Glucocorticoid receptors: ATP and cell cycle dependence, phosphorylation, and hormone resistance.
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Adenosine Triphosphate↗
Biomedical subjects
L M Hu
Publications and source records attributed to L M Hu.
[Participation of septum on the increase of plasma cortisol level induced by stimulation of the greater splanchnic nerve of cats].
Experimens were performed on 36 cats, showing that stimulation of the central end of the left major splanchnic nerve induced an increase of the plasma cortisol level. The centripetal effect was abolished by lesioning the septal nuclei, just as the same as injection of propranolol into the nuclei; but phentolamine had no such preventive action. All the findings show that it is the noradrenergic beta-receptor system of the septal nuclei that exerts a considerable effect on cortisol secretion.
Adrenergic Antagonists↗
Glucocorticoid receptors in ATP-depleted cells. Dephosphorylation, loss of hormone binding, HSP90 dissociation, and ATP-dependent cycling.
Dependence of hormone binding to glucocorticoid receptors (GRs) on cellular ATP levels suggested that GRs traverse an ATP-dependent cycle, and without ATP accumulate in forms that cannot bind hormone. Such "null" receptors (NRs) were identified in ATP-depleted WEHI-7 cells, where they are tightly associated with the nuclear fraction and partly dephosphorylated. With WCL2 cells (Chinese hamster ovary cells with overexpressed GRs) depleted of ATP with azide, we have now identified dephosphorylated sites on NRs, studied possible roles of phosphorylation using GR mutants, and measured association with the 90-kDa heat shock protein (hsp90). Most NRs in WCL2 cells are dephosphorylated at serines 220 and 234, but GRs with those serines mutated to alanines do not resemble NRs since they bind hormone. They do not associate strongly with nuclei. On azide treatment, however, mutated GRs lose hormone binding capacity faster than normal GRs. Association of hsp90 (and presumably other heat shock proteins) with cytosolic GRs is drastically reduced by azide treatment, sufficient to account for decreased hormone binding. We conclude that: (a) dephosphorylation of GRs does not yield NRs, but may weaken association with hsp90. (b) The postulated ATP-dependent GR cycle can be accounted for by dissociation, and ATP-dependent reconstitution, of GR-hsp90 complexes. (c) ATP depletion blocks reconstitution of complexes. Uncomplexed GRs may accumulate as one form of NR; they are probably also the precursors for other forms of NR.
Adenosine Triphosphate↗
Kinetics of glucocorticoid receptor phosphorylation in intact cells. Evidence for hormone-induced hyperphosphorylation after activation and recycling of hyperphosphorylated receptors.
Glucocorticoid receptors are basally phosphorylated in the absence of hormone and become hyperphosphorylated after hormone treatment of intact cells. To determine the sequence of changes which the receptor undergoes following hormone binding, we analyzed the kinetics of receptor phosphorylation in WEHI-7 mouse thymoma cells and in stably transfected Chinese hamster ovary cells that overexpress the mouse receptor. No major differences were found between these two cell types. Cells were preincubated with 32P(i) and [35S] methionine to label the receptors metabolically. The phosphate content of the receptor protein was determined from the ratio of 32P to 35S in radioactive gel slices after immunopurification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Hormone-induced increases in phosphorylation were seen as early as 5 min after adding hormone and persisted for 20 h. Analysis of newly formed cytosolic and nuclear-bound activated (DNA-binding) receptors showed that activation precedes hyperphosphorylation. Nonactivated receptors, both unliganded and hormone-liganded, also became hyperphosphorylated but more slowly than activated receptors. The rate of receptor dephosphorylation, determined by chasing with unlabeled P(i), was much slower than the rate of phosphorylation or of hormone dissociation and appeared to be slightly increased by agonists and by the antagonist RU486 (which does not cause hyperphosphorylation). Mutant WEHI-7 cells lacking cAMP-dependent protein kinase activity gave basal and hormone-induced receptor phosphorylation indistinguishable from wild type cells. We conclude that (a) the substrate for hormone-dependent hyperphosphorylation is the activated hormone-receptor complex; (b) most hyperphosphorylated receptors are recycled and reutilized in hyperphosphorylated form; (c) control of receptor phosphorylation may not be cell-specific; (d) cAMP-dependent protein kinase is not involved directly or indirectly in phosphorylating major sites on the receptor in vivo.
