Increase In Receptor Levels Research Articles

High level expression of functional full length and truncated glucocorticoid receptor in Chinese hamster ovary cells. Demonstration of ligand-induced down-regulation of expressed receptor mRNA and protein

Full length human glucocorticoid receptor and truncated receptor derivatives lacking the major amino-terminal trans-activating domain were expressed in stably transfected Chinese hamster ovary (CHO) cells. The receptors were co-expressed together with human metallothionein IIa, and the expression levels were amplified in the presence of increasing concentrations of metal. In amplified cells, both synthesized receptor forms showed the expected molecular weights, as assayed by affinity labeling and immunoblotting. They were expressed at concentrations of about 350,000-520,000 molecules/cell which corresponds to a 10-fold increase in receptor levels as compared to rat liver cells. The hormone (agonist or antagonist) binding properties of the expressed proteins were very similar to those characteristic of authentic glucocorticoid receptors in tissues or cultured cells. Moreover, the expressed proteins specifically recognized a glucocorticoid-response element sequence motif in in vitro protein-DNA binding experiments. The activation of a glucocorticoid-responsive reporter gene by the expressed full length receptor was dramatic (about 75-fold) and strictly ligand-dependent. In contrast, the expressed amino-terminal deletion mutant exhibited considerably weaker functional activity but showed normal hormone-binding properties. Upon exposure to dexamethasone in vivo, the expressed receptor mRNAs and proteins were down-regulated about 2- to 6-fold, indicating that regulatory signals important for autoregulation may be contained within structures corresponding to the ligand and DNA-binding domains. Transcription from the expression vector was not negatively regulated from the hormone, strongly arguing that receptor down-regulation was due to a post-transcriptional mechanism. In conclusion, this expression system should be a useful tool for further structural and functional studies of the receptor, including the biochemistry of its activation from a cryptic to a functional species, and its ligand-dependent autoregulation.

Effects of Growth and Insulin Treatment on the Levels of Insulin Receptors and Their mRNA in HEP G2 Cells

We have studied the variations in the number of insulin receptor and insulin receptor mRNA levels in (Hep G2) cells in response to growth and insulin treatment. The levels of insulin receptors are relatively low in growing cells. After approximately 5 days in culture, if cells are not refed they cease to divide and the number of receptors/cell increases, reaching 4 times the initial values by the 9th day. Refeeding the cells completely prevented both growth arrest and the increase in insulin receptor number. Insulin added daily to cells at 0.33 microM caused receptor down-regulation but did not prevent a 3-fold increase in binding with growth arrest. Pulse-chase studies of metabolically labeled ([35S]methionine) cells showed that the receptor degradation rate (apparent t 1/2, 18-20 h) was comparable in rapidly growing versus growth-arrested cells. The increased receptor level in non-refed cells is not due to generation of a soluble factor by confluent cells, nor is it caused by depletion of insulin, glucose, or insulin-like growth factor I from the culture medium. The levels of insulin receptor mRNA measured on Northern blots increased in growth-arrested cells in parallel to the increase in receptor number. The mRNA value begins to increase from the 3rd day in culture and by the 9th day reaches a level 6.0 times that on the 3rd day. Chronic insulin-induced receptor down-regulation did not alter insulin receptor mRNA levels at any time point studied. These data demonstrate that the increase in insulin receptor number/cell in growth-arrested cells is paralleled by an increase in insulin receptor mRNA content with no change in the receptor degradation rates. This suggests that the increase in the number of insulin receptors is due to enhanced receptor synthesis due to increased receptor mRNA content. Conversely, down-regulation of the insulin receptor does not affect the level of insulin receptor mRNA and thus must be due to increased receptor degradation.

Turnover of epidermal growth factor binding sites in mouse mammary epithelial cells

An in vitro method of studying epidermal growth factor (EGF) receptors in mouse mammary epithelial cells in serum-free collagen gel culture has been developed. Binding of EGF averaged 108 ± 19 fmol/mg DNA in cells isolated from freshly dissociated virgin mammary tissue. Initial binding values were maintained in cells cultured in the presence of 0.1 ng/ml EGF, but decreased in either 0 ng/ml or 10 ng/ml EGF. Addition of either chloroquine (100 μM) or ammonium chloride (10 mM) to the culture medium increased receptor levels 10-fold. Cycloheximide (0.1 μg/ml), ouabain (3 mM), and actinomycin D (5 × 10 −2 μg/ml) each decreased receptor levels, in some cases by as much as 80%. Both methylamine (10 mM) and dinitrophenol (0.1 mM) had no significant effect. These studies suggest that the net level of EGF receptors in these target cells is the result of an equilibrium between synthesis and degradation. The difference between the effects of the compounds tested on either receptor degradation or synthesis in comparison to cell growth, may be indicative that receptor degradation is not linked to cell proliferation.

