Graduate Thesis Or Dissertation
 

Early events in hydrocortisone induced glutamine synthetase activity in differentiating chick neural retina

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  • Glutamine synthetase in the embryonic chick neural retina shows a 50 fold increase in activity between the 16th and 17th day of embryonic development. This increase in enzyme activity results from de novo enzyme synthesis thus making it a useful marker of differential gene expression. A precocious rise in activity can be induced in as early as 10-day retinas by various corticosteroids, hydrocortisone being particularly effective. This induction requires both RNA and protein synthesis. Certain questions about the induction mechanism arose which this study attempts to answer: (1) Does hydrocortisone initiate the glutamine synthetase response by a trigger-type mechanism so that after removal of the steroid, the response continues? (2) How is intracellular hormone uptake related to induction? (3) How do induction response to hydrocortisone and uptake of the hormone vary with tissue differentiation? (4) Can a time limitation be described for the sensitivity to inhibitors of protein synthesis? In vitro tissue cultures of 10-day chick neural retina were exposed to hydrocortisone for 2 and 6 hr, after which the tissue was washed with media and replaced in culture without inducer for a 24 hr total culture time. Glutamine synthetase activity levels in these cultures were approximately two-thirds of the level obtained from cultures exposed to the hormone continuously during the 24 hr culture period. After 2 or 6 hr of culture in the presence of tritiated hydrocortisone, the tissue retains approximately two-thirds of the steroid obtained with continuous exposure to the labelled hormone. The correlation between these two factors suggests that glutamine synthetase activity increases are initiated in response to the action of an intracellularly retained pool of hydrocortisone. Neural retinas from older embryos (14- and 17-day) were cultured in the presence of hydrocortisone for short intervals (2 or 6 hr) as above. Glutamine synthetase activity in these older embryos showed less relative inducibility after short term hydrocortisone exposure than 10-day embryos. In 14- and 17-day embryos, the activity increased to only 25% and 1% of their respective 24 hr control levels. The uptake of tritiated hydrocortisone per cell remained the same for 10-, 14-, and 17-day embryonic retinas suggesting the development during differentiation of some step in the hormone action process which reduces the response to intracellularly bound hormone. A two hour treatment with inhibitors of protein synthesis, puromycin and cycloheximide, added to the cultures with the inducer, prevented the induced rise in glutamine synthetase activity after 24 hr of culture. Retinas regained their inducibility when hydrocortisone was added again to the cultures after removal of the inhibitor. Cycloheximide did not interfere with tritiated hydrocortisone uptake. If the 2 hr cycloheximide treatment was delayed until 6 or 8 hr after addition of hydrocortisone, glutamine synthetase activity increased without the readdition of hydrocortisone. Thus, there is a step which is sensitive to inhibitors of protein synthesis only during the first 6 hr after the inducer is added. The evidence suggests that an early phase of hydrocortisone action is responsible for the subsequent glutamine synthetase activity increase in chick neural retina. Before 6 hr, (1) most cellular uptake occurs, (2) hydrocortisone exerts its effect, so that with subsequent washing and reculturing without inducer, glutamine synthetase activity continues to increase, (3) a step changing with differentiation occurs which reduces the responsiveness to retained hydrocortisone, and (4) a step sensitive to inhibitors of protein synthesis occurs.
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