A hypothesis of the disc-sphere transformation of the erythrocytes between glass surfaces and of related observations

Erythrocytes suspended at a low hematocrit in a non-buffered isotonic saline change from biconcave discs to spheres between glass surfaces of a slide and of a coverslip with the echinocyte as an intermediate. A pH increase is a major factor responsible for this disc-sphere transformation or glass effect. It is also observed between surfaces made of various polymers and of mica provided that the distance between them is controlled (0.1 mm). The glass effect is antagonized by serum, plasma, serum albumin, ammonium salts and CO2. It is not observed above a 1-2% hematocrit, but is enhanced by gamma-globulins. The sites of reappearance of the spicules are the same and the order of their disappearance is the inverse of the order of their reappearance during the repetitive cycle of the disc-sphere transformation and reversal when a small glass rod is alternatively approached near a site on the erythrocyte surface and withdrawn. A mechanism of erythrocyte shape control has been previously hypothesized in which Band 3 (AE1), the anion exchange protein, plays a central role. Specifically, decrease and increase of the ratio of its outward-facing conformation (Band 3o) and inward-facing conformation (Band 3i) contract and relax the membrane skeleton, promoting the echinocytosis and stomatocytosis, respectively. The Band 3o/Band 3i equilibrium ratio is determined by the Donnan equilibrium ratio of Cl-, HCO3- and H+ (r=Cl(i)-/Cl(o)-=HCO3i-/HCO3o-=Ho+/Hi+), increasing with it. The mechanism could explain by a change of the Donnan ratio the above observations with the assumptions that polymers are permeable to CO2 and that an unstirred layer slows the propagation of the change occurring at the site of approach of the glass rod to peripheral sites. The presence of HCO3- in serum or plasma may be the basis for the absence of the glass effect in these fluids.     

A basis of echinocytosis and stomatocytosis in the disc-sphere transformations of the erythrocyte

A mechanism of erythrocyte shape control has been previously hypothesized in which Band 3, the anion exchange protein, controls the shape. In essence, the mechanism hypothesizes that the membrane skeleton is used to generate different shapes and the alternate influx and efflux of anions mediated by Band 3, which recruit Band 3 to an inward-facing and an outward-facing conformation, contract and relax the skeleton by folding and unfolding spectrin. Spectrin is bound to Band 3 by the intermediary of ankyrin. The mechanism is shown to be consistent with rapid shape deformations of the erythrocyte in blood circulation. We have examined whether the mechanism could provide a basis of echinocytosis and stomatocytosis in disc-sphere transformations of the erythrocyte induced by a wide variety of agents. These agents were classified into four groups: lipids of the bilayer, Donnan equilibrium modifiers, Band 3 anion transport inhibitors and integral membrane protein modifiers. Evidence is presented that the lipids play a secondary function in the control of the erythrocyte shape, as indicated by the mechanism. Two possible functions of the lipids are suggested with respect to the mechanism. Without exception, echinocytogenic and stomatocytogenic Donnan equilibrium modifiers decrease and increase the equilibrium ratio of chloride (Cl-(i)/Cl-(o)), respectively, as predicted by the mechanism. Echinocytosis produced by competitive anion transport inhibitors slowly transported inward by Band 3 and by affinity labels of Band 3 is compatible with the mechanism. Evidence is presented which indicates that echinocytosis and stomatocytosis induced by amphiphilic drugs and detergents occur by inhibition of the Band 3 anion transport. Finally, echinocytosis and stomatocytosis induced by non-covalent and covalent modifiers of integral membrane proteins such as agglutinins and digestive enzymes are consistent with the mechanism.     

The basis of echinocytosis of the erythrocyte by glucose depletion

Echinocytosis of erythrocytes by glucose depletion is attributed to adenosine triphosphate depletion, but its process still remains unknown. A mechanism of control of the erythrocyte shape has been previously proposed in which the anion exchanger Band 3, linked to flexible membrane skeleton, has a pivotal role. Recruitments of its inward facing (Band 3(i) ) and outward facing (Band 3(o) ) conformations contract and relax the membrane skeleton, thus promoting echinocytosis and stomatocytosis, respectively. The Band 3(o) /Band 3(i) equilibrium ratio increases with the increase of the Donnan equilibrium ratio, and preferential inward and outward transport by Band 3 of substrates slowly transported are echinocytogenic and stomatocytogenic, respectively. The mechanism suggests the following process. The major organic phosphate 2,3-bisphosphoglycerate is catabolized to lactate to form inorganic phosphate, 3-phosphoglycerate, and adenosine triphosphate. The last two products can be reversibly transformed into 1,3-bisphosphoglycerate and adenosine diphosphate by the glycolytic enzyme phosphoglycerate kinase, thus allowing 2,3-bisphosphoglycerate formation by 2,3-bisphosphoglycerate synthase/phosphatase. The catabolic and cyclic processes initially oppose echinocytosis by increasing the Donnan ratio and outward transport of slowly transported inorganic phosphate by Band 3 (its basic form is transported with a hydrogen ion). Echinocytosis occurs when inward transport of this product becomes predominant. This process can rationalize direct and indirect observations.     

