New CRF family peptides and functional analysis of stress system

促肾上腺皮质激素释放因子(CRF)是在下丘脑室旁核(PVN)产生的应激反应，刺激促肾上腺皮质激素的释放。我们证实了雌激素受体β(ER-β)的优势表达，并发现生理剂量的雌激素(E_2)和ER-β激动剂能刺激下丘脑4B细胞CRF基因的转录。雌二醇通过ERβ刺激IL-6转录活性，进而刺激IL-6m RNA和蛋白水平。我们还发现，IL-6处理显著降低了细胞存活率。因此，这些数据表明，E2对下丘脑4B细胞CRF基因和IL-6的产生有重要的调节作用。微量荧光实验表明，CRF(0.2nM)和urocortin(Ucn)1(0.2nM)提高了[Ca~(2+)]_i的水平。CRF和UCN-1的作用均可被非选择性CRF受体拮抗剂Astressin减弱。选择性CRF_2…-30更多的受体拮抗剂可增强UCN_1升高[Ca~(2+)]_i的作用，而加入UCN_3可抑制CRF对[Ca~(2+)]_i升高的作用。总之，CRF_2受体的激活可拮抗CRF_1受体刺激的Ca~(2+)内流。为了确定GRK2在CRF诱导的小鼠促肾上腺皮质激素细胞CRF1受体脱敏中的作用，将显性负性突变型GRK2基因导入AtT-20细胞。CRF通过CRF_1受体脱敏cAMP依赖的反应。在转染显性负性突变型GRK2的AtT-20细胞中，CRF对CRF_1受体的脱敏作用明显低于对照组。这些结果表明，GRK2参与了CRF诱导的ATT-20细胞CRF_1受体的脱敏，蛋白激酶A途径也可能在CRF对皮质营养细胞CRF_1受体的脱敏中起重要作用。较少

Corticotropin-releasing factor (CRF) is produced in the hypothalamic paraventricular nucleus (PVN) in response to stress and stimulates the release of adrenocorticotropic hormone in the corticotrophs. We demonstrated the dominant expression of estrogen receptor typeβ(ERβ) and found that a physiologically relevant dose of estradiol (E2) and an Erβ agonist stimulated CRF gene transcription in hypothalamic 4B cells. E2 stimulated interleukin (IL)-6 transcriptional activity via ERβ, and subsequently the levels of IL-6 mRNA and protein. We also found that treatment with IL-6 significantly reduced cell viability. Thus, these data suggest the important effects of E2 on the regulation of CRF gene and IL-6 production via Erβ in hypothalamic 4B cells.Microfluorimetric experiments showed that CRF (0.2 nM) and urocortin (UCN) 1 (0.2 nM) elevated [Ca^<2+>]_I levels. Both CRF and UCN 1 effects were attenuated by astressin, a non-selective CRF receptor antagonist. Antisauvagine-30, a selective CRF_2 … More receptor antagonist, appeared to enhance the UCN 1 effect on the elevation of [Ca^<2+>]_i. The CRF effect on the elevation of [Ca^<2+>]_i was inhibited by the addition of UCN 3. Taken together, the activation of CRF_2 receptor antagonizes the CRF_1 receptor-stimulated Ca^<2+> influx.We also found that G protein-coupled receptor kinase (GRK) 2 (but not GRK3) mRNA and protein were expressed in rat anterior pituitary cells and AtT-20 cells (a line of mouse corticotroph tumor cells). In order to determine the role of GRK2 in CRF-induced desensitization of CRF_1 receptor in mouse corticotrophs, AtT-20 cells were transfected with a dominant negative mutant GRK2 construct. CRF desensitized the cAMP-dependent response by CRF_1 receptor. Desensitization of CRF_1 receptor by CRF was significantly less in AtT-20 cells transfected with the dominant negative mutant GRK2 construct compared with desensitization in control AtT-20 cells. These results suggest that GRK2 is involved in CRF-induced desensitization of CRF_1 receptor in AtT-20 cells, and protein kinase A pathway may also have an important role in desensitization of CRF_1 receptor by CRF seen in cortivotrophic cells. Less