scholarly journals Cystine transport in purified rat liver lysosomes

1986 ◽  
Vol 236 (3) ◽  
pp. 671-677 ◽  
Author(s):  
A J Jonas
Keyword(s):  
Rat Liver ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Proton Pumping ◽  
Proton Gradient ◽  
Ionophore A23187 ◽  
Ph Gradients ◽  
Bivalent Cations ◽  
Liver Lysosomes ◽  
Rat Liver Lysosomes

Amino acid efflux from highly purified rat liver lysosomes exposed to the methyl ester derivatives of leucine, methionine, tyrosine and cystine was examined. The lysosomal efflux of leucine, methionine and tyrosine was unaffected by the presence of MgATP, whereas cystine efflux was stimulated by MgATP. Exposure of lysosomes to 2 mM-MgATP resulted in lysosomal acidification and a 0.5 pH unit increase in the lysosomal pH gradient through the action of a proton-pumping ATPase. Cystine efflux was also stimulated when the lysosomal proton gradient was increased through changes in buffer pH. Decreasing the lysosomal proton gradient with ionophores resulted in diminished cystine efflux. Bivalent cations had no effect on the lysosomal efflux of leucine, methionine and tyrosine. However, cystine efflux was stimulated by the presence of bivalent cations even when the lysosomal proton gradient was minimized. Cation-stimulated cystine efflux was inhibited by the presence of the calcium ionophore A23187, which altered the lysosomal membrane potential. Cystine efflux from lysosomes appears to be uniquely dependent on pH gradients and cation concentrations.

scholarly journals Inhibition of neutrophil oxidative metabolism by lysosomotropic weak bases

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 334-342 ◽  
Author(s):  
B Styrt ◽  
MS Klempner
Keyword(s):  
Phorbol Myristate Acetate ◽  
Oxidative Metabolism ◽  
Respiratory Burst ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Enzyme Complex ◽  
Myristate Acetate ◽  
Ph Gradients ◽  
Weak Bases

Abstract Maintenance of an acidic intralysosomal compartment may be relevant to multiple aspects of neutrophil function. The effect of lysosomal alkalinization on the neutrophil respiratory burst was studied by measuring cytochrome c reduction in response to soluble stimuli in the presence of lysosomotropic weak bases. The weak bases chloroquine, ammonium chloride, methylamine, and clindamycin all raised the intralysosomal pH and inhibited neutrophil oxidative metabolism at concentrations ranging from 0.1 to 100 mmol/L. Inhibition was dose dependent for each base and correlated significantly with the degree of lysosomal alkalinization. Concentrations that did not alkalinize the lysosome did not inhibit the respiratory burst. Inhibition by weak bases was seen when oxidative metabolism was stimulated by phorbol myristate acetate, calcium ionophore A23187, formyl-methionyl-leucyl- phenylalanine, opsonized zymosan, or sodium fluoride. Increasing the stimulus concentration (from 5 ng/mL to 5 micrograms/mL phorbol myristate acetate and from 0.5 to 1 mumol/L A23187) diminished or abolished inhibition by weak bases. Washing the cells after incubation with bases and before stimulation substantially reversed the inhibition. None of the bases impaired detection of superoxide in a cell-free xanthine-xanthine oxidase assay. Other indexes of oxidative metabolism, including oxygen consumption and hydrogen peroxide release, were also inhibited by weak bases. Analysis of particulate NADPH oxidase activity from neutrophils stimulated in the presence of bases suggested that these cells assemble a subnormal amount of an enzyme complex with normal kinetic characteristics. Lysosomotropic weak bases alkalinized the neutrophil lysosome and produced inhibition of oxidative metabolism that was dose related, was not stimulus specific, and was largely reversed by washing the cells before stimulation. A possible explanation would be altered assembly of the enzyme complex involved in respiratory burst activation as a consequence of impaired granule/plasma membrane fusion in the presence of diminished transmembrane pH gradients.

scholarly journals The calcium ionophore A23187 increases the tight-junctional permeability in rat liver

1988 ◽  
Vol 256 (3) ◽  
pp. 1039-1041 ◽  
Author(s):  
K S Kan ◽  
R Coleman
Keyword(s):  
Horseradish Peroxidase ◽  
Rat Liver ◽  
Bile Flow ◽  
Calcium Ionophore ◽  
Peak Height ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Hepatocellular Damage ◽  
Junctional Permeability ◽  
Second Peak

