Acidosis defense mechanisms in the preimplantation stages of embryos in BALB/c strain mice.

Regulation of intracellular pH (pHi) is an important homeostatic function of cells. There are three major pHi regulatory mechanisms: the HCO3-/Cl- exchanger (AE), which alleviates alkalosis, and the Na+/H+ exchanger (NHE) and Na+,HCO3-/Cl- exchanger (NDBCE), both of which counteract acidosis. NHE activity, which is high at the germinal vesicle stage of oocyte, is inhibited during meiotic maturation, while this inhibition is abolished when the oocyte reaches the pronuclear (PN) stage of the zygote. On the other hand, we have previously found that NDBCE performs complementary regulation against acidosis during meiotic maturation. Additionally, we found that AE activity, which is a defense mechanism against alkalosis, gradually decreases during preimplantation period of embryonic development. Considering that NHE activity is inhibited during meiotic maturation and AE activity gradually decreases during embryonic development stages, we investigated whether NHE and NDBCE activities, both of which act against acidosis, functionally change from the PN zygote to the blastocyst stage of the embryo and identified these pH-regulating proteins at the molecular level in mice of the Balb/c strain. PN zygotes, two-cell (2-c), four-cell (4-c), morula and blastocyst stage embryos were obtained from 5-8-week-old, sexually mature female Balb/c mice by using the classical superovulation procedure. pHi was recorded by using the microspectrofluorometric technique on zygotes and embryos simultaneously loaded with the pH-sensitive fluorophore, 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The activities of NHE and NDBCE were determined from the recovery curve of induced-acidosis in bicarbonate-free and bicarbonate-containing media, respectively. Specific inhibitors such as cariporide (1 µM), S3226 (1 and 10 µM), EIPA (1, 5, and 25 µM), and amiloride (1 mM) were used to functionally identify NHE isoforms, and the nonspecific inhibitor 4,4'-diisocyanatostilbene-2,2' disulphonic acid, disodium salt (DIDS) was used to confirm NDBCE activity. The isoforms of the pHi-regulatory proteins were also identified by molecular biology using real-time PCR. We found that NHE activity was high at all embryonic stages, and differences between stages were not significant. Functional and molecular findings indicated that isoforms of NHE 1 and 5 are present in the blastocyst, whereas isoforms of NHE 1, 3, and 4 are functional at earlier embryonic stages. Although the contribution of NDBCE activity to recovery from induced-acidosis was detected at all embryonic stages, it was significant only in the PN zygote and the 2-c embryo. This finding was confirmed by molecular analysis, which detected the expression of SLC4A8 encoding NDBCE at all embryonic stages. In conclusion, NHE is an active and important defense mechanism against acidosis and is encoded by at least two protein isoforms in all stages of the Balb/c strain of mice. NDBCE has a supportive function in all embryonic stages, especially in the PN zygote and the 2-c embryo. Preimplantation stage embryos have effective mechanisms to defend against acidosis in response to their metabolic end products (increased acid load) and the acidic environment in utero.

Roles of Cl­/HCO3­ anion exchanger 2 in the physiology and pathophysiology of the digestive system (Review).

Cl­/HCO3­ anion exchangers (AEs), which are members of the solute carrier 4 family, contribute to the exchange of one intracellular HCO3­ for one extracellular Cl­. AE2, a vital subtype of the Cl­/HCO3­ exchangers, is expressed widely in various cells and tissues in mammals and serves essential roles in the pathophysiological processes of the cardiovascular system and renal tubular reabsorption. Recently, research on the function of AE2 in the digestive system shed new light on its roles in the regulation of cellular and organ physiology. AE2 not only participates in gastric acid secretion, but also mediates bile secretion and digestive cancer development. The aim of the present review was to describe the role of AE2 in the physiology and pathophysiology of the digestive system, with the aim of guiding clinical diagnosis and treatment.

Trichomonas vaginalis infection impairs anion secretion in vaginal epithelium.

