RESUMO
Cultured fibroblasts obtained from patients with tissue resistance to 1,25-dihydroxyvitamin D3 (vitamin D3--dependent rickets, type II) contain normal, low, or undetectable concentrations of this hormone's receptor protein as measured by a ligand-binding assay. Extracts from these cells were evaluated for receptors by immunoassay with a recently developed monoclonal antibody to the chick receptor. The results show that a protein sedimenting at 3.7S and recognizable by the antibody exists in comparable concentrations in cells from both normal and resistant patients, irrespective of the hormone-binding abnormalities of the cells. This implies that deficiencies in hormone binding associated with inherited tissue resistance to 1,25-dihydroxyvitamin D3 probably arise from structural variations in the receptor molecule and not from defective receptor synthesis.
Assuntos
Fibroblastos/análise , Hipofosfatemia Familiar/metabolismo , Receptores de Esteroides/análise , Anticorpos Monoclonais , Células Cultivadas , Humanos , Radioimunoensaio , Ensaio Radioligante , Receptores de Calcitriol , Pele/citologiaRESUMO
The androgen resistance syndromes are generally felt to be due to quantitative or qualitative abnormalities of the androgen receptor. Some patients with testicular feminization have no demonstrable fibroblast cytosol androgen binding, whereas others have androgen binding in cultured fibrobalsts that is thermolabile or fails to be stabilized by sodium molybdate. I describe here familial incomplete testicular feminization associated with reduced nuclear androgen retention. Fibroblasts, cultured from pubic skin biopsies of two phenotypic female 46XY siblings, were assayed for whole cell and nuclear uptake of [(3)H]dihydrotestosterone in dispersed, intact cells. Whole cell binding of [(3)H]dihydrotestosterone at 22 degrees C in the patients' fibroblasts was in the normal range. However, no high affinity, saturable binding of [(3)H]dihydrotestosterone was demonstrable in crude nuclear pellets prepared from the patients' fibroblasts incubated at 37 degrees C with the hormone. Incubating the patients' cells with [(3)H]methyltrienolone or examining the nuclear uptake of [(3)H]dihydrotestosterone in these cells at 22 degrees C did not alter these findings. Although cytosol from the patients' cells revealed a quantitatively diminished 8S peak for [(3)H]dihydrotestosterone after centrifugation on sodium molybdate-containing sucrose gradients, there was no peak of (3)H in the 4S region from 0.3 M KCl nuclear extracts of the patients' cells after they had been incubated with [(3)H]dihydrotestosterone at 37 degrees C. Although whole cell binding studies at 37 degrees C showed minimally diminished androgen binding in the patients' cells compared with binding at 22 degrees C, Griffin (1979. J. Clin. Invest.64: 1624-1631.) has demonstrated thermolability of the androgen receptors in fibroblasts also cultured from these patients. The observations with intact cells coupled with the diminished cytosol 8S peak of [(3)H]dihydrotestosterone on sucrose gradients indicate that these patients have cytosol androgen receptors that are qualitatively abnormal physicochemically, the physiologic consequence of which is failure of nuclear androgen localization. Thus, although the underlying defect in the pathogenesis of the androgen resistance in these patients appears to reside in the androgen receptor, the crucial biologic manifestation of the molecular lesion is impaired nuclear androgen retention. These experiments, therefore, suggest that assessment of nuclear [(3)H]dihydrotestosterone uptake is an effective indicator of the functional integrity of the androgen receptor system in patients with various forms of androgen insensitivity and provides additional insights to those obtained by thermolability or cytosol sucrose gradient studies.
