Na+/H+-exchange inhibition by cariporide is compensated via Na+,HCO3--cotransport and has no net growth consequences for ErbB2-driven breast carcinomas.

Defense against intracellular acidification of breast cancer tissue depends on net acid extrusion via Na+,HCO3--cotransporter NBCn1/Slc4a7 and Na+/H+-exchanger NHE1/Slc9a1. NBCn1 is increasingly recognized as breast cancer susceptibility protein and promising therapeutic target, whereas evidence for targeting NHE1 is discordant. Currently, selective small molecule inhibitors exist against NHE1 but not NBCn1. Cellular assays-with some discrepancies-link NHE1 activity to proliferation, migration, and invasion; and disrupted NHE1 expression can reduce triple-negative breast cancer growth. Studies on human breast cancer tissue associate high NHE1 expression with reduced metastasis and-in some molecular subtypes-improved patient survival. Here, we evaluate Na+/H+-exchange and therapeutic potential of the NHE1 inhibitor cariporide/HOE-642 in murine ErbB2-driven breast cancer. Ex vivo, cariporide inhibits net acid extrusion in breast cancer tissue (IC50 = 0.18 µM) and causes small decreases in steady-state intracellular pH (pHi). In vivo, we deliver cariporide orally, by osmotic minipumps, and by intra- and peritumoral injections to address the low oral bioavailability and fast metabolism. Prolonged cariporide administration in vivo upregulates NBCn1 expression, shifts pHi regulation towards CO2/HCO3--dependent mechanisms, and shows no net effect on the growth rate of ErbB2-driven primary breast carcinomas. Cariporide also does not influence proliferation markers in breast cancer tissue. Oral, but not parenteral, cariporide elevates serum glucose by ∼1.5 mM. In conclusion, acute administration of cariporide ex vivo powerfully inhibits net acid extrusion from breast cancer tissue but lowers steady-state pHi minimally. Prolonged cariporide administration in vivo is compensated via NBCn1 and we observe no discernible effect on growth of ErbB2-driven breast carcinomas.

The role of HCO3- in propionate-induced anion secretion across rat caecal epithelium.

Propionate, a metabolite from the microbial fermentation of carbohydrates, evokes a release of epithelial acetylcholine in rat caecum resulting in an increase of short-circuit current (Isc) in Ussing chamber experiments. The present experiments were performed in order to characterize the ionic mechanisms underlying this response which has been thought to be due to Cl- secretion. As there are regional differences within the caecal epithelium, the experiments were conducted at oral and aboral rat corpus caeci. In both caecal segments, the propionate-induced Isc (IProp) was inhibited by > 85%, when the experiments were performed either in nominally Cl-- or nominally HCO3--free buffer. In the case of Cl-, the dependency was restricted to the presence of Cl- in the serosal bath. Bumetanide, a blocker of the Na+-K+-2Cl--cotransporter, only numerically reduced IProp suggesting that a large part of this current must be carried by an ion other than Cl-. In the aboral caecum, IProp was significantly inhibited by mucosally administered stilbene derivatives (SITS, DIDS, DNDS), which block anion exchangers. Serosal Na+-free buffer reduced IProp significantly in the oral (and numerically also in aboral) corpus caeci. RT-PCR experiments revealed the expression of several forms of Na+-dependent HCO3--cotransporters in caecum, which might underlie the observed Na+ dependency. These results suggest that propionate sensing in caecum is coupled to HCO3- secretion, which functionally would stabilize luminal pH when the microbial fermentation leads to an increase in the concentration of short-chain fatty acids in the caecal lumen.

The functional association between the sodium/bicarbonate cotransporter (NBC) and the soluble adenylyl cyclase (sAC) modulates cardiac contractility.

The soluble adenylyl cyclase (sAC) was identified in the heart as another source of cyclic AMP (cAMP). However, its cardiac physiological function is unknown. On the other hand, the cardiac Na+/HCO3- cotransporter (NBC) promotes the cellular co-influx of HCO3- and Na+. Since sAC activity is regulated by HCO3-, our purpose was to investigate the potential functional relationship between NBC and sAC in the cardiomyocyte. Rat ventricular myocytes were loaded with Fura-2, Fluo-3, or BCECF to measure Ca2+ transient (Ca2+i) by epifluorescence, Ca2+ sparks frequency (CaSF) by confocal microscopy, or intracellular pH (pHi) by epifluorescence, respectively. Sarcomere or cell shortening was measured with a video camera as an index of contractility. The NBC blocker S0859 (10 µM), the selective inhibitor of sAC KH7 (1 µM), and the PKA inhibitor H89 (0.1 µM) induced a negative inotropic effect which was associated with a decrease in Ca2+i. Since PKA increases Ca2+ release through sarcoplasmic reticulum RyR channels, CaSF was measured as an index of RyR open probability. The generation of CaSF was prevented by KH7. Finally, we investigated the potential role of sAC activation on NBC activity. NBC-mediated recovery from acidosis was faster in the presence of KH7 or H89, suggesting that the pathway sAC-PKA is negatively regulating NBC function, consistent with a negative feedback modulation of the HCO3- influx that activates sAC. In summary, the results demonstrated that the complex NBC-sAC-PKA plays a relevant role in Ca2+ handling and basal cardiac contractility.