Trichomonas vaginalis is a common protozoan parasite, which causes trichomoniasis associated with severe adverse reproductive outcomes. However, the underlying pathogenesis has not been fully understood. As the first line of defense against invading pathogens, the vaginal epithelial cells are highly responsive to environmental stimuli and contribute to the formation of the optimal luminal fluid microenvironment. The cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel widely distributed at the apical membrane of epithelial cells, plays a crucial role in mediating the secretion of Cl- and HCO3-. In this study, we investigated the effect of T. vaginalis on vaginal epithelial ion transport elicited by prostaglandin E2 (PGE2), a major prostaglandin in the semen. Luminal administration of PGE2 triggered a remarkable and sustained increase of short-circuit current (ISC) in rat vaginal epithelium, which was mainly due to Cl- and HCO3- secretion mediated by the cAMP-activated CFTR. However, T. vaginalis infection significantly abrogated the ISC response evoked by PGE2, indicating impaired transepithelial anion transport via CFTR. Using a primary cell culture system of rat vaginal epithelium and a human vaginal epithelial cell line, we demonstrated that the expression of CFTR was significantly down-regulated after T. vaginalis infection. In addition, defective Cl- transport function of CFTR was observed in T. vaginalis-infected cells by measuring intracellular Cl- signals. Conclusively, T. vaginalis restrained exogenous PGE2-induced anion secretion through down-regulation of CFTR in vaginal epithelium. These results provide novel insights into the intervention of reproductive complications associated with T. vaginalis infection such as infertility and disequilibrium in vaginal fluid microenvironment.

Genetic disruption of slc4a10 alters the capacity for cellular metabolism and vectorial ion transport in the choroid plexus epithelium.

BACKGROUND: Genetic disruption of slc4a10, which encodes the sodium-dependent chloride/bicarbonate exchanger Ncbe, leads to a major decrease in Na+-dependent HCO3- import into choroid plexus epithelial cells in mice and to a marked reduction in brain intraventricular fluid volume. This suggests that Ncbe functionally is a key element in vectorial Na+ transport and thereby for cerebrospinal fluid secretion in the choroid plexus. However, slc4a10 disruption results in severe changes in expression of Na+,K+-ATPase complexes and other major transport proteins, indicating that profound cellular changes accompany the genetic manipulation. METHODS: A tandem mass tag labeling strategy was chosen for quantitative mass spectrometry. Alterations in the broader patterns of protein expression in the choroid plexus in response to genetic disruption of Ncbe was validated by semi-quantitative immunoblotting, immunohistochemistry and morphometry. RESULTS: The abundance of 601 proteins were found significantly altered in the choroid plexus from Ncbe ko mice relative to Ncbe wt. In addition to a variety of transport proteins, particularly large changes in the abundance of proteins involved in cellular energy metabolism were detected in the Ncbe ko mice. In general, the abundance of rate limiting glycolytic enzymes and several mitochondrial enzymes were reduced following slc4a10 disruption. Surprisingly, this was accompanied by increased ATP levels in choroid plexus cells, indicating that the reduction in capacity for energy metabolism was adaptive to high ATP rather than causal for a decreased capacity for ion and water transport. Ncbe-deficient cells also had a reduced cell area and decreased K+ content. CONCLUSION: Our findings suggest that the lack of effective Na+-entry into the epithelial cells of the choroid plexus leads to a profound change in the cellular phenotype, shifting from a high-rate secretory function towards a more dormant state; similar to what is observed during ageing or Alzheimer's disease.

Early Hearing Loss upon Disruption of Slc4a10 in C57BL/6 Mice.

The unique composition of the endolymph with a high extracellular K+ concentration is essential for sensory transduction in the inner ear. It is secreted by a specialized epithelium, the stria vascularis, that is connected to the fibrocyte meshwork of the spiral ligament in the lateral wall of the cochlea via gap junctions. In this study, we show that in mice the expression of the bicarbonate transporter Slc4a10/Ncbe/Nbcn2 in spiral ligament fibrocytes starts shortly before hearing onset. Its disruption in a C57BL/6 background results in early onset progressive hearing loss. This hearing loss is characterized by a reduced endocochlear potential from hearing onset onward and progressive degeneration of outer hair cells. Notably, the expression of a related bicarbonate transporter, i.e., Slc4a7/Nbcn1, is also lost in spiral ligament fibrocytes of Slc4a10 knockout mice. The histological analysis of the spiral ligament of Slc4a10 knockout mice does not reveal overt fibrocyte loss as reported for Slc4a7 knockout mice. The ultrastructural analysis, however, shows mitochondrial alterations in fibrocytes of Slc4a10 knockout mice. Our data suggest that Slc4a10 and Slc4a7 are functionally related and essential for inner ear homeostasis.

