Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Effects and receptors of glucocorticoids in rat thymus cells and human peripheral lymphocytes Allan Munck , Gerald R. Crabtree & Kendall A. Smith To cite this article: Allan Munck , Gerald R. Crabtree & Kendall A. Smith (1978) Effects and receptors of glucocorticoids in rat thymus cells and human peripheral lymphocytes, Journal of Toxicology and Environmental Health, 4:2-3, 409-425, DOI: 10.1080/15287397809529668 To link to this article: http://dx.doi.org/10.1080/15287397809529668
Published online: 19 Oct 2009.
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Citing articles: 8 View citing articles
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Date: 05 November 2015, At: 21:01
EFFECTS AND RECEPTORS OF GLUCOCORTICOIDS IN RAT THYMUS CELLS AND HUMAN PERIPHERAL LYMPHOCYTES Allan Munck, Gerald R. Crabtree, Kendall A. Smith
Downloaded by [Universite Laval] at 21:01 05 November 2015
Departments of Physiology and Medicine, Dartmouth Medical School, Hanover, New Hampshire
Glucocorticoids are widely used for therapeutic purposes and have many toxic side effects. It seems likely that their physiological, therapeutic, and toxic effects are exerted through similar receptors and may be inherently inseparable. Lymphoid cells are targets for all these effects. With rat thymus cells in vitro, glucocorticoids immediately bind to cytoplasmic receptors, which are translocated to the nucleus, where they apparently induce messenger RNA for specific proteins that rapidly inhibit glucose transport. Protein and RNA metabolism are inhibited more slowly and eventually the cells die. The rate of formation of nuclear complexes and the timing of the hypothetical RNA-synthetic step are such as to suggest that the hormone-receptor complexes stimulate RNA synthesis in proportion to the rate at which they bind to nuclear sites rather than in proportion to the number of nuclear sites occupied. With normal peripheral human lymphocytes the rates of formation of hormonereceptor complexes are similar to those in rat thymocytes. The rate of onset of inhibition of glucose transport is lower, however, as is the rate of cytolysis. In human peripheral lymphocytes stimulated to undergo blast transformation with concanavalin A there is a dramatic increase in the number of glucocorticoid receptor sites per cell. This increase may be associated with a stage of the normal cell cycle, the mitogen stimulus inducing partial synchronization of the cell population. It has not been found, contrary to widespread belief, that mitogen stimulation renders cells insensitive to glucocorticoids.
INTRODUCTION Among the steroid hormones, glucocorticoids are probably the ones that have found widest application as therapeutic agents for the treatment of conditions, such as rheumatoid arthritis and lymphocytic leukemias, that do not seem to be caused by a deficiency or other imbalance of the hormone itself. The doses required in such applications often result in toxic effects of the kind that characterize Cushing's syndrome. In contrast to many of the therapeutic effects, the toxic effects of the glucocorticoids We are grateful for the excellent technical assistance of Rosemary Foley and Susan Kennedy. This work was supported by research grants AM 03535, CA 17323, and CA 17646 from the National Institutes of Health, U.S. Public Health Service. Requests for reprints should be sent to Allan Munck, Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755.
