Intravesicular Acidification Research Articles

Intravesicular Acidification Regulates Lipopolysaccharide Inflammation and Tolerance through TLR4 Trafficking

TLRs recognize pathogen components and drive innate immune responses. They localize at either the plasma membrane or intracellular vesicles such as endosomes and lysosomes, and proper cellular localization is important for their ligand recognition and initiation of signaling. In this study, we disrupted ATP6V0D2, a component of vacuolar-type H+ adenosine triphosphatase (V-ATPase) that plays a central role in acidification of intracellular vesicles, in a macrophage cell line. ATP6V0D2-deficient cells exhibited reduced cytokine production in response to endosome-localized, nucleic acid-sensing TLR3, TLR7, and TLR9, but enhanced inflammatory cytokine production and NF-κB activation following stimulation with LPS, a TLR4 agonist. Moreover, they had defects in internalization of cell surface TLR4 and exhibited enhanced inflammatory cytokine production after repeated LPS stimulation, thereby failing to induce LPS tolerance. A component of the V-ATPase complex interacted with ARF6, the small GTPase known to regulate TLR4 internalization, and ARF6 deficiency resulted in prolonged TLR4 expression on the cell surface. Taken together, these findings suggest that ATP6V0D2-dependent intravesicular acidification is required for TLR4 internalization, which is associated with prevention from excessive LPS-triggered inflammation and induction of tolerance.

Advances in Use of Capsule-Based Fluorescent Sensors for Measuring Acidification of Endocytic Compartments in Cells with Altered Expression of V-ATPase Subunit V1G1.

Acidification of eukaryotic cell compartments is accomplished by vacuolar H+-ATPases (V-ATPases), large multisubunit complexes able to pump protons into the lumen of organelles or in the extracellular medium. V-ATPases are involved in a number of physiological cellular processes, and thus regulation of V-ATPase activity is of crucial importance for the cell. Indeed, dysfunction of V-ATPase or alterations of acidification have been recently recognized as key factors in a variety of human diseases. In this study, we applied capsule-based pH sensors and a real-time tracking method for investigating the role of the V1G1 subunit of V-ATPases in regulating the activity of the proton pump. We first constructed stable cell lines overexpressing or silencing the subunit V1G1. Second, we used fluorescent capsule-based pH sensors to monitor acidification before and during internalization by modified and control living cells. By using a simple real-time method for tracking capsule internalization, we were able to identify different capsule acidification levels with respect to each analyzed cell and to establish the kinetics for each. The intracellular pH measurements indicate a delay in acidification in either V1G1-overexpressing or V1G1-silenced cells compared to controls. Finally, in an independent set of experiments, we applied transmission electron microscopy and confocal fluorescence microscopy to further investigate the internalization of the capsules. Both analyses confirm that capsules are engulfed in acidic vesicular structures in modified and control cell lines. The use of capsule-based pH sensors allowed demonstration of the importance of the V1G1 subunit in V-ATPase activity concerning intravesicular acidification. We believe that the combined use of these pH-sensor system and such a real-time method for tracking their internalization path would contribute to systematically measure the proton concentration changes inside the endocytic compartments in various cell systems. This approach would provide fundamental information regarding molecular mechanisms and factors that regulate intracellular acidification, vesicular trafficking, and cytoskeletal reorganizations.

Characterization of V-ATPase inhibitor-induced secretion of cysteine-rich with EGF-like domains 2

