Bicarbonate-chloride Exchange Research Articles

SLC4A2 anion exchanger promotes tumour cell malignancy via enhancing net acid efflux across golgi membranes

Proper functioning of each secretory and endocytic compartment relies on its unique pH micro-environment that is known to be dictated by the rates of V-ATPase-mediated H+ pumping and its leakage back to the cytoplasm via an elusive “H+ leak” pathway. Here, we show that this proton leak across Golgi membranes is mediated by the AE2a (SLC4A2a)-mediated bicarbonate-chloride exchange, as it is strictly dependent on bicarbonate import (in exchange for chloride export) and the expression level of the Golgi-localized AE2a anion exchanger. In the acidic Golgi lumen, imported bicarbonate anions and protons then facilitate a common buffering reaction that yields carbon dioxide and water before their egress back to the cytoplasm via diffusion or water channels. The flattened morphology of the Golgi cisternae helps this process, as their high surface-volume ratio is optimal for water and gas exchange. Interestingly, this net acid efflux pathway is often upregulated in cancers and established cancer cell lines, and responsible for their markedly elevated Golgi resting pH and attenuated glycosylation potential. Accordingly, AE2 knockdown in SW-48 colorectal cancer cells was able to restore these two phenomena, and at the same time, reverse their invasive and anchorage-independent growth phenotype. These findings suggest a possibility to return malignant cells to a benign state by restoring Golgi resting pH.

Reversal of Cancer Cell Invasiveness Via Reprogramming Their Golgi pH Homeostasis

Cancer invasiveness and metastatic spread are the main causes of cancer-associated deaths, yet successful therapies for their prevention are currently non-existing. Here we exploit cancer cell reprogramming, a strategy developed earlier to prevent tumour growth, to show that cancer cell invasiveness can be reversed via restoring their Golgi pH homeostasis and normal glycosylation status. Specifically, we demonstrate that AE2a (SLC4A2a)-mediated bicarbonate-chloride exchange controls proton leakage across Golgi membranes via a well-known buffering reaction that produces water and carbon dioxide from luminal protons and bicarbonate anions. Its forced overexpression in non-malignant cells, or upregulation in certain cancer cells in vitro and in vivo, also coincides with elevated Golgi resting pH, the synthesis of invasion and metastasis-promoting glycans and a shorter life span of cancer patients. Finally, AE2 knockdown in cancer cells blocked their invasiveness via restoring their Golgi pH homeostasis and normal glycosylation status. Thus, cancer metastasis is a reversible process that can be targeted by addressing organelle pH homeostasis.

Asymmetry of inverted-topology repeats in the AE1 anion exchanger suggests an elevator-like mechanism.

The membrane transporter anion exchanger 1 (AE1), or band 3, is a key component in the processes of carbon-dioxide transport in the blood and urinary acidification in the renal collecting duct. In both erythrocytes and the basolateral membrane of the collecting-duct α-intercalated cells, the role of AE1 is to catalyze a one-for-one exchange of chloride for bicarbonate. After decades of biochemical and functional studies, the structure of the transmembrane region of AE1, which catalyzes the anion-exchange reaction, has finally been determined. Each protomer of the AE1 dimer comprises two repeats with inverted transmembrane topologies, but the structures of these repeats differ. This asymmetry causes the putative substrate-binding site to be exposed only to the extracellular space, consistent with the expectation that anion exchange occurs via an alternating-access mechanism. Here, we hypothesize that the unknown, inward-facing conformation results from inversion of this asymmetry, and we propose a model of this state constructed using repeat-swap homology modeling. By comparing this inward-facing model with the outward-facing experimental structure, we predict that the mechanism of AE1 involves an elevator-like motion of the substrate-binding domain relative to the nearly stationary dimerization domain and to the membrane plane. This hypothesis is in qualitative agreement with a wide range of biochemical and functional data, which we review in detail, and suggests new avenues of experimentation.