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.     

Mouse Ae1 E699Q mediates SO42-i/anion-o exchange with [SO42-]i-dependent reversal of wild-type pHo sensitivity

The SLC4A1/AE1 gene encodes the electroneutral Cl(-)/HCO(3)(-) exchanger of erythrocytes and renal type A intercalated cells. AE1 mutations cause familial spherocytic and stomatocytic anemias, ovalocytosis, and distal renal tubular acidosis. The mutant mouse Ae1 polypeptide E699Q expressed in Xenopus oocytes cannot mediate Cl(-)/HCO(3)(-) exchange or (36)Cl(-) efflux but exhibits enhanced dual sulfate efflux mechanisms: electroneutral exchange of intracellular sulfate for extracellular sulfate (SO(4)(2-)(i)/SO(4)(2-)(o) exchange), and electrogenic exchange of intracellular sulfate for extracellular chloride (SO(4)(2-)(i)/Cl(-)(o) exchange). Whereas wild-type AE1 mediates 1:1 H(+)/SO(4)(2-) cotransport in exchange for either Cl(-) or for the H(+)/SO(4)(2-) ion pair, mutant Ae1 E699Q transports sulfate without cotransport of protons, similar to human erythrocyte AE1 in which the corresponding E681 carboxylate has been chemically converted to the alcohol (hAE1 E681OH). We now show that in contrast to the normal cis-stimulation by protons of wild-type AE1-mediated SO(4)(2-) transport, both SO(4)(2-)(i)/Cl(-)(o) exchange and SO(4)(2-)(i)/SO(4)(2-)(o) exchange mediated by mutant Ae1 E699Q are inhibited by acidic pH(o) and activated by alkaline pH(o). hAE1 E681OH displays a similarly altered pH(o) dependence of SO(4)(2-)(i)/Cl(-)(o) exchange. Elevated [SO(4)(2-)](i) increases the K(1/2) of Ae1 E699Q for both extracellular Cl(-) and SO(4)(2-), while reducing inhibition of both exchange mechanisms by acid pH(o). The E699Q mutation also leads to increased potency of self-inhibition by extracellular SO(4)(2-). Study of the Ae1 E699Q mutation has revealed the existence of a novel pH-regulatory site of the Ae1 polypeptide and should continue to provide valuable paths toward understanding substrate selectivity and self-inhibition in SLC4 anion transporters.     

Novel regulation of cystic fibrosis transmembrane conductance regulator (CFTR) channel gating by external chloride

The cystic fibrosis transmembrane conductance regulator (CFTR) is vital for Cl(-) and HCO(3)(-) transport in many epithelia. As the HCO(3)(-) concentration in epithelial secretions varies and can reach as high as 140 mm, the lumen-facing domains of CFTR are exposed to large reciprocal variations in Cl(-) and HCO(3)(-) levels. We have investigated whether changes in the extracellular anionic environment affects the activity of CFTR using the patch clamp technique. In fast whole cell current recordings, the replacement of 100 mm external Cl(-) ((Cl(o)(-))) with HCO(3)(-), Br(-), NO(3)(-), or aspartate(-) inhibited inward CFTR current (Cl(-) efflux) by approximately 50% in a reversible manner. Lowering Cl(o)(-) alone by iso-osmotic replacement with mannitol also reduced Cl(-) efflux to a similar extent. The maximal inhibition of CFTR current was approximately 70%. Raising cytosolic calcium shifted the Cl(-) dose-inhibition curve to the left but did not alter the maximal current inhibition observed. In contrast, a reduction in the internal [Cl(-)] neither inhibited CFTR nor altered the block caused by reduced Cl(o)(-). Single channel recordings from outside-out patches showed that lowering Cl(o)(-) markedly reduced channel open probability with little effect on unitary conductance. Together, these results indicate that alterations in Cl(o)(-) alone and not the Cl(-)/HCO(3)(-) ratio regulate the gating of CFTR. Physiologically, our data have implications for current models of epithelial HCO(3)(-) secretion and for the control of pH at epithelial cell surfaces.     

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.     

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.     

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.     

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.     