Calcineurin/P38MAPK/HSP27-dependent pathways are involved in the attenuation of postischemic mitochondrial injury afforded by sodium bicarbonate co-transporter (NBCe1) inhibition.

The electrogenic sodium bicarbonate co-transporter isoform 1 (NBCe1) plays an important role in ischemia-reperfusion injury. The cardioprotective action of an antibody directed to the extracellular loop 3 (a-L3) of NBCe1 was previously demonstrated by us. However, the role of a-L3 on mitochondrial post-ischemic alterations has not yet been determined. In this study, we aimed to elucidate the effects of a-L3 on post-ischemic mitochondrial state and dynamics analysing the involved mechanisms. Isolated rat hearts were assigned to the following groups: 1) Non-ischemic control (NIC): 110 min of perfusion; 2) Ischemic control (IC): 30 min of global ischemia and 60 min of reperfusion (R); 3) a-L3: a-L3 was administered during the initial 10 min of R; 4) SB + a-L3: SB202190 (p38MAPK inhibitor) plus a-L3. Infarct size (IS) was measured by TTC staining. Developed pressure (LVDP), maximal velocities of rise and decay of LVP (+dP/dt max, -dP/dt max) and end-diastolic pressure (LVEDP) of the left ventricle were used to assess systolic and diastolic function. Mitochondrial Ca2+ response (CaR), Ca2+ retention capacity (CRC), membrane potential (ΔΨm) and MnSOD levels were measured. The expression of P-p38MAPK, calcineurin, P-HSP27, P-Drp1, Drp1, and OPA1 were determined. a-L3 decreased IS, improved post-ischemic recovery of myocardial function, increased P-p38MAPK, P-HSP27, P-Drp1, cytosolic Drp1, and OPA1 expression and decreased calcineurin. These effects were abolished by p38MAPK inhibition with SB. These data show that NBCe1 inhibition by a-L3 limits the cell death, improves myocardial post-ischemic contractility and mitochondrial state and dynamic through calcium decrease/calcineurin inhibition-mediated p38MAPK activation and p38MAPK/HSP27-dependent pathways. Thus, we demonstrated that a-L3 is a potential therapeutic strategy in post-ischemic alterations.

Exploring the autoinhibitory domain of the electrogenic Na+ /HCO3- transporter NBCe1-B, from residues 28 to 62.

KEY POINTS: Slc4a4 (mouse) encodes at least five variants of the electrogenic sodium/bicarbonate transporter NBCe1. The initial 41 cytosolic amino acids of NBCe1-A and -D are unique; NBCe1-A has high activity. The initial 85 amino acids of NBCe1-B, -C and -E are unique; NBCe1-B and -C have low activity. Previous work showed that deleting residues 1-85 or 40-62 of NBCe1-B, or 1-87 of NBCe1-C, eliminates autoinhibition. These regions also include binding determinants for IRBIT (inositol trisphosphate (IP3 )-receptor binding protein released with IP3 ), which relieves autoinhibition. Here, systematically replacing/deleting residues 28-62, we find that only the nine amino acid cationic cluster (residues 40-48) of NBCe1-B is essential for autoinhibition. IRBIT stimulates all but one low-activity construct. We suggest that electrostatic interactions - which IRBIT presumably interrupts - between the cationic cluster and the membrane or other domains of NBCe1 play a central role in tempering the activity of NBCe1-B in the pancreas, brain and other organs. ABSTRACT: Variant B of the electrogenic Na+ /HCO3- cotransporter (NBCe1-B) contributes to the vectorial transport of HCO3- in epithelia (e.g. pancreatic ducts) and to the maintenance of intracellular pH in the central nervous systems (e.g. astrocytes). NBCe1-B has very low basal activity due to an autoinhibitory domain (AID) located, at least in part, in the unique portion (residues 1-85) of the cytosolic NH2 -terminus. Previous work has shown that removing 23 amino acids (residues 40-62) stimulates NBCe1-B. Here, we test the hypothesis that a cationic cluster of nine consecutive positively charged amino acids (residues 40-48) is a necessary part of the AID. Using two-electrode voltage clamping of Xenopus oocytes, we assess the activity of human NBCe1-B constructs in which we systematically replace or delete residues 28-62, which includes the cationic cluster. We find that replacing or deleting all residues within the cationic cluster markedly increases NBCe1-B activity (i.e. eliminates autoinhibition). On the background of a cationic clusterless construct, systematically restoring Arg residues restores autoinhibition in two distinct quanta, with one to three Arg residues restoring ∼50%, and four or more Arg residues restoring virtually all autoinhibition. Systematically deleting residues before the cluster reduces autoinhibition by, at most, a small amount. Replacing or deleting residues after the cluster has no effect. For constructs with low NBCe1 activity (but good surface expression, as assessed by biotinylation), co-expression with super-IRBIT (lacking PP1-binding site) restores full activity (i.e. relieves autoinhibition). In summary, the cationic cluster is a necessary component of the AID of NBCe1-B.

