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.     

Purification and Characterization of l-Aminoacylase from Alcaligenes denitrificans DA181

The l-aminoacylase produced intracellularly by Alcaligenes denitrificans DA181 was puritied to homogeneity. This enzyme had an apparent molecular weight of 80,000, and was composed of two subunits of identical molecular weight. Its isoelectric point was pH 5.1. The optimal reaction temperature and pH were 65°C and 8.0, respectively. This enzyme showed specificity toward N-acetyl-derivative of hydrophobic l-amino acids with N-acetyl-l-valine as the favored substrate, followed by N-acetyl-l-alanine.     

Reduced contribution from Na+/H+ exchange to acid extrusion during anoxia in adult rat hippocampal CA1 neurons

The effect of anoxia on Na+/H+ exchange activity was examined in acutely isolated adult rat hippocampal CA1 neurons loaded with the H+-sensitive fluorophore, BCECF. Five-minute anoxia imposed under nominally HCO3-/CO2-free conditions induced a fall in pHi, the magnitude of which was smaller following prolonged exposure to medium in which N-methyl-D-glucamine (NMDG+) was employed as an extracellular Na+ (Na(+)(o)) substitute. Also consistent with the possibility that Na+/H+ exchange becomes inhibited soon after the induction of anoxia, rates of Na(+)(o)-dependent pHi recovery from internal acid loads imposed during anoxia were slowed, compared to rates of Na(+)(o)-dependent pHi recovery observed prior to anoxia. At the time at which rates of pHi recovery were reduced during anoxia, cellular adenosine triphosphate (ATP) levels had fallen to 35% of preanoxic levels, suggesting that ATP depletion might contribute to the observed inhibition of Na+/H+ exchange. In support, incubation of neurons with 2-deoxyglucose and antimycin A under normoxic conditions induced a fall in cellular ATP levels that was also associated with reduced Na(+)(o)-dependent rates of pHi recovery from imposed acid loads; conversely, pre-treatment with 10 mm creatine attenuated the effects of anoxia to reduce both ATP levels and Na(+)(o)-dependent rates of pHi recovery from internal acid loads. Taken together, the results are consistent with the possibility that functional Na+/H+ exchange activity in adult rat CA1 neurons declines soon after the onset of anoxia, possibly as a result of anoxia-induced falls in intracellular ATP.     

Apical and basolateral Na/H exchange in cultured murine proximal tubule cells (MCT): Effect of parathyroid hormone (PTH)

Summary Kidney proximal tubule Na/H exchange is inhibited by PTH. To analyze further the cellular mechanisms involved in this regulation we have used MCT cells (a culture of SV-40 immortalized mouse cortical tubule cells) grown on permeant filter supports. Na/H exchange was measured using single cell fluorescence microscopy (BCECF) and phosphate transport (measured for comparisons) by tracer techniques. MCT cells express apical and basolateral Na/H exchangers which respond differently to inhibition by ethylisopropylamiloride and by dimethylamiloride, the basolateral membrane transporter being more sensitive. Apical membrane Na/H exchange was inhibited by PTH (10^–8m; by an average of 25%); similar degrees of inhibition were observed when cells were exposed either to forskolin, 8-bromo-cAMP or phorbol ester. Basolateral membrane Na/H exchange was stimulated either by incubation with PTH (to 129% above control levels) or by addition of phorbol ester (to 120% above control levels); it was inhibited after exposure to either forskolin or 8-bromo-cAMP. The above effects of PTH and phorbol ester (apical and basolateral) were prevented by preincubation of cells with protein kinase C antagonists, staurosporine and calphostin C; both compounds did not affect forskolin or 8-bromo-cAMP induced effects. PTH also inhibited apical Na-dependent phosphate influx (29% inhibition at 10^–8m); it had no effect on basolateral phosphate fluxes (Na-dependent and Na-independent). Incubation with PTH (10^–8m) resulted in a rapid and transient increase in [Ca²⁺]_i (measured with the fluorescent indicator, fura-2), due to stimulation of a Ca²⁺ release from intracellular stores. Exposure of MCT cells to PTH did not elevate cellular levels of cAMP. Taken together, these results suggest that PTH utilizes in MCT cells the phospholipase C/protein kinase C pathway to differently control Na/H exchangers (apical vs. basolateral) and to inhibit apical Na/P_i cotransport.This work was supported by the Swiss National Science Foundation (Grant No. 32-30785.91), the Stiftung für wissenschaftliche Forschung an der Universität Zürich, the Hartmann-Müller Stiftung, the Sandoz-Stiftung, the Roche Research Foundation and the Geigy-Jubiläumsstiftung. We are grateful to Denise Rossi and Christa Knellwolf for their excellent secretarial assistance.     