Cholesterol homeostasis in guinea pigs fed saturated and polyunsaturated fat diets

Whole body sterol balance, hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, hepatic low-density lipoprotein (LDL) receptor levels and net tissue cholesterol concentrations were determined in guinea pigs fed either a corn oil- or lard-based purified diet for 6–7 weeks. In comparison to the saturated lard diet, the polyunsaturated corn oil diet resulted in a 34% reduction in plasma total cholesterol levels ( P<0.02) and a 40% lower triacylglycerol level ( P<0.02). Feeding the corn oil diet altered very-low-density lipoprotein (VLDL) and LDL composition; the percent cholesterol ester in both particles was decreased and the relative percentages of VLDL triacylglycerol and LDL phospholipid increased. The ratio of surface to core components of LDL from corn oil-fed guinea pigs was significantly higher compared to LDL from animals fed lard. Dietary fat quality had no effect on fecal neutral or acidic steroid excretion, net tissue accumulation of cholesterol, whole body cholesterol synthesis or gallbladder bile composition. Consistent with these results was the finding that fat quality did not alter either expressed (non-phosphorylated) or total hepatic HMG-CoA reductase activities. The hepatic concentrations of free and esterified cholesterol were significantly increased in corn oil-fed animals, as were cholesterol concentrations in intestine, adipose tissue, muscle and total carcass. Analysis of receptor-mediated LDL binding to isolated hepatic membranes demonstrated that the polyunsaturated corn-oil based diet caused a 1.9-fold increase in receptor levels ( P<0.02). The data indicate that the hypocholesterolemic effects of dietary polyunsaturated fat in the guinea pig are not attributable to changes in endogenous cholesterol synthesis or catabolism but rather may result from a redistribution of plasma cholesterol to body tissue due to an increase in tissue LDL receptors.

Accumulation of epidermal growth factor receptors in retinoic acid-treated fetal rat lung cells is due to enhanced receptor synthesis.

125I-Epidermal growth factor (EGF) binding capacity in fetal rat lung (FRL) cells is increased approximately 2 to 3-fold within 18 h of retinoic acid addition. Analysis of 125I-EGF binding assays at 0 C reveals approximately 25,000 receptors per cell, while analysis of growth factor binding to retinoic acid-treated cells demonstrates an increase in receptor levels to approximately 70,000 receptors per cell with no detectable changes in receptor affinities. We show by immunoprecipitation of 35S-methionine labeled EGF receptors that retinoic acid addition produces an increase in the accumulation of EGF receptor protein. Using brief pulses of 35S-methionine, an increase in EGF receptor synthesis can be identified within 3 h after retinoic acid addition. These results are the first to demonstrate that a retinoic acid-induced increase in 125I-EGF binding capacity is due to increased EGF receptor protein synthesis. Also, we find that a transient decrease in the rate of EGF receptor turnover occurs when retinoic acid is initially added to FRL cells. On the basis of our data, we conclude that the retinoic acid-induced accumulation of EGF receptors in FRL cells is primarily due to increased receptor synthesis. The effect of retinoic acid on EGF receptor turnover may be a secondary factor, influencing the rate at which receptors accumulate.

