|Abstract or Summary
- 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.