Barry H. Ginsberg

Regulation of the glucocorticoid receptor in human lymphocytes

The presence of a glucocorticoid receptor in human lymphocytes is well established, but factors affecting its regulation have not been described. Using a competitive binding whole cell assay, we have examined the binding of [3H]-dexamethasone at 24 and 37 degrees C in untreated normal subjects and in healthy subjects taking various glucocorticoid preparations. At 24 degrees C normal human lymphocytes had 6000 binding sites/cell and a dissociation constant of 4 x 10(-9) M. The administration of 1 mg of dexamethasone, 5 mg of prednisone, and 37.5 mg of cortisone acetate resulted in a 30% decrease in binding sites after 1 week with no change in binding affinity. No changes in the number of binding sites was noted before 1 week and the diminished number persisted for 1 week after discontinuation of glucocorticoid treatment. Lymphocytes from hospitalized patients taking 40-60 mg of dexamethasone daily demonstrated the same change in number of binding sites that was seen in normal subjects taking 1 mg of dexamethasone. When binding assays were carried out at physiologic temperature there was the same decrease in number of binding sites after dexamethasone administration, and in addition, there was a two-fold increase in binding affinity. Glucocorticoid administration results in a time-dependent decrease in the number of lymphocyte glucocorticoid binding sites that is independent of the type of glucocorticoid administered. This is the first in vivo demonstration that glucocorticoids modulate their own receptors in man.

THE NATURE OF IN VITRO HYDROXYLATION OF l‐TRYPTOPHAN BY BRAIN TISSUE

EARLIER attempts (RENSON, WEISSBACH and UDENFRIEND, 1962) failed to show any evidence for hydroxylation of tryptophan to 5-hydroxytryptophan (5-HTP) by brain tissue; even recently it was thought (YUWILER, GELLER and SLATER, 1965) that 5-HTP found in the brain originated from other organs. In a detailed study, evidence was presented for the enzymic hydroxylation of tryptophan to 5-HTP by brain tissue in viuo (GAL, POCZIK and MARSHALL, 1963; GAL, MORGAN, CHATTERJEE and MARSHALL, 1964). While the present project was in progress the hydroxylation of tryptophan by dog brain homogenates in vitro was reported (GRAHAME-SMITH, 1964). It was further noted that most of the enzymic activity, following fractionation of the brain tissue in 0.25 M-sucrose, resided in the mitochondria (GAL, 1965). In another preliminary report (NAKAMURA, ICHIYAMA and HAYAISHI, 1965) a 100-fold purification of tryptophan hydroxylase was indicated. The present study describes the occurrence and localization of and the conditions affecting the hydroxylation irt vitro of tryptophan in brain tissue.

myo‐Inositol Metabolism in 41 A3 Neuroblastoma Cells: Effects of High Glucose and Sorbitol Levels

Abstract: Neuroblastoma cells were used to determine the effect of high carbohydrate and polyol levels on myo‐inositol metabolism. The presence of elevated concentrations of glucose or sorbitol caused a significant decrease in both inositol accumulation and incorporation into phospholipid. These conditions, however, did not alter the accumulation of the other phospholipid head groups or the growth rate and water content of the cells. Two weeks of growth in either of the modified conditions was necessary to obtain a maximal effect on inositol incorporation. In contrast, growth in elevated concentrations of fructose, mannitol, or dulcitol had no effect on inositol metabolism. The reduced inositol accumulation and incorporation into lipids seen with glucose or sorbitol supplementation resulted in a decrease in the total phosphatidylinositol content of the cell without changing the levels of the other phospholipids. Kinetic analysis of cells grown in the presence of elevated glucose indicated that V1max for inositol uptake was significantly decreased with little change in the K1max. These data suggest that glucose decreases myo‐inositol uptake in this system by noncompetitive inhibition. Cells grown in the presence of increased glucose also had elevated levels of intracellular sorbitol and decreased levels of myo‐inositol. These results suggest that the high levels of glucose and sorbitol which exist in poorly regulated diabetes may be at least partially responsible for diabetic neuropathy via a reduction in the cellular content of myo‐inositol and phosphatidylinositol. This system may be a useful model to determine the effect of reduced inositol phospholipid levels on neural cell function.

Mitogenic activity of glia maturation factor. Interaction with insulin and insulin-like growth factor-II.

