Polarization of Na(+)/H(+) and Cl(-)/HCO (3)(-) exchangers in migrating renal epithelial cells

Cell migration is crucial for processes such as immune defense, wound healing, or the formation of tumor metastases. Typically, migrating cells are polarized within the plane of movement with lamellipodium and cell body representing the front and rear of the cell, respectively. Here, we address the question of whether this polarization also extends to the distribution of ion transporters such as Na(+)/H(+) exchanger (NHE) and anion exchanger in the plasma membrane of migrating cells. Both transporters are required for locomotion of renal epithelial (Madin-Darby canine kidney, MDCK-F) cells and human melanoma cells since their blockade reduces the rate of migration in a dose-dependent manner. Inhibition of migration of MDCK-F cells by NHE blockers is accompanied by a decrease of pH(i). However, when cells are acidified with weak organic acids, migration of MDCK-F cells is normal despite an even more pronounced decrease of pH(i). Under these conditions, NHE activity is increased so that cells are swelling due to the accumulation of organic anions and Na(+). When exclusively applied to the lamellipodium, blockers of NHE or anion exchange inhibit migration of MDCK-F cells as effectively as when applied to the entire cell surface. When they are directed to the cell body, migration is not affected. These data are confirmed immunocytochemically in that the anion exchanger AE2 is concentrated at the front of MDCK-F cells. Our findings show that NHE and anion exchanger are distributed in a polarized way in migrating cells. They are consistent with important contributions of both transporters to protrusion of the lamellipodium via solute uptake and consequent volume increase at the front of migrating cells.     

Identification of the carbonic anhydrase II binding site in the Cl(-)/HCO(3)(-) anion exchanger AE1

The human Cl(-)/HCO(3)(-) anion exchanger (AE1) possesses a binding site within its 33 residue carboxyl-terminal region (Ct) for carbonic anhydrase II (CAII). The amino acid sequence comprising this CAII binding site was determined by peptide competition and by testing the ability of truncation and point mutants of the Ct sequence to bind CAII with a sensitive microtiter plate binding assay. A synthetic peptide consisting of the entire 33 residues of the Ct (residues 879-911) could compete with a GST fusion protein of the Ct (GST-Ct) for binding to immobilized CAII, while a peptide consisting of the last 16 residues (896-911) could not. A series of truncation mutants of the GST-Ct showed that the terminal 21 residues of AE1 were not required for binding CAII. Removal of four additional residues (887-890) from the Ct resulted in loss of CAII binding. Acidic residues in this region (D887ADD) were critical for binding since mutating this sequence in the GST-Ct to DAAA, AAAA, or NANN caused loss of CAII binding. A GST-Ct construct mutated to D887ANE, the homologous sequence in AE2, could bind CAII. AE2 is a widely expressed anion exchanger and has a homologous Ct region with 60% sequence identity to AE1. A GST fusion protein of the 33 residue Ct of AE2 could bind to CAII similarly to the Ct of AE1. Tethering of CAII to an acidic motif within the Ct of anion exchangers may be a general mechanism for promoting bicarbonate transport across cell membranes.     

Intracellular pH regulatory mechanism in human atrial myocardium: functional evidence for Na(+)/H(+) exchanger and Na(+)/HCO(3)(-) symporter