TGF-ß signaling directly regulates transcription and functional expression of the electrogenic sodium bicarbonate cotransporter 1, NBCe1 (SLC4A4), via Smad4 in mouse astrocytes.

The electrogenic sodium bicarbonate cotransporter NBCe1 (SLC4A4) expressed in astrocytes regulates intracellular and extracellular pH. Here, we introduce transforming growth factor beta (TGF-ß) as a novel regulator of NBCe1 transcription and functional expression. Using hippocampal slices and primary hippocampal and cortical astrocyte cultures, we investigated regulation of NBCe1 and elucidated the underlying signaling pathways by RT-PCR, immunoblotting, immunofluorescence, intracellular H(+ ) recording using the H(+ ) -sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein, mink lung epithelial cell (MLEC) assay, and chromatin immunoprecipitation. Activation of TGF-ß signaling significantly upregulated transcript, protein, and surface expression of NBCe1. These effects were TGF-ß receptor-mediated and suppressed following inhibition of JNK and Smad signaling. Moreover, 4-aminopyridine (4AP)-dependent NBCe1 regulation requires TGF-ß. TGF-ß increased the rate and amplitude of intracellular H+ changes upon challenging NBCe1 in wild-type astrocytes but not in cortical astrocytes from Slc4a4-deficient mice. A Smad4 binding sequence was identified in the NBCe1 promoter and Smad4 binding increased after activation of TGF-ß signaling. The data show for the first time that NBCe1 is a direct target of TGF-ß/Smad4 signaling. Through activation of the canonical pathway TGF-ß acts directly on NBCe1 by binding of Smad4 to the NBCe1 promoter and regulating its transcription, followed by increased protein expression and transport activity.

Mechanisms of CO2/H+ Sensitivity of Astrocytes.

Ventral regions of the medulla oblongata of the brainstem are populated by astrocytes sensitive to physiological changes in PCO2/[H+]. These astrocytes respond to decreases in pH with elevations in intracellular Ca2+ and facilitated exocytosis of ATP-containing vesicles. Released ATP propagates Ca2+ excitation among neighboring astrocytes and activates neurons of the brainstem respiratory network triggering adaptive increases in breathing. The mechanisms linking increases in extracellular and/or intracellular PCO2/[H+] with Ca2+ responses in chemosensitive astrocytes remain unknown. Fluorescent imaging of changes in [Na+]i and/or [Ca2+]i in individual astrocytes was performed in organotypic brainstem slice cultures and acute brainstem slices of adult rats. It was found that astroglial [Ca2+]i responses triggered by decreases in pH are preceded by Na+ entry, markedly reduced by inhibition of Na+/HCO3- cotransport (NBC) or Na+/Ca2+ exchange (NCX), and abolished in Na+-free medium or by combined NBC/NCX blockade. Acidification-induced [Ca2+]i responses were also dramatically reduced in brainstem astrocytes of mice deficient in the electrogenic Na+/HCO3- cotransporter NBCe1. Sensitivity of astrocytes to changes in pH was not affected by inhibition of Na+/H+ exchange or blockade of phospholipase C. These results suggest that in pH-sensitive astrocytes, acidification activates NBCe1, which brings Na+ inside the cell. Raising [Na+]i activates NCX to operate in a reverse mode, leading to Ca2+ entry followed by activation of downstream signaling pathways. Coupled NBC and NCX activities are, therefore, suggested to be responsible for functional CO2/H+ sensitivity of astrocytes that contribute to homeostatic regulation of brain parenchymal pH and control of breathing. SIGNIFICANCE STATEMENT: Brainstem astrocytes detect physiological changes in pH, activate neurons of the neighboring respiratory network, and contribute to the development of adaptive respiratory responses to the increases in the level of blood and brain PCO2/[H+]. The mechanisms underlying astroglial pH sensitivity remained unknown and here we show that in brainstem astrocytes acidification activates Na+/HCO3- cotransport, which brings Na+ inside the cell. Raising [Na+]i activates the Na+/Ca2+ exchanger to operate in a reverse mode leading to Ca2+ entry. This identifies a plausible mechanism of functional CO2/H+ sensitivity of brainstem astrocytes, which play an important role in homeostatic regulation of brain pH and control of breathing.

