Regulation of the initiation of pancreatic digestive enzyme protein synthesis by cholecystokinin in rat pancreas in vivo  M.Julia Bragado, *, Mitsuo Tashiro, *, John A. Williams, *,‡  Gastroenterology  Volume 119, Issue 6, Pages 1731-1739 (December 2000) DOI: 10.1053/gast.2000.20242 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Identification of eIF4E in pancreatic acini and separation of phosphorylated and unphosphorylated forms. (A) Western blot of eIF4E expression in pancreatic acini, pancreatic homogenates (Panc), and 2 cell lines (SKN and NIH 3T3). (B) IEF gel separation and Western blot of phosphorylated (-P) and unphosphorylated forms of eIF4E in acini and in vivo pancreas (Panc). Alkaline phosphatase treatment was used to confirm the identity of the 2 forms. Gastroenterology 2000 119, 1731-1739DOI: (10.1053/gast.2000.20242) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 CCK enhances the phosphorylation of pancreatic eIF4E in vivo and in NIH 3T3 cells bearing CCK-A receptors in vitro. (A) Rats were injected with varying doses of CCK; 15 minutes later, the pancreas was removed and homogenized. Analysis of total eIF4E after SDS-PAGE was performed and the phosphorylation state of eIF4E by IEF was assessed as in Figure 1. The top panel shows representative Western blots, and the bottom panel shows the mean and SE for eIF4E phosphorylation for 5–8 rats with duplicate samples analyzed for each rat. (B) NIH 3T3 cells bearing CCK-A receptors were treated with 1 nmol/L CCK and washed, and lysates were prepared and subjected to SDS-PAGE and IEF followed by Western blotting. Results show both representative samples and pooled data for eIF4E phosphorylation from at least 3 experiments with each time point evaluated in triplicate in each experiment. *P < 0.05 and **P < 0.01 vs. control (0 CCK or 0 minute) in all cases. Gastroenterology 2000 119, 1731-1739DOI: (10.1053/gast.2000.20242) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 PHAS-I phosphorylation is enhanced by CCK injection in vivo in a time- and dose-dependent manner. (A) Time course of PHAS-I phosphorylation after injection with 5 μg/kg CCK. (B) Dose response to CCK measured 15 minutes after CCK injection. In both panels, representative Western blots are shown along with the pooled data for 4–7 rats. **P < 0.01 vs. control (0 minute or 0 CCK). Gastroenterology 2000 119, 1731-1739DOI: (10.1053/gast.2000.20242) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Identification of initiation factor eIF4G and increase of its association with eIF4E by CCK in rat pancreas. (A) Expression of eIF4G in pancreatic lysates by Western blotting analysis. (B) Dose dependence of CCK stimulation of eIF4G-eIF4E association measured 15 minutes after CCK administration by coimmunoprecipitation (see Materials and Methods) followed by Western blotting. Pooled data for coimmunoprecipitated eIF4G are the mean and SE for 4–6 rats per point. **P < 0.01 vs. 0 CCK. Gastroenterology 2000 119, 1731-1739DOI: (10.1053/gast.2000.20242) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Endogenous CCK released after feeding of camostat enhances the (A) phosphorylation of PHAS-I, (B) association of eIF4G with eIF4E, and (C) phosphorylation of eIF4E. In all cases, camostat was administered by intragastric bolus at 100 mg/kg; control animals received a similar volume of saline. After 30 minutes, pancreas was removed, homogenized, and dissolved in appropriate buffer. A representative Western blot is shown at the top; bars show mean and SE from 4 to 6 rats. **P < 0.01 vs. vehicle by unpaired t test. Gastroenterology 2000 119, 1731-1739DOI: (10.1053/gast.2000.20242) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Proposed schematic diagram for the control of the initiation phase of protein synthesis in an exocrine pancreas cell by CCK. Mechanisms not fully characterized in rat exocrine pancreas are represented as dashed arrows. Known regulatory mechanisms in pancreas and proteins studied in this work are represented with bold letters. Phosphorylated proteins are represented with circled P. Gastroenterology 2000 119, 1731-1739DOI: (10.1053/gast.2000.20242) Copyright © 2000 American Gastroenterological Association Terms and Conditions