PAT-1 (Slc26a6) is the predominant apical membrane Cl-/HCO3- exchanger in the upper villous epithelium of the murine duodenum

Basal HCO(3)(-) secretion across the duodenum has been shown in several species to principally involve the activity of apical membrane Cl(-)/HCO(3)(-) exchanger(s). To investigate the identity of relevant anion exchanger(s), experiments were performed using wild-type (WT) mice and mice with gene-targeted deletion of the following Cl(-)/HCO(3)(-) exchangers localized to the apical membrane of murine duodenal villi: Slc26a3 [down-regulated in adenoma (DRA)], Slc26a6 [putative anion transporter 1 (PAT-1)], and Slc4a9 [anion exchanger 4 (AE4)]. RT-PCR of the isolated villous epithelium demonstrated PAT-1, DRA, and AE4 mRNA expression. Using the pH-sensitive dye BCECF, anion exchange rates were measured across the apical membrane of epithelial cells in the upper villus of the intact duodenal mucosa. Under basal conditions, Cl(-)/HCO(3)(-) exchange activity was reduced by 65-80% in the PAT-1(-) duodenum, 30-40% in the DRA(-) duodenum, and <5% in the AE4(-) duodenum compared with the WT duodenum. SO(4)(2-)/HCO(3)(-) exchange was eliminated in the PAT-1(-) duodenum but was not affected in the DRA(-) and AE4(-) duodenum relative to the WT duodenum. Intracellular pH (pH(i)) was reduced in the PAT-1(-) villous epithelium but increased to WT levels in the absence of CO(2)/HCO(3)(-) or during methazolamide treatment. Further experiments under physiological conditions indicated active pH(i) compensation in the PAT-1(-) villous epithelium by combined activities of Na(+)/H(+) exchanger 1 and Cl(-)-dependent transport processes at the basolateral membrane. We conclude that 1) PAT-1 is the major contributor to basal Cl(-)/HCO(3)(-) and SO(4)(2-)/HCO(3)(-) exchange across the apical membrane and 2) PAT-1 plays a role in pH(i) regulation in the upper villous epithelium of the murine duodenum.     

A molecular mechanism for aberrant CFTR-dependent HCO(3)(-) transport in cystic fibrosis

Aberrant HCO(3)(-) transport is a hallmark of cystic fibrosis (CF) and is associated with aberrant Cl(-)-dependent HCO(3)(-) transport by the cystic fibrosis transmembrane conductance regulator (CFTR). We show here that HCO(3)(-) current by CFTR cannot account for CFTR-activated HCO(3)(-) transport and that CFTR does not activate AE1-AE4. In contrast, CFTR markedly activates Cl(-) and OH(-)/HCO(3)(-) transport by members of the SLC26 family DRA, SLC26A6 and pendrin. Most notably, the SLC26s are electrogenic transporters with isoform-specific stoichiometries. DRA activity occurred at a Cl(-)/HCO(3)(-) ratio > or =2. SLC26A6 activity is voltage regulated and occurred at HCO(3)(-)/Cl(-) > or =2. The physiological significance of these findings is demonstrated by interaction of CFTR and DRA in the mouse pancreas and an altered activation of DRA by the R117H and G551D mutants of CFTR. These findings provide a molecular mechanism for epithelial HCO(3)(-) transport (one SLC26 transporter-electrogenic transport; two SLC26 transporters with opposite stoichiometry in the same membrane domain-electroneutral transport), the CF-associated aberrant HCO(3)(-) transport, and reveal a new function of CFTR with clinical implications for CF and congenital chloride diarrhea.     