Intracellular pH (pH(i)) exerts considerable influence on cardiac contractility and rhythm. Over the last few years, extensive progress has been made in understanding the system that controls pH(i) in animal cardiomyocytes. In addition to the housekeeping Na(+)-H(+) exchanger (NHE), the Na(+)-HCO(3)(-) symporter (NHS) has been demonstrated in animal cardiomyocytes as another acid extruder. However, whether the NHE and NHS functions exist in human atrial cardiomyocytes remains unclear. We therefore investigated the mechanism of pH(i) recovery from intracellular acidosis (induced by NH(4)Cl prepulse) using intracellular 2',7'-bis(2-carboxethyl)-5(6)-carboxy-fluorescein fluorescence in human atrial myocardium. In HEPES (nominally HCO(3)(-)-free) Tyrode solution, pH(i) recovery from induced intracellular acidosis could be blocked completely by 30 microM 3-methylsulfonyl-4-piperidinobenzoyl, guanidine hydrochloride (HOE 694), a specific NHE inhibitor, or by removing extracellular Na(+). In 3% CO(2)-HCO(3)(-) Tyrode solution, HOE 694 only slowed the pH(i) recovery, while addition of HOE 694 together with 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (an NHS inhibitor) or removal of extracellular Na(+) inhibited the acid extrusion entirely. Therefore, in the present study, we provided evidence that two acid extruders involved in acid extrusion in human atrial myocytes, one which is HCO(3)(-) independent and one which is HCO(3)(-) dependent, are mostly likely NHE and NHS, respectively. When we checked the percentage of contribution of these two carriers to pH(i) recovery following induced acidosis, we found that the activity of NHE increased steeply in the acid direction, while that of NHS did not change. Our present data indicate for the first time that two acid extruders, NHE and NHS, exist functionally and pH(i) dependently in human atrial cardiomyocytes.     

Involvement of the Na+-independent Cl-/HCO3- exchange (AE) isoform in the compensation of myocardial Na+/H+ isoform 1 hyperactivity in spontaneously hypertensive rats

Enhanced activity of Na+/H+ isoform 1 (NHE-1) and the Na+-independent Cl-/HCO3- exchange (AE) is a feature of the hypertrophied myocardium in spontaneously hypertensive rats (SHR). The present study explored the possibility that sustained intracellular acidosis due to increased myocardial acid loading through AE causes NHE-1 enhancement. To this aim, SHR were treated for 2 weeks with a rabbit polyclonal antibody against an AE3 isoform that was recently developed and proven to have inhibitory effects on myocardial AE activity. We then compared the AE activity in the left ventricle papillary muscles isolated from untreated SHR with antiAE3-treated SHR; AE activity was measured in terms of the rate of intracellular pH recovery after an intracellular alkali load was introduced. AE activity was diminished by approximately 70% in SHR treated with the antiAE3 antibody, suggesting that the AE3 isoform is a major carrier of acid-equivalent influx in the hypertrophied myocardium. However, the antibody treatment failed to normalize NHE-1 activity that remained elevated in the myocardium of normotensive rats. The data therefore rule out the possibility that NHE-1 hyperactivity in hypertensive myocardium was due to sustained intracellular acidosis induced by increased AE activity that characterizes SHR myocardial tissue.     

How pH regulates a pH regulator: a regulatory hot spot in the N-terminal cytoplasmic domain of the AE2 anion exchanger

Regulation of cell pH and cell volume require homeostatic control of intracellular cations and anions. Bicarbonate transporters play an important role in these cellular functions. The SLC4 and SLC26 gene families both encode bicarbonate transporter polypeptides. The SLC4 gene family includes four Na⁺-independent chloride-bicarbonate exchanger genes and multiple Na⁺-bicarbonate cotransporter and Na⁺-dependent anion-exchanger genes. The acute regulatory properties of the recombinant polypeptides encoded by these genes remain little studied. The most extensively studied among them are the Na⁺-independent anion exchangers AE1, AE2, and AE3. The widely expressed AE2 anion exchanger participates in recovery from alkaline load and in regulatory cell volume increase following shrinkage. AE2 can also be regulated by the ammonium ion. These properties are not shared by the closely related AE1 anion exchanger of the erythrocyte and the renal collecting duct Type A intercalated cell. Structure-function studies of recombinant proteins involving chimeras, deletions, and point mutations have delineated regions of AE2, which are important in the exhibition of the regulatory properties absent from AE1. These include regions of the transmembrane domain and the N-terminal cytoplasmic domain. Noncontiguous regions in the middle of the N-terminal cytoplasmic domain are of particular importance for acute regulation by several types of stimulus.     

