pH Compartment Research Articles

Acidification of lysosomes and endosomes.

Lysosomes, endosomes, and a variety of other intracellular organelles are acidified by a family of unique proton pumps, termed the vacuolar H(+)-ATPases, that are evolutionarily related to bacterial membrane proton pumps and the F1-F0 H(+)-ATPases that catalyze ATP synthesis in mitochondria and chloroplasts. The electrogenic vacuolar H(+)-ATPase is responsible for generating electrical and chemical gradients across organelle membranes with the magnitude of these gradients ultimately determined by both proton pump regulatory mechanisms and, more importantly, associated ion and organic solute transporters located in vesicle membranes. Analogous to Na+, K(+)-ATPase on the cell membrane, the vacuolar proton pump not only acidifies the vesicle interior but provides a potential energy source for driving a variety of coupled transporters, many of them unique to specific organelles. Although the basic mechanism for organelle acidification is now well understood, it is already apparent that there are many differences in both the function of the proton pump and the associated transporters in different organelles and different cell types. These differences and their physiologic and pathophysiologic implications are exciting areas for future investigation.

Relationship between pH and the availability of dissolved inorganic nitrogen in the zooxanthella-giant clam symbiosis

Giant clams of the family Tridacnidae live in symbiosis with intercellular dinoflagellates of the genus Symbiodinium, often referred to by the trivial name “zooxanthellae.” Intact clams take up both ammonium and nitrate when they are offered separately. When zooxanthellae were removed from the mantle and incubated in hemolymph, they took up ammonium, and they took up nitrate when ammonium concentrations were low enough (< 1 PM) not to inhibit uptake. The pH of hemolymph collected from the venous sinuses (immediately below the mantle tissue where most of the zooxanthellae reside) varied on a diel cycle, with highest values (-8.1) measured during the middle of the day and lowest (-7.2) detected at night. Zooxanthellae are probably responsible for the alkalization of the hemolymph since the increase in pH is coincident with maximum light levels and photosynthesis. Natural concentrations of ammonium in the hemolymph of freshly collected clams were inversely correlated with pH, with highest values detected at night when pH was lowest. Rates of diffusion of ammonia from seawater (pH 8.1) into animal compartments of lower pH were proportional to the pH differential. Diffusion of ammonia over a pH gradient, with diel variation in pH, may act to regulate availability of nitrogen to the algal symbionts in all zooxanthellae symbioses, including reef corals, thereby controlling the timing and rate of algal division.

The potent mitogen Pasteurella multocida toxin is highly resistant to proteolysis but becomes susceptible at lysosomal pH

The susceptibility of the potent mitogen Pasteurella multocida toxin (PMT) to various proteases was investigated. PMT at a toxin to protease molar ratio of 1:1 was resistant to 8 of the 11 proteases tested after one hour. With longer incubation, PMT remained resistant to 7 proteases, and this correlated with a retention of biological activity, indicating that PMT might not require proteolytic cleavage at least until it bound to a cell receptor. Previous evidence had suggested that PMT is processed in the cell via an endosome or lysosome. We have shown that PMT became susceptible to proteolysis when the pH was lowered to 5 or below. This supports the previous suggestion that PMT is processed via a low pH compartment in the cell.

Transferrin is made and bound by photoreceptor cells

Retinal pigment epithelial cells, which form one aspect of the blood-retinal barrier, control the access of blood-borne components such as diferric transferrin to the neural retina. It has recently been shown that RPE cells remove iron from diferric transferrin in a low pH compartment and subsequently release it in a low molecular weight form that can be chelated by apo-transferrin (Hunt and Davis: J. Cell Physiol. 152:102-110, 1992). It is now shown that photoreceptor cells can bind diferric transferrin to receptors on their inner segments. Moreover, polymerase chain reaction and in situ hybridization show that cells of the neural retina, particularly photoreceptors, make apo-transferrin.