An impaired biliary bicarbonate umbrella may be involved in dysregulated autophagy in primary biliary cholangitis.

Dysregulated autophagy may be a central player in trehe pathogenesis of primary biliary cholangitis (PBC) by inducing autoimmune processes via abnormal expression of mitochondrial antigens such as pyruvate dehydrogenase complex, E2 component (PDC-E2) and also by inducing cellular senescence in biliary epithelial cells (BECs) in bile duct lesions in PBC. We examined the association of an impaired "biliary bicarbonate umbrella" due to dysfunction of anion exchanger 2 (AE2) with dysregulated autophagy and cellular senescence in PBC. The expression of AE2 was examined in cultured BECs treated with bile acids such as glycochenodeoxycholic acid (GCDC) and tauro-ursodeoxycholic acid (TUDCA), various cytokines (IL-4, IL-13, IFNγ, TNFα, TGFß), and serum deprivation. The effect of AE2 knockdown using siRNA on autophagy, cell surface expression of PDC-E2, and cellular senescence was also examined. The expression of AE2 and its association with autophagy-related markers and senescent markers p16INK4a and p21WAF1/Cip1 were immunohistochemically determined in livers taken from the patients with PBC (n = 50) and 69 control diseased and normal livers. The expression of AE2 was significantly induced in the cultured BECs shortly treated with GCDC and other stresses, whereas it was significantly decreased in senescent BECs induced by GCDC and other stresses (p < 0.05). Dysregulated autophagy, cell surface expression of PDC-E2, and cellular senescence were significantly increased by knockdown of AE2 (p < 0.05). The expression of AE2 was significantly decreased in cholangitis in PBC, compared to control livers (p < 0.05). The decreased expression of AE2 was correlated with dysregulated autophagy, abnormal expression of PDC-E2, and cellular senescence in bile duct lesions in PBC. In conclusion, an impaired biliary bicarbonate umbrella may be involved in the pathogenesis of PBC by inducing dysregulated autophagy.

SLC26A3 (DRA) prevents TNF-alpha-induced barrier dysfunction and dextran sulfate sodium-induced acute colitis.

SLC26A3 encodes a Cl-/HCO3- ion transporter that is also known as downregulated in adenoma (DRA) and is involved in HCO3-/mucus formation. The role of DRA in the epithelial barrier has not been previously established. In this study, we investigated the in vivo and in vitro mechanisms of DRA in the colon epithelial barrier. Immunofluorescence (IF) and co-immunoprecipitation (co-IP) studies reveal that DRA binds directly to tight junction (TJ) proteins and affects the expression of TJ proteins in polarized Caco-2BBe cells. Similarly, DRA colocalizes with ZO-1 in the intestinal epithelium. Knockdown or overexpression of DRA leads to alterations in TJ proteins and epithelial permeability. In addition, TNF-α treatment downregulates DRA by activating NF-кB and subsequently affecting intestinal epithelial barrier integrity. Furthermore, overexpression of DRA partly reverses the TNF-α-induced damage by stabilizing TJ proteins. Neutralization of TNF-α in dextran sulfate sodium (DSS)-induced colitis mice demonstrates improved the outcomes, and the therapeutic effect of the TNF-α neutralizing mAb is mediated in part by the preservation of DRA expression. These data suggest that DRA may be one of the therapeutic targets of TNF-α. Moreover, DRA delivered by adenovirus vector significantly prevents the exacerbation of colitis and improves epithelial barrier function by promoting the recovery of TJ proteins in DSS-treated mice. In conclusion, DRA plays a role in protecting the epithelial barrier and may be a therapeutic target in gut homeostasis.

A Mathematical Model Supports a Key Role for Ae4 (Slc4a9) in Salivary Gland Secretion.