The mitogenic activity of glia maturation factor (GMF) was tested on sparse-cultured cells. GMF stimulates the growth rate of normal astroblasts and fibroblasts grown in the presence of fetal calf serum (FCS), and raises the saturation density of the cells over what is imposed by the corresponding serum concentrations. GMF has no mitogenic effect in the complete absence of serum. The mitogenicity of GMF is also demonstrable in defined media where certain serum components are present. In particular, GMF in combination with the defined medium N2 partially mimics the proliferative effect of serum alone. Insulin, an ingredient of N2, can substitute for the complete N2 formula. Insulin-like growth factor-II (IGF-II), in turn, can substitute for insulin. The interaction of GMF with insulin or IGF-II can be demonstrated in a sequential manner, suggesting that GMF is a competence factor. Since insulin is required at a concentration well above the physiologic serum level, and must be used at a dose 1000 times higher than IGF-II, we suspected that insulin acts on IGF-II receptors. This was substantiated by the demonstration of IGF-II receptors and the absence of detectable insulin receptors on the astroblasts. The combined effect of IGF-II and GMF mimics the combined effect of 10% FCS and GMF, in both growth rate and saturation density.

Effect of Increased Glucose Levels on Na+/K+‐Pump Activity in Cultured Neuroblastoma Cells

Abstract: Neuroblastoma cells were used to analyze the effect of elevated glucose levels on myo‐inositol metabolism and Na+/K+‐pump activity. The activity of the Na+/K+ pump in neuroblastoma cells is almost totally sensitive to ouabain inhibition. Culturing neuroblastoma cells in 30 mM glucose caused a significant decrease in Na+/K+‐pump activity, myo‐inositol metabolism, and myo‐inositol content, compared to cells grown in the presence of 30 mM fructose. Glucose supplementation also caused a large intracellular accumulation of sorbitol. The aldose reductase inhibitor sorbinil prevented the abnormalities in myo‐inositol metabolism and partially restored Na+/K+‐pump activity in neuroblastoma cells cultured in the presence of elevated glucose levels. These results suggest that the accumulation of sorbitol by neuroblastoma cells exposed to elevated concentrations of extracellular glucose causes a decrease in myo‐inositol metabolism and these abnormalities are associated with a reduction in Na+/K+‐pump activity.

Reconstitutiom of the Solubilized Insulin Receptor in Phospholipid Vesicles1

The insulin receptor was solubilized from turkey erythrocyte membranes by extraction with 1% β-octylglucopyranoside. Insulin binding was enhanced when the solubilized material was reconstituted in phospholipid vesicles. The affinity of the reconstituted vesicles for various insulins was similar to that of the intact membranes: porcine insulin > proinsulin > desoctapeptide insulin. A curvilinear Scatchard plot was obtained for insulin binding to the reconstituted system at 15d`C. A high affinity association constant of 1.4 X 109 M-1 was obtained from the Scatchard plot. This is a four-fold increase over the value for the turkey erythrocyte membrane, which contains more highly saturated phospholipids. This suggests that the insulin receptor may be sensitive to the lipid composition of the membranes in which it is embedded.

Characterization of an insulin receptor in human Y79 retinoblastoma cells

Abstract: Cultured human Y79 retinoblastoma cells bind [125I]iodoinsulin in a manner similar to that of other CNS and peripheral tissues. The only difference noted between the insulin binding properties of the Y79 cells and other CNS preparations is that insulin binding to Y79 cells is down‐regulated by prolonged exposure of the cells to insulin. By contrast, studies with the various brain preparations indicate that the brain insulin receptor is not down‐regulated by circulating levels of insulin. Insulin binding to Y79 cells exhibits negative cooperativity, has a pH optimum of 7.8, is responsive to cations, and gives a curvilinear Scatchard plot. Y79 cell insulin binding capacity is 26 fmol/100 μg of cell protein, corresponding to about 125,000 binding sites per cell. These findings are the first to report insulin binding in a human cell line of retinal origin. The characterization of the insulin binding in this cell line may facilitate an understanding of the relationship between insulin and its specific functions in the human retina.

