scholarly journals Characterization of the swelling-induced alkalinization of endocytotic vesicles in fluorescein isothiocyanate-dextran-loaded rat hepatocytes

1995 ◽  
Vol 309 (1) ◽  
pp. 19-24 ◽  
Author(s):  
R Schreiber ◽  
D Häussinger
Keyword(s):  
Fluorescein Isothiocyanate ◽  
Rat Hepatocytes ◽  
Free Calcium ◽  
Independent Mechanism ◽  
Free Calcium Concentration ◽  
Cytosolic Free Calcium Concentration ◽  
Fluorescein Isothiocyanate Dextran ◽  
Induced Changes ◽  
Vesicular Compartment

Short-term cultivated rat hepatocytes were allowed to endocytose fluorescein isothiocyanate (FITC)-coupled dextran and the apparent vesicular pH (pHves) was measured by single-cell fluorescence. After 2 h of exposure to FITC-dextran, the apparent pH in the vesicular compartments accessible to endocytosed FITC-dextran was 6.01 +/- 0.05 (n = 39) in normo-osmotic media. Hypo-osmotic exposure increased, whereas hyper-osmotic exposure decreased apparent pHves. by 0.18 +/- 0.02 (n = 26) and 0.12 +/- 0.01 (n = 23) respectively. Incubation of the cells with unlabelled dextran for 2h before a 2-h FITC-dextran exposure had no effect on apparent pHves and its osmosensitivity. When, however, hepatocytes were exposed to unlabelled dextran for 5 h after a 2 h exposure to FITC-dextran, in order to allow transport of endocytosed FITC-dextran to late endocytotic/lysosomal compartments, apparent pHves. decreased to 5.38 +/- 0.04 (n = 12) and the apparent pH in the vesicular compartment containing the dye was no longer sensitive to aniso-osmotic exposure. These findings indicate that the osomosensitivity of pHves. is apparently restricted to early endocytotic compartments. Aniso-osmotic regulation of apparent pHves. in freshly FITC-loaded hepatocytes was not accompanied by aniso-osmolarity-induced changes of the cytosolic free calcium concentration, and neither vasopressin nor extracellular ATP, which provoked a marked Ca2+ signal, affected apparent pHves. Dibutyryl-cyclic AMP (cAMP) or vanadate (0.5 mmol/l) were without effect on apparent pHves. and its osmosensitivity. However, pertussis toxin-treatment or genistein (but not daidzein) or the erbstatin analogue methyl 2,5-dihydroxycinnamate fully abolished the osmo-sensitivity of apparent pHves., but did not affect apparent pHves. It is concluded that regulation of pHves. by cell volume occurs in early endocytotic compartments, but probably not in lysosomes, and is mediated by a G-protein and tyrosine kinase-dependent, but Ca2+- and cAMP-independent mechanism.

scholarly journals Effects of aniso-osmolarity and hydroperoxides on intracellular pH in isolated rat hepatocytes as assessed by (2′,7′)-bis(carboxyethyl)-5(6)-carboxyfluorescein and fluorescein isothiocyanate-dextran fluorescence

1994 ◽  
Vol 303 (1) ◽  
pp. 113-120 ◽  
Author(s):  
R Schreiber ◽  
B Stoll ◽  
F Lang ◽  
D Häussinger
Keyword(s):  
Fluorescein Isothiocyanate ◽  
Rat Hepatocytes ◽  
Cell Swelling ◽  
Cell Shrinkage ◽  
Isolated Rat Hepatocytes ◽  
Proton Gradients ◽  
Fluorescein Isothiocyanate Dextran ◽  
And Oxidative Stress ◽  
Vesicular Compartment ◽  
Isolated Rat

Freshly isolated rat hepatocytes were plated for 4-6 h and either loaded with (2′,7)-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) or allowed to endocytose fluorescein isothiocyanate (FITC)-coupled dextran in order to study the effects of aniso-osmotic exposure and oxidative stress on cytosolic (pHcyt) and apparent vesicular pH (pHves) by single-cell fluorescence recordings. In the presence of normo-osmotic (305 mosmol/l) medium pHcyt was 7.23 +/- 0.03 (n = 108), whereas an apparent pH of 6.07 +/- 0.02 (n = 156) was found in the vesicular compartment accessible to endocytosed FITC-dextran. Substitution of 60 mM NaCl against 120 mM raffinose had no effect on pHcyt or apparent pHves, whereas addition of NH4Cl increased both pHcyt and apparent pHves. Hypo-osmotic cell swelling lowered pHcyt, whereas simultaneously apparent pHves increased. These effects were rapidly reversible upon re-institution of normo-osmotic media. Similarly, an increase of apparent pHves was observed when cell swelling was induced by Ba2+, glutamine or histidine. Conversely, hyperosmotic cell shrinkage due to addition of NaCl or raffinose led to a cytosolic alkalinization and a vesicular acidification. Both, H2O2 (0.2 mmol/l) and t-butyl-hydroperoxide (0.2 mmol/l) were without effect on pHcyt, but lowered apparent pHves by about 0.2 pH units. Ba2+ (1 mmol/l) diminished the acidifying effect of the hydroperoxides by about 50%. Pretreatment of the cells with colchicine, but not with lumicolchicine, largely abolished the effects of aniso-osmolarity and hydroperoxides on pHves. The data suggest that hepatocellular hydration affects the proton gradients built up across the membranes of endocytotic FITC-dextran-accessible compartments in a microtubule-dependent way. They further suggest that hydroperoxides induce vesicular acidification in a colchicine- and Ba(2+)-sensitive way. Because hydroperoxides induce Ba(2+)-sensitive cell shrinkage [Hallbrucker, Ritter, Lang, Gerok and Häussinger (1992) Eur. J. Biochem. 211, 449-458], the results are compatible with the view that hydroperoxide-induced cell shrinkage mediates vesicular acidification. It is concluded that modulation of vesicular pH by the hepatocellular hydration state may play a role in triggering some metabolic changes in response to cell swelling/shrinkage.