Downregulated in adenoma and putative anion transporter are regulated by CFTR in cultured pancreatic duct cells

The mechanism of the pancreatic ductal HCO secretion defect in cystic fibrosis (CF) is not well defined. However, a lack of apical Cl(-)/HCO exchange may exist in CF. To test this hypothesis, we examined the expression of Cl(-)/HCO exchangers in cultured pancreatic duct epithelial cells with physiological features prototypical of CF [CFPAC-1 cells lacking a functional CF transmembrane conductance regulator (CFTR)] or normal duct cells (CFPAC-1 cells transfected with functional wild-type CFTR, CFPAC-WT). Cl(-)/HCO exchange activity, assayed with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in cells grown on coverslips, increased about twofold in cells transfected with functional CFTR. This correlated with increased apical (36)Cl influx in cells expressing functional CFTR and grown on permeable support. Northern hybridizations indicated the induction of downregulated in adenoma (DRA) in cells expressing functional CFTR. The expression of putative anion transporter PAT1 also increased significantly in cells expressing functional CFTR. DRA was detected at high levels in native mouse pancreas by Northern hybridization and localized to the apical domain of the duct cells by immunohistochemical studies. In conclusion, CFTR upregulates DRA and PAT1 expression in cultured pancreatic duct cells. We propose that the pancreatic HCO secretion defect in CF patients is partly due to the downregulation of apical Cl(-)/HCO exchange activity mediated by DRA (and possibly PAT1).     

Chloride conductance of CFTR facilitates basal Cl-/HCO3- exchange in the villous epithelium of intact murine duodenum

Villi of the proximal duodenum are situated for direct exposure to gastric acid chyme. However, little is known about active bicarbonate secretion across villi that maintains the protective alkaline mucus barrier, a process that may be compromised in cystic fibrosis (CF), i.e., in the absence of a functional CF transmembrane conductance regulator (CFTR) anion channel. We investigated Cl(-)/HCO(3)(-) exchange activity across the apical membrane of epithelial cells located at the midregion of villi in intact duodenal mucosa from wild-type (WT) and CF mice using the pH-sensitive dye BCECF. Under basal conditions, the Cl(-)/HCO(3)(-) exchange rate was reduced by approximately 35% in CF compared with WT villous epithelium. Cl(-)/HCO(3)(-) exchange in WT and CF villi responded similarly to inhibitors of anion exchange, and membrane depolarization enhanced rates of Cl(-)(out)/HCO(3)(-)(in) exchange in both epithelia. In anion substitution studies, anion(in)/HCO(3)(-)(out) exchange rates were greater in WT epithelium using Cl(-) or NO(3)(-), but decreased to the level of the CF epithelium using the CFTR-impermeant anion, SO(4)(2-). Similarly, treatment of WT epithelium with the CFTR-selective blocker glybenclamide decreased the Cl(-)/HCO(3)(-) exchange rate to the level of CF epithelium. The mRNA expression of Slc26a3 (downregulated in adenoma) and Slc26a6 (putative anion exchanger-1) was similar between WT and CF duodena. From these studies of murine duodenum, we conclude 1) characteristics of Cl(-)/HCO(3)(-) exchange in the villous epithelium are most consistent with Slc26a6 activity, and 2) Cl(-) channel activity of CFTR facilitates apical membrane Cl(-)(in)/HCO(3)(-)(out) exchange by providing a Cl(-) "leak" under basal conditions.     

The intracellular pH-regulatory HCO3-/Cl- exchanger in the mouse oocyte is inactivated during first meiotic metaphase and reactivated after egg activation via the MAP kinase pathway

The HCO(3)(-)/Cl(-) exchanger is quiescent in the unfertilized mouse egg but is highly active in regulating intracellular pH in the early embryo and required for normal development. We show here that the HCO(3)(-)/Cl(-) exchanger is active in first meiotic prophase (GV) oocyte but inactivated during meiotic metaphase before the MI to MII transition. Reactivation does not occur until the activated egg enters interphase. A quiescent HCO(3)(-)/Cl(-) exchanger is not simply a general feature of metaphase, because activity did not decrease during first mitotic metaphase. Inactivation of the HCO(3)(-)/Cl(-) exchanger during MI coincided with the activation of MAP kinase (MAPK), whereas its reactivation coincided with the loss of MAPK activity after egg activation. Maintaining high MAPK activity after egg activation prevented the normal reactivation of the HCO(3)(-)/Cl(-) exchanger. Inactivating MAPK in unfertilized MII eggs resulted in HCO(3)(-)/Cl(-) exchanger activation. Preventing MAPK activation during first meiotic metaphase prevented the inactivation of HCO(3)(-)/Cl(-) exchange. Conversely, activating MAPK in the GV oocyte resulted in inactivation of HCO(3)(-)/Cl(-) exchange. These results imply that the HCO(3)(-)/Cl(-) exchanger in mouse oocytes is negatively regulated by MAPK. Thus, suppression of pH-regulatory mechanisms during meiosis is a novel function of MAPK and cytostatic factor activity in the oocyte.     

