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CELLULAR AND MOLECULAR

Dominant Role for Calpain in Thromboxane-Induced Neuromicrovascular Endothelial Cytotoxicity

Departments of Pediatrics and Pharmacology, Centre de Recherche de l'Hôpital Ste-Justine, Université de Montréal, Montréal, Québec, Canada (C.Q., F.S., M.H.B., D.C., I.L., S.B., F.G., M.S., A.K., P.H., A.P., S.C.); Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec, Canada (D.C., S.B., M.S., A.K., S.C.); Department of Biochemistry, Université de Montréal, Montréal, Québec, Canada (C.Q.); Department of Pharmacology, Université de Sherbrooke, Sherbrooke, Québec, Canada (F.G.); and Institut National de la Santé et Recherche Médicale, Unite 598, Paris, France (F.S.)

Thromboxane A₂ (TXA₂) is an important lipid mediator generated during oxidative stress and implicated in ischemic neural injury. This autacoid was recently shown to partake in this injury process by directly inducing endothelial cytotoxicity. We explored the mechanisms for this TXA₂-evoked neural microvascular endothelial cell death. Stable TXA₂ mimetics 5-heptenoic acid, 7-[6-(3-hydroxy-1-octenyl)-2-oxabicyclo[2.2.1]hept-5-yl]-[1R-[1,4,5(Z),6,(1E,3S)]]-9,11-dedioxy-9,11-methanolpoxy (U-46619) [as well as [1S-[1,2(Z),3(1E,3S^*),4]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.1.1]-hept-2-yl]-5-heptenoic acid; I-BOP] induced a retinal microvascular degeneration in rat pups in vivo and in porcine retinal explants ex vivo and death of porcine brain endothelial cells (in culture). TXA₂ dependence of these effects was corroborated by antagonism using the selective TXA₂ receptor blocker (-)-6,8-difluoro-9-p-methyl-sulfonyl-benzyl-1,2,3,4-tetrahydrocarbazol-1-yl-acetic acid (L670596). In all cases, neurovascular endothelial cell death was prevented by pan-calpain and specific m-calpain inhibitors but not by caspase-3 or pan-caspase inhibitors. Correspondingly, TXA₂ (mimetics) augmented generation of known active m-calpain (but not µ-calpain) form and increased the activity of m-calpain (cleavage of fluorogenic substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin; and of -spectrin into specific fragments) but not of pan-caspase or specific caspase-3 (respectively, using sulforhodamine-Val-Arg-Asp-fluoromethyl ketone and detecting its active 17- and 12-kDa fragments). Interestingly, these effects were phospholipase C (PLC)-dependent [associated with increase in inositol triphosphate and inhibited by PLC blocker 1-[6-[[17-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122 [GenBank] )] and required calcium but were not associated with increased intracellular calcium. U-46619-induced calpain activation resulted in translocation of Bax to the mitochondria, loss of polarization of the latter (using potentiometric probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide; JC-1) and in turn release of cytochrome c into the cytosol and depletion of cellular ATP; these effects were all blocked by calpain inhibitors. Overall, this work identifies (specifically) m-calpain as a dominant protease in TXA₂-induced neurovascular endothelial cell death.

Address correspondence to: Dr. Sylvain Chemtob, Department of Pediatrics, Ophthalmology, and Pharmacology, Research Centre, Ste-Justine Hospital, 3175 Cote Ste-Catherine Montreal, Quebec H3T 1C5, Canada. E-mail: sylvain.chemtob{at}umontreal.ca

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