The effect of an increase in intracellular Ca2+ concentration on tight-junctional permeability in rat liver was studied by using the calcium ionophore A23187. Infusion of 100 microliters of dimethyl sulphoxide containing various amounts of A23187 over 30 min into isolated perfused livers was followed by a pulse of horseradish peroxidase (HRP) under single-pass conditions. The first biliary HRP peak, a measure of junctional permeability, was increased 4-fold with 100 micrograms of A23187. There were, however, no significant effects on bile flow or on aspartate aminotransferase leakage as compared with the control at this dosage, and thus the increase in junctional permeability was occurring without evidence of appreciable cholestatic or hepatocellular damage. Higher dosages of A23187, however, caused not only an increase in HRP peak height but also changes in bile flow and increases in aminotransferase leakage, indicating more extensive effects at these higher dosages. A second peak of HRP secretion, occurring 20-25 min after the HRP pulse, was also elevated approx. 3.5-fold; this may indicate that pinocytosis and transcellular movement of HRP are also increased under these conditions.

scholarly journals Glycogenolytic effects of the calcium ionophore A23187, but not of vasopressin or angiotensin, in foetal-rat hepatocytes

1984 ◽  
Vol 220 (2) ◽  
pp. 441-445 ◽  
Author(s):  
M Freemark ◽  
S Handwerger
Keyword(s):  
Cyclic Amp ◽  
Rat Liver ◽  
Glycogen Synthesis ◽  
Calcium Ionophore ◽  
Rat Hepatocytes ◽  
Glycogen Metabolism ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Foetal Rat ◽  
20 Nm

Vasopressin, angiotensin and phenylephrine stimulate glycogenolysis in postnatal rat liver by a Ca2+-mediated mechanism not involving cyclic AMP. To determine whether these hormones promote glycogenolysis in foetal liver, we have examined their effects, and those of the Ca2+ ionophore A23187, on glycogen metabolism in cultured foetal-rat hepatocytes. Vasopressin and angiotensin (0.1 nM-0.1 microM) had no effects on either glycogen synthesis (as assessed by [14C]glucose incorporation into glycogen) or phosphorylase a activity. However, A23187 at 1 and 10 microM inhibited glycogen synthesis by 31.3 and 89.1% respectively (both P less than 0.001) and stimulated phosphorylase a activity by 66.9 and 184.1% respectively (both P less than 0.01). Incubation of cells in Ca2+-deficient medium attenuated the effects of 10 microM-A23187 on glycogen synthesis and abolished the effects of 1 microM-A23187. As in postnatal liver, glucagon (1 and 20 nM) and isoprenaline (1 and 10 microM), which activate adenylate cyclase, inhibited glycogen synthesis and stimulated phosphorylase a activity in foetal hepatocytes. The minimal effective concentration of phenylephrine was 10 times that of isoprenaline. These results indicate striking differences in the ontogeny of cyclic AMP-mediated and Ca2+-mediated processes which regulate hepatic glycogenolysis. Since increases in cytosolic Ca2+ induce glycogenolysis in foetal-rat liver, the weak or absent responses to vasopressin, angiotensin and the alpha-adrenergic agonists may result from defects in hormone-receptor binding or in post-receptor events leading to the mobilization of intracellular Ca2+ stores.

scholarly journals Inhibition of neutrophil oxidative metabolism by lysosomotropic weak bases

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 334-342
Author(s):  
B Styrt ◽  
MS Klempner
Keyword(s):  
Phorbol Myristate Acetate ◽  
Oxidative Metabolism ◽  
Respiratory Burst ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Enzyme Complex ◽  
Myristate Acetate ◽  
Ph Gradients ◽  
Weak Bases