We develop a mathematical model of a salivary gland acinar cell with the objective of investigating the role of two [Formula: see text] exchangers from the solute carrier family 4 (Slc4), Ae2 (Slc4a2) and Ae4 (Slc4a9), in fluid secretion. Water transport in this type of cell is predominantly driven by [Formula: see text] movement. Here, a basolateral [Formula: see text] adenosine triphosphatase pump (NaK-ATPase) and a [Formula: see text]-[Formula: see text]-[Formula: see text] cotransporter (Nkcc1) are primarily responsible for concentrating the intracellular space with [Formula: see text] well above its equilibrium potential. Gustatory and olfactory stimuli induce the release of [Formula: see text] ions from the internal stores of acinar cells, which triggers saliva secretion. [Formula: see text]-dependent [Formula: see text] and [Formula: see text] channels promote ion secretion into the luminal space, thus creating an osmotic gradient that promotes water movement in the secretory direction. The current model for saliva secretion proposes that [Formula: see text] anion exchangers (Ae), coupled with a basolateral [Formula: see text] ([Formula: see text]) (Nhe1) antiporter, regulate intracellular pH and act as a secondary [Formula: see text] uptake mechanism (Nauntofte in Am J Physiol Gastrointest Liver Physiol 263(6):G823-G837, 1992; Melvin et al. in Annu Rev Physiol 67:445-469, 2005. https://doi.org/10.1146/annurev.physiol.67.041703.084745 ). Recent studies demonstrated that Ae4 deficient mice exhibit an approximate [Formula: see text] decrease in gland salivation (Peña-Münzenmayer et al. in J Biol Chem 290(17):10677-10688, 2015). Surprisingly, the same study revealed that absence of Ae2 does not impair salivation, as previously suggested. These results seem to indicate that the Ae4 may be responsible for the majority of the secondary [Formula: see text] uptake and thus a key mechanism for saliva secretion. Here, by using 'in-silico' Ae2 and Ae4 knockout simulations, we produced mathematical support for such controversial findings. Our results suggest that the exchanger's cotransport of monovalent cations is likely to be important in establishing the osmotic gradient necessary for optimal transepithelial fluid movement.

Loss-of-activity-mutation in the cardiac chloride-bicarbonate exchanger AE3 causes short QT syndrome.

Patients with short QT syndrome (SQTS) may present with syncope, ventricular fibrillation or sudden cardiac death. Six SQTS susceptibility genes, encoding cation channels, explain <25% of SQTS cases. Here we identify a missense mutation in the anion exchanger (AE3)-encoding SLC4A3 gene in two unrelated families with SQTS. The mutation causes reduced surface expression of AE3 and reduced membrane bicarbonate transport. Slc4a3 knockdown in zebrafish causes increased cardiac pHi, short QTc, and reduced systolic duration, which is rescued by wildtype but not mutated SLC4A3. Mechanistic analyses suggest that an increase in pHi and decrease in [Cl-]i shortened the action potential duration. However, other mechanisms may also play a role. Altered anion transport represents a mechanism for development of arrhythmia and may provide new therapeutic possibilities.

Bicarbonate in cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF, mucoviscidosis) is caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), which is a chloride and bicarbonate channel necessary for fluid secretion and extracellular alkalization. For a long time, research concentrated on abnormal Cl- and Na+ transport, but neglected bicarbonate as a crucial factor in CF. METHODS: The present short review reports early findings as well as recent insights into the role of CFTR for bicarbonate transport and its defects in CF. RESULTS: The available data indicate impaired bicarbonate transport not only in pancreas, intestine, airways, and reproductive organs, but also in salivary glands, sweat duct and renal tubular epithelial cells. Defective bicarbonate transport is closely related to the impaired mucus properties and mucus blocking in secretory organs of CF patients, causing the life threatening lung disease. CONCLUSIONS: Apart from the devastating lung disease, abrogated bicarbonate transport also leads to many other organ dysfunctions, which are outlined in the present review.

Ion Transport by Ameloblasts during Amelogenesis.