AE4 is a DIDS-sensitive Cl(-)/HCO(-)(3) exchanger in the basolateral membrane of the renal CCD and the SMG duct

The renal cortical collecting duct (CCD) plays an important role in systemic acid-base homeostasis. The beta-intercalated cells secrete most of the HCO(-)(3), which is mediated by a luminal, DIDS-insensitive, Cl(-)/HCO(-)(3) exchange. The identity of the luminal exchanger is a matter of debate. Anion exchanger isoform 4 (AE4) cloned from the rabbit kidney was proposed to perform this function (Tsuganezawa H et al. J Biol Chem 276: 8180-8189, 2001). By contrast, it was proposed (Royaux IE et al. Proc Natl Acad Sci USA 98: 4221-4226, 2001) that pendrin accomplishes this function in the mouse CCD. In the present work, we cloned, localized, and characterized the function of the rat AE4. Northern blot and RT-PCR showed high levels of AE4 mRNA in the CCD. Expression in HEK-293 and LLC-PK(1) cells showed that AE4 is targeted to the plasma membrane. Measurement of intracellular pH (pH(i)) revealed that AE4 indeed functions as a Cl(-)/HCO(-)(3) exchanger. However, AE4 activity was inhibited by DIDS. Immunolocalization revealed species-specific expression of AE4. In the rat and mouse CCD and the mouse SMG duct AE4 was in the basolateral membrane. By contrast, in the rabbit, AE4 was in the luminal and lateral membranes. In both, the rat and rabbit CCD AE4 was in alpha-intercalated cells. Importantly, localization of AE4 was not affected by the systemic acid-base status of the rats. Therefore, we conclude that expression and possibly function of AE4 is species specific. In the rat and mouse AE4 functions as a Cl(-)/HCO(-)(3) exchanger in the basolateral membrane of alpha-intercalated cells and may participate in HCO(-)(3) absorption. In the rabbit AE4 may contribute to HCO(-)(3) secretion.     

Extracellular HCO(3)(-) dependence of electrogenic Na/HCO(3) cotransporters cloned from salamander and rat kidney

We studied the extracellular [HCOabstract (3) (-)] dependence of two renal clones of the electrogenic Na/HCO(3) cotransporter (NBC) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (DeltaV(m)) elicited by the NBC cloned from the kidney of the salamander Ambystoma tigrinum (akNBC) and by the NBC cloned from the kidney of rat (rkNBC). We used a two-electrode voltage clamp to measure the change in current (DeltaI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCOabstract (3 )(-)/CO(2) (0.33-99 mM HCOabstract (3)(-), pH(o) 7.5) elicited an immediate, DIDS (4, 4-diisothiocyanatostilbene-2,2-disulfonic acid)-sensitive and Na(+)-dependent hyperpolarization (or outward current). In DeltaV(m) experiments, the apparent K(m ) for HCOabstract (3)(-) of akNBC (10. 6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent K(m) for HCOabstract (3)(-) of rkNBC was less (6.5 mM). Because it has been reported that SOabstract (3)(=)/HSO abstract (3)(-) stimulates Na/HCO(3 ) cotransport in renal membrane vesicles (a result that supports the existence of a COabstract (3)(=) binding site with which SOabstract (3)(=) interacts), we examined the effect of SOabstract (3)(=)/HSO abstract (3)(-) on rkNBC. In voltage-clamp studies, we found that neither 33 mM SOabstract (4)(=) nor 33 mM SOabstract (3) (=)/HSOabstract (3)(-) substantially affects the apparent K(m) for HCO abstract (3)(-). We also used microelectrodes to monitor intracellular pH (pH(i)) while exposing rkNBC-expressing oocytes to 3.3 mM HCOabstract (3 )(-)/0.5% CO(2). We found that SO abstract (3)(=)/HSOabstract (3 )(-) did not significantly affect the DIDS-sensitive component of the pH(i) recovery from the initial CO(2 )-induced acidification. We also monitored the rkNBC current while simultaneously varying [CO(2)](o), pH(o), and [COabstract (3)(=)](o) at a fixed [HCOabstract (3)(-)](o) of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [COabstract (3)(=)](o ). However, a pH titration curve nicely fitted the data expressed as current versus pH(o). Thus, rkNBC expressed in Xenopus oocytes does not appear to interact with SOabstract (3 )(=), HSOabstract (3)(-), or COabstract (3)(=).     