We previously demonstrated that cysteine-rich with EGF-like domains 2 (CRELD2), a novel ER stress-inducible factor, is a secretory glycoprotein; however, the stimuli that induce CRELD2 secretion have not yet been characterized. In this study, we found that the perturbation of intravesicular acidification of cytoplasmic organelles in HEK293 cells stably expressing wild-type (wt) CRELD2 induced its secretion. In particular, Concanamycin A (CMA) and Bafilomycin A1 (Baf), inhibitors of vacuolar ATPase (V-ATPase), increased the secretion of CRELD2 without relying on its C-terminal structure. The levels of secretion of EGFP-fused CRELD2 (SP-EGFP-CRELD2), which consists of EGFP following the putative signal peptide (SP) sequence of CRELD2, from COS7 cells transiently transfected with this construct were also increased after each of the treatments, but their intracellular localization was barely affected by CMA treatment. Transient overexpression of 78-kDa glucose-regulated protein (GRP78) and protein disulfide isomerase (PDI) also increased the secretion of CRELD2 from HEK293 cells expressing wt CRELD2, whereas the perturbation of intravesicular acidification did not alter the expression of GRP78 and PDI in the HEK293 cells. We further studied the roles of intracellular calcium ions and the Golgi apparatus in the secretion of CRELD2 from HEK293 cells in which intravesicular acidification was perturbed. The treatment with calcium ionophore increased the secretion of wt CRELD2, while that with BAPTA-AM, an intracellular calcium chelator, did not reduce the CMA-induced CRELD2 secretion. By contrast, treatment with brefeldin A (BFA), which inhibits the transportation of proteins from the ER to the Golgi apparatus, almost completely abolished the secretion of wt CRELD2 from the HEK293 cells. In conclusion, we demonstrated that the intravesicular acidification by V-ATPase regulates the secretion of CRELD2 without relying on the balance of intracellular calcium ions and the expression of ER chaperones such as GRP78 and PDI. These findings concerning the role of V-ATPases in modulating the secretion of CRELD2, a novel ER stress-inducible secretory factor, may provide new insights into the prevention and treatment of certain ER stress-related diseases.

The V-ATPase a2-subunit as a putative endosomal pH-sensor

V-ATPase (vesicular H(+)-ATPase)-driven intravesicular acidification is crucial for vesicular trafficking. Defects in vesicular acidification and trafficking have recently been recognized as essential determinants of various human diseases. An important role of endosomal acidification in receptor-ligand dissociation and in activation of lysosomal hydrolytic enzymes is well established. However, the molecular mechanisms by which luminal pH information is transmitted to the cytosolic small GTPases that control trafficking events such as budding, coat formation and fusion are unknown. Here, we discuss our recent discovery that endosomal V-ATPase is a pH-sensor regulating the degradative pathway. According to our model, V-ATPase is responsible for: (i) the generation of a pH gradient between vesicular membranes; (ii) sensing of intravesicular pH; and (iii) transmitting this information to the cytosolic side of the membrane. We also propose the hypothetical molecular mechanism involved in function of the V-ATPase a2-subunit as a putative pH-sensor. Based on extensive experimental evidence on the crucial role of histidine residues in the function of PSPs (pH-sensing proteins) in eukaryotic cells, we hypothesize that pH-sensitive histidine residues within the intra-endosomal loops and/or C-terminal luminal tail of the a2-subunit could also be involved in the pH-sensing function of V-ATPase. However, in order to identify putative pH-sensitive histidine residues and to test this hypothesis, it is absolutely essential that we increase our understanding of the folding and transmembrane topology of the a-subunit isoforms of V-ATPase. Thus the crucial role of intra-endosomal histidine residues in pH-dependent conformational changes of the V-ATPase a2-isoform, its interaction with cytosolic small GTPases and ultimately in its acidification-dependent regulation of the endosomal/lysosomal protein degradative pathway remain to be determined.

Trafficking of Streptococcus uberis in bovine mammary epithelial cells

Results from our laboratory showed that Streptococcus uberis internalized bovine mammary epithelial cells by exploiting host cell cytoskeleton and signal transduction mechanisms. It was also shown that S. uberis survived intracellularly for up to 120 h and capable of transcytose bovine mammary epithelial cells. To define mechanisms and strategies used by S. uberis to move through host cells and survive intracellularly, internalization studies using specific inhibitors, double immunofluorescence labeling and confocal laser microscopy were conducted. When bovine mammary epithelial cells were treated with inhibitors of endocytic vesicle acidification, the number of intracellular S. uberis was similar to untreated controls. When selective inhibitors of lipid rafts/caveolae or receptor-mediated endocytosis were used, a significantly lower number of intracellular S. uberis was detected compared with untreated controls. However, when the effect of inhibitors of receptor-mediated endocytosis and lipid rafts/caveolae were compared, the latter induced the lowest S. uberis internalization values suggesting a preferential exploitation of caveolae-mediated endocytosis. Since caveloae-dependent intracellular trafficking does not include intravesicular acidification or lysosome fusion; these results suggest that by exploiting preferential intracellular trafficking pathways in bovine mammary epithelial cells, S. uberis avoids intracellular bactericidal mechanisms. Such a strategy would allow S. uberis to persist intracellularly and may explain how persistent intramammary infections occur.

