thapsigargin and 2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein

Hyperammonemia is a key factor in the pathogenesis of hepatic encephalopathy (HE) as well as other metabolic encephalopathies, such as those associated with inherited disorders of urea cycle enzymes and in Reye's syndrome. Acute HE results in increased brain ammonia (up to 5 mM), astrocytic swelling, and altered glutamatergic function. In the present study, using fluorescence imaging techniques, acute exposure (10 min) of ammonia (NH4+/NH3) to cultured astrocytes resulted in a concentration-dependent, transient increase in [Ca2+]i. This calcium transient was due to release from intracellular calcium stores, since the response was thapsigargin-sensitive and was still observed in calcium-free buffer. Using an enzyme-linked fluorescence assay, glutamate release was measured indirectly via the production of NADH (a naturally fluorescent product when excited with UV light). NH4+/NH3 (5 mM) stimulated a calcium-dependent glutamate release from cultured astrocytes, which was inhibited after preincubation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester but unaffected after preincubation with glutamate transport inhibitors dihydrokainate and DL-threo-beta-benzyloxyaspartate. NH4+/NH3 (5 mM) also induced a transient intracellular alkaline shift. To investigate whether the effects of NH4+/NH3 were mediated by an increase in pH(i), we applied trimethylamine (TMA+/TMA) as another weak base. TMA+/TMA (5 mM) induced a similar transient increase in both pH(i) and [Ca2+]i (mobilization from intracellular calcium stores) and resulted in calcium-dependent release of glutamate. These results indicate that an acute exposure to ammonia, resulting in cytosolic alkalinization, leads to calcium-dependent glutamate release from astrocytes. A deregulation of glutamate release from astrocytes by ammonia could contribute to glutamate dysfunction consistently observed in acute HE.

Topics: Adenosine Triphosphate; Amino Acid Transport System X-AG; Ammonia; Animals; Aspartic Acid; Astrocytes; Calcium; Cells, Cultured; Dose-Response Relationship, Drug; Egtazic Acid; Endoplasmic Reticulum; Fluoresceins; Glutamic Acid; Hydrogen-Ion Concentration; Kainic Acid; Methylamines; Mice; Microscopy, Fluorescence; Spectrometry, Fluorescence; Thapsigargin; Ultraviolet Rays

Phospholipid scrambling, the disruption of normal plasma-membrane asymmetry, occurs during apoptotic and necrotic cell death and during the activation of platelets and neutrophils. It is currently believed that phospholipid scrambling is triggered simply by increases in bulk cytosolic [Ca(2+)]. We have presented evidence previously that the styryl dye FM1-43 is sensitive to phospholipid scrambling in Jurkat human leukaemic T-lymphocytes. Here we have used FM1-43, in combination with fura 2 and the Ca(2+)-elevating agents ionomycin and thapsigargin, in imaging experiments to test the idea that increases in bulk cytosolic [Ca(2+)] stimulate scrambling. Intracellular Ca(2+) increases of approximately 2 microM accompanied ionomycin-stimulated scrambling in approximately 50% of cells, and scrambling occurred in >99% of cells in which intracellular Ca(2+) rose to 4 microM. Chelating intracellular Ca(2+) with bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid or EGTA suppressed both ionomycin-stimulated intra cellular Ca(2+) increases and scrambling, demonstrating that intracellular Ca(2+) increases are necessary for ionomycin-stimulated scrambling. However, elevating intracellular Ca(2+) to 2-4 microM with thapsigargin, a drug that depletes intracellular Ca(2+) stores and triggers Ca(2+) entry via Ca(2+)-release-activated Ca(2+) channels, did not trigger scrambling, as assessed with either FM1-43 or FITC-labelled annexin V. These results suggest that increases in intracellular [Ca(2+)] are necessary but not sufficient to stimulate scrambling in lymphoyctes, and indicate that ionomycin has an additional effect that is required to stimulate scrambling.