Predicting the behaviour and selectivity of fluorescent probes for lysosomes and related structures by means of structure-activity models

Cultured rat fibroblasts were exposed to 50 fluorescent probes of varied physicochemical characteristics. Probe concentrations, fluorochrome excitation wavelength and period of illumination, and cell-probe contact time were varied. Structure-activity relationships defining a number of classes of fluorescent probes for lysosomes and related processes and compartments were demonstrated. Numerical specifications are now available for several familiar classes of probes: (a) acidotropic weak bases, used as markers for low pH compartments; (b) markers of adsorptive pinocytosis, involving non-specific protein binding; (c) markers for fluid phase pinocytosis; and (d) viability stains involving intralysosomal enzymic activity. Two novel classes of probes have also been specified numerically: (a) acid-precipitated weak acids, as markers for low pH compartments; and (b) lipid-binding markers of adsorptive pinocytosis. Overall, these structure-activity models provide a tool for predicting whether or not compounds enter cells; and whether they accumulate in lysosomes and related compartments. Pathways of entry are also predicted. This tool should permit design and selection of improved probes, and provide a better understanding of existing reagents. Moreover these models are expected to be applicable to interactions between any non-polymeric xenobiotic with lysosomes and related compartments.

Transplacental distribution of weak acids in mice: Accumulation in compartments of high pH

The transplacental distribution of three weak acids was measured in mice on gestational day 11. The ratio of drug concentration in maternal, embryonic, and extraembryonic compartments compared to maternal plasma followed a consistent hierarchy such that amniotic fluid greater than or equal to exocoelomic fluid greater than embryo greater than embryo plasma greater than maternal skeletal muscle. This distribution pattern correlates well with pH in these compartments and suggests that pH gradients between compartments are an influential factor in determining weak acid drug disposition during pregnancy.

Uptake and concentration of bioactive macromolecules by K562 cells via the transferrin cycle utilizing an acid-labile transferrin conjugate

The transferrin cycle was used to attempt the import of bioactive macromolecules into cells with the aid of an acid-labile cross-linking agent. Anti-tetanus F(ab')2 fragments were iodinated and then conjugated to transferrin with a newly developed acid-labile cleavable cross-linking reagent, bismaleimidoethoxy propane, following thiolation of both proteins. Noncleavable conjugates were also prepared. At saturating conjugate concentrations, the uptake rate for both conjugates averaged over the first 2 h is about 6.5 fmol/million cells/min. Incubation of loaded cells in fresh medium for 30 min and analysis of cell pellets and supernatants reveal that 1) of the previously cell-associated label, only intact conjugate (about 50% of the label) is returned to the medium; 2) most of the remaining cell-associated material for the cleavable conjugate is chromatographically coincident with free Fab with some contribution from free F(ab')2 fragments. In contrast, the cell pellets loaded with noncleavable conjugates contained intact transferrin-F(ab'), conjugates. These results are consistent with transferrin receptor-mediated uptake of acid-labile conjugate followed by hydrolysis in acidified endosomes and resulting in concentration of free F(ab')2 and Fab within a prelysosomal intracellular compartment. A protein shuttle such as transferrin may therefore be used with ketal based acid-labile cross-linkers to load foreign molecules into an intracellular compartment. In addition, these data provide independent confirmation of the low pH compartment within the transferrin cycle. This new methodology is applicable to other cases of receptor/ligand trafficking to report low pH compartments independent of morphological analysis. Since transferrin receptors are overexpressed in tumors, antineoplastic agents could be targeted to tumors as transferrin acid-labile conjugates. This import system might be particularly useful in combatting the tumor cell export of antitumor agents occurring in multidrug resistance.

Distribution of endocytosed molecules to intracellular acidic environments correlates with immunotoxin activity

We have investigated the internalization to low pH intracellular compartments of transferrin (Tfn), diphtheria toxin (DT) and of anti-cell surface antibodies (MAb) by a cytofluorometric assay based on low pH quenching of fluorescein (FITC) emission. FITC-labelled Tfn, anti-CD3, anti-CD5 and anti-Thy 1.2 MAb internalization resulted in a progressively lower FITC quenching effect. Following internalization, a distinction could be made between molecules that enter low pH compartments without undergoing intracellular degradation (e.g., Tfn, anti-CD3 MAb) and molecules that are internalized through low pH organelles and are then degraded within the cell (e.g., DT). A strict correlation was observed between quenching of internalized FITC-protein fluorescent emission and the cytotoxic activity of DT-based immunotoxins (IT).

