Endothelin 1 (ET-1), the most potent vasoconstrictive peptide among other agents has been implicated in cerebrovascular disorders (e.g., vasospasm, hypertension, and/or ischemia). Recently we demonstrated a functional interaction between ET-1 and nitric oxide (NO) manifested in postischemic cerebral hypoperfusion. Since these studies suggested that the interplay between ET-1 and NO occurs at the level of the endothelial ETA receptor, we investigated the effect of NO on ET-1 receptor binding sites in the endothelium obtained from human brain microvessels (HBMEC). ET-1 binding experiments were performed on HBMEC cultivated in 96-well microtiter plates in the presence or absence of novel NO donors: NOR1, NOR3 or NOR4 (half-life 1.8, 30, and 60 min, respectively) in medium 199 containing 0.1% bovine serum albumin for 4 hours at 37oC. The nonspecific binding was determined in the presence of ET-1 receptor antagonist, BQ123 (1 uM). NOR was the only NO donor which dose- dependently decreased [125] ET-1 binding to HBMEC. The greatest reduction (28.4%) in [I 125]ET-1 binding sites was observed at a concentration of 50 uM of NOR 3 (p less than 0.05). The Scatchard analysis of saturation binding data revealed that NOR 3 significantly decreased the binding capacity (Bmax) by about 20% (40. plus or minus 2.4 vs controls 50.0 plus or minus 5.6 fmol/mg protein and apparent dissociation constant (KD) by 25% (30.8 plus/minus 2.2 versus controls 40.2 plus/minus 5.1 pM). The findings indicate that the known and unique resistance of ET-1 to dissociate from its ligand receptor complex can be affected by NO. Furthermore, the results indicate that NOR down- regulates and sensitizes ETA receptors on HBMEC. These findings support the existence of interactions between NO and ET-1 and indicate the possible involvement of the ETA-receptor site in at least one of the vascular responses to these vasoactive substances.
