Search

The calcium-sensing receptor (CaSR), a G protein-coupled receptor, regulates Ca 2+ concentration in plasma by regulating parathyroid hormone secretion. In other tissues, it is reported to play roles in cellular differentiation and migration and in secretion and absorption. We reported previously that CaSR can be conditionally deleted in the mouse esophagus. This conditional knockout (KO) ( Eso CaSR -/- ) model showed a significant reduction in the levels of adherens and tight junction proteins and had a marked buildup of bacteria on the luminal esophageal surface. To further examine the role of CaSR, we used RNA sequencing to determine gene expression profiles in esophageal epithelia of control and Eso CaSR -/- mice RNA Seq data indicated upregulation of gene sets involved in DNA replication and cell cycle in Eso CaSR -/- . This is accompanied by the downregulation of gene sets involved in the innate immune response and protein homeostasis including peptide elongation and protein trafficking. Ingenuity pathway analysis (IPA) demonstrated that these genes are mapped to important biological networks including calcium and Ras homologus A (RhoA) signaling pathways. To further explore the bacterial buildup in Eso CaSR -/- esophageal tissue, 16S sequencing of the mucosal-associated bacterial microbiome was performed. Three bacterial species, g_Rodentibacter , s _Rodentibacter_unclassified , and s_Lactobacillus_hilgardi were significantly increased in Eso CaSR -/- . Furthermore, metagenomic analysis of 16S sequences indicated that pathways related to oxidative phosphorylation and metabolism were downregulated in Eso CaSR -/- tissues. These data demonstrate that CaSR impacts major pathways of cell proliferation, differentiation, cell cycle, and innate immune response in esophageal epithelium. The disruption of these pathways causes inflammation and significant modifications of the microbiome. NEW & NOTEWORTHY Calcium-sensing receptor (CaSR) plays a significant role in maintaining the barrier function of esophageal epithelium. Using RNA sequencing, we show that conditional deletion of CaSR from mouse esophagus causes upregulation of genes involved in DNA replication and cell cycle and downregulation of genes involved in the innate immune response, protein translation, and cellular protein synthesis. Pathway analysis shows disruption of signaling pathways of calcium and actin cytoskeleton. These changes caused inflammation and esophageal dysbiosis.

Background: The relationship between proton-pump inhibitor (PPI) use and chronic kidney disease (CKD) progression remains controversial. Specifically, there is a lack of data evaluating renal outcomes in established CKD patients. The aim of our study is to determine the risk of progression to end-stage kidney disease (ESKD) or death amongst CKD patients on PPI, histamine-2 receptor blocker (H2B), or no anti-acid therapy., Methods: Using our CKD registry, we evaluated the relationship between PPI and H2B use and outcomes amongst patients with CKD (eGFR < 60), with at least 2 PCP visits in the year prior. A Cox proportional hazards model was used to evaluate the relationship between medication groups and overall mortality, while competing risks regression models were used to determine the risk of ESKD with death as a competing risk., Results: 25,455 patients met inclusion criteria and were stratified according to medication group: no antacid therapy (15,961), PPI use (8646), or H2B use (848). At 4 years, the cumulative incidence of ESKD with death as a competing risk was 2.0% (95% CI: 1.7, 2.4), 1.5% (0.8, 2.8), and 1.6%(1.4, 1.9) among PPI, H2B, and no medication respectively (P = 0.22). The cumulative incidence of death with ESKD as a competing risk was 17.6% (95% CI: 16.6, 18.6), 16.7% (13.7, 19.8), and 17.3% (16.6, 18.0) (P = 0.71)., Conclusions: Use of PPI in a CKD population was not associated with increased mortality or progression to ESKD when compared to H2 blocker and to no acid suppressing therapy., (© 2021. The Author(s).)

The Ussing chamber was developed in 1949 by Hans Ussing and quickly became a powerful tool to study ion and solute transport in epithelia. The chamber has two compartments strictly separating the apical and basolateral sides of the tissue under study. The two sides of the tissue are connected via electrodes to a modified electrometer/pulse generator that allows measurement of electrical parameters, namely, transepithelial voltage, current, and resistance. Simultaneously, permeability of the tissue to specific solutes or markers can be monitored by using tracers or isotopes to measure transport from one side of the tissue to the other. In this chapter, we will describe the use of the Ussing chamber to study the barrier properties of the mouse esophageal epithelium. We will also briefly describe the use of the modified Ussing chamber to simultaneously study transepithelial and cellular electrophysiology in the rabbit esophageal epithelium. Lastly, we will cover the use of the Ussing chamber to study bicarbonate secretion in the pig esophagus. These examples highlight the versatility of the Ussing chamber technique in investigating the physiology and pathophysiology of epithelia including human biopsies.

Calcium-sensing receptor (CaSR) is the molecular sensor by which cells respond to small changes in extracellular Ca 2+ concentrations. CaSR has been reported to play a role in glandular and fluid secretion in the gastrointestinal tract and to regulate differentiation and proliferation of skin keratinocytes. CaSR is present in the esophageal epithelium, but its role in this tissue has not been defined. We deleted CaSR in the mouse esophagus by generating keratin 5 CreER;CaSR Flox+/+ compound mutants, in which loxP sites flank exon 7 of CaSR gene. Recombination was initiated with multiple tamoxifen injections, and we demonstrated exon 7 deletion by PCR analysis of genomic DNA. Quantitative real-time PCR and Western blot analyses showed a significant reduction in CaSR mRNA and protein expression in the knockout mice ( Eso CaSR -/- ) as compared with control mice. Microscopic examination of Eso CaSR -/- esophageal tissues showed morphological changes including elongation of the rete pegs, abnormal keratinization and stratification, and bacterial buildup on the luminal epithelial surface. Western analysis revealed a significant reduction in levels of adherens junction proteins E-cadherin and β catenin and tight junction protein claudin-1, 4, and 5. Levels of small GTPase proteins Rac/Cdc42, involved in actin remodeling, were also reduced. Ussing chamber experiments showed a significantly lower transepithelial resistance in knockout (KO) tissues. In addition, luminal-to-serosal-fluorescein dextran (4 kDa) flux was higher in KO tissues. Our data indicate that CaSR plays a role in regulating keratinization and cell-cell junctional complexes and is therefore important for the maintenance of the barrier function of the esophagus. NEW & NOTEWORTHY The esophageal stratified squamous epithelium maintains its integrity by continuous proliferation and differentiation of the basal cells. Here, we demonstrate that deletion of the calcium-sensing receptor, a G protein-coupled receptor, from the basal cells disrupts the structure and barrier properties of the epithelium.

