Effects of dexamethasone and 5-bromodeoxyuridine on the synthesis of amylase mRNA during pancreatic development in vitro.

Abstract

The effects of the glucocorticoid, dexamethasone, and the thymidine analog, Q-bromodeoxyuridine (BrdUrd), on steady state levels of the predominant mRNAs syn- thesized during in vitro development of 14-day pan- creatic rudiments were examined. Amylase mRNA lev- els were measured by two specific assays: hybridiza- tion with amylase cDNA and specific immunoprecipi- tation of amylase synthesized in the rabbit reticulocyte lysate translation system. The levels of other frequent pancreatic mRNAs were examined qualitatively by hy- bridization with cDNA synthesized from total adult pancreas polyadenylated RNA and by comparison of sodium dodecyl sulfate-polyacrylamide gel patterns of in vitro translation products. Both hybridization and translation analyses indicate that total RNA isolated from dexamethasone-treated embryonic pancreases contains about twice as much amylse mRNA as does total RNA isolated from controls. Amylase specific activity is about 4-fold greater in dexamethasone-treated pancreases than in controls. Thus, dexamethasone increases amylase specific activ- ity in part by increasing the steady state level of amy- lase mRNA. Amylase mRNA synthesized in the pres- ence of dexamethasone is identified as pancreatic am- ylase mRNA (rather than an amylase mRNA of differ- ent nucleotide sequence normally synthesized in an- other tissue such as the parotid gland or liver) by the thermal stability of the cDNA-RNA hybrids. Dexameth- asone does not change significantly the amount of total mRNA relative to non-mRNA sequences or steady state levels of most other frequent mRNAs. RNA isolated from BrdUrd-treated embryonic pan- creases contains about 3% of the control value of amy- lase mRNA and greatly reduced levels (<8%) of mRNAs which code primarily for other exocrine proteins. At the levels of BrdUrd employed the specific activities of amylase and other exocrine proteins are reduced to about 3% of control values. The total amount of mRNA relative to non-mRNA sequences is relatively unaf- fected. Thus, BrdUrd inhibits the synthesis of pancreas * This research was supported by National Science Foundation grant BMS 72-02222. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be herebv marked