Ca2+ activates cystic fibrosis transmembrane conductance regulator- and Cl- -dependent HCO3 transport in pancreatic duct cells

Pancreatic duct cells secrete bicarbonate-rich fluids, which are important for maintaining the patency of pancreatic ductal trees as well as intestinal digestive function. The bulk of bicarbonate secretion in the luminal membrane of duct cells is mediated by a Cl(-)-dependent mechanism (Cl(-)/HCO(3)(-) exchange), and we previously reported that the mechanism is CFTR-dependent and cAMP-activated (Lee, M. G., Choi, J. Y., Luo, X., Strickland, E., Thomas, P. J., and Muallem, S. (1999) J. Biol. Chem. 274, 14670-14677). In the present study, we provide comprehensive evidence that calcium signaling also activates the same CFTR- and Cl(-)-dependent HCO(3)(-) transport. ATP and trypsin evoked intracellular calcium signaling in pancreatic duct-derived cells through the activation of purinergic and protease-activated receptors, respectively. Cl(-)/HCO(3)(-) exchange activity was measured by recording pH(i) in response to [Cl(-)](o) changes of the perfusate. In perfusate containing high concentrations of K(+), which blocks Cl(-) movement through electrogenic or K(+)-coupled pathways, ATP and trypsin highly stimulated luminal Cl(-)/HCO(3)(-) exchange activity in CAPAN-1 cells expressing wild-type CFTR, but not in CFPAC-1 cells that have defective (DeltaF508) CFTR. Notably, adenoviral transfection of wild-type CFTR in CFPAC-1 cells completely restored the stimulatory effect of ATP on luminal Cl(-)/HCO(3)(-) exchange. In addition, the chelation of intracellular calcium by 1,2-bis(2-aminophenoxy)ethane-N,N,N,N'-tetraacetic acid (BAPTA) treatment abolished the effect of calcium agonists on luminal Cl(-)/HCO(3)(-) exchange. These results provide a molecular basis for calcium-induced bicarbonate secretion in pancreatic duct cells and highlight the importance of CFTR in epithelial bicarbonate secretion induced by various stimuli.     

Enhanced formation of a HCO3- transport metabolon in exocrine cells of Nhe1-/- mice

Cl(-) influx across the basolateral membrane is a limiting step in fluid production in exocrine cells and often involves functionally linked Cl(-)/HCO(3)(-) (Ae) and Na(+)/H(+) (Nhe) exchange mechanisms. The dependence of this major Cl(-) uptake pathway on Na(+)/H(+) exchanger expression was examined in the parotid acinar cells of Nhe1(-/-) and Nhe2(-/-) mice, both of which exhibited impaired fluid secretion. No change in Cl(-)/HCO(3)(-) exchanger activity was detected in Nhe2-deficient mice. Conversely, Cl(-)/HCO(3)(-) exchanger activity increased nearly 4-fold in Nhe1-deficient mice, despite only minimal or any change in mRNA and protein levels of the anion exchanger Ae2. Acetazolamide completely blocked the increase in Cl(-)/HCO(3)(-) exchanger activity in Nhe1-null mice suggesting that increased anion exchange required carbonic anhydrase activity. Indeed, the parotid glands of Nhe1(-/-) mice expressed higher levels of carbonic anhydrase 2 (Car2) polypeptide. Moreover, the enhanced Cl(-)/HCO(3)(-) exchange activity was accompanied by an increased abundance of Car2.Ae2 complexes in the parotid plasma membranes of Nhe1(-/-) mice. Anion exchanger activity was also significantly reduced in Car2-deficient mice, consistent with an important role of a putative Car2.Ae2 HCO(3)(-) transport metabolon in parotid exocrine cell function. Increased abundance of this HCO(3)(-) transport metabolon is likely one of the multiple compensatory changes in the exocrine parotid gland of Nhe1(-/-) mice that together attenuate the severity of in vivo electrolyte and acid-base balance perturbations.     