Plasma membrane of Beta vulgaris storage root shows high water channel activity regulated by cytoplasmic pH and a dual range of calcium concentrations

Plasma membrane vesicles isolated by two-phase partitioning from the storage root of Beta vulgaris show atypically high water permeability that is equivalent only to those reported for active aquaporins in tonoplast or animal red cells (Pf=542 microm s(-1)). The values were determined from the shrinking kinetics measured by stopped-flow light scattering. This high Pf was only partially inhibited by mercury (HgCl2) but showed low activation energy (Ea) consistent with water permeation through water channels. To study short-term regulation of water transport that could be the result of channel gating, the effects of pH, divalent cations, and protection against dephosphorylation were tested. The high Pf observed at pH 8.3 was dramatically reduced by medium acidification. Moreover, intra-vesicular acidification (corresponding to the cytoplasmic face of the membrane) shut down the aquaporins. De-phosphorylation was discounted as a regulatory mechanism in this preparation. On the other hand, among divalent cations, only calcium showed a clear effect on aquaporin activity, with two distinct ranges of sensitivity to free Ca2+ concentration (pCa 8 and pCa 4). Since the normal cytoplasmic free Ca2+ sits between these ranges it allows for the possibility of changes in Ca2+ to finely up- or down-regulate water channel activity. The calcium effect is predominantly on the cytoplasmic face, and inhibition corresponds to an increase in the activation energy for water transport. In conclusion, these findings establish both cytoplasmic pH and Ca2+ as important regulatory factors involved in aquaporin gating.

Blue Native/SDS-PAGE Analysis Reveals Reduced Expression of the mClCA3 Protein in Cystic Fibrosis Knock-out Mice

Cystic fibrosis (CF) is a frequent autosomal recessive disorder caused by mutation of a gene encoding a multifunctional transmembrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), located in the apical membrane of epithelial cells lining exocrine glands. In an attempt to get a more complete picture of the pleiotropic effects of the CFTR defect on epithelial cells and particularly on the membrane compartment, a bidimensional blue native (BN)/SDS-PAGE-based proteomic approach was used on colonic crypt samples from control and CFTR knock-out mice (cftr-/-). This approach overcomes the difficulties of membrane protein analysis by conventional two-dimensional PAGE and is able to resolve multiprotein complexes. Used here for the first time on crude membrane proteins that were extracted from murine colonic crypts, BN/SDS-PAGE allows effective separation of protein species and complexes of various origins, including mitochondria, plasma membrane, and intracellular compartments. The major statistically significant difference in protein maps obtained with samples from control and cftr-/- mice was unambiguously identified as mClCA3, a member of a family of calcium-activated chloride channels considered to be key molecules in mucus secretion by goblet cells. On the basis of this finding, we evaluated the overall expression and localization of mClCA3 in the colonic epithelium and in the lung of mice by immunoblot analysis and immunohistochemistry. We found that mClCA3 expression was significantly decreased in the colon and lung of the cftr-/- mice. In an ex vivo assay, we found that the Ca2+-dependent (carbachol-stimulated) glycoprotein secretion strongly inhibited by the calcium-activated chloride channel blocker niflumic acid (100 microm) was impaired in the distal colon of cftr-/- mice. These results support the conclusion that a ClCA-related function in the CF colon depends on CFTR expression and may be correlated with the impaired expression of mClCA3.

Chloride channels as tools for developing selective insecticides.

Ligand-gated chloride channels underlie inhibition in excitable membranes and are proven target sites for insecticides. The gamma-aminobutyric acid (GABA(1)) receptor/chloride ionophore complex is the primary site of action for a number of currently used insecticides, such as lindane, endosulfan, and fipronil. These compounds act as antagonists by stabilizing nonconducting conformations of the chloride channel. Blockage of the GABA-gated chloride channel reduces neuronal inhibition, which leads to hyperexcitation of the central nervous system, convulsions, and death. We recently investigated the mode of action of the silphinenes, plant-derived natural compounds that structurally resemble picrotoxinin. These materials antagonize the action of GABA on insect neurons and block GABA-mediated chloride uptake into mouse brain synaptoneurosomes in a noncompetitive manner. In mammals, avermectins have a blocking action on the GABA-gated chloride channel consistent with a coarse tremor, whereas at longer times and higher concentrations, activation of the channel suppresses neuronal activity. Invertebrates display ataxia, paralysis, and death as the predominant signs of poisoning, with a glutamate-gated chloride channel playing a major role. Additional target sites for the avermectins or other chloride channel-directed compounds might include receptors gated by histamine, serotonin, or acetylcholine.The voltage-sensitive chloride channels form another large gene family of chloride channels. Voltage-dependent chloride channels are involved in a number of physiological processes including: maintenance of electrical excitability, chloride ion secretion and resorption, intravesicular acidification, and cell volume regulation. A subset of these channels is affected by convulsants and insecticides in mammals, although the role they play in acute lethality in insects is unclear. Given the wide range of functions that they mediate, these channels are also potential targets for insecticide development.