Hypomineralization of developing enamel is associated with changes in ameloblast modulation during the maturation stage. Modulation (or pH cycling) involves the cyclic transformation of ruffle-ended (RE) ameloblasts facing slightly acidic enamel into smooth-ended (SE) ameloblasts near pH-neutral enamel. The mechanism of ameloblast modulation is not clear. Failure of ameloblasts of Cftr-null and anion exchanger 2 ( Ae2)-null mice to transport Cl- into enamel acidifies enamel, prevents modulation, and reduces mineralization. It suggests that pH regulation is critical for modulation and for completion of enamel mineralization. This report presents a review of the major types of transmembrane molecules that ameloblasts express to transport calcium to form crystals and bicarbonates to regulate pH. The type of transporter depends on the developmental stage. Modulation is proposed to be driven by the pH of enamel fluid and the compositional and/or physicochemical changes that result from increased acidity, which may turn RE ameloblasts into SE mode. Amelogenins delay outgrowth of crystals and keep the intercrystalline space open for diffusion of mineral ions into complete depth of enamel. Modulation enables stepwise removal of amelogenins from the crystal surface, their degradation, and removal from the enamel. Removal of matrix allows slow expansion of crystals. Modulation also reduces the stress that ameloblasts experience when exposed to high acid levels generated by mineral formation or by increased intracellular Ca2+. By cyclically interrupting Ca2+ transport by RE ameloblasts and their transformation into SE ameloblasts, proton production ceases shortly and enables the ameloblasts to recover. Modulation also improves enamel crystal quality by selectively dissolving immature Ca2+-poor crystals, removing impurities as Mg2+ and carbonates, and recrystallizing into more acid-resistant crystals.

Double Knockout of the Na+-Driven Cl-/HCO3- Exchanger and Na+/Cl- Cotransporter Induces Hypokalemia and Volume Depletion.

We recently described a novel thiazide-sensitive electroneutral NaCl transport mechanism resulting from the parallel operation of the Cl-/HCO3- exchanger pendrin and the Na+-driven Cl-/2HCO3- exchanger (NDCBE) in ß-intercalated cells of the collecting duct. Although a role for pendrin in maintaining Na+ balance, intravascular volume, and BP is well supported, there is no in vivo evidence for the role of NDCBE in maintaining Na+ balance. Here, we show that deletion of NDCBE in mice caused only subtle perturbations of Na+ homeostasis and provide evidence that the Na+/Cl- cotransporter (NCC) compensated for the inactivation of NDCBE. To unmask the role of NDCBE, we generated Ndcbe/Ncc double-knockout (dKO) mice. On a normal salt diet, dKO and single-knockout mice exhibited similar activation of the renin-angiotensin-aldosterone system, whereas only dKO mice displayed a lower blood K+ concentration. Furthermore, dKO mice displayed upregulation of the epithelial sodium channel (ENaC) and the Ca2+-activated K+ channel BKCa. During NaCl depletion, only dKO mice developed marked intravascular volume contraction, despite dramatically increased renin activity. Notably, the increase in aldosterone levels expected on NaCl depletion was attenuated in dKO mice, and single-knockout and dKO mice had similar blood K+ concentrations under this condition. In conclusion, NDCBE is necessary for maintaining sodium balance and intravascular volume during salt depletion or NCC inactivation in mice. Furthermore, NDCBE has an important role in the prevention of hypokalemia. Because NCC and NDCBE are both thiazide targets, the combined inhibition of NCC and the NDCBE/pendrin system may explain thiazide-induced hypokalemia in some patients.

The Effect and Underlying Mechanism of Ethanol on Intracellular H(+) -Equivalent Membrane Transporters in Human Aorta Smooth Muscle Cells.

