Todisco A, Ramamoorthy S, Witham T, Pausawasdi N, Srinivasan S, Dickinson C J, Askari F K, Krametter D
Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0682, USA.
Am J Physiol Gastrointest Liver Physiol. 2001 Feb;280(2):G298-307. doi: 10.1152/ajpgi.2001.280.2.G298.
Gastrin (G17) has a CCK-B receptor-mediated growth-promoting effect on the AR42J rat acinar cell line. We examined whether G17 inhibits apoptosis induced by serum withdrawal of AR42J cells and CHO-K1 cells stably expressing CCK-B receptors (CHO-K1/CCK-B cells). Cellular apoptosis was measured by flow cytometry and the terminal deoxynucleotidyltransferase-mediated dUTP-FITC nick end-labeling method. Serum withdrawal induced AR42J and CHO-K1/CCK-B cell apoptosis. Addition of 10 nM G17 reversed these effects. We examined the action of G17 (10 nM) on phosphorylation and activation of protein kinase B/Akt, a kinase known to promote cell survival. Akt phosphorylation and activation were measured by kinase assays and Western blots with an anti-phospho-Akt antibody. G17 stimulated Akt phosphorylation and activation. G17 induction of Akt phosphorylation was inhibited by the phosphoinositide 3-kinase (PI 3-kinase) inhibitors LY-294002 (10 microM) and wortmannin (200 nM) but not by the mitogen-activated protein kinase kinase 1 inhibitor PD-98059 (50 microM). To study the role of p38 kinase in G17 signaling to Akt, we examined the effect of G17 on p38 kinase activation and phosphorylation using kinase assays and Western blots with an anti-phospho-p38 kinase antibody. G17 induced p38 kinase activity at doses and with kinetics similar to those observed for Akt induction. The p38 kinase inhibitor SB-203580 inhibited G17 induction of Akt phosphorylation and activation at a concentration (10 microM) 10-fold higher than necessary to block p38 kinase (1 microM), suggesting the possible involvement of kinase activities other than p38 kinase. Transduction of AR42J cells with the adenoviral vector Adeno-dn Akt, which overexpresses an inhibitor of Akt, reversed the antiapoptotic action of G17. In conclusion, G17 promotes AR42J cell survival through the induction of Akt via PI 3-kinase and SB-203580-sensitive kinase activities.
胃泌素（G17）对AR42J大鼠腺泡细胞系具有胆囊收缩素B（CCK - B）受体介导的促生长作用。我们研究了G17是否能抑制血清剥夺诱导的AR42J细胞以及稳定表达CCK - B受体的CHO - K1细胞（CHO - K1/CCK - B细胞）的凋亡。通过流式细胞术和末端脱氧核苷酸转移酶介导的dUTP - FITC缺口末端标记法检测细胞凋亡。血清剥夺诱导AR42J和CHO - K1/CCK - B细胞凋亡。添加10 nM G17可逆转这些效应。我们研究了G17（10 nM）对蛋白激酶B/Akt磷酸化和激活的作用，Akt是一种已知可促进细胞存活的激酶。通过激酶分析和使用抗磷酸化Akt抗体的蛋白质印迹法检测Akt的磷酸化和激活情况。G17刺激Akt的磷酸化和激活。G17诱导的Akt磷酸化被磷酸肌醇3激酶（PI 3激酶）抑制剂LY - 294002（10 microM）和渥曼青霉素（200 nM）抑制，但未被丝裂原活化蛋白激酶激酶1抑制剂PD - 98059（50 microM）抑制。为了研究p38激酶在G17向Akt信号转导中的作用，我们使用激酶分析和使用抗磷酸化p38激酶抗体的蛋白质印迹法检测G17对p38激酶激活和磷酸化的影响。G17诱导p38激酶活性的剂量和动力学与观察到的Akt诱导相似。p38激酶抑制剂SB - 203580在浓度（10 microM）比阻断p38激酶所需浓度（1 microM）高10倍时，抑制了G17诱导的Akt磷酸化和激活，这表明除p38激酶外可能还有其他激酶活性参与其中。用腺病毒载体Adeno - dn Akt转导AR42J细胞，该载体过表达Akt抑制剂，可逆转G17的抗凋亡作用。总之，G17通过PI 3激酶和对SB - 203580敏感的激酶活性诱导Akt，从而促进AR42J细胞存活。
