A novel nanogel-based fluorescent probe for ratiometric detection of intracellular pH values

A new nanogel indicator (NGI) for ratiometric fluorescent detection of intracellular pH values has been reported. In aqueous solution, the NGI can be used for ratiometric sensing of pH changes with a large hypsochromic shift of 100 nm in the green–red region and reversible. Furthermore, the NGI has been used to calibrate the cytosol pH in living cells.

Involvement of ATP-dependentPseudomonasExotoxin Translocation from a Late Recycling Compartment in Lymphocyte Intoxication Procedure

Pseudomonas exotoxin (PE) is a cytotoxin which, after endocytosis, is delivered to the cytosol where it inactivates protein synthesis. Using diaminobenzidine cytochemistry, we found over 94% of internalized PE in transferrin (Tf) -positive endosomes of lymphocytes. When PE translocation was examined in a cell-free assay using purified endocytic vesicles, more than 40% of endosomal125I-labeled PE was transported after 2 h at 37°C, whereas a toxin inactivated by point mutation in its translocation domain was not translocated. Sorting of endosomes did not allow cell-free PE translocation, whereas active PE transmembrane transport was observed after > 10 min of endocytosis when PE and fluorescent-Tf were localized by confocal immunofluorescence microscopy within a rab5-positive and rab4- and rab7-negative recycling compartment in the pericentriolar region of the cell. Accordingly, when PE delivery to this structure was inhibited using a 20°C endocytosis temperature, subsequent translocation from purified endosomes was impaired. Translocation was also inhibited when endosomes were obtained from cells labeled with PE in the presence of brefeldin A, which caused fusion of translocation-competent recycling endosomes with translocation-incompetent sorting elements. No PE processing was observed in lymphocyte endosomes, the full-sized toxin was translocated and recovered in an enzymatically active form. ATP hydrolysis was found to directly provide the energy required for PE translocation. Inhibitors of endosome acidification (weak bases, protonophores, or bafilomycin A1) when added to the assay did not significantly affect125I-labeled PE translocation, demonstrating that this transport is independent of the endosome-cytosol pH gradient. Nevertheless, when125I-labeled PE endocytosis was performed in the presence of one of these molecules, translocation from endosomes was strongly inhibited, indicating that exposure to acidic pH is a prerequisite for PE membrane traversal. When applied during endocytosis, treatments that protect cells against PE intoxication (low temperatures, inhibitors of endosome acidification, and brefeldin A) impaired125I-labeled PE translocation from purified endosomes. We conclude that PE translocation from a late receptor recycling compartment is implicated in the lymphocyte intoxication procedure.

Effects of Ion Transport Inhibition on Rat Mandibular Gland Secretion

The effects of substituting gluconate for extracellular Cl, and of treatment with various ion transport blockers, on cytosol pH (pHi) and secretion by the acetylcholine stimulated rat mandibular gland were studied in vitro. Gluconate replacement increased pHi from 7.12 ± 0.02 to 7.27 ± 0.04, caused secretory rate to fall by 75%, and increased salivary HC03 from 14 ± 0.9 mmollL to 67 ± 1.5 mmoll L. Furosemide (I mmol/L), which blocks Na-K-2Cl symports and Cl-HCO 3 antiports, had effects similar to those of gluconate replacement, except that secretion was reduced only by 59%. Bumetanide (1 mmoll L), which blocks only Na-K-2Cl symports, caused a 67% reduction in secretion rate, but it had little effect on pHi and caused only a small rise in salivary HCO3 concentration. SITS (1 mmol/L), which blocks Cl-HCO3 antiports, increased pHi to 7.26 ± 0.03 and induced a small rise in the secretory rate. Methazolamide and acetazolamide (1 mmol/L), both of which inhibit carbonic anhydrase and may also block anion channels, increased pHi to 7.43 ± 0.02 and 7.20 ± 0.03, respectively, but had no effect on secretory rate, and reduced salivary HC03 slightly. Ba (3 mmol/L), tetraethylammonium (10 mmoll L), and decamethonium (5 mmol/L) all caused marked but reversible reductions in secretory rate, consistent with the known actions of these agents on K channels. Ba, however, also appeared to act as a Ca antagonist, an action that it seemed to share with Mn ions (5 mmoll L). The results are interpreted in terms of a secretion model based on the presence, in the baso-lateral plasma membranes of the secretory cells, not only of a Na-K-2Cl symport, but also of Na-H and Cl-HC03 antiports, contributing, respectively, to secretion in the proportions 8:5:3.

The mechanism of inhibition of ureogenesis by acidosis

In perfused rat liver a decrease of cytosol pH, determined with pH-sensitive microelectrodes7 from 7.2 to 6.85 is associated with a 50% fall in ureogenesis from ammonium chloride. In isolated rat hepatocytes the fall in ureogenesis due to acidosis is associated with decrease in the mitochondrial and cytosolic concentration of citrulline. Limitation of carbamoyl phosphate synthesis and thus citrulline supply could be responsible for the inhibition of ureogenesis observed.

The regulation of oat coleoptile phosphoenolpyruvate carboxylase and malic enzyme activities by H+ and metabolites. Kinetic evidence for and against a metabolic pH-stat

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) and malic enzyme (EC 1.1.1.40) activities in soluble protein extracts of Avena coleoptiles were investigated as functions of pH. The presence of malic enzyme activity was confirmed by radiochemical assays which identified the product of the forward reaction and spectrophotometric assays which demonstrated that reduction or oxidation of NADP required all the substrates and cofactors necessary for the forward or reverse reactions. In neither assay could NADP or Mn2+ be replaced by NAD or Mg2+. Activity was independent of pH (6.0 to 7.7) at malate concentrations less than 0.1 mM. At higher concentrations the pH increase raised activity by at least 100%, and substrate inhibition by malate resulted in activity at 0.97 mM malate which was less than that at 0.5 mM malate. The increase in malic enzyme activity with rising pH can be attributed to a decrease in substrate inhibition and a decrease in Km,app (Mn2+). pH increases between 7.0 and 7.6 increased Vmax and phosphoenolpyruvate limited phosphoenolpyruvate carboxylase (PEPC) activity by over 200%, and decreased the Km,app (Mg2+). Inhibition of PEPC and malic enzyme by various carboxylic acids and phosphorylated sugars decreased as the assay pH rose. The results are discussed in light of contrasting suggestions concerning the role of these enzymes in cytosol pH regulation.