Na+/H+ exchange subtype 1 inhibition during extracellular acidification and hypoxia in glioma cells

Lactacidosis is a common feature of ischaemic brain tissue, but its role in ischaemic neuropathology is still not fully understood. Na(+)/H(+) exchange, a mechanism involved in the regulation of intracellular pH (pH(i)), is activated by low pH(i). The role of Na(+)/H(+) exchange subtype 1 was investigated during extracellular acidification and subsequent pH recovery in the absence and presence of (4-isopropyl-3-methylsulphonyl-benzoyl)-guanidine methanesulfonate (HOE642, Cariporid), a new selective and powerful inhibitor of the Na(+)/H(+) exchanger subtype 1 (NHE-1). It was compared for normoxia and hypoxia in two glioma cell lines (C6 and F98). pH(i) was monitored by fluorescence spectroscopy using the intracellularly trapped pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Alterations in glial cell metabolism were characterized using high-resolution (1)H, (13)C and (31)P NMR spectroscopy of perchloric acid extracts. NHE-1 contributed to glial pH regulation, especially at pathologically low pH(i) values. NHE-1 inhibition with HOE642 during acidification caused exacerbated metabolic disorders which were prolonged during extracellular pH recovery. However, NHE-1 inhibition during hypoxia protected the energy state of glial cells.     

Na+-dependent HCO 3 − transport and Na+/H+ exchange regulate pH i in human ciliary muscle cells

Summary We investigated intracellular pH (pH _i ) regulation in cultured human ciliary muscle cells by means of the pH-sensitive absorbance of 5(and 6)-carboxy-4,5-dimethylfluorescein (CDMF). The steady-state pH _i was 7.09±0.04 (n = 12) in CO₂/ HCO ₃ ^– -buffered and 6.86±0.03 (n = 12) in HEPES-buffered solution. Removal of extracellular sodium for 6 min acidified the cells by 1.11±0.06 pH units (n = 12) in the presence of CO₂/ HCO ₃ ^– and by 0.91±0.05 pH units (n = 8) in its absence. Readdition of external sodium resulted in a rapid pH _i recovery, which was almost completely amiloride-sensitive in the absence of CO₂/ HCO ₃ ^– but only slightly influenced by amiloride in its presence. Application of DIDS under steady-state conditions significantly acidified the ciliary muscle cells by 0.25±0.02 (n = 4) in 6 min, while amiloride had no effect. The pH _i recovery after an intracellular acid load was completely dependent on extracellular sodium. In HEPES-buffered solution the pH _i recovery was almost completely mediated by Na⁺/H⁺ exchange, since it was blocked by amiloride (1 mmol/liter). In contrast, a marked amilorideinsensitive pH _i recovery was observed in CO₂/HCO ₃ ^– -buffered solution which was mediated by chloride-independent and chloride-dependent Na⁺ HCO ₃ ^– cotransport. This recovery, inhibited by DIDS (0.2 mmol/liter). was also observed if the cells were preincubated in chloride-free solution for 4 hr. Analysis of the sodium dependence of the pH _i recovery after NH₄Cl prepulse revealed V _max = 0.57 pH units/min, K _m= 39.7 mmol/liter extracellular sodium for the amiloride-sensitive component and V _max = 0.19 pH units/min, K _m= 14.3 mmol/liter extracellular sodium for the arniloride-insensitive component. We conclude that Na⁺/H⁺ exchange and chloride-independent and chloride-dependent Na⁺HCO ₃ ^– cotransport are involved in the pH _i regulation of cultured human ciliary muscle cells.The expert technical assistance of Astrid Krolik is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft grant DFG Wi 328/11.     