Increased epidermal growth factor receptor in multidrug‐resistant human neuroblastoma cells

Multidrug-resistant human neuroblastoma cell lines obtained by selection with vincristine or actinomycin D from two independent clonal lines, SH-SY5Y and MC-IXC, have 3- to 30-fold more cell surface epidermal growth factor (EGF) receptors than the drug-sensitive parental cells as indicated by EGF binding assays and immunoprecipitation, affinity-labeling, and phosphorylation studies. Reversion to drug sensitivity in one line was accompanied by a return to the parental level of EGF receptor. SH-EP cells, a clone derived from the same neuroblastoma cell line as SH-SY5Y but which displays melanocyte rather than neuronal lineage markers, also express significantly more EGF receptor than SH-SY5Y cells. By nucleic acid hybridization analysis with a molecularly cloned probe, increased receptor level in multidrug-resistant cells was shown to be the result of higher levels of EGF receptor mRNA in drug-resistant than in drug-sensitive cells. The increased steady state amount of specific RNA did not result from amplification of receptor-encoding genes. A small difference was observed in the electrophoretic mobility under denaturing conditions of EGF receptor immunoprecipitated from drug-resistant and drug-sensitive cells. Quantitative and qualitative modulation of the EGF receptor might reflect alterations in the transformation and/or differentiation phenotype of the resistant cells or might result from unknown selective pressures associated with the development of multidrug resistance.

Evidence that the self-induced metabolism of 1,25-dihydroxyvitamin D-3 limits the homologous up-regulation of its receptor in rat osteosarcoma cells

The osteoblast-like osteosarcoma cell line UMR-106 has been shown to possess high-affinity receptors for 1,25-dihydroxyvitamin D (1,25-(OH) 2D 3). Also, these cells metabolize 1,25-(OH) 2D 3 to more polar metabolites. As previously demonstrated (Pols, H.A.P., et al. (1987) Biochim. Biophys. Acta 931, 115–119) the time course of specific binding of 1,25-(OH) 2D 3 in intact UMR-106 cells was found to be characterised by (a) an ascending phase, representing association with receptor, (b) a maximum at 90–120 min and (c) a rapid descending phase, closely associated with a decrease of medium 1,25-(OH) 2D 3 due to the metabolism of the hormone. The purpose of the present study was to investigate further the self-induced metabolism of 1,25-(OH) 2D 3 in relation to the homologous up-regulation of its receptor in these cells. Inhibition of metabolism of 1,25-(OH) 2D 3 with ketoconazole resulted, after a lag-time of about 90 min, in a sharp increase of receptor accumulation. This increase in receptor level in the presence of ketoconazole was blocked by coincubation with cycloheximide and actinomycin D. Preincubation experiments with unlabeled 1,25-(OH) 2D 3 showed that the elevation of hormone binding was 1,25-(OH) 2D 3-concentration dependent (ED 50 200–300 pM). Addition of ketoconazole during these preincubations resulted in an even more pronounced accumulation of receptors, whereby the ED 50 (50–60 pM) was comparable with the dissociation constant of the 1,25-(OH) 2D 3 receptor (41.3 ± 4.3pM). In summary, these data support the concept that the self-induced metabolism of 1,25-(OH) 2D 3 has a dual effect: (1) directly, by the regulation of the cellular concentration of and, consequently, receptor occupancy by the active form of vitamin D and (2) indirectly by its ability to modulate the ligand-dependent regulation of the 1,25-(OH) 2D 3.

Function and regulation of a murine macrophage-specific IgG Fc receptor, Fc gamma R-alpha.

Ligand binding specificities of two cloned murine Fc gamma Rs (Fc gamma R-alpha, Fc gamma R-beta [9]) were determined by gene transfer into Fc gamma R negative cell lines. Both receptors were expressed as full-length molecules capable of IgG immune complex binding that was inhibitable by the mAb 2.4G2. The ligand binding profiles of these receptors were indistinguishable whereby both bound immune-complexed mouse IgG1, IgG2a, and IgG2b, but not IgG3. Neither receptor could bind monomeric IgG2a, indicating these receptors to be low-affinity IgG Fc receptors. Accumulation of the Fc gamma R-alpha mRNA can be induced with murine IFN-gamma at a concentration of 200 U/ml in the macrophage-like cell lines RAW 264.7 and J774a. The time course for induction indicates that the mRNA accumulation is transient but does not return to the uninduced level even after 50 h of treatment. Fc gamma R-beta mRNA was not induced by IFN-gamma, rather its expression was down modulated in mouse peritoneal macrophages. Both RAW and J774a cells lines exhibited increased receptor levels after IFN-gamma stimulation as measured by 125I-2.4G2 and ligand binding. In the absence of IFN-gamma, the RAW and J774a cell lines were minimally phagocytic, while P388D1 cells were actively phagocytic. In the presence of IFN-gamma, however, RAW 264.7 and J774a cells were induced to become actively phagocytic. Induction of Fc gamma R-alpha mRNA and protein by IFN-gamma may be part of the process by which macrophages become activated to engulf antibody-coated particles.