Assuntos
Transtornos do Desenvolvimento Sexual/metabolismo , Receptores Androgênicos/análise , Receptores de Esteroides/análise , Adolescente , Síndrome de Resistência a Andrógenos/metabolismo , Neoplasias da Mama/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Centrifugação com Gradiente de Concentração , Citosol/análise , Di-Hidrotestosterona/metabolismo , Transtornos do Desenvolvimento Sexual/genética , Fibroblastos/metabolismo , Humanos , Masculino , Receptores Androgênicos/genéticaRESUMO
UNLABELLED: We evaluated the interaction of [3H]1,25(OH)2D3 with skin fibroblasts cultured from normal subjects or from affected members of six kindreds with rickets and resistance to 1-alpha, 25(OH)2D [1,25(OH)2D]. We analyzed two aspects of the radioligand interaction; nuclear uptake with dispersed, intact cells at 37 degrees C and binding at 0 degrees C with soluble extract ("cytosol") prepared from cells disrupted in buffer containing 300 mM KCl and 10 mM sodium molybdate. With normal fibroblasts the affinity and capacity of nuclear uptake of [3H]1,25(OH)2D3 were 0.5 nM and 10,300 sites per cell, respectively; for binding with cytosol these were 0.13 nM and 8,900 sites per cell, respectively. The following four patterns of interaction with [3H]1,25(OH)2D3 were observed with cells cultured from affected patients: (a) two kindreds; cytosol binding and whole-cell nuclear uptake both unmeasurable; (b) one kindred, decreased capacity and normal affinity both for binding in cytosol and for nuclear uptake in whole cells; (c) two kindreds, normal or nearly normal capacity and affinity of binding in cytosol but unmeasurable whole-cell nuclear uptake; and (d) one kindred, normal capacity and affinity of both cytosol binding and whole-cell nuclear uptake. In all cases where the radioligand bound with high affinity in nucleus or cytosol, the nucleus- or cytosol-associated radioligand exhibited normal sedimentation velocity on sucrose density gradients. When two kindreds exhibited similar patterns (i.e. pattern a or c) with the analyses of cultured fibroblasts, clinical features in affected members suggested that the underlying genetic defects were not identical. IN CONCLUSION: (a) Fibroblasts cultured from human skin manifest nuclear uptake and cytosol binding of [3H]1,25(OH)2D3 that is an expression of the genes determining these processes in target tissues. (b) Based upon data from clinical evaluations and from analyses of cultured fibroblasts, severe resistance to 1,25(OH)2D resulted from five or six distinct genetic mutations in six kindreds.
Assuntos
Fibroblastos/citologia , Receptores de Esteroides/genética , Núcleo Celular/metabolismo , Citosol/efeitos da radiação , Resistência a Medicamentos , Humanos , Ensaio Radioligante , Receptores de Calcitriol , Pele/citologiaRESUMO
Deficient activity of the guanine nucleotide regulatory protein (G unit), an integral component of the membrane-bound adenylate cyclase complex, has been implicated as the biochemical lesion in many patients with pseudohypoparathyroidism (PHP) type I. In addition to renal resistance to parathyroid hormone in this disorder, there is decreased responsiveness of diverse tissues to hormones that act via 3',5'-cyclic AMP (cAMP). To assess whether a deficiency of G units could account for impaired adenylate cyclase activity, we studied cAMP production in intact cultured fibroblasts and fibroblast plasma membranes from five patients with PHP in response to several activators of adenylate cyclase. The number of G units in PHP fibroblast membranes, measured by cholera toxin-dependent [(32)P]ADP ribosylation of G-unit peptides, as well as the G-unit activity, determined by the ability of detergent extracts to reconstitute adenylate cyclase activity in G-unit-deficient S49 CYC(-) membranes, were found to be markedly reduced compared with control membranes (43 and 40%, respectively), The activation of fibroblast membrane adenylate cyclase by effectors that act directly through the G unit (guanosine triphosphate, guanosine 5'-0-[3-thiotriphosphate] [GTP-gamma-S], NaF) was significantly greater in control membranes than in membranes from patients with PHP. Moreover, we found that hormone (prostaglandin E(1)) stimulated adenylate cyclase activity was also greater in control membranes than in PHP membranes. Neither the apparent affinity of membrane adenylate cyclase for GTP-gamma-S (apparent K(m) =5 X 10(-8) M) nor the rate of enzyme activation by GTP-gamma-S