Hypercapnia and subsequent respiratory acidosis are serious complications in many patients with respiratory disorders. The acute response to hypercapnia is buffering of H + by hemoglobin and cellular proteins but this effect is limited. The chronic response is renal compensation that increases HCO 3 - reabsorption, and stimulates urinary excretion of titratable acids (TA) and NH 4 + . However, the main effective pathway is the excretion of NH 4 + in the collecting duct. Our hypothesis is that, the renal NH 3 /NH 4 + transporters, Rhbg and Rhcg, in the collecting duct mediate this response. The effect of hypercapnia on these transporters is unknown. We conducted in vivo experiments on mice subjected to chronic hypercapnia. One group breathed 8% CO 2 and the other breathed normal air as control (0.04% CO 2 ). After 3 days, the mice were euthanized and kidneys, blood, and urine samples were collected. We used immunohistochemistry and Western blot analysis to determine the effects of high CO 2 on localization and expression of the Rh proteins, carbonic anhydrase IV, and pendrin. In hypercapnic animals, there was a significant increase in urinary NH 4 + excretion but no change in TA. Western blot analysis showed a significant increase in cortical expression of Rhbg (43%) but not of Rhcg. Expression of CA-IV was increased but pendrin was reduced. These data suggest that hypercapnia leads to compensatory upregulation of Rhbg that contributes to excretion of NH 3 /NH 4 + in the kidney. These studies are the first to show a link among hypercapnia, NH 4 + excretion, and Rh expression., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Renal Rhbg is localized to the basolateral membrane of intercalated cells and is involved in NH 3 /NH 4 + transport. The structure of Rhbg is not yet resolved; however, a high-resolution crystal structure of AmtB, a bacterial homolog of Rh, has been determined. We aligned the sequence of Rhbg to that of AmtB and identified important sites of Rhbg that may affect transport. Our analysis positioned three conserved amino acids, histidine 183 (H183), histidine 342 (H342), and tryptophan 230 (W230), within the hydrophobic pore where they presumably serve to control NH 3 transport. A fourth residue, phenylalanine 128 (F128) was positioned at the upper vestibule, presumably contributing to recruitment of NH 4 + We generated three mutations each of H183, H342, W230, and F128 and expressed them in frog oocytes. Immunolabeling showed that W230 and F128 mutants were localized to the cell membrane, whereas H183 and H342 staining was diffuse and mostly intracellular. To determine function, we compared measurements of NH 3 /NH 4 + and methyl amine (MA)/methyl ammonium (MA + )-induced currents, intracellular pH, and surface pH (pHs) among oocytes expressing the mutants, Rhbg, or injected with H 2 O. In H183 and W230 mutants, NH 4 + -induced current and intracellular acidification were inhibited compared with that of Rhbg, and MA-induced intracellular alkalinization was completely absent. Expression of H183A or W230A mutants inhibited NH 3 /NH 4 + - and MA/MA + -induced decrease in pHs to the level observed in H 2 O-injected oocytes. Mutations of F128 did not significantly affect transport of NH 3 or NH 4 + These data demonstrated that mutating H183 or W230 caused loss of function but not F128. H183 and H342 may affect membrane expression of the transporter.

In this study we characterized ammonia and ammonium (NH3/NH4(+)) transport by the rhesus-associated (Rh) glycoproteins RhAG, Rhbg, and Rhcg expressed in Xenopus oocytes. We used ion-selective microelectrodes and two-electrode voltage clamp to measure changes in intracellular pH, surface pH, and whole cell currents induced by NH3/NH4(+) and methyl amine/ammonium (MA/MA(+)). These measurements allowed us to define signal-specific signatures to distinguish NH3 from NH4(+) transport and to determine how transport of NH3 and NH4(+) differs among RhAG, Rhbg, and Rhcg. Our data indicate that expression of Rh glycoproteins in oocytes generally enhanced NH3/NH4(+) transport and that cellular changes induced by transport of MA/MA(+) by Rh proteins were different from those induced by transport of NH3/NH4(+). Our results support the following conclusions: 1) RhAG and Rhbg transport both the ionic NH4(+) and neutral NH3 species; 2) transport of NH4(+) is electrogenic; 3) like Rhbg, RhAG transport of NH4(+) masks NH3 transport; and 4) Rhcg is likely to be a predominantly NH3 transporter, with no evidence of enhanced NH4(+) transport by this transporter. The dual role of Rh proteins as NH3 and NH4(+) transporters is a unique property and may be critical in understanding how transepithelial secretion of NH3/NH4(+) occurs in the renal collecting duct.

The calcium-sensing receptor (CaSR), a G-protein-coupled receptor, plays a role in glandular and fluid secretion in the gastrointestinal tract, and regulates differentiation and proliferation of epithelial cells. We examined the expression of CaSR in normal and pathological conditions of human esophagus and investigated the effect of a CaSR agonist, cinacalcet (CCT), and antagonist, calhex (CHX), on cell growth and cell-cell junctional proteins in primary cultures of porcine stratified squamous esophageal epithelium. We used immunohistochemistry and Western analysis to monitor expression of CaSR and cell-cell adhesion molecules, and MTT assay to monitor cell proliferation in cultured esophageal cells. CCT treatment significantly reduced proliferation, changed the cell shape from polygonal to spindle-like, and caused redistribution of E-cadherin and β-catenin from the cell membrane to the cytoplasm. Furthermore, it reduced expression of β-catenin by 35% (P < 0.02) and increased expression of a proteolysis cleavage fragment of E-cadherin, Ecad/CFT2, by 2.3 folds (P < 0.01). On the other hand, CHX treatment enhanced cell proliferation by 27% (P < 0.01), increased the expression of p120-catenin by 24% (P < 0.04), and of Rho, a GTPase involved in cytoskeleton remodeling, by 18% (P < 0.03). In conclusion, CaSR is expressed in normal esophagus as well as in Barrett's, esophageal adenocarcinoma, squamous cell carcinoma, and eosinophilic esophagitis. Long-term activation of CaSR with CCT disrupted the cadherin-catenin complex, induced cytoskeletal remodeling, actin fiber formation, and redistribution of CaSR to the nuclear area. These changes indicate a significant and complex role of CaSR in epithelial remodeling and barrier function of esophageal cells., (© 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Eosinophilic esophagitis (EoE), an inflammatory atopic disease of the esophagus, causes massive eosinophil infiltration, basal cell hyperplasia, and sub-epithelial fibrosis. To elucidate cellular and molecular factors involved in esophageal tissue damage and remodeling, we examined pinch biopsies from EoE and normal pediatric patients. An inflammation gene array confirmed that eotaxin-3, its receptor CCR3 and interleukins IL-13 and IL-5 were upregulated. An extracellular matrix (ECM) gene array revealed upregulation of CD44 & CD54, and of ECM proteases (ADAMTS1 & MMP14). A cytokine antibody array showed a marked decrease in IL-1α and IL-1 receptor antagonist and an increase in eotaxin-2 and epidermal growth factor. Western analysis indicated reduced expression of intercellular junction proteins, E-cadherin and claudin-1 and increased expression of occludin and vimentin. We have identified a number of novel genes and proteins whose expression is altered in EoE. These findings provide new insights into the molecular mechanisms of the disease., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Acid-base balance and potassium disorders are often clinically linked. Importantly, acid-base disorders alter potassium transport. In general, acidosis causes decreased K(+) secretion and increased reabsorption in the collecting duct. Alkalosis has the opposite effects, often leading to hypokalemia. Potassium disorders also influence acid-base homeostasis. Potassium depletion causes increased H(+) secretion, ammoniagenesis and H-K-ATPase activity. Hyperkalemia decreases ammoniagenesis and NH4(+) transport in the thick ascending limb. Some combined potassium and acid-base disorders involve indirect factors such as aldosterone, impaired renal function, volume depletion, and diarrhea. In summary, disorders of potassium and acid-base homeostasis are mechanistically linked and clinically important., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The mammalian Rh glycoproteins belong to the solute transporter family SLC42 and include RhAG, present in red blood cells, and two non-erythroid members RhBG and RhCG that are expressed in various tissues, including kidney, liver, skin and the GI tract. The Rh proteins in the red blood cell form an "Rh complex" made up of one D-subunit, one CE-subunit and two RhAG subunits. The Rh complex has a well-known antigenic effect but also contributes to the stability of the red cell membrane. RhBG and RhCG are related to the NH4(+) transporters of the yeast and bacteria but their exact function is yet to be determined. This review describes the expression and molecular properties of these membrane proteins and their potential role as NH3/NH4(+) and CO2 transporters. The likelihood that these proteins transport gases such as CO2 or NH3 is novel and significant. The review also describes the physiological importance of these proteins and their relevance to human disease., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The esophageal submucosal glands (SMG) secrete HCO(3)(-) and mucus into the esophageal lumen, where they contribute to acid clearance and epithelial protection. This study characterized the ion transport mechanisms linked to HCO(3)(-) secretion in SMG. We localized ion transporters using immunofluorescence, and we examined their expression by RT-PCR and in situ hybridization. We measured HCO(3)(-) secretion by using pH stat and the isolated perfused esophagus. Using double labeling with Na(+)-K(+)-ATPase as a marker, we localized Na(+)-coupled bicarbonate transporter (NBCe1) and Cl(-)-HCO(3)(-) exchanger (SLC4A2/AE2) to the basolateral membrane of duct cells. Expression of cystic fibrosis transmembrane regulator channel (CFTR) was confirmed by immunofluorescence, RT-PCR, and in situ hybridization. We identified anion exchanger SLC26A6 at the ducts' luminal membrane and Na(+)-K(+)-2Cl(-) (NKCC1) at the basolateral membrane of mucous and duct cells. pH stat experiments showed that elevations in cAMP induced by forskolin or IBMX increased HCO(3)(-) secretion. Genistein, an activator of CFTR, which does not increase intracellular cAMP, also stimulated HCO(3)(-) secretion, whereas glibenclamide, a Cl(-) channel blocker, and bumetanide, a Na(+)-K(+)-2Cl(-) blocker, decreased it. CFTR(inh)-172, a specific CFTR channel blocker, inhibited basal HCO(3)(-) secretion as well as stimulation of HCO(3)(-) secretion by IBMX. This is the first report on the presence of CFTR channels in the esophagus. The role of CFTR in manifestations of esophageal disease in cystic fibrosis patients remains to be determined.

