Insulin Receptor Sub-Types in a Human Lymphoid-Derived Cell Line (IM-9): Differential Regulation by Insulin,Dexamethasone and Monensin

Abstract

The cells of the human IM-9 lymphocyte-derived line contain a sub-population of insulin binding sites which differ from classical insulin binding sites in their higher binding affinity for insulin-like growth factor II (IGF-II) and insulin-like growth factor I (IGF-I). These atypical insulin binding sites are identified on IM-9 cells by [¹²⁵I]IGF-II binding.

To determine whether the atypical and classical insulin receptors of IM-9 cells were subject to different modes of in vivo regulation, we treated IM-9 cells with agents known to alter the surface expression of insulin receptors - insulin, dexamethasone and monensin. We then measured insulin and IGF-II binding to the surface of the washed cells.

Pretreatment of IM-9 cells with 1 μM insulin for 20 h at 37°C induced a 44–48% decrease in the number of high affinity insulin binding sites, but no change in the number of IGF-II binding sites. In contrast, the surface expression of both insulin and IGF-II binding sites (classical and atypical insulin receptors) increased 1.3 to 1.7-fold after treatment with dexamethasone (200 nM) and decreased 30 to 45% after monensin (1 μM). These results suggest that atypical and classical insulin receptors are differentially susceptible to down-regulation by insulin.     

Characterization of insulin receptors in isolated epithelial cells of rat ventral prostate: effect of fasting

Insulin receptors have been characterized in rat prostatic epithelial cells by using [125I]insulin and a variety of physicochemical conditions. The binding data at equilibrium (2 h at 15 degrees C) could be interpreted in terms of two populations of insulin receptors: a class of receptors with high affinity (Kd = 2.16 nM) and low binding capacity (28.0 fmol mg-1 protein), and another class of receptors with low affinity (Kd = 0.29 microM) and high binding capacity (1.43 pmol mg-1 protein). Proinsulin exhibited a 63-fold lower affinity than insulin for binding sites whereas unrelated peptides were ineffective. The specific binding of insulin increased by about 50 per cent after 96 h of fasting; this increase could be explained by an increase of both the number of the high affinity-low capacity sites and the affinity of the low affinity-high capacity sites. These results together with previous studies on insulin action at the prostatic level strongly suggest that insulin may exert a physiological role on the prostatic epithelium.     

Dexamethasone stimulates insulin receptor synthesis in cultured rat hepatocytes

The ability of the glucocorticoid dexamethasone to modulate the insulin receptor was examined directly in primary cultures of hepatocytes prepared from adult male rats. Hepatocytes were cultured in a defined medium in the presence and absence of dexamethasone, 0.1 microM. The exposure of hepatocytes to dexamethasone resulted in a time-dependent (steady state by 32 h) increase in insulin binding in both intact hepatocytes and Triton X-100-soluble extracts (total insulin receptor content). The enhanced insulin binding found in soluble extracts of dexamethasone-treated hepatocytes was the result of an increase in insulin receptor number without a change in receptor affinity. In order to assess the mechanism by which dexamethasone "up-regulates" the insulin receptor, the heavy isotope density-shift technique was used to analyze insulin receptor turnover in control and dexamethasone-treated hepatocytes. Hepatocytes were initially cultured for 32 h in standard culture media containing only "light" (14C, 12C, 1H) amino acids. In hepatocytes exposed to dexamethasone, a 417% increase in insulin binding in Triton X-100-soluble extracts was observed. After 32 h, when steady state binding is achieved in dexamethasone-treated cultures, parallel cultures of hepatocytes incubated in the absence and presence of dexamethasone were washed and subsequently cultured in media containing "heavy" amino acids (15N, 13C, 2H). The time-dependent disappearance of light insulin receptor (receptor degradation) and appearance of heavy insulin receptor (receptor synthesis) were monitored using CsCl gradients to resolve the two density species of receptor. At steady state, the rate of receptor synthesis (k8) was 2.94 and 0.62 fmol of insulin bound h-1 in dexamethasone-treated and control hepatocytes, respectively. In contrast to this large increase in the rate of receptor synthesis observed in dexamethasone-treated cells, the first order rate constant for decay (k d) was the same in dexamethasone-treated (0.074 h-1) and in control (0.077 h-1) hepatocytes. We therefore conclude that glucocorticoid-induced up-regulation of the insulin receptor in the liver is due to stimulation of insulin receptor synthesis.     