Novel role for pendrin in orchestrating bicarbonate secretion in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing airway serous cells

In most HCO(3)(-)-secreting epithelial tissues, SLC26 Cl(-)/HCO(3)(-) transporters work in concert with the cystic fibrosis transmembrane conductance regulator (CFTR) to regulate the magnitude and composition of the secreted fluid, a process that is vital for normal tissue function. By contrast, CFTR is regarded as the only exit pathway for HCO(3)(-) in the airways. Here we show that Cl(-)/HCO(3)(-) anion exchange makes a major contribution to transcellular HCO(3)(-) transport in airway serous cells. Real-time measurement of intracellular pH from polarized cultures of human Calu-3 cells demonstrated cAMP/PKA-activated Cl(-)-dependent HCO(3)(-) transport across the luminal membrane via CFTR-dependent coupled Cl(-)/HCO(3)(-) anion exchange. The pharmacological and functional profile of the luminal anion exchanger was consistent with SLC26A4 (pendrin), which was shown to be expressed by quantitative RT-PCR, Western blot, and immunofluorescence. Pendrin-mediated anion exchange activity was confirmed by shRNA pendrin knockdown (KD), which markedly reduced cAMP-activated Cl(-)/HCO(3)(-) exchange. To establish the relative roles of CFTR and pendrin in net HCO(3)(-) secretion, transepithelial liquid secretion rate and liquid pH were measured in wild type, pendrin KD, and CFTR KD cells. cAMP/PKA increased the rate and pH of the secreted fluid. Inhibiting CFTR reduced the rate of liquid secretion but not the pH, whereas decreasing pendrin activity lowered pH with little effect on volume. These results establish that CFTR predominately controls the rate of liquid secretion, whereas pendrin regulates the composition of the secreted fluid and identifies a critical role for this anion exchanger in transcellular HCO(3)(-) secretion in airway serous cells.     

Mechanism of glucocorticoid-mediated reversal of inhibition of Cl(-)/HCO(-)(3) exchange during chronic ileitis

In the normal ileum, coupled NaCl absorption occurs via the dual operation of Na(+)/H(+) and Cl(-)/HCO(-)(3) exchange on the brush-border membrane (BBM) of villus cells. In a rabbit model of chronic small intestinal inflammation we determined the cellular mechanism of inhibition of NaCl absorption and the effect of steroids on this inhibition. Cl(-)/HCO(-)(3) but not Na(+)/H(+) exchange was reduced in the BBM of villus cells during chronic ileitis. Cl(-)/HCO(-)(3) exchange was inhibited secondary to a decrease in the affinity for Cl(-) rather than an alteration in the maximal rate of uptake of Cl(-) (V(max)). Methylprednisolone (MP) stimulated Cl(-)/HCO(-)(3) exchange in the normal ileum by increasing the V(max) of Cl(-) uptake rather than altering affinity for Cl(-). MP reversed the inhibition of Cl(-)/HCO(-)(3) exchange in rabbits with chronic ileitis. However, MP alleviated the Cl(-)/HCO(-)(3) exchange inhibition by restoring the affinity for Cl(-) rather than altering the V(max) of Cl(-) uptake. These data suggest that glucocorticoids mediate the alleviation of Cl(-)/HCO(-)(3) exchange inhibition in chronically inflamed ileum by reversing the same mechanism that was responsible for inhibition of this transporter rather than exerting a direct effect on the transporter itself, as was the case in normal ileum.     

