Involvement of P2X7 receptors in the hypoxia-induced death of rat retinal neurons.
OBJECTIVE
To investigate the hypoxia-induced death of rat retinal neurons and to determine whether P2X(7) activation is involved in this type of neuronal death.


METHODS
Cultured retinal neurons from fetal rats were used. The effects and time course of various degrees of hypoxia (1%-5% O(2)) in the death of retinal neurons, were examined. The effects of P2X(7) antagonists, oxidized adenosine triphosphate (oxidized ATP; 30-100 microM), and brilliant blue G (BBG; 100 nM-10 microM) on hypoxia-induced neuronal death, including apoptosis, were assessed by using trypan blue exclusion, TUNEL assays, and cleaved caspase-3 immunoreactivity. Immunocytochemical analysis was performed to determine whether these neurons express P2X(7) receptors. The effects of P2X(7) receptor stimulation, induced by the P2X(7) agonist benzoyl- benzoyl-ATP (BzATP), on neuronal viability and intracellular Ca(2+) levels ([Ca(2+)](i)) were examined.


RESULTS
Retinal neuronal death increased according to the degree of hypoxia and became more severe after 12 hours. Both oxidized ATP and BBG significantly decreased hypoxia-induced neuronal death. Immunocytochemistry demonstrated that P2X(7) receptors were expressed by the cultured retinal neurons. ATP and BzATP caused P2X(7) receptor-dependent neuronal death in a dose-dependent manner and led to a sustained increase in [Ca(2+)](i), with BzATP being more effective than ATP. These effects were hypoxia-induced factor-1alpha- independent and were prevented by oxidized ATP.


CONCLUSIONS
The results suggest that the death of retinal neurons can be triggered by hypoxia and that P2X(7) activation is involved in the hypoxia-induced death of retinal neurons. P2X(7) antagonists can prevent hypoxia-induced damage in retinal neurons.
Sugiyama
Tetsuya
T
Department of Ophthalmology, Osaka Medical College, Takatsuki, Osaka, Japan. tsugiyama@poh.osaka-med.ac.jp
Oku
Hidehiro
H
Shibata
Maho
M
Fukuhara
Masayuki
M
Yoshida
Hideyo
H
Ikeda
Tsunehiko
T
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005260:Female; D000860:Hypoxia / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors; D020287:In Situ Nick-End Labeling; D011247:Pregnancy; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D051381:Rats; D017208:Rats, Wistar; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058486:Receptors, Purinergic P2X7; D055351:Retinal Neurons / Q000378:metabolism* / Q000473:pathology*; D012394:Rosaniline Dyes / Q000494:pharmacology
C497444:Hif1a protein, rat; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C546470:P2rx7 protein, rat; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D012394:Rosaniline Dyes; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; C017199:2',3'-dialdehyde ATP; D000255:Adenosine Triphosphate; D053148:Caspase 3; C004692:coomassie Brilliant Blue; D002118:Calcium