title: Dominant role for calpain in thromboxane-induced neuromicrovascular endothelial cytotoxicity.
abstract: Thromboxane A(2) (TXA(2)) 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 TXA(2)-evoked neural microvascular endothelial cell death. Stable TXA(2) mimetics 5-heptenoic acid, 7-[6-(3-hydroxy-1-octenyl)-2-oxabicyclo[2.2.1]hept-5-yl]-[1R-[1alpha,4alpha,5beta(Z),6alpha,(1E,3S)]]-9,11-dedioxy-9alpha,11alpha-methanolpoxy (U-46619) [as well as [1S-[1alpha,2alpha(Z),3beta(1E,3S(*)),4alpha]]-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). TXA(2) dependence of these effects was corroborated by antagonism using the selective TXA(2) 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, TXA(2) (mimetics) augmented generation of known active m-calpain (but not mu-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 alpha-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-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122)] 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 TXA(2)-induced neurovascular endothelial cell death.
authors.affiliation: Department of Pediatrics, Centre de Recherche de l'Hôpital Ste-Justine, Université de Montréal, Québec, Canada.
authors.forename: Christiane
authors.initials: C
authors.lastname: Quiniou
authors.forename: Florian
authors.initials: F
authors.lastname: Sennlaub
authors.forename: Martin H
authors.initials: MH
authors.lastname: Beauchamp
authors.forename: Daniella
authors.initials: D
authors.lastname: Checchin
authors.forename: Isabelle
authors.initials: I
authors.lastname: Lahaie
authors.forename: Sonia
authors.initials: S
authors.lastname: Brault
authors.forename: Fernand
authors.initials: F
authors.lastname: Gobeil
authors.forename: Mirna
authors.initials: M
authors.lastname: Sirinyan
authors.forename: Amna
authors.initials: A
authors.lastname: Kooli
authors.forename: Pierre
authors.initials: P
authors.lastname: Hardy
authors.forename: Alexey
authors.initials: A
authors.lastname: Pshezhetsky
authors.forename: Sylvain
authors.initials: S
authors.lastname: Chemtob
mesh_terms: D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000378:metabolism / Q000502:physiology*; D002227:Carbazoles / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D007295:Inositol Phosphates / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D008833:Microcirculation / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000037:antagonists & inhibitors*; D012162:Retinal Degeneration / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D012171:Retinal Vessels / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology
chemical_list: D002227:Carbazoles; D007295:Inositol Phosphates; D044244:Receptors, Thromboxane A2, Prostaglandin H2; C061299:L 670596; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D002154:Calpain; D002118:Calcium