Normalization of epidermal growth factor receptor and transforming growth factor production in drug resistant variants derived from adenovirus transformed cells.

Variants (G2, G5) resistant to the cancer chemotherapeutic drug methylglyoxal bis (guanylhydrazone) (MGBG) were isolated from adenovirus type 2 transformed rat brain cells (F4; Sircar et al., 1987). Although at least one of these variants continued to express the adenovirus Ela and Elb transforming proteins, they both exhibited a detransformed phenotype as witnessed by flat morphology, loss of anchorage independent growth, and tumor forming capacity. Reverse transformation suggested the possibility of changes in growth factor receptors and the production of transforming growth factors. To test this possibility, we investigated the status of epidermal growth factor receptors (EGF-r) and transforming growth factor alpha (TGF-alpha) production in F4, G2 and G5 cells. The level of 125I-labeled EGF binding to intact drug resistant cells increased by 2- to 3-fold compared to the transformed parental cell. Scatchard analysis suggests that increased binding was the result of increased receptor levels rather than altered affinity of receptor for ligand. The production of growth factors which compete with 125I-labeled EGF binding declined in the detransformed G2 and G5 cells to a level intermediate between transformed (F4) and normal cells (FR3T3). EGF-receptor increase and the complementary decrease in growth factor production in the drug resistant variants may be associated with detransformation.

Development of vasoactive intestinal peptide-responsive adenylate cyclase during enterocytic differentiation of Caco-2 cells in culture. Evidence for an increased receptor level.

The purpose of this work was to study vasoactive intestinal peptide (VIP) receptors and the adenylate cyclase response to VIP upon enterocytic differentiation of the human colon adenocarcinoma cell line Caco-2 in culture. The VIP-stimulated enzyme activity is very low, e.g. 20% above basal activity in undifferentiated cells (day 5) and is enhanced markedly at confluency reaching a maximum, e.g. 270%, above basal activity in fully differentiated cells (day 30). VIP potency is also slightly enhanced, the EC50 of VIP ranging from 0.31 nM at day 5 to 0.07 nM at day 30. Modifications of the adenylate cyclase system are not responsible for the development of VIP response. Indeed, forskolin-stimulated adenylate cyclase activity is unchanged during differentiation supporting no alteration of the enzyme catalytic subunit. The same holds true for NaF and guanosine 5'-(beta, gamma-imido)trisphosphate, indicating a constant activity of the guanine nucleotide regulatory unit which mediates hormonal stimulation of adenylate cyclase (Ns). This is further supported by the similar extent of cholera toxin-catalyzed [32P]ADP-ribosylation of the Ns protein that is observed during differentiation. In sharp contrast, a dramatic increase of VIP receptor concentration is observed ranging from 32 fmol/mg of protein at day 5 to 414 fmol/mg of protein at day 30. This is confirmed by affinity cross-linking experiments showing an increased specific incorporation of 125I-VIP in a major 66,000-dalton component during differentiation. A slight increase in receptor affinity is also observed during differentiation with Kd ranging from 0.39 nM at day 5 to 0.08 nM at day 30. These data indicate that one population of VIP receptors accumulates during Caco-2 cell differentiation, representing the crucial event in the development of adenylate cyclase response to the peptide.

Ligand-dependent regulation of the 1,25-dihydroxyvitamin D3 receptor in rat osteosarcoma cells.

The specific binding of radiolabeled 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to intact rat osteosarcoma (ROS 17/2) cells was followed for 24 h. In the presence of 0.5-1.5 nM 1,25(OH)2D3, hormone binding increased over a period of 12 h, from 1.1 X 10(4) to 1.3 X 10(5) receptors/cell. The elevated level of hormone binding persisted through 24 h provided that the initial concentration of hormone was maintained. The concentration dependence of this increase in receptor level was centered between 10 and 30 pM 1,25(OH)2D3, and the binding at 12 h exhibited the metabolite specificity expected for a 1,25(OH)2D3 receptor. The t 1/2 values for the disappearance of unoccupied and occupied receptors were roughly the same, approximately 2.7 h; therefore, the increase in hormone binding was not due to receptor stabilization. In comparison, hormone-receptor complexes appeared to dissociate with a t 1/2 of 1 h. alpha-Amanitin treatment reduced the magnitude of receptor accumulation by 50-60%, indicating that mRNA synthesis was required to achieve the maximal response. Ligand-dependent regulation of cellular receptor levels provides a mechanism for amplifying the primary hormonal signal and is predicted to influence the kinetics, magnitude, and dose dependence of cellular responses.