Rapid Cl−/HCO3− exchange kinetics of AE1 in HEK293 cells and hereditary stomatocytosis red blood cells

Anion exchanger 1 (AE1) or band 3 is a membrane protein responsible for the rapid exchange of chloride for bicarbonate across the red blood cell membrane. Nine mutations leading to single amino-acid substitutions in the transmembrane domain of AE1 are associated with dominant hereditary stomatocytosis, monovalent cation leaks, and reduced anion exchange activity. We set up a stopped-flow spectrofluorometry assay coupled with flow cytometry to investigate the anion transport and membrane expression characteristics of wild-type recombinant AE1 in HEK293 cells, using an inducible expression system. Likewise, study of three stomatocytosis-associated mutations (R730C, E758K, and G796R), allowed the validation of our method. Measurement of the rapid and specific chloride/bicarbonate exchange by surface expressed AE1 showed that E758K mutant was fully active compared with wild-type (WT) AE1, whereas R730C and G796R mutants were inactive, reinforcing previously reported data on other experimental models. Stopped-flow analysis of AE1 transport activity in red blood cell ghost preparations revealed a 50% reduction of G796R compared with WT AE1 corresponding to a loss of function of the G796R mutated protein, in accordance with the heterozygous status of the AE1 variant patients. In conclusion, stopped-flow led to measurement of rapid transport kinetics using the natural substrate for AE1 and, conjugated with flow cytometry, allowed a reliable correlation of chloride/bicarbonate exchange to surface expression of AE1, both in recombinant cells and ghosts and therefore a fine comparison of function between different stomatocytosis samples. This technical approach thus provides significant improvements in anion exchange analysis in red blood cells.

Anion exchanger 1: Protean function and associations

Anion exchanger 1 (AE1) is the most abundant protein on the erythrocyte membrane and is also present on the basolateral surface of the alpha intercalated cell in the distal nephron. Mutations can cause either hereditary haemolytic red cell diseases, or hereditary distal renal tubular acidosis. Classically it mediates the electroneutral exchange of chloride for bicarbonate, as well as comprising an important mechanical component of the red cell membrane. It is increasingly recognised that it plays many other roles too: alternative anion transport, such as sulphate transport and proton and sulphate symport, associations with other erythrocyte membrane proteins as part of the AE1 macrocomplex, regulation of glycolysis and more recently cation transport through the so-called 'leak' pathway. These new functions and associations are reviewed in health and disease, and the role of AE1 as a putative regulator of cell volume is discussed.

Human Plasma Membrane Anion Exchanger, AE1: Over‐expression in Saccharomyces cerevisiae

Anion exchanger 1 (AE1) facilitates the exchange of chloride for bicarbonate across the plasma membrane of renal and erythrocyte membranes. The 55 kDa membranedomain of AE1 alone is responsible for its transport function, however a high resolution structure of this domain has not been solved. The limiting factor to obtaining acrystal structure is the availability of homogenous, purified AE1. Here we developed a novel expression and efficient purification system for AE1, using S. cerevisiaeto allow for large‐scale expression, while maintaining proper folding of AE1. The expression construct consists of the AE1 membrane domain (AE1MD), residues 388–911 of human AE1, followed by the 9 C‐terminal amino acids of rhodopsin. Expression was under the control of the constitutive promoter and transcriptionalterminator of the yeast H+‐ATPase gene. AE1MD was expressed in S. cerevisiae, strain BJ5457, to 0.3 mg AE1MD per litre of culture. We characterized the cellularlocalization of AE1MD by confocal immunofluorescence microscopy and sucrose gradient ultracentrifugation. AE1MD structure was assessed by binding to animmobilized anion exchange inhibitor (SITS‐Affi‐gel). Once solubilized by the detergent n‐dodecyl‐β‐D‐maltoside, AE1MD was purified with a 1D4 antibody affinitycolumn, which recognizes the rhodopsin tag on AE1MD. This research is supported by the Canadian Institutes of Health Research.

Effect of Cytosolic Hereditary Spherocytosis Mutations of Human Erythrocyte Anion Exchanger 1 on Its Interaction with Cytoskeletal Protein 4.2.

