In vivo studies:Previously, we demonstrated that hypoperfusion injury of the hippocampus CA1 region following transient global ischemia is ameliorated by postischemic treatment with the endothelin (ETA) receptor antagonist Ro 61-1790. Present investigations evaluated the effect of Ro 61-1790 in SHR subjected to middle cerebral artery occlusion for 24 hr. The findings indicate that Ro 61-1790 significantly attenuated the severe reductions in perfusion observed in control rats following focal cerebral ischemia. Furthermore, the improvement in perfusion was associated with a significant decrease in total lesion volume that can be maintained 24 hr postischemia. These results strongly suggest a causative relationship between preservation of tissue perfusion and prevention of tissue injury. These findings support the hypothesis that selective blockade of ETA receptor-mediated vasoconstriction helps to sustain tissue perfusion in peri- ischemic/penumbral tissue at a level sufficient to maintain tissue viability.In vitro studies:The investigations have been designed to elucidate the putative mechanisms that might be involved in the pathogenic responses observed in ischemia/reperfusion injury in vivo. For the present study, endothelium (EC) derived from human brain capillaries was used to characterize the postreceptor pathway and signal transduction mechanisms involved in the calcium response to nitric oxide (NO) and endothelin-1 (ET-1). ET-1 dose-dependently stimulated intracellular calcium accumulation through activation of ETA receptors. Inhibition of nitric oxide synthase (NOS) activity doubled the level of ET-1-induced intracellular calcium accumulation. Pretreatment with 0.1-50 uM Nor-1 (NO donor) dose-dependently inhibited (29.4-67.8%, respectively) ET-1-induced calcium accumulation. Comparable inhibition was observed in EC pretreated with 8-bromo-cGMP, implicating cGMP in the inhibitory pathway. Pretreatment with ODQ, an inhibitor of guanylyl cyclase, prevented the Nor-1 effect which supports this contention. An analogous response observed in EC pretreated with Rp-8-pCPT-cGMPS, an inhibitor of protein kinase G, indicates that this event is medaited through the cGMP/kinase system. Similar to ET-1, Mas7 (activator of GTP-binding protein), IP3, and thapsigargin (inhibitor of Ca-ATPase) also stimualted intracellular calcium accumulation; Nor-1 and cGMP also inhibited these effects. In addition, both NO and ET-1 induced changes in the cytoskeletal arrangements of F-actin and vimentin.These findings represent the first demonstration of an autocrine/paracrine relationship between NO and ET- 1 in capillary endothelium. This interaction occurs at the level of the ET receptor and downstream second messenger pathway leading to rapid changes in intracellular calcium. These results indicate that NO is involved in abrogating the responses to ET-1. In addition, simultaneous changes in the cytoskeleton are observed; however, the precise mechanism remains to be clarified. - endothelial cells, NO, ET-1, calcium accumulation, signal transduction, cytoskeleton, BBB, stroke, cerebral ischemia