Topics: Annexin A5; Calcium; Calcium Chloride; Calcium-Transporting ATPases; Cytosol; Egtazic Acid; Fluoresceins; Fluorescent Dyes; Humans; Ionomycin; Jurkat Cells; Kinetics; Microscopy, Fluorescence; Phospholipids; Pyridinium Compounds; Quaternary Ammonium Compounds; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Spectrometry, Fluorescence; T-Lymphocytes; Thapsigargin

Intracellular free calcium concentration ([Ca2+]i) and intracellular pH (pHi) were monitored in Ehrlich ascites tumor cells using Fura-2 or 2',7',-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), or both probes in combination. An increase in [Ca2+]i induced by thrombin or bradykinin, agonists known to elicit transient cell shrinkage in these cells, evoked a transient intracellular acidification, followed by an alkalinization. The latter was due to activation of a Na+/H+ exchanger and was inhibited under conditions preventing agonist-induced cell shrinkage without preventing the increase in [Ca2+]i. In contrast, a smaller, slower increase in [Ca2+]i elicited by thapsigargin did not cause cell shrinkage, and did not activate the Na+/H+ exchanger. Exposure to hypertonic solution was not associated with an increase in [Ca2+]i, but elicited an intracellular alkalinization similar to that induced by thrombin or bradykinin, via activation of the Na+/H+ exchanger. Thus, activation of the exchanger by the Ca2+-mobilizing agonists is suggested to be secondary to the cell shrinkage induced by these compounds. NH4Cl-induced intracellular alkalinization resulted in an increase in [Ca2+]i, apparently via stimulation of Ca2+ influx, whereas shrinkage-induced intracellular alkalinization did not stimulate Ca2+ influx. Thus, cell shrinkage appears to inhibit the Ca2+ influx otherwise resulting from alkalosis. In agreement with that notion, thapsigargin-induced Ca2+ influx was inhibited by cell shrinkage.

Topics: Ammonium Chloride; Animals; Bradykinin; Calcium; Carcinoma, Ehrlich Tumor; Cell Size; Fluoresceins; Fluorescent Dyes; Fura-2; Hydrogen-Ion Concentration; Hypertonic Solutions; Sodium-Hydrogen Exchangers; Thapsigargin; Thrombin

1. We have investigated interactions between intracellular pH (pHi) and the intracellular free calcium concentration ([Ca2+]i) in collagenase-isolated rat lacrimal acinar cells. The fluorescent dyes fura-2 and 2',7'-bis(carboxyethyl)-5-carboxyfluorescein (BCECF) were used to measure [Ca2+]i and pHi, respectively. 2. Application of the weak base NH4Cl alkalinized the cytosol and caused a dose-dependent increase in [Ca2+]i. Trimethylamine (TMA) also alkalinized the cytosol and increased [Ca2+]i. The increase in [Ca2+]i evoked by NH4Cl or TMA was much smaller than that evoked by the secretory agonist acetylcholine (ACh). 3. Application of NH4Cl also increased [Ca2+]i in cells bathed in Ca(2+)-free medium, indicating that NH4Cl released Ca2+ from an intracellular pool. 4. Ammonium chloride had no effect on [Ca2+]i in cells bathed in Ca(2+)-free medium if agonist-sensitive intracellular Ca2+ pools had been depleted with either ACh or the microsomal Ca(2+)-ATPase inhibitor 2,5-di(tert-butyl)hydroquinone. Treatment of cells with NH4Cl in Ca(2+)-free medium reduced the amount of Ca2+ released by ACh. These results suggest that NH4Cl released Ca2+ from the same intracellular pool released by ACh. 5. Calcium release from the agonist-sensitive pool was also triggered when the cytosol was alkalinized by removing the weak acid acetate. 6. Ammonium chloride caused a modest increase in inositol phosphate production, suggesting that NH4Cl may have released stored Ca2+ via an increase in the intracellular inositol 1,4,5-trisphosphate concentration. 7. The increase in [Ca2+]i evoked by NH4Cl was not sustained even in the presence of extracellular Ca2+. In contrast, when a low dose of ACh was used to evoke intracellular Ca2+ release of similar magnitude, sustained Ca2+ entry was observed. 8. Alkalinizing the cytosol appeared to partially inhibit Ca2+ entry triggered by thapsigargin or by ACh. 9. We suggest that alkalinizing the cytoplasm in unstimulated lacrimal acinar cells can release Ca2+ from the intracellular agonist-sensitive Ca2+ pool. However, releasing stored Ca2+ via alkalinization does not appear to trigger significant Ca2+ entry, perhaps because intracellular alkalinization inhibits either the Ca2+ entry pathway or the mechanism which couples the entry pathway to store depletion.