409 Journal of Toxicology and Environmental Health, 4:409-425,1978 Copyright© 1978 by Hemisphere Publishing Corporation
0098-4108/78/0402-0409$ 2.25
Downloaded by [Universite Laval] at 21:01 05 November 2015
410
A. MUNCK ETAL.
appear in most cases to be exaggerated manifestations of their normal physiological actions. Unfortunately, despite massive efforts on the part of the chemists and endocrinologists of an earlier era who achieved spectacular success in separating glucocorticoid from mineralocorticoid activity, it has not proved possible to synthesize steroids in which the beneficial therapeutic activities of glucocorticoids are present without the toxic effects. Consequently, although we are far from a complete understanding of any of the actions of the glucocorticoids, we are forced to assume that most if not all of them—physiological, pharmacological, therapeutic, and toxic—are mediated through similar specific glucocorticoid binding sites. It remains to be seen whether all these sites are parts of glucocorticoid receptors similar to those that have now been identified and associated with physiological effects in many tissues (Munck and Leung, 1977). Lymphoid cells, the main subject of this paper, are targets for both therapeutic and toxic actions of glucocorticoids. In lymphoid cells these actions may be not only inseparable but indistinguishable. For example, the lympholytic actions that are regarded as undesirable side effects in many clinical applications may be directly responsible for the benefits of glucocorticoid treatment in certain forms of leukemia. Even normal physiological levels of glucocorticoids apparently cause lymphocytolysis, or at least suppression of lymphoid tissues, since removal of the adrenals is generally followed by hypertrophy of the lymphoid organs. It therefore seems likely that in the actions of glucocorticoids on lymphoid tissues, the physiological, therapeutic, and toxic effects of the hormones are produced not only through the same receptors but through the same mechanisms, the differences between them being differences of degree or of clinical context. GLUCOCORTICOID INHIBITION OF GLUCOSE TRANSPORT BY RAT THYMUS CELLS Our interest in lymphoid cells developed originally from studies on the effects of glucocorticoids on carbohydrate metabolism. From observations with whole animals that suggested that glucocorticoids inhibit glucose uptake in peripheral tissues, we were led to test for and demonstrate such an effect first with adipose tissue and subsequently with rat thymus cells. This is one of the more widespread effects of glucocorticoids, having also been observed in several other peripheral tissues (Munck, 1971). With rat thymus cells in suspension as our experimental system, we have investigated in some detail the mechanism of the inhibition. Figure 1 summarizes schematically a number of observations that indicate that the effect is primarily a result of inhibition of glucose transport. Exposure of cells incubated at 37°C to 1 /JLM cortisol for about 1 h decreases the rate of disappearance of glucose from the medium, lowers the level inside the
GLUCOCORTICOIDS IN RAT THYMUS CELLS AND HUMAN LYMPHOCYTES
THYMUS
411
CELL
methyl t 3-0glucose
Downloaded by [Universite Laval] at 21:01 05 November 2015
FIGURE 1. Outline of evidence that cortisol affects glucose transport in thymus cells. Abbreviations: G, glucose; G6P, glucose 6-phosphate; 3OMg, 3-O-methylglucose. See text for details.
cell of glucose 6-phosphate (G6P), reduces lactate output, and, perhaps most significantly, reduces the rate of transport of the nonmetabolizable glucose analog 3-O-methylglucose. Transport of glucose, furthermore, appears to be the rate-limiting step in glucose metabolism, since there is little free glucose inside the cells (Munck, 1971; Munck and Leung, 1977). In Fig. 2 we show the effect on G6P levels of adding glucose to cells that have been incubated without glucose and with or without cortisol for 20 min at 37°C. Within 5 min the G6P levels have stabilized, exhibiting a significant cortisol effect (Munck, 1968; Mosher et al., 1971). With this 5-min glucose pulse we explored the early time course of trie hormone effect and found that the first signs of the effect appear with a pulse from 15 to 20 min after the addition of cortisol. Subsequently we have developed an even shorter (1 or 2 min) and simpler pulse method, using radioactive 3-O-methylglucose or glucose and measuring the radioactivity taken up in the cells (Munck and Zyskowski, 1975). The time course of inhibition of glucose transport measured with this
Control -O— Cortisol.lO M(20min) Glucose added at 0 min 0
I
0
lOmin
FIGURE 2. Effect of cortisol on levels of glucose 6-phosphate (G6P) generated in rat thymus cells by addition of glucose. Thymus cell suspensions were incubated for 20 min with or without (control) cortisol and without substrate. Glucose was added to give a concentration of 5.5 rnM. The incubations were continued, and samples were removed at intervals for G6P analysis. The values for 0 min were obtained with aliquots taken just before glucose addition. Each point is the mean from triplicate incubations. Standard errors are smaller than the symbols (from Mosher et al., 1971).
A. MUNCK ETAL.
412
TEMPERATURE SENSITIVE—--
SPECIFIC -RNAfs:
30
[3H] Cortisol bound to
% inhibition
SPECIFIC -PROTEIN(S)-
Inhibition of glucose transport
I< JO
V °
V
receptors (dpm)
5 10 15 20 25 " 4 0 0 80 Minutes after addition of cortisol at 37 C
Downloaded by [Universite Laval] at 21:01 05 November 2015
Y777777, -Cortisol +Act. D Vi.'i'i 7/777 +CycloheximideC 20°C
20
General inhibition of protein synthesis
10
120
i.'.'//;. / / / / / / / / / /
EZ3 blocks, or •
fails to block, early cortisol effects
FIGURE 3. Time course in rat thymus cell suspensions at 37°C of cortisol-receptor complex formation, cortisol-induced inhibition of glucose transport, and inhibition of protein synthesis. Kinetics of receptor complex formation were determined with [ 3 H] cortisol at about 0.1 \iM. Inhibitory metabolic effects were produced with about 1 pM cortisol. Glucose transport was measured with 2-min pulses of radioactive hexose, initiated at the times indicated. Shaded segments of the horizontal bars in the lower part of the figure indicate roughly the time intervals during which emergence of the cortisol effect on glucose metabolism can be blocked by treatment with cortexolone (which displaces cortisol from the glucocorticoid receptors), Actinomycin D, and cycloheximide, and delayed by lowering temperature. Open bars indicate periods during which these treatments have no effect. At the top of the figure is the sequence of steps by which it is hypothesized that the cortisol-receptor complex leads to synthesis of a specific protein that inhibits glucose transport (from Munck et al., 1978).