scholarly journals In Vitro Activity of Acanthamoeba castellanii on Human Platelets and Erythrocytes

2008 ◽  
Vol 77 (2) ◽  
pp. 733-738 ◽  
Author(s):  
A. Mattana ◽  
L. Alberti ◽  
G. Delogu ◽  
P. L. Fiori ◽  
P. Cappuccinelli
Keyword(s):  
Hemolytic Activity ◽  
Acanthamoeba Castellanii ◽  
Human Platelets ◽  
Free Calcium ◽  
Plasma Membrane Permeability ◽  
Human Red Blood Cells ◽  
Free Calcium Concentration ◽  
Cytosolic Free Calcium Concentration

ABSTRACT The effect of Acanthamoeba on human platelets and erythrocytes has not been fully elucidated. This paper reports that cell-free supernatants prepared from A. castellanii can activate human platelets, causing both a significant increase in the cytosolic free-calcium concentration and platelet aggregation. In addition, we demonstrated that platelet activation depends on the activity of ADP constitutively secreted into the medium by trophozoites. This study also showed that A. castellanii can affect human red blood cells, causing hemolysis, and provided evidence that hemolysis occurs in both contact-dependent and contact-independent ways; there are differences in kinetics, hemolytic activity, and calcium dependency between the contact-dependent and contact-independent mechanisms. Partial characterization of contact-independent hemolysis indicated that ADP does not affect the plasma membrane permeability of erythrocytes and that heat treatment of amoebic cell-free supernatant abolishes its hemolytic activity. These findings suggest that some heat-labile molecules released by A. castellanii trophozoites are involved in this phenomenon. Finally, our data suggest that human platelets and erythrocytes may be potential cell targets during Acanthamoeba infection.

scholarly journals Intracellular calcium mobilization and activation of the Na+/H+ exchanger in platelets

1993 ◽  
Vol 290 (2) ◽  
pp. 617-622 ◽  
Author(s):  
E Poch ◽  
A Botey ◽  
J Gaya ◽  
A Cases ◽  
F Rivera ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Fluorescent Dyes ◽  
Extracellular Calcium ◽  
Free Calcium ◽  
Regulatory Relationship ◽  
Cytosolic Ph ◽  
Intracellular Calcium Mobilization ◽  
Free Calcium Concentration ◽  
Cytosolic Free Calcium Concentration ◽  
Intracellular Alkalinization

The aim of the present study was to evaluate the regulatory relationship between the cytosolic free calcium concentration ([Ca2+]i and cytosolic pH (pHi). [Ca2+]i and pHi were measured using the fluorescent dyes fura-2 and BCECF [2′,7′-bis-(carboxyethyl)-5,6-carboxyfluorescein] respectively. In a medium with 1 mmol/l extracellular calcium, thrombin (2.5 units/ml) induced an increment in [Ca2+]i of 638 +/- 31 nmol/l (n = 5) and an intracellular alkalinization of 0.14 +/- 0.01 pH units (n = 8). Both responses were dependent on the concentration of thrombin, displaying a sigmoidal dose-response pattern. The intracellular alkalinization was dependent upon extracellular Na+ and was amiloride-sensitive, indicating that it was mediated by activation of the Na+/H+ exchanger. When extracellular calcium was chelated with EGTA prior to the addition of thrombin, the intracellular alkalinization was not affected (0.15 +/- 0.02 at 2.5 units/ml thrombin, n = 8). Under these circumstances, the [Ca2+]i increment represents mobilization from internal stores, reaching 157 +/- 42 nmol/l at 2.5 units/ml thrombin. When platelets were preloaded with the intracellular calcium chelator MAPTAM (1,2-bis-5-methylaminophenoxylethane-NNN'-tetraacetoxymethyl acetate) to block the increase in [Ca2+]i induced by thrombin, no increment in pHi was observed. Moreover, MAPTAM-loaded calcium-depleted platelets had a basal pHi that was more acidic than in the presence of 1 mmol/l extracellular calcium (6.93 +/- 0.09 versus 7.14 +/- 0.01, n = 26, P < 0.001). Ionomycin induced an elevation of [Ca2+]i that was accompanied by a concomitant increase in pHi, which was Na(+)-dependent and amiloride-sensitive. [Ca2+]i and pHi increases induced by ionomycin were both dependent on the concentration of ionomycin. In conclusion, an increase in [Ca2+]i is necessary for the agonist-induced activation of the Na+/H+ exchanger in platelets. Non-agonist-induced increases in [Ca2+]i seems to prompt activation of the exchanger. In addition, Ca(2+)-depleted platelets have a more acidic basal pHi, indicating that the basal level of [Ca2+]i is also important for maintaining the basal pHi.

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