Comparative characterization of the two primary pumps, H+‐ATPase and Na+‐ATPase, in the plasma membrane of the marine alga Tetraselmis viridis

The transport characteristics of the plasma membrane H+‐ATPase (PMHA) and Na+‐ATPase (PMNA) from marine unicellular green alga Tetraselmis viridis Rouch. were studied using sealed plasma membrane vesicles isolated from this species. The activities of the ATPases were investigated by monitoring the ATP‐dependent pH changes in the vesicle lumen. PMHA operation led to acidification of the vesicle lumen, whereas Na+ translocation into plasma membrane vesicles catalysed by PMNA was accompanied by H+ efflux, namely the alkalization of the vesicle lumen (Balnokin et al., FEBS Lett 462: 402–406, 1999). The intravesicular acidification and alkalization were detected with the ΔpH probe acridine orange and the pH probe pyranine, respectively. PMHA and PMNA were found to operate in distinct pH regions, maximal activity of PMHA being observed at pH 6.5 and that of PMNA at pH 7.8. Kinetic studies revealed that the ATPases have similar affinities to their primary substrate, MgATP complex (an apparent Km = 34 ± 6.2 µM for PMHA and 73 ± 8.7 µM for PMNA). At the same time, the ATPases were differently affected by free Mg2+ and ATP. Free Mg2+ appeared to be a mixed‐type inhibitor for PMNA (Ki′ = 210 µM) but it did not suppress PMHA. Conversely, free ATP markedly suppressed PMHA being a mixed‐type inhibitor (Ki′ = 330 µM), but PMNA was affected by free ATP only slightly. Furthermore, the ATPases substantially differed in their sensitivities to the inhibitors of membrane ATPases, such as orthovanadate, N‐ethylmaleimide and N,N′‐dicyclohexylcarbodiimide. The differences found in the properties of the PMHA and PMNA are discussed in terms of regulation of their activities and their capacity to be involved in cytosolic ion homeostasis in T. viridis cells.

Characterisation of intestinal peptide transporter of the Antarctic haemoglobinless teleost Chionodraco hamatus.

H(+)/peptide cotransport was studied in brush-border membrane vesicles (BBMV) from the intestine of the haemoglobinless Antarctic teleost Chionodraco hamatus by monitoring peptide-dependent intravesicular acidification with the pH-sensitive dye Acridine Orange. Diethylpyrocarbonate-inhibited intravesicular acidification was specifically achieved in the presence of extravesicular glycyl-L-proline (Gly-L-Pro) as well as of glycyl-L-alanine (Gly-L-Ala) and D-phenylalanyl-L-alanine (D-Phe-L-Ala). H(+)/Gly-L-Pro cotransport displayed saturable kinetics, involving a single carrier system with an apparent substrate affinity (K(m,app)) of 0.806+/-0.161 mmol l(-1). Using degenerated primers from eel and human (PepT1) transporter sequence, a reverse transcription-polymerase chain reaction (RT-PCR) signal was detected in C. hamatus intestine. RT-PCR paralleled kinetic analysis, confirming the hypothesis of the existence of a PepT1-type transport system in the brush-border membranes of icefish intestine. Functional expression of H(+)/peptide cotransport was successfully performed in Xenopus laevis oocytes after injection of poly(A)(+) RNA (mRNA) isolated from icefish intestinal mucosa. Injection of mRNA stimulated D-Phe-L-Ala uptake in a dose-dependent manner and an excess of glycyl-L-glutamine inhibited this transport. H(+)/peptide cotransport in the Antarctic teleost BBMV exhibited a marked difference in temperature optimum with respect to the temperate teleost Anguilla anguilla, the maximal activity rate occurring at approximately 0 degrees C for the former and 25 degrees C for the latter. Temperature dependence of icefish and eel intestinal mRNA-stimulated uptake in the heterologous system (oocytes) was comparable.