Measurement of 1,25-dihydroxyvitamin D3 receptor turnover by dense amino acid labeling: changes during receptor up-regulation by vitamin D metabolites.

We have previously demonstrated that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors are up-regulated by exposure to 24,25-(OH)2D3 and other vitamin D metabolites in several mammalian cell cultures systems as well as in vivo in rats. The goal of the current study was to determine how changes in receptor turnover could give rise to increased receptor levels after treatment with 24,25-(OH)2D3, whether by an increased rate of synthesis or a decreased rate of degradation. Receptor turnover was studied by the dense amino acid shift technique in cultured pig kidney cells (LLC-PK1). In control cells, the half-life of the 1,25-(OH)2D3 receptor was estimated to be 4.3 +/- 0.4 h, with a degradation rate constant (kD) of 0.16 +/- 0.01 h-1. In parallel experiments, cells were treated with 24,25-(OH)2D3 for about 48 h to reach a new up-regulated steady state before the measurement of receptor turnover. In up-regulated cells, the receptor half-life was prolonged to 8.9 +/- 1.0 h and the kD was 0.07 +/- 0.01 h-1. Assuming a steady state, the values obtained for total receptor levels and the degradation rate allow calculation of the synthesis rate (kS) at equilibrium. Control cells synthesized receptors at the rate of 3.4 +/- 0.4 vs. up-regulated cells at 4.9 +/- 0.5 fmol mg protein-1 h-1. In conclusion, the data indicate that up-regulation of receptors after exposure of cells to vitamin D metabolites results predominantly from a prolongation of receptor half-life in addition to a relatively smaller increase in the rate of receptor synthesis.

Down-regulation of thyroid hormone nuclear receptor levels by l-triiodothyronine in cultured glial C6 cells

l-Triiodothyronine (T3) produced a time- and dose-dependent depletion of nuclear thyroid hormone receptor levels in C6 cells, a rat glioma cell line. Receptor number diminished by 30–40% after a 48 h incubation with concentrations of T3 that saturate the nuclear receptor. The nuclear binding curve obtained in cells incubated for 48 h with T3 was shifted leftward of the curve obtained after a 3 h incubation, which indicates an apparent increase in receptor affinity after long-term incubation with T3. However, this change probably represents a further equilibration of the hormone, since the dissociation rate from the nuclei was similar in C6 cells after long- and short-term incubation with T3. The effect of T3 was further demonstrated in C6 cells incubated with short-chain fatty acids. Butyrate and isobutyrate increased receptor levels, and T3 partially decreased the response to these compounds. These findings suggest the existence of a desensitization process by which C6 glial cells would be protected against an excess of thyroid hormone.

Interactions between estrogen and EGF in uterine growth and function.

The rat uterus contains specific, high-affinity EGF receptors which possess a tyrosine kinase activity. As demonstrated autoradiographically, these receptors are present in the epithelial, stromal and myometrial cells of the uterus. Estrogen treatment in vivo produces a 2-3-fold increase in EGF receptor levels in the immature rat, the immature mouse and the ovariectomized adult rat; furthermore, EGF receptor levels vary throughout the estrus cycle in concert with levels of occupied nuclear estrogen receptor. This estrogen-induced increase in EGF receptor is preceded by an increase in the level of EGF receptor mRNA as judged by Northern blot analysis. In general, there is a good correlation between estrogen-induced DNA synthesis and EGF receptor levels in the uterus, although in certain situations EGF receptor levels are elevated without a subsequent increase in DNA synthesis. These observations suggest that an increase in tissue EGF receptor levels is important in estrogen-induced uterine growth, but that this increase in receptor levels alone is not sufficient to stimulate DNA synthesis. In addition to its possible role in tissue growth, we have shown very recently that EGF causes contraction of myometrial smooth muscle in a completely in vitro organ bath system. The qualitative nature of this contractile response is distinct from that produced by other classical uterotonic agents. The physiological significance of this uterine response to EGF remains to be elucidated.

