Supersensitivity of P2X receptors in cerebrocortical cell cultures after in vitro ischemia.
Neuronally enriched primary cerebrocortical cultures were exposed to glucose-free medium saturated with argon (in vitro ischemia) instead of oxygen (normoxia). Ischemia did not alter P2X7 receptor mRNA, although serum deprivation clearly increased it. Accordingly, P2X7 receptor immunoreactivity (IR) of microtubuline-associated protein 2 (MAP2)-IR neurons or of glial fibrillary acidic protein (GFAP)-IR astrocytes was not affected; serum deprivation augmented the P2X7 receptor IR only in the astrocytic, but not the neuronal cell population. However, ischemia markedly increased the ATP- and 2'-3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP)-induced release of previously incorporated [3H]GABA. Both Brilliant Blue G and oxidized ATP inhibited the release of [3H]GABA caused by ATP application; the Brilliant Blue G-sensitive, P2X7 receptor-mediated fraction, was much larger after ischemia than after normoxia. Whereas ischemic stimulation failed to alter the amplitude of ATP- and BzATP-induced small inward currents recorded from a subset of non-pyramidal neurons, BzATP caused a more pronounced increase in the frequency of miniature inhibitory postsynaptic currents (mIPSCs) after ischemia than after normoxia. Brilliant Blue G almost abolished the effect of BzATP in normoxic neurons. Since neither the amplitude of mIPSCs nor that of the muscimol-induced inward currents was affected by BzATP, it is assumed that BzATP acts at presynaptic P2X7 receptors. Finally, P2X7 receptors did not enhance the intracellular free Ca2+ concentration either in proximal dendrites or in astrocytes, irrespective of the normoxic or ischemic pre-incubation conditions. Hence, facilitatory P2X7 receptors may be situated at the axon terminals of GABAergic non-pyramidal neurons. When compared with normoxia, ischemia appears to markedly increase P2X7 receptor-mediated GABA release, which may limit the severity of the ischemic damage. At the same time we did not find an accompanying enhancement of P2X7 mRNA or protein expression, suggesting that receptors may become hypersensitive because of an increased efficiency of their transduction pathways.
Wirkner
Kerstin
K
Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany.
Köfalvi
Attila
A
Fischer
Wolfgang
W
Günther
Albrecht
A
Franke
Heike
H
Gröger-Arndt
Helke
H
Nörenberg
Wolfgang
W
Madarász
Emília
E
Vizi
E Sylvester
ES
Schneider
Dietmar
D
Sperlágh
Beáta
B
Illes
Peter
P
D015763:2-Amino-5-phosphonovalerate / Q000494:pharmacology; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000779:Anesthetics, Local / Q000494:pharmacology; D000818:Animals; D001640:Bicuculline / Q000494:pharmacology; D002110:Caffeine / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002452:Cell Count / Q000379:methods; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D003839:Deoxyadenosines / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004558:Electric Stimulation / Q000379:methods; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D016257:Fura-2 / Q000378:metabolism; D018756:GABA Antagonists / Q000494:pharmacology; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D066298:In Vitro Techniques; D007511:Ischemia / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D018613:Microscopy, Confocal / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D018408:Patch-Clamp Techniques / Q000379:methods; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013779:Tetrodotoxin / Q000494:pharmacology; D013997:Time Factors; D014227:Triazines / Q000494:pharmacology; D014316:Tritium / Q000378:metabolism; D014970:Xanthines / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D000779:Anesthetics, Local; D003839:Deoxyadenosines; D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D018756:GABA Antagonists; D005904:Glial Fibrillary Acidic Protein; C489787:MAP2 protein, rat; D008869:Microtubule-Associated Proteins; C546470:P2rx7 protein, rat; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D014227:Triazines; D014970:Xanthines; D014316:Tritium; C081320:8-(3-chlorostyryl)caffeine; C042810:N(6)-methyl-2'-deoxyadenosine; D002110:Caffeine; D013779:Tetrodotoxin; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D005680:gamma-Aminobutyric Acid; C012842:Cibacron Blue F 3GA; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D015763:2-Amino-5-phosphonovalerate; D000255:Adenosine Triphosphate; C051360:1,3-dipropyl-8-cyclopentylxanthine; D002118:Calcium; D016257:Fura-2; D001640:Bicuculline