García-Tornadú Isabel, Rubinstein Marcelo, Gaylinn Bruce D, Hill David, Arany Edith, Low Malcolm J, Díaz-Torga Graciela, Becu-Villalobos Damasia
Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and University of Buenos Aires, V. Obligado 2490, 1428 Buenos Aires, Argentina.
J Endocrinol. 2006 Sep;190(3):611-9. doi: 10.1677/joe.1.06902.
Recently, the importance of the dopaminergic D2 receptor (D2R) subtype in normal body growth and neonatal GH secretion has been highlighted. Disruption of D2R alters the GHRH-GH-IGF-I axis and impairs body growth in adult male mice. The D2R knockout (KO) dwarf mouse has not been well characterized; we therefore sought to determine somatotrope function in the adult pituitary. Using immunohistochemistry and confocal microscopy, we found a significant decrease in the somatotrope population in pituitaries from KO mice (P=0.043), which was paralleled by a decreased GH output from pituitary cells cultured in vitro. In cells from adult mice the response amplitude to GHRH differed between genotypes (lower in KO), but this difference was less dramatic after taking into account the lower basal release and hormone content in the KO cells. Furthermore, there were no significant differences in cAMP generation in response to GHRH between genotypes. By Western blot, GHRH-receptor in pituitary membranes from KO mice was reduced to 46% of the level found in wildtype (WT) mice (P=0.016). Somatostatin induced a concentration-dependent decrease in GH and prolactin (PRL) secretion in both genotypes, and 1x10(-7) M ghrelin released GH in cells from both genotypes (P=0.017) in a proportionate manner to basal levels. These results suggest that KO somatotropes maintain a regulated secretory function. Finally, we tested the direct effect of dopamine on GH and PRL secretion in cells from both genotypes at 20 days and 6 months of life. As expected, we found that dopamine could reduce PRL levels at both ages in WT mice but not in KO mice, but there was no consistent effect of the neurotransmitter on GH release in either genotype at the ages studied. The present study demonstrates that in the adult male D2R KO mouse, there is a reduction in pituitary GH content and secretory activity. Our results point to an involvement of D2R signaling at the hypothalamic level as dopamine did not release GH acting at the pituitary level either in 1-month-old or adult mice. The similarity of the pituitary defect in the D2R KO mouse to that of GHRH-deficient models suggests a probable mechanism. A loss of dopamine signaling via hypothalamic D2Rs at a critical age causes the reduced release of GHRH from hypophyseotropic neurons leading to inadequate clonal expansion of the somatotrope population. Our data also reveal that somatotrope cell number is much more sensitive to changes in neonatal GHRH input than their capacity to develop properly regulated GH-secretory function.
最近，多巴胺能D2受体（D2R）亚型在正常身体生长和新生儿生长激素（GH）分泌中的重要性已得到凸显。D2R的破坏会改变生长激素释放激素（GHRH）-GH-胰岛素样生长因子I（IGF-I）轴，并损害成年雄性小鼠的身体生长。D2R基因敲除（KO）侏儒小鼠的特征尚未得到充分描述；因此，我们试图确定成年垂体中生长激素细胞的功能。通过免疫组织化学和共聚焦显微镜，我们发现KO小鼠垂体中生长激素细胞数量显著减少（P = 0.043），体外培养的垂体细胞中GH分泌量也相应减少。在成年小鼠的细胞中，对GHRH的反应幅度在不同基因型之间存在差异（KO小鼠较低），但考虑到KO细胞较低的基础释放量和激素含量后，这种差异不太明显。此外，不同基因型对GHRH反应时cAMP生成没有显著差异。通过蛋白质印迹法，KO小鼠垂体膜中的GHRH受体水平降至野生型（WT）小鼠的46%（P = 0.016）。生长抑素在两种基因型中均诱导GH和催乳素（PRL）分泌呈浓度依赖性下降，1×10⁻⁷ M胃饥饿素在两种基因型的细胞中均释放GH（P = 0.017），且与基础水平成比例。这些结果表明，KO生长激素细胞维持着调节分泌功能。最后，我们测试了多巴胺对20日龄和6月龄两种基因型小鼠细胞中GH和PRL分泌的直接影响。正如预期的那样，我们发现多巴胺在WT小鼠的两个年龄段均可降低PRL水平，但在KO小鼠中则不然，并且在所研究的年龄段，该神经递质对两种基因型的GH释放均无一致影响。本研究表明，成年雄性D2R KO小鼠垂体中的GH含量和分泌活性降低。我们的结果表明，D2R信号在下丘脑水平起作用，因为在1月龄或成年小鼠中，多巴胺作用于垂体水平均不释放GH。D2R KO小鼠垂体缺陷与GHRH缺乏模型的相似性提示了一种可能的机制。关键年龄时通过下丘脑D2R的多巴胺信号缺失导致促垂体神经元释放GHRH减少，从而导致生长激素细胞群体的克隆扩增不足。我们的数据还表明，生长激素细胞数量对新生儿GHRH输入变化的敏感性远高于其发育出正常调节的GH分泌功能的能力。