BACKGROUND: The presence of intracellular pH (pHi ) regulators, including Na(+) -H(+) exchanger (NHE), Na(+) -HCO3- co-transporter (NBC), Cl(-) /OH(-) exchanger (CHE), and Cl(-) /HCO3- exchanger (AE), have been confirmed in many mammalian cells. Alcohol consumption is associated with increased risk of cardiovascular disorder. The aims of the study were to identify the possible transmembrane pHi regulators and to explore the effects of ethanol (EtOH) (10 to 300 mM) on the resting pHi and pHi regulators in human aorta smooth muscle cells (HASMCs). METHODS: HASMCs were obtained from patients undergoing heart transplant. The pHi was measured by microspectrofluorimetry with the pH-sensitive dye, BCECF-AM. RESULTS: The following results are obtained. (i) In cultured HASMCs, the resting pHi was 7.19 ± 0.04 and 7.13 ± 0.02 for HEPES- and CO2 /HCO3--buffered solution, respectively. (ii) Two different Na(+) -dependent acid-equivalent extruders, including NHE and Na(+) -coupled HCO3- transporter, functionally coexisted. (iii) Two different Cl(-) -dependent acid loaders (CHE and AE) were functionally identified. (iv) EtOH induced a biphasic, concentration-dependent change in resting pHi (+0.25 pH unit at 100 mM but only +0.05 pH unit at 300 mM) in bicarbonate-buffered solution, while caused a concentration-dependent decrease in resting pHi (-0.06 pH unit at 300 mM) in HEPES-buffered solution. (v) The effect of EtOH on NHE activity was also biphasic: increase of 40% at lower concentration of 10 mM, followed by decrease of 30% at higher concentration of 300 mM. (vi) The increase in Na(+) -coupled HCO3- transporter activity by EtOH was concentration dependent. (vii) The effect of EtOH on CHE and AE activities was both biphasic: increase of ~25% at 30 mM, followed by decrease of 10 to 25% at 100 mM, and finally increase of 15 to 20% at 300 mM. CONCLUSIONS: This study demonstrated that 2 acid extruders and 2 acid loaders coexisted functionally in HASMCs and that EtOH induced a biphasic, concentration-dependent change in resting pHi by altering the activity of the 2 acid extruders, NHE and Na(+) -coupled HCO3- transporter, and the 2 acid loaders, CHE and AE.

Ameloblast Modulation and Transport of Cl⁻, Na⁺, and K⁺ during Amelogenesis.

Ameloblasts express transmembrane proteins for transport of mineral ions and regulation of pH in the enamel space. Two major transporters recently identified in ameloblasts are the Na(+)K(+)-dependent calcium transporter NCKX4 and the Na(+)-dependent HPO4 (2-) (Pi) cotransporter NaPi-2b. To regulate pH, ameloblasts express anion exchanger 2 (Ae2a,b), chloride channel Cftr, and amelogenins that can bind protons. Exposure to fluoride or null mutation of Cftr, Ae2a,b, or Amelx each results in formation of hypomineralized enamel. We hypothesized that enamel hypomineralization associated with disturbed pH regulation results from reduced ion transport by NCKX4 and NaPi-2b. This was tested by correlation analyses among the levels of Ca, Pi, Cl, Na, and K in forming enamel of mice with null mutation of Cftr, Ae2a,b, and Amelx, according to quantitative x-ray electron probe microanalysis. Immunohistochemistry, polymerase chain reaction analysis, and Western blotting confirmed the presence of apical NaPi-2b and Nckx4 in maturation-stage ameloblasts. In wild-type mice, K levels in enamel were negatively correlated with Ca and Cl but less negatively or even positively in fluorotic enamel. Na did not correlate with P or Ca in enamel of wild-type mice but showed strong positive correlation in fluorotic and nonfluorotic Ae2a,b- and Cftr-null enamel. In hypomineralizing enamel of all models tested, 1) Cl(-) was strongly reduced; 2) K(+) and Na(+) accumulated (Na(+) not in Amelx-null enamel); and 3) modulation was delayed or blocked. These results suggest that a Na(+)K(+)-dependent calcium transporter (likely NCKX4) and a Na(+)-dependent Pi transporter (potentially NaPi-2b) located in ruffle-ended ameloblasts operate in a coordinated way with the pH-regulating machinery to transport Ca(2+), Pi, and bicarbonate into maturation-stage enamel. Acidification and/or associated physicochemical/electrochemical changes in ion levels in enamel fluid near the apical ameloblast membrane may reduce the transport activity of mineral transporters, which results in hypomineralization.

The role of pendrin in renal physiology.