Na+, HCO3--cotransporter NBCn1 increases pHi gradients, filopodia, and migration of smooth muscle cells and promotes arterial remodelling.

AIMS: Arterial remodelling can cause luminal narrowing and obstruct blood flow. We tested the hypothesis that cellular acid-base transport facilitates proliferation and migration of vascular smooth muscle cells (VSMCs) and enhances remodelling of conduit arteries. METHODS AND RESULTS: [Formula: see text]-cotransport via NBCn1 (Slc4a7) mediates net acid extrusion and controls steady-state intracellular pH (pHi) in VSMCs of mouse carotid arteries and primary aortic explants. Carotid arteries undergo hypertrophic inward remodelling in response to partial or complete ligation in vivo, but the increase in media area and thickness and reduction in lumen diameter are attenuated in arteries from NBCn1 knock-out compared with wild-type mice. With [Formula: see text] present, gradients for pHi (∼0.2 units magnitude) exist along the axis of VSMC migration in primary explants from wild-type but not NBCn1 knock-out mice. Knock-out or pharmacological inhibition of NBCn1 also reduces filopodia and lowers initial rates of VSMC migration after scratch-wound infliction. Interventions to reduce H(+)-buffer mobility (omission of [Formula: see text] or inhibition of carbonic anhydrases) re-establish axial pHi gradients, filopodia, and migration rates in explants from NBCn1 knock-out mice. The omission of [Formula: see text] also lowers global pHi and inhibits proliferation in primary explants. CONCLUSION: Under physiological conditions (i.e. with [Formula: see text] present), NBCn1-mediated [Formula: see text] uptake raises VSMC pHi and promotes filopodia, VSMC migration, and hypertrophic inward remodelling. We propose that axial pHi gradients enhance VSMC migration whereas global acidification inhibits VSMC proliferation and media hypertrophy after carotid artery ligation. These findings support a key role of acid-base transport, particularly via NBCn1, for development of occlusive artery disease.

Regulating effect of TongXie-YaoFang on colonic epithelial secretion via Cl- and HCO3- channel.

AIM: To investigate the pharmacological effect of TongXie-YaoFang (TXYF) formula, a Chinese herbal formula, on Diarrhea-predominant irritable bowel syndrome (D-IBS) rats. METHODS: In a neonatal maternal separation plus restraint stress (NMS + RS) model of D-IBS, male Sprague Dawley rats were randomly divided into two groups (NMS + RS group and TXYF-formula group) with no handlings were used as controls (NH group). Starting from postnatal day 60, rats in TXYF-formula group were administered TXYF-formula (4.92 g/100 g bodyweight) orally twice a day for 14 consecutive days while NH group and NMS + RS group were given distilled water. Using short-circuit current technology, we observed 5-HT-induced changes of current across ion channels, such as cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, epithelial Na+ channel (ENaC), Ca2+-dependent Cl- channel (CACC), Na+-K+-2Cl- co-transporter (NKCC), and Na+-HCO3- co-transporter (NBC), in the colonic epithelium of three groups after exposure to drugs and specific blockers with a Power Lab System (AD Instruments International). RESULTS: Under basal conditions, the changes of short-circuit current (∆Isc, µA/cm2) induced by 5-HT were similar in NH group and TXYF-formula group, and both higher than NMS + RS group (70.86 µA/cm2 ± 12.32 µA/cm2, 67.67 µA/cm2 ± 11.68 µA/cm2vs 38.8 µA/cm2 ± 7.25 µA/cm2, P < 0.01, respectively). When CACC was blocked by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid, 5-HT-induced ∆Isc was smaller in NMS + RS group than in NH group and TXYF-formula group, respectively (48.41 µA/cm2 ± 13.15 µA/cm2vs 74.62 µA/cm2 ± 10.73 µA/cm2, 69.22 µA/cm2 ± 11.7 µA/cm2, P < 0.05, respectively). The similar result could be obtained when ENaC was blocked by Amiloride (44.69 µA/cm2 ± 12.58 µA/cm2vs 62.05 µA/cm2 ± 11.26 µA/cm2, 62.11 µA/cm2 ± 12.01 µA/cm2, P < 0.05, respectively). However, when CFTR Cl- channel was blocked by 1,1-dimethyl piperidinium chloride (DPC), 5-HT-induced ∆Isc did not significantly differ in three groups (42.28 µA/cm2 ± 10.61 µA/cm2vs 51.48 µA/cm2 ± 6.56 µA/cm2vs 47.75 µA/cm2 ± 7.99 µA/cm2, P > 0.05, respectively). The similar results could also be obtained in three groups when NBC and NKCC were respectively blocked by their blockers. CONCLUSION: TXYF-formula can regulate the Cl- and HCO3- secretion of colonic mucosa via CFTR Cl- channel, Cl-/HCO3- exchanger, NBC and NKCC co-transporters.