Anion exchanger 1 (AE1, Band 3) is the predominant membrane protein of erythrocytes. Human AE1 has two functionally independent domains: its 52 kDa C-terminal membrane domain catalyzes the exchange of chloride for bicarbonate across the membrane while its 43 kDa N-terminal cytosolic domain (cdb3) anchors the membrane to the cytoskeleton, giving the red cell its stability and flexibility. Several proteins bind to cdb3 including cytoskeletal protein 4.2, ankyrin, glycolytic enzymes and deoxyhemoglobin. Three mutations in cdb3 (E40K, G130R and P327R) are associated with the hemolytic anemia hereditary spherocytosis (HS) and decreased levels of erythrocyte protein 4.2 while maintaining a normal amount of AE1 at the red cell membrane. Wild-type and mutant cdb3 proteins were expressed in E. coli and purified and it was shown through a variety of biophysical methods that these three HS mutations do not cause major structural changes in this domain. Each of these mutations introduces a positive charge at the surface of the protein that is predominantly negatively charged, which may have an effect on protein interactions while maintaining its native folded structure. Full-length wild-type AE1 or HS mutants were co-expressed with protein 4.2 in HEK-293 cells in order to study their interaction in a mammalian cell line. All three HS mutant proteins were expressed at similar levels to wild-type in these cells and were shown to be present at the membrane using immunofluorescence and confocal microscopy. These proteins were also co-expressed in LLCPK-1 cells for the purpose of co-localization studies using immunofluorescence. A series of GST fusion proteins of protein 4.2 domains were designed based on a homology model of protein 4.2 and regions of the protein known to interact with AE1. These fusion proteins were expressed in E. coli and purified on glutathione-Sepharose resin and were used to study their interaction with purified wild-type and HS mutant cdb3 proteins in vitro. Blot overlay analysis showed that a protein 4.2 GST fusion protein containing a putative β-hairpin region (Asp145 to Glu203) binds specifically to wild-type cdb3 while GST does not. It is hypothesized that since these three HS mutations do not cause major structural changes in cdb3, the decreased level of protein 4.2 in the red cells of these patients is a result of impaired binding that occurs due to these mutation sites.

SLC26 Chloride/Base Exchangers in the Kidney in Health and Disease

Solute-linked carrier 26 (SLC26) isoforms are members of a large, conserved family of anion exchangers, many of which display highly restricted and distinct tissue distribution. Cloning experiments have identified 10 SLC26 genes or isoforms (SLC26A1-11). Except for SLC26A5 (prestin), all function as anion exchangers with versatility with respect to transported anions. Modes of transport mediated by SLC26 members include the exchange of chloride for bicarbonate, hydroxyl, sulfate, formate, iodide, or oxalate with variable specificity. Other anion exchange modes not involving chloride also have been reported for some of the members of this family. Several members of SLC26 isoforms are expressed in the kidney. These include SLC26A1 (SAT1), SLC26A4 (pendrin), SLC26A6 (putative anion transporter [PAT1] or chloride/formate exchange [CFEX]), SLC26A7, and SLC26A11. Each isoform displays a specific nephron segment distribution with a distinct subcellular localization. Coupled to expression studies and examination of genetically engineered mice deficient in various SLC26 isoforms, the evolving picture points to important roles for the SLC26 family in chloride absorption, vascular volume homeostasis, acid-base regulation, and oxalate excretion in the kidney. This review summarizes recent advances in the identification and characterization of SLC26 family members, with specific emphasis on their distribution and role in kidney physiology. Specifically, the roles of A4 (pendrin), A6 (PAT1), and A7 (PAT2) in chloride homeostasis, oxalate excretion, and acid-base balance are discussed.

Expression and interaction of two compound heterozygous distal renal tubular acidosis mutants of kidney anion exchanger 1 in epithelial cells

Kidney AE1 (kAE1) is a glycoprotein responsible for the electroneutral exchange of chloride for bicarbonate, promoting the reabsorption of bicarbonate into the blood by alpha-intercalated cells of the collecting tubule. Mutations occurring in the gene encoding kAE1 can induce defects in urinary acidification resulting in distal renal tubular acidosis (dRTA). We expressed two kAE1 dRTA mutants, A858D, a mild dominant mutation, and DeltaV850, a recessive mutation, in epithelial Madin-Darby canine kidney (MDCK) cells. Individuals heterozygous with wild-type (WT) kAE1 either did not display any symptoms of dRTA (DeltaV850/WT) or displayed a mild incomplete form of dRTA (A858D/WT), while compound heterozygotes (DeltaV850/A858D) had dRTA. We found that the A858D mutant was slightly impaired in the endoplasmic reticulum (ER) exit but could target to the basolateral membrane of polarized MDCK cells. Despite an altered binding to an inhibitor affinity resin, anion transport assays showed that the A858D mutant was functional at the cell surface. The DeltaV850 mutant showed altered binding to the affinity resin but was predominantly retained in the ER, resulting in undetectable AE1 expression at the basolateral membrane. When coexpressed in MDCK cells, the WT protein, and to a lesser extent the A858D mutant, enhanced the cell surface expression of the DeltaV850 mutant. The DeltaV850 mutant also affected the cell surface expression of the A858D mutant. Compound heterozygous (A858D/DeltaV850) patients likely possess a decreased amount of functional anion exchangers at the basolateral membrane of their alpha-intercalated cells, resulting in impaired bicarbonate transport into the blood and defective acid transport into the urine.

