Effects of dexmedetomidine on hippocampal focal adhesion kinase tyrosine phosphorylation in physiologic and ischemic conditions.
BACKGROUND
Dexmedetomidine is a potent and selective alpha2-adrenoceptor agonist that exhibits a broad pattern of actions, including sedation, analgesia, and neuroprotection. Some of these actions (e.g., neuroprotection) may require targets involved in long-term cellular changes. The authors hypothesized that dexmedetomidine increases the expression of active (autophosphorylated) focal adhesion kinase (FAK), a nonreceptor tyrosine kinase playing a pivotal role in cellular plasticity and survival. Therefore, we examined the cellular mechanisms involved in this effect and its sensitivity to oxygen-glucose deprivation (OGD) in rat hippocampal slices.


METHODS
The effects of dexmedetomidine on phospho-tyrosine FAK phosphorylation were studied first with or without various pharmacologic agents in normoxic conditions, and second in a model of pharmacologic preconditioning of slices subjected to 30 min of OGD followed by 1 h of reperfusion. FAK phosphorylation and caspase-3 activation were examined by immunoblotting. Neuronal death was assessed by propidium iodide fluorescence.


RESULTS
Dexmedetomidine produced a dose-related increase in FAK phosphorylation (187 +/- 4%, mean +/- SD, from basal level, EC50 = 0.2 microm; 95% confidence interval, 0.09-0.5 microm). This effect was stereoselective and was completely blocked by yohimbine and the combination of the cyclic monophosphate permeant analog 8 bromo cyclic monophosphate and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. It was mimicked by the protein kinase A inhibitor H 89. In contrast, prazosin and the protein kinase C inhibitors chelerythrine and bisindolylmaleimide I were ineffective. OGD induced a significant increase in immunoreactivity of the cleaved caspase-3 17-kd fragment (417 +/- 22; P < 0.001), a decrease in FAK phosphorylation (78 +/- 12% of control; P < 0.05), and production of significant neuronal death. In OGD conditions, a preconditioning application of dexmedetomidine (0.2 microm, 20-min application, 3 h before anoxia) significantly reduced neuronal death and cleaved caspase-3 expression and significantly attenuated the decrease in phosphorylated FAK content. The dexmedetomidine-induced reduction in caspase-3 expression was significantly decreased by the Src tyrosine kinase inhibitor PP2.


CONCLUSIONS
Dexmedetomidine exhibits a preconditioning effect against ischemic injury in hippocampal slices subjected to OGD. Increase in phosphorylation of FAK via stimulation of alpha2 adrenoceptors and decrease in cleaved caspase-3 expression correlate with dexmedetomidine-induced cell survival.
Dahmani
Souhayl
S
Institut National de la Santé et de la Recherche Médicale U 676, Hôpital Robert Debré, Paris, France. dahmanis@hotmail.com
Rouelle
Danielle
D
Gressens
Pierre
P
Mantz
Jean
J
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002545:Brain Ischemia / Q000201:enzymology; D053148:Caspase 3; D061945:Caspase Inhibitors; D002452:Cell Count; D016923:Cell Death; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D020927:Dexmedetomidine / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005947:Glucose / Q000172:deficiency; D006624:Hippocampus / Q000187:drug effects / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006993:Hypnotics and Sedatives / Q000494:pharmacology*; D000860:Hypoxia / Q000503:physiopathology; D019194:Ischemic Preconditioning; D008297:Male; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D017207:Rats, Sprague-Dawley; D018341:Receptors, Adrenergic, alpha-2 / Q000187:drug effects; D014443:Tyrosine / Q000378:metabolism*
D000316:Adrenergic alpha-Agonists; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D006993:Hypnotics and Sedatives; D018341:Receptors, Adrenergic, alpha-2; D014443:Tyrosine; D020927:Dexmedetomidine; D051416:Focal Adhesion Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C505410:Casp3 protein, rat; D053148:Caspase 3; D005947:Glucose