Animals↗
Glucocorticoid receptors: ATP-dependent cycling and hormone-dependent hyperphosphorylation.
The dependence of hormone binding to glucocorticoid receptors (GRs) on cellular ATP levels led us to propose that GRs normally traverse an ATP-dependent cycle, possibly involving receptor phosphorylation, and that without ATP they accumulate in a form that cannot bind hormone. We identified such a form, the null receptor, in ATP-depleted cells. GRs are basally phosphorylated, and become hyperphosphorylated after treatment with hormone (but not RU486). In mouse receptors we have identified 7 phosphorylated sites, all in the N-terminal domain. Most are on serines and lie within a transactivation region. The time-course of hormone-induced hyperphosphorylation indicates that the primary substrates for hyperphosphorylation are the activated receptors; unliganded and hormone-liganded nonactivated receptors become hyperphosphorylated more slowly. After dissociation of substrates for hyperphosphorylation are the activated receptors; unliganded and hormone-liganded nonactivated receptors become hyperphosphorylated more slowly. After dissociation of hormone, most receptors appear to be recycled and reutilized in hyperphosphorylated form. From these and related observations, we have concluded that the postulated ATP-dependent cycle can be accounted for by hormone-induced or spontaneous dissociation of receptor-Hsp90 complexes, followed by reassociation of unliganded receptors with Hsp90 via an ATP-dependent reaction like that demonstrated in cell-free systems. Other steroid hormone receptors might traverse a similar cycle. Four of the 7 phosphorylated sites in the N-terminal domain are in consensus sequences for p34cdc2 kinases important in cell cycle regulation. This observation, along with the known cell cycle-dependence of sensitivity to glucocorticoids and other evidence, point to a role for receptor phosphorylation in controlling responses to glucocorticoids through the cell cycle.
Adenosine Triphosphate↗
Terbutaline does not improve lung function in preterm rabbits.
OBJECTIVE: We used the premature rabbit model of surfactant deficiency to test the hypothesis that perinatal administration of terbutaline would lead to increased secretion of surfactant into the alveolar space and increase lung compliance during mechanical ventilation. STUDY DESIGN: Fetuses underwent delivery at a gestational age of 28 days (term 31 days) followed by mechanical ventilation. Fetuses were subdivided into four treatment protocols: control, fetuses given terbutaline at birth, fetuses of mothers given terbutaline 1 hour before delivery, and fetuses of mothers given terbutaline intramuscularly 12 hours before delivery. Dynamic compliance was determined. After this, alveolar lavage fluid was obtained for phosphatidylcholine content determination. Some fetuses were killed at birth and their alveolar lavage phosphatidylcholine was determined. RESULTS: Among the fetuses undergoing mechanical ventilation, perinatal terbutaline exposure did not alter either dynamic compliance or alveolar lavage phosphatidylcholine. Mechanical ventilation was associated with large increases in alveolar lavage phosphatidylcholine content. CONCLUSION: Perinatal beta-adrenergic agonist exposure does not alter in vivo lung function following preterm delivery.
Animals↗
Intratubular germ cell neoplasia in infantile yolk sac tumor. Verification by tandem repeat sequence in situ hybridization.