Phosphate binder therapy for attainment of K/DOQI™ bone metabolism guidelines

Phosphate binder therapy for attainment of K/DOQI™ bone metabolism guidelines

Carboxyl-terminal truncations of human anion exchanger impair its trafficking to the plasma membrane.

The human anion exchanger AE1 (Band 3) is an abundant glycoprotein localized in plasma membrane of red cells and is responsible for the electro-neutral exchange of chloride for bicarbonate. In order to determine the role of the carboxyl-terminal tail of AE1 in its expression, function and trafficking to the plasma membrane, we generated a series of five constructs encoding truncation mutants missing the last 5 (Delta5), 11 (Delta11), 15 (Delta15), 20 (Delta20) or 35 (Delta35) amino-acids. In transiently transfected HEK 293 cells, immunoblotting of whole cell extracts showed that all the proteins were expressed at the same level as full-length AE1, except Delta20 and particularly Delta35, which showed a reduced expression. Furthermore, the last 15 amino-acids were not required for AE1 folding in the membrane, since Delta5, Delta11 and Delta15 were able to bind to an inhibitor affinity matrix, while Delta20 and Delta35 exhibited poor binding. Immunofluorescence and deglycosylation results showed that Delta15 and Delta11 were retained intracellularly, whereas a lower amount of Delta5 compared with WT trafficked to the plasma membrane. These results indicate that an intact C-terminal tail of human AE1 is important for efficient AE1 trafficking to the plasma membrane.

Control of cell pH in immature primitive red cells from chick embryo

1. 1. The intracellular pH in primitive red cells from 4 day chick embryos was measured with the digitonin null-point method and the fluorescent indicator SNARF-1. At physiological pH e of 8.0 red cell pH is 7.39 at day 4. 2. 2. The calculated proton equilibrium potential of − 38 mV is in good agreement with previous measurements of E m (Engelke et al., 1988) and supports the conclusion that the E m is dominated by a proton conductance. 3. 3. The sodium-proton exchanger is present in primitive red cells but quiescent under physiological conditions. 4. 4. The results indicate that the bicarbonate-chloride exchange via Band 3 protein is impaired.

Immunoregulation of Endometrial and Jejunal Epithelia Sensitized by Infection

The hypothesis was tested that the uterus of the rat orally infected with the parasite Trichinella spiralis becomes hypersensitized and that subsequent antigenic challenge affects functions in the endometrial epithelium. Results of experiments comparing the immunological responsiveness of isolated rat uterus with that of the jejunum supports our hypothesis. Antigenic challenge of uterus mounted in Ussing-type chambers causes an elevation in transuterine short circuit current (Isc) of 6.4 +/- 0.8 microA/cm2. The transduction of the antigenic signal to elicit the electrophysiological response involves 5-hydroxytryptamine (5-HT) working through a nerve-independent pathway. The antigen-stimulated rise in Isc peaks approximately 3 min after challenge. The uterine response is blocked by diisothiocyanostilbene-2,2'-disulfonic acid, an inhibitor of bicarbonate-chloride exchange. The antigen-evoked change in jejunal Isc is biphasic, peaking at 1.5 and approximately 4.0 min after challenge, and is about 10-fold greater in magnitude than the Isc in the uterus. The transductive pathway in the jejunum involves 5-HT, histamine and prostaglandin acting partly through intrinsic nerves. The jejunal response to antigen is inhibited by diphenylamine-2-carboxylate, a chloride channel blocker. Changes in net ion transport which are primed by infection and evoked by antigen are apparently triggered by local anaphylaxis in both the uterus and jejunum.