Inhibition of monocarboxylate transporter by N-cyanosulphonamide S0859.

The synthetic compound N-cyanosulphonamide S0859 has been described as a selective inhibitor of sodium-bicarbonate cotransporters (NBC, SLC4) in mammalian heart (Ch'en et al., 2008). First, for comparison, the electrogenic human NBCe1 (SLC4A4) was heterologously expressed in Xenopus laevis oocytes, where its transport activity was inhibited by S0859 with an IC50 of 9µM. The activity of monocarboxylate transporter (MCT) isoforms 1, 2, and 4 (SLC16A1, SLC16A7, SLC16A3), which transport lactate, pyruvate and ketone bodies, were also heterologously expressed in Xenopus oocytes, and their transport activity was similarly and reversibly inhibited by S0859 with an IC50 of 4-10µM. Partial inhibition of lactate transport by S0859 (50µM) was also obtained in cultured astrocytes of mice. Thus, S0859 appears to be an inhibitor of anion transport with a broader spectrum than previously thought, and may also interfere with cellular metabolite uptake/release.

Functional interaction between bicarbonate transporters and carbonic anhydrase modulates lactate uptake into mouse cardiomyocytes.

Blood-derived lactate is a precious energy substrate for the heart muscle. Lactate is transported into cardiomyocytes via monocarboxylate transporters (MCTs) together with H(+), which couples lactate uptake to cellular pH regulation. In this study, we have investigated how the interplay between different acid/base transporters and carbonic anhydrases (CA), which catalyze the reversible hydration of CO2, modulates the uptake of lactate into isolated mouse cardiomyocytes. Lactate transport was estimated both as lactate-induced acidification and as changes in intracellular lactate levels measured with a newly developed Förster resonance energy transfer (FRET) nanosensor. Recordings of intracellular pH showed an increase in the rate of lactate-induced acidification when CA was inhibited by 6-ethoxy-2-benzothiazolesulfonamide (EZA), while direct measurements of lactate flux demonstrated a decrease in MCT transport activity, when CA was inhibited. The data indicate that catalytic activity of extracellular CA increases lactate uptake and counteracts intracellular lactate-induced acidification. We propose a hypothetical model, in which HCO3 (-), formed from cell-derived CO2 at the outer surface of the cardiomyocyte plasma membrane by membrane-anchored, extracellular CA, is transported into the cell via Na(+)/HCO3 (-) cotransport to counteract intracellular acidification, while the remaining H(+) stabilizes extracellular pH at the surface of the plasma membrane during MCT activity to enhance lactate influx into cardiomyocytes.

Functional characterization of intracellular pH regulators responsible for acid extrusion in human radial artery smooth muscle cells.