Glucocorticoids increase cholecystokinin receptors and amylase secretion in pancreatic acinar AR42J cells

We recently reported in AR42J pancreatic acinar cells that glucocorticoids increased the synthesis, cell content, and mRNA levels for amylase (Logsdon, C.D., Moessner, A., Williams, J.A., and Goldfine, I.D. (1985) J. Cell Biol. 100, 1200-1208). In addition, in these cells glucocorticoids increased the volume density of secretory granules and rough endoplasmic reticulum. In the present study we investigate the effects of glucocorticoids on the receptor binding and biological effects of cholecystokinin (CCK) on AR42J cells. Treatment with 10 nM dexamethasone for 48 h increased the specific binding of 125I-CCK. This increase in binding was time-dependent, with maximal effects occurring after 48 h, and dose-dependent, with a one-half maximal effect elicited by 1 nM dexamethasone. Other steroid analogs were also effective and their potencies paralleled their relative effectiveness as glucocorticoids. Analyses of competitive binding experiments conducted at 4 degrees C to minimize hormone internalization and degradation revealed the presence of a single class of CCK binding sites with a Kd of approximately 6 nM and indicated that dexamethasone treatment nearly tripled the number of CCK receptors/cell with little change in receptor affinity. Treatment with 10 nM dexamethasone increased both basal amylase secretion and the amylase released in response to CCK stimulation. In addition, dexamethasone increased the sensitivity of the cells to CCK. The glucocorticoid decreased the concentration of CCK required for one half-maximal stimulation of amylase secretion from 35 +/- 6 to 8 +/- 1 pM. These data indicate, therefore, that glucocorticoids induce an increase in the number of CCK receptors in AR42J cells, and this increase leads to enhanced sensitivity to CCK.     

Alterations in the responsiveness of diabetic fibroblasts to insulin

Fibroblastic cultures from the skin of nondiabetic and diabetic (db/db) mice have been used to investigate alterations in the biological responses of diabetic cells to insulin. Confluent cultures from the skin of both nondiabetic and diabetic animals possess specific receptors for insulin. Diabetic fibroblasts exhibit only 36% as much specific binding of insulin as nondiabetic fibroblasts, because of a decrease in the total number of binding sites, without a change in binding affinity. Insulin caused a time- and dose-dependent increase in the rate of 2-deoxy D-glucose (dGlc) uptake and in ornithine decarboxylase (ODC) activity of both nondiabetic and diabetic fibroblasts. In nondiabetic cells, half-maximal increase in dGlc uptake was obtained with 0.3 nM insulin, and a maximum increase of 120% was obtained with 4.1 nM insulin. In contrast, diabetic cultures required 0.8 nM insulin for a half-maximal increase in dGlc uptake, and maximum stimulation with 4.1 nM insulin was only 50% above control levels. With 4-fold higher insulin concentrations, ODC activity of diabetic cells was only 40% that of nondiabetic cells. In nondiabetic cells, down regulation of insulin receptors by insulin abolished the ability of insulin to stimulate dGlc uptake. These results demonstrate that cells cultured from diabetic animals, which possess a decreased number of insulin receptors, also exhibit decreased stimulation of deoxy D-glucose uptake and ornithin decarboxylase activity by insulin.     

Insulin down-regulates insulin receptor number and up-regulates insulin receptor affinity in cells expressing a tyrosine kinase-defective insulin receptor

