2--7--bis-(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl-ester and Acidosis

In cholangiocarcinogenesis, chronic inflammation and oxidative stress play a key role. The Na(+)/H(+) exchanger (NHE) forms a potential link between control of intra- and pericellular pH and tumor development. Therefore, the effects of oxidant stress were determined by the use of tert-butyl hydroperoxide (t-BOOH) on Na(+)/H(+) exchange in a biliary epithelial cancer cell line (Mz-Cha-1). The cells were exposed to the hydroperoxide and the rate of recovery from acidosis was determined by the use of the pH-sensitive fluorochrome 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF/AM). t-BOOH reduced Na(+)/H(+) exchange activity in a dose-dependent manner. At 4 mM t-BOOH, Na(+)/H(+) exchange activity was virtually absent. This was accompanied by an increase in cytotoxicity (MTT assay). Glutathione repletion and intracellular Ca(++) chelation partially restored the Na(+)/H(+) exchange activity. Hydroperoxide seemed neither to alter the intracellular signal transduction pathways (cAMP and Ca(++) oscillations) nor the membrane distribution of the exchanger (immunostaining). Decrease in Na(+)/H(+) exchange activity in this model of oxidant stress may represent an early perturbation of membrane function, and the functional integrity of Na(+)/H(+) exchange could therefore be dependent on the glutathione redox system.

Topics: Acidosis; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Calcium; Cation Transport Proteins; Cell Line, Tumor; Chelating Agents; Cholangiocarcinoma; Cyclic AMP; Dose-Response Relationship, Drug; Fluoresceins; Glutathione; Humans; Oncogene Proteins, Fusion; Oxidative Stress; Proto-Oncogene Proteins c-myc; Signal Transduction; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; tert-Butylhydroperoxide

During cerebral ischemia, dysregulated glutamate release activates N-methyl-d-aspartate (NMDA) receptors which promotes excitotoxicity and intracellular acidosis. Ischemia also induces cellular adenosine (ADO) release, which activates ADO receptors and reduces neuronal injury. The aim of this research was to determine if decreasing intracellular pH (pH(i)) enhances ADO release from neurons. Rat forebrain neurons were incubated with NMDA, acetate, propionate, 5-(N)-ethyl-N-isopropyl amiloride (EIPA) or low pH buffer. pH(i) was determined with the fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and cellular release of ADO was assayed. NMDA decreased pH(i) and increased ADO release from neurons. Acetate and propionate decreased pH(i) and evoked ADO release from neurons. EIPA, an inhibitor of sodium hydrogen exchanger 1 (NHE1), enhanced the acidosis in neurons but did not enhance ADO release. Decreasing extracellular pH (pH(e)) to 6.8 or 6.45 significantly decreased pH(i) in neurons, but was not consistently associated with increased ADO release. The main finding of this study was that acidosis per se did not enhance ADO release from neurons.

Topics: Acetates; Acidosis; Adenosine; Amiloride; Analysis of Variance; Animals; Cells, Cultured; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Excitatory Amino Acid Agonists; Fluoresceins; Hydrogen-Ion Concentration; Methylamines; Models, Biological; N-Methylaspartate; Neurons; Neuroprotective Agents; Prosencephalon; Purines; Rats; Time Factors; Tritium

This study sought to investigate effects of short-chain fatty acids and CO2 on intracellular pH (pHi) and mechanisms that mediate pHi recovery from intracellular acidification in cultured ruminal epithelial cells of sheep. pHi was studied by spectrofluorometry using the pH-sensitive fluorescent indicator 2',7'-bis (carboxyethyl)-5(6')-carboxyfluorescein acetoxymethyl ester (BCECF/AM). The resting pHi in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution was 7.37 +/- 0.03. In HEPES-buffered solution, a NH4+/NH3-prepulse (20 mM) or addition of butyrate (20 mM) led to a rapid intracellular acidification (P < 0.05). Addition of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA: 10 microM) or HOE-694 (200 microM) inhibited pHi recovery from an NH4+/NH3-induced acid load by 58% and 70%, respectively. pHi recovery from acidification by butyrate was reduced by 62% and 69% in the presence of EIPA (10 microM) and HOE-694 (200 microM), respectively. Changing from HEPES-(20 mM) to CO2/HCO3(-)-buffered (5%/20 mM) solution caused a rapid decrease of pHi (P < 0.01), followed by an effective counter-regulation. 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 100 microM) blocked the pHi recovery by 88%. The results indicate that intracellular acidification by butyrate and CO2 is effectively counter-regulated by an Na+/H+ exchanger and by DIDS-sensitive, HCO3(-)-dependent mechanism(s). Considering the large amount of intraruminal weak acids in vivo, both mechanisms are of major importance for maintaining the pHi homeostasis of ruminal epithelial cells.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acidosis; Amiloride; Animals; Anti-Arrhythmia Agents; Bicarbonates; Biological Transport; Butyrates; Carbon Dioxide; Cell Culture Techniques; Cells, Cultured; Epithelial Cells; Fluoresceins; Guanidines; Hydrogen-Ion Concentration; Rumen; Sheep; Sodium-Hydrogen Exchangers; Sulfones