Rhbg is a membrane glycoprotein that is involved in NH(3)/NH(4)(+) transport. Several models have been proposed to describe Rhbg, including an electroneutral NH(4)(+)/H(+) exchanger, a uniporter, an NH(4)(+) channel, or even a gas channel. In this study, we characterized the pH sensitivity of Rhbg expressed in Xenopus oocytes. We used two-electrode voltage clamp and ion-selective microelectrodes to measure NH(4)(+)-induced [and methyl ammonium (MA(+))] currents and changes in intracellular pH (pH(i)), respectively. In oocytes expressing Rhbg, 5 mM NH(4)Cl (NH(3)/NH(4)(+)) at extracellular pH (pH(o)) of 7.5 induced an inward current, decreased pH(i), and depolarized the cell. Raising pH(o) to 8.2 significantly enhanced the NH(4)(+)-induced current and pH(i) changes, whereas decreasing bath pH to 6.5 inhibited these changes. Lowering pH(i) (decreased by butyrate) also inhibited the NH(4)(+)-induced current and pH(i) decrease. In oocytes expressing Rhbg, 5 mM methyl amine hydrochloride (MA/MA(+)), often used as an NH(4)Cl substitute, induced an inward current, a pH(i) increase (not a decrease), and depolarization of the cell. Exposing the oocyte to MA/MA(+) at alkaline bath pH (8.2) enhanced the MA(+)-induced current, whereas lowering bath pH to 6.5 inhibited the MA(+) current completely. Exposing the oocyte to MA/MA(+) at low pH(i) abolished the MA(+)-induced current and depolarization; however, pH(i) still increased. These data indicate that 1) transport of NH(4)(+) and MA/MA(+) by Rhbg is pH sensitive; 2) electrogenic NH(4)(+) and MA(+) transport are stimulated by alkaline pH(o) but inhibited by acidic pH(i) or pH(o); and 3) electroneutral transport of MA by Rhbg is likely but is less sensitive to pH changes.

Rhbg is a nonerythroid membrane glycoprotein belonging to the Rh antigen family. In the kidney, Rhbg is expressed at the basolateral membrane of intercalated cells of the distal nephron and is involved in NH4+ transport. We investigated the substrate specificity of Rhbg by comparing transport of NH3/NH4+ with that of methyl amine (hydrochloride) (MA/MA+), often used to replace NH3/NH4+, in oocytes expressing Rhbg. Methyl amine (HCl) in solution exists as neutral methyl amine (MA) in equilibrium with the protonated methyl ammonium (MA+). To assess transport, we used ion-selective microelectrodes and voltage-clamp experiments to measure NH3/NH4+- and MA/MA+-induced intracellular pH (pH(i)) changes and whole cell currents. Our data showed that in Rhbg oocytes, NH3/NH4+ caused an inward current and decrease in pH(i) consistent with electrogenic NH4+ transport. These changes were significantly larger than in H2O-injected oocytes. The NH3/NH4+-induced current was not inhibited in the presence of barium or in the absence of Na+. In Rhbg oocytes, MA/MA+ caused an inward current but an increase (rather than a decrease) in pH(i). MA/MA+ did not cause any changes in H2O-injected oocytes. The MA/MA+-induced current and pH(i) increase were saturated at higher concentrations of MA/MA+. Amiloride inhibited MA/MA+-induced current and the increase in pH(i) in oocytes expressing Rhbg but had no effect on control oocytes. These results indicate that MA/MA+ is transported by Rhbg but differently than NH3/NH4+. The protonated MA+ is likely a direct substrate whose transport resembles that of NH4+. Transport of electroneutral MA is also enhanced by expression of Rhbg.

Tegaserod, a 5-HT4 partial agonist, was shown to reduce esophageal acid exposure in patients with gastroesophageal reflux disease; however, its mechanism of action is poorly understood. Therefore, we have examined the effect of tegaserod on luminal bicarbonate and mucin secretion in the isolated perfused pig esophagus. We also studied its role in esophageal protection using SMG-bearing pig esophagus in comparison to the rabbit esophagus, which is devoid of them. The tissues were mounted in Ussing chambers, and acid injury was replicated by exposing the lumen to acid (pH 1.6) or acid/pepsin (pH 2.5). In pig esophagus, tegaserod increased bicarbonate secretion, but had no effect on basal mucin secretion. In Ussing chambers, tegaserod reduced injury to pig, but not rabbit esophagus exposed to acid (pH 2.5) plus pepsin. These results indicate that tegaserod stimulates SMG bicarbonate secretion, an effect that likely accounts for the observed protection against acid-pepsin injury to pig, but not rabbit, esophagus.

