Dominant role for calpain in thromboxane-induced neuromicrovascular endothelial cytotoxicity.

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.

Redox-dependent effects of nitric oxide on microvascular integrity in oxygen-induced retinopathy.

Opposing effects have been ascribed to nitric oxide (NO) on retinal microvascular survival. We investigated whether changes in the redox state may contribute to explain apparent conflicting actions of NO in a model of oxygen-induced retinal vasoobliteration. Retinal microvascular obliteration was induced by exposing 7-day-old rat pups (P7) for 2 or 5 days to 80% O(2). The redox state of the retina was assessed by measuring reduced glutathione and oxidative and nitrosative products malondialdehyde and nitrotyrosine. The role of NO on vasoobliteration was evaluated by treating animals with nitric oxide synthase (NOS) inhibitors (N-nitro-l-arginine; L-NA) and by determining NOS isoform expression and activity; the contribution of nitrosative stress was also determined in animals treated with the degradation catalyst of peroxynitrite FeTPPS or with the superoxide dismutase mimetic CuDIPS. eNOS, but not nNOS or iNOS, expression and activity were increased throughout the exposure to hyperoxia. These changes were associated with an early (2 days hyperoxia) decrease in reduced glutathione and increases in malondialdehyde and nitrotyrosine. CuDIPS, FeTPPS, and L-NA treatments for these 2 days of hyperoxia nearly abolished the vasoobliteration. In contrast, during 5 days exposure to hyperoxia when the redox state rebalanced, L-NA treatment aggravated the vasoobliteration. Interestingly, VEGFR-2 expression was respectively increased by NOS inhibition after short-term (2 days) exposure to hyperoxia and decreased during the longer hyperoxia exposure. Data disclose that the dual effects of NO on newborn retinal microvascular integrity in response to hyperoxia in vivo depend on the redox state and seem mediated at least in part by VEGFR-2.

Modulation of pro-inflammatory gene expression by nuclear lysophosphatidic acid receptor type-1.

Lysophosphatidic acid (LPA) is a bioactive molecule involved in inflammation, immunity, wound healing, and neoplasia. Its pleiotropic actions arise presumably by interaction with their cell surface G protein-coupled receptors. Herein, the presence of the specific nuclear lysophosphatidic acid receptor-1 (LPA1R) was revealed in unstimulated porcine cerebral microvascular endothelial cells (pCMVECs), LPA1R stably transfected HTC4 rat hepatoma cells, and rat liver tissue using complementary approaches, including radioligand binding experiments, electron- and cryomicroscopy, cell fractionation, and immunoblotting with three distinct antibodies. Coimmunoprecipitation studies in enriched plasmalemmal fractions of unstimulated pCMVEC showed that LPA1Rs are dually sequestrated in caveolin-1 and clathrin subcompartments, whereas in nuclear fractions LPA1R appeared primarily in caveolae. Immunofluorescent assays using a cell-free isolated nuclear system confirmed LPA1R and caveolin-1 co-localization. In pCMVEC, LPA-stimulated increases in cyclooxygenase-2 and inducible nitric-oxide synthase RNA and protein expression were insensitive to caveolea-disrupting agents but sensitive to LPA-generating phospholipase A2 enzyme and tyrosine kinase inhibitors. Moreover, LPA-induced increases in Ca2+ transients and/or iNOS expression in highly purified rat liver nuclei were prevented by pertussis toxin, phosphoinositide 3-kinase/Akt inhibitor wortmannin and Ca2+ chelator and channel blockers EGTA and SK&F96365, respectively. This study describes for the first time the nucleus as a potential organelle for LPA intracrine signaling in the regulation of pro-inflammatory gene expression.

PGE(2)-mediated eNOS induction in prolonged hypercapnia.

PURPOSE: Because prostaglandins (PGs) are implicated in acute hypercapnia-induced hyperemia, this study was conducted to test the hypothesis that prolonged hypercapnia may cause a sustained increase in retinal blood flow (RBF) through a PG-dependent induction of endothelial nitric oxide synthase (eNOS). METHODS: Time-dependent RBF (microsphere technique), PGE(2), nitrite (NO(2)(-)), and NOS protein (reduced nicotinamide adenine dinucleotide phosphate [NADPH]-diaphorase staining) production were measured in hypercapnia (6% CO(2))-treated piglets. From the same species, PGE(2), eNOS mRNA, NOS protein, and vasomotor responses were measured in eyecup preparations, as were Ca(2+) transients in neuroretinovascular endothelial cells. RESULTS: Hypercapnia caused biphasic (at 0.5 hours and 6-8 hours) increases in RBF that were abolished with normalization of the pH. The early phase (0.5 hour) was associated with an increase in PGE(2) levels and the latter phase (6-8 hours) with an increase in NO(2)(-) and NOS protein. Inhibition of cyclooxygenase by diclofenac prevented the early and late increase in RBF. NOS inhibitor L-nitro-arginine prevented only the latter. Hypercapnic acidosis increased retinal PGE(2) levels and eNOS-dependent vasorelaxation ex vivo. The ex vivo time course of eNOS mRNA expression corresponded with the late-phase increase in RBF and was blocked by the transcription inhibitor actinomycin D and the receptor-operated Ca(2+) channel blocker SK&F96365. In neuroretinovascular cells, acidosis increased Ca(2+) transients, which were inhibited by SK&F96365, but not diclofenac. CONCLUSIONS: This study discloses a previously unexplored mechanism for late retinal hyperemia during sustained hypercapnia that appears secondary to the induced expression of eNOS mediated by PGE(2).

Regulation of eNOS expression in brain endothelial cells by perinuclear EP(3) receptors.

We reported upregulation of endothelial nitric oxide synthase (eNOS) by PGE(2) in tissues and presence of perinuclear PGE(2) receptors (EP). We presently studied mechanisms by which PGE(2) induces eNOS expression in cerebral microvessel endothelial cells (ECs). 16,16-Dimethyl PGE(2) and selective EP(3) receptor agonist M&B28767 increased eNOS expression in ECs and the NO-dependent vasorelaxant responses induced by substance P on cerebral microvessels. These effects could be prevented by prostaglandin transporter blocker bromcresol green and actinomycin D. EP(3) immunoreactivity was confirmed on plasma and perinuclear membrane of ECs. M&B28767 increased eNOS RNA expression in EC nuclei, and this effect was augmented by overexpression of EP(3) receptors. M&B28767 also induced increased phosphorylation of Erk-1/2 and Akt, as well as changes in membrane potential revealed by the potentiometric fluorescent dye RH421, which were prevented by iberiotoxin; perinuclear K(Ca) channels were detected, and their functionality corroborated by NS1619-induced Ca(2+) signals and nuclear membrane potential changes. Moreover, pertussis toxin, Ca(2+) chelator, and channel blockers EGTA, BAPTA, and SK&F96365, as well as K(Ca) channel blocker iberiotoxin, protein-kinase inhibitors wortmannin and PD 98059, and NF-kappaB inhibitor pyrrolidine dithiocarbamate prevented M&B28767-induced increase in Ca(2+) transients and/or eNOS expression in EC nuclei. We describe for the first time that PGE(2) through its access into cell by prostaglandin transporters induces eNOS expression by activating perinuclear EP(3) receptors coupled to pertussis toxin-sensitive G proteins, a process that depends on nuclear envelope K(Ca) channels, protein kinases, and NF-kappaB; the roles for nuclear EP(3) receptors seem different from those on plasma membrane.