Abstract
This study used the rat pancreatoma AR42J as a model system to investigate the possible regulation of cholesterol esterase biosynthesis in pancreas. Initial experiments were performed to verify the synthesis of pancreatic cholesterol esterase by the AR42J cells. Results indicated that this pancreatoma cell line synthesized two forms of cholesterol esterase (Mr = 71,000 and 74,000). The 74-kDa protein is most likely the precursor protein, and the 71-kDa protein is the matured enzyme secreted by the AR42J cells. The synthesis and secretion of cholesterol esterase were stimulated 2-5-fold by incubating the cells with 0.5-4 nM of cholecystokinin or 0.5-2 nM of secretin. Analysis of the RNA isolated from the hormone-stimulated cells revealed that stimulation of cholesterol esterase biosynthesis was not due to an increase in cholesterol esterase mRNA. Furthermore, inhibition of transcription with actinomycin D has no effect on the hormone-induced cholesterol esterase biosynthesis. Therefore, the mechanism of hormone stimulation was not dependent on de novo RNA synthesis. In vitro translation studies showed that the cholesterol esterase mRNA isolated from stimulated AR42J cells were translated more efficiently than those from control cells. Thus, the increased cholesterol esterase biosynthesis induced by gastric hormones was most likely mediated by posttranscriptional modification of the cholesterol esterase mRNA.
Highlights
This studyused the rat pancreatoma AR42J as a important for catalyzing the lymphatic absorptionof dietary model system to investigate the possible regulation of cholesterol and fat-soluble vitamins
In view of previous observations that thleevels of most pancreaticdigestive enzymes are regulated by the composition of the diet and by gastric hormones synthesized in thgeut [3,4,5,6,7], it ispossible that cholesterol esterase biosynthesis may be regulated in a similar manner
The purpose of this investigation is to use the rat pancreatoma AR42J cells as a model system to tion with actinomycin D has no effect on the hormone- determine the effect of gastric hormones, such ascholecystoinduced cholesterol esterase biosynthesis
Summary
100, andproteaseinhibitors as describedabove.Nonspecifically hound proteins were removed from the Protein A-agarose heads by washing twice with the same buffer containing 1 M NaCl and twice with the same buffer containin0g.1% SDS. Effects of Gastric Hormoneson Cholesterol Esterase Biosyn- Experiments were performedto determineif the gastric thesis-In order to determine thepossible 'hormonal regula- hormonescan induce the secretion of cholesterol esterase tion of cholesterol esterase biosynthesis, the AR42J cells were from the cells In these studies, the AR42J cells were preinpulse-labeled with [""Slmethioninein thepresence of varying cubated with [:"S]methionine for 30 min andthen chased concentrations of CCK or secretin. Hybridizashowed that addition of CCK and secretin during the chase tion of the RNA from AR42J cells with rat actin cDNA period resultedin a concentration-dependent accumulationof revealedasingle band corresponding to an mRNA of 2.1 radiolabeledcholesterol esterasein cell lysateandinthe kilobases (Fig. 8).The specificity of these hybridization studmedium The results reported here were obtained by averaging of three separate experimentsi S.D
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