We examined the effect of insulin treatment on HTC cells transfected with large numbers of either normal insulin receptors (HTC-IR) or insulin receptors defective in tyrosine kinase (HTC-IR/M-1030). In both HTC-IR and HTC-IR/M-1030 cells, 20 h of insulin treatment (1 microM) at 37 degrees C resulted in a 65% decrease in the number of binding sites with a reciprocal 6-fold increase in affinity. In contrast, treatment with 10 nM insulin (20 h, 37 degrees C) also increased receptor affinity but had a smaller effect on the number of binding sites. 125I-Insulin binding to soluble receptors from HTC-IR and HTC-IR/M-1030 cells pretreated with insulin showed results similar to those obtained in intact cells. In both HTC-IR and HTC-IR/M-1030 cells, insulin enhanced insulin receptor degradation. In HTC-IR/M-1030 cells a 1-h incubation with insulin did not change receptor number and had only a small effect on receptor affinity; also there was no effect of insulin after a 20-h incubation at 15 degrees C. Inhibiting protein synthesis by pretreatment with cycloheximide (100 microM) did not block either the decrease in receptor number or the increase in receptor affinity. Both HTC-IR and HTC-IR/M-1030 cells exhibited a very slow rate of insulin and insulin receptor internalization and no differences were seen in this parameter when HTC-IR cells were compared to HTC-IR/M-1030 cells. These studies indicate, therefore, that in cells expressing kinase-defective insulin receptors, insulin down-regulates insulin receptor number via enhanced receptor degradation, and up-regulates receptor affinity. These effects were time- and temperature-dependent, but not dependent on new protein synthesis, and suggest that activation of tyrosine kinase may not be a prerequisite for certain mechanisms whereby insulin regulates its receptor.     

Time dependent effects of dexamethasone on serum insulin level and insulin receptors in rat liver and erythrocytes

The effects of glucocorticoid excess on regulation of insulin receptors were investigated in dexamethasone-treated rats. Glucocorticoid excess was produced by administration of dexamethasone (0.5 mg/100 g b.w.) 30 min, 4, 12, 18, 24, 42 or 70 h before experiments. This treatment caused time-dependent changes of glucose and insulin concentration in blood, as well as in amounts of specific insulin binding and insulin receptors of liver cells and erythrocytes. The time intervals in which dexamethasone produced the increase in insulin concentration were accompanied with decrease in insulin binding to receptors in membranes of liver cells, while significant changes in insulin binding to receptors of erythrocytes were not observed under the same experimental conditions. The effect is maximal 18 and 42 h after dexamethasone treatment that increase insulin blood level by about 85% and 60%, respectively. Receptor analysis revealed that changes in specific binding of insulin could be due to significant changes in amount of binding sites on cell surface rather than to mild alteration in receptor affinity. These findings suggest that besides the changes in insulin level, the alterations in insulin receptor number and affinity may play a major role in the states of altered insulin sensitivity which accompany glucocorticoid excess.     

Modulation by dexamethasone of insulin binding and insulin receptor mRNA levels in U-937 human promonocytic cells.

Treatment with 5 x 10(-6) M dexamethasone stimulated insulin binding in human promonocytic U-937 cells. When curvilinear Scatchard plots were examined according to the one-site model, only changes in affinity, but not in receptor numbers, were observed. However, when the two-site model was applied, an increase in both the affinity and the number of the high affinity-low capacity sites was observed, with maximum values at 15 h. By contrast, the low affinity-high capacity sites did not undergo significant alterations. Northern blot assays revealed two insulin receptor-related mRNAs of approximately 11 and 7 kb in size. Dexamethasone increased the levels of these RNAs, following similar kinetics to those of high affinity receptor expression. This suggests that the 11 and 7 kb species carry information for high affinity insulin receptors, and that in U-937 cells the expression of this receptor subclass is primarily regulated at the mRNA level.     

Uptake and receptor binding of dexamethasone in cultured 7800 C1 hepatoma cells in relation to regulation of cell growth and peroxisomal beta-oxidation

1. Uptake and binding of dexamethasone to glucocorticoid receptor has been studied in Morris hepatoma 7800 C1 cells in relation to its effect on cell growth and peroxisomal beta-oxidation. 2. Intact cells showed saturable, specific dexamethasone binding of limited capacity and Scatchard analysis revealed one single class of binding sites with equilibrium dissociation constant (Kd) of 0.24 nM similar to other glucocorticoid receptors. However, the binding capacity of 24 fmol/mg cell protein is less than 5% of previously reported values. 3. Uptake of [3H]dexamethasone by intact cells was temperature dependent giving a linear Arrhenius plot with a calculated energy of activation of 58.5 kJ mol-1 x degree-1. 4. Cytosol fractions had specific binding proteins for glucocorticoid hormones with sedimentation coefficient of ca 7S. No specific binding sites for [3H]dexamethasone was demonstrated in purified membrane fractions. 5. Dexamethasone and the synthetic fatty acid analogue tetradecylthio acetic acid (TTA) both inhibited the growth of the 7800 C1 cells and induced the peroxisomal acyl-CoA oxidase activity. A combination of the two compounds gave additive effects. Both these effects of dexamethasone and TTA were counteracted by insulin. 6. We conclude that dexamethasone induces growth inhibition and enzyme induction by binding to functional intracellular glucocorticoid receptors. The action of dexamethasone is consistent with a dissolution in the membrane from where it diffuses passively into the cell and binds to specific receptors in an energy dependent step. 6. The synergistic action of dexamethasone and TTA and the counteraction exerted by insulin are not due to changes in the dexamethasone receptor affinity or binding capacity.     

