Increases in [Ca2+]i and changes in intracellular pH during chemical anoxia in mouse neocortical neurons in primary culture.
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.
Jørgensen
N K
NK
Biochemical Department, The August Krogh Institute, Copenhagen, Denmark. nkjorgensen@yahoo.com
Petersen
S F
SF
Damgaard
I
I
Schousboe
A
A
Hoffmann
E K
EK
D015763:2-Amino-5-phosphonovalerate / Q000494:pharmacology; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015687:Cell Hypoxia / Q000502:physiology*; D002478:Cells, Cultured; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005452:Fluoresceins; D016257:Fura-2; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008815:Mice, Inbred Strains; D019579:Neocortex / Q000166:cytology / Q000187:drug effects / Q000196:embryology / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019810:Sodium Azide / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000502:physiology
D018691:Excitatory Amino Acid Antagonists; D005452:Fluoresceins; D017923:Sodium-Hydrogen Exchangers; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D015763:2-Amino-5-phosphonovalerate; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D019810:Sodium Azide; D005947:Glucose; D002118:Calcium; D016257:Fura-2; C039614:ethylisopropylamiloride