Early events in the induction of glutamine synthetase activity by hydrocortisone in embryonic chick neural retina

Summary Primary cultures of 10-day embryonic chick neural retinas were used to investigate early aspects of the mechanism of hydrocortisone action on glutamine synthetase activity. As little as 2 hr of hydrocortisone exposure served to initiate significant increases in the glutamine synthetase activity levels assayed after 24 hr culture. Time course studies indicated that the increase in glutamine synthetase activity observed after 24 hr in culture resulted from a two-phase rise in activity and that cycloheximide was effective in suppressing the second-phase rise. Additional inhibition studies demonstrated that the second-phase increase in enzyme activity required continuous protein synthesis during the initial 6 hr. The evidence suggests a mechanism of hydrocortisone action involving the production of a protein which is important for the induction of glutamine synthetase activity by hydrocortisone. This work was supported by a National Science Foundation (U.S.A.) Training Grant.     

Early events in the induction of glutamine synthetase activity by hydrocortisone in embryonic chick neural retina.

Primary cultures of 10-day embryonic chick neural retinas were used to investigate early aspects of the mechanism of hydrocortisone action on glutamine synthetase activity. As little as 2 hr of hydrocortisone exposure served to initiate significant increases in the glutamine synthetase activity levels assayed after 24 hr culture. Time course studies indicated that the increase in glutamine synthetase activity observed after 24 hr in culture resulted from a two-phase rise in activity and that cycloheximide was effective in suppressing the second-phase rise. Additional inhibition studies demonstrated that the second-phase increase in enzyme activity required continuous protein synthesis during the initial 6 hr. The evidence suggests a mechanism of hydrocortisone action involving the production of a protein which is important for the induction of glutamine synthetase activity by hydrocortisone.     

Glutamine synthetase induction in chick embryo retina monolayers

Induction of glutamine synthetase (GS) by cortisol has been shown to occur in monolayer cultures of cells obtained by enzymatic dissociation of retinas from 8- and 12-day-old chick embryos with papain (0.1%) or trypsin (0.25%). Although essentially single cells when plated, monolayers obtained by enzymatic dissociation show significant aggregation by 4--6 h. Monolayers prepared by mechanical dispersion (cells forced through successively smaller gage needles) are minimally inducible, perhaps owing to poor viability in such cultures. Storage at 4 degrees C for 24 h prior to treatment with cortisol significantly elevated both basal GS activity and inducibility in whole (but not in monolayer) retina cultures.     

Glutamine synthetase induction in chick embryo retina monolayers

Induction of glutamine synthetase (GS) by cortisol has been shown to occur in monolayer cultures of cells obtained by enzymatic dissociation of retinas from 8- and 12-day-old chick embryos with papain (0.1%) or trypsin (0.25%). Although essentially sigle cells when plated, monolayers obtained by enzymatic dissociation show significant aggregation by 4–6 h. Monolayers prepared by mechanical dispersion (cells forced through successively smaller gage needles) are minimally inducible, perhaps owing to poor viability in such cultures. Storage at 4°C for 24 h prior to treatment with cortisol significantly elevated both basal GS activity and inducibility in whole (but not in monolayer) retina cultures.     

Observations on the histochemically demonstrable liver glycogen phosphorylase activity and native glycogen under different nutritional conditions

Summary In this study a histochemical demonstration of glycogen phosphorylase activity and native glycogen in the livers from normally fed, overfed and starved rats was performed.It was found that the amount and localization of phosphorylase activity well corresponded to the amount and localization of the native glycogen. A change of the glycogen content in the liver also resulted in a change of the histochemically demonstrable liver glycogen phosphorylase activity.It is concluded that the presence of tissue bound glycogen and undissolved glycogenphosphorylase complexes are necessary for positive histochemical demonstration of liver glycogen phosphorylase activity.This work was supported by a grant from the Finnish Veterinary Medical Foundation.     