Specific glucocorticoid binding in human uterine tissues, placenta and fetal membranes

The binding of [3H]dexamethasone to cytosol fractions of human myometrium, endometrium, decidua, chorion, amnion and placenta has been studied. All tissues examined contained high affinity, low capacity binding sites with high specificity for glucocorticoids. Maximum specific binding of [3H]dexamethasone was reached after about 10 h at 0-4 degrees C and remained stable for at least the next 12 h. Sucrose density gradient analysis showed that the binding macromolecules sedimented at 7.9 S in hypotonic solutions and at 4.35 in solutions containing 0.4 M KCl. In the presence of sodium molybdate, the sedimentation coefficients shifted both in the absence and presence of 0.4 M KCl to 8.9 and 5.7 S, respectively. The apparent equilibrium dissociation constants (Kd) of the glucocorticoid binding sites were similar in most tissues, ranging between 1 and 6 nM, with the exception of the placenta in which the binding sites showed a higher Kd (13-22 nM). In all tissues studied, the binding affinities were similar in nonpregnant and pregnant patients and in patients at different stages of pregnancy or in labor. The concentration of the binding sites in the different tissues ranged from 11 to 268 fmol/mg protein, higher concentrations being found in myometrium, placenta and amnion and lower concentrations found in endometrium, chorion and decidua. The number of binding sites was higher in the myometrium of nonpregnant than pregnant women, but was similar in the myometrium of women at term pregnancy before or during labor. In the placenta, the number of binding sites increased significantly from early pregnancy to midpregnancy, while in chorion, amnion and decidua the number of binding sites did not change during pregnancy. It is concluded that human uterine tissues, placenta and fetal membranes contain specific binding sites with properties characteristic of glucocorticoid receptors suggesting that these tissues may respond directly to glucocorticoids.     

Antibody Bingling Boyding to the Juxtamembr are Ahch of the Insulin Receptor Alters Reotr Affinity

Abstract

The effect of three antibodies that interact with distinct regions of the insulin receptor (the a subunit (83-7), the juxtamembrane region near tyrosine 960 (960) or the carboxy terminal region of the I3 subunit (CT-1)) on insulin binding was examined. Detergent-solubilized insulin receptors from IM-9 cells immobilized on Sepharose beads by 960 antisera bound 2-3 times more IWinsulin tracer (25-60 pM) than receptors immobilized with either 83-7 or CT-1. &-incubation of solubilized receptors with either 83-7 or 960 resulted in equivalent depletion (90%) of insulin binding activity from solubilized IM-9 cell extracts, suggesting that both antibodies were in excess and capable of binding a similar population of receptors. Antibody 960, but not CT-1 or 83-7, also increased insulin binding 2 fold to solubilized receptors precipitated with polyethylene glycol. To determine whether the altered binding observed with antibody 960 was due to increased affinity of the receptor for insulin or appearance of more insulin binding sites, binding studies were performed over a wide range of insulin concentrations. Analysis of the resulting binding curves indicated that 960 increased the affinity of the receptor for insulin 3 fold over control (b= 0.3 nM for 960, and 0.9 nM for 83-7, respectively). The antibody 960 also specifically increased insulin binding to intact, saponin-permeabilized IM-9 cell membranes. These results indicate that binding of 960 antibody to the juxtamembrane region of the insulin receptor alters the affmity of the receptor for insulin. Since tyrosine 960 in the juxtamembrane region has been suggested to play a role in receptor signalling, changes in receptor conformation in this region that are likely to account for the change in affinity may play a role in signal transduction.     