Intracellular pH (pHi) is a critical factor influencing many important cellular functions. Acid extrusion carriers such as an Na⁺/H⁺ exchanger (NHE) Na⁺/HCO3⁻ cotransporter (NBC) and monocarboxylate transporters (MCT) can be activated when cells are in an acidic condition (pHi < 7.1). Human radial artery smooth muscle cells (HRASMC) is an important conduit in coronary artery bypass graft surgery. However, such far, the pHi regulators have not been characterized in HRASMCs. We therefore investigated the mechanism of pHi recovery from intracellular acidosis and alkalosis, induced by NH4Cl-prepulse and Na-acetate-prepulse, respectively, using intracellular 2',7'-bis(2-carboxethyl)-5(6)- carboxy-fluorescein (BCECF)-fluorescence in HRASMCs. Cultured HRASMCs were derived from the segments of human radial artery that were obtained from patients undergoing bypass grafting. The resting pHi is 7.22 ± 0.03 and 7.17 ± 0.02 for HEPES- (nominally HCO3⁻-free) and CO2/HCO3⁻- buffered solution, respectively. In HEPES-buffered solution, a pHi recovery from induced intracellular acidosis could be blocked completely by 30 µM HOE 694 (3-methylsulfonyl-4-piperidinobenzoyl, guanidine hydrochloride) a specific NHE inhibitor, or by removing [Na⁺]0. In 3% CO2/HCO3⁻-buffered solution, HOE 694 slowed the pHi recovery from the induced intracellular acidosis only, while adding together with DIDS (a specific NBC inhibitor) or removal of [Na⁺]0 entirely inhibited the acid extrusion. Moreover, α-cyano-4-hydroxycinnamate (CHC; a specific blocker of MCT) blocked the lactate-induced pHi changes. In conclusion, we demonstrate, for the first time, that 3 different pHi regulators responsible for acid extruding, i.e. NHE and NBC, and MCT, are functionally co-existed in cultured HRASMCs.

The electrogenic cardiac sodium bicarbonate co-transporter (NBCe1) contributes to the reperfusion injury.

BACKGROUND: Although the participation of the electrogenic sodium/bicarbonate cotransporter (NBCe1) in the recovery from an intracellular acid load is recognized, its role in ischemia-reperfusion is still unclear. METHODS AND RESULTS: Our objective was to assess the role of NBCe1 in reperfusion injury. We use selective functional antibodies against extracellular loop 3 (a-L3) and loop 4 (a-L4) of NBCe1. a-L3 inhibits and a-L4 stimulates NBCe1 activity. Isolated rat hearts were submitted to 40 min of coronary occlusion and 1 h of reperfusion. a-L3, a-L4 or S0859--selective Na(+)-HCO3(-) co-transport inhibitor--were administered during the initial 10 min of reperfusion. The infarct size (IS) was measured by triphenyltetrazolium chloride staining technique. Postischemic systolic and diastolic functions were also assessed. a-L3 and S0859 treatments decreased significantly (P < .05) the IS (16 ± 3% for a-L3 vs. 32 ± 5% in hearts treated with control nonimmune serum and 19 ± 3% for S0859 vs. 39 ± 2% in untreated hearts). Myocardial function during reperfusion improved after a-L3 treatment, but it was not modified by S0859. The infusion of a-L4 did not modify neither the IS nor myocardial function. CONCLUSIONS: The NBCe1 hyperactivity during reperfusion leads to Na(+) and Ca(2+) loading, conducing to Ca(2+) overload and myocardial damage. Consistently, we have shown herein that the selective NBCe1 blockade with a-L3 exerted cardioprotection. This beneficial action strongly suggests that NBCe1 could be a potential target for the treatment of coronary disease.

Identification of functionally distinct Na-HCO3 co-transporters in colon.

Na-HCO3 cotransport (NBC) regulates intracellular pH (pHi) and HCO3 secretion in rat colon. NBC has been characterized as a 5,5'-diisothiocyanato-2-2'-stilbene (DIDS)-sensitive transporter in several tissues, while the colonic NBC is sensitive to both amiloride and DIDS. In addition, the colonic NBC has been identified as critical for pHi regulation as it is activated by intravesicular acid pH. Molecular studies have identified several characteristically distinct NBC isoforms [i.e. electrogenic (NBCe) and electroneutral (NBCn)] that exhibit tissue specific expression. This study was initiated to establish the molecular identity and specific function of NBC isoforms in rat colon. Northern blot and reverse transcriptase PCR (RT-PCR) analyses revealed that electrogenic NBCe1B or NBCe1C (NBCe1B/C) isoform is predominantly expressed in proximal colon, while electroneutral NBCn1C or NBCn1D (NBCn1C/D) is expressed in both proximal and distal colon. Functional analyses revealed that amiloride-insensitive, electrogenic, pH gradient-dependent NBC activity is present only in basolateral membranes of proximal colon. In contrast, amiloride-sensitive, electroneutral, [H(+)]-dependent NBC activity is present in both proximal and distal colon. Both electrogenic and electroneutral NBC activities are saturable processes with an apparent Km for Na of 7.3 and 4.3 mM, respectively; and are DIDS-sensitive with apparent Ki of 8.9 and 263.8 µM, respectively. In addition to Na-H exchanger isoform-1 (NHE1), pHi acidification is regulated by a HCO3-dependent mechanism that is HOE694-insensitive in colonic crypt glands. We conclude from these data that electroneutral, amiloride-sensitive NBC is encoded by NBCn1C/D and is present in both proximal and distal colon, while NBCe1B/C encodes electrogenic, amiloride-insensitive Na-HCO3 cotransport in proximal colon. We also conclude that NBCn1C/D regulates HCO3-dependent HOE694-insensitive Na-HCO3 cotransport and plays a critical role in pHi regulation in colonic epithelial cells.

