Role In Regulation Of Intracellular pH Research Articles

Potential Involvement of NHE1 in COVID‐19 Related Pulmonary Damage

In some moderate and severe COVID‐19 infections lung scarring similar to fibrosis have been observed in survivors, many of which have persistent functional and radiographic abnormalities at the initial follow‐up suggesting a possible shared pathobiology between fibrotic lung disease and COVID‐19‐ lung disease. Fibrosis is an inflammatory response to pulmonary infections that activates fibroblasts and produces excessive extracellular matrix proteins that stiffen cells, leading to reduction in lung capacity and breathing deficiency. Several factors involved in the “cytokine storm” also are pre‐fibrotic leading to the advanced disease. This includes interleukin 6 and others. The focus of this study is on sodium hydrogen exchanger isoform 1 (NHE1) that plays a key role in intracellular pH regulation and cell movement and its effect on fibrosis development. Both cytokines IL‐1 and IL‐6 are used to show one measure of cell restructuring and stiffness by measuring stress fiber formation. To examine the role of NHE1 in fibroblast response, both IL‐1 and IL‐6 cells were treated with proinflammatory cytokines in the presence and absence of NHE1 inhibitor (EIPA). This study will provide an initial insight to the relationship between NHE1 and proinflammatory response for future experiments.

Slc4a11 disruption causes duct cell loss and impairs NaCl reabsorption in female mouse submandibular glands.

Slc4a11, a member of the Slc4 HCO3 − transporter family, has a wide tissue distribution. In mouse salivary glands, the expression of Slc4a11 mRNA was more than eightfold greater than the other nine members of the Slc4 gene family. The Slc4a11 protein displayed a diffuse subcellular distribution in both the acinar and duct cells of mouse submandibular glands (SMG). Slc4a11 disruption induced a significant increase in the Na+ and Cl− concentrations of stimulated SMG saliva, whereas it did not affect the fluid secretion rate in response to either β‐adrenergic or cholinergic receptor stimulation. Heterologous expressed mouse Slc4a11 acted as a H+/OH− transporter that was uncoupled of Na+ or Cl− movement, and this activity was blocked by ethyl‐isopropyl amiloride (EIPA) but not 4,4′‐Diisothiocyanato‐2,2′‐stilbenedisulfonic acid (DIDS). Slc4a11 disruption revealed that Slc4a11 does not play a major role in intracellular pH regulation in mouse salivary gland cells. In contrast, NaCl reabsorption was impaired in the SMG saliva of female compared to male Slc4a11 null mice, which correlated with the loss of duct cells and a decrease in expression of the duct‐cell‐specific transcription factor Ascl3. Together, our results suggest that Slc4a11 expression regulates the number of ducts cells in the mouse SMG and consequently NaCl reabsorption.

Ahcyl2 upregulates NBCe1-B via multiple serine residues of the PEST domain-mediated association.

Inositol-1,4,5-triphosphate [IP3] receptors binding protein released with IP3 (IRBIT) was previously reported as an activator of NBCe1-B. Recent studies have characterized IRBIT homologue S-Adenosylhomocysteine hydrolase-like 2 (AHCYL2). AHCYL2 is highly homologous to IRBIT (88%) and heteromerizes with IRBIT. The two important domains in the N-terminus of AHCYL2 are a PEST domain and a coiled-coil domain which are highly comparable to those in IRBIT. Therefore, in this study, we tried to identify the role of those domains in mouse AHCYL2 (Ahcyl2), and we succeeded in identifying PEST domain of Ahcyl2 as a regulation region for NBCe1-B activity. Site directed mutagenesis and coimmunoprecipitation assay showed that NBCe1-B binds to the N-terminal Ahcyl2-PEST domain, and its binding is determined by the phosphorylation of 4 critical serine residues (Ser151, Ser154, Ser157, and Ser160) in Ahcyl2 PEST domain. Also we revealed that 4 critical serine residues in Ahcyl2 PEST domain are indispensable for the activation of NBCe1-B using measurement of intracellular pH experiment. Thus, these results suggested that the NBCe1-B is interacted with 4 critical serine residues in Ahcyl2 PEST domain, which play an important role in intracellular pH regulation through NBCe1-B.