Measurement of intracellular pH in fungal hyphae using BCECF and digital imaging microscopy: Evidence for a primary proton pump in the plasmalemma of a marine fungus

ABSTRACT The facultative marine fungus, Dendryphiella salina, has the most negative membrane potential yet recorded for a marine organism. The ionic basis for this is thought to be through the action of a primary proton pump, though there exists the possibility of electrogenic pumping of Na+ or Cl−, given the high ambient concentration of these ions. Fluorescence ratio imaging microscopy with the pH-sensitive fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5(and-6) carboxyfluorescein (BCECF) has been used to estimate intracellular pH. Hyphae loaded readily with BCECF after incubation with the acetoxymethyl ester (BCECF/AM). Mean resting intracellular pH (pHi) was 7.3, calculated by comparing 490/450 nm fluorescence ratios with in vivo calibration curvesobtained by pH equilibration using nigericin. Distinct pH compartments could be observed, corresponding to cytoplasmic and smaller vacuolar compartments. Sodium azide reversibly reduced pHi by an average of 0.51 of a pH unit, though the response varied between individual hyphae. Inhibiting the plasmalemma ATPase with orthovanadate also reversibly decreased pHi. The results support the presence of a proton pump in the plasmamembrane. The energetic and evolutionary implications are discussed.

Biochemical and genetic analysis of the biosynthesis, sorting, and secretion of Dictyostelium lysosomal enzymes

Dictyostelium discoideum is a useful system to study the biosynthesis of lysosomal enzymes because of the relative ease with which it can be manipulated genetically and biochemically. Previous studies have revealed that lysosomal enzymes are synthesized in vegetatively growing amoebae as glycosylated precursor polypeptides that are phosphorylated and sulfated on their N-linked oligosaccharide side-chains upon arrival in the Golgi complex. The precursor polypeptides are membrane associated until they are proteolytically processed and deposited as soluble mature enzymes in lysosomes. In this paper we review biochemical experiments designed to determine the roles of post-translational modification, acidic pH compartments, and proteolytic processing in the transport and sorting of lysosomal enzymes. We also describe molecular genetic approaches that are being employed to study the biosynthesis of these enzymes. Mutants altered in the sorting and secretion of lysosomal enzymes are being analyzed biochemically, and we describe recent efforts to clone the genes coding for three lysosomal enzymes in order to better understand the molecular mechanisms involved in the targeting of these enzymes.

Manipulating the intracellular environment of hippocampal slices: pH and high-energy phosphates

The intracellular energetic environment of rat hippocampal slices was manipulated by bolstering ATP levels following the addition of adenosine to the incubation medium, or by manipulating intracellular pH. Addition of 8 mM adenosine to the incubation medium increased total tissue adenylate and ATP content, but did not prolong electrical function during anoxia. Further, it resulted in long-lasting alterations in normoxic evoked responses. Intracellular pH (pH i ) was changed by manipulating the bicarbonate/CO 2 ratio of the incubation medium, or by adding amiloride, a hydrogen/sodium antiport blocker. Estimates of intracellular pH using the creatine kinase equilibrium agree with those obtained by Neutral red scanning spectrophotometry in control conditions. However, only Neutral red indicated an acidification with amiloride treatment, while the creatine kinase equilibrium was preferentially affected by hypercapnia, suggesting the presence of at least two pH compartments in hippocampal brain slices. These manipulations cannot be carried out easily in vivo, and provide a means of determining the importance of metabolic changes on neural function during anoxia.

