Serradeil-Le Gal C, Bourrié B, Raufaste D, Carayon P, Garcia C, Maffrand J P, Le Fur G, Casellas P
Sanofi Recherche, Biochimie Exploratoire, Toulouse, France.
Biochem Pharmacol. 1994 Feb 11;47(4):633-41. doi: 10.1016/0006-2952(94)90125-2.
The effects of SR 49059, a new non-peptide, selective arginine vasopressin (AVP), V1a antagonist, were investigated both on AVP's receptors and on the mitogenic effects of AVP on Swiss 3T3 fibroblasts. We characterized the AVP V1a receptors on Swiss 3T3 cell membranes using the new highly specific AVP V1a 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 Kd of 40 +/- 20 pM and a Bmax 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 V1a antagonist d(CH2)5Tyr(Me)AVP and by the synthetic V1a antagonist, SR 49059 (IC50 in the nanomolar range) while OPC-21268, another non-peptide compound, was about 100-fold less potent. Both DDAVP, a selective V2 agonist, and oxytocin exhibited low affinity (IC50 > 1 microM) in agreement with the AVP V1a nature of the site identified on Swiss 3T3 cells. In addition, the broad-spectrum antiproliferative agent [Arg6, D-Trp7,9, MePhe8] substance P (6-11), was also able to interact at 3T3 AVP V1a receptors (IC50 = 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 (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)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 G1/G0 into the S/G2M phase, as evidenced by cell cycle analysis using a cytofluorometer. In summary, SR 49059, through direct interaction at AVP V1a receptors, exerts the most potent antiproliferative effect yet described for any V1a antagonist on Swiss 3T3 cells.
研究了新型非肽类选择性精氨酸加压素（AVP）V1a拮抗剂SR 49059对AVP受体以及AVP对瑞士3T3成纤维细胞促有丝分裂作用的影响。我们使用新型高特异性的AVP V1a放射性碘化配体125I-线性AVP拮抗剂对瑞士3T3细胞膜上的AVP V1a受体进行了表征。125I-线性AVP拮抗剂的特异性结合具有饱和性、时间依赖性和可逆性。鉴定出一类高亲和力结合位点，其表观解离常数（Kd）为40±20 pM，最大结合容量（Bmax）为63±20 fmol/mg蛋白。AVP、肽类V1a拮抗剂d(CH2)5Tyr(Me)AVP和合成V1a拮抗剂SR 49059（IC50在纳摩尔范围内）均以浓度依赖性方式强力抑制125I-线性AVP拮抗剂与其受体的结合，而另一种非肽类化合物OPC-21268的效力则低约100倍。选择性V2激动剂去氨加压素（DDAVP）和催产素均表现出低亲和力（IC50>1 microM），这与在瑞士3T3细胞上鉴定出的位点的AVP V1a性质一致。此外，广谱抗增殖剂[Arg6，D-Trp7,9，MePhe8]P物质（6-11）也能够与3T3 AVP V1a受体相互作用（IC50 = 395±170 nM）。通过[3H]胸苷掺入评估，AVP对静止瑞士3T3细胞的促有丝分裂作用被SR 40959选择性、立体特异性且强力抑制（IC50 = 14±2 nM），而OPC-21268在高达220 nM时无活性。在抑制AVP诱导的DNA合成方面，SR 49059甚至比d(CH2)5Tyr(Me)AVP高效约六倍。此外，SR 49059完全抑制了瑞士3T3成纤维细胞的增殖，因为它完全阻断了AVP刺激的3T3细胞从G1/G0期进入S/G2M期的生长，这通过使用细胞荧光仪进行的细胞周期分析得到证明。总之，SR 49059通过直接与AVP V1a受体相互作用，对瑞士3T3细胞发挥了迄今为止所描述的任何V1a拮抗剂中最有效的抗增殖作用。