PSD‐95 interacts with NBCn1 and enhances channel‐like activity without affecting cotransport function

NBCn1 plays an essential role in intracellular pH regulation and transepithelial bicarbonate movement in the body. NBCn1 also has sodium channel‐like activity uncoupled to Na/HCO3 cotransport. We previously reported that NBCn1 interacts with the postsynaptic density protein PSD‐95 in the brain. Here, we elucidated the structural determinant and functional consequence of NBCn1/PSD‐95 interaction. In rat hippocampal CA3 neurons, NBCn1 was localized to the postsynaptic membranes of both dendritic shafts and spines and occasionally to the presynaptic membranes. A GST/NBCn1 fusion protein containing the C‐terminal 131 amino acids of NBCn1 pulled down PSD‐95 from rat brain lysates, whereas GST/NBCn1‐ΔETSL (deletion of the last four amino acids) and GST/NBCn2 lacking the same ETSL did not. NBCn1 and PSD‐95 were coimmunoprecipitated in HEK 293 cells. PSD‐95 has negligible effects on intracellular pH changes mediated by NBCn1 in HEK 293 cells and Xenopus oocytes. However, PSD‐95 increased an ionic conductance produced by NBCn1 channel‐like activity. This increase was abolished by NBCn1‐ΔETSL or by the peptide containing the last 15 amino acids of NBCn1. Our data suggest that PSD‐95 interacts with NBCn1 and increases its channel‐like activity while negligibly affecting Na/HCO3 cotransport. We propose that the channel‐like activity may occur via an intermolecular cavity of multimeric NBCn1 proteins.

HVCN1 contributes to superoxide production independent of intracellular pH regulation

HVCN1 is a voltage‐gated ion channel, required for maximal superoxide (O2−) production by NADPH oxidase during the respiratory burst in macrophages (MØ). During activation of NADPH oxidase, H+ ions are produced resulting in cellular acidification. By extruding H+, HVCN1 maintains the respiratory burst by preventing feedback inhibition of the oxidase by low pH. Recent evidence from our laboratory indicates that HVCN1 may also contribute to superoxide formation by additional mechanisms. We hypothesized that ‘HVCN1 acts to enhance NADPH oxidase activity independent of its role in intracellular pH regulation’. Intracellular pH was clamped using nigericin (100μM)/K+(135μM) in isolated peritoneal MØ from the rat at pH 8.0, 7.5, 7.0 or 6.5. Maximal O2− production (L‐012 luminescence (AU)) in MØ in response to (PMA 100μM) was linearly related to intracellular pH (r2=0.97). Addition of Zn2+ (100μM), to specifically inhibit HVCN1, significantly reduced maximal O2− production by >; 50% at all pH levels (Table 1). As these reductions in O2− production could not be explained by changes in intracellular pH, we conclude that HVCN1 enhances O2− production in MØ by pH independent mechanisms.

Effect of Simultaneously Replacing Putative TM6 and TM12 of Human NBCe1-A with Those from NBCn1 on Surface Abundance in Xenopus Oocytes

HCO₃⁻ translocation across the plasma membrane via the electrogenic Na/HCO₃⁻ cotransporter NBCe1 plays an important role in intracellular pH regulation and transepithelial HCO₃⁻ transport. However, the structural determinants of transporter function remain largely unknown. A previous study showed that the putative fourth extracellular loop (EL4) plays an essential role in determining the electrogenicity of NBCe1. In the present study, we generated eight new chimeras of human NBCe1-A and NBCn1-A. All possess the putative NBCe1 EL4 and are electrogenic. Chimera O, in which the putative sixth transmembrane segment (TM6) and EL5 through the C terminus (Ct) of NBCe1 was replaced by corresponding NBCn1 sequence, produces the smallest hyperpolarization (1-2 mV) when CO₂/HCO₃⁻ is added to the extracellular solution. Biotinylation experiments show that O has a very low abundance at the plasma membrane. However, chimeras in which we simultaneously replaced the putative TM6 and smaller subdomains of the EL5-Ct region for the NBCn1 sequence were strongly electrogenic except for chimera T, in which we replaced TM6 and TM12 of NBCe1 with the corresponding regions of NBCn1. T exhibited greatly reduced transporter surface expression compared to wild-type NBCe1-A, while retaining at least some electrogenic character. We hypothesize that putative TM6 and TM12 are part of a functional unit and that if the two TMs are replaced by those of the same transporter type, high surface expression would require that the surrounding TMs are also from the same transporter type.

Regulation of Na+/H+ exchanger 1 allosteric balance by its localization in cholesterol‐ and caveolin‐rich membrane microdomains

