thapsigargin and trimethylamine

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

This study focuses on the potential interrelationships between changes in pH and capacitative calcium entry in stimulated platelets and on the participation of SOCs in the control of intracellular pH (pH(i)). Extracellular acidification reduces the Mn(2+) entry, measured by the slope of the quenching of FURA 2 fluorescence at the isoemissive wavelength of 360 nm. In thrombin-stimulated platelets Mn(2+) entry is reduced by acidosis (pH(o) = 6.89) to 17 +/- 4% of control (pH(o) = 7.32). In platelets treated with thapsigargin (TG) to induce the opening of store-operated channels (SOCs) the rate of quenching was reduced by acidosis to 31 +/- 5 % of control. Calcium entry was measured as the peak of [Ca(2+)](i) response to extracellular calcium readmission after mobilization of calcium from intracellular stores. Changes in pH(o) of platelet suspension media markedly alters the calcium entry evoked by thrombin that reach a 16 +/- 6 % of control in acidosis (pH(o) = 6.89) and 150 +/- 15% of control in alkalosis (pH(o) = 7.62). The SERCA inhibitor TG was used to study the effect of pH(o) on Ca(2+) influx. Acidosis decreases and alkalosis increases the capacitative calcium entry to 22 +/- 4 % and 129 +/- 1% of control respectively. These changes in pH(o) also produced changes in pH(i). Treatment of platelets with titrated solutions of trimethylamine causes intracellular alkalinization without changes in pH(o) increasing the capacitative calcium entry to 120 +/- 5%. TG itself produces an intracellular alkalinization that is further increased by calcium entry. Blockage of the Na(+)/H(+) exchanger reverted TG effect on pH(i) without changes in capacitative calcium entry.

Topics: Blood Platelets; Calcium; Cell Membrane; Electric Capacitance; Fura-2; Humans; Hydrogen-Ion Concentration; Manganese; Methylamines; Sodium-Hydrogen Exchangers; Spectrometry, Fluorescence; 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