Effects of attachment substrates on the growth and differentiation of LLC-PK1 cells

Summary The growth and differentiation of an established renal epithelial cell line, LLC-PK₁, on membrane bound mussel adhesive protein (MAP), collagen, and extracellular matrix (ECM) in serum-containing medium was studied. Cell attachment and growth on uncoated- vs. protein-coated cellulose nitrate and acetate membranes did not differ significantly, and confluence was achieved on all membranes. However, cells remained in a single monolayer only when plated on collagen or ECM. LLC-PK₁ monolayers grown on ECM-coated membranes displayed the highest transepitheliald-glucose transport (333 ± 22 ng·cm⁻²·min⁻¹) whereas cells plated on collagen-coated membranes displayed the lowest (94 ± 23 ng·cm⁻²·min⁻¹). Glucose flux values increased with age of the culture, reaching a plateau at 28 d postseeding. These results indicate that the underlying substratum and cell age can affect differentiation of renal epithelial cells in vitro.     

Inhibition and redistribution of NHE3, the apical Na+/H+ exchanger, by Clostridium difficile toxin B

NHE3, the apical isoform of the Na(+)/H(+) exchanger, is central to the absorption of salt and water across the intestinal epithelium. We report that treatment of epithelial cells with toxin B of Clostridium difficile, a diarrheal pathogen, causes a pronounced inhibition of NHE3 activity, with little effect on the basolateral NHE1 isoform. Depression of NHE3 activity is accompanied by the translocation of apical exchangers to a subapical endomembrane compartment. Treatment of cells with toxin B increased the fraction of exchangers that were solubilized by nonionic detergents and induced dephosphorylation and extensive redistribution of ezrin. The Rho-kinase inhibitor, Y-27632, also altered the distribution and activity of NHE3. We suggest that inactivation of Rho-family GTPases by clostridial toxin B alters the interaction between NHE3 and the microvillar cytoskeleton, possibly by impairing the ability of ezrin to bridge the exchangers to filamentous actin. Detachment of NHE3 from the actin skeleton would facilitate its internalization, resulting in net disappearance from the apical surface. The consequent inhibition of transport is likely to contribute to the diarrheal effects of C. difficile.     

Interactions of external and internal H+ and Na+ with Na+/Na+ and Na+/H+ exchange of rabbit red cells: Evidence for a common pathway