A mathematical model of blood-interstitial acid-base balance: application to dilution acidosis and acid-base status

We developed mathematical models that predict equilibrium distribution of water and electrolytes (proteins and simple ions), metabolites, and other species between plasma and erythrocyte fluids (blood) and interstitial fluid. The models use physicochemical principles of electroneutrality in a fluid compartment and osmotic equilibrium between compartments and transmembrane Donnan relationships for mobile species. Across the erythrocyte membrane, the significant mobile species Cl? is assumed to reach electrochemical equilibrium, whereas Na(+) and K(+) distributions are away from equilibrium because of the Na(+)/K(+) pump, but movement from this steady state is restricted because of their effective short-term impermeability. Across the capillary membrane separating plasma and interstitial fluid, Na(+), K(+), Ca2(+), Mg2(+), Cl?, and H(+) are mobile and establish Donnan equilibrium distribution ratios. In each compartment, attainment of equilibrium by carbonates, phosphates, proteins, and metabolites is determined by their reactions with H(+). These relationships produce the recognized exchange of Cl(-) and bicarbonate across the erythrocyte membrane. The blood submodel was validated by its close predictions of in vitro experimental data, blood pH, pH-dependent ratio of H(+), Cl?, and HCO?? concentrations in erythrocytes to that in plasma, and blood hematocrit. The blood-interstitial model was validated against available in vivo laboratory data from humans with respiratory acid-base disorders. Model predictions were used to gain understanding of the important acid-base disorder caused by addition of saline solutions. Blood model results were used as a basis for estimating errors in base excess predictions in blood by the traditional approach of Siggaard-Andersen (acid-base status) and more recent approaches by others using measured blood pH and Pco? values. Blood-interstitial model predictions were also used as a basis for assessing prediction errors of extracellular acid-base status values, such as by the standard base excess approach. Hence, these new models can give considerable insight into the physicochemical mechanisms producing acid-base disorders and aid in their diagnoses.     

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.     

SLC26A7 is a Cl- channel regulated by intracellular pH

Members of the SLC26 transporter family play an essential role in several epithelial functions, as revealed by diseases associated with mutations in members of the family. Several members were shown to function as Cl(-) and HCO(3)(-) transporters that likely play an important role in epithelial Cl(-) absorption and HCO(3)(-) secretion. However, the mechanism of most transporters is not well understood. SLC26A7 is a member of the SLC26 transporter family reported to be expressed in the basolateral membrane of the cortical collecting duct and parietal cells and functions as a coupled Cl(-)/HCO(3)(-) exchanger. In the present work we examined the transport properties of SLC26A7 to determine its transport characteristics and electrogenicity. We found that when expressed in Xenopus oocytes or HEK293 cells SLC26A7 functions as a pH(i)-regulated Cl(-) channel with minimal OH(-)/HCO(3)(-) permeability. Expression of SLC26A7 in oocytes or HEK293 cells generated a Cl(-) current with linear I/V and an instantaneous current that was voltage- and time-independent. Based on measurement of reversal potential the selectivity of SLC26A7 is NO(3)(-)>Cl(-)=Br(-)=I(-)>SO(4)(2-)=Glu(-), although I(-) partially inhibited the current. Incubating the cells with HCO(3)(-) or butyrate acidified the cytosol and increased the selectivity of SLC26A7 for Cl(-). Measurement of membrane potential and pH(i) showed minimal OH(-) and HCO(3)(-) transport by SLC26A7 when the cells were incubated in Cl(-)-containing or Cl(-)-free media. The activity of SLC26A7 was inhibited by all inhibitors of anion transporters tested, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, diphenylamine-2-carboxylic acid, and glybenclamide. These findings reveal that SLC26A7 functions as a unique Cl(-) channel that is regulated by intracellular H(+).     

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.     

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.     

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.     

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.     

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.     

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.     

Volume regulation mechanisms in Rana castebeiana cardiac tissue under hyperosmotic stress

Volume changes of cardiac tissue under hyperosmotic stress in Rana catesbeiana were characterized by the identification of the osmolytes involved and the possible regulatory processes activated by both abrupt and gradual changes in media osmolality (from 220 to 280mosmol/kg H(2)O). Slices of R. catesbeiana cardiac tissue were subjected to hyperosmotic shock, and total tissue Na(+), K(+), Cl(-) and ninhydrin-positive substances were measured. Volume changes were also induced in the presence of transport inhibitors to identify osmolyte pathways. The results show a maximum volume loss to 90.86+/-0.73% of the original volume (measured as 9% decrease in wet weight) during abrupt hyperosmotic shock. However, during a gradual osmotic challenge the volume was never significantly different from that of the control. During both types of hyperosmotic shock, we observed an increase in Na(+) but no significant change in Cl(-) contents. Additionally, we found no change in ninhydrin-positive substances during any osmotic challenge. Pharmacological analyses suggest the involvement of the Na(+)/H(+) exchanger, and perhaps the HCO(3)(-)/Cl(-) exchanger. There is indirect evidence for decrease in Na(+)/K(+)-ATPase activity. The Na(+) fluxes seem to result from Mg(2+) signaling, as saline rich in Mg(2+) enhances the regulatory volume increase, followed by a higher intracellular Na(+) content. The volume maintenance mechanisms activated during the gradual osmotic change are similar to that activated by abrupt osmotic shock.