Involvement of Nha1 antiporter in regulation of intracellular pH in Saccharomyces cerevisiae

The Nha1 antiporter is involved in regulation of intracellular pH in Saccharomyces cerevisiae. We report that deletion of the NHA1 gene resulted in an increase of cytoplasmic pH in cells suspended in water or acidic buffers. Addition of KCl or NaCl to exponentially growing cells lowered the internal pH but the difference between cells with or without NHA1 was maintained. Addition of KCl to starved cells resulted in much higher alkalinization of cytoplasmic pH in a strain lacking Nha1p compared to the wild-type or Nha1p-overexpressing strains. The H+/K+(Na+) exchange mechanism of Nha1p was confirmed in reconstituted plasma membrane vesicles.     

An intramolecular transport metabolon: fusion of carbonic anhydrase II to the COOH terminus of the Cl(-)/HCO(3)(-)exchanger, AE1

Anion exchanger 1 (AE1) is the plasma membrane Cl(-)/HCO(3)(-) exchanger of erythrocytes. Carbonic anhydrases (CA) provide substrate for AE1 by catalyzing the reaction, H(2)O + CO(2) ? HCO(3)(-) + H(+). The physical complex of CAII with AE1 has been proposed to maximize anion exchange activity. To examine the effect of CAII catalysis on AE1 transport rate, we fused either CAII-wild type or catalytically inactive CAII-V143Y to the cytoplasmic COOH terminus of AE1 to form AE1.CAII and AE1.CAII-V143Y, respectively. When expressed in transfected human embryonic kidney 293 cells, AE1.CAII had a similar Cl(-)/HCO(3)(-) exchange activity to AE1 alone, as assessed by the flux of H(+) equivalents (87 ± 4% vs. AE1) or rate of change of intracellular Cl(-) concentration (93 ± 4% vs. AE1), suggesting that CAII does not activate AE1. In contrast, AE1.CAII-V143Y displayed transport rates for H(+) equivalents and Cl(-) of 55 ± 2% and of 40 ± 2%, versus AE1. Fusion of CAII to AE1 therefore reduces anion transport activity, but this reduction is compensated for during Cl(-)/HCO(3)(-) exchange by the presence of catalytically active CAII. Overexpression of free CAII-V143Y acts in a dominant negative manner to reduce AE1-mediated HCO(3)(-) transport by displacement of endogenous CAII-wild type from its binding site on AE1. To examine whether AE1.CAII bound endogenous CAII, we coexpressed CAII-V143Y along with AE1 or AE1.CAII. The bicarbonate transport activity of AE1 was inhibited by CAII-V143Y, whereas the activity of AE1.CAII was unaffected by CAII-V143Y, suggesting impaired transport activity upon displacement of functional CAII from AE1 but not AE1.CAII. Taken together, these data suggest that association of functional CAII with AE1 increases Cl(-)/HCO(3)(-) exchange activity, consistent with the HCO(3)(-) transport metabolon model.     

Na+-independent Cl(-)-HCO-3- exchange in Madin-Darby canine kidney cells. Role in intracellular pH regulation