Evidence against aquaporin‐1‐dependent CO2 permeability in lung and kidney

AQP1-dependent CO2 transport has been suggested from the increased CO2 permeability in Xenopus oocytes expressing AQP1. Potential implications of this finding include AQP1-facilitated CO2 exchange in mammalian lung and HCO3-/CO2 transport in kidney proximal tubule. We reported previously that: (a) CO2 permeability in erythrocytes was not affected by AQP1 deletion, (b) CO2 permeability in liposomes was not affected by AQP1 reconstitution despite a 100-fold increased water permeability, and (c) CO2 blow-off by the lung in living mice was not impaired by AQP1 deletion. We extend these observations by direct measurement of CO2 permeabilities in lung and kidney. CO2 transport across the air-space-capillary barrier in isolated perfused lungs was measured from changes in air-space fluid pH in response to addition/removal of HCO3-/CO2 from the pulmonary artery perfusate. The pH was measured by pleural surface fluorescence of a pH indicator (BCECF-dextran) in the air-space fluid. Air-space fluid pH equilibrated rapidly (t(1/2) approximately 6 s) in response to addition/removal of HCO3-/CO2. However, the kinetics of pH change was not different in lungs of mice lacking AQP1, AQP5 or AQP1/AQP5 together, despite an up to 30-fold reduction in water permeability. CO2 transport across BCECF-loaded apical membrane vesicles from kidney proximal tubule was measured from the kinetics of intravesicular acidification in response to rapid mixing with a HCO3-/CO2 solution. Vesicles rapidly acidified (t(1/2) approximately 10 ms) in response to HCO3-/CO2 addition. However the acidification rate was not different in kidney vesicles from AQP1-null mice despite a 20-fold reduction in water permeability. The results provide direct evidence against physiologically significant transport of CO2 by AQP1 in mammalian lung and kidney.

Endocytosed SP-A and surfactant lipids are sorted to different organelles in rat type II pneumocytes.

Intracellular transport of endocytosed surfactant protein A (SP-A) and lipid was investigated in isolated rat type II cells. After internalization, SP-A and lipid are taken up via the coated-pit pathway and reside in a common compartment, positive for the early endosomal marker EEA1 but negative for the lamellar body marker 3C9. SP-A then recycles rapidly to the cell surface via Rab4-associated recycling vesicles. Internalized lipid is transported toward a Rab7-, CD63-, 3C9-positive compartment, i.e., lamellar bodies. Inhibition of calmodulin led to inhibition of uptake and transport out of the EEA1-positive endosome and thus of resecretion of both components. Inhibition of intravesicular acidification (bafilomycin A1) led to decreased uptake of both surfactant components. It inhibited transport out of early endosomes for lipid only, not for SP-A. We conclude that in type II cells, endocytosed SP-A and lipid are transported toward a common early endosomal compartment. Thereafter, both components dissociate. SP-A is rapidly recycled to the cell surface and does not enter classic lamellar bodies. Lipid is transported toward lamellar bodies.

Amine uptake and peptide hormone secretion: APUD cells in a new landscape.