Histochemical studies on glycogen synthetase activity by autoradiographic method

Summary Glycogen synthetase (uridine diphosphate glucose-glycogen glucosyl transferase) was studied in different organs by a histoautoradiographic method and by usual staining methods. This activity was found to be present in muscles and liver of different animals. Human skin also showed some activity. Human liver and myocardium showed the highest activity.In the present study, it was found that the glucose-6-phosphate dependent form (D-form) of the glycogen synthetase predominates over the glucose-6-phosphate independent form (I-form) in all the organs except hamster liver where the I-form predominates.Addition of calcium chloride in the incubation medium, to prevent phosphorolytic breakdown of the newly synthesized glycogen, does not improve the reaction. No glucose is incorporated into glycogen from ¹⁴C-glucose-6-phosphate of the incubation medium for glycogen synthetase. Fixation in absolute alcohol at –20° is recommended for tissues where cytolysis is caused by the incubation medium.     

Development of phosphodiesterase activity in the chick retina

Significant cyclic GMP phosphodiesterase activity is apparent in the early stages of development of the chick neural retina. By day 8, specific activity drops by approximately two-thirds. After day 14, a sharp rise in activity is observed, continuing through the time of hatching. Cyclic AMP phosphodiesterase activity is considerably lower and does not markedly change over the embryonic period.     

Interferon suppresses steroid-inducible glutamine synthetase biosynthesis in embryonic chick neural retina

Effect of chick interferon on the biosynthesis of glutamine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2) was studied in the embryonic chick neural retina cultures induced for the enzyme activity by hydrocortisone. The retinal enzyme radioactively labelled with [³H]leucine was precipitated by specific antibody against the enzyme isolated from adult chick liver. The immunological determination offered evidence that the suppressive effect of interferon on the hormonal induction of the enzyme was primarily due to reduced rate of its synthesis and accumulation.     

Intracellular localization and size of glycogen particles in glycogen synthesized under histochemical conditions.

During the investigation of the histochemical synthesis of glycogen particles from glucose 1-phosphate by the phosphorylase-branching glycosyltransferase system in various tissue cells, it was observed that focal synthesis localized in a certain area of the cytoplasm occurred in some cells. This differed from the usual synthesis in which particles of similar size were synthesized within the cytoplasm. Otherwise, cytoplasmic particles of various size were also synthesized in other cells under the same histochemical condition. The possible significance of the presence of these patterns in glycogen synthesis is discussed.     

Interferon suppresses glutamine synthetase induction in chick embryonic neural retina.

Two different proteins precipitable with antiserum to albumin exist in liver. One is albumin, the other is precursor albumin. Liver cells in suspension contain mainly precursor, but secrete only albumin. In subcellular fractions isolated from liver homogenate, 95.3% of anti-albumin precipitable protein in the rough endoplasmic reticulum, 51.4% in the smooth endoplasmic reticulum, 33.5% in the Golgi apparatus and 0% in the supernatant fraction was precursor albumin. The results suggest that albumin precursor is synthesized in the rough endoplasmic reticulum and converted into albumin in the smooth endoplasmic reticulum and the Golgi apparatus.     

Interferon suppresses steroid-inducible glutamine synthetase biosynthesis in embryonic chick neural retina.

Effect of chick interferon on the biosynthesis of glutamine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2) was studied in the embryonic chick neural retina cultures induced for the enzyme activity by hydrocortisone. The retinal enzyme radioactively labelled with [3H]leucine was precipitated by specific antibody against the enzyme isolated from adult chick liver. The immunological determination offered evidence that the suppressive effect of interferon on the hormonal induction of the enzyme was primarily due to reduced rate of its synthesis and accumulation.     

Glutamate decarboxylase activity in chick brain and retina

We have studied the effect of Triton-X-100 on glutamate decarboxylase (GAD) activity in brain and retina from chick embryos of 12 and 16 days' incubation and from chicks 4–6 weeks old. GAD activity was measured in five different homogenization media. Triton-X-100 inhibited the enzyme by about 60% in both brain and retina of 12-day embryos and by about 50% in 16-day embryos, independently of the homogenization medium. In chicks only about 20% inhibition by the detergent was observed in brain whereas no effect was found in retina. These results indicate that the evaluation of the experimental conditions of enzyme assays at different ages is essential for developmental studies of GAD activity in nervous tissue.