Abstract
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-[1α,4α,5β(Z),6α,(1E,3S)]]-9,11-dedioxy-9α,11α-methanolpoxy (U-46619) [as well as [1S-[1α,2α(Z),3β(1E,3S*),4α]]-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 α-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-[[17β-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 TXA2-induced neurovascular endothelial cell death.
Footnotes
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This study was supported in parts by grants from the Canadian Institute of Health Research, the Heart and Stroke Foundation of Québec, and the March of Dimes. C.Q. is a recipient of a scholarship from the Heart and Stroke Foundation of Canada. F.G. is a recipient of a Junior 1 scholarship from and a researcher of the Canada Foundation for Innovation. S.C. is a Canada Research Chair recipient.
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doi:10.1124/jpet.105.093898.
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ABBREVIATIONS: TXA2, thromboxane A2; TP, thromboxane receptor; Calp, calpain; U-46619, 5-heptenoic acid, 7-[6-(3-hydroxy-1-octenyl)-2-oxabicyclo[2.2.1]hept-5-yl]-[1R-[1α,4α,5β(Z),6α,(1E,3S)]]-9,11-dedioxy-9α,11α-methanolpoxy; IBOP, [1S-[1α,2α(Z),3β(1E,3S*),4α]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.1.1]-hept-2-yl]-5-heptenoic acid; L670596, (-)-6,8-difluoro-9-p-methylsulfonyl-benzyl-1,2,3,4-tetrahydrocarbazol-1-yl-acetic acid; MTT, 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide; U73122, 1-[6-((17β-3-methoxyestro-1,3,5(10)-trien-17-yl)-amino)hexyl]-1H-pyrrole-2,5-dione; TRITC, tetramethylrhodamine B isothiocyanate; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; PI, propidium iodide; LDH, lactate dehydrogenase; SR, sulforhodamine; AMC, 7-amino-4-methylcoumarin; [Ca2+]i, intracellular calcium concentration; PKC, protein kinase C; Z, N-benzyloxycarbonyl; Suc-LLVY-AMC, N-succinyl-Leu-Leu-Val-Tyr-7-AMC; DEVD, Asp-Glu-Val-Asp; fmk, fluoromethyl ketone; VAD, Val-Arg-Asp.
- Received August 9, 2005.
- Accepted October 6, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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