Abnormal insulin binding and membrane physical properties of a Friend erythroleukemia clone resistant to dimethylsulfoxide-induced differentiation

We have compared insulin binding, plasma membrane fluidity, and phospholipid composition of three different Friend erythroleukemia clones, a wild type (FLC) a mutant (R3) and the revertant to wild type F+. The R3 clone is a non-differentiating DMSO-resistant clone (R3) and has altered membrane fluidity and dramatically altered insulin-binding properties. The receptor of R3 bound insulin as if it possessed a single class of low affinity receptors that lacks the property of negative cooperativity. The Scatchard plot is linear and there is no ligand-induced acceleration of dissociation. The Hill coefficient for R3 is 1, implying no cooperativity, whereas the Hill coefficient for the two DMSO-inducible clones, (FLC and F+) is 0.3, implying negative cooperativity. In addition, the insulin receptor of R3 has a decreased affinity for insulin, manifested as a 40-fold increase in the amount of insulin required to compete for half of the tracer binding (41 nM for R3 vs. 1 nM for FLC and F+). Computer-fitted Scatchard plots analyzed by the negative cooperativity model reveal that R3 has 95 000 receptor sites/cell, with a high affinity constant Ke of 0.016 nM-1, and a low affinity constant, Kf of 0.012 nM-1. Both DMSO-inducible clones have about 40 000 receptor sites/cell with Ke of 0.11 nM-1 and Kf of 0.02 nM-1. Electron spin resonance measurements with the 5-nitroxy stearate spin probe demonstrate that R3 had a more fluid plasma membrane than the FLC and F+ clones. The lipid composition of R3 is different from that of the DMSO-inducible clones. The weight ratio for unsaturated fatty acids to saturated fatty acids for R3 is 2.5, and the FLC clone has a lower ratio of 1.9. These results are consistent with our earlier findings in FLC that very high membrane fluidity is associated with alterations in the binding properties of the insulin receptor.

Modification of membrane physical properties, biological response and insulin binding in Friend cells by low serum concentration

The effect of low serum concentration on plasma membrane fluidity and lipid composition, differentiation and insulin binding was investigated in three Friend erythroleukemia clones. Both FLC (clones No. 745) and F(+) (Ostertag F4N) Friend erythroleukemia cells can be induced to differentiate and to produce hemoglobin when exposed to DMSO. Clone R(3) (Ostertag F4-D5-1) is a DMSO-resistant clone when grown under normal conditions (15% serum) but could undergo differentiation with accumulation of protoporphyrin IX upon induction with DMSO when grown in low serum concentration (2.5% serum). Electron spin resonance measurements of the order parameters (S) and S(T parallel) demonstrate that R(3) has a more fluid plasma membrane than the FLC and F(+). The order parameters of the outer hyperfine splittings S(T parallel) at 37 degrees C are 0.60 +/- 0.009, 0.62 +/- 0.008 and 0.64 +/- 0.009 for R(3), F(+) and FLC, respectively. We have used the insulin receptors as a model for a polypeptide hormone receptor associated with the plasma membrane of the Friend clones. Insulin binding studies demonstrated that the receptor of R(3) had a decreased affinity for insulin manifest as a 10-fold increase in the amount of insulin required to compete for half of the tracer binding (18 nM for R(3) vs. 2 nM for FLC and F(+)). Computer-fit Scatchard plot analysis by the negative cooperativity model reveal that R(3) possessed a similar number of sites/cell (about 70,000) as the FLC or F(+) cells, with similar high and low affinities. Growing the DMSO-resistant clone R(3) in low serum concentration caused a decrease in receptor number by 35%, and an increase in receptor affinity to that seen with the differentiable clones. Thus, the abnormal properties of the plasma membrane and insulin receptor of the DMSO-resistant clone in our earlier report (Simon et al. (1984) Biochim. Biophys. Acta 803, 39-47) were partially reversed by growing the cells in a low serum concentration, restoring the cellular response to the differentiation agent.

Synthesis and Regulation of Receptors for Polypeptide Hormones

In a dynamic environment the key to survival is adaptation. Of the adaptive responses an organism generates in response to its changing environment, many are generated via the endocrine system. Hormones, the messengers of the endocrine system, have access to most cells, but their effect on any specific site will be modified by local factors. The hormones react with their target cells via specific hormone receptors either at the cell surface or intracellularly and the receptor-hormone complex then generates a signal that regulates cellular metabolism. Among many possible local factors that influence hormonal action, two important sites of regulation are alterations in the reaction of the hormone with the hormone receptor and transmission of the signal of complex formation.