Cotransporter NBCn1 Research Articles

Sodium/bicarbonate cotransporter NBCn1/slc4a7 increases cytotoxicity in magnesium depletion in primary cultures of hippocampal neurons

Growing evidence suggests that pharmacological inhibition of Na/H exchange and Na/HCO(3) transport provides protection against damage or injury in cardiac ischemia. In this study, we examined the contribution of the sodium/bicarbonate cotransporter NBCn1 (slc4a7) to cytotoxicity in cultured hippocampal neurons of rats. In neurons exposed to extracellular pH (pH(o)) ranging from 6.2 to 8.3, NBCn1 protein expression increased by fivefold at pH < 6.5 compared to the expression at pH(o) 7.4. At pH(o) 6.5, the intracellular pH of neurons was approximately 1 unit lower than that at pH 7.4. Immunochemistry showed a marked increase in NBCn1 immunofluorescence in plasma membranes and cytosol of the soma as well as in dendrites, at pH(o) 6.5. NBCn1 expression also increased by 40% in a prolonged Mg(2+)-free incubation at normal pH(o). Knockdown of NBCn1 in neurons had negligible effect on cell viability. The effect of NBCn1 knockdown on cytotoxicity was then determined by exposing neurons to 0.5 mm glutamate for 10 min and measuring lactate dehydrogenase (LDH) release from neurons. Compared to normal incubation (pH(o) 7.2 for 6 h) after glutamate exposure, acidic incubation (pH(o) 6.3 for 6 h) reduced cytotoxicity by 75% for control neurons and 78% for NBCn1-knockdown neurons. Thus, both controls and knockdown neurons showed acidic protection from cytotoxicity. However, in Mg(2+)-free incubation after glutamate exposure, NBCn1 knockdown progressively attenuated cytotoxicity. This attenuation was unaffected by acidic preincubation before glutamate exposure. We conclude that NBCn1 has a dynamic upregulation in low pH(o) and Mg(2+) depletion. NBCn1 is not required for acidic protection, but increases cytotoxicity in Mg(2+)-free conditions.

Antibody-independent localization of the electroneutral Na+-HCO3− cotransporter NBCn1 (slc4a7) in mice

The expression pattern of the electroneutral Na(+)-HCO(3)(-)cotransporter NBCn1 (slc4a7) was investigated by beta-galactosidase staining of mice with a LacZ insertion into the NBCn1 gene. This method is of particular value because it is independent of immunoreactivity. We find that the NBCn1 promoter is active in a number of tissues where NBCn1 has previously been functionally or immunohistochemically identified, including a broad range of blood vessels (vascular smooth muscle cells and endothelial cells), kidney thick ascending limb and medullary collecting duct epithelial cells, the epithelial lining of the kidney pelvis, duodenal enterocytes, choroid plexus epithelial cells, hippocampus, and retina. Kidney corpuscles, colonic mucosa, and nonvascular smooth muscle cells (from the urinary bladder, trachea, gastrointestinal wall, and uterus) were novel areas of promoter activity. Atrial but not ventricular cardiomyocytes were stained. In the brain, distinct layers of the cerebral cortex and cerebellar Purkinje cells were stained as was the dentate nucleus. No staining of skeletal muscle or cortical collecting ducts was observed. RT-PCR analyses confirmed the expression of NBCn1 and beta-galactosidase in selected tissues. Disruption of the NBCn1 gene resulted in reduced NBCn1 expression, and in bladder smooth muscle cells, reduced amiloride-insensitive Na(+)-dependent HCO(3)(-) influx was observed. Furthermore, disruption of the NBCn1 gene resulted in a lower intracellular steady-state pH of bladder smooth muscle cells in the presence of CO(2)/HCO(3)(-) but not in its nominal absence. We conclude that NBCn1 has a broad expression profile, supporting previous findings based on immunoreactivity, and suggest several new tissues where NBCn1 may be important.

Vacuolar H+-ATPase expression is increased in acid-secreting intercalated cells in kidneys of rats with hypercalcaemia-induced alkalosis

