Endothelium, which is controlled by a variety of circulating and locally released mediators plays a central role in regulating vascular tone and blood flow. Nitric oxide (NO) and endothelin-1 (ET-1) are the most potent endothelium-derived vasoactive substances which have been considered as main contributors in maintaining vascular tone and hemostasis. Studies both in vitro and in vivo showed that a functional interaction between NO and ET-1 exists at the level of ET-1 receptors. Micovascular endothelial cells (HBMEC) were isolated from human brain tissue which was removed for treatment of idiopathic epilepsy. HBMEC were <95% Factor VIII-positive and were grown to confluence on 24-well plates, washed and incubated with a fluorescent calcium indicator (2.5 uM fluo-3/A) for 90 min at 37 deg C. The effects of ET-1 on calcium accumulation in capillary EC cultures were measured in the presence or absence of NOR-1. Staining with Texas Red-X phalloidin was used to assess changes in F-actin cytoskeleton. ET-1 stimulated intracellular calcium accumulation; selective ETA (but not ETB) receptor antagonists inhibited this effect. The inhibition of nitric oxide synthase with L-NAME doubled the ET-1-induced calcium accumulation. Pretreatment with the NO donor, NOR-1 (0.1-50 uM) dose-dependently decreased calcium accumulation induced by 20 nM ET-1. Reduced calcium accumulation was also observed with 8-bromo-cGMP, implicating cGMP in this response. ODQ, an inhibitor of guanylyl cyclase, prevented NOR-1 from reducing the ET-1-stimulated accumulation of calcium. The pretreatment of EC with Rp-8-pCPT-cGMPS, an inhibitor of protein kinase G (PKG), exhibited a similar response. In addition, in capillary EC, both ET-1 and NO induced rearrangement of F-actin filaments. The precise mechanism responsible for this effect is currently being investigated. These separate and joint responses of NO and ET-1 inducate that EC have the intrinsic machinery and capacity needed to control and contribute to the regulation of microvascular tone, microcirculation, and blood-brain barrier function. These results indicate a role for the cGMP/PKG system in the cross-talk between NO and ET-1. These experiuments are currently being performed with capillary EC and are comparing the responses obtained with microvascular EC. In addition, these studies are being extended to include examination of the effect of hypoxia-ischemia on the above parameters as well as other indicators associated with the pathophysiologic response.
