6-cyano-7-nitroquinoxaline-2-3-dione and 2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein

Intracellular pH (pH(i)) is an important factor for understanding cellular processes associated with the response of central neurons to metabolic disturbances such as anoxia or ischemia. In the present study, pH(i) was fluorometrically measured in 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescin (BCECF)-filled, voltage-clamped dorsal vagal neurons (DVN) of brainstem slices from rats during metabolic disturbances activating ATP-sensitive K(+) (K(ATP)) channels. Chemical anoxia induced by cyanide, rotenone or p-trifluoromethoxy-phenylhydrazone (FCCP) decreased pH(i) by >0.4 pH units. Untreated neurons with normal pH(i) baseline (7.2) responded to glucose-free superfusate after a delay of 7-16 min with a progressive fall of pH(i). In contrast, pH(i) increased by >0.2 pH units after approximately 10 min in cells that had a mean pH(i) of 6.8 due to incomplete recovery from a CN(-)induced acid load prior to glucose depletion. Metabolic arrest, induced by cyanide in glucose-free solution after 30 min preincubation in glucose-free saline, caused a progressive glutamate-mediated inward current with no change of pH(i). Upon metabolic arrest, depolarization-evoked pH(i) decreases ( approximately 0.2 pH units) were abolished, whereas glucose-free superfusate slightly delayed their recovery without major effects on amplitude. The glucose-dependent pH(i) fall coincided with activation of the K(ATP) channel-mediated outward current, while K(ATP) currents due to anoxia or metabolic arrest could reach their maximum in the absence of a major pH(i) change. The results indicate that the anoxic pH(i) decrease is due to enhanced glycolysis and lactate formation with often no obvious effect on K(ATP) channel activity. The origin of glucose-dependent acidosis and its relation to K(ATP) channel activity remain to be determined.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Brain Stem; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cyanides; Drug Interactions; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; Fluoresceins; Glucose; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Intracellular Space; Male; Membrane Potentials; Membrane Proteins; Neurons; Patch-Clamp Techniques; Potassium Channels; Rats; Rotenone; Time Factors; Vagus Nerve; Valine

The effect of chemical anoxia (azide) in the presence of glucose on the free intracellular Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) in mouse neocortical neurons was investigated using Fura-2 and BCECF. Anoxia induced a reversible increase in [Ca2+]i which was significantly inhibited in nominally Ca2+-free medium. A change in pHo (8.2 or 6.6), or addition of NMDA and non-NMDA receptor antagonists (D-AP5 and CNQX) in combination, significantly reduced the increase in [Ca2+]i, pointing to a protective effect of extracellular alkalosis or acidosis, and involvement of excitatory amino acids. An initial anoxia-induced acidification was observed under all experimental conditions. In the control situation, this acidification was followed by a recovery/alkalinization of pHi in about 50% of the cells, a few cells showed no recovery, and some showed further acidification. EIPA, an inhibitor of Na+/H+ exchangers, prevented alkalinization, pointing towards anoxia-induced activation of a Na+/H+ exchanger. In a nominally Ca2+-free medium, the initial acidification was followed by a significant alkalinization. At pHo 8.2, the alkalinization was significantly increased, while at pHo 6.2, the initial acidification was followed by further acidification in about 50% of the cells, and by no further change in the remaining cells.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Amiloride; Animals; Calcium; Cell Hypoxia; Cells, Cultured; Excitatory Amino Acid Antagonists; Extracellular Space; Female; Fluoresceins; Fura-2; Glucose; Hydrogen-Ion Concentration; Mice; Mice, Inbred Strains; Neocortex; Neurons; Sodium Azide; Sodium-Hydrogen Exchangers