The submucosal glands (SMGs) of the pig esophagus, like the human, secrete mucin and bicarbonate, which help in luminal acid clearance and epithelial protection. The aim of this study was to characterize histochemically the esophageal SMGs and a primary culture obtained from these glands. Tissues and cultures were stained with hematoxylin and eosin, periodic acid Schiff, Alcian blue, lectins, or cytokeratins. In the perfused esophagus, addition of carbachol increased mucin secretion by approximately 2-fold. The results indicate that [1] a method for culturing SMG cells was developed; [2] conventional staining indicates the presence of sulfated, acidic, and neutral mucopolysaccharides in glands and cultures; [3] lectin binding indicates the presence of N-acetyl glucosamine, N-acetyl neuraminic acid, N-acetyl galactosamine, and alpha-L: -fucose in mucous cells and cultures; [4] cytokeratin and lectin staining indicated similarities with Barrett epithelium (columnar metaplasia of the esophagus); and [5] cholinergic agonists enhance mucin secretion and this could play a significant role in esophageal protection.

The recently cloned, non-erythrocyte Rh glycoproteins (Rhbg and Rhcg) are expressed in the intercalated cells of the renal collecting duct. The apical Rhcg and the basolateral Rhbg are likely involved in NH3 and/or NH4+ transport, yet the characteristics of this transport are not yet certain. In this study we investigated the mechanism of NH4+ transport by Rhbg and Rhcg expressed in Xenopus oocytes. We used a two-electrode voltage-clamp and ion-selective microelectrodes to measure NH4+-induced currents (I(NH4)) and changes in pHi, respectively. In oocytes expressing Rhcg, exposure to bath [NH4+] of 2.5-20 mM induced inward currents that were slightly more than those in H2O-injected (control) oocytes. I-V plots in the presence of NH4+ showed a small increase in slope conductance only at positive potentials. On the other hand, in oocytes expressing Rhbg, 5 mM NH4+ induced an inward I(NH4) of -79 nA, decreased pHi (DeltapHi) by 0.13 at a rate (dpHi/dt) of -2 7 x 10(-4) pH/s and depolarized the cell by 45 mV. These changes were significantly more than those in control oocytes. I-V plots in the presence of NH4+ showed substantial increase in conductance. Amiloride (1 mM) inhibited I(NH4), DeltapHi and dpHi/dt in oocytes expressing Rhbg but not in control oocytes. Raising bath [NH4+] in increments from 1 to 20 mM elicited a faster dpHi/dt, a larger decrease in pHi and a larger depolarization. Net NH4+ flux by Rhbg (estimated from dpHi/dt) was proportional to [NH4+] gradient and followed saturation kinetics with an apparent Km of 2.3 mM. Methyl ammonium (5 mM) induced a current of -63 nA in Rhbg oocytes but did not cause any change in control oocytes. These data indicate that: 1) Rhbg transport of NH4+ is electrogenic. 2) Methyl ammonium is transported by Rhbg. 3) NH4+ transport by Rhbg is saturated at high concentrations with Michaelis-Menten kinetics.

The mammalian esophagus has the capacity to secrete a HCO(3)(-) and mucin-rich fluid in the esophageal lumen. These secretions originate from the submucosal glands (SMG) and can contribute to esophageal protection against refluxed gastric acid. The cellular mechanisms by which glandular cells achieve these secretions are largely unknown. To study this phenomenon, we used the pH-stat technique to measure luminal alkali secretion in an isolated, perfused pig esophagus preparation. Immunohistochemistry was used to localize receptors and transporters involved in HCO(3)(-) transport. The SMG-bearing esophagus was found to have significant basal alkali secretion, predominantly HCO(3)(-), which averaged 0.21 +/- 0.04 microeq.h(-1).cm(-2). This basal secretion was doubled when stimulated by carbachol but abolished by HCO(3)(-) or Cl(-) removal. Basal- and carbachol-stimulated secretions were also blocked by serosal application of atropine, pirenzipine, DIDS, methazolamide, and ethoxzolamide. The membrane-impermeable carbonic anhydrase inhibitor benzolamide, applied to the serosal bath, partially inhibited basal HCO(3)(-) secretion and blocked the stimulation by carbachol. Immunohistochemistry using antibodies to M(1) cholinergic receptor or carbonic anhydrase-II enzyme showed intense labeling of duct cells and serous demilunes but no labeling of mucous cells. Labeling with an antibody to Na(+)-(HCO(3)(-))(n) (rat kidney NBC) was positive in ducts and serous cells, whereas labeling for Cl(-)/HCO(3)(-) exchanger (AE2) was positive in duct cells but less pronounced in serous cells. These data indicate that duct cells and serous demilunes of SMG play a role in HCO(3)(-) secretion, a process that involves M(1) cholinergic receptor stimulation. HCO(3)(-) transport in these cells is dependent on cytosolic and serosal membrane-bound carbonic anhydrase. HCO(3)(-) secretion is also dependent on serosal Cl(-) and is mediated by DIDS-sensitive transporters, possibly NBC and AE2.

Rhbg is one of two recently cloned nonerythroid glycoproteins belonging to the Rh antigen family. Rhbg is expressed in basolateral membranes of intercalated cells of the kidney cortical collecting duct and some other cell types of the distal nephron and may function as NH(4)(+) transporters. The aim of this study was to characterize the role of Rhbg in transporting NH(4)(+). To do so, we expressed Rhbg in Xenopus laevis oocytes. Two-electrode voltage-clamp and H(+)-selective microlectrodes were used to measure NH(4)(+) currents, current-voltage plots, and intracellular pH (pH(i)). In oocytes expressing Rhbg, 5 mM NH(4)(+) induced an inward current of 93 +/- 7.7 nA (n = 20) that was significantly larger than that in control oocytes of -29 +/- 7.1 nA (P < 0.005). Whole cell conductance, at all tested potentials (-60 to +60 mV), was significantly more in oocytes expressing Rhbg compared with H(2)O-injected oocytes. In Rhbg oocytes, 5 mM NH(4)(+) depolarized the oocyte by 28 +/- 3.6 mV and decreased pH(i) by 0.30 +/- 0.04 at a rate of -20 +/- 2.5 x 10(-4) pH/s. In control oocytes, 5 mM NH(4)(+) depolarized V(m) by only 20 +/- 5.8 mV and pH(i) decreased by 0.07 +/- 0.01 at a rate of -2.7 +/- 0.6 x 10(-4) pH/s. Raising bath [NH(4)(+)] in increments from 1 to 20 mM elicited a proportionally larger decrease in pH(i) (DeltapH(i)), larger depolarization (DeltaV(m)), and a faster rate of pH(i) decrease. Bathing Rhbg oocytes in 20 mM NH(4)(+) induced an inward current of 140 +/- 7 nA that was not significantly different from 178 +/- 23 nA induced in H(2)O-injected (control) oocytes. The rate of pH(i) decrease induced by increasing external [NH(4)(+)] was significantly faster in Rhbg than in H(2)O-injected oocytes at all external NH(4)(+) concentrations. In oocytes expressing Rhbg, net NH(4)(+) influx (estimated from NH(4)(+)-induced H(+) influx) as a function of external [NH(4)(+)] saturated at higher [NH(4)(+)] with a V(max) of approximately 30.8 and an apparent K(m) of 2.3 mM (R(2) = 0.99). These data strongly suggest that Rhbg is a specific electrogenic transporter of NH(4)(+).

