Neuroprotective effects of tacrolimus (FK506) in a model of ischemic cortical cell cultures: role of glutamate uptake and FK506 binding protein 12 kDa.
BACKGROUND
The mechanisms underlying the neuroprotective effects of the immunosuppressant tacrolimus, observed in vivo, remain unclear. Here we quantify these effects in vitro, and evaluate the potential involvement of the glutamate and/or immunophilin FK506 binding protein 12 kDa in tacrolimus-induced neuroprotection.


METHODS
Primary cultures of neurons and astrocytes from rat cerebral cortex were subjected to transient oxygen-glucose deprivation. Neuronal injury was evaluated by cell counting after immunostaining experiments, lactate dehydrogenase release and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction. The involvement of the immunophilin FK506 binding protein 12 kDa was explored using an anti-FK506 binding protein 12 kDa antibody, (3-3-pyridyl)-1-propyl(2 s)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidine carboxylate and rapamycin. Extracellular glutamate and glutamate uptake were respectively measured by high performance liquid chromatography and l-[3H]glutamate incorporation.


RESULTS
When added during either oxygen-glucose deprivation or reoxygenation, FK506 (50-500 pM) offered significant neuroprotection. During oxygen-glucose deprivation, it was able to reverse the oxygen-glucose deprivation-induced increase in extracellular glutamate and decrease in glutamate uptake and this effect was reversed in the presence of threo-3-methyl glutamate, a specific inhibitor of glutamate transporter-1. Blocking FK506 binding protein 12 kDa inhibited the neuroprotection induced by tacrolimus added during either oxygen-glucose deprivation or reoxygenation. Tacrolimus-induced neuroprotection was also reversed in the presence of rapamycin, an immunosuppressant FK506 binding protein 12 kDa ligand devoid of neuroprotective properties and (3-3-pyridyl)-1-propyl(2 s)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidine carboxylate, a non-immunosuppressant ligand of FK506 binding protein 12 kDa, exerteing neuroprotective effects.


CONCLUSIONS
The beneficial effects of tacrolimus during in vitro ischemia/reperfusion seem to indicate the restoration of a glutamate transporter-1-mediated activity and could be mediated by a FK506 binding protein 12 kDa pathway.
Labrande
C
C
Laboratoire de Pharmacodynamie, Faculté de Pharmacie, Université de la Méditerranée, Marseille, France.
Velly
L
L
Canolle
B
B
Guillet
B
B
Masmejean
F
F
Nieoullon
A
A
Pisano
Pascale
P
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000473:pathology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D005947:Glucose / Q000172:deficiency; D018698:Glutamic Acid / Q000378:metabolism; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D009474:Neurons / Q000187:drug effects* / Q000473:pathology; D018696:Neuroprotective Agents / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000188:drug therapy*; D016559:Tacrolimus / Q000494:pharmacology*; D022021:Tacrolimus Binding Proteins / Q000187:drug effects* / Q000378:metabolism
D018696:Neuroprotective Agents; D018698:Glutamic Acid; D022021:Tacrolimus Binding Proteins; D005947:Glucose; D016559:Tacrolimus