APUD cells were so named by Pearse (1969) as a result of his attempt to produce a synthesis relating a number of observations concerning the histochemistry and function of peptide-secreting endocrine cells, particularly those of the gastrointestinal tract. One of these properties was that peptide-secreting endocrine cells appeared to share an ability to take up precursors of biologically active amines, to produce active amine through subsequent intracellular decarboxylation and then store the amine product in secretory vesicles, hence APUD (amine precursor uptake and decarboxylation). This widespread phenomenon was initially described by Pearse himself (1966) for the calcitonin-secreting parafollicular C cells of dog thyroid using 5-hydroxytryptophan as the amine precursor. In that paper the author noted that such an uptake was also found in other peptide hormone-secreting cells, for example in the corticotrophs of the anterior pituitary gland and in the β-cells of the endocrine pancreas. However, these clear-cut observations became hidden beneath the much larger edifice that the APUD concept became, with APUD cells (eventually including more than 40 cell types) defined as a ‘diffuse neuroendocrine system … the third branch of the nervous system acting within the second, the autonomic nervous system in the control of all the intestinal organs’ and with a developmental lineage that was defined as necessarily being from neuroectoderm. This monumental hypothesis has not stood the rigour of experimental test using modern methods of cell and developmental biology. However, if the grand theory itself has been found wanting (see for example Andrew et al. 1998), the original observations that led to the idea of the APUD cell have certainly been confirmed and yet have remained neglected for much of the last decade. Why should cells that are undoubtedly peptide-secreting endocrine cells have the general property of handling amines in a way characteristic of neurones? In this issue of The Journal of Physiology, Blackmore et al. shed a fascinating new perspective on these early findings by showing that functionally the observed pattern of secreted peptide hormones (which emerge as processed fragments from larger polypeptide precursors) results from the ability of these cells to take up amines. The connection arises because of two phenomena which share a common molecular event - in one case the result of, in the other the cause of the link. Because peptide hormone processing is carried out by intracellular endopeptidases within secretory vesicles and because the enzymes responsible for site-specific activity (the pro-hormone convertases) are themselves highly pH sensitive, the pattern of peptide release will necessarily depend upon secretory vesicular pH. Blackmore et al. (2001) have now shown that the pH within this organelle is itself regulated by monoamines through a simple link. Amine transport across the vesicular membrane occurs through transporters that are proton-coupled antiporters. Hence addition of amine to the cell, by activating amine transport and accumulation within the vesicles reduces intravesicular acidification; this alters the processing of secreted peptide, thus establishing the link between amine uptake and peptide hormone secretion. The conclusion from this study is so unambiguous because of the imaginative combination of modern experimental strategies adopted by the authors. Using confocal microscopy to measure intravesicular pH (with a pH-sensitive green fluorescent protein targeted to secretory vesicles) in cells in situ and pharmacological tools to isolate the contribution of specific vesicular monoamine transporters, the authors make a combined attack using a genetically engineered cell line. A pancreatic β-cell line (a nice touch here is that the authors manage to acknowledge materials received from both Drs Banting and Best!) was stably transfected with cDNA constructs expressing both the vesicular monoamine transporter VMAT2 and the peptide hormone precursor progastrin, a peptide that is well known to be cleaved into a number of specific smaller fragments (e.g. G34 and G17) with differing biological effects and properties depending on the specific product released. Moreover gastrin is a peptide for which cleavage-product-specific antibodies have been developed, allowing peptide processing as a function of amine transport-dependent intravesicular pH to be monitored. The results are clear-cut: expression of the vesicular monoamine transporter alters peptide processing in these cells and the mechanism for this coupling does indeed appear to be altered intravesicular pH. Wilmer (1960) observed in his classic monograph Cytology and Evolution that ‘… in its evolutionary processes Nature tends to build on what is already present and does so by a continual process of modification. A plurality of purpose may arise as a consequence of some basic embryological processes which probably have an earlier and originally a functional origin' or as Jacob (see Crick, 1988) put it more succinctly, ‘evolution is a tinkerer’. The paper by Blackmore et al. (2001) exemplifies this particularly clearly by showing how a novel process linking neuronal and endocrine cell function emerges from such evolutionary tinkering with the cellular expression and properties of an intracellular vesicular monoamine transporter.

Characterisation of the H(+)/peptide cotransporter of eel intestinal brush-border membranes.

H(+)/peptide cotransport in brush-border membrane vesicles (BBMVs) from eel (Anguilla anguilla) intestine was studied by measuring d-[(3)H]-phenylalanyl-l-alanine uptake and by monitoring peptide-dependent intravesicular acidification using the pH-sensitive dye Acridine Orange. d-[(3)H]-phenylalanyl-l-alanine influx was greatly stimulated by an inside-negative membrane potential and enhanced by an inwardly directed H(+) gradient. In parallel, vesicular H(+) influx was significantly increased in the presence of extravesicular d-phenylalanyl-l-alanine or a series of glycyl and l-prolyl peptides. H(+)/peptide cotransport displayed saturable kinetics involving a single carrier system with apparent substrate affinities of 0.9-2.6 mmol l(-1) depending on the particular peptide. All substrates tested competed with this system. Pre-incubation of BBMVs with dipeptides prevented diethylpyrocarbonate inhibition of transport activity, suggesting that the substrates mask histidine residues involved in the catalytic function of the transporter. Using human PepT1-specific primers, a reverse transcription-polymerase chain reaction (RT-PCR) signal was detected in eel intestine. Our results suggest that, in eel intestine, a brush-border membrane 'low-affinity'-type H(+)/peptide cotransport system is present that shares kinetic features with the mammalian intestinal PepT1-type transporters.