AE anion exchangers in atrial tumor cells

Intracellular pH homeostasis and intracellular Cl(-) concentration in cardiac myocytes are regulated by anion exchange mechanisms. In physiological extracellular Cl(-) concentrations, Cl(-)/HCO(3)(-) exchange promotes intracellular acidification and Cl(-) loading sensitive to inhibition by stilbene disulfonates. We investigated the expression of AE anion exchangers in the AT-1 mouse atrial tumor cell line. Cultured AT-1 cells exhibited a substantial basal Na(+)-independent Cl(-)/HCO(3)(-) (but not Cl(-)/OH(-)) exchange activity that was inhibited by DIDS but not by dibenzamidostilbene disulfonic acid (DBDS). AT-1 cell Cl(-)/HCO(3)(-) activity was stimulated two- to threefold by extracellular ATP and ANG II. AE mRNAs detected by RT-PCR in AT-1 cells included brain AE3 (bAE3), cardiac AE3 (cAE3), AE2a, AE2b, AE2c1, AE2c2, and erythroid AE1 (eAE1), but not kidney AE1 (kAE1). Cultured AT-1 cells expressed AE2, cAE3, and bAE3 polypeptides, which were detected by immunoblot and immunocytochemistry. An AE1-like epitope was detected by immunocytochemistry but not by immunoblot. Both bAE3 and cAE3 were present in intact AT-1 tumors. Cultured AT-1 cells provide a useful system for the study of mediators and regulators of Cl(-)/HCO(3)(-) exchange activity in an atrial cell type.     

Development and characterization of secretin-stimulated secretion of cultured rat cholangiocytes

We sought to develop a cholangiocyte cell culture system that has preservation of receptors, transporters, and channels involved in secretin-induced secretion. Isolated bile duct fragments, obtained by enzyme perfusion of normal rat liver, were seeded on collagen and maintained in culture up to 18 wk. Cholangiocyte purity was assessed by staining for gamma-glutamyl transpeptidase (gamma-GT) and cytokeratin-19 (CK-19). We determined gene expression for secretin receptor (SR), cystic fibrosis transmembrane conductance regulator, Cl(-)/HCO(3)(-) exchanger, secretin-stimulated cAMP synthesis, Cl(-)/HCO(3) exchanger activity, secretin-stimulated Cl(-) efflux, and apical membrane-directed secretion in polarized cells grown on tissue culture inserts. Cultured cholangiocytes were all gamma-GT and CK-19 positive. The cells expressed SR and Cl(-)/HCO(3)(-) exchanger, and secretin-stimulated cAMP synthesis, Cl(-)/HCO(3)(-) exchanger activity, and Cl(-) efflux were similar to freshly isolated cholangiocytes. Forskolin (10(-4) M) induced fluid accumulation in the apical chamber of tissue culture inserts. In conclusion, we have developed a novel cholangiocyte line that has persistent HCO(3)(-), Cl(-), and fluid transport functions. This cell system should be useful to investigators who study cholangiocyte secretion.     

SLC26 anion exchangers of guinea pig pancreatic duct: molecular cloning and functional characterization