The Rhesus (Rh) glycoproteins, originally described in human blood cells, are mostly recognized for their immunogenic characteristics and importance in pregnancy. The Rh proteins in the red blood cell are expressed as an "Rh complex" made up of one D-subunit, one CE-subunit and two Rh-associated glycoprotein (RhAG) subunits. In addition to its antigenic property, the Rh complex is thought to contribute to membrane stability and structure of red blood cells. The exact function is yet to be determined. Recently, two non-erythroid Rh glycoproteins were cloned from mice (Rhcg and Rhbg) and humans (RhCG and RhBG). RhCG is expressed at the membrane surface alone with no apparent need for heteromeric interaction with other glycoproteins. It is more similar to RhAG than to Rh CE/D, occurs late in development and is expressed abundantly and broadly in kidney and testis. In the kidney RhCG is localized to the apical cell membrane of the collecting duct. Rhbg and its human analog (RhBG) are expressed mainly in liver, skin and the kidney tubules. In the kidney collecting duct, Rhbg is localized to the basolateral membrane. Based on structural similarities to the methylammonium and ammonium permease/ammonium (MEP/Amt) transporters in yeast and their sequence homology, these proteins probably function as NH(4)(+) transporters. An initial study has indicated that RhAG or RhCG promote efflux of NH(4)(+), whereas another study has suggested that RhAG functions as an NH(4)(+)-H(+) exchanger. Evidence for such a function is still circumstantial and data indicating that Rh proteins function as NH(4)(+) transporters are indirect.

We investigated Cl(-) transport pathways in the apical and basolateral membranes of rabbit esophageal epithelial cells (EEC) using conventional and ion-selective microelectrodes. Intact sections of esophageal epithelium were mounted serosal or luminal side up in a modified Ussing chamber, where transepithelial potential difference and transepithelial resistance could be determined. Microelectrodes were used to measure intracellular Cl(-) activity (a), basolateral or apical membrane potentials (V(mBL) or V(mC)), and the voltage divider ratio. When a basal cell was impaled, V(mBL) was -73 +/- 4.3 mV and a(i)(Cl) was 16.4 +/- 2.1 mM, which were similar in presence or absence of bicarbonate. Removal of serosal Cl(-) caused a transient depolarization of V(mBL) and a decrease in a(i)(Cl) of 6.5 +/- 0.9 mM. The depolarization and the rate of decrease of a(i)(Cl) were inhibited by approximately 60% in the presence of the Cl(-)-channel blocker flufenamate. Serosal bumetanide significantly decreased the rate of change of a(i)(Cl) on removal and readdition of serosal Cl(-). When a luminal cell was impaled, V(mC) was -65 +/- 3.6 mV and a was 16.3 +/- 2.2 mM. Removal of luminal Cl(-) depolarized V(mC) and decreased a by only 2.5 +/- 0.9 mM. Subsequent removal of Cl(-) from the serosal bath decreased a(i)(Cl) in the luminal cell by an additional 6.4 +/- 1.0 mM. A plot of V(mBL) measurements vs. log a(i)(Cl)/log a(o)(Cl) (a(o)(Cl) is the activity of Cl(-) in a luminal or serosal bath) yielded a straight line [slope (S) = 67.8 mV/decade of change in a(i)(Cl)/a(o)(Cl)]. In contrast, V(mC) correlated very poorly with log a/a (S = 18.9 mV/decade of change in a/a). These results indicate that 1) in rabbit EEC, a(i)(Cl) is higher than equilibrium across apical and basolateral membranes, and this process is independent of bicarbonate; 2) the basolateral cell membrane possesses a conductive Cl(-) pathway sensitive to flufenamate; and 3) the apical membrane has limited permeability to Cl(-), which is consistent with the limited capacity for transepithelial Cl(-) transport. Transport of Cl(-) at the basolateral membrane is likely the dominant pathway for regulation of intracellular Cl(-).

Proton-translocating vacuolar ATPases (H+V-ATPase) are increasingly recognized as essential components of most eukaryotic cells. This electrogenic transporter is present in the cell membranes of many differentiated cell types and in the membranes of many subcellular organelles. The primary active pump is a multi-subunit enzyme with a membrane-bound component (V0 domain) and an intracellular catalytic component (V1 domain). The V0 domain is responsible for proton translocation and the V1 domain is responsible for ATP hydrolysis. All the subunits of the H+V-ATPase are now identified and many of their structural and molecular properties are characterized. The H+V-ATPase plays an important role in many physiological processes such as receptor-mediated transport, endocytosis, protein degradation and processing, and intracellular trafficking. In the cell membranes, it contributes to regulation of intracellular pH, secretion of acid, and generation of transmembrane electrical gradients that serve as a driving force for transport across the membrane of these cells. The role of this transporter is perhaps most significant in the kidney where it has been demonstrated in almost all segments of the nephron. H+V-ATPase in the apical membranes contributes significantly to proximal tubule bicarbonate reabsorption and is chiefly responsible for H+ secretion in the distal portions of the nephron. Basolateral H+V-ATPase in other cell types drives luminal HCO3- secretion. Regulation of distal nephron H+V-ATPase is predominantly via shuttling of transporters into and out of the surface membrane.

The purpose of this study was to investigate the direct effect of NH(3)/NH on mouse epithelial Na(+) channels (mENaC) expressed in Xenopus oocytes. Two-electrode voltage-clamp and ion-selective microelectrodes were used to measure the Na(+) current, intracellular pH (pH(i)), and ion activities in oocytes expressing mENaC. In oocytes expressing mENaC, removal of external Na(+) reversibly hyperpolarized membrane potential by 129 +/- 5.3 mV in the absence of 20 mM NH(4)Cl but only by 100 +/- 7.8 mV in its presence. Amiloride completely inhibited the changes in membrane potential. In oocytes expressing mENaC, butyrate (20 mM) caused a decrease in pH(i) (0.43 +/- 0.07) similar to the NH(4)Cl-induced pH(i) decrease (0.47 +/- 0.12). Removal of Na(+) in the presence of butyrate caused hyperpolarization that was not significantly different from that in the absence of butyrate at high pH(i) (in the absence of NH(4)Cl). Removal of external Na(+) resulted in an outward current of 3.7 +/- 0.8 microA (at -60 mV). The magnitude of this change in current was only 2.7 +/- 0.7 microA when Na(+) was removed in the presence of NH(4)Cl. In oocytes expressing mENaC, NH(4)Cl also caused a decrease in whole cell conductance at negative potential and an outward current at positive potential. In the presence of amiloride, steady-state current and the change in current caused by removal of Na(+) were not different from zero. These results indicate that NH(4)Cl inhibits Na(+) transport when mENaC is expressed in oocytes. The inhibition of voltage changes is not due to intracellular acidification caused by NH(4)Cl. Permeability and selectivity of ENaC to NH may play a role.

The aim of this study was to determine whether expressing aquaporin (AQP)-1 could affect transport of NH(3). Using ion-selective microelectrodes, the experiments were conducted on frog oocytes (cells characterized by low NH(3) permeability) expressing AQP1. In H(2)O-injected oocytes, exposure to NH(3)/NH (20 mM, pH 7.5) caused a sustained cell acidification and no initial increase in pH(i) (as expected from NH(3) influx), and the cell depolarized to near 0 mV. The absence of Na(+), the presence of Ba(2+), or raising bath pH (pH(B)) did not inhibit the magnitude of the pH(i) decrease or result in an initial increase in pH(i) when NH(3)/NH was added. However, after the cell was acidified (because of NH(3)/NH), raising pH(B) to 8.0 caused a slow increase in pH(i) but had no effect on membrane potential. The changes in pH(i) with raising pH(B) did not occur in the absence of NH(3)/NH. In AQP1 oocytes, exposure to NH(3)/NH usually resulted in little or no change in pH(i), and in the absence of Na(+) there was a small increase in pH(i) (the cell still depolarized to near 0 mV). However, after exposure to NH(3)/NH, raising pH(B) to 8.0 caused pH(i) to increase more than two times faster than in control oocytes. This increase in pH(i) is likely the result of increased NH(3) entry and not the result of NH transport. These results indicate that 1) the oocyte membrane, although highly permeable to NH, has a significant NH(3) permeability and 2) NH(3) permeability is enhanced by AQP1.