Modulation of thyroid hormone nuclear receptors by short-chain fatty acids in glial C6 cells. Role of histone acetylation.

We have studied the effect of butyrate and other short-chain fatty acids on thyroid hormone nuclear receptors in C6 cells, a rat glioma cell line. Exposure of C6 cells to butyrate leads to increased levels of L-triiodothyronine (T3) in the nuclear and extranuclear compartments. The rise in nuclear binding is not merely a reflection of the higher cellular hormone content, and Scatchard analysis of T3 binding to isolated nuclei reveals that butyrate increases receptor number without changing affinity. The effect on the receptor is quantitatively important: a 48-h incubation with 2 mM butyrate increases nuclear binding by 2-3-fold, and 5 mM butyrate by 3-5-fold. Other short-chain fatty acids were found to similarly influence both nuclear receptor and extranuclear T3 levels with the following potency: butyrate greater than valerate greater than propionate greater than acetate. On the contrary, ketone bodies were ineffective. Butyrate increases receptor levels by decreasing receptor degradation, since the apparent t1/2 of receptor disappearance increased by approximately 3-fold in cells incubated with 2 mM butyrate for 48 h. The regulation of receptor number might be secondary to an action of butyrate on regions of the chromatin to which the receptor associates. We then examined the effect of butyrate on histone acetylation. The fatty acid had little effect in increasing the level of multiacetylated forms of H3 and H4 histone when studied in acid-urea gels, but it markedly inhibited the turnover of [3H] acetate from the histone fraction. There was a striking similarity in the dose-response of butyrate for increasing receptor levels and inhibiting histone deacetylation. Furthermore, a very close correlation between receptor levels and [3H]acetate release was also found when different short-chain fatty acids were used. We thus conclude that the effect of butyrate on the receptor could be explained by a modification of the chromatin structure of C6 cells secondary to acetylation.

Action of the colony-stimulating factor, CSF-1.

Colony-stimulating factor 1 (CSF-1) is a glycoprotein growth factor that specifically regulates the survival, proliferation and differentiation of mononuclear phagocytes and their precursors via a cell surface receptor selectively expressed on these cell types. The purified receptor is a single glycosylated polypeptide, Mr 165 000, which exhibits CSF-1-dependent autophosphorylation in tyrosine. CSF-1 alone regulates cells of the mononuclear phagocytic series (CSF-1-dependent colony-forming unit [CFU-C]----monoblast----promonocyte----monocyte----macrophage). However, the presence of a multipotent haemopoietic cell growth factor, haemopoietin-1, permits CSF-1 to stimulate precursors of CFU-C to proliferate and differentiate to macrophages. Precursors of CFU-C possess low levels of the CSF-1 receptor but there is an increase in receptor levels on CFU-C just before their differentiation to adherent, proliferating mononuclear phagocytes. As the timing of this developmentally associated increase in receptor expression coincides with the acquisition of responsiveness to CSF-1 alone, it is an early indicator of determination to the mononuclear phagocytic lineage.

Modulation of hepatic 2,3,7,8-TCDD cytosolic receptor levels by PCBs and organochlorine pollutants

Administration of several persistent PCB congeners to male Wistar rats demonstrated a structure-dependent elevation of hepatic cytosolic 2,3,7,8-TCDD receptor levels. 2,2′,4,4′5,5′-Hexachlorobiphenyl and several related compounds which exhibit low affinity for the receptor protein increased receptor levels to over 300% compared to the control animals. Aroclor 1254 and 2,3,3′,4,4′,5′-hexachlorobiphenyl both bind with moderate affinity to the receptor protein (10 uM) and effect some elevation of receptor levels. In contrast, 3,3′,4,4′,5-pentachlorobiphenyl, a toxic PCB with high in vitro binding affinity (10 uM) does not alter hepatic receptor levels. Time course studies, demonstrated that 2,2′,4,4′,5,5′-hexachlorobiphenyl elevated hepatic cytosolic receptor levels for at least 14 days with the maximum elevation occurring 7 days after initial exposure to this PCB congener. In contrast, dose and time-response studies with hexachlorobenzene, several DDT analogs and heptachlor expoxide gave variable results with respect to hepatic 2,3,7,8-TCDD receptor modulation. For the PCBs, it was apparent that the most active congeners exhibited low affinity for the receptor and were phenobarbitone (PB)-like inducers of microsomal monooxygenases. Although all the organochlorine insecticides which were tested also were PB-type monooxygenase enzyme inducers only the DDT analogs modulated hepatic receptor levels.