Summary We have studied the kinetic properties of rabbit red cell (RRBC) Na⁺/Na⁺ and Na⁺/H⁺ exchanges (EXC) in order to define whether or not both transport functions are conducted by the same molecule. The strategy has been to determine the interactions of Na⁺ and H⁺ at the internal (i) and external (o) sites for both exchanges modes. RRBC containing varying Na _i and H _l were prepared by nystatin and DIDS treatment of acid-loaded cells. Na⁺/Na⁺ EXC was measured as Na _o -stimulated Na⁺ efflux and Na⁺/H⁺ EXC as Na _o -stimulated H⁺ efflux and pH _o -stimulated Na⁺ influx into acid-loaded cells.The activation of Na⁺/Na⁺ EXC by Na _o at pH _i 7.4 did not follow simple hyperbolic kinetics. Testing of different kinetic models to obtain the best fit for the experimental data indicated the presence of high (K _m 2.2 mM) and low affinity (K _m 108 mM) sites for a single- or two-carrier system. The activation of Na⁺/H⁺ EXC by Na _o (pH _i 6.6, Na _i <1 mM) also showed high (K _m 11 mM) and low (K _m 248 mM) affinity sites. External H⁺ competitively inhibited Na⁺/Na⁺ EXC at the low affinity Na _o site (K _H 52 nM) while internally H⁺ were competitive inhibitors (pK 6.7) at low Na _i and allosteric activators (pK 7.0) at high Na _i .Na⁺/H₊ EXC was also inhibited by acid pH _o and allosterically activated by H _i (pK 6.4). We also established the presence of a Na _i regulatory site which activates Na⁺/H⁺ and Na⁺/Na⁺ EXC modifying the affinity for Na _o of both pathways. At low Na _i , Na⁺/Na⁺ EXC was inhibited by acid pH _i and Na⁺/H⁺ stimulated but at high Na _i , Na⁺/Na⁺ EXC was stimulated and Na⁺/H⁺ inhibited being the sum of both pathways kept constant. Both exchange modes were activated by two classes of Na _o sites,cis-inhibited by external H _o , allosterically modified by the binding of H⁺ to a H _i regulatory site and regulated by Na _i . These findings are consistent with Na⁺/Na⁺ EXC being a mode of operation of the Na⁺/H⁺ exchanger.Na⁺/H⁺ EXC was partially inhibited (80–100%) by dimethyl-amiloride (DMA) but basal or pH _i -stimulated Na⁺/Na⁺ EXC (pH _i 6.5, Na _i 80 mM) was completely insensitive indicating that Na⁺/Na⁺ EXC is an amiloride-insensitive component of Na⁺/H⁺ EXC. However, Na⁺ and H⁺ efflux into Na-free media were stimulated by cell acidification and also partially (10 to 40%) inhibited by DMA: this also indicates that the Na⁺/H⁺ EXC might operate in reverse or uncoupled modes in the absence of Na⁺/Na⁺ EXC.In summary, the observed kinetic properties can be explained by a model of Na⁺/H⁺ EXC with several conformational states, H _i and Na _i regulatory sites and loaded/unloaded internal and external transport sites at which Na⁺ and H⁺ can compete. The occupancy of the H⁺ regulatory site induces a conformational change and the occupancy of the Na _i regulatory site modulates the flow through both pathways so that it will conduct Na⁺/H⁺ and/or Na⁺/Na⁺ EXC depending on the ratio of internal Na⁺:H⁺.     

NHE基因家族的研究进展

Na⁺/H⁺交换泵(Na⁺/H⁺ exchanger, NHE)是存在于所有脊椎动物细胞中的重要跨膜蛋白,该蛋白质涉及细胞的多种功能,包括细胞内pH值调节、细胞体积的控制以及离子转运等.目前已克隆了五个亚型NHE的cDNA,它们构成了脊椎动物细胞离子转运泵的一个基因家族. 这五个亚型的表达水平及活性可受多种因素的调节.在肿瘤、高血压及糖尿病等疾病中,已发现NHE-1亚型的表达水平和活性显著增高.因此,研究NHE-1的转录及活性调节机制,将可能为这些疾病的诊治提供新的手段.     

Role of intracellular sodium activity in the control of contraction in cardiac purkinje fibers

The role of intracellular sodium activity (a_Naⁱ) in the control of force was studied in sheep cardiac Purkinje fibers exposed to norepinephrine (NE) and high [Ca]_o in the absence and presence of overdrive or of a low concentration of strophanthidin. Both NE and high [Ca]_o decrease a_Naⁱ and increase force, while overdrive increases and low strophanthidin decreases both parameters. In the presence of NE, overdrive increases a_Naⁱ less than force and is followed by a more pronounced undershoot in a_Naⁱ and force. In contrast, in high [Ca]_o overdrive increases a_Naⁱ more than force and is followed by a less pronounced undershoot in a_Naⁱ and force than in NE. High [Ca]_o increases force to a peak, but then the decreasing a_Naⁱ reduces force. In all these conditions, a_Naⁱ determines force changes during recovery from overdrive. NE and high [Ca]_o decrease a_Naⁱ less and increase force more in low strophanthidin. Thus, changes in a_Naⁱ modulate the increase in force due to increased Ca influx and control force development when Ca influx is either unchanged (low strophanthidin) or has reached a steady state (high [Ca]_o, recovery from overdrive).     