latter assay is shown in Fig. 3. It is characterized by the abrupt appearance of inhibition after 15-20 min exposure to cortisol, followed by a slower increase. Other general metabolic effects of the glucocorticoids on thymus cells develop considerably more slowly. For example, inhibition of protein synthesis does not become evident until after about 1 h (Young et al., 1971). Measurable cell lysis takes about 8 h (Leung and Munck, 1975), although structural changes can be observed by electron microscopy after 1-2 h (Burton et al., 1967). The causal relationships between the early effects on glucose uptake and the later inhibitory and lytic effects remain obscure (Munck and Leung, 1977). EARLIEST STEPS IN THE ACTIONS OF GLUCOCORTICOIDS ON THYMUS CELLS The rapid effect of cortisol on glucose uptake can be blocked by Actinomycin D and cycloheximide, inhibitors of RNA and protein
Downloaded by [Universite Laval] at 21:01 05 November 2015
GLUCOCORTICOIDS IN RAT THYMUS CELLS AND HUMAN LYMPHOCYTES
413
synthesis, respectively. The time course of sensitivity to these inhibitors is revealing. As illustrated schematically by the horizontal bars in the lower part of Fig. 3, Actinomycin D blocks the hormone effect (assayed after 30 min) if it is present during the first 5 min of exposure to cortisol, but not if it is added after 5 min (Mosher et al., 1971). Cordycepin acts similarly (Young et al., 1974). After 5 min cortisol itself is not essential: it can be washed out or displaced by using the antiglucocorticoid cortexolone without affecting the magnitude of the effect after 30 min (Mosher et al., 1971). Cycloheximide, as well as puromycin, inhibits only if it is present from about 15 to 25 min, precisely the time interval during which the cortisol inhibition begins to appear (Hallahan et al., 1973b). From these observations we have hypothesized that cortisol rapidly induces a burst of synthesis of messenger RNA (mRNA) that after 15 min brings about synthesis of a protein that directly or indirectly inhibits glucose transport (Munck, 1971; Hallahan et al., 1973b). During the interval from 5 to 15 min, when neither inhibitor is effective, the process is highly temperature-sensitive. We assume that during this interval the hypothetical mRNA is transported from the nucleus to the ribosomes. RATES OF FORMATION OF "CYTOPLASM IC" NUCLEAR GLUCOCORTICOID-RECEPTOR COMPLEXES IN RAT THYMUS CELLS On addition of [ 3 H]cortisol to thymus cells at 37°C, the hormone becomes bound to cytoplasmic receptors, which are rapidly translocated to nuclear-bound form. The kinetics of formation of these forms are illustrated by the first two curves in Fig. 3 (Munck and Brinck-Johnsen, 1968; Wira and Munck, 1974; Munck and Foley, 1976). They show that the cytoplasmic complex is formed first, preceding by about 1 min the formation of nuclear complex. We have measured directly the rate of transformation of cytoplasmic to nuclear complex (Fig. 4) and find that the overall reaction of activation and translocation has a half-time of about 30 s in intact cells at 37°C (Wira and Munck, 1974). We have repeated all these observations using [3H]dexamethasone with similar results (Munck and Foley, 1976), except that since the dissociation rate constant of dexamethasone is smaller than that of cortisol, dexamethasone takes longer to reach a steady state. These studies on association agree well with the generally accepted model of formation of steroid hormone-receptor complexes, and in particular with the role of the cytoplasmic complex as an obligatory intermediate in formation of the nuclear complex. How the hormone and receptor eventually leave the nucleus is not known, but the prevalent view appears to be that dissociation takes place by simple reversal of the reactions of association. Several observations we have made suggest that the true picture may
A. MUNCK ET AL.
414 1
i.o
1
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Effects and receptors of glucocorticoids in rat thymus cells and human peripheral lymphocytes Allan Munck , Gerald R. Crabtree & Kendall A. Smith To cite this article: Allan Munck , Gerald R. Crabtree & Kendall A. Smith (1978) Effects and receptors of glucocorticoids in rat thymus cells and human peripheral lymphocytes, Journal of Toxicology and Environmental Health, 4:2-3, 409-425, DOI: 10.1080/15287397809529668 To link to this article: http://dx.doi.org/10.1080/15287397809529668
Published online: 19 Oct 2009.