The Na+/H+ exchanger 1, which plays an essential role in intracellular pH regulation in most tissues, is also known to be a key actor in both proliferative and apoptotic processes. Its activation by H+ is best described by the Monod-Wyman-Changeux model: the dimeric NHE-1 oscillates between a low and a high affinity conformation, the balance between the two forms being defined by the allosteric constant L(0). In this study, influence of cholesterol- and caveolin-rich microdomains on NHE-1 activity was examined by using cholesterol depleting agents, including methyl-beta-cyclodextrin (MBCD). These agents activated NHE-1 by modulating its L(0) parameter, which was reverted by cholesterol repletion. This activation was associated with NHE-1 relocation outside microdomains, and was distinct from NHE-1 mitogenic and hormonal stimulation; indeed MBCD and serum treatments were additive, and serum alone did not change NHE-1 localization. Besides, MBCD activated a serum-insensitive, constitutively active mutated NHE-1 ((625)KDKEEEIRK(635) into KNKQQQIRK). Finally, the membrane-dependent NHE-1 regulation occurred independently of Mitogen Activated Protein Kinases, especially Extracellular Regulated Kinase activation, although this kinase was activated by MBCD. In conclusion, localization of NHE-1 in membrane cholesterol- and caveolin-rich microdomains constitutes a novel physiological negative regulator of NHE-1 activity.

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.)

Na+/H+ exchangers in the human eccrine sweat duct.

Using an anti-NHE1 antibody, we demonstrate the presence of a Na+/H+ exchanger of isoform 1 (NHE1) in the human eccrine sweat duct. A strong staining was observed at the basolateral membrane of the outer cell layer (NHE1basal), at the junction between inner and outer cells layers (NHE1inter), and along the lateral membranes (NHE1later) of all cells of the duct. At the luminal membrane, no staining was demonstrated either for NHE1 or NHE3. To investigate Na+/H+ mediated proton transport, straight sweat duct portions were isolated and perfused in vitro under HCO3-free conditions. In the presence of basolateral 5-ethyl-N-isopropyl amiloride (EIPA), an acidification of 0.29 +/- 0.03 pH units was observed, whereas no effect was observed with luminal EIPA. Bath sodium removal generated a stronger acidification (0.41 +/- 0.09 pH units). Removal of luminal sodium (in the absence or presence of basolateral EIPA), or low luminal chloride, led to an alkalinization, presumably due to a decrease in intracellular sodium, strongly suggesting functional activity of NHE1inter. We therefore conclude that in the sweat duct, NHE1 plays a major role in intracellular pH regulation.

Structural determinants and significance of regulation of electrogenic Na(+)-HCO(3)(-) cotransporter stoichiometry.

Na(+)-HCO(3)(-) cotransporters play an important role in intracellular pH regulation and transepithelial HCO(3)(-) transport in various tissues. Of the characterized members of the HCO(3)(-) transporter superfamily, NBC1 and NBC4 proteins are known to be electrogenic. An important functional property of electrogenic Na(+)-HCO(3)(-) cotransporters is their HCO(3)(-):Na(+) coupling ratio, which sets the transporter reversal potential and determines the direction of Na(+)-HCO(3)(-) flux. Recent studies have shown that the HCO(3)(-):Na(+) transport stoichiometry of NBC1 proteins is either 2:1 or 3:1 depending on the cell type in which the transporters are expressed, indicating that the HCO(3)(-):Na(+) coupling ratio can be regulated. Mutational analysis has been very helpful in revealing the molecular mechanisms and signaling pathways that modulate the coupling ratio. These studies have demonstrated that PKA-dependent phosphorylation of the COOH terminus of NBC1 proteins alters the transport stoichiometry. This cAMP-dependent signaling pathway provides HCO(3)(-) -transporting epithelia with an efficient mechanism for modulating the direction of Na(+)-HCO(3)(-) flux through the cotransporter.

Identification and Localization of Acid-base Transporters in the Conjunctival Epithelium

Acid-base transporters of rabbit and porcine conjunctival epithelia were identified and localized with immunoblotting and immunohistochemical techniques using specific antibodies against carriers commonly found in epithelia, i.e. the Cl−/HCO<$>^{\\minus}_{3}<$>exchanger (AE2), Na+/H+exchanger (NHE-1, -2, -3) and the electrogenic Na+-(n)HCO<$>^{\\minus}_{3}<$>cotransporter (NBC). Western blot analysis demonstrated that anti-AE2 reacted with an approximate 170kDa protein in both rabbit and pig cell membranes prepared from separately isolated bulbar and palpebral conjunctivae. NHE1 was similarly identified in these distinct conjunctival regions but results with anti-NBC were ambiguous. Histochemical examinations indicated that the AE2 and NHE1 proteins reside on the basolateral surfaces of the plasma membrane throughout the multilayered tissue. The immunostaining of porcine cryosections for AE2 and rabbit sections for NHE1 was specific, because of its abolishment following either pre-absorption with the corresponding peptide or omission of the primary antibody. Screening with anti-NBC produced weak staining of the sections that appeared to be non-specific. For confirmation of these results, the acid-base transporters present in rabbit cell cultures of conjunctival epithelia were ascertained from the changes in intracellular pH (pHi) evoked upon sequential superfusion with media of altered composition. This approach readily obtained Na+- and Cl−-dependent pHieffects consistent with the existence of Cl−/HCO<$>^{\\minus}_{3}<$>and Na+/H+exchange activities. Evidence for the presence of NBC could not be acquired, thereby substantiating the observations from the immunodetection techniques. The identity and location of the antiporters that were found suggested that these elements could contribute to transcellular Cl−transport in the basolateral-to-apical direction. To test this possibility, the effects of AE and/or NHE inhibition were determined on the bumetanide-insensitive Cl−-dependent short-circuit current across rabbit conjunctivae freshly isolated in Ussing-type chambers. These experiments revealed that such current is indeed sustained by the antiporters. Results with acetazolamide further suggested that the contribution of the acid-base transporters towards transepithelial Cl−secretion is variable and dependent upon individual rates of metabolic CO2production. Overall, the present study provides an initial identification of the acid-base transporters present in the conjunctiva. Besides their likely role in intracellular pH regulation, the parallel, basolateral expression of AE2 and NHE1 indicates that these elements do not directly contribute to the pH of the tear film but may complement the Na+-2Cl−-K+cotransporter in effectuating Cl−secretion.