Pendrin is a Na(+)-independent Cl(-)/HCO3(-) exchanger that localizes to type B and non-A, non-B intercalated cells, which are expressed within the aldosterone-sensitive region of the nephron, i.e., the distal convoluted tubule, the connecting tubule, and the cortical collecting duct. Type B cells mediate Cl(-) absorption and HCO3(-) secretion primarily through pendrin-mediated Cl(-)/HCO3(-) exchange. At least in some treatment models, pendrin acts in tandem with the Na(+)-dependent Cl(-)/HCO3(-) exchanger (NDCBE) encoded by Slc4a8 to mediate NaCl absorption. The pendrin-mediated Cl(-)/HCO3(-) exchange process is greatly upregulated in models of metabolic alkalosis, such as following aldosterone administration or dietary NaHCO3 loading. It is also upregulated by angiotensin II. In the absence of pendrin [Slc26a4 (-/-) or pendrin null mice], aldosterone-stimulated NaCl absorption is reduced, which lowers the blood pressure response to aldosterone and enhances the alkalosis that follows the administration of this steroid hormone. Pendrin modulates aldosterone-induced Na(+) absorption by changing ENaC abundance and function through a kidney-specific mechanism that does not involve changes in the concentration of a circulating hormone. Instead, pendrin changes ENaC abundance and function at least in part by altering luminal HCO3(-) and ATP concentrations. Thus, aldosterone and angiotensin II also stimulate pendrin expression and function, which likely contributes to the pressor response of these hormones. This review summarizes the contribution of the Cl(-)/HCO3(-) exchanger pendrin in distal nephron function.

Evaluation of intracellular pH regulation and alkalosis defense mechanisms in preimplantation embryos.

Intracellular pH (pHi) regulation is an important homeostatic function of cells. There are three major pHi-regulatory mechanisms: HCO3(-)/Cl(-) exchanger (anion exchanger [AE]), which alleviates alkalosis, and the Na(+)/H(+) and Na(+),HCO3(-)/Cl(-) exchangers, both of which alleviate acidosis. We hypothesized that there would be developmental changes in pHi-regulatory activity in preimplantation embryos as conditions in the oviduct are alkaline but acidic in the uterus. This study focused on the AE mechanism in pronuclear (PN) zygotes, two-cell (2-c), four-cell (4-c), morula, and blastocyst stage embryos from Balb/c mice. Microspectrofluorometry was used to monitor changes in pHi in embryos subjected to Cl(-)-free media in presence or absence of an AE inhibitor, DIDS, and in embryos recovering from NH4Cl-induced alkalosis. Real-time polymerase chain reaction was used to identify AE isoforms. The pHi changes were greatest in PN zygotes (0.086 ± 007 pHU/min) but fell as embryos developed to the 2-c, 4-c, morula, and blastocyst stages (0.063 ± 006; 0.035 ± 007; 0.024 ± 004, and 0.014 ± 004 pHU/min, respectively). DIDS significantly reduced the rise in pHi caused by Cl(-) removal in all embryos; the finding pointed out that this pHi changes are due to AE activity. But DIDS only inhibited the recovery responses of PN zygote, 2-c and 4-c embryos but not morula or blastocyst stage embryos. In bicarbonate-containing medium, all embryos recovered from induced alkalosis but only the morula and blastocyst stages could fully compensate from ammonium induced-alkalosis in bicarbonate-free medium. The finding showed that commonly used ammonium pulse method to investigate AE function against alkalosis is not suitable for morula and blastocyst embryonic stages. All embryos expressed SLC4A2 and SLC4A4 coding for AE-2 and AE-4, but none expressed either AE-1 or AE-3. The gradual change in the response to alkalosis in preimplantation embryos may be adaptations to their normal in vivo environment, where the early embryos are located in the alkaline oviduct, whereas the morula and blastocyst move into the acidic uterus.

NBCe1 as a model carrier for understanding the structure-function properties of Na⁺ -coupled SLC4 transporters in health and disease.

SLC4 transporters are membrane proteins that in general mediate the coupled transport of bicarbonate (carbonate) and share amino acid sequence homology. These proteins differ as to whether they also transport Na(+) and/or Cl(-), in addition to their charge transport stoichiometry, membrane targeting, substrate affinities, developmental expression, regulatory motifs, and protein-protein interactions. These differences account in part for the fact that functionally, SLC4 transporters have various physiological roles in mammals including transepithelial bicarbonate transport, intracellular pH regulation, transport of Na(+) and/or Cl(-), and possibly water. Bicarbonate transport is not unique to the SLC4 family since the structurally unrelated SLC26 family has at least three proteins that mediate anion exchange. The present review focuses on the first of the sodium-dependent SLC4 transporters that was identified whose structure has been most extensively studied: the electrogenic Na(+)-base cotransporter NBCe1. Mutations in NBCe1 cause proximal renal tubular acidosis (pRTA) with neurologic and ophthalmologic extrarenal manifestations. Recent studies have characterized the important structure-function properties of the transporter and how they are perturbed as a result of mutations that cause pRTA. It has become increasingly apparent that the structure of NBCe1 differs in several key features from the SLC4 Cl(-)-HCO3 (-) exchanger AE1 whose structural properties have been well-studied. In this review, the structure-function properties and regulation of NBCe1 will be highlighted, and its role in health and disease will be reviewed in detail.

