EFFECT OF A NEW, POTENT, NONPEPTIDE V-1A VASOPRESSIN ANTAGONIST, SR 49059, ON THE BINDING AND THE MITOGENIC ACTIVITY OF VASOPRESSIN ON SWISS 3T3 CELLS

The effects of SR 49059, a new non-peptide, selective arginine vasopre ssin (AVP), V-1a antagonist, were investigated both on AVP's receptors and on the mitogenic effects of AVP on Swiss 3T3 fibroblasts. We char acterized the AVP V-1a receptors on Swiss 3T3 cell membranes using the new highly specific AVP V-1a radioiodinated ligand, I-125-linear AVP antagonist. Specific binding of the I-125- linear AVP antagonist was s aturable, time-dependent and reversible. A single class of high affini ty binding sites was identified with an apparent K-d of 40 +/- 20 pM a nd a B-max of 63 +/- 20 fmol/mg protein. I-125-Linear AVP antagonist b inding to its receptors was potently inhibited in a concentration-depe ndent manner by AVP, by the peptide V-1a antagonist d(CH2)(5)Tyr(Me)AV P and by the synthetic V-1a antagonist, SR 49059 (IC50 in the nanomola r range) while OPC-21268, another non-peptide compound, was about 100- fold less potent. Both DDAVP, a selective VI agonist, and oxytocin exh ibited low affinity (IC50 > 1 mu M) in agreement with the AVP V-1a nat ure of the site identified on Swiss 3T3 cells. In addition, the broad- spectrum antiproliferative agent [Arg(6), D-Trp(7,9), MePhe(8)] substa nce P (6-11), was also able to interact at 3T3 AVP V-1a receptors (IC5 0 = 395 +/- 170 nM). The mitogenic effects of AVP on quiescent Swiss 3 T3 cells, assessed through [H-3]thymidine incorporation, were selectiv ely, stereospecifically and strongly inhibited by SR 40959 (IC50 = 14 +/- 2 nM) while OPC-21268 was inactive up to 220 nM. SR 49059 was even about six times more efficient than d(CH2)(5)Tyr(Me)AVP in inhibiting AW-induced DNA synthesis. Moreover, SR 49059 fully inhibited Swiss 3T 3 fibroblast proliferation since it completely blocked AVP-stimulated 3T3 cell growth from the G1/G0 into the S/G(2)M phase, as evidenced by cell cycle analysis using a cytofluorometer. In summary, SR 49059, th rough direct interaction at AVP V-1a receptors, exerts the most potent antiproliferative effect yet described for any V-1a antagonist on Swi ss 3T3 cells.