RESUMEN
Previous studies have demonstrated that functional interaction between endothelin (ET)-1 and nitric oxide (NO) involves changes in Ca(2+) mobilization and cytoskeleton in human brain microvascular endothelial cells. The focus of this investigation was to examine the possible existence of analogous interplay between these vasoactive substances and elucidate their signal transduction pathways in human brain capillary endothelial cells. The results indicate that ET-1-stimulated Ca(2+) mobilization in these cells is dose-dependently inhibited by NOR-1 (an NO donor). This inhibition was prevented by ODQ (an inhibitor of guanylyl cyclase) or Rp-8-CPT-cGMPS (an inhibitor of protein kinase G). Treatment of endothelial cells with 8-bromo-cGMP reduced ET-1-induced Ca(2+) mobilization in a manner similar to that observed with NOR-1 treatment. In addition, NOR-1 or cGMP reduced Ca(2+) mobilization induced by mastoparan (an activator of G protein), inositol 1,4,5-trisphosphate, or thapsigargin (an inhibitor of Ca(2+)-ATPase). Interestingly, alterations in endothelial cytoskeleton (actin and vimentin) were associated with these effects. The data indicate for the first time that the cGMP-dependent protein kinase colocalizes with actin. These changes were accompanied by altered levels of phosphorylated vasodilator-stimulated phosphoprotein, which were elevated in endothelial cells incubated with NOR-1 and significantly reduced by ODQ or Rp-8-CPT-cGMPS. The findings indicate a potential mechanism by which the functional interrelationship between ET-1 and NO plays a role in regulating capillary tone, microcirculation, and blood-brain barrier function.
Asunto(s)
Encéfalo/irrigación sanguínea , Circulación Cerebrovascular/fisiología , Endotelina-1/metabolismo , Endotelio Vascular/metabolismo , Óxido Nítrico/metabolismo , Actinas/efectos de los fármacos , Actinas/metabolismo , Arginina/farmacología , Encéfalo/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Moléculas de Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Proteínas Quinasas Dependientes de GMP Cíclico/efectos de los fármacos , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Relación Dosis-Respuesta a Droga , Antagonistas de los Receptores de Endotelina , Endotelina-1/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Inmunohistoquímica , Proteínas de Microfilamentos , Donantes de Óxido Nítrico/farmacología , Fosfoproteínas/efectos de los fármacos , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Receptor de Endotelina A , Receptores de Endotelina/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Reactive oxygen species (ROS) were shown to play a role in altering blood-brain barrier (BBB) permeability and formation of brain edema induced by trauma and/or ischemia. 2-arachidonoyl glycerol (2-AG), a novel, potent vasodilatory and cytoprotective endocannabinoid has been implicated to act as an antioxidative agent. This study examines: 1) the possible 2-AG modulation of BBB injury and edema formation induced by closed head injury (CHI); and 2) comparable effects between 2-AG and 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TPL), a known antioxidant nitroxide on endothelial Ca2+ and cytoskeletal responses to H2O2 (ROS). 2-AG treatment reduced the CHI-induced increase in BBB permeability and brain edema. The endothelial H2O2-stimulated Ca2+ mobilization and cytoskeleton (vimentin) rearrangement was modified by either 2-AG or TPL. These findings provide evidence of 2-AG antioxidant activity and are consistent with the involvement of ROS in the pathomechanism of CHI-induced BBB injury and brain edema.