Gram-scale solution-phase synthesis of selective sodium bicarbonate co-transport inhibitor S0859: in vitro efficacy studies in breast cancer cells.

Na(+)-coupled HCO(3)(-) transporters (NBCs) mediate the transport of bicarbonate ions across cell membranes and are thus ubiquitous regulators of intracellular pH. NBC dysregulation is associated with a range of diseases; for instance, NBCn1 is strongly up-regulated in a model of ErbB2-dependent breast cancer, a malignant and widespread cancer with no targeted treatment options, and single-nucleotide polymorphisms in NBCn1 genetically link to breast cancer development and hypertension. The N-cyanosulfonamide S0859 has been shown to selectively inhibit NBCs, and its availability on the gram scale is therefore of significant interest to the scientific community. Herein we describe a short and efficient synthesis of S0859 with an overall yield of 45 % from commercially available starting materials. The inhibitory effect of S0859 on recovery of intracellular pH after an acid load was verified in human and murine cancer cell lines in Ringer solutions. However, S0859 binds very strongly to components in plasma, and accordingly, measurements on isolated murine tissues showed no effect of S0859 at concentrations up to 50 µM.

IRBIT reduces the apparent affinity for intracellular Mg²âº in inhibition of the electrogenic Na⁺-HCO3⁻ cotransporter NBCe1-B.

The electrogenic Na(+)-HCO(3)(-) cotransporter NBCe1-B can be regulated by intracellular Mg(2+) (Mg(2+)(i)). We previously reported that under whole-cell voltage-clamp conditions, bovine NBCe1-B (bNBCe1-B) currents heterologously expressed in mammalian cells are strongly inhibited by Mg(2+)(i), and the inhibition is likely mediated by electrostatic interaction and relieved by truncation of the cytosolic NBCe1-B specific N-terminal region. Intriguingly, NBCe1-B-like currents natively expressed in bovine parotid acinar (BPA) cells are much less sensitive to Mg(2+)(i) inhibition than bNBCe1-B currents. Here, we hypothesized that this apparent discrepancy may involve IRBIT, a previously identified NBCe1-B-interacting protein. RT-PCR, Western blot and immunofluorescence confocal microscopy revealed that IRBIT was not only expressed in the cytosol, but also colocalized with NBCe1-B in the region of plasma membranes of BPA cells. IRBIT was coimmunoprecipitated with NBCe1-B by an anti-NBCe1 antibody in bovine parotid cell lysate. Whole-cell patch-clamp experiments showed that coexpression of IRBIT lowered the Mg(2+)(i) sensitivity of bNBCe1-B currents stably expressed in HEK293 cells. Collectively, these results suggest that IRBIT may reduce the apparent affinity for Mg(2+)(i) in inhibition of NBCe1-B activity in mammalian cells.

Physiology, pharmacology and pathophysiology of the pH regulatory transport proteins NHE1 and NBCn1: similarities, differences, and implications for cancer therapy.

The Na⁺/H⁺-exchanger 1, NHE1 (SLC9A1) and the electroneutral Na⁺,HCO3⁻ cotransporter NBCn1 (SLC4A7) are coexpressed in a wide range of tissues. Under normal physiological conditions these transporters play an ostensibly similar role, namely that of net acid extrusion after cellular acidification. In addition, they have been implicated in multiple other cellular processes, including regulation of transepithelial transport, cell volume, cell death/survival balance, and cell motility. In spite of their apparent functional similarity, the two transporters also serve distinctly different functions and are differentially regulated. Here, we provide an update on the basic structure, function, regulation, physiology and pharmacology of NHE1 and NBCn1, with particular focus on the factors responsible for their functional similarities and differences. Finally, we highlight recent findings implicating these transporters in cancer development, and discuss issues relating to NHE1 and NBCn1 as potential targets in cancer treatment.

Relief of autoinhibition of the electrogenic Na-HCO(3) [corrected] cotransporter NBCe1-B: role of IRBIT vs.amino-terminal truncation.