Submit your article to this journal
Article views: 2
View related articles
Citing articles: 8 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [Universite Laval]
Date: 05 November 2015, At: 21:01
EFFECTS AND RECEPTORS OF GLUCOCORTICOIDS IN RAT THYMUS CELLS AND HUMAN PERIPHERAL LYMPHOCYTES Allan Munck, Gerald R. Crabtree, Kendall A. Smith
Downloaded by [Universite Laval] at 21:01 05 November 2015
Departments of Physiology and Medicine, Dartmouth Medical School, Hanover, New Hampshire
Glucocorticoids are widely used for therapeutic purposes and have many toxic side effects. It seems likely that their physiological, therapeutic, and toxic effects are exerted through similar receptors and may be inherently inseparable. Lymphoid cells are targets for all these effects. With rat thymus cells in vitro, glucocorticoids immediately bind to cytoplasmic receptors, which are translocated to the nucleus, where they apparently induce messenger RNA for specific proteins that rapidly inhibit glucose transport. Protein and RNA metabolism are inhibited more slowly and eventually the cells die. The rate of formation of nuclear complexes and the timing of the hypothetical RNA-synthetic step are such as to suggest that the hormone-receptor complexes stimulate RNA synthesis in proportion to the rate at which they bind to nuclear sites rather than in proportion to the number of nuclear sites occupied. With normal peripheral human lymphocytes the rates of formation of hormonereceptor complexes are similar to those in rat thymocytes. The rate of onset of inhibition of glucose transport is lower, however, as is the rate of cytolysis. In human peripheral lymphocytes stimulated to undergo blast transformation with concanavalin A there is a dramatic increase in the number of glucocorticoid receptor sites per cell. This increase may be associated with a stage of the normal cell cycle, the mitogen stimulus inducing partial synchronization of the cell population. It has not been found, contrary to widespread belief, that mitogen stimulation renders cells insensitive to glucocorticoids.
INTRODUCTION Among the steroid hormones, glucocorticoids are probably the ones that have found widest application as therapeutic agents for the treatment of conditions, such as rheumatoid arthritis and lymphocytic leukemias, that do not seem to be caused by a deficiency or other imbalance of the hormone itself. The doses required in such applications often result in toxic effects of the kind that characterize Cushing's syndrome. In contrast to many of the therapeutic effects, the toxic effects of the glucocorticoids We are grateful for the excellent technical assistance of Rosemary Foley and Susan Kennedy. This work was supported by research grants AM 03535, CA 17323, and CA 17646 from the National Institutes of Health, U.S. Public Health Service. Requests for reprints should be sent to Allan Munck, Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755.