The secretin-stimulated human pancreatic duct secretes HCO(3)(-)-rich fluid essential for normal digestion. Optimal stimulation of pancreatic HCO(3)(-) secretion likely requires coupled activities of the cystic fibrosis transmembrane regulator (CFTR) anion channel and apical SLC26 Cl(-)/HCO(3)(-) exchangers. However, whereas stimulated human and guinea pig pancreatic ducts secrete ～140 mM HCO(3)(-) or more, mouse and rat ducts secrete ～40-70 mM HCO(3)(-). Moreover, the axial distribution and physiological roles of SLC26 anion exchangers in pancreatic duct secretory processes remain controversial and may vary among mammalian species. Thus the property of high HCO(3)(-) secretion shared by human and guinea pig pancreatic ducts prompted us to clone from guinea pig pancreatic duct cDNAs encoding Slc26a3, Slc26a6, and Slc26a11 polypeptides. We then functionally characterized these anion transporters in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. In Xenopus oocytes, gpSlc26a3 mediated only Cl(-)/Cl(-) exchange and electroneutral Cl(-)/HCO(3)(-) exchange. gpSlc26a6 in Xenopus oocytes mediated Cl(-)/Cl(-) exchange and bidirectional exchange of Cl(-) for oxalate and sulfate, but Cl(-)/HCO(3)(-) exchange was detected only in HEK 293 cells. gpSlc26a11 in Xenopus oocytes exhibited pH-dependent Cl(-), oxalate, and sulfate transport but no detectable Cl(-)/HCO(3)(-) exchange. The three gpSlc26 anion transporters exhibited distinct pharmacological profiles of (36)Cl(-) influx, including partial sensitivity to CFTR inhibitors Inh-172 and GlyH101, but only Slc26a11 was inhibited by PPQ-102. This first molecular and functional assessment of recombinant SLC26 anion transporters from guinea pig pancreatic duct enhances our understanding of pancreatic HCO(3)(-) secretion in species that share a high HCO(3)(-) secretory output.     

Slc26a6 regulates CFTR activity in vivo to determine pancreatic duct HCO3- secretion: relevance to cystic fibrosis

Fluid and HCO(3)(-) secretion are vital functions of the pancreatic duct and other secretory epithelia. CFTR and Cl(-)/HCO(3)(-) exchange activity at the luminal membrane are required for these functions. The molecular identity of the Cl(-)/HCO(3)(-) exchangers and their relationship with CFTR in determining fluid and HCO(3)(-) secretion are not known. We show here that the Cl(-)/HCO(3)(-) exchanger slc26a6 controls CFTR activity and ductal fluid and HCO(3)(-) secretion. Unexpectedly, deletion of slc26a6 in mice and measurement of fluid and HCO(3)(-) secretion into sealed intralobular pancreatic ducts revealed that deletion of slc26a6 enhanced spontaneous and decreased stimulated secretion. Remarkably, inhibition of CFTR activity with CFTR(inh)-172, knock-down of CFTR by siRNA and measurement of CFTR current in WT and slc26a6(-/-) duct cells revealed that deletion of slc26a6 resulted in dis-regulation of CFTR activity by removal of tonic inhibition of CFTR by slc26a6. These findings reveal the intricate regulation of CFTR activity by slc26a6 in both the resting and stimulated states and the essential role of slc26a6 in pancreatic HCO(3)(-) secretion in vivo.     

Native and recombinant Slc26a3 (downregulated in adenoma, Dra) do not exhibit properties of 2Cl-/1HCO3- exchange

The recent proposal that Dra/Slc26a3 mediates electrogenic 2Cl(-)/1HCO(3)(-) exchange suggests a required revision of classical concepts of electroneutral Cl(-) transport across epithelia such as the intestine. We investigated 1) the effect of endogenous Dra Cl(-)/HCO(3)(-) activity on apical membrane potential (V(a)) of the cecal surface epithelium using wild-type (WT) and knockout (KO) mice; and 2) the electrical properties of Cl(-)/(OH(-))HCO(3)(-) exchange by mouse and human orthologs of Dra expressed in Xenopus oocytes. Ex vivo (36)Cl(-) fluxes and microfluorometry revealed that cecal Cl(-)/HCO(3)(-) exchange was abolished in the Dra KO without concordant changes in short-circuit current. In microelectrode studies, baseline V(a) of Dra KO surface epithelium was slightly hyperpolarized relative to WT but depolarized to the same extent as WT during luminal Cl(-) substitution. Subsequent studies indicated that Cl(-)-dependent V(a) depolarization requires the anion channel Cftr. Oocyte studies demonstrated that Dra-mediated exchange of intracellular Cl(-) for extracellular HCO(3)(-) is accompanied by slow hyperpolarization and a modest outward current, but that the steady-state current-voltage relationship is unaffected by Cl(-) removal or pharmacological blockade. Further, Dra-dependent (36)Cl(-) efflux was voltage-insensitive in oocytes coexpressing the cation channels ENaC or ROMK. We conclude that 1) endogenous Dra and recombinant human/mouse Dra orthologs do not exhibit electrogenic 2Cl(-)/1HCO(3)(-) exchange; and 2) acute induction of Dra Cl(-)/HCO(3)(-) exchange is associated with secondary membrane potential changes representing homeostatic responses. Thus, participation of Dra in coupled NaCl absorption and in uncoupled HCO(3)(-) secretion remains compatible with electroneutrality of these processes, and with the utility of electroneutral transport models for predicting epithelial responses in health and disease.     