The opossum esophagus, like that of humans, contains a network of submucosal glands with the capacity to secrete bicarbonate ions into the esophageal lumen. To evaluate the role of these glands in protecting the epithelial surface from acid insult, we measured the lumen-to-surface pH gradient in opossum esophagus at different luminal pH and compared it to that of rabbit esophagus, an organ devoid of submucosal glands. Sections of opossum and rabbit esophageal epithelium were mounted luminal side up in a modified Ussing chamber. pH-sensitive microelectrodes, positioned within 5 microm of the epithelial cell surface, were used to monitor surface pH during perfusion with solutions of different pH. At luminal pH 7. 5, the pH(s) of both opossum and rabbit were similar (pH(s) = 7.5). Lowering luminal pH from 7.5 to 3.5 in opossum decreased pH(s) to 4.2+/-0.16, a value significantly higher than pH of perfusate, whereas in rabbit this maneuver decreased pH(s) to 3.69+/-0.08, a value not significantly different from pH of perfusate. In opossum but not in rabbit, addition of carbachol to the serosal solution increased basal pH(s) to 7.8+/- 0.1 and significantly blunted the decline in pH(s) on perfusion with acidic Ringer solution (pH 3.5), with pH(s) falling to 5.6+/-0.45. The effect of carbachol on surface buffering was inhibited by prior treatment with atropine. Luminal acidification to pH 2.0 in opossum (as in rabbit) abolished the lumen-to-surface pH gradient even after addition of serosal carbachol. We conclude that the presence of submucosal glands in esophagus contributes through bicarbonate secretion to creation of a lumen-to-surface pH gradient. Although this gradient can be modulated by carbachol, its capacity to buffer (and therefore to protect) the epithelial surface against back-diffusing H(+) is limited and dissipated at pH 2.0.

The M-1 cell line, derived from the mouse cortical collecting duct (CCD), is being used as a mammalian model of the CCD to study Na+ transport. The present studies aimed to further define the role of various hormones in affecting Na+ transport in M-1 cells grown in defined media. M-1 cells on permeable support, in serum-free media, developed amiloride-sensitive current 4-5 days after seeding. As expected for the involvement of epithelial Na+ channels, alpha-, beta-, and gamma-subunits of the epithelial Na+ channel were identified by RT-PCR. Either dexamethasone (Dex, 10-100 nM) or aldosterone (Aldo, 10(-6)-10(-7) M) for 24 h stimulated transport. Cells grown in the presence of Aldo and Dex had higher transport than with Dex alone. Spironolactone added to Dex media decreased transport. The acute effects of hormones reported to inhibit Na+ transport in CCD were also examined. Epidermal growth factor, phorbol esters, and increased intracellular Ca2+ with thapsigargin did not alter transport. Arginine vasopressin caused a transient increase in transport (probably Cl- secretion), which was not amiloride sensitive. Also, the protease inhibitor aprotinin decreased Na+ transport; in aprotinin-treated cells, trypsin stimulated transport. This study demonstrates that adrenal steroids (Dex > Aldo) stimulate Na+ transport in M-1 cells. At least part of this response may represent activation of mineralocorticoid receptors based on an additive effect of Dex and Aldo, as well as inhibition by spironolactone. Responses to immediate-acting hormones is limited. However, an endogenous protease activity, which activates Na+ transport, is present in these cells.

We examined the effect of norepinephrine (NE) on intracellular pH (pHi) and activity of Na+ (aNai) in the isolated perfused kidney proximal tubule of Ambystoma, using single-barreled voltage and ion-selective microelectrodes. In control HCO-3 Ringer, addition of 10(-6) M NE to the bath reversibly depolarized the basolateral membrane potential (V1), the luminal membrane potential (V2), and the transepithelial potential difference (V3) and increased pHi by 0. 14 +/- 0.02. These effects were mimicked by isoproterenol but were abolished after pretreatment with SITS or in the absence of CO2/HCO-3. Removal of bath Na+ depolarized V1 and V2, hyperpolarized V3, and decreased pHi. These effects are largely mediated by the electrogenic Na+-(HCO-3)n cotransporter. In the presence of NE, the effects of Na+ removal on membrane potential differences and the rate of change of pHi were significantly smaller. Reducing bath HCO-3 concentration from 10 to 2 mM at constant CO2 (pH 6.8) depolarized V1 and V2, decreased pHi, and lowered aNai. These changes are also due to Na+-(HCO-3)n. In the presence of NE, reducing bath [HCO-3] caused a smaller depolarizations of V1 and V2, and the rate of pHi decrease was significantly reduced. Our results indicate: 1) NE causes an increase in pHi; 2) the NE-induced alkalinization is mediated by a SITS-sensitive and HCO-3-dependent transporter on the basolateral membrane; and 3) in the presence of NE, the reduced effects caused by basolateral HCO-3 changes or Na+ removal are indicative of an inhibitory effect of NE on Na+-(HCO-3)n cotransport.

It is generally accepted that gases such as CO2 cross cell membranes by dissolving in the membrane lipid. No role for channels or pores in gas transport has ever been demonstrated. Here we ask whether expression of the water channel aquaporin-1 (AQP1) enhances the CO2 permeability of Xenopus oocytes. We expressed AQP1 in Xenopus oocytes by injecting AQP1 cRNA, and we assessed CO2 permeability by using microelectrodes to monitor the changes in intracellular pH (pHi) produced by adding 1.5% CO2/10 mM HCO3- to (or removing it from) the extracellular solution. Oocytes normally have an undetectably low level of carbonic anhydrase (CA), which eliminates the CO2 hydration reaction as a rate-limiting step. We found that expressing AQP1 (vs. injecting water) had no measurable effect on the rate of CO2-induced pHi changes in such low-CA oocytes: adding CO2 caused pHi to fall at a mean initial rate of 11.3 x 10(-4) pH units/s in control oocytes and 13.3 x 10(-4) pH units/s in oocytes expressing AQP1. When we injected oocytes with water, and a few days later with CA, the CO2-induced pHi changes in these water/CA oocytes were more than fourfold faster than in water-injected oocytes (acidification rate, 53 x 10(-4) pH units/s). Ethoxzolamide (ETX; 10 microM), a membrane-permeant CA inhibitor, greatly slowed the pHi changes (16.5 x 10(-4) pH units/s). When we injected oocytes with AQP1 cRNA and then CA, the CO2-induced pHi changes in these AQP1/CA oocytes were approximately 40% faster than in the water/CA oocytes (75 x 10(-4) pH units/s), and ETX reduced the rates substantially (14.7 x 10(-4) pH units/s). Thus, in the presence of CA, AQP1 expression significantly increases the CO2 permeability of oocyte membranes. Possible explanations include 1) AQP1 expression alters the lipid composition of the cell membrane, 2) AQP1 expression causes overexpression of a native gas channel, and/or 3) AQP1 acts as a channel through which CO2 can permeate. Even if AQP1 should mediate a CO2 flux, it would remain to be determined whether this CO2 movement is quantitatively important.