The development of the glucocorticoid receptor system in the rat limbic brain. I. Ontogeny and autoregulation

In order to characterize the development of the glucocorticoid receptor system in the brain, we examined [ 3H]dexamethasone binding in rat pups at various ages. Using an in vitro, cytosol, receptor assay we found evidence for low levels of glucocorticoid receptors perinatally with a subsequent increase in receptor concentrations that began by about the end of the first week of life. We have also shown that receptors during this period have a ligand specificity similar to that of receptors in adult animals. The postnatal increase in receptor levels parallels an increase in circulating corticosterone titers. Thus, receptor and hormone levels increase coincidentally. In adult animals, however, increasing levels of corticosterone are associated with a decrease in receptor levels and vice versa, such that corticosterone is thought to regulate its own receptor (i.e. autoregulation). This suggested an absence of autoregulation during development. We then determined hippocampal receptor concentrations of rats treated for 5 days with corticosterone, or adrenalectomized (ADX) 5 days prior to assay, examining whether up- or down-regulation occurs throughout development. In adults corticosterone treatment decreased (−45%) and long-term adrenalectomy increased (211%) glucocorticoid receptor concentrations. In contrast, at the youngest age tested (Day 10), the effects of manipulations of corticosterone titers on receptor concentrations were negligible. The potential for autoregulation emerged gradually throughout development. Thus, it appears that corticosterone regulation of its own receptors emerges only by about the time of puberty, and that this permits an increase in receptor levels to occur despite the concurrently increasing levels of circulating corticosterone.

Narcolepsy: Cholinergic receptor changes in an animal model

An inbred colony of narcoleptic doberman pinschers has been analyzed for muscarinic receptor levels in 19 discrete brain regions. In comparison to age-matched controls, receptors were generally elevated in the brainstem and reduced in forebrain areas. No changes in receptor binding affinity were detected. The increased receptor levels found in the brainstem suggest that cholinoceptive neurons in this region are hypersensitive and may be involved in the initiation of cataplexy and other aspects of the narcolepsy syndrome.

1 alpha,25-dihydroxyvitamin D3 receptors in cultured rat osteoblast-like cells. Glucocorticoid treatment increases receptor content.

The direct actions of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) in target cells are initiated by the binding of hormone to specific receptor sites. The aims of this study were to characterize the receptor for 1,25(OH)2D3 in cultured rat osteoblast-like (OB) cells and to determine whether these receptors are regulated by either the culture cycle itself or glucocorticoid treatment. The 1,25(OH)2D3 receptor in rat OB cells exhibited the same apparent binding affinity (Kd = 0.1 nM) and sedimentation coefficient (3.2 S) as mouse OB cells and receptors in other target organs. However, the receptor concentration in rat OB cells was substantially lower than mouse OB cells (approximately 20%). The concentration of receptors in rat OB cells did not show a correlation with the rate of DNA synthesis and therefore did not exhibit an endogenous rhythm in receptor concentration as was previously seen in mouse OB cells. Also in contrast to mouse cells, where glucocorticoids caused a decrease in receptor level, dexamethasone induced a marked increase in receptor binding throughout the culture cycle. This increase was due to an increase in receptor number with no change in receptor affinity. The change was glucocorticoid-specific, dose-dependent with half-maximal stimulation occurring between 1.3 and 13 nM and exhibited a latent period of at least 4 h. The independence of receptor concentration from DNA synthesis rate was established by assessing receptors after stimulating cell proliferation with epidermal growth factor and inhibiting it with hydroxyurea. Neither treatment altered basal 1,25(OH)2D3 receptor concentration or prevented the marked increase in receptor levels elicited by dexamethasone. We conclude that although the biochemical characteristics of 1,25(OH)2D3 receptors are indistinguishable in rat and mouse OB cells, there are genuine species differences in the regulation of the receptor number as it relates to DNA synthesis rate and response to glucocorticoids.