Effects of PACAP on the gene expression of catecholamine synthesizing enzymes.

PACAP对儿茶酚胺合成酶基因表达的影响。

基本信息

批准号：
06671000
负责人：
ISOBE Kazumasa
金额：
$ 1.22万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

Pituitary adenylate cyclase-activating polypeptide (PACAP), a potent stimulant for catecholamine secretion, stimulated catecholamine production in cultured porcine adrenal medullary chromaffin cells. PACAP induced dose-and time-dependent increases in mRNAs for the catecholamine synthesizing enzymes, tyrosine hydroxylase (TH) and dopamine b-hydroxylase (DBH), with maximal 6-and 4-fold increases occuring at 8 to 16h, respectively. The half-maximally and maximally effective PACAP concentrations for stimulation of TH and DBH gene expression were 0.5 and 0.3 nM,respectively. The TH-protein level also showed an increase over the unstimulated basal level at 16 to 24h in PACAP-stimulated cells.We previously demonstrated that PACAP activates both phospholipase C and adenylate cyclase in adrenal medullary cells. Addition of forskolin alone induced increases in mRNA expression of both TH and DBH.The phosphodiesterase inhibior 3-isobutyl-1-methylxanthine potentiated the induction of TH and DBH mRNA by PACAP.Addition of the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) also caused increases in TH and DBH mRNA levels. In protein kinase C-downregulated cells pretrearted with PMA for 24h, the stimulatory effect of PACAP on TH and DBH gene expression was diminished. These results suggest that cAMP and protein kinase C mediated the PACAP-induced TH and DBH gene expression. Removal of extracellular Ca2+with EGTA enhanced the PACAP-induced increases in both cellular cAMP and mRNA levels of TH and DBH mRNAs.In conclusion, the present study indicates that PACAP coordinately up-regulates the gene expression of both TH and DBH by activating the cAMP and protein kinase C signaling pathway, leading to stimulation of catecholamine synthesis, while Ca2+negatively regulates TH and DBH gene expression in adrenal medullaru cells.

垂体腺苷酸环化酶激活多肽 (PACAP) 是儿茶酚胺分泌的有效刺激剂，可刺激培养的猪肾上腺髓质嗜铬细胞中儿茶酚胺的产生。 PACAP 诱导儿茶酚胺合成酶、酪氨酸羟化酶 (TH) 和多巴胺 b-羟化酶 (DBH) mRNA 呈剂量和时间依赖性增加，最大增加分别在 8 至 16 小时时增加 6 倍和 4 倍。刺激 TH 和 DBH 基因表达的半最大和最大有效 PACAP 浓度分别为 0.5 和 0.3 nM。在 PACAP 刺激的细胞中，16 至 24 小时，TH 蛋白水平也显示出比未刺激的基础水平有所增加。我们之前证明 PACAP 激活肾上腺髓质细胞中的磷脂酶 C 和腺苷酸环化酶。单独添加毛喉素可诱导 TH 和 DBH mRNA 表达增加。磷酸二酯酶抑制剂 3-异丁基-1-甲基黄嘌呤可增强 PACAP 对 TH 和 DBH mRNA 的诱导作用。添加蛋白激酶 C 激活剂佛波醇 12-肉豆蔻酸酯 13-乙酸酯(PMA) 还导致 TH 和 DBH mRNA 水平增加。在用 PMA 预处理 24 小时的蛋白激酶 C 下调细胞中，PACAP 对 TH 和 DBH 基因表达的刺激作用减弱。这些结果表明 cAMP 和蛋白激酶 C 介导 PACAP 诱导的 TH 和 DBH 基因表达。用 EGTA 去除细胞外 Ca2+ 增强了 PACAP 诱导的细胞 cAMP 和 TH 和 DBH mRNA mRNA 水平的增加。总之，本研究表明 PACAP 通过激活cAMP 和蛋白激酶 C 信号通路，导致儿茶酚胺合成的刺激，而 Ca2+ 负向调节肾上腺髓质细胞中的 TH 和 DBH 基因表达。