum and the mechanisms of acid-base transport that are revealed by the CF condition. Ion substitution, channel blocker and pH stat studies comparing duodena from wild-type and CF mice indicate that CFTR mediates a HCO3- conductance across the apical membrane of the epithelium. In the presence of a favorable cell-to-lumen HCO3- gradient, the CFTR-mediated HCO3- current accounts for ~ 80 % of stimulated HCO3- secretion. Exposure of the duodenal mucosa to acidic pH reveals another role of CFTR in facilitating HCO3- secretion via an electroneutral, DIDS-sensitive Cl-/HCO3- exchange process. In CF duodenum, other apical membrane acid-base transporters retain function, thereby affording limited control of transepithelial pH adjustment. Activity of a Cl--dependent anion exchanger provides near-constant HCO3- secretion in CF intestine, but under basal conditions the magnitude of secretion is lessened by simultaneous activity of a Na+/H+ exchanger (NHE). During cAMP stimulation of CF duodenum, a small increase in net base secretion is measured but the change results from cAMP inhibition of NHE activity rather than increased HCO3- secretion. Interestingly, a small inward current that is sensitive to the anion channel blocker, NPPB, is also activated during cAMP stimulation of the CFTR-null intestine but the identity of the current is yet to be resolved. Studies to identify the proteins involved in non-CFTR mediated HCO3- secretion are on-going and potentially will provide targets to correct deficient HCO3- secretion in the CF intestine.

Duodenal Mucosal Bicarbonate Secretion 

Jon I. Isenberg, Birgitta Mårtensson, Vijaya Pratha, Gunnar Flemström# and Daniel L. Hogan
Department of Medicine, University of California at San Diego, San Diego, CA #Physiology Department, University of Uppsala Biomedical Center, Uppsala, Sweden

In spite of gastric [H+] reaching >130 mM in humans, there is a sharp (6 log units) increase in luminal pH from the gastric antrum to the most proximal duodenum. This is in large part due to duodenal epithelial HCO3- secretion. This presentation represents a gradual progression over the past 15 years. Initial studies revealed that the in vivo human duodenum secreted HCO3- at rest (approximately 1 mmole/hour) and identified a number of agonists (that increase HCO3- output by about 2-fold) including: luminal acidification, prostaglandin E2, cAMP, and VIP; inhibitors, such as: acetazolamide, non-steroidal anti-inflammatory drugs, cigarette smoke and others. Of clinical relevance, duodenal ulcer patients infected with Helicobacter pylori (HP) have impaired epithelial HCO3- secretion; and, after eradication of HP, duodenal epithelial HCO3- returns towards normal. This work has been extended to quantitation of human duodenal HCO3- transport in vitro. Of note, brief bathing of normal human duodenal mucosa with HP water extract also causes a diminution in basal and stimulated epithelial HCO3- secretion. Thus, there appears to be an unidentified constituent of pathogenic strains of HP that impair duodenal HCO3- transport. To define the cellular acid/base transporters present and their respective roles in duodenal epithelial HCO3- transport experiments, were performed with BCECF-loaded isolated duodenal enterocytes from rat, rabbit and human. At least four major duodenal acid/base transporters are present: i) amiloride-sensitive NHEs which extrude acid; ii) a basolateral Na:(n)HCO3 cotransporter which imports HCO3-; iii) an apical Cl-/HCO3- exchanger which exports HCO3-; and, iv) CFTR, and potentially other anion conductances, which conduct HCO3-. We have recently focused on attempting to unravel the role(s) of CFTR in duodenal HCO3- transport utilizing: CFTR (-/-) mice duodenum in vivo, human duodenal biopsies obtained from patients with cystic fibrosis (CF), as well as CFTR and CFTR-DF508 transfected cells. In brief, these studies indicate that CFTR conducts HCO3- that is stimulated further (pHi/t) by agonists of cAMP or cGMP (STa). STa stimulated HCO3- secretion in CF mucosae both in the in vitro human and in vivo CFTR (-/-) mouse duodenum via mechanisms that require further study. Further, utilizing both cell-attached and excised patches of duodenal enterocytes (rat and human) there appears to be a linear conductance of about 4.8 pS stimulated by ATP and inhibited by glibenclamide suggesting functional CFTR in duodenal surface epithelial cells. These will be discussed further.

Duodenal Intracellular Bicarbonate and the ‘CF Paradox’

Jonathan D. Kaunitz
Department of Medicine,UCLA School of Medicine, West LA VAMC, Los Angeles, CA, USA

Duodenal HCO3- secretion, which is believed to neutralize acid within the mucus gel, is the most studied defense mechanism. In general, HCO3- secretion rate and mucosal injury susceptibility correlate closely. Recent studies suggest that luminal acid can lower intracellular pH (pHi) of duodenal epithelial cells and that HCO3- secretion is unchanged during acid stress. Furthermore, peptic ulcers are rare in cystic fibrosis (CF), although, with impaired HCO3- secretion, increased ulcer prevalence is predicted, giving rise to the ‘CF Paradox’. We thus tested the hypothesis that duodenal epithelial cell protection occurs as the result of pHi regulation rather than by neutralization of acid by HCO3- in the pre-epithelial mucus. Cellular acidification during luminal acid perfusion, and unchanged HCO3- secretion during acid stress are inconsistent with pre-epithelial acid neutralization by secreted HCO3-. Furthermore, inhibition of HCO3- secretion by NPPB despite preservation of pHi and protection from acid-induced injury further question the pre-epithelial acid neutralization hypothesis. This decoupling of HCO3- secretion and injury susceptibility by NPPB (and possibly by CF) further suggest that cellular buffering, rather than HCO3- exit into the mucus, is of primary importance for duodenal mucosal protection, and may account for the lack of peptic ulceration in CF patients.