The role of plasma membrane Cl(-)-HCO-3-exchange in regulating intracellular pH (pHi) was examined in Madin-Darby canine kidney cell monolayers. In cells bathed in 25 mM HCO-3, pH 7.4, steady state pHi was 7.10 +/- 0.03 (n = 14) measured with the fluorescent pH probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. Following acute alkaline loading, pHi recovered exponentially in approximately 4 min. The recovery rate was significantly decreased by Cl- or HCO-3 removal and in the presence of 50 microM 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS). Na+ removal or 10(-3) M amiloride did not inhibit the pHi recovery rate after an acute alkaline load. Following acute intracellular acidification, the pHi recovery rate was significantly inhibited by 10(-3) M amiloride but was not altered by Cl- removal or 50 microM DIDS. At an extracellular pH (pHo) of 7.4, pHi remained unchanged when the cells were bathed in either Cl- free media, HCO-3 free media, or in the presence of 50 microM DIDS. As pHo was increased to 8.0, steady state pHi was significantly greater than control in Cl(-)-free media and in the presence of 50 microM DIDS. It is concluded that Madin-Darby canine kidney cells possess a Na+-independent Cl(-)-HCO-3 exchanger with a Km for external Cl- of approximately 6 mM. The exchanger plays an important role in pHi regulation following an elevation of pHi above approximately 7.1. Recovery of pHi following intracellular acidification is mediated by the Na+/H+ antiporter and not the anion exchanger.     

Cl(-)/HCO(3)(-) exchange is acetazolamide sensitive and activated by a muscarinic receptor-induced [Ca(2+)](i) increase in salivary acinar cells

Large volumes of saliva are generated by transepithelial Cl(-) movement during parasympathetic muscarinic receptor stimulation. To gain further insight into a major Cl(-) uptake mechanism involved in this process, we have characterized the anion exchanger (AE) activity in mouse serous parotid and mucous sublingual salivary gland acinar cells. The AE activity in acinar cells was Na(+) independent, electroneutral, and sensitive to the anion exchange inhibitor DIDS, properties consistent with the AE members of the SLC4A gene family. Localization studies using a specific antibody to the ubiquitously expressed AE2 isoform labeled acini in both parotid and sublingual glands. Western blot analysis detected an approximately 170-kDa protein that was more highly expressed in the plasma membranes of sublingual than in parotid glands. Correspondingly, the DIDS-sensitive Cl(-)/HCO(3)(-) exchanger activity was significantly greater in sublingual acinar cells. The carbonic anhydrase antagonist acetazolamide markedly inhibited, whereas muscarinic receptor stimulation enhanced, the Cl(-)/HCO(3)(-) exchanger activity in acinar cells from both glands. Intracellular Ca(2+) chelation prevented muscarinic receptor-induced upregulation of the AE, whereas raising the intracellular Ca(2+) concentration with the Ca(2+)-ATPase inhibitor thapsigargin mimicked the effects of muscarinic receptor stimulation. In summary, carbonic anhydrase activity was essential for regulating Cl(-)/HCO(3)(-) exchange in salivary gland acinar cells. Moreover, muscarinic receptor stimulation enhanced AE activity through a Ca(2+)-dependent mechanism. Such forms of regulation may play important roles in modulating fluid and electrolyte secretion by salivary gland acinar cells.     

Expression of Na+/HCO3- cotransporter and its role in pH regulation in mouse parotid acinar cells

Ion transporters such as Na(+)/H(+) exchanger (NHE), Cl(-)/HCO(3)(-) exchanger (AE), and Na(+)/HCO(3)(-) cotransporter (NBC) are known to contribute to the intracellular pH (pH(i)) regulation during agonist-induced stimulation. This study examined the mechanisms for the pH(i) regulation in the mouse parotid and sublingual acinar cells using the fluorescent pH-sensitive probe, BCECF. The pH(i) recovery from agonist-induced acidification in the sublingual acinar cells was completely blocked by EIPA, a NHE inhibitor. However, the parotid acinar cells required DIDS, a NBC1 inhibitor, in addition to EIPA in order to block the pH(i) recovery. Moreover, RT-PCR analysis detected the expression of pancreatic NBC1 (pNBC1) only in the parotid acinar cells. These results provide strong evidence that the mechanisms for the pH(i) regulation are different in the two types of acinar cells, and pNBC1 contributes to pH(i) regulation in the parotid acinar cells, whereas NHE is likely to be the exclusive pH(i) regulator in the sublingual acinar cells.     