The effect of norepinephrine (NE) on mechanisms of cellular Na+ transport in the isolated, perfused proximal tubule of Ambystoma tigrinum was examined. Single-barreled voltage and ion-selective microelectrodes were used to determine basolateral (V1), luminal (V2), and transepithelial (V3) membrane potentials and intracellular Na+ activity (alpha Nai). In CO2/HCO3- control solution, addition of NE (10(-6) M) to the bath caused depolarizations of V1, V2, and V3 are decreased alpha Nai. These effects were mimicked by isoproterenol and inhibited by propranolol. Addition of NE in the absence of luminal Na+ and substrates did not cause any changes in V1, V2, V3, or alpha Nai. NE did not affect the changes in membrane potential difference (PD) or alpha Nai caused by removal and readdition of luminal substrates and/or Na+. To study the effect of NE on Na-K-adenosinetriphosphatase (Na-K-ATPase), the pump was inhibited by external K+ removal and then reactivated by readdition of 12 mM K+ to the bath in the presence and absence of NE. Reactivation of the pump caused hyperpolarization of membrane PDs, and alpha Nai recovered monotonically in 3-5 min. The peak hyperpolarizations of V1 and V2 (approximately 1 min) were significantly larger in the presence of NE. During the first 3 min, and also at the same alpha Nai, the rate of decrease of alpha Nai was significantly faster in the presence of NE. In conclusion, these results show a direct effect of NE on cell membrane PDs and alpha Nai in the kidney proximal tubule. Most likely, beta-receptors are involved in mediating the action of NE. Neither Na/H exchange nor Na-substrate cotransport at the luminal membrane are affected by NE. On the other hand, NE activates Na-K-ATPase.

We examined H+ and HCO3- transport mechanisms that are involved in the regulation of intracellular pH of Schwann cells. Primary cultures of Schwann cells were prepared from the sciatic nerves of 1-3-day-old rats. pHi of single cells attached to cover slips was continuously monitored by measuring the absorbance spectra of the pH-sensitive dye dimethylcarboxyfluorescein incorporated intracellularly. The average pHi of neonatal Schwann cells bathed in HEPES mammalian solution was 7.17 +/- 0.02 (n = 32). In the nominal absence of HCO3-, pHi spontaneously recovered from an acute acid load induced by exposing the Schwann cells to 20 mM NH4+ (NH4+ prepulse). This pHi recovery from the acute acid load was totally inhibited in the absence of external Na+ or in the presence of 1 mM amiloride. In both cases, the pHi recovery was readily restored upon readdition of external Na+ or removal of amiloride. In the steady-state, addition of amiloride caused a small and slow decrease in pHi which was readily reversed upon removal of amiloride. In the presence of HCO3-, removal of external Cl- caused pHi to rapidly and reversibly increase by 0.23 +/- 0.03 (n = 15) and the initial rate of alkalinization was 20.6 +/- 2.7 x 10(-4) pH/sec. In the absence of external Na+, removal of bath Cl- still caused pHi to increase by 0.15 +/- 0.02 and the initial rate of pHi increase was not significantly altered. In the nominal absence of HCO3-, removal of bath Cl- caused pHi to increase very slightly (0.05 +/- 0.01) with an initial dpHi/dt of only 4.4 +/- 0.2 x 10(-4) pH/sec (n = 4). Addition of 100 microM DIDS did not inhibit the pHi increase caused by removal of bath Cl-. These data indicate that 1) Rat Schwann cells regulate their pHi via an Na-H exchange mechanism which is moderately active at steady-state pHi. 2) In the presence of HCO3-, there is a Na-independent Cl-HCO3 (base) exchanger which also contributes to regulation of intracellular pH in Schwann cells.

We used the absorbance spectrum of the pH-sensitive dye dimethylcarboxyfluorescein to monitor intracellular pH (pHi) in the isolated perfused S3 segment of the rabbit proximal tubule, and examined the effect on pHi of switching from a HEPES to a CO2/HCO3- buffer in the lumen and/or the bath (i.e., basolateral solution). Solutions were titrated to pH 7.40 at 37 degrees C. With 10 mM acetate present bilaterally (lumen and bath), this causing steady-state pHi to be rather high (approximately 7.45), bilaterally switching the buffer from 32 mM HEPES to 5% CO2/25 mM HCO3- caused a sustained fall in pHi of approximately 0.26. However, with acetate absent bilaterally, this causing steady-state pHi to be substantially lower (approximately 6.9), bilaterally switching to CO2/HCO3- caused a transient pHi fall (due to the influx of CO2), followed by a sustained rise to a level approximately 0.18 higher than the initial one. The remainder of the experiments was devoted to examining this alkalinization in the absence of acetate. Switching to CO2/HCO3- only in the lumen caused a sustained pHi fall of approximately 0.15, whereas switching to CO2/HCO3- only in the bath caused a transient fall followed by a sustained pHi increase to approximately 0.26 above the initial value. This basolateral CO2/HCO3(-)-induced alkalinization was not inhibited by 50 microM DIDS applied shortly after CO2/HCO3- washout, but was slowed approximately 73% by DIDS applied more than 30 min after CO2/HCO3- washout. The rate was unaffected by 100 microM bilateral acetazolamide, although this drug greatly reduced CO2-induced pHi transients. The alkalinization was not blocked by bilateral removal of Na+ per se, but was abolished at pHi values below approximately 6.5. The alkalinization was also unaffected by short-term bilateral removal of Cl- or SO4=. Basolateral CO2/HCO3- elicited the usual pHi increase even when all solutes were replaced, short or long-term (> 45 min), by N-methyl-D-glucammonium/glucuronate (NMDG+/Glr-). Luminal CO2/HCO3- did not elicit a pHi increase in NMDG+/Glr-. Although the sustained pHi increase elicited by basolateral CO2/HCO3- could be due to a basolateral HCO3- uptake mechanism, net reabsorption of HCO3- by the S3 segment, as well as our ACZ data, suggest instead that basolateral CO2/HCO3- elicits the sustained pHi increase either by inhibiting an acid-loading process or stimulating acid extrusion across the luminal membrane (e.g., via an H+ pump).

Intracellular potassium and membrane potential were measured simultaneously by means of double-barrelled liquid ion-exchange microelectrodes in single fibers of rat thigh muscle in vivo in rats maintained in seven different metabolic states. The K+ equilibrium potential (EK) was more negative than the simultaneously measured membrane potential (Em) in the normal state by 18.4 mV. K+ loading, acute and chronic, resulted in depolarization of Em due to increased serum K+ (hyperkalemia) with no increase in intracellular K+. K+ depletion resulted in hyperpolarization of Em as plasma K+ decreased proportionately more than intracellular K+. Low Na+ diet had no effect. Intracellular K+ was decreased in acute acidosis but not in the chronic state. Thus K+ depletion and acute acidosis are associated with intracellular K+ decrease. The fact that hyperpolarization exists in the former and not the latter is a reflection that hypokalemia accompanies the former condition. The hyperpolarizing states of K+ depletion and chronic acidosis are accompanied by decreased excitability and muscle weakness.

