Figure 7
Functional role of the PLC/IP3/PKC and CaM/CaMK-II cascades in PACAP- and Forskolin-induced PRL promoter activity. αT3-1 cells were transiently transfected with pPRL(−1156).LUC for 6 h by using lipofectamine. After 18 h recovery, the cells were incubated with respective drugs. (A) αT3-1 cells over-expressed pPRL(−1156).LUC were treated with oPACAP38 (10 nM, 24 hrs) in the presence or absence of PLC inhibitor Edelfosine (20 μM). (B) Transfected αT3-1 cells were labeled with 2 μCi/well of myo-[3 H] inositol (DuPont/NEN) in myo-inositol free DMEM medium containing 10% fetal bovine serum and then treated with oPACAP38, VIP, and GnRH for 45 mins. The total IP production were analyzed by detecting the radio-labelled inositol incorporation. Then, αT3-1 cells over-expressed pPRL(−1156).LUC were treated with oPACAP38 (10 nM, 24 hrs) in the presence or absence of (C) IP3 receptor inhibitor 2-APB (100 μM), (D) PKC inhibitor GF109203 (1 μM), (E) CaM antagonist calmidazolium (1 μM) and (F) CaM KinaseII inhibitor KN62 (5 μM). In parallel, the transfected cells were challenged with Forskolin (100 nM, 24 hrs) in the presence or absence of CaM antagonist calmidazolium (G, 1 μM) and CaM KinaseII inhibitor KN62 (H, 5 μM). After drug treatment, cell lysate was prepared for dual-luciferase measurement. Data presented were expressed as percentage of control by conversing the ratio of firefly and renilla luciferase in the same sample. Data presented are expressed as mean ± SEM (n = 4) and different letters denote a significant difference at p < 0.05 (ANOVA followed by Fisher’s LSD Test).