was significantly different in fibroblast membranes from control subjects and patients with PHP. In contrast to the notable differences in hormone and G-unit-activated adenylate cyclase shown in fibroblast membranes from PHP patients and control subjects, the intrinsic catalytic activity of membranes, as determined by forskolin-stimulated adenylate cyclase, was not significantly different in the two groups. Intact fibroblasts derived from patients with PHP accumulated significantly (P 0.001) less cAMP (46+/-21 pmol cAMP/mcg DNA, n = 5) than cells from normal individuals (170+/-51 pmol cAMP/mcg DNA, n = 11) when stimulated with PGE(1). PGE(1)-stimulated accumulation of cAMP by intact fibroblast monolayers correlated closely with PGE(1) plus GTP-activated membrane adenylate cyclase activity in both patients and controls (r = 0.97, P < 0.001). Our data show that, in patients with PHP, (a) fibroblast membranes show a decreased complement of G units, (b) membrane catalytic activity is normal, but adenylate cyclase activity is reduced when stimulated by hormone or by effectors which activate the G unit, (c) the ability of cells to accumulate cAMP in response to hormone stimulation is reduced, and (d) reduced membrane adenylate cyclase activity correlates well with impaired cellular cAMP synthesis. These results, taken together, indicate that a deficiency of G-unit activity can impair synthesis of cAMP by both intact and broken cells, and may explain the resistance of multiple tissues to hormones that act via cAMP observed in PHP.
Assuntos
AMP Cíclico/biossíntese , Fibroblastos/metabolismo , Pseudo-Hipoparatireoidismo/metabolismo , Receptores de Superfície Celular/deficiência , Adenilil Ciclases/metabolismo , Alprostadil , Membrana Celular/metabolismo , Células Cultivadas , Feminino , Fibroblastos/efeitos dos fármacos , Proteínas de Ligação ao GTP , Nucleotídeos de Guanina/farmacologia , Humanos , Prostaglandinas E/farmacologia , Pseudo-Hipoparatireoidismo/etiologia , Fluoreto de Sódio/farmacologiaRESUMO
UNLABELLED: 1,25(OH)2D3 induces 25(OH)D3-24-hydroxylase (24-OHase) in cultured skin fibroblasts from normal subjects. We evaluated 24-OHase induction by 1,25(OH)2D3 in skin fibroblasts from 10 normal subjects and from four unrelated patients with hereditary resistance to 1,25(OH)2D or vitamin D-dependent rickets type II (DD II). Fibroblasts were preincubated with varying concentrations of 1,25(OH)2D3 for 15 h and were then incubated with 0.5 microM [3H]25(OH)D3 at 37 degrees C for 30 min; lipid extracts of the cells were analyzed for [3H]24,25(OH)2D3 by high performance liquid chromatography and periodate oxidation. Apparent maximal [3H]24,25(OH)2D3 production in normal cell lines was 9 pmol/10(6) cells per 30 min and occurred after induction with 10(-8) M 1,25(OH)2D3. 24-OHase induction was detectable in normal fibroblasts at approximately 3 X 10(-10) M 1,25(OH)2D3. [3H]24,25(OH)2D3 formation after exposure to 1,25(OH)2D3 was abnormal in fibroblasts from all four patients with DD II. In fibroblasts from two patients with DD II, [3H]24,25(OH)2D3 formation was unmeasurable (below 0.2 pmol/10(6) cells per 30 min) at 1,25(OH)2D3 concentrations up to 10(-6) M. Fibroblasts from the other two patients with DD II required far higher than normal concentrations of 1,25(OH)2D3 for detectable [3H]24,25(OH)2D3 induction. In one, [3H]24,25(OH)2D3 production reached 2.9 pmol/10(6) cells per 30 min at 10(-6) M 1,25(OH)2D3 (30% normal maximum at 10(-6) M 1,25(OH)2D3). In the other, [3H]24,25(OH)2D3 production achieved normal levels, 7.3 pmol/10(6) cells per 30 min after 10(-6) M 1,25(OH)2D3. The two patients whose cells had a detectable 24-OHase induction by 1,25(OH)2D3 showed a calcemic response to high doses of calciferols in vivo. Our current observations correlate with these two patients' responsiveness to calciferols in vivo and suggest that their target organ defects can be partially or completely overcome with extremely high concentrations of 1,25(OH)2D3. The two patients whose cells showed no detectable 24-OHase induction in vitro failed to show a calcemic response to high doses of calciferols in vivo. IN CONCLUSION: (a) the measurement of 24-OHase induction by 1,25(OH)2D3 in cultured skin fibroblasts is a sensitive in vitro test for defective genes in the 1,25(OH)2D effector pathway. (b) This assay provides a useful tool for characterizing the target tissue defects in DD II and predicting response to calciferol therapy.