cAMP Stimulation of HCO3- Secretion Across Airway Epithelia

Michael J. Welsh and Jeffrey J. Smith
HHMI and University of Iowa College of Medicine, Iowa City, IA, USA

To test for the presence of HCO3- transport across airway epithelia, we measured short-circuit current in primary cultures of canine and human airway epithelia bathed in a Cl--free, HCO3-/CO2-buffered solution. cAMP agonists stimulated a secretory current that was likely carried by HCO3- because it was absent in HCO3--free solutions. In addition, the cAMP-stimulated current was inhibited by the carbonic anhydrase inhibitor, acetazolamide, and by the apical addition of a blocker of CFTR, diphenylamine-2-carboxylate. The current was dependent on Na+ because it was inhibited by removing Na+ from the submucosal solution and by inhibition of the Na+-K-ATPase with ouabain. The cAMP-stimulated current was absent in cystic fibrosis (CF) airway epithelia. These data suggest that cAMP agonists can stimulate HCO3- secretion across airway epithelia and that CFTR may provide a conductive pathway for HCO3- movement across the apical membrane.

Selective Activation of CFTR Cl- and HCO3- Conductances
M.M. Reddy# and P. M. Quinton#*

#Department of Pediatrics, UCSD School of Medicine, La Jolla, CA, USA, and *Biomedical Sciences, UC Riverside, Riverside, CA, USA


While CFTR is well known to function as a Cl- channel, some mutations in the channel protein causing cystic fibrosis (CF) disrupt another vital physiological function, HCO3- transport. Pathological implications of derailed HCO3- transport are clearly demonstrated by the pancreatic destruction that accompanies certain mutations in CF. Despite the crucial role of HCO3- in buffering pH, little is known about the relationship between the cause of CF pathology and the molecular defects arising from specific mutations. Using electrophysiological techniques on basilaterally a-toxin permeabilized preparations of microperfused native sweat ducts, we investigated whether: a.) CFTR can act as a HCO3- conductive channel, b.) different conditions for stimulating CFTR can alter its selectivity for HCO3- vs. Cl-, and c.) pancreatic insufficiency correlates with HCO3- conductance in different CFTR mutations. We show that under some conditions, CFTR can conduct HCO3-. HCO3- conductance in the apical plasma membranes of the sweat duct appears to be mediated by CFTR and not by any other Cl- channel because HCO3 conductance is abolished when CFTR is: a.) deactivated by removing cAMP and ATP, b.) blocked by application of 1 mM DIDS in the cytoplasmic bath, and c.) absent in the plasma membranes of DF508 CF ducts. Further, the HCO3-/Cl- selectivity of CFTR appears to be dependent on the conditions of stimulating CFTR. That is, CFTR activated by cAMP+ATP appears to conduct both HCO3- and Cl- (with an estimated selectivity ratio of 0.2 to 0.5). However, we found that in the apparent, complete absence of cAMP and ATP, cytoplasmic glutamate can activate CFTR Cl- conductance without any HCO3- conductance. Glutamate activated CFTR can be induced to conduct HCO3- by the addition of ATP without cAMP. The non-hydrolysable AMP-PNP cannot substitute for ATP in activating this HCO3- conductance. We also found that a heterozygous R117H/DF508 CFTR sweat duct retained significant HCO3- conductance while a homozygous DF508/DF508 CFTR sweat duct showed virtually no HCO3- conductance. While we suspect that the conditions described here are not optimal for selectively activating CFTR Cl- and HCO3- conductances, we surmise that CFTR may be subject to dramatic alterations in its conductance to at least these two anions under physiological conditions which require distinctly different physiological functions. That is, physiologically, CFTR may exhibit Cl- conductance with and/or without HCO3- conductance. We also surmise that the severity of the pathogenesis in CF is closely related to the phenotypic ability of a mutant CFTR to express a HCO3- conductance.

Abnormally Acidic Airway Surface Liquid on Cultured Cystic Fibrosis Bronchial Epithelium Reflects Defective Transepithelial HCO3- Transport

Raymond D. Coakley and Richard C. Boucher
The Cystic Fibrosis/Pulmonary Research and Treatment Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina USA

Many critical biological processes in the lung exhibit pH-dependence. CFTR's putative role in apical epithelial bicarbonate conductance could thus contribute to abnormal lung defense in CF by affecting airway surface liquid pH (pHASL). We have investigated pH and ionic composition of ASL on polarized normal and CF primary bronchial epithelia grown at an air-liquid interface. CF and normal epithelia isotonically absorb and acidify Kreb's Ringer Bicarbonate solution (KBR) on the apical surface without accumulating lactate. ASL acidification and HCO3- depletion are more rapid on CF vs normal cultures, resulting in lower pHASL at 24 hours. A component of ASL acidification is K+-dependent and mediated by activity of K+H+ATPase, which we identified at a molecular level in the apical membrane of normal and CF tissues. Similar ASL K+ depletion rates in CF and normal cultures indicate that K+H+ATPase activity is not different in CF, suggesting a role for abnormal HCO3- secretion in the genesis of acidic CF ASL. A basolateral HCO3--dependent component of recovery (alkalinization) from lumenally-applied acidic ASL characterizes normal cultures, but is defective in CF cultures, suggesting impaired transepithelial HCO3- movement. Raising intracellular cAMP in normal and disease control tissues results in ASL alkalinization, whereas this maneuver elicits acidification of ASL on CF cultures, consistent with defective CFTR-dependent apical HCO3- secretion. Paracellular permselectivity experiments support an additional paracellular route for transepithelial HCO3- transport in both CF and normal cultures. We conclude that pHASL on cultured human CF bronchial epithelia is abnormally regulated. Acidic ASL could contribute to the abnormal host defense in the CF lung.