Factors affecting gas transfer in respiratory organs of vertebrates

The ability of a gas-exchange organ to transfer respiratory gases (O2 and CO2) between the respiratory medium (air or water) and blood is quantitatively characterized by its transfer conductance, usually termed diffusing capacity, D. The problems in defining and determining D are reviewed. In a blood-perfused gas-exchange organ it is useful to consider the ratio [Formula: see text] ([Formula: see text], blood flow; β, effective solubility) which determines the relative role of diffusion limitation. Poor gas transfer may be due to functional inhomogeneities, and not to low D values. Besides the well-known ventilation–perfusion inequality, other types of inhomogeneity involving diffusion resistances in the medium may be operative. Determination of D in functionally inhomogeneous gas-exchange organs is difficult because of both modeling and measurement problems. In blood to medium transfer of CO2, particular features have been noted. First, the equilibration of CO2 between medium and blood appears to be slower than expected on the basis of high physical solubility, owing to the slowness of some steps in the CO2 exchange process (dehydration of carbonic acid, bicarbonate–chloride exchange of red cells). Second, there is controversial evidence for equilibration of pulmonary capillary blood to a CO2 partial pressure lower than that in lung gas.

Molecular mechanisms of bone resorption by the osteoclast

The osteoclast is a multinucleated cell that is actively engaged in the synthesis of lysosomal enzymes, their vectorial transport toward the apical membrane, and the secretion of these enzymes at its apical pole. These secreted enzymes are targeted to the apical ruffled-border membrane by mechanisms that involve cation-independent mannose-6-phosphate receptors. These receptors bind to an enzyme-linked mannose-6-phosphate recognition marker in the Golgi complex, and the enzyme-ligand-receptor complex, carried within small coated transport vesicles, dissociates upon reaching the low pH established in the bone-resorbing compartment by the osteoclast. The apical bone-resorbing compartment is sealed off by the attachment of the osteoclast to the calcified matrix and is actively acidified by the osteoclast. The plasma membrane of the cell is divided into distinct domains. The apical membrane at the ruffled-border shares common antigenic determinants with lysosomal and endosomal membranes, including a 100 kD protein and proton pumps that may be involved in the acidification of the extracellular resorbing compartment. The basolateral membrane is highly enriched in carbonic anhydrase, and bicarbonate-chloride exchange appears to regulate the intracellular pH of this cell. These observations are consistent with a scheme in which, in the low pH environment of the bone-resorbing lacuna produced by the osteoclast, the mineral phase dissolves, exposing the organic matrix to the action of the secreted enzymes. The activity of these enzymes is in turn presumably favored by the acidic milieu. All constituents of the matrix, whether mineral or organic, then would be reduced to their elemental forms (ions and amino acids) extracellularly.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of renal tubular acidification.

Both bicarbonate retrieval from the filtrate as well as the net excretion of acid depend upon hydrogen ion secretion by the tubular epithelium. Hydrogen ion secretion is mediated either by sodium-hydrogen exchange, an electroneutral and secondary active process, or by hydrogen ion secretion, a directly electrogenic and primary active process. Extrusion of hydrogen ions across the apical cell membrane is accompanied by electrogenic bicarbonate transfer across the basolateral cell membrane. Both luminal and peritubular pH exert a strong influence upon acidification by altering the gradient against which hydrogen transport or base exit occur. In the distal nephron, both hydrogen ion secretion and bicarbonate secretion may occur. These transport operations have been shown to be mediated by subgroups of intercalated cells in which hydrogen pumps and bicarbonate-chloride exchange processes are located either in the apical or basolateral cell membranes. Regulation of acidification involves several factors: the rate of luminal buffer delivery, sodium and chloride delivery, the luminal and peritubular pH and pCO2, the electrical potential, mineralocorticoids and the state of the potassium balance.

Hypothesis Revisited

SummaryAn earlier hypothesis based on the results of pancreatic studies in cystic fibrosis (CF) patients without steatorrhoea suggesting that the basic defect was related to an abnormality in control of electrolyte movement, particularly the bicarbonate ion, and water in exocrine secretory tissue is reviewed in light of recent advances in knowledge of secretory mechanisms. Evidence for defective bicarbonate–chloride exchange in a variety of CF exocrine tissues other than the pancreas is cited. Recent recognition of the close relationship between bicarbonate and mucus secretion and their stimulation by prostaglandin E2 is discussed. Consideration is given to the possibility of altered metabolism of PGE2 because of abnormal handling of essential fatty acids in CF, and an attempt is made to relate such a defect to the recent confirmation of oligosaccharide side chain differences such as an increased fucose–sialic acid ratio in CF gastrointestinal mucus and other glycoproteins. It is suggested that membrane permeability to chloride, bicarbonate–chloride exchange, and movement of water through extra‐and intracellular exocrine tissues depends on modifications of the carbohydrate structure of glycoproteins during secretion and that these changes are stimulated by co‐ordination of cell messengers PGE2, calcium, and cyclic AMP. Disturbed PGE2 metabolism in CF could inhibit these changes rendering resulting secretions dry, concentrated, and readily able to block ducts or tubes.