Maintenance of an acidic intralysosomal compartment may be relevant to multiple aspects of neutrophil function. The effect of lysosomal alkalinization on the neutrophil respiratory burst was studied by measuring cytochrome c reduction in response to soluble stimuli in the presence of lysosomotropic weak bases. The weak bases chloroquine, ammonium chloride, methylamine, and clindamycin all raised the intralysosomal pH and inhibited neutrophil oxidative metabolism at concentrations ranging from 0.1 to 100 mmol/L. Inhibition was dose dependent for each base and correlated significantly with the degree of lysosomal alkalinization. Concentrations that did not alkalinize the lysosome did not inhibit the respiratory burst. Inhibition by weak bases was seen when oxidative metabolism was stimulated by phorbol myristate acetate, calcium ionophore A23187, formyl-methionyl-leucyl- phenylalanine, opsonized zymosan, or sodium fluoride. Increasing the stimulus concentration (from 5 ng/mL to 5 micrograms/mL phorbol myristate acetate and from 0.5 to 1 mumol/L A23187) diminished or abolished inhibition by weak bases. Washing the cells after incubation with bases and before stimulation substantially reversed the inhibition. None of the bases impaired detection of superoxide in a cell-free xanthine-xanthine oxidase assay. Other indexes of oxidative metabolism, including oxygen consumption and hydrogen peroxide release, were also inhibited by weak bases. Analysis of particulate NADPH oxidase activity from neutrophils stimulated in the presence of bases suggested that these cells assemble a subnormal amount of an enzyme complex with normal kinetic characteristics. Lysosomotropic weak bases alkalinized the neutrophil lysosome and produced inhibition of oxidative metabolism that was dose related, was not stimulus specific, and was largely reversed by washing the cells before stimulation. A possible explanation would be altered assembly of the enzyme complex involved in respiratory burst activation as a consequence of impaired granule/plasma membrane fusion in the presence of diminished transmembrane pH gradients.

Growth factors and the primary cilium in BALB/c 3T3 cells

1987 ◽  
Vol 45 ◽  
pp. 830-831
Author(s):  
R. W. Tucker ◽  
N. S. More ◽  
S. Jayaraman
Keyword(s):  
Growth Factors ◽  
Primary Cilium ◽  
Cultured Cells ◽  
Morphological Changes ◽  
Calcium Ionophore ◽  
Growth Stimulation ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
3T3 Cells ◽  
Microtubule Depolymerization

The mechanisms by which polypeptide growth factors Induce DNA synthesis in cultured cells is not understood, but morphological changes Induced by growth factors have been used as clues to Intracellular messengers responsible for growth stimulation. One such morphological change has been the transient disappearance of the primary cilium, a “9 + 0” cilium formed by the perinuclear centriole in interphase cells. Since calcium ionophore A23187 also produced both mitogenesis and ciliary changes, microtubule depolymerization might explain ciliary disappearance monitored by indirect immunofluorescence with anti-tubulin antibody. However, complete resorption and subsequent reformation of the primary cilium occurs at mitosis, and might also account for ciliary disappearance induced by growth factors. To settle this issue, we investigated the ultrastructure of the primary cilium using serial thin-section electron microscopy of quiescent BALB/c 3T3 cells before and after stimulation with serum.

Inhibition of Platelet Aggregation by Ethanol in Vitro Shows Specificity for Aggregating Agent Used and Is Influenced by Platelet Lipid Composition

1982 ◽  
Vol 48 (01) ◽  
pp. 049-053 ◽  
Author(s):  
C G Fenn ◽  
J M Littleton
Keyword(s):  
Platelet Aggregation ◽  
Lipid Composition ◽  
Saturated Fatty Acids ◽  
Calcium Ionophore ◽  
Cholesterol Content ◽  
Membrane Lipid ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Increased Sensitivity

SummaryEthanol at physiologically tolerable concentrations inhibited platelet aggregation in vitro in a relatively specific way, which may be influenced by platelet membrane lipid composition. Aggregation to collagen, calcium ionophore A23187 and thrombin (low doses) were often markedly inhibited by ethanol, adrenaline and ADP responses were little affected, and aggregation to exogenous arachidonic acid was actually potentiated by ethanol. Aggregation to collagen, thrombin and A23187 was inhibited more by ethanol in platelets enriched with saturated fatty acids than in those enriched with unsaturated fats. Platelets enriched with cholesterol showed increased sensitivity to ADP, arachidonate and adrenaline but this increase in cholesterol content did not appear to influence the inhibition by ethanol of platelet responses. The results suggest that ethanol may inhibit aggregation by an effect on membrane fluidity and/or calcium mobilization resulting in decreased activity of a membrane-bound phospholipase.

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