Na+/H+ Exchange in isolated hamster enterocytes: Its major role in intracellular pH regulation

The intracellular pH (pHi) of isolated hamster intestinal cells was determined with three techniques: the null-point method with digitonin, the distribution of the weak acid 5,5-dimethyloxazolidine2,4-dione, and the trapped fluorescent indicator 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein. In physiologic saline, the pHi determined with the fluorescent dye 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein was 7.00 ± 0.03 (n = 20) but was 6.68 ± 0.04 (n = 25) when there was no Na+ in the medium, indicating an important role of external Na+ in maintaining pHi at neutral level. When Na+ was added to an incubation medium lacking the ion, the pHi increased. The time-course of this alkalinization depended on the Na+ concentration, whereas K+ had only a slight effect. Amiloride (1 mM) completely inhibited the Na+ effect, thereby showing the basic role of the Na+/H+ antiport in the regulation of pHi. The effect of 4-acetamido-4′isothiocyanostilbene-2, 2′-disulfonic acid and furosemide showed that there also exists an anionic component in the process, probably of the Cl−/OH− (HCO3post−) exchange type. These results show that the amiloride-sensitive Na+/H+ antiport plays an important but not exclusive role in maintaining the pHi neutral or slightly alkaline in isolated intestinal cells from the hamster.

Apical and basolateral Na+/H+ exchange in the rabbit outer medullary thin descending limb of Henle: role in intracellular pH regulation.

The present study was designed to investigate the apical and basolateral transport processes responsible for intracellular pH regulation in the thin descending limb of Henle. Rabbit thin descending limbs of long-loop nephrons were perfused in vitro and intracellular pH (pHi) was measured using BCECF. Steady-state pHi in HEPES buffered solutions (pH 7.4) was 7.18 +/- 0.03. Following the removal of luminal Na+, pHi decreased at a rate of 1.96 +/- 0.37 pH/min. In the presence of luminal amiloride (1 mM), the rate of decrease of pHi was significantly less, 0.73 +/- 0.18 pH/min. Steady-state pHi decreased 0.18 pH units following the addition of amiloride (1 mM) to the lumen (Na+ 140 mM lumen and bath). When Na+ was removed from the basolateral side of the tubule, pHi decreased at a rate of 0.49 +/- 0.05 pH/min. The rate of decrease of pHi was significantly less in the presence of 1 mM basolateral amiloride, 0.29 +/- 0.04 pH/min. Addition of 1 mM amiloride to the basolateral side (Na+ 140 mM lumen and bath) caused steady-state pHi to decrease significantly by 0.06 pH units. When pHi was acutely decreased to 5.87 +/- 0.02 following NH4Cl removal (lumen, bath), pHi failed to recover in the absence of Na+ (lumen, bath). Addition of 140 mM Na+ to the lumen caused pHi to recover at a rate of 2.17 +/- 0.59 pH/min. The rate of pHi recovery was inhibited 93% by 1 mM luminal amiloride. When 140 mM Na+ was added to the basolateral side, pHi recovered only partially at 0.38 +/- 0.07 pH/min. Addition of 1 mM basolateral amiloride inhibited the recovery of pHi by 97%. The results demonstrate that the rabbit thin descending limb of long-loop nephrons possesses apical and basolateral Na+/N+ antiporters. In the steady state, the rate of Na+-dependent H+ flux across the apical antiporter exceeds the rate of Na+-dependent H+ flux via the basolateral antiporter. Recovery of pHi following acute intracellular acidification is Na+ dependent and mediated primarily by the luminal antiporter.

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.