Feedback inhibition of NaCl entry inNecturus gallbladder epithelial cells

Summary WhenNecturus gallbladder epithelium is treated with ouabain the cells swell rapidly for 20–30 minutes then stabilize at a cell volume 30% greater than control. The cells then begin to shrink slowly to below control size. During the initial rapid swelling phase cell Na activity, measured with microelectrodes, rises rapidly. Calculations of the quantity of intracellular Na suggest that the volume increase is due to NaCl entry. Once the peak cell volume is achieved, the quantity of Na in the cell does not increase, suggesting that NaCl entry has been inhibited. We tested for inhibition of apical NaCl entry during ouabain treatment either by suddenly reducing the NaCl concentration in the mucosal bath or by adding bumetanide to the perfusate. Both maneuvers caused rapid cell shrinkage during the initial phase of the ouabain experiment, but had no effect on cell volume if performed during the slow shrinkage period. The lack of sensitivity to the composition of the mucosal bath during the shrinkage period occurred because of apparent feedback inhibition of NaCl entry. Another maneuver, reduction of the Na in the serosal bath to 10mm, also resulted in inhibition of apical NaCl uptake. The slow shrinkage which occurred after one or more hours of ouabain treatment was sensitive to the transmembrane gradients for K and Cl across the basolateral membrane and could be inhibited by bumetanide. Thus during pump inhibition inNecturus gallbladder epithelium cell Na and volume first increase due to continuing NaCl entry and then cell volume slowly decreases due to inhibition of the apical NaCl entry and activation of basolateral KCl exit.     

Membrane surface charge dictates the structure and function of the epithelial Na+/H+ exchanger

The Na(+)/H(+) exchanger NHE3 plays a central role in intravascular volume and acid-base homeostasis. Ion exchange activity is conferred by its transmembrane domain, while regulation of the rate of transport by a variety of stimuli is dependent on its cytosolic C-terminal region. Liposome- and cell-based assays employing synthetic or recombinant segments of the cytosolic tail demonstrated preferential association with anionic membranes, which was abrogated by perturbations that interfere with electrostatic interactions. Resonance energy transfer measurements indicated that segments of the C-terminal domain approach the bilayer. In intact cells, neutralization of basic residues in the cytosolic tail by mutagenesis or disruption of electrostatic interactions inhibited Na(+)/H(+) exchange activity. An electrostatic switch model is proposed to account for multiple aspects of the regulation of NHE3 activity.     

Na超负荷与Na/H交换的关系——在等容收缩离体大鼠心脏的研究

心脏富氧灌流30min稳定后随机分为四组:(1)对照组:富氧灌流75min;(2)低血流缺氧组:低血流缺氧45min后,再富氧灌流30min;(3)Ouabain组:于低血流缺氧过程中,溶Ouabain(200μmol/L)于K—H液中,余同组(2);(4)Ouabain+Amiloride组:除在低血流缺氧期给0.5mmol/L amiloride外,余同组(3)。与低血流缺氧组相比,Ouabain可引起再灌注时心肌Na的明显增加并伴有心室功能的抑制,Amiloride可明显减轻这一损害作用。这表明,Na/K ATPase活性的抑制与再灌注心肌的Na超载有关,而这一作用的机制可能是由于Na/H交换的激活所引起。     

Macromolecular crowding and volume perception in dog red cells

To differentiate whether the primary volume signal in dog red cells arises from a change in cell configuration or the concentration and dilution of cell contents, we prepared resealed ghosts that had the same surface area and hemoglobin concentration as intact cells but less than 1/3 their volume. Shrinkage of both intact cells and resealed ghosts triggered Na/H exchange. Activation of this transporter in the two preparations correlated closely with cytosolic protein concentration but not at all with volume. The Na/H exchanger was more sensitive to shrinkage in albumin-loaded resealed ghosts than in intact cells or ghosts containing only hemoglobin. Similar results were obtained for the swelling-induced [K-Cl] cotransporter. We believe perception of cell volume originates with changes in cytoplasmic protein concentration. We think the kinases and phosphatases that control the activation of membrane transporters in response to cell swelling or shrinkage are regulated by the mechanism of macromolecular crowding.     