Effects of Ethanol on Receptor-Mediated Endocytosis in the Liver

M cVICKER, B. L. AND C. A. CASEY. Effects of ethanol on receptor-mediated endocytosis in the liver. ALCOHOL 19(3) 255–260, 1999.—Ethanol administration impairs multiple aspects in the process of receptor-mediated endocytosis (RME) in the liver. Studies from our laboratory over the last 10 years have carefully examined RME by the hepatocyte-specific asialoglycoprotein receptor (ASGP-R). We have identified a time course for ethanol-induced defects in RME and established that many of the impairments occur initially in the centrilobular region of the liver and as early as one week after ethanol administration. Impaired intravesicular acidification in ethanol-fed animals has been identified, and these defects in acidification could alter multiple protein trafficking pathways including RME. In addition to altered acidification, altered receptor function (including receptor inactivation) could also contribute to impaired trafficking. Current studies in our laboratory are aimed at an examination of posttranslational modifications in the receptor (acylation and phosphorylation) that are known to affect its function. A role for the ASGP-R in the process of alcoholic apoptosis is also being examined because proper functioning of the ASGP-R is thought to be important in clearance of apoptotic cells.

Cadmium inhibits vacuolar H+-ATPase and endocytosis in rat kidney cortex

The mechanism of cadmium (Cd)-induced damage in the mammalian proximal tubule that is manifested by defects in reabsorption of various compounds, is poorly understood. A vacuolar H(+)-ATPase (V-ATPase) in proximal tubule (PT) brush border and intracellular vesicles may be affected by Cd, and this may influence intracellular vesicle trafficking and reabsorption of the filtered proteins. We studied the effects of Cd on V-ATPase and endocytosis in rat renal PT in vivo and on acidification mechanisms in isolated renal cortical organelles in vitro. The V-ATPase activity in brush border membrane (BBM) from Cd-intoxicated rats was 40% lower compared to that in control animals. Immunofluorescence studies in cortical tissue sections and Western blot studies in BBM from Cd-treated rats showed a strongly decreased abundance of the 31 kDa and 70 kDa V-ATPase subunits. Functional studies in vivo showed a dramatically diminished endocytosis of fluorescein-labeled dextran in PT cells from Cd-treated animals, whereas morphological studies revealed a loss of endocytic invaginations and subapical vesicles in the same cells. In studies in vitro, Cd inhibited V-ATPase activity in a concentration- and time-dependent manner in both BBM and endocytic vesicles, whereas in endocytic vesicles, Cd inhibited ATP-driven intravesicular acidification and accelerated the dissipation of transmembrane pH gradients. We conclude that Cd may impair acidification in cell organelles by (a) causing a loss of V-ATPase protein in their limiting membranes, (b) inhibiting the intrinsic V-ATPase activity, and (c) dissipating the transmembrane pH gradient. This may inhibit endocytosis of filtered proteins and impair vesicle-mediated recycling of some membrane transporters, thus contributing to the loss of reabsorptive capacity of the PT.

Direct Measurement of ATP-Dependent Proton Concentration Changes and Characterization of a K+-Stimulated ATPase in Pea Chloroplast Inner Envelope Vesicles.

Inner envelope membrane vesicles prepared from pea (Pisum sativum L. var Laxton's Progress No. 9) chloroplasts have K+-stimulated ATPase activity with a pH optimum of 8.4. ATP addition to inner envelope vesicles loaded with pyranine caused a decrease in pyranine fluorescence that was consistent with internal acidification. The transmembrane pH change induced by the addition of 5 mM ATP was about 0.4 unit. Measurement of phosphate released by ATP hydrolysis paralleled the pH change, indicating that intravesicular acidification was linked to ATPase activity. Vanadate, molybdate, N-ethylmaleimide, and dithiothreitol inhibited ATP-dependent vesicle acidification completely, whereas ATPase activity was only partially inhibited. These data indicate that pea chloroplast inner envelope vesicles contain a proton translocating ATPase and that the pyranine-loading method can be utilized to study directly ATP-dependent H+ transport across these membranes.

ATP-dependent transport of tetraethylammonium by endosomes isolated from rat renal cortex