Hypothesis Revisited

An earlier hypothesis based on the results of pancreatic studies in cystic fibrosis (CF) patients without steatorrhoea suggesting that the basic defect was related to an abnormality in control of electrolyte movement, particularly the bicarbonate ion, and water in exocrine secretory tissue is reviewed in light of recent advances in knowledge of secretory mechanisms. Evidence for defective bicarbonate-chloride exchange in a variety of CF exocrine tissues other than the pancreas is cited. Recent recognition of the close relationship between bicarbonate and mucus secretion and their stimulation by prostaglandin E2 is discussed. Consideration is given to the possibility of altered metabolism of PGE2 because of abnormal handling of essential fatty acids in CF, and an attempt is made to relate such a defect to the recent confirmation of oligosaccharide side chain differences such as an increased fucose-sialic acid ratio in CF gastrointestinal mucus and other glycoproteins. It is suggested that membrane permeability to chloride, bicarbonate-chloride exchange, and movement of water through extra- and intracellular exocrine tissues depends on modifications of the carbohydrate structure of glycoproteins during secretion and that these changes are stimulated by coordination of cell messengers PGE2, calcium, and cyclic AMP. Disturbed PGE2 metabolism in CF could inhibit these changes rendering resulting secretions dry, concentrated, and readily able to block ducts or tubes.

Fast bicarbonate-chloride exchange between brain cells and brain extracellular fluid in respiratory acidosis.

The extracellular pH, PCO2, and [Cl-] at the surface of the brain cortex, expiratory PCO2 and arterial blood pressure were continuously recorded in anaesthetized and artificially ventilated cats. The observations from such a preparation were: 1. In response to a nearly step increase in end-tidal PCO2, the brain ECF pH, PCO2, [Cl-] and calculated [HCO-3] changed in the form of a nearly mono-exponential time function after a delay of 5-7 s. 2. The time constants of the changes in the extracellular pH, PCO2, [Cl-] and [HCO-3] were in the range of 30-40 s. 3. The extracellular [HCO-3] increased markedly at an initial rate of 4.22 mmol.1(-1) . min-1 after 36 s. 4. This increase occurred almost simultaneously with a decrease in the extracellular [Cl-]. An [HCO-3]-[Cl-] exchange ratio was determined which very closely approached one. It is concluded that the brain extracellular bicarbonate concentration in respiratory acidosis increases because the H+ formed from the hydrated CO2 reacts with the intracellular buffers of brain cells, mainly glial cells, and HCO-3 inside the cell is formed and exchanged for Cl- outside the cell similar to the HCO-3/Cl- exchange which occurs between red cells and blood plasma during CO2 loading. The described time constants of the anion exchange represent the wash in or wash out time of CO2 in a tissue containing intracellular buffer.

Effect of the absence of bicarbonate upon intracellular pH and calcium fluxes in pancreatic islet cells

The removal of extracellular HCO − 3 together with a decrease in pCO 2, in order to maintain a normal extracellular pH, caused a sustained increase of intracellular pH in rat pancreatic islets. This increase was more marked in glucose-deprived than in glucose-stimulated islets, and was associated with a facilitation of 45Ca efflux from the glucose-deprived islets. Such a facilitation was slightly reduced in the absence of extracellular Ca 2+ and abolished at low extracellular Na + concentration. It failed to occur in glucose-stimulated islets, whether in the presence or absence of extracellular Ca 2+. The removal of HCO − 3 and decrease in the pCO 2 also reduced the magnitude of both the secondary rise in 45Ca efflux and stimulation of insulin release normally evoked by an increase in glucose concentration. These findings suggest that changes in intracellular pH affect both the outflow of Ca 2+ from islet cells as mediated by Na +-Ca 2+ countertransport and the inflow of Ca 2+ by gated Ca 2+ channels. The experimental data are also compatible with the view that islet cells are equipped with an active process of bicarbonate-chloride exchange involved in the regulation of intracellular pH.