Effects of the slow calcium-channel blocker verapamil on phosphatic metabolism of crystalline lens

The effects of calcium-channel blockade on phosphate metabolism in the rabbit lens ex vivo was studied using phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. Rates-of-change of the intralenticular pH and the following phosphatic metabolites in the lens were determined: ATP, ADP, inorganic orthophosphate, sugar phosphates, nucleoside diphosphosugars, phosphodiesters, dinucleotides, and an uncharacterized resonance peak at 6 delta in the 31P spectrum. Incubation in 20, 200 or 2000 micrograms/ml verapamil led to the same qualitative changes in the lens's metabolic profile at each different concentration; only the rates of metabolite change were altered significantly by verapamil concentration. Compared with the control, the entire orthophosphate resonance band increased relative to the signals of ATP, and the alpha-glycerophosphate and nucleoside monophosphate resonances increased in relation to inorganic orthophosphate, and these two resonance groups shifted relative to each other so that they coalesced, indicating that the two groups of signals arose from compounds in relatively different pH compartments. In addition to altering metabolic rates, verapamil reduced the intralenticular pH of the sugar phosphate and inorganic orthophosphate pools with respect to those containing the nucleoside monophosphates; the pH-change difference was about one order of magnitude smaller for the mononucleotide pool. Despite the metabolic alterations and changes in pH during verapamil incubation, lens clarity was unchanged.

The role of the diphtheria toxin receptor in cytosol translocation.

The role of the receptor in the transport of diphtheria toxin (DT) to the cytosol was examined. A point-mutant form of DT, CRM 107 (CRM represents cross-reacting material), that has an 8,000-fold lower affinity for the DT receptor than native toxin was conjugated to transferrin and monoclonal antibodies specific for the cell-surface receptors T3 and Thy1. Conjugating the binding site-inactivated CRM 107 to new binding moieties reconstituted full toxicity, indistinguishable from native DT linked to the same ligand, indicating that the entry activity of the DT B chain can be fully separated from the receptor binding function. Like DT, the toxin conjugates exhibited a dose-dependent lag period before first-order inactivation of protein synthesis. Inactivation of the binding site of the toxin portion of the conjugate was found to have no effect on the kinetics of protein synthesis inactivation. The receptor used by the toxin determined the length of the lag period relative to the killing rate. Comparing the potency of CRM 107 conjugates with native DT, standardized for receptor occupancy, shows that new receptors can be as or more efficient than the DT receptor in transporting DT to the cytosol. The transferrin-CRM 107 conjugate, unlike native DT, was highly toxic to murine cells. All the data presented are consistent with a model that the DT receptor, other than initiating rapid internalization of the toxin to low pH compartments, is unnecessary for transport of the toxin to the cytosol and that membrane translocation activity is expressed by the DT B subunit independent of the receptor-binding site.

Intracellular digestion of reovirus particles requires a low pH and is an essential step in the viral infectious cycle.

Lysosomotropic drugs such as NH4Cl have been useful for studying the role of low pH in early events in virus infection. NH4Cl blocks the production of infectious progeny virus in mammalian reovirus-infected cells. The inhibitory effect of NH4Cl is mediated by an inhibition of intracellular digestion of reovirus outer capsid proteins. In vitro digestion of viral outer capsid proteins produces infectious partially uncoated particles, called intermediate subviral particles, which are no longer inhibited by the presence of NH4Cl. These results indicate that proteolytic processing of reovirus outer capsid proteins takes place in a low pH compartment of the cell and is an essential step in the viral infectious cycle.

Lysosomal involvement in cellular intoxication with Clostridium difficile toxin B

The process of internalisation of Clostridium difficile toxin B into human lung fibroblasts was further studied, with the aim of elucidating the fate of endocytosed toxin. Development of the toxin-induced cytopathogenic effect was reversibly inhibited at 18 °C and in the presence of 200 m m KCl or 1–20 mM benzyl alcohol, i.e. at conditions when the fusion between endosomes and lysosomes is prevented. Fibroblasts treated with toxin at 37 °C but transferred to 18 °C within 10 min were also completely protected, whereas transfer to 18 °C later during the latency resulted in only partial protection. KCl was also protective upon addition after the toxin binding step. Inhibitors of lysosomal proteases, such as chymostatin, leupeptin and antipain, prevented the appearance of the cytopathogenic effect, when present during toxin exposure or added after the toxin binding step. Chinese hamster ovary cell mutants, defective in acidification of their endosomes, were resistant to toxin B, whereas wildtype cells were sensitive. The resistance was not overcome by applying a low extracellular pH. The results suggest that exposure to a low pH compartment is necessary but not sufficient for entry of active toxin B to the cytosol. In addition to a low pH, a fusion of toxin-containing endosomes with lysosomes and a further processing of the toxin by lysosomal proteases is required for cellular intoxication.