We studied the role of basolateral HCO3- transport in the regulation of intracellular pH (pHi) in the isolated perfused S3 segment of the rabbit proximal tubule. pHi was calculated from absorbance spectra of the pH-sensitive dye dimethylcarboxyfluorescein. Solutions were normally buffered to pH 7.4 at 37 degrees C with 25 mM HCO3- 5% CO2. pHi fell by approximately 0.17 when luminal [HCO3-] was lowered to 5 mM at fixed PCO2 (i.e., reducing pH to 6.8) but by approximately 0.42 when [HCO3-] in the bath (i.e., basolateral solution) was lowered to 5 mM. The pHi decrease elicited by reducing bath [HCO3-] was substantially reduced by removal of Cl- or Na+, suggesting that components of basolateral HCO3- transport are Cl- and/or Na+ dependent. We tested for the presence of basolateral Cl-HCO3 exchange by removing bath Cl-. This caused pHi to increase by approximately 0.23, with an initial rate of approximately 100 X 10(-4) pH/s. Although the initial rate of this pHi increase was not reduced by removing Na+ bilaterally, it was substantially lowered by the nominal removal of HCO3- from bath and lumen or by the addition of 0.1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) to the bath. The results thus suggest that a Na-independent Cl-HCO3 exchanger is present at the basolateral membrane. We tested for the presence of basolateral Na-HCO3 cotransport by removing bath Na+. This caused pHi to fall reversibly by approximately 0.26 with initial rates of pHi decline and recovery being approximately 30 and approximately 41 X 10(-4) pH/s, respectively. Although the bilateral removal of Cl- had no effect on these rates, the nominal removal of HCO3- or the presence of DIDS substantially slowed the pHi changes. Thus, in addition to a Cl-HCO3 exchanger, the basolateral membrane of the S3 proximal tubule also appears to possess a Na-HCO3 cotransport mechanism. The data do not rule out the possibility of other basolateral HCO3- transporters.

We used the absorbance spectrum of 4',5'-dimethyl-5-(and 6) carboxyfluorescein to measure intracellular pH (pHi) in the isolated, perfused S3 segment of the rabbit proximal tubule. Experiments were conducted in HCO3- -free solutions. pHi recovered from an acid load imposed by an NH4+ prepulse, indicating the presence of one or more active acid-extrusion mechanisms. Removal of Na+ from bath and lumen caused pHi to decrease by approximately 0.6, whereas Na+ readdition caused complete pHi recovery. Removal of Na+ from the bath caused only a slow pHi decrease that was enhanced about fourfold when Na+ was subsequently removed from the lumen also. Similarly, the pHi recovery produced by the readdition of Na+ to the bath and lumen was about ninefold faster than when Na+ was returned to the bath only. Amiloride (1-2 mM) inhibited the pHi recovery that was elicited by returning 15 or 29 mM Na+ to lumen by only approximately 30%. However, in the absence of external acetate (Ac-), 1 mM amiloride inhibited approximately 66% of the pHi recovery induced by the readdition of 29 mM Na+ to the lumen only. The removal of external Ac- reduced the pHi recovery rate from an NH4+-induced acid load by approximately 47%, and that elicited by Na+ readdition, by approximately 67%. Finally, when bilateral removal of Na+ was maintained for several minutes, pHi recovered from the initial acidification, slowly at first, and then more rapidly, eventually reaching a pHi approximately 0.1 higher than the initial one. This Na+-independent pHi recovery was not significantly affected by lowering [HEPES]o from 32 to 3 mM or by adding N'N'-dicyclohexylcarbodiimide (10(-4) M) to the lumen, but it was reduced approximately 57% by iodoacetate (0.5 mM) plus cyanide (1 mM). We conclude that in the nominal absence of HCO3-, three transport systems contribute to acid extrusion by S3 cells: (a) a Na+-independent mechanism, possibly an H+ pump; (b) a Na-H exchanger, confined primarily to the luminal membrane; and (c) an Ac- and luminal Na+-dependent mechanism. The contribution of these three mechanisms to total acid extrusion, assessed by the rapid readdition of Na+, was approximately 13, approximately 30, and approximately 57%, respectively.

Traditional models of acid-base transport and intracellular pH (pHi) regulation in the renal proximal tubule have been based on the existence of a Na+/H+ exchanger at the luminal membrane and a simple HCO3- conductance at the basolateral membrane. Our recent work, in which we used pH-sensitive microelectrodes or dyes to monitor pHi in isolated renal tubules perfused in the nominal absence of HCO3-, has demonstrated the existence of a novel mechanism of acid extrusion in amphibian and mammalian proximal tubule cells. The salamander proximal tubule, for example, possesses an electroneutral Na+ monocarboxylate (Na+-X-) co-transporter, but only at the luminal membrane. It also possesses an electroneutral H+-X- co-transporter, but only at the blood side or basolateral membrane. In the presence of lactate, the luminal Na+-lactate co-transporter mediates a net influx of lactate, driven by the Na+ gradient. The cell-to-blood lactate gradient, in turn, drives the coupled efflux of H+ and lactate across the basolateral membrane. The net effect is the reabsorption of lactate, the luminal uptake of Na+ and the basolateral extrusion of H+. Acid extrusion mediated by this monocarboxylate system in the salamander is comparable in magnitude to that mediated by the Na+/H+ exchanger. In the S3 segment of the rabbit proximal tubule, a similar monocarboxylate system (studied with acetate instead of lactate) extrudes acetate at twice the rate of the Na+/H+ exchanger. Thus, monocarboxylate transport, at least in the nominal absence of HCO3-, can have a major impact on pHi regulation.

We monitored intracellular pH (pHi) in isolated perfused S3 segments of the rabbit proximal tubule, and studied the effect of acetate (Ac-) transport on pHi. pHi was calculated from the absorbance spectrum of 4',5'-dimethyl-5-(and 6) carboxyfluorescein trapped intracellularly. All solutions were nominally HCO3(-)-free. Removal of 10 mM Ac- from bath and lumen caused pHi to rapidly rise by approximately 0.2, and then to decline more slowly to a value approximately 0.35 below the initial one. Removal of only luminal Ac- caused pHi changes very similar to those resulting from bilateral removal of Ac-. When Ac- was removed from bath only, pHi rose rapidly at first, and then continued to rise more slowly. Readdition of Ac- to bath caused pHi to rapidly fall to a value slightly higher than the one prevailing before the removal of Ac- from the bath. In experiments in which Ac- was first removed from both bath and lumen, readdition of 10 mM Ac- to only lumen caused a rapid but small acidification, followed by a slower alkalinization that brought the pHi near the value that prevailed before the bilateral removal of Ac-. The alkalinizing effects caused by the readdition of 10 or 0.5 mM Ac- were indistinguishable. When Ac- was returned to only lumen in the absence of luminal Na+, there was a small and rapid pHi decrease, but no pHi recovery. Removal of Na+ from bath did not affect the pHi transients caused by the addition of Ac- to lumen. In experiments in which Ac- was first removed bilaterally, readdition of Ac- to only bath caused a large and sustained drop in pHi, whereas the subsequent removal of Ac- from the bath caused a slight alkalinization. These pHi changes caused by readdition or removal of Ac- from baths were unaffected by the absence of external Na+. We conclude that there is a Na+/Ac- cotransporter at the luminal membrane, and pathways for acetic acid transport at both luminal and basolateral membranes. The net effect of Ac- transport on pHi is to alkalinize the cell as a result of the luminal entry of Na+/Ac-, which is followed by the luminal and basolateral exit of acetic acid.