Hypercalcaemia is known to be associated with systemic metabolic alkalosis, although the underlying mechanism is uncertain. Therefore, we aimed to examine whether hypercalcaemia was associated with changes in the expression of acid-base transporters in the kidney. Rats were infused with human parathyroid hormone (PTH, 15 microg kg(-1) day(-1)), or vehicle for 48 h using osmotic minipumps. The rats treated with PTH developed hypercalcaemia and exhibited metabolic alkalosis (arterial HCO: 31.1 +/- 0.8 vs. 28.1 +/- 0.8 mmol L(-1) in controls, P < 0.05, n = 6), whereas the urine pH of 6.85 +/- 0.1 was significantly decreased compared with the pH of 7.38 +/- 0.1 in controls (P < 0.05, n = 12). The observed alkalosis was associated with a significantly increased expression of the B1-subunit of the H(+)-ATPase in kidney inner medulla (IM, 233 +/- 45% of the control level). In contrast, electroneutral Na(+)-HCO cotransporter NBCn1 and Cl(-)/HCO anion exchanger AE2 expression was markedly reduced in the inner stripe of the outer medulla (to 26 +/- 9% and 65 +/- 6%, respectively). These findings were verified by immunohistochemistry. (1) hypercalcaemia-induced metabolic alkalosis was associated with increased urinary excretion of H(+); (2) the increased H(+)-ATPase expression in IM may partly explain the enhanced urinary acidification, which is speculated to prevent stone formation because of hypercalciuria and (3) the decreased expression of outer medullary AE2 suggests a compensatory reduction of the transepithelial bicarbonate transport.

Colony-Stimulating Factor-1 Increases Osteoclast Intracellular pH and Promotes Survival via the Electroneutral Na/HCO3 Cotransporter NBCn1

Colony-stimulating factor-1 (CSF-1) promotes the survival of osteoclasts, short-lived cells that resorb bone. Although a rise in intracellular pH (pH(i)) has been linked to inhibition of apoptosis, the effect of CSF-1 on pH(i) in osteoclasts has not been reported. The present study shows that, in the absence of CO(2)/HCO(3)(-), CSF-1 causes little change in osteoclast pH(i). In contrast, exposing these cells to CSF-1 in the presence of CO(2)/HCO(3)(-) causes a rapid and sustained cellular alkalinization. The CSF-1-induced rise in pH(i) is not blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, an inhibitor of HCO(3)(-) transporters but is abolished by removing extracellular sodium. This inhibition profile is similar to that of the electroneutral Na/HCO(3) cotransporter NBCn1. By RT-PCR, NBCn1 transcripts are present in both osteoclasts and osteoclast-like cells (OCLs), and by immunoblotting, the protein is present in OCLs. Moreover, CSF-1 promotes osteoclast survival in the presence of CO(2)/HCO(3)(-) buffer but not in its absence. Preventing the activation of NBCn1 markedly attenuates the ability of CSF-1 to 1) block activation of caspase-8 and 2) prolong osteoclast survival. Inhibiting caspase-3 or caspase-8 in OCLs prolongs osteoclast survival to the same extent as does CSF-1. This study provides the first evidence that osteoclasts express a CSF-1-regulated Na/HCO(3) cotransporter, which may play a role in cell survival.

Characterization of a polyclonal antibody directed against the amino terminus of the human sodium‐coupled bicarbonate transporter NCBE

The SLC4 family of bicarbonate transporters includes the products of ten genes. These transporters play an essential role in intracellular-pH regulation, transepithelial HCO3 −transport, and CO2 transport by the RBC. Because the sequences of SLC4 members are highly conserved, it is important for the study of the SLC4 proteins to develop antibodies that can distinguish different members of the family. NCBE is an electroneutral Na+-coupled HCO3 −transporter that is mainly expressed in central nervous system. Here we characterized the specificity of a new antibody that we generated in a rabbit against a fusion protein consisting of maltose-binding protein (MBP) and the first 135 amino acids at the N-terminus of human NCBE. The two SLC4 proteins most closely related to NCBE are the human Na+-driven Cl-HCO3 exchanger NDCBE and the electroneutral Na/HCO3 cotransporter NBCn1. By western blotting, the antiserum at a dilution of 1:10,000 recognized a purified peptide representing the first 124 N-terminal amino acids (aa) of NCBE, but not comparable peptides for NDCBE (116 aa) or NBCn1 (123 aa). Also by western blotting, the antiserum at a dilution of 1:2000 recognized full-length NCBE expressed in Xenopus oocytes, but not full-length versions of either NDCBE or NBCn1 expressed in oocytes. Thus, the antiserum is specific for NCBE and does not cross react with the two SLC4 members that are most highly homologous to NCBE. (This work is supported by NS18400.)