31P NMR studies of the intact perfused rat heart: A novel analytical approach for determining functional-metabolic correlates, temporal relationships, and intracellular actions of cardiotoxic chemicals nondestructively in an intact organ model

Intact hearts isolated from adult male, Sprague-Dawley rats were perfused under standardized conditions in an apparatus designed for use in a high-resolution nuclear magnetic resonance (NMR) spectrometer system. Myocardial phosphatic metabolite concentrations (ATP, PCr, P i, and phosphomonoesters) and intracellular pH were determined sequentially at timed intervals coincident with the functional assessments of the intact heart by phosphorus-31 ( 31 P) NMR spectroscopic methods. Myocardial functional and metabolic parameters were unaffected by sustained control perfusion (2 hr). The negative inotropic actions of cadmium were associated with significant changes in the chemical environment of inorganic phosphate ( P i) within the cells. This initial cellular response to cadmium, which correlated with the onset and magnitude of the contractile disturbances, appeared to represent the formation of an acidic, intracellular P i pool (pH, 6.0). This pH compartment reached a steady state during the period in which maximal changes in contractile function were manifested, and before cellular ATP and PCr concentrations were altered. These findings are consistent with the interpretation that the functional deficits caused by cadmium originated primarily from changes in the chemical environment experienced by intracellular metabolites, rather than changes in the amounts of cellular high energy substrates. In contrast, the time-dependent negative inotropic effects of arsenate were proportional to the loss of cellular ATP stores. Intracellular pH was not affected in these hearts. A distinctive metabolic finding associated with the cardiotoxicity of arsenate was the time-dependent accumulation of previously undetected phosphatic metabolites in the arsenate-treated hearts. Efforts to chemically identify these metabolites proved inconclusive; however, existing evidence suggests the possibility that these phosphorus-containing compounds may be arsenophosphate derivatives of naturally occurring cellular metabolites. The present findings provide experimental evidence demonstrating that toxicologic assessments in an intact organ model are feasible using whole organ 31 P NMR spectroscopic methods and that meaningful, new insights regarding the biochemical mechanisms responsible for the cardiotoxic actions of xenobiotic agents can be obtained by this analytical approach.

Insulin and Insulin Receptor Uptake into Rat Liver: Chloroquine Action on Receptor Recycling

In the present study, the effect of chloroquine on both insulin and receptor distribution was examined in vivo. Insulin injection (25 nmol/100 g body wt) caused a marked accumulation of both insulin and its receptor in purified hepatic Golgi fractions by 15 min postinjection. Percoll fractionation of parent Golgi fractions resolved two endocytic components of low (rho = 1.040-1.050) and high (rho = 1.053-1.064) density in which the relative distribution of insulin binding sites was unaltered by chloroquine. Chloroquine significantly accumulated in the high-density region of the Percoll gradient consistent with this being a low pH compartment. 125I-insulin accumulated first in the low-density (1 min) and subsequently in the high-density region (5-10 min) of Percoll-subfractionated Golgi fractions. Chloroquine treatment caused marked accumulation of 125I-insulin in the high-density compartment with substantial retention of radiolabel therein at 20 min postinjection. 125I-insulin extracted from the Percoll fractions was comparably intact in control and chloroquine-treated rats. These data suggest that the chloroquine-accumulating, high-density compartment of hepatic Golgi fractions is the site of dissociation of internalized insulin-receptor complexes before degradation of the ligand and receptor recycling.