Possible role of carbonic anhydrase, V-H(+)-ATPase, and Cl(-)/HCO3- exchanger in electrogenic ion transport across the gills of the euryhaline crab Chasmagnathus granulatus

We studied the participation of carbonic anhydrase (CA), V-H(+)-ATPase, and Cl(-)/HCO3- exchanger in electrogenic ion absorption through the gills of Chasmagnathus granulatus. CA activity was measured in anterior gills and posterior gills after acclimation to 2 per thousand, 10 per thousand, 30 per thousand (about seawater), and 45 per thousand salinity. The highest CA specific activity was detected in the microsomal fraction in anterior gills, and in the cytosolic fraction, in posterior ones. Both fractions were strongly induced by decreasing salinity only in posterior gills. Perfusion of posterior gills from crabs acclimated to either 2 per thousand or 10 per thousand with acetazolamide inhibited CA activity almost completely. In posterior gills from crabs acclimated to 2 per thousand and perfused with 20 per thousand saline (iso-osmotic for these crabs), acetazolamide reduced transepithelial potential difference (V(te)) by 47%, further addition of ouabain enhanced the effect to 88%. Acetazolamide had no effect in the same gills perfused with 30 per thousand saline (iso-osmotic for seawater acclimated crabs). Bafilomycin A1 and SITS (inhibitors of V-H(+)-ATPase and Cl(-)/HCO3-) reduced V(te) by 15-16% in gills perfused with normal 20 per thousand saline, and by 77% and 45%, respectively when they were applied in Na-free 20 per thousand saline, suggesting the participation of those transporters and cytosolic CA in electrogenic ion absorption.     

Dual role of CO2/HCO3(-) buffer in the regulation of intracellular pH of three-dimensional tumor growths

Intracellular pH (pH(i)), a major modulator of cell function, is regulated by acid/base transport across membranes. Excess intracellular H(+) ions (e.g. produced by respiration) are extruded by transporters such as Na(+)/H(+) exchange, or neutralized by HCO(3)(-) taken up by carriers such as Na(+)-HCO(3)(-) cotransport. Using fluorescence pH(i) imaging, we show that cancer-derived cell lines (colorectal HCT116 and HT29, breast MDA-MB-468, pancreatic MiaPaca2, and cervical HeLa) extrude acid by H(+) efflux and HCO(3)(-) influx, largely sensitive to dimethylamiloride and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), respectively. The magnitude of HCO(3)(-) influx was comparable among the cell lines and may represent a constitutive element of tumor pH(i) regulation. In contrast, H(+) efflux varied considerably (MDA-MB-468 > HCT116 > HT29 > MiaPaca2 > HeLa). When HCO(3)(-) flux was pharmacologically inhibited, acid extrusion in multicellular HT29 and HCT116 spheroids (～10,000 cells) was highly non-uniform and produced low pH(i) at the core. With depth, acid extrusion became relatively more DIDS-sensitive because the low extracellular pH at the spheroid core inhibits H(+) flux more than HCO(3)(-) flux. HCO(3)(-) flux inhibition also decelerated HCT116 spheroid growth. In the absence of CO(2)/HCO(3)(-), acid extrusion by H(+) flux in HCT116 and MDA-MB-468 spheroids became highly non-uniform and inadequate at the core. This is because H(+) transporters require extracellular mobile pH buffers, such as CO(2)/HCO(3)(-), to overcome low H(+) ion mobility and chaperone H(+) ions away from cells. CO(2)/HCO(3)(-) exerts a dual effect: as substrate for membrane-bound HCO(3)(-) transporters and as a mobile buffer for facilitating extracellular diffusion of H(+) ions extruded from cells. These processes can be augmented by carbonic anhydrase activity. We conclude that CO(2)/HCO(3)(-) is important for maintaining uniformly alkaline pH(i) in small, non-vascularized tumor growths and may be important for cancer disease progression.     