The strong association of intratubular germ cell neoplasia (ITGCN) with adult germ cell testicular tumors is well known, but studies noting the absence of ITGCN in certain germ cell neoplasms such as spermatocytic seminoma, childhood teratoma, and infantile yolk sac tumor (YST) have raised the issue of whether these latter neoplasms follow a different path of tumorigenesis, accounting for their more benign behavior. A case study illustrating the association of ITGCN with infantile YST is presented to challenge this hypothesis. In addition to the usual characteristic features that included strong cytoplasmic glycogen deposits, and focal placental alkaline phosphatase immunoreactivity, the atypical intratubular germ cells manifested triploidy by in situ hybridization using as probe a telomeric tandem repeat sequence, p1-79, specific to chromosome 1. The invasive YST cells, in contrast, showed evidence of tetraploidy by both in situ hybridization and flow and image cytometric studies, excluding the possibility that the atypical intratubular germ cells represented intratubular invasion by adjacent YST. These findings challenge the belief that the infantile YST follows a different path of tumorigenesis than its adult germ cell counterpart and suggest other hypotheses that might better explain its more benign behavior.
Blotting, Southern↗
Effects of dideoxyforskolin on proteoglycan synthesis and structure in embryonic chick chondrocyte cultures.
1,9-Dideoxyforskolin inhibits proteoglycan synthesis and xyloside-initiated glycosaminoglycan (GAG) synthesis in chick embryo chondrocytes. Dideoxyforskolin does not affect the length of xyloside-initiated GAG chains secreted into the medium but chains from the dense proteoglycan secreted into the medium appear slightly longer. Incorporation of labeled serine into the dense proteoglycan and subsequent digestion with Pronase revealed a dramatic decrease in percent of total radioactivity associated with GAG chains in the proteoglycan from cultures treated with forskolin or dideoxyforskolin. These observations suggest that these diterpenes have a specific inhibitory effect on chain initiation reactions and thus may be useful tools in the study of proteoglycan synthesis and processing.
Animals↗
Effect of forskolin on synthesis of xyloside-initiated glycosaminoglycans in embryonic chick chondrocytes.
The synthesis of sulfated glycosaminoglycan (GAG) chains has been studied in the presence of various concentrations of the artificial acceptor 4-methylumbelliferyl beta-D-xyloside and of forskolin. Sulfated GAG chains formed in the presence of forskolin had a smaller hydrodynamic radius than controls, as revealed by chromatography on Sepharose CL-6B. Sulfated GAGs from both control and treated cultures behaved identically when chromatographed on DEAE.
Animals↗
[A new therapy of hemophilia with ranitidine].
20 cases of hemophilia, including ten of hemophilia A , nine of hemophilia B and one of von Willebrand's disease (VWD), were treated with ranitidine. The results revealed that the levels of both factor VIII and IX were increased and the clinical symptoms of bleeding were ameliorated. The level of factor VIII in hemophilia A ranged from 5.27 +/- 3.94% before treatment and rose to 14.68 +/- 4.70% during the treatment period (P less than 0.001). The level of the factor IX in the patients with hemophilia B increased from 4.42 +/- 3.01% before treatment to 20.33 + 9.31% under treatment (P less than 0.001). Tn the patient with VWD the level of the factor VIII rose but the level of von Willebrand factor did not change. The drug has no side effect. The results of our study suggests that ranitidine therapy is effective and safe in hemophilia.
Adolescent↗
Metabolic effects of forskolin in chick chondrocytes.
The effects of forskolin on parameters of energy metabolism and proteoglycan synthesis have been investigated in chick embryo sternal chondrocyte cultures. After 8 h exposure to 100 microM forskolin, ATP levels and oxygen consumption were unaltered. Protein synthesis was unaffected up to 50 microM forskolin and protein degradation was unaffected by forskolin up to 100 microM. In contrast, incorporation of the proteoglycan precursors, 35SO4 and [3H]glucosamine, was more sensitive to forskolin. Inhibition was linear with dose between 10 and 100 microM, reaching 70% at 100 microM. Incorporation of 35SO4 into glycosaminoglycan chains initiated on an artificial beta-xyloside acceptor was inhibited in the same manner. cAMP accumulation was maximal at 10 microM forskolin, a concentration which did not alter proteoglycan synthesis. We conclude that a major, acute effect of forskolin in these short-term experiments is inhibition of proteoglycan synthesis in a cAMP-independent manner.
Adenosine Triphosphate↗
Injury and remodeling of pulmonary veins by high oxygen. A morphometric study.