Topics: Acetic Acid; Acetylcholine; Ammonium Chloride; Animals; Calcium; Cytoplasm; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fluoresceins; Fluorescent Dyes; Fura-2; Hydrogen-Ion Concentration; Lacrimal Apparatus; Methylamines; Phosphatidylinositols; Rats; Rats, Wistar; Thapsigargin

We investigated the relationship between intracellular Ca2+ and pH homeostasis in Madin-Darby canine kidney-focus (MDCK-F) cells, a cell line exhibiting spontaneous oscillations of intracellular Ca2+ concentration (Ca2+i). Ca2+i and intracellular pH (pHi) were measured with the fluorescent dyes Fura-2 and BCECF by means of video imaging techniques. Ca2+ influx from the extracellular space into the cell was determined with the Mn2+ quenching technique. Cells were superfused with HEPES-buffered solutions. Under control conditions (pH 7.2), spontaneous Ca2+i oscillations were observed in virtually all cells investigated. Successive alkalinization and acidification of the cytoplasm induced by an ammonia ion prepulse had no apparent effect on Ca2+i oscillations. On the contrary, changes of extracellular pH value strongly affected Ca2+i oscillations. Extracellular alkalinization to pH 7.6 completely suppressed oscillations, whereas extracellular acidification to pH 6.8 decreased their frequency by 40%. Under the same conditions, the respective pHi changes were less than 0.1 pH units. However, experiments with the Mn2+ quenching technique revealed that extracellular alkalinization significantly reduced Ca2+ entry from the extracellular space. Large increases of Ca2+i triggered by the blocker of the cytoplasmic Ca(2+)-ATPase, thapsigargin, had no effect on pHi. We conclude: intracellular Ca2+ homeostasis in MDCK-F cells is pH dependent. pH controls Ca2+ homeostasis mainly by effects on the level of Ca2+ entry across the plasma membrane. On the contrary, the intracellular pH value seems to be insensitive to rap changes of Ca2+i.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Cell Line, Transformed; Cell Membrane; Cell Membrane Permeability; Dogs; Dose-Response Relationship, Drug; Fluoresceins; Fura-2; Homeostasis; Hydrogen-Ion Concentration; Kidney; Terpenes; Thapsigargin

Maintenance of calcium homeostasis is a critical activity of eukaryotic cells. Homeostatic pathways stabilize intracellular free calcium concentrations ([Ca2+]i) at the resting level and provide the source of mobilized calcium for cellular activation. We have measured calcium release from intracellular pools within bloodstream forms of Trypanosoma brucei to better understand homeostatic pathways which operate in these organisms. Fura-2 and 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein were used to quantitate [Ca2+]i and intracellular pH (pHi), respectively. We report that the tumor promoter, thapsigargin, elevated [Ca2+]i by 50-75 nM. Mn2+ quench experiments demonstrated that the source of calcium was intracellular. No change in pHi was associated with the release of calcium from this compartment. In contrast, nigericin released approximately three-fold more calcium than thapsigargin from a pH-sensitive, intracellular pool. The nigericin-sensitive pool was nonmitochondrial. The effects of thapsigargin and nigericin on [Ca2+]i were additive, regardless of the order in which the treatment was given. We conclude that at least two pools of exchangeable calcium occur in bloodstream forms of T. brucei. One pool is sensitive to thapsigargin and apparently resides within the endoplasmic reticulum, while the nigericin-sensitive pool is nonmitochondrial and is of unknown origin.

Topics: Animals; Calcium; Carcinogens; Fluoresceins; Fura-2; Hydrogen-Ion Concentration; Nigericin; Terpenes; Thapsigargin; Trypanosoma brucei brucei