The regulation of intracellular pH in monkey kidney epithelial cells (BSC-1). Roles of Na+/H+ antiport, Na+-HCO3(-)-(NaCO3-) symport, and Cl-/HCO3- exchange

Using the pH-sensitive absorbance of 5 (and 6)-carboxy-4',5'- dimethylfluorescein, we investigated the regulation of cytoplasmic pH (pHi) in monkey kidney epithelial cells (BSC-1). In the absence of HCO3-, pHi is 7.15 +/- 0.1, which is not significantly different from pHi in 28 mM HCO3-, 5% CO2 (7.21 +/- 0.07). After an acid load, the cells regulate pHi in the absence of HCO3- by a Na+ (or Li+)-dependent, amiloride-inhibitable mechanism (indicative of Na+/H+ antiport). In 28 mM HCO3-, while still dependent on Na+, this regulation is only blocked in part by 1 mM amiloride. A partial block is also observed with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (1 mM). With cells pretreated with DIDS, 1 mM amiloride nearly totally inhibits this regulation. Cl- had no effect on pHi regulation in the acidic range. In HCO3(-)-free saline, Na+ removal leads to an amiloride-insensitive acidification, which is dependent on Ca2+. In 28 mM HCO3-, Na+ (and Ca2+) removal led to a pronounced reversible and DIDS-sensitive acidification. When HCO3- was lowered from 46 to 10 mM at constant pCO2 (5%), pHi dropped by a DIDS-sensitive mechanism. Identical changes in pHo (7.6 to 6.9) in the nominal absence of HCO3- led to smaller changes of pHi. In the presence but not in the absence of HCO3-, removal of Cl- led to a DIDS-sensitive alkalinization. This was also observed in the nominal absence of Na+, which leads to a sustained acidification. It is concluded that in nominally bicarbonate-free saline, the amiloride-sensitive Na+/H+ antiport is the predominant mechanism of pHi regulation at acidic pHi, while being relatively inactive at physiological values of pHi. In bicarbonate saline, two other mechanisms effect pHi regulation: a DIDS-sensitive Na+-HCO3- symport, which contributes to cytoplasmic alkalinization, and a DIDS-sensitive Cl-/HCO3- exchange, which is apparently independent of Na+.     

IRBIT reduces the apparent affinity for intracellular Mg(2+) in inhibition of the electrogenic Na(+)-HCO(3)(-) 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.     

Measurements of intracellular pH in single LLC-PK1 cells: Recovery from an acid load via basolateral Na+/H+ exchange

Summary LLC-PK₁ cells (a continuous epithelioid cell line with renal characteristics) are examined by microspectrofluorometry as single cells, in order to determine the mechanism of intracellular pH (pH _i ) recovery from an acid load imposed by ammonium preincubation and removal (NH₄ prepulse). Initial experiments evaluate the intracellular K⁺ levels through a null point analysis of total cellular K⁺ with flame photometry. The response of BCECF (a pH-sensitive fluorescent dye) is then calibrated, using saturating concentrations of nigericin to cause defined changes in pH _i . For experiments with the microspectrofluorometer, LLC-PK₁ cells were grown on either glass coverslips or filters (the latter attached to plastic coverslips with a hole under the filter). The cells on glass coverslips demonstrate a Na⁺-dependent recovery from an (NH₄ prepulse) acid load which is sensitive to 1 M ethylisopropylamiloride. They also demonstrate a set point of activation of Na⁺/H⁺ exchange. When examined for changes in pH _i due to changes in membrane potential, plasma membrane proton conductance could not be detected at resting pH _i . Cells grown on filters also demonstrate a pH _i recovery from an acid load which is Na⁺ dependent and ethylisopropylamiloride sensitive, but in this configuration, the majority of cells (22/23 preparations) require Na⁺ at the basolateral membrane for rapid pH _i recovery. The morphology and polarity of the cells grown on permeable supports appears normal at the electron-microscopic level. The results are not affected by changes in cell seeding density or collagen treatment of the filters.