9509289 Narayan The importance of endothelin-l (ET-l) as a potent vasoconstrictor and mitogen in cardiovascular and pulmonary systems has become well established. The presence of both endothelin and endothelin binding sites in the brain also has been documented; however, the role of endothelin in the central nervous system is obscure. The overall objective of this research is to elucidate, at the molecular level, the role of endothelin and its receptor(s) in the central nervous system. Competitive binding experiments using endothelin receptor agonists and antagonists with SCP cells suggest the presence of two classes of binding sites for ET-l: a high affinity site with an ICso of 0.9 nM and a super-high affinity site with an ICso of 0.1 pM. Both binding sites exhibit the pharmacological characteristics of the ETA receptor subtype, but the super-high affinity binding site is unique in that ligand-receptor interactions with this extremely high affinity have not been previously documented. The results of the binding studies suggest that the SCP cells contain two distinct receptor molecules encoded by two separate mRNAs or that a single receptor molecule can exist in two affinity states. The immediate goal of this research is to clone the normal, high affinity as well as the novel, super-high affinity receptor cDNAs from SCP cells. The working hypothesis is that this novel receptor maintains sufficient homology with the normal ETA receptor to facilitate the cloning. The cloned receptor will be expressed in a heterologous cell line and its ligand binding properties will be determined. The predicted results are that two clones that share sequence homology and exhibit different binding affinities will be obtained or that one clone that yields two binding affinities will be produced. With the receptor clone(s) in hand, it will be possible to examine the distribution of the receptor in different parts of the brain and cell types within the brain. This information will be extremely valuable in determin ing the physiological role and possibly the mode of action of endothelin in the central nervous system. %%% The imortance of endothelin-l (ET-l) as a potent vasoconstrictor and mitogen in cardiovascular and pulmonary systems has become well established. The presence of both endothelin and endothelin binding sites in the brain also has been documented; however, the role of endothelin in the central nervous system is obscure. The objective of this research is to begin to elucidatethe role of endothelin and its receptor(s) in the central nervous system by cloning the normal, high affinity as well as the novel, super-high affinity endothelin receptor cDNAs from sheep choroid plexus (SCP) cells. With the receptor clone(s) in hand, it will be possible to examine the distribution of the receptor in different parts of the brain and cell types within the brain. This information will be extremely valuable in determining the physiological role and possibly the mode of action of endothelin in the central nervous system. ***
