Langouche L, Roudbaraki M, Pals K, Denef C
Laboratory of Cell Pharmacology, University of Leuven, Medical School, Campus Gasthuisberg, B-3000 Leuven, Belgium.
Endocrinology. 2001 Jan;142(1):257-66. doi: 10.1210/endo.142.1.7878.
The melanocortin (MC) gamma3MSH is a peptide that can be generated from the N-terminal domain of POMC and is believed to signal through the MC3 receptor. We recently showed that it induces a sustained rise in intracellular free calcium levels (Ca(2+)) in a subpopulation of pituitary cells, particularly in the lactosomatotroph lineage. In the present study we report that gamma3MSH and some analogs increase Ca(2+) in the GH- and PRL-secreting GH3 cell line and evaluate on the basis of pharmacological experiments and gene expression studies which MC receptor may be involved. A dose as low as 1 pM gamma3MSH induced an oscillating Ca(2+) increase in a significant percentage of GH3 cells. Increasing the dose recruited an increasing number of responding cells; a maximum was reached at 0.1 nM. gamma2MSH, alphaMSH, and NDP-alphaMSH displayed a similar effect. SHU9119, an MC3 and MC4 receptor antagonist, and an MC5 receptor agonist, did not affect the number of cells showing a Ca(2+) rise in response to gamma3MSH. SHU9119 had also no effect when added alone. MTII, a potent synthetic agonist of the MC3, MC4, and MC5 receptor as well as an N-terminally extended recombinant analog of gamma3MSH showed low potency in increasing Ca(2+) in GH3 cells, but high potency in stimulating cAMP accumulation in HEK 293 cells stably transfected with the MC3 receptor. In contrast, a peptide corresponding to the gamma2MSH sequence of POMC-A of Acipenser transmontanus increased Ca(2+) in GH3 cells, but was about 50 times less potent than gamma2- or gamma3MSH in stimulating cAMP accumulation in the MC3 receptor expressing HEK 293 cells. By means of RT-PCR performed on a RNA extract from GH3 cells, the messenger RNA of the MC2, MC3, and MC4 receptor was undetectable, but messenger RNA of the MC5 receptor was clearly present. These data suggest that the GH3 cell line does not mediate the effect of gamma3MSH through the MC3 receptor. The involvement of the MC5 receptor is unlikely, but cannot definitely be excluded. The findings animate the hypothesis that there exists a second, hitherto unidentified, MC receptor that displays high affinity for gamma3MSH.
促黑素（MC）γ3MSH是一种可从阿黑皮素原（POMC）的N端结构域产生的肽，据信它通过MC3受体发挥信号作用。我们最近发现，它能使垂体细胞亚群，特别是泌乳生长激素细胞系中的细胞内游离钙水平（[Ca(2+)]i）持续升高。在本研究中，我们报告γ3MSH和一些类似物可使分泌生长激素（GH）和催乳素（PRL）的GH3细胞系中的[Ca(2+)]i升高，并通过药理学实验和基因表达研究来评估可能涉及哪种MC受体。低至1 pM的γ3MSH就能使相当比例的GH3细胞中的[Ca(2+)]i产生振荡性升高。增加剂量会使响应细胞数量增加；在0.1 nM时达到最大值。γ2MSH、αMSH和NDP-αMSH表现出类似的效果。MC3和MC4受体拮抗剂以及MC5受体激动剂SHU9119并不影响因γ3MSH而出现[Ca(2+)]i升高的细胞数量。单独添加SHU9119也没有效果。MTII是MC3、MC4和MC5受体的强效合成激动剂，也是γ3MSH的N端延伸重组类似物，它在增加GH3细胞中[Ca(2+)]i方面效力较低，但在刺激稳定转染MC3受体的人胚肾293（HEK 293）细胞中cAMP积累方面效力较高。相比之下，对应于虹鳟鱼POMC-A的γ2MSH序列的一种肽能使GH3细胞中的[Ca(2+)]i升高，但在刺激表达MC3受体的HEK 293细胞中cAMP积累方面，其效力比γ2 - 或γ3MSH低约50倍。通过对GH3细胞的RNA提取物进行逆转录聚合酶链反应（RT-PCR），未检测到MC2、MC3和MC4受体的信使核糖核酸（mRNA），但明显存在MC5受体的mRNA。这些数据表明，GH3细胞系不是通过MC3受体介导γ3MSH的作用。MC5受体参与的可能性不大，但不能完全排除。这些发现支持了这样一种假说，即存在一种迄今未被识别的第二种MC受体，它对γ3MSH具有高亲和力。
