Viguerie N, Tahiri-Jouti N, Ayral A M, Cambillau C, Scemama J L, Bastié M J, Knuhtsen S, Estève J P, Pradayrol L, Susini C
INSERM U 151, CHU Rangueil, Toulouse, France.
Endocrinology. 1989 Feb;124(2):1017-25. doi: 10.1210/endo-124-2-1017.
Somatostatin has been demonstrated to negatively regulate pancreatic growth in vivo. In this study we used the AR4-2J rat pancreatic acinar tumor cell line to investigate the effect of a stable somatostatin analog, SMS 201-995 (SMS) on cell proliferation. SMS induced an antiproliferative effect on both serum or epidermal growth factor (EGF)-induced cell proliferation; exposure of the cells for 48 h to SMS caused a slight inhibition of serum-induced proliferation (maximal inhibition, 26%) and abolished the growth-promoting effect of EGF. Maximal effect was observed with 10 nM SMS, and half-maximal (IC50) effect with 0.06-0.1 nM SMS. Binding studies with an iodinated derivative of SMS, [125I-Tyr3]SMS, revealed the presence of a single class of high affinity binding sites on AR4-2J plasma membranes with an equilibrium dissociation constant of 0.2 +/- 0.03 nM and a binding site number of 1.1 +/- 0.07 pmol/mg protein. Addition of the nonhydrolyzable GTP analog, guanosine 5-[gamma-thio] triphosphate (GTP gamma S), increased the rate of dissociation of the specifically bound peptide in agreement with the coupling of somatostatin receptors with a GTP-binding regulatory protein. The good agreement between the IC50 for SMS inhibition of cell proliferation and the apparent Kd for binding indicates that the characterized binding sites are the somatostatin receptors that mediate the antiproliferative effect of SMS. When cells were grown in serum-free medium EGF stimulated AR4-2J cell proliferation with half-maximal (ED50) and maximal effects at 0.6 and 10 nM EGF, respectively. This stimulatory effect of EGF was mediated by specific receptors, since binding studies with [125I]EGF indicated that AR4-2J cells contained a single class of EGF receptors (13,000 sites/cell), with an affinity constant for [125I]EGF (Kd = 0.9 +/- 0.09 nM) close to the ED50 for EGF stimulation of cell growth. To examine if SMS-induced growth inhibition involved a cAMP-dependent mechanism we first studied the effect of SMS on cAMP production. SMS had no effect on basal cAMP, but completely inhibited VIP-stimulated cAMP production with an IC50 of 0.2 nM. Pertussis toxin, which is known to abolish the inhibitory effect of somatostatin on adenylate cyclase activity in AR4-2J cells, did not reverse the ability of SMS to inhibit cell proliferation as well as EGF-induced cell proliferation. These data indicate that the antiproliferative effect of SMS does not involve the GTP-binding protein-mediated negative coupling of somatostatin receptors to adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)
生长抑素已被证明在体内对胰腺生长具有负调节作用。在本研究中，我们使用AR4-2J大鼠胰腺腺泡肿瘤细胞系来研究一种稳定的生长抑素类似物SMS 201-995（SMS）对细胞增殖的影响。SMS对血清或表皮生长因子（EGF）诱导的细胞增殖均具有抗增殖作用；将细胞暴露于SMS 48小时会轻微抑制血清诱导的增殖（最大抑制率为26%），并消除EGF的促生长作用。在10 nM SMS时观察到最大效应，在0.06 - 0.1 nM SMS时观察到半数最大效应（IC50）。用SMS的碘化衍生物[125I-Tyr3]SMS进行的结合研究表明，AR4-2J质膜上存在一类单一的高亲和力结合位点，平衡解离常数为0.2±0.03 nM，结合位点数为1.1±0.07 pmol/mg蛋白。添加不可水解的GTP类似物鸟苷5-[γ-硫代]三磷酸（GTPγS）会增加特异性结合肽的解离速率，这与生长抑素受体与GTP结合调节蛋白的偶联一致。SMS抑制细胞增殖的IC50与结合的表观Kd之间的良好一致性表明，所表征的结合位点是介导SMS抗增殖作用的生长抑素受体。当细胞在无血清培养基中生长时，EGF刺激AR4-2J细胞增殖，在0.6 nM和10 nM EGF时分别产生半数最大效应（ED50）和最大效应。EGF的这种刺激作用是由特异性受体介导的，因为用[125I]EGF进行的结合研究表明，AR4-2J细胞含有一类单一的EGF受体（13,000个位点/细胞），对[125I]EGF的亲和常数（Kd = 0.9±0.09 nM）接近EGF刺激细胞生长的ED50。为了研究SMS诱导的生长抑制是否涉及cAMP依赖性机制，我们首先研究了SMS对cAMP产生的影响。SMS对基础cAMP无影响，但能完全抑制VIP刺激的cAMP产生，IC50为0.2 nM。已知百日咳毒素可消除生长抑素对AR4-2J细胞腺苷酸环化酶活性的抑制作用，但它并不能逆转SMS抑制细胞增殖以及EGF诱导的细胞增殖的能力。这些数据表明，SMS的抗增殖作用不涉及GTP结合蛋白介导的生长抑素受体与腺苷酸环化酶的负偶联。（摘要截短至400字）