An anti-NH2-terminal antibody localizes NBCn1 to heart endothelia and skeletal and vascular smooth muscle cells

The electroneutral sodium bicarbonate cotransporter NBCn1 or NBC3 was originally cloned from rat aorta and from human skeletal muscle. NBCn1 (or NBC3) has been localized to the basolateral membrane of various epithelia, but thus far it has been impossible to detect the protein in these tissues by using anti-COOH-terminal antibodies. Hence an antibody was developed against the NH2-terminus of NBCn1 and was validated by peptide recognition and immunoblotting on positive control tissues and by binding of an approximately 180-kDa protein in the rat kidney, cerebrum, cerebellum, and duodenum. In addition, an approximately 180-kDa immunoreactive band appeared using samples from the aorta, heart ventricles and atria, mesenteric arteries, lung, spleen, liver, pancreas, and epididymis. Immunohistochemical analysis confirmed the previously described labeling in the kidney, duodenum, and the choroid plexus. The anti-NH2-terminal antibody localized NBCn1 to the plasma membrane domains of endothelia and smooth muscle cells in small mesenteric and renal arteries, as well as the capillaries of the heart ventricles, spleen, and salivary glands. NBCn1 was also detected in neuromuscular junctions and vasculature in skeletal muscle. Analysis of variable NBCn1 splicing by RT-PCR revealed that an NH2-terminal sequence, the cassette III, seems absent from cardiovascular NBCn1 and that both cassettes I and III are variable in most epithelia, whereas cassette II is absent from epithelial NBCn1. Thus the development of the NH2-terminal antibody allowed the localization of NBCn1 protein to major cardiovascular tissues where NBCn1 mRNA was previously detected. The NBCn1 is a likely candidate for mediating the reported electroneutral Na+-HCO3(-) cotransport in vascular smooth muscle.

NKCC1 and NHE1 are abundantly expressed in the basolateral plasma membrane of secretory coil cells in rat, mouse, and human sweat glands

In isolated sweat glands, bumetanide inhibits sweat secretion. The mRNA encoding bumetanide-sensitive Na(+)-K(+)-Cl(-) cotransporter (NKCC) isoform 1 (NKCC1) has been detected in sweat glands; however, the cellular and subcellular protein localization is unknown. Na(+)/H(+) exchanger (NHE) isoform 1 (NHE1) protein has been localized to both the duct and secretory coil of human sweat duct; however, the NHE1 abundance in the duct was not compared with that in the secretory coil. The aim of this study was to test whether mRNA encoding NKCC1, NKCC2, and Na(+)-coupled acid-base transporters and the corresponding proteins are expressed in rodent sweat glands and, if expressed, to determine the cellular and subcellular localization in rat, mouse, and human eccrine sweat glands. NKCC1 mRNA was demonstrated in rat palmar tissue, including sweat glands, using RT-PCR, whereas NKCC2 mRNA was absent. Also, NHE1 mRNA was demonstrated in rat palmar tissue, whereas NHE2, NHE3, NHE4, electrogenic Na(+)-HCO(3)(-) cotransporter 1 NBCe1, NBCe2, electroneutral Na(+)-HCO(3)(-) cotransporter NBCn1, and Na(+)-dependent Cl(-)/HCO(3)(-) exchanger NCBE mRNA were not detected. The expression of NKCC1 and NHE1 proteins was confirmed in rat palmar skin by immunoblotting, whereas NKCC2, NHE2, and NHE3 proteins were not detected. Immunohistochemistry was performed using sections from rat, mouse, and human palmar tissue. Immunoperoxidase labeling revealed abundant expression of NKCC1 and NHE1 in the basolateral domain of secretory coils of rat, mouse, and human sweat glands and low expression was found in the coiled part of the ducts. In contrast, NKCC1 and NHE1 labeling was absent from rat, mouse, and human epidermis. Immunoelectron microscopy demonstrated abundant NKCC1 and NHE1 labeling of the basolateral plasma membrane of mouse sweat glands, with no labeling of the apical plasma membranes or intracellular structures. The basolateral NKCC1 of the secretory coils of sweat glands would most likely account for the observed bumetanide-sensitive NaCl secretion in the secretory coils, and the basolateral NHE1 is likely to be involved in Na(+)-coupled acid-base transport.

NBCn1 is a basolateral \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Na}^{+}\mathrm{-HCO}_{3}^{-}\) \end{document} cotransporter in rat kidney inner medullary collecting ducts

Primary cultures of rat inner medullary collecting duct (IMCD) cells Na(+) dependently import HCO(3)(-) across the basolateral membrane through an undefined transport protein. We used RT-PCR, immunoblotting, and immunohistochemistry to identify candidate proteins for this basolateral Na(+)-HCO(3)(-) cotransport. The mRNA encoding the electroneutral Na(+)-HCO(3)(-) cotransporter NBCn1 was detected as the only Na(+)-HCO(3)(-) cotransporter in the rat inner medulla (IM) among the five characterized Na(+)-dependent HCO(3)(-) transporters. The mRNA of a yet uncharacterized transporter-like protein, BTR1, was also present in the IM, but its expression in microdissected tubules seemed restricted to the thin limbs of Henle's loop. Immunoblotting confirmed the presence of NBCn1 as an approximately 180-kDa protein of the rat IM. Anti-NBCn1 immunolabeling was confined to the basolateral plasma membrane domain of IMCD cells in the papillary two-thirds of the IM. Consistent with the presence of NBCn1, IMCD cells possessed stilbene-insensitive, Na(+)- and HCO(3)(-)-dependent pH recovery after acidification, as assessed by fluorescence microscopy using a pH-sensitive intracellular dye. In furosemide-induced alkalotic rats, NBCn1 protein abundance was decreased in both the IM and inner stripe of outer medulla (ISOM) as determined by immunoblotting and immunohistochemistry. In contrast, NBCn1 abundance in the IM and ISOM was unchanged in NaHCO(3)-loaded animals, and the NBCn1 abundance increased only in the ISOM after NH(4)Cl loading. In conclusion, NBCn1 is a basolateral Na(+)-HCO(3)(-) cotransporter of IMCD cells and is differentially regulated in IMCD and medullary thick ascending limb.