Breathing 87% oxygen at normobaric pressure for 28 days injuries and remodels the wall of distal pulmonary veins (less than or equal to 150 mu). Occluded vessels are evident, as are vessel remnants in which wall integrity is lost (obliterated vessels). Significantly more veins have a muscular or partially muscular wall than normal (P less than or equal to 0.001 for veins in each size category less than or equal to 150 mu, chi-square test). In some veins new muscle develops between an external and internal lamina but in many it develops within the intima, beneath the endothelium and adluminal to a single lamina. Small veins (20-25 mu in ED) with a muscular or partially muscular wall are present only in the hyperoxic lung. Increase in the percent medial thickness (%MT) of veins indicates lumen narrowing: this is relatively greater in the smallest veins. Reduction in the cross-sectional area of venous segments that are immediately postcapillary, by lumen narrowing or occlusion, contributes to the restriction of the pulmonary vascular bed by hyperoxia.
Animals↗
The effect of heparin on the haemodynamic and structural response in the rat to acute and chronic hypoxia.
In the mouse, heparin administered intermittently, has been shown to reduce the right ventricular hypertrophy (RVH) caused by hypoxia. We have investigated in the rat the effect of heparin on the haemodynamic and pulmonary vascular structural remodelling produced by hypoxia, with special reference to the new muscularization of peripheral arteries. Heparin at one of two doses (30 and 50 u/kg/h) was administered by continuous intravenous infusion from a miniosmotic pump to rats during 10 days exposure to hypobaric hypoxia and its effect examined on mean pulmonary artery pressure (PPa), RVH and, using morphometric techniques, vascular structural remodelling. Hypoxia produced the haemodynamic and structural changes previously described in this model. Heparin had no significant effect on PPa; a slight reduction in RVH was seen in the high-dose heparin group. After heparin, the narrowing of the axial pulmonary artery lumen caused by hypoxia was less: heparin reduced the proportion of arteries that became muscularized, particularly at alveolar duct level where the pericyte is the precursor smooth muscle cell. Heparin did not diminish the increase in medial thickness or reduction in external diameter of muscular arteries. Some rats, after chronic hypoxia, did not respond to an acute hypoxic challenge yet were no different from 'responders' in other haemodynamic and structural features. Including all rats, the mean acute pressor response to hypoxia was unaffected by heparin: taking only responder rats, a trend was apparent that heparin reduced the rise in PPa on acute hypoxic challenge.
Animals↗
The effects of intrauterine pneumonectomy in lambs. A morphometric study of the remaining lung at term.
A morphometric study was made of the remaining lung of term lambs that had undergone left pneumonectomy at 60, 80, 100, or 120 days gestation. All pneumonectomy lambs showed some compensatory growth since right lung volume to body weight ratio was higher in all lambs after pneumonectomy than in term controls. In 3 lambs operated at 80, 100, and 120 days, respectively, however, compensatory volume increase was more complete than in the other lamb of each age pair or the 2 lambs operated at 60 days. Despite individual variation, the potential for compensatory lung growth seems greater in the canalicular and later stages than in the pseudoglandular. Measurements indicating distal air-space size (volume density of alveoli, volume density of alveolar duct, surface density of alveolar epithelium, numerical density of alveoli, and mean linear intercept) were similar in all animals, but total alveolar surface area and number increased proportionally to lung volume. The fact that the remaining lung compensates by increasing alveolar number rather than by enlarging air spaces is a model of human developmental abnormalities in which the alveolar number at birth is greater than normal (polyalveolar). At the intraacinar level, a higher than normal proportion of muscularized arteries was found in lambs operated early in gestation and in lambs with smaller compensatory lung growth: an inverse relationship was seen--the lower the lung volume the higher the proportion of muscular arteries. The cause for this is unknown, but may result from the relatively increased flow to a vascular bed smaller than that of two normal lungs.
Angiography↗
A clinical and experimental study of acupuncture treatment of tertian malaria.
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Acupuncture Therapy↗