Selective effect of castration on the anterior pituitary VIP receptor of male rats

We investigated the effect of surgical castration of male rats on the binding of [Tyr(¹²⁵I)¹⁰]VIP to receptors on the anterior pituitary gland, superior mesenteric artery, brain, liver, and prostate gland. In anterior pituitary membranes the maximum number of VIP binding sites was increased whereas binding affinity was decreased 24 hours following castration. In particular, the high affinity equilibrium dissociation constant (K_D) increased from 0.13±0.02 nM (mean±SEM) to 0.67±0.07 nM and the maximum number of high affinity binding sites (B_max) increased from 71±9 to 470±112 fmol/mg protein. No significant change was observed in the other tissues. Anesthesia or sham operation did not alter the anterior pituitary VIP receptor binding parameters. The changes in the VIP receptor 24 hours after castration were prevented by prior injection of testosterone. These findings demonstrate tissue-selective alterations to the anterior pituitary VIP receptor by castration that are likely mediated by withdrawal of testosterone.     

Presence of insulin receptors in cultured glial C6 cells. Regulation by butyrate.

The presence of insulin receptor and its regulation by butyrate and other short-chain fatty acids was studied in C6 cells, a rat glioma cell line. Intact C6 cells bind 125I-insulin in a rapid, reversible and specific manner. Scatchard analysis of the binding data gives typical curvilinear plots with apparent affinities of approx. 6 nM and 70 nM for the low-affinity (approx. 90% of total) and high-affinity (approx. 10% of total) sites respectively. Incubation with butyrate results in a time- and dose-dependent decrease of insulin binding to C6 cells. A maximal effect was found with 2 mM-butyrate that decreased the receptor by 40-70% after 48 h. Butyrate decreased numbers of receptors of both classes, but did not significantly alter receptor affinity. Other short-chain fatty acids, as well as keto acids, had a similar effect, but with a lower potency. Cycloheximide caused an accumulation of insulin receptors at the cell surface, since insulin binding increased and receptor affinity did not change after incubation with the inhibitor. Simultaneous addition of butyrate and cycloheximide abolished the loss of receptors produced by the fatty acid. In cells preincubated with butyrate, cycloheximide also produced a large increase in receptor numbers, showing that in the absence of new receptor synthesis a large pool of receptors re-appears at the surface of butyrate-treated cells.     

Regulatory relation between insulin receptor and its functional responses in primary cultured hepatocytes of adult rats

The relation between changes of insulin receptor and various metabolic responses were studied in adult rat hepatocytes in primary culture. In cells cultured for 3 h without insulin, the number of high affinity sites and the dissociation constant (Kd) of insulin receptor, determined from a Scatchard plot, were 1.05 x 10(5) sites/cell and 1.5 x 10(-9) M, respectively. The receptor number increased 2-fold, but the Kd value remained constant during 2-days culture in insulin-free medium (up-regulation). Addition of dexamethasone (Dex), growth hormone, glucagon or triiodothyronine did not change the number of insulin receptors or the Kd value. In contrast, 1-day culture in insulin (1 x 10(-7) M) medium decreased the receptor number by half (down-regulation) without change of the Kd value. Short-term responses of glycogenesis, amino acid transport and lipogenesis by insulin increased as the receptor number increased. In these cases, the sensitivity to insulin (Ka: half dose for the maximum response) did not change in cells with different receptor numbers, but the maximum response changed. These results show that hepatocytes, unlike adipocytes, do not have spare receptors of insulin. During down-regulation, the receptor number decreased by only half, but the insulin responses were lost almost completely. The receptor number returned to the normal level after culture in insulin-free medium for 12 h, but recovery of the responses took longer, suggesting that for the insulin response not only change of receptor number, but also other regulatory mechanisms for post-receptor processes, such as desensitization, are involved.     