409 Journal of Toxicology and Environmental Health, 4:409-425,1978 Copyright© 1978 by Hemisphere Publishing Corporation
0098-4108/78/0402-0409$ 2.25
Downloaded by [Universite Laval] at 21:01 05 November 2015
410
A. MUNCK ETAL.
appear in most cases to be exaggerated manifestations of their normal physiological actions. Unfortunately, despite massive efforts on the part of the chemists and endocrinologists of an earlier era who achieved spectacular success in separating glucocorticoid from mineralocorticoid activity, it has not proved possible to synthesize steroids in which the beneficial therapeutic activities of glucocorticoids are present without the toxic effects. Consequently, although we are far from a complete understanding of any of the actions of the glucocorticoids, we are forced to assume that most if not all of them—physiological, pharmacological, therapeutic, and toxic—are mediated through similar specific glucocorticoid binding sites. It remains to be seen whether all these sites are parts of glucocorticoid receptors similar to those that have now been identified and associated with physiological effects in many tissues (Munck and Leung, 1977). Lymphoid cells, the main subject of this paper, are targets for both therapeutic and toxic actions of glucocorticoids. In lymphoid cells these actions may be not only inseparable but indistinguishable. For example, the lympholytic actions that are regarded as undesirable side effects in many clinical applications may be directly responsible for the benefits of glucocorticoid treatment in certain forms of leukemia. Even normal physiological levels of glucocorticoids apparently cause lymphocytolysis, or at least suppression of lymphoid tissues, since removal of the adrenals is generally followed by hypertrophy of the lymphoid organs. It therefore seems likely that in the actions of glucocorticoids on lymphoid tissues, the physiological, therapeutic, and toxic effects of the hormones are produced not only through the same receptors but through the same mechanisms, the differences between them being differences of degree or of clinical context. GLUCOCORTICOID INHIBITION OF GLUCOSE TRANSPORT BY RAT THYMUS CELLS Our interest in lymphoid cells developed originally from studies on the effects of glucocorticoids on carbohydrate metabolism. From observations with whole animals that suggested that glucocorticoids inhibit glucose uptake in peripheral tissues, we were led to test for and demonstrate such an effect first with adipose tissue and subsequently with rat thymus cells. This is one of the more widespread effects of glucocorticoids, having also been observed in several other peripheral tissues (Munck, 1971). With rat thymus cells in suspension as our experimental system, we have investigated in some detail the mechanism of the inhibition. Figure 1 summarizes schematically a number of observations that indicate that the effect is primarily a result of inhibition of glucose transport. Exposure of cells incubated at 37°C to 1 /JLM cortisol for about 1 h decreases the rate of disappearance of glucose from the medium, lowers the level inside the
GLUCOCORTICOIDS IN RAT THYMUS CELLS AND HUMAN LYMPHOCYTES
THYMUS
411
CELL
methyl t 3-0glucose
Downloaded by [Universite Laval] at 21:01 05 November 2015
FIGURE 1. Outline of evidence that cortisol affects glucose transport in thymus cells. Abbreviations: G, glucose; G6P, glucose 6-phosphate; 3OMg, 3-O-methylglucose. See text for details.
cell of glucose 6-phosphate (G6P), reduces lactate output, and, perhaps most significantly, reduces the rate of transport of the nonmetabolizable glucose analog 3-O-methylglucose. Transport of glucose, furthermore, appears to be the rate-limiting step in glucose metabolism, since there is little free glucose inside the cells (Munck, 1971; Munck and Leung, 1977). In Fig. 2 we show the effect on G6P levels of adding glucose to cells that have been incubated without glucose and with or without cortisol for 20 min at 37°C. Within 5 min the G6P levels have stabilized, exhibiting a significant cortisol effect (Munck, 1968; Mosher et al., 1971). With this 5-min glucose pulse we explored the early time course of trie hormone effect and found that the first signs of the effect appear with a pulse from 15 to 20 min after the addition of cortisol. Subsequently we have developed an even shorter (1 or 2 min) and simpler pulse method, using radioactive 3-O-methylglucose or glucose and measuring the radioactivity taken up in the cells (Munck and Zyskowski, 1975). The time course of inhibition of glucose transport measured with this
Control -O— Cortisol.lO M(20min) Glucose added at 0 min 0
I
0
lOmin
FIGURE 2. Effect of cortisol on levels of glucose 6-phosphate (G6P) generated in rat thymus cells by addition of glucose. Thymus cell suspensions were incubated for 20 min with or without (control) cortisol and without substrate. Glucose was added to give a concentration of 5.5 rnM. The incubations were continued, and samples were removed at intervals for G6P analysis. The values for 0 min were obtained with aliquots taken just before glucose addition. Each point is the mean from triplicate incubations. Standard errors are smaller than the symbols (from Mosher et al., 1971).
A. MUNCK ETAL.
412
TEMPERATURE SENSITIVE—--
SPECIFIC -RNAfs:
30
[3H] Cortisol bound to
% inhibition
SPECIFIC -PROTEIN(S)-
Inhibition of glucose transport
I< JO
V °
V
receptors (dpm)
5 10 15 20 25 " 4 0 0 80 Minutes after addition of cortisol at 37 C
Downloaded by [Universite Laval] at 21:01 05 November 2015
Y777777, -Cortisol +Act. D Vi.'i'i 7/777 +CycloheximideC 20°C
20
General inhibition of protein synthesis
10
120
i.'.'//;. / / / / / / / / / /
EZ3 blocks, or •
fails to block, early cortisol effects
FIGURE 3. Time course in rat thymus cell suspensions at 37°C of cortisol-receptor complex formation, cortisol-induced inhibition of glucose transport, and inhibition of protein synthesis. Kinetics of receptor complex formation were determined with [ 3 H] cortisol at about 0.1 \iM. Inhibitory metabolic effects were produced with about 1 pM cortisol. Glucose transport was measured with 2-min pulses of radioactive hexose, initiated at the times indicated. Shaded segments of the horizontal bars in the lower part of the figure indicate roughly the time intervals during which emergence of the cortisol effect on glucose metabolism can be blocked by treatment with cortexolone (which displaces cortisol from the glucocorticoid receptors), Actinomycin D, and cycloheximide, and delayed by lowering temperature. Open bars indicate periods during which these treatments have no effect. At the top of the figure is the sequence of steps by which it is hypothesized that the cortisol-receptor complex leads to synthesis of a specific protein that inhibits glucose transport (from Munck et al., 1978).