Dynamic control of cystic fibrosis transmembrane conductance regulator Cl(-)/HCO3(-) selectivity by external Cl(-)

HCO(3)(-) secretion is a vital activity in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing epithelia. However, the role of CFTR in this activity is not well understood. Simultaneous measurements of membrane potential and pH(i) and/or current in CFTRexpressing Xenopus oocytes revealed dynamic control of CFTR Cl(-)/HCO(3)(-) permeability ratio, which is regulated by external Cl(-) (Cl(-)(o)). Thus, reducing external Cl(-) from 110 to 0-10 mm resulted in the expected increase in membrane potential, but with no corresponding OH(-) or HCO(3)(-) influx. Approximately 3-4 min after reducing Cl(o)(-) to 0 mm, an abrupt switch in membrane potential occurs that coincided with an increased rates of OH(-) and HCO(3)(-) influx. The switch in membrane permeability to OH(-)/HCO(3)(-) can also be recorded as a leftward shift in the reversal potential. Furthermore, an increased rate of OH(-) influx in response to elevating pH(o) to 9.0 was observed only after the switch in membrane potential. The time to switch increased to 11 min at Cl(o)(-) of 5 mm. Conversely, re-addition of external Cl(-) after the switch in membrane potential did not stop HCO(3)(-) influx, which continued for about 3.9 min after Cl(-) addition. Importantly, addition of external Cl(-) to cells incubated in Cl(-)-free medium never resulted in HCO(3)(-) efflux. Voltage and current clamp experiments showed that the delayed HCO(3)(-) transport is electrogenic. These results indicate that CFTR exists in two conformations, a Cl(-) only and a Cl(-) and OH(-)/HCO(3)(-) permeable state. The switch between the states is controlled by external Cl(-). Accordingly, a different tryptic pattern of CFTR was found upon digestion in Cl(-)-containing and Cl(-)-free media. The physiological significance of these finding is discussed in the context of HCO(3)(-) secretion by tissues such as the pancreas and salivary glands.     

An intramolecular transport metabolon: fusion of carbonic anhydrase II to the COOH terminus of the Cl(-)/HCO(3)(-)exchanger, AE1

Anion exchanger 1 (AE1) is the plasma membrane Cl(-)/HCO(3)(-) exchanger of erythrocytes. Carbonic anhydrases (CA) provide substrate for AE1 by catalyzing the reaction, H(2)O + CO(2) ? HCO(3)(-) + H(+). The physical complex of CAII with AE1 has been proposed to maximize anion exchange activity. To examine the effect of CAII catalysis on AE1 transport rate, we fused either CAII-wild type or catalytically inactive CAII-V143Y to the cytoplasmic COOH terminus of AE1 to form AE1.CAII and AE1.CAII-V143Y, respectively. When expressed in transfected human embryonic kidney 293 cells, AE1.CAII had a similar Cl(-)/HCO(3)(-) exchange activity to AE1 alone, as assessed by the flux of H(+) equivalents (87 ± 4% vs. AE1) or rate of change of intracellular Cl(-) concentration (93 ± 4% vs. AE1), suggesting that CAII does not activate AE1. In contrast, AE1.CAII-V143Y displayed transport rates for H(+) equivalents and Cl(-) of 55 ± 2% and of 40 ± 2%, versus AE1. Fusion of CAII to AE1 therefore reduces anion transport activity, but this reduction is compensated for during Cl(-)/HCO(3)(-) exchange by the presence of catalytically active CAII. Overexpression of free CAII-V143Y acts in a dominant negative manner to reduce AE1-mediated HCO(3)(-) transport by displacement of endogenous CAII-wild type from its binding site on AE1. To examine whether AE1.CAII bound endogenous CAII, we coexpressed CAII-V143Y along with AE1 or AE1.CAII. The bicarbonate transport activity of AE1 was inhibited by CAII-V143Y, whereas the activity of AE1.CAII was unaffected by CAII-V143Y, suggesting impaired transport activity upon displacement of functional CAII from AE1 but not AE1.CAII. Taken together, these data suggest that association of functional CAII with AE1 increases Cl(-)/HCO(3)(-) exchange activity, consistent with the HCO(3)(-) transport metabolon model.     