Dexamethasone inhibition of rat hepatoma cell growth and cell cycle traverse is reversed by insulin

(1) The growth of 7800 C1 Morris hepatoma cells was inhibited by dexamethasone. The inhibition was detectable at 1 nM and half-maximal effect was obtained with approx. 13 nM dexamethasone. About 80% growth inhibition was obtained with 250 nM of the hormone and the growth rate was normalized on cessation of treatment. (2) These hepatoma cells contain dexamethasone receptors with equilibrium dissociation constant of 0.24 nM and a capacity of 24 fmol/mg cell protein. Treatment of the cells with insulin did not change these dexamethasone binding properties. Binding experiments showed that 2, 10 and 100% of the receptors were occupied when the cells were incubated with 1 nM, 7 nM and 250 nM dexamethasone, respectively. (3) Insulin completely counteracted the growth inhibition by dexamethasone and antagonized the induction of peroxisomal acyl-CoA oxidase and tyrosine aminotransferase caused by the glucocorticoid. (4) Micro-flow fluorometry showed that the cultures had a major diploid DNA stem line and a minor tetraploid stem line. Changes in diploid, tetraploid and S phase cells of the diploid stem line were scored. Dexamethasone reduced the proportion of cells in S phase and of tetraploid cells. Insulin partly reversed the action of dexamethasone in S phase, but prevented the reduction in tetraploid cells caused by dexamethasone. (5) The mitotic rate was significantly reduced by dexamethasone and this effect was reversed by insulin. (6) Continuous [3H]methyl-thymidine labelling showed a growth fraction of unity in all treatment groups. (7) It is concluded that dexamethasone induces growth inhibition by reducing the G1-S transition. Insulin is able to counteract this effect and increase the rate of DNA synthesis.     

Characterization and Regulation of Insulin Receptors in Rat Brain

An in vitro receptor binding assay, using filtration to separate bound from free [125I]insulin, was developed and used to characterize insulin receptors on membranes isolated from specific areas of rat brain. The kinetic and equilibrium binding properties of central receptors were similar to those of hepatic receptors. The binding profiles in all tissues were complex and were consistent with binding in multiple steps or to multiple sites. Similar binding properties were found among receptors in olfactory tubercle/bulb, cerebral cortex, hippocampus, striatum, hypothalamus, and cerebellum. High affinity [125I]insulin binding sites (KD = 3-11 nM) were distributed evenly between membranes isolated from P1 and P2 fractions of these brain areas, with the exception of the olfactory tubercle in which binding to P2 membranes was four-fold greater (Bmax = 150 fmol/mg protein). One difference between insulin receptors in brain and peripheral target tissues, however, was observed. Following exposure to 0.17 microM insulin for 3 h at 37 degrees C, the number of specific [125I]insulin binding sites on adipocytes decreased by 40%, while the number of binding sites on minces of cerebral cortex/olfactory tubercle remained constant. The results suggest that although the binding characteristics of central and peripheral insulin receptors are similar, these receptors do not appear to be regulated in the same manner.     

Down-regulation of insulin receptors in the heart: studies on primary cultured adult cardiac myocytes

Primary cultured cardiac myocytes from adult rats have been used to study insulin receptor regulation. Culturing of cells in the presence of insulin induced a dose-dependent down-regulation of insulin binding with a maximal effect of 35% at an insulin concentration of 1.7 X 10(-7) mol/l. The number of high-affinity sites decreased from 110 000 to 70 000 sites per cell in control and down-regulated cells, respectively, with no change in the apparent affinity constant. Down-regulation was found to be rapid (t 1/2 = 3 h) and fully reversible. Culturing of cells in the presence of cycloheximide (0.1 mmol/l) or Tris (35 mmol/l) resulted in a further time-dependent increase in insulin-induced receptor loss with no effect on insulin binding to control cells. The action of both agents was found to be additive reaching a down-regulation of 51% after a culture period of 16 h. Recovery of insulin binding activity after removal of insulin remained unaffected in the presence of cycloheximide, whereas Tris inhibited this process by 74%. In conclusion our results show that the concept of insulin-induced receptor down regulation can be extended to the adult heart muscle. Moreover the data suggest involvement of protein synthesis and receptor recycling in this process.     