Two maneuvers known to stimulate electrogenic sodium bicarbonate cotransporter 1 (NBCe1) activity are 1) deletion from the cytosolic amino-terminus (Nt) of NBCe1-C of an 87-amino acid sequence that contains an autoinhibitory domain (AID); and 2) binding of the protein IRBIT to elements within the same 87-amino acid module in a different variant, NBCe1-B. Helpful to understanding the relationship between these two phenomena would be an appreciation of the relative magnitude of stimulation caused by each maneuver for the same NBCe1 variant. In the present study, we performed two-electrode voltage-clamp on Xenopus oocytes expressing human NBCe1-B constructs, with and without human IRBIT constructs. We find that removal of the AID stimulates NBCe1-B to the same extent as coexpression of wild-type IRBIT. The potency of wild-type IRBIT apparently is reduced by the action of endogenous oocyte protein phosphatases: a mutant IRBIT that cannot be influenced by the action of protein phosphatase-1 stimulates NBCe1-B to an extent 50% greater than can be achieved by removal of the NBCe1-B AID. Thus the stimulatory effect of IRBIT cannot be explained solely by masking of autoinhibitory determinants within the AID. Finally, we find that an NBCe1-B construct that lacks amino acid residues 2-16 of the Nt is fully autoinhibited, but cannot be stimulated by IRBIT, indicating that autoinhibitory and IRBIT-binding determinants within the cytosolic Nt are not identical.

The Na+/H+ exchanger NHE1, but not the Na+, HCO3(-) cotransporter NBCn1, regulates motility of MCF7 breast cancer cells expressing constitutively active ErbB2.

We and others have shown central roles of the Na(+)/H(+) exchanger NHE1 in cell motility. The aim of this study was to determine the roles of NHE1 and of the Na(+), HCO(3)(-) cotransporter NBCn1 in motility of serum-starved MCF-7 breast cancer cells expressing constitutively active ErbB2 (ΔNErbB2). ΔNErbB2 expression elicited NBCn1 upregulation, Ser(703)-phosphorylation of NHE1, and NHE1-inhibitor (EIPA)-sensitive pericellular acidification, in conjunction with increased expression of ß1 integrin and ERM proteins. Active ERM proteins and NHE1 colocalized strongly to invadopodial rosettes, the diameter of which was increased by ΔNErbB2. Adhesion and migration on collagen-I were augmented by ΔNErbB2, unaffected by the NBC inhibitor S0859, and further stimulated by EIPA in a manner potentiated by PI3K-Akt-inhibition. These findings demonstrate that NHE1 inhibition can enhance cancer cell motility, adding an important facet to the understanding of NHE1 in cancer.

Intracellular acidification is associated with changes in free cytosolic calcium and inhibition of action potentials in rat trigeminal ganglion.

The effect of intracellular acidification and subsequent pH recovery in sensory neurons has not been well characterized. We have studied the mechanisms underlying Ca(2+)-induced acidification and subsequent recovery of intracellular pH (pH(i)) in rat trigeminal ganglion neurons and report their effects on neuronal excitability. Glutamate (500 µM) and capsaicin (1 µM) increased intracellular Ca(2+) concentration ([Ca(2+)](i)) with a following decrease in pH(i). The recovery of [Ca(2+)](i) to the prestimulus level was inhibited by LaCl(3) (1 mM) and o-vanadate (10 mM), a plasma membrane Ca(2+)/ATPase (PMCA) inhibitor. Removal of extracellular Ca(2+) also completely inhibited the acidification induced by capsaicin. TRPV1 was expressed only in small and medium sized trigeminal ganglion neurons. mRNAs for Na(+)/H(+) exchanger type 1 (NHE1), pancreatic Na(+)-HCO(3)(-) cotransporter type 1 (pNBC1), NBC3, NBC4, and PMCA types 1-3 were detected by RT-PCR. pH(i) recovery was significantly inhibited by pretreatment with NHE1 or pNBC1 siRNA. We found that the frequency of action potentials (APs) was dependent on pH(i). Application of the NHE1 inhibitor 5'-(N-ethyl-N-isopropyl) amiloride (5 µM) or the pNBC1 inhibitor 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid (500 µM) delayed pH(i) recovery and decreased AP frequency. Simultaneous application of 5'-(N-ethyl-N-isopropyl) amiloride and 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid almost completely inhibited APs. In summary, our results demonstrate that the rise in [Ca(2+)](i) in sensory neurons by glutamate and capsaicin causes intracellular acidification by activation of PMCA type 3, that the pH(i) recovery from acidification is mediated by membrane transporters NHE1 and pNBC1 specifically, and that the activity of these transporters has direct consequences for neuronal excitability.