Relationship of delta-aminolevulinic acid-induced protoporphyrin IX levels to mitochondrial content in neoplastic cells in vitro

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a small conductance cAMP-activated chloride ion channel. In the CF pancreatic duct, mutations in CFTR cause a reduction in bicarbonate secretion. This is thought to result from CFTR operating in parallel with a chloride-bicarbonate (Cl(-)/HCO(-)(3)) exchanger, located in the apical membrane of pancreatic duct cells. The molecular basis of this Cl(-)/HCO(-)(3) exchanger has not been identified. A combination of screening cDNA libraries, RNase protection, and 5' RACE analysis was used to identify Cl(-)/HCO(-)(3) exchangers in human fetal pancreas. An AE2 Cl(-)/HCO(-)(3) exchanger was shown to be expressed in human fetal pancreas from the midtrimester of gestation, at a time when CF-associated pathology commences. In addition, an AE1 Cl(-)/HCO(3) was identified in fetal pancreas but was absent from the adult pancreas and cultured ductal epithelial cells from fetal and adult pancreas.     

Granulosa cells regulate intracellular pH of the murine growing oocyte via gap junctions: development of independent homeostasis during oocyte growth

Oocytes grow within ovarian follicles in which the oocyte is coupled to the surrounding granulosa cells by gap junctions. It was previously found that small growing oocytes isolated from juvenile mice and freed of their surrounding granulosa cells (denuded) lacked the ability to regulate their intracellular pH (pH(i)), did not exhibit the pH(i)-regulatory HCO(3)(-)/Cl(-) and Na(+)/H(+) exchange activities found in fully-grown oocytes, and had low pH(i). However, both exchangers became active as oocytes grew near to full size, and, simultaneously, oocyte pH(i) increased by approximately 0.25 pH units. Here, we show that, in the more physiological setting of the intact follicle, oocyte pH(i) is instead maintained at approximately 7.2 throughout oocyte development, and the growing oocyte exhibits HCO(3)(-)/Cl(-) exchange, which it lacks when denuded. This activity in the oocyte requires functional gap junctions, as gap junction inhibitors eliminated HCO(3)(-)/Cl(-) exchange activity from follicle-enclosed growing oocytes and substantially impeded the recovery of the oocyte from an induced alkalosis, implying that oocyte pH(i) may be regulated by pH-regulatory exchangers in granulosa cells via gap junctions. This would require robust HCO(3)(-)/Cl(-) exchange activity in the granulosa cells, which was confirmed using oocytectomized (OOX) cumulus-oocyte complexes. Moreover, in cumulus-oocyte complexes with granulosa cells coupled to fully-grown oocytes, HCO(3)(-)/Cl(-) exchange activity was identical in both compartments and faster than in denuded oocytes. Taken together, these results indicate that growing oocyte pH(i) is controlled by pH-regulatory mechanisms residing in the granulosa cells until the oocyte reaches a developmental stage where it becomes capable of carrying out its own homeostasis.