Novel vasopressin receptor ligands.

Studies indicating a diversity of receptors for the hormone [8-arginine]vasopressin (AVP) have benefitted from the development of a range of selective AVP analogues [l]. One such ligand, [1-(/3-mercapto-/3,/3-cyclopentamethylenepropionic acid, [2]) is a highly potent and selective antagonist for the Vla receptor subtype expressed by liver, smooth muscle, brain and WRK 1 cells. We have synthesized a novel ligand, namely ~ ( C H ~ ) ~ T ~ K ( M ~ ) ~ L ~ S N H ~ ~ A V P (ALVP) which is amenable to the attachment of functional groups onto the L ~ S N H ~ ~ c-amino group via reaction with N-Hydroxysuccinimide (NHS) esters. Using this strategy we have produced the biotinylated Vla-selective antagonist d(CH2) gTyr(Me) 2Lys (Nc -biotinamido~aproate)NH2~AVP (ALBtnAVP). ALVP was prepared using solid phase synthesis with subsequent disulphide bond formation by air oxidation and purification by reverse phase h.p.1.c. ALBtnVP was produced by reacting ALVP with biotinamidocaproate NHS ester (Sigma) followed by purification using h.p.1.c. Rat liver membranes containing the Vla receptor were made as previous described [3]. Bovine kidney medullary membranes containing V2 receptors were prepared essentially as in [4] except that buffers contained bacitracin (lmg/ml) and the final preparation was frozen and stored in the Hepes based buffer used for the production of liver membranes [3]. Competition binding assays used [ 3H]d(CH2)5Tyr(Me)2AVP (56.2 Ci/mmol) as the tracer ligand for the Via receptor (kd 0.28nM) and [3H]AVP (67.7Ci/mmol) for the v2 receptor (kd 0.68nM) as previously reported [3]. Glycogen phosphorylase a was assayed in freshly isolated rat hepatocytes using [14C] glucose-lphosphate [3]. To measure inositol lipid breakdown WRK 1 cells maintained and equilibrium labelled with my0-[2-~H] inositol [5] were stimulated with AVP and accumulated inositol phosphates extracted and separated by ion exchange chromatography [6]. 2 0-methyltyrosine) ] AVP (d( CH2) 5Tyr (Me) 2AVP,