During renal organic cation secretion by some species, intracellular concentrations greatly exceed the 10- to 15-fold ratio predicted by the potential-driven mechanism thought to mediate their basolateral uptake. Free cytoplasmic organic cation concentrations within the tubular cells might be decreased through sequestration within intracellular organelles. The data reported here show that endosomal vesicles isolated from rat renal cortex take up tetraethylammonium (TEA) by an ATP-dependent mechanism. Addition of 0.2-5 mM ATP to the medium stimulated uptake 5- to 10-fold at 5 min and 20-fold at 60 min. More than 80% of the ATP-dependent uptake was associated with an osmotically active space. The nonhydrolyzable ATP analogue, adenosine 5'-O-(3-thiotriphosphate), did not stimulate TEA uptake. Mg2+ and Cl- were required for stimulation. Uptake was inhibited by several organic cations, including TEA itself. Uptake was also inhibited by inhibitors of intravesicular acidification, e.g., monensin and N-ethylmaleimide. Furthermore, the ATP requirement could be bypassed by establishing a pH gradient (inside acidic). These data show that endosomal TEA accumulation is mediated by proton/TEA exchange and is driven by the pH gradient maintained by H(+)-adenosinetriphosphatase. This potent sequestration mechanism may play an important role in organic cation secretion.

H(+)-ATPases of renal cortical and medullary endosomes are differentially sensitive to Sch-28080 and omeprazole

Adenosinetriphosphatase (ATPase) activity stimulated by K+ and inhibited by Sch-28080 (SCH), omeprazole (OME), and vanadate has been measured in microsomes from mammalian renal medulla and attributed to a kidney isoform of the H(+)-K(+)-ATPase. To determine whether the H(+)-K(+)-ATPase inhibitors could also inhibit the vacuolar (V)-type H(+)-adenosinetriphosphatase (H(+)-ATPase, i.e., H+ pump) in mammalian intracellular vesicles, we examined their effects on bafilomycin-sensitive acidification in renal cortical vesicles (CEV) and medullary endocytic vesicles (MEV). Rats were injected with fluorescein isothiocyanate-labeled dextran, and labeled endosomes were enriched from kidney tissue homogenates by differential and Percoll density gradient centrifugation. In the CEV, the V-type H+ pump was inhibited 25% by SCH and 30% by OME (100 microM each). Whereas the inhibition by OME was concentration and time dependent, the inhibition by SCH was only concentration dependent. Inhibition by these compounds was similar in the presence of 50 mM K+ (in = out) and in the complete absence of K+, thus ruling out a significant involvement of H(+)-K(+)-ATPase-mediated acidification. Inhibition, however, was not observed with 10 microM SCH and OME. The sensitivity of the V-type H+ pump to 100 microM SCH and OME in CEV was confirmed by the comparable inhibitions of intravesicular acidification observed in acridine orange fluorescence quench studies and by inhibition of Pi liberation in an ATPase assay. We also found that the V-type H+ pump in isolated rat liver endosomes is sensitive to 100 microM SCH and OME to a similar degree. In the MEV, acidification was only weakly affected by 100 microM SCH and OME, thus suggesting that H(+)-ATPases in endosomes from cortical and medullary tubules are different, possibly due to a previously described selective expression of subunit isoforms. Our finding indicates the importance of using low concentrations (< 10 microM) of OME and SCH in studies of H(+)-K(+)-ATPase in nongastric tissues to avoid misinterpretation of the data due to nonspecific inhibition of V-type H(+)-ATPases.

Enzymatic and functional evidence for adaptation of the vacuolar H(+)-ATPase in proximal tubule apical membranes from rats with chronic metabolic acidosis.

The present work examined the effects of chronic metabolic acidosis on the vacuolar proton-translocating adenosine triphosphatase (H(+)-ATPase) activity both in rat renal cortical homogenates and in their luminal membranes. Moreover, to assess the effect of acidosis on H+ transport by the apical H(+)-ATPase, we have developed a detergent-dilution procedure, resulting in the formation of sealed vesicles having this enzyme at their external surface. NH4Cl loading for 4 days had no effect on homogenates H(+)-ATPase activity, estimated with either N-ethylmaleimide or bafilomycin A1. In contrast, H(+)-ATPase activities were increased significantly by about 30% in both native apical membranes prepared by Ca2+ aggregation and detergent-treated luminal vesicles from acidotic animal. Kinetic analysis revealed that this stimulation was solely through changes in the Vmax for ATP. In membranes prepared by Mg2+ aggregation, acidosis also caused significant stimulation of the H(+)-ATPase activity. In addition, the initial rate of ATP-induced intravesicular acidification was 25% higher in reoriented H(+)-ATPase vesicles from acidotic rats, whereas passive proton permeability was identical in both groups. Finally, both vesicle enrichments and yields of luminal markers and de-enrichments and yields of intracellular membrane markers were identical in the two groups. These results provide enzymatic and functional evidence suggesting that chronic acidosis induces an adaptative change in the rat brush border H(+)-ATPase.