Modulation of apical Na permeability of the toad urinary bladder by intracellular Na,Ca, and H

Summary The Na conductance of the apical membrane of the toad urinary bladder was measured at different concentrations of Na both in the external medium and in the cell. Bladders were bathed in high K-sucrose medium to reduce basal-lateral resistance and voltage, and the transepithelial currents measured under voltage-clamp conditions. Amiloride was used as a specific blocker of the apical Na channel. At constant external Na, the internal Na concentration was increased by blocking the basallateral Na pump with ouabain. With high Na activity in the mucosal medium (86mm), increases in intracellular Na activity from 10 to over 40mm increased the amiloride-sensitive slope conductance at zero voltage while apical Na permeability, estimated from current-voltage plots using the constant field equation, decreased by less than 20%. Lowering the serosal Ca concentration from 1 to 0.1mm had no effect on the change inP _Na with increasing Na_c, but increasing serosal Ca to 5mm enhanced the reduction inP _Na with increasing Na _c , presumably by increasing Ca influx into the cell.P _Na was also reduced by serosal vanadate (0.5mm), a putative blocker of ATP-dependent Ca extrusion from the cell, and by acute exposure to CO₂, which presumably acidifies the cytoplasm. Current-voltage relationships of the amiloridesensitive transport pathway were also measured in the absence of a Na gradient across the apical membrane. These plots show that outward current passes through the channels somewhat less easily than does inward current. The shape of theI-V relationships was not significantly altered by changes in cellular Na, Ca or H, indicating that the effects of these ions onP _Na are voltage independent.     

The low molecular weight inhibitor of NCX1 interacts with a cytosolic domain that differs from the ion-transport site of the Na/Ca exchanger

The endogenous inhibitory factor (NCX(IF)) of the cardiac Na/Ca exchanger (NCX1) is a low molecular weight substance, which has a strong capacity to modulate the ventricle muscle contractility. Previously, we have shown that NCX(IF) can completely inhibit either the forward (Na(i)-dependent Ca-uptake) or reverse (Na(o)-dependent Ca-release) mode of Na/Ca exchange as well as its partial reaction, the Ca/Ca exchange. Although the preliminary studies have shown that NCX(IF) can rapidly (within few milliseconds) interact with a putative inhibitory site of the Na/Ca exchanger protein (or within its vicinity), it was not clear whether the NCX(IF) can directly interact with the ion transport sites of the exchanger protein or the interaction site of NCX(IF) is distinct from the ion-binding/transport site of NCX1. In order to segregate between these possibilities the NCX(IF) was tested for its capacity to compete with Ca at the cytosolic side by using the preparation of sarcolemma vesicles having predominantly the inside-out orientation. For this goal, the initial rates of Na(i)-dependent (45)Ca-uptake were measured in the presence of extravesicular (cytosolic) NCX(IF) under conditions in which the concentration of extravesicular Ca was varied (2-200 microM) and intravesicular Na was kept fixed at saturating concentration (160 mM). Under these conditions the NCX(IF) results in several fold decrease in V(max) values, while having no significant effect on the K(m). Taking into account the molecular weight of 350-550 Da (derived from the gel-filtration and mass-spectra data), the experimentally measured inhibitory potency of NCX(IF) can be estimated as the IC(50) = 0.3-0.6 microM. Therefore, it is concluded that the NCX(IF) is reasonably potent blocker, which interacts with cytosolic domain thereby preventing the ion-translocation (and not ion-binding) events.