latter assay is shown in Fig. 3. It is characterized by the abrupt appearance of inhibition after 15-20 min exposure to cortisol, followed by a slower increase. Other general metabolic effects of the glucocorticoids on thymus cells develop considerably more slowly. For example, inhibition of protein synthesis does not become evident until after about 1 h (Young et al., 1971). Measurable cell lysis takes about 8 h (Leung and Munck, 1975), although structural changes can be observed by electron microscopy after 1-2 h (Burton et al., 1967). The causal relationships between the early effects on glucose uptake and the later inhibitory and lytic effects remain obscure (Munck and Leung, 1977). EARLIEST STEPS IN THE ACTIONS OF GLUCOCORTICOIDS ON THYMUS CELLS The rapid effect of cortisol on glucose uptake can be blocked by Actinomycin D and cycloheximide, inhibitors of RNA and protein
Downloaded by [Universite Laval] at 21:01 05 November 2015
GLUCOCORTICOIDS IN RAT THYMUS CELLS AND HUMAN LYMPHOCYTES
413
synthesis, respectively. The time course of sensitivity to these inhibitors is revealing. As illustrated schematically by the horizontal bars in the lower part of Fig. 3, Actinomycin D blocks the hormone effect (assayed after 30 min) if it is present during the first 5 min of exposure to cortisol, but not if it is added after 5 min (Mosher et al., 1971). Cordycepin acts similarly (Young et al., 1974). After 5 min cortisol itself is not essential: it can be washed out or displaced by using the antiglucocorticoid cortexolone without affecting the magnitude of the effect after 30 min (Mosher et al., 1971). Cycloheximide, as well as puromycin, inhibits only if it is present from about 15 to 25 min, precisely the time interval during which the cortisol inhibition begins to appear (Hallahan et al., 1973b). From these observations we have hypothesized that cortisol rapidly induces a burst of synthesis of messenger RNA (mRNA) that after 15 min brings about synthesis of a protein that directly or indirectly inhibits glucose transport (Munck, 1971; Hallahan et al., 1973b). During the interval from 5 to 15 min, when neither inhibitor is effective, the process is highly temperature-sensitive. We assume that during this interval the hypothetical mRNA is transported from the nucleus to the ribosomes. RATES OF FORMATION OF "CYTOPLASM IC" NUCLEAR GLUCOCORTICOID-RECEPTOR COMPLEXES IN RAT THYMUS CELLS On addition of [ 3 H]cortisol to thymus cells at 37°C, the hormone becomes bound to cytoplasmic receptors, which are rapidly translocated to nuclear-bound form. The kinetics of formation of these forms are illustrated by the first two curves in Fig. 3 (Munck and Brinck-Johnsen, 1968; Wira and Munck, 1974; Munck and Foley, 1976). They show that the cytoplasmic complex is formed first, preceding by about 1 min the formation of nuclear complex. We have measured directly the rate of transformation of cytoplasmic to nuclear complex (Fig. 4) and find that the overall reaction of activation and translocation has a half-time of about 30 s in intact cells at 37°C (Wira and Munck, 1974). We have repeated all these observations using [3H]dexamethasone with similar results (Munck and Foley, 1976), except that since the dissociation rate constant of dexamethasone is smaller than that of cortisol, dexamethasone takes longer to reach a steady state. These studies on association agree well with the generally accepted model of formation of steroid hormone-receptor complexes, and in particular with the role of the cytoplasmic complex as an obligatory intermediate in formation of the nuclear complex. How the hormone and receptor eventually leave the nucleus is not known, but the prevalent view appears to be that dissociation takes place by simple reversal of the reactions of association. Several observations we have made suggest that the true picture may
A. MUNCK ET AL.
414 1
i.o
1