CAMP-mediated regulation of murine intestinal/pancreatic Na+/HCO3- cotransporter subtype pNBC1.

Basolateral Na(+)-HCO(3)(-) cotransport is essential for intestinal anion secretion, and indirect evidence suggests that it may be stimulated by a rise of intracellular cAMP. We therefore investigated the expression, activity, and regulation by cAMP of the Na(+)-HCO(3)(-) cotransporter isoforms NBC1 and NBCn1 in isolated murine colonic crypts. Na(+)-HCO(3)(-) transport rates were measured fluorometrically in BCECF-loaded crypts, and mRNA expression levels and localization were determined by semiquantitative PCR and in situ hybridization. Acid-activated Na(+)-HCO(3)(-) cotransport rates were 5.07 +/- 0.7 mM/min and increased by 62% after forskolin stimulation. NBC1 mRNA was more abundant in colonic crypts than in surface cells, and crypts expressed far more NBC1 than NBCn1. To investigate whether the cAMP-induced Na(+)-HCO(3)(-) cotransport activation was secondary to secretion-associated changes in HCO(3)(-) or cell volume, we measured potential forskolin-induced changes in intracellular pH and assessed Na(+)-HCO(3)(-) transport activity in CFTR -/- crypts (in which no forskolin-induced cell shrinkage occurs). We found 30% reduced Na(+)-HCO(3)(-) transport rates in CFTR -/- compared with CFTR +/+ crypts but similar Na(+)-HCO(3)(-) cotransport activation by forskolin. These studies establish the existence of an intracellular HCO(3)(-) concentration- and cell volume-independent activation of colonic NBC by an increase in intracellular cAMP.

Regulation of intracellular pH in Calu-3 human airway cells.

The Calu-3 human cell line exhibits features of submucosal gland serous cells and secretes HCO(3)(-). The aim of this study was to identify the HCO(3)(-) transporters present in these cells by studying their role in the regulation of intracellular pH (pH(i)). Calu-3 cells were grown on coverslips, loaded with the pH-sensitive fluorescent dye BCECF, and their fluorescence intensity monitored as an indication of pH(i). Cells were acidified with NH(4)Cl (25 mM, 1 min) and pH(i) recovery recorded. In the absence of HCO(3)(-), initial recovery was 0.208 +/- 0.016 pH units min(-1) (n = 37). This was almost abolished by removal of extracellular Na(+) and by amiloride (1 mM), consistent with the activity of a Na(+)-H(+) exchanger (NHE). In the presence of HCO(3)(-) and CO(2), recovery (0.156 +/- 0.018 pH units min(-1)) was abolished (reduced by 91.8 +/- 6.7 %, n = 7) by removal of Na(+) but only attenuated (by 63.3 +/- 5.8 %, n = 9) by amiloride. 4,4-Dinitrostilbene-2,2-disulfonic acid (DNDS) inhibited recovery by 45.8 +/- 5.0 % (n = 7). The amiloride-insensitive recovery was insensitive to changes in membrane potential, as confirmed by direct microelectrode measurements, brought about by changing extracellular [K(+)] in the presence of either valinomycin or the K(+) channel opener 1-EBIO. In addition, forskolin (10 microM), which activates the cystic fibrosis transmembrane conductance regulator Cl(-) conductance in these cells and depolarises the cell membrane, had no effect on recovery. Removal of extracellular Cl(-) trebled pH(i) recovery rates, suggesting that an electroneutral, DNDS-sensitive, Cl(-)-HCO(3)(-) exchanger together with a NHE may be involved in pH(i) regulation and HCO(3)(-) secretion in these cells. RT-PCR detected the expression of the electrogenic Na(+)-HCO(3)(-) cotransporter NBC1 and the Cl(-)-HCO(3)(-) exchanger (AE2) but not the electroneutral Na(+)-HCO(3)(-) cotransporter NBCn1.

Intracellular pH.

Intracellular pH.