Effect of a new, potent, non-peptide V 1a vasopressin antagonist, SR 49059, on the binding and the mitogenic activity of vasopressin on swiss 3T3 cells

Abstract

The effects of SR 49059, a new non-peptide, selective arginine vasopressin (AVP), V 1a, antagonist, were investigated both on AVP's receptors and on the mitogenic effects of AVP on Swiss 3T3 fibroblasts. We characterized the AVP V 1a, receptors on Swiss 3T3 cell membranes using the new highly specific AVP V 1a, radioiodinated ligand, 125I-linear AVP antagonist. Specific binding of the 125I-linear AVP antagonist was saturable, time-dependent and reversible. A single class of high affinity binding sites was identified with an apparent K d of 40 ± 20 pM and a B max of 63 ± 20 fmol/mg protein. 125I-Linear AVP antagonist binding to its receptors was potently inhibited in a concentration-dependent manner by AVP, by the peptide V 1Aa antagonist d(CH 2)5Tyr(Me)AVP and by the synthetic V 1a antagonist, SR 49059 ( ic 50 in the nanomolar range) while OPC-21268, another non-peptide compound, was about 100-fold less potent. Both DDAVP, a selective V 2 agonist, and oxytocin exhibited low affinity ( ic 50 > 1 μ M) in agreement with the AVP V 1a, nature of the site identified on Swiss 3T3 cells. In addition, the broad-spectrum antiproliferative agent [Arg 6spd D-Trp7' 7,9, MePhe 8] substance P (6–11), was also able to interact at 3T3 AVP V 1a, receptors ( ic 50 = 395 ± 170 nM) . The mitogenic effects of AVP on quiescent Swiss 3T3 cells, assessed through [ 3H]thymidine incorporation, were selectively, stereospecifically and strongly inhibited by SR 40959 ( ic 50 = 14 ± 2 nM) while OPC-21268 was inactive up to 220 nM. SR 49059 was even about six times more efficient than d(CH 22) 5Tyr(Me)AVP in inhibiting AVP-induced DNA synthesis. Moreover, SR 49059 fully inhibited Swiss 3T3 fibroblast proliferation since it completely blocked AVP-stimulated 3T3 cell growth from the Gl/GO into the S/G 2M phase, as evidenced by cell cycle analysis using a cytofluorometer. In summary, SR 49059, through direct interaction at AVP V 1a receptors, exerts the most potent antiproliferative effect yet described for any V[1a] antagonist on Swiss 3T3 cells.