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

The effects of the glucocorticoid, dexamethasone, and the thymidine analogue, 5-bromodeoxyuridine (BrdUrd), on steady state levels of the predominant mRNAs synthesized during in vitro development of 14-day pancreatic rudiments were examined. Amylase mRNA levels were measured by two specific assays: hybridization with amylase cDNA and specific immunoprecipitation of amylase synthesized in the rabbit reticulocyte lysate translation system. The levels of other frequent pancreatic mRNAs were examined qualitatively by hybridization 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 amylases indicate that total RNA isolated from dexamethasone-treated embryonic pancreases contains about twice as much amylase 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 activity in part by increasing the steady state level of amylase mRNA. Amylase mRNA synthesized in the presence of dexamethasone is identified as pancreatic amylase mRNA (rather than an amylase mRNA of different nucleotide sequence normally synthesized in another tissue such as the parotid gland or liver) by the thermal stability of the cDNA-RNA hybrids. Dexamethasone 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 pancreases contains about 3% of the control value of amylase 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 unaffected. Thus, BrdUrd inhibits the synthesis of pancreas specific proteins by inhibiting the accumulation of their mRNAs.