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.     

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.     

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.     

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.     

Expression of v-src in embryonic neural retina alters cell adhesion, inhibits histogenesis, and prevents induction of glutamine synthetase.

Using Rous sarcoma virus as the vector, v-src or c-src genes were introduced into 6-day chicken embryo retina tissue in organ culture and their effects on retina development were investigated. Overexpression of c-src in many of the cells had no noticeable effect on retina development. In contrast, infection with v-src resulted in abnormal histogenesis and inhibition of differentiation. Although only a portion of the cells in infected tissue expressed the oncogene and displayed the transformation phenotype, the other cells were also hindered from becoming normally positioned and organized. Therefore, presence of oncogene-transformed cells within the tissue hindered organization and development of adjacent nontransformed cells. Failure of normal cell relationships impeded induction by cortisol of glutamine synthetase in Muller glia, which requires contact associations of the glia cells with neurons. The transformed cells tended to assemble into chaotic clusters, suggesting that their adhesiveness and contact affinities had become altered. This was confirmed by aggregation experiments with dissociated cells which showed that adhesiveness of transformed cells was greatly reduced and that they had lost the ability to cohere with nontransformed cells. In binary mixtures of transformed and nontransformed cells, the two sorted out into separate aggregates. Transformed cells formed loose clusters devoid of tissue architecture; aggregates of nontransformed cells became organized into retinotypic structures, and glutamine synthetase was inducible. Our findings suggest that the mechanisms of cell adhesion and cell affinities are a key target of v-src activity in infected cells and that modification of the cell surface may be a leading factor in other cellular changes characteristic of the v-src transformation phenotype.     

The induction of glutamine synthetase in cell aggregates of embryonic neural retina: correlations with differentation and multicellular organization

A rapid increase in the synthesis and accumulation of the enzyme glutamine synthetase (GS) in the neural retina of the chick embryo characterizes the functional differentiation and maturation of this tissue. A precocious increase of GS can be induced in the embryonic retina by hydrocortisone and related corticosteroids. This paper presents evidence that the responsiveness of neural retina cells to GS induction by the hormonal inducer is dependent on histotypic associations and organization. This was demonstrated, using retina from embryos of different ages, by comparing GS induction in cultures of intact retina tissue with that in aggregates of retina cells and in monolayer cultures of retina cells.     

Ligatin from embryonic chick neural retina

Ligatin, a filamentous protein previously found in suckling rat ileum, has been purified from plasma membranes of embryonic chick neural retina. The isolated plasma membranes are covered in part by 4.5-nm filaments that can be released from the membranes by treatment with Ca++. Subsequent dialysis against EGTA followed by sieve chromatography results in purification of the 10,000-dalton ligatin monomer. When labeled either with radioisotopes or with fluorescamine, the monomer is shown to electrophorese as a single discrete band in polyacrylamide gels. However, during standard fixing and staining procedures it diffuses from the gels and thus is not visualized. Ligatin's amino acid composition is distinguished by its high content of polar residues, especially Glx and Asx, and by the presence of phosphorylated serine. Upon re-addition of Ca++, purified ligatin monomers polymerize to form filaments 3 nm in Diam, identical to those formed by purified ileal ligatin. However, in both retina and ileum, the filaments observed on plasma membranes are greater than 3 nm in Diam. In ileum, this enlargement results from ligatin's function as a baseplate for the attachment of another protein, a beta-N-acetylhexosaminidase, to the cell surface. In retina, a corresponding difference in diameter between filaments seen in vivo and those formed from repolymerized ligatin alone and the co-solubilization of other proteins with ligatin suggest that ligatin may also function there as a baseplate for other cell surface proteins. The proteins associated with ligatin in retina differ morphologically from beta-N-acetylhexosaminidase and do not possess this enzymatic activity.     

Glutamine synthetase induction in embryonic neural retina. Interactions of receptor-hydrocortisone complexes with cell nuclei.

In the neural retina of the chick embryo, hydrocortisone (HC) elicits differential gene expression resulting in the induction of glutamine synthetase (GS), which is an enzyme marker of differentiation in the retina. The relationship between nuclear binding of receptor-hydrocortisone (R-HC) complexes and GS induction was investigated in cultures of retina tissue from 12-day chick embryos. The number of HC binding sites in the cytoplasm was estimated as 1650+/-200 per retina cell; there are approximately 1500+/-100 acceptor sites for R-HC per retina nucleus. GS induction in the retina became detectable only after R-HC bound to more than 40% of the nuclear acceptors sites; increased binding coincided with higher induction levels, until complete site saturation was attained; Proflavine, which blocks preferentially and completely GS induction in the retina by interfering in the nucleus with the enzyme-inducing action of the hormone, reduced nuclear binding of R-HC by only 20%; thus, only part of the R-HC that binds in the nucleus appears to be directly involved in eliciting the induction of GS. Within one hour after exposure of the retina to an inducing dose of HC, there was translocation of HC and HC-receptors (as R-HC complexes) from the cytoplasm into the nucleus and saturation of nuclear accepegan to decline; in 12 h, it was reduced to 50% of the initial saturation level. Since, during this time, the enzyme activity to increase, persistence of the induced state depends on association of the hormone with only a portion of the sites in the nucleus to which it can bind. The decrease in the amount of bound HC in the nuclei of induced cells was accompanied by an increase in the level of HC receptors in the cytoplasm. About 50% of this increase could be prevented by cycloheximide; this suggests that the reappearance of HC receptors in the cell cytoplasm may be due, at least in part, to de novo synthesis of HC receptors.     

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.     

Cortisol receptors and inducibility of glutamine synthetase in embryonic retina

Glutamine synthetase (GS) is a marker enzyme for Müller glia cells in neural retina. In chick embryo retina GS begins to increase sharply on the 16th day of development, but can be precociously induced by premature supply of the inducer, cortisol, already on the 8th day. At this stage GS inducibility is low, but it increases progressively with embryonic age. We investigated whether there was a corresponding age-dependent increase of cortisol-binding molecules (cortisol receptors) and found that their level is highest in the early retina and decreases with development. In light of this inverse relationship, we examined whether functional characteristics of these receptors change with age, but detected no differences. In in vitro tests, receptors from older retina translocated cortisol into nuclei from young retina, and vice versa, with similar effectiveness. Also, cortisol receptors from liver cells (which differ from retina receptors) can translocate the hormone into retina nuclei, and vice versa. These findings indicate that translocation of cortisol receptors is neither tissue-specific or age-dependent, nor is it conditional on the total amount of receptors normally present in cells. Therefore, the age-dependent increase of GS inducibility in embryonic retina cannot be directly related to quantitative or functional differences of cortisol receptors and is evidently controlled primarily at the gene level. The very large amount of cortisol-binding molecules in early embryonic retina raises the possibility that they play some role in early differentiation of retina cells unrelated to hormone binding.