New CRF family peptides and functional analysis of stress system

促肾上腺皮质激素释放因子（CRF）是下丘脑室旁核（PVN）在应激反应中产生的，刺激促肾上腺皮质激素的释放。我们证实了雌激素受体β型（ERβ）的显性表达，并发现生理相关剂量的雌二醇（E2）和ERβ激动剂可刺激下丘脑4B细胞中CRF基因的转录。E2通过ERβ刺激白细胞介素(IL)-6转录活性，进而刺激IL-6 mRNA和蛋白水平。我们还发现，用IL-6治疗可显著降低细胞活力。因此，这些数据表明E2在下丘脑4B细胞中通过Erβ调控CRF基因和IL-6产生的重要作用。微荧光实验显示，CRF （0.2 nM）和urocortin (UCN) 1 （0.2 nM）升高了[Ca^<2+>]_I水平。应激素（一种非选择性的CRF受体拮抗剂）减弱了CRF和UCN 1的作用。选择性CRF_2 - More受体拮抗剂Antisauvagine-30可增强UCN - 1对[Ca^<2+>]_i升高的作用。CRF对[Ca^<2+>]_i升高的影响被ucn3的加入所抑制。综上所述，CRF_2受体的激活可拮抗CRF_1受体刺激的Ca^<2+>内流。我们还发现G蛋白偶联受体激酶（GRK） 2（而不是GRK3） mRNA和蛋白在大鼠垂体前细胞和AtT-20细胞（一种小鼠促皮质性肿瘤细胞）中表达。为了确定GRK2在crf诱导的小鼠促皮质细胞CRF_1受体脱敏中的作用，我们用GRK2显性阴性突变体转染at -20细胞。CRF通过CRF_1受体使camp依赖性反应脱敏。与对照at -20细胞相比，转染显性负突变体GRK2构建的at -20细胞中CRF对CRF_1受体的脱敏作用明显减少。这些结果表明，GRK2参与了CRF诱导at -20细胞中CRF_1受体的脱敏，蛋白激酶A途径也可能在促皮质营养细胞中CRF对CRF_1受体的脱敏中起重要作用。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