Cyclic AMP and alkaline pH downregulate carbonic anhydrase 2 in mouse fibroblasts.

BACKGROUND: The hydration of CO2 catalyzed by the ubiquitous carbonic anhydrase 2 (Ca2) is central for bicarbonate transport, bone metabolism and acid-base homeostasis in metazoans. There is evidence that in some tissues Ca2 expression can be acutely induced by cAMP, whereas in other cell types it is unresponsive to cAMP-mediated transcriptional activation. METHODS: We isolated fibroblasts from wild type and mice lacking the ubiquitous chloride/bicarbonate exchanger (Ae2a,b(-/-) mice). In these cells the regulation of carbonic anhydrase 2 by cAMP was studied. RESULTS: We show that Ca2 expression is strongly inhibited by chronic incubation with dibutyryl-cAMP, forskolin or alkaline pH in cultured mouse fibroblasts. Furthermore, fibroblasts obtained from anion exchanger 2 deficient (Ae2a,b(-/-)) mice, which display intracellular alkalosis and increased cAMP production, express less than 10% of control Ca2 mRNA and protein. Surprisingly, inhibition of the bicarbonate-sensitive soluble adenylyl cyclase (sAC) was found to reduce CA2 expression instead of increasing it. CONCLUSIONS: CA2 expression is strongly regulated by intracellular pH and by cAMP, suggesting a role for soluble adenylyl cyclase. Regulation occurs in opposite directions which may be explained by an incoherent feedforward loop consisting of activation by pCREB and repression by ICER.

SLC26 Cl-/HCO3- exchangers in the kidney: roles in health and disease.

Solute-linked carrier 26 (SLC26) isoforms constitute a conserved family of anion transporters with 10 distinct members. Except for SLC26A5 (prestin), all can operate as multifunctional anion exchangers, with three members (SLC26A7, SLC26A9, and SLC26A11) also capable of functioning as chloride channels. Several SLC26 isoforms can specifically mediate Cl(-)/HCO(3)(-) exchange. These include SLC26A3, A4, A6, A7, A9, and A11, which are expressed in the kidney except for SLC26A3 (DRA), which is predominantly expressed in the intestine. SLC26 Cl(-)/HCO(3)(-) exchanger isoforms display unique nephron segment distribution patterns with distinct subcellular localization in the kidney tubules. Together with studies in pathophysiologic states and the examination of genetically engineered mouse models, the evolving picture points to important roles for the SLC26 family in health and disease states. This review summarizes recent advances in the characterization of the SLC26 Cl(-)/HCO(3)(-) exchangers in the kidney with emphasis on their essential role in diverse physiological processes, including chloride homeostasis, oxalate excretion and kidney stone formation, vascular volume and blood pressure regulation, and acid-base balance.

The SLC4 family of bicarbonate (HCO3⁻) transporters.

The SLC4 family consists of 10 genes (SLC4A1-5; SLC4A7-11). All encode integral membrane proteins with very similar hydropathy plots-consistent with 10-14 transmembrane segments. Nine SLC4 members encode proteins that transport HCO3(-) (or a related species, such as CO3(2-)) across the plasma membrane. Functionally, eight of these proteins fall into two major groups: three Cl-HCO3 exchangers (AE1-3) and five Na(+)-coupled HCO3(-) transporters (NBCe1, NBCe2, NBCn1, NBCn2, NDCBE). Two of the Na(+)-coupled transporters (NBCe1, NBCe2) are electrogenic; the other three Na(+)-coupled HCO3(-) transporters and all three AEs are electroneutral. In addition, two other SLC4 members (AE4, SLC4A9 and BTR1, SLC4A11) do not yet have a firmly established function. Most, though not all, SLC4 members are functionally inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). SLC4 proteins play important roles many modes of acid-base homeostasis: the carriage of CO2 by erythrocytes, the transport of H(+) or HCO3(-) by several epithelia, as well as the regulation of cell volume and intracellular pH.