Effects of trypsin on binding of [3H]epinephrine and [3H]-dihydroergocryptine to rat liver plasma membranes. Evidence for interconversion of binding sites

Treatment of liver plasma membranes with trypsin at low concentrations (1 to 2 microgram/mg of protein) caused at 3- to 4-fold increase in alpha-specific [3H]epinephrine binding. The change was due to an increase in the number of high affinity binding sites, with no change in the dissociation constant. With increasing trypsin concentrations, the dissociation constant was decreased and there was a progressive loss of binding. Elastase, papain, and thermolysin caused similar effects, whereas the thrombin, leucine aminopeptidase, phospholipase A2, phospholipase C, phospholipase D, and detergents did not cause an increase in [EH]epinephrine binding. The increase in epinephrine high affinity binding sites was correlated with a loss of high affinity [3H]-dihydroergocryptine binding sites which also bind [3H]epinephrine with low affinity (El-Refai, M. F., Blackmore, P. F., and Exton, J. H. (1979) J. Biol. Chem. 254, 4375-4386). Incubation of membranes with the alpha blockers dihydroergocryptine (50 nM) and phenoxybenzamine (20 nM) prior to protease treatment diminished the increase in [3H]epinephrine binding induced by trypsin (1.5 microgram/mg). The concentration dependence and time course of trypsin actions on 70 nM [3H]epinephrine binding and 10 nM [3H]dihydroergocryptine binding are consistent with a trypsin-mediated conversion of low affinity epinephrine binding sites to high affinity epinephrine binding sites.     

Dexamethasone increases expression of mannose receptors and decreases extracellular lysosomal enzyme accumulation in macrophages

Macrophages express a mannose-specific pinocytosis receptor that binds and internalizes lysosomal hydrolases. Treatment of rat bone marrow-derived macrophages with dexamethasone resulted in a concentration- and time-dependent increase in mannose-receptor activity. The dexamethasone effect was maximal at 24 h. Half-maximal effects were observed at a dexamethasone concentration of 2.5 X 10(-9) M. With 125I-beta-glucuronidase as ligand, a 2.5-fold increase in uptake rate was observed in dexamethasone-treated cells, with no change in Kuptake (2.5 X 10(-7) M beta-glucuronidase). Cell surface binding (4 degrees C) was elevated 2.6-fold following dexamethasone treatment. The increase in ligand binding appeared to be due to an increase in number of sites with no change in affinity. Cycloheximide suppressed the dexamethasone-mediated rise in receptor number, while cycloheximide alone had little effect on receptor activity over 16 h. These results suggest that dexamethasone stimulates synthesis of mannose receptors in macrophages. Extracellular accumulation of hexosaminidase was sharply reduced by dexamethasone treatment, and corresponded with the rise in mannose-receptor activity. Extracellular levels of hexosaminidase from untreated macrophages were modestly increased by the presence of mannan, while the extracellular activity from dexamethasone-treated cells was increased significantly by mannan. Extracellular hexosaminidase, released from zymosan-treated macrophages, was dramatically reduced by dexamethasone pretreatment. Enzyme released from zymosan-stimulated macrophages was efficiently endocytosed by dexamethasone-treated cells in co-culture experiments, and this endocytosis was blocked by the addition of mannan. These results suggest that the mannose receptor of macrophages may play a role in regulating extracellular levels of lysosomal enzymes via a secretion-recapture mechanism.     

Glucocorticoids upregulate the high affinity receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes in vitro.

Glucocorticoids were shown to induce a time- and dose-dependent increment of specific [125I]VIP-binding on human mononuclear leucocytes in culture. Cortisol (0.5 microM) increased specific [125I]VIP-binding to 132% of control after 48 h preincubation, to 162% after 96 h, and to 175% after 144 h. Dexamethasone (0.5 microM) increased specific [125I]VIP-binding to 140%, 194% and 210% after the same time periods. Analysis of the binding data revealed an increase in Bmax to 119% by cortisol (0.5 microM, 48 h) and to 194% by dexamethasone (0.5 microM, 48 h), and no change in Kd for the high affinity receptor after preincubation. The number of low affinity binding sites for VIP was also increased by glucocorticoids. However, in contrast to the high affinity receptor, low affinity binding sites were initially downregulated in culture, and glucocorticoids induced a restitution to number and affinity close to those obtained for freshly isolated leucocytes. This increase in low affinity binding sites was blocked by actinomycin D, in contrast to the high affinity receptor upregulation which was independent of de novo protein synthesis. Furthermore, corresponding to the glucocorticoid induced high affinity receptor upregulation, an increase in VIP stimulated cyclic AMP production was observed. The results of this study suggest that leucocyte responsiveness to VIP can be influenced by glucocorticoids.