Assuntos
Calcitriol/farmacologia , Sistema Enzimático do Citocromo P-450 , Ergocalciferóis/fisiologia , Fibroblastos/enzimologia , Raquitismo/enzimologia , Esteroide Hidroxilases/biossíntese , Alopecia/etiologia , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Indução Enzimática/efeitos dos fármacos , Ergocalciferóis/uso terapêutico , Humanos , Hipercalcemia/etiologia , Raquitismo/classificação , Raquitismo/complicações , Pele/citologia , Vitamina D3 24-HidroxilaseRESUMO
We have studied a man suspected of having primary cortisol resistance on the basis of high 24-h mean plasma cortisol levels (27.4 micrograms/dl) and no stigmata of Cushing's syndrome. His son had slightly elevated 24-h mean plasma cortisol levels (9.9 micrograms/dl; normal 7.52 micrograms/dl). Both had high plasma protein unbound cortisol and increased urinary free cortisol. Plasma ACTH concentration was high, and both were resistant to adrenal suppression by dexamethasone. The father appeared to have mineralocorticoid excess resulting in hypertension, hypokalemia, and metabolic alkalosis. This was found to be due to markedly elevated plasma levels of deoxycorticosterone and corticosterone. The son, who was normotensive, had mildly increased plasma corticosterone and normal deoxycorticosterone levels. To study the apparent end-organ resistance to cortisol, we examined the glucocorticoid receptor in the white cells and fibroblasts of these patients. In both tissues, using both whole cell and cytosol assays, the glucocorticoid receptor was found to have reduced affinity for dexamethasone. In the cytoxol assays, a reduced receptor number was found as well. We conclude that cortisol resistance is a rare familial syndrome owing to an abnormal glucocorticoid receptor with a decreased affinity for cortisol.
Assuntos
Hiperfunção Adrenocortical/sangue , Hidrocortisona/sangue , Receptores de Glucocorticoides/metabolismo , Receptores de Esteroides/metabolismo , Hiperfunção Adrenocortical/complicações , Hormônio Adrenocorticotrópico/sangue , Adulto , Aldosterona/urina , Alcalose/sangue , Alcalose/complicações , Ritmo Circadiano , Corticosterona/sangue , Desoxicorticosterona/sangue , Dexametasona/sangue , Humanos , Hidrocortisona/urina , Hipopotassemia/sangue , Hipopotassemia/complicações , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
Saturable binding of androgens, glucocorticoids, and triiodothyronine was found in the 64-24 hormone-responsive rat mammary carcinoma cell line. Androgen receptors had a dissociation content (Kd) for methyltrienolone of 3.4 X 10(-10) M and a binding capacity of approximately 10,000 sites/cell in whole cells. 5 alpha-[3H]dihydrotestosterone (DHT) was specifically taken up into approximately 2,150 nuclear sites with an affinity of 8.3 X 10(-10) M when nuclei were isolated from whole cells incubated with [3H]DHT. Sucrose gradient centrifugation of cytosol prepared from these cells revealed a displaceable [3H]DHT-binding component which migrated at 8S. Sedimentation analysis with high salt gradients of nuclear extracts from cells incubated with [3H]DHT revealed a peak of radioactivity in the 4S region which was abolished by coincubation of the cells with excess nonradioactive methyltrienolone. Receptors for [3H]dexamethasone were more abundant (approximately 50,000 sites/cell) in whole cells and had a Kd of 7.5 X 10(-9) M, but the number of nuclear binding sites was similar to that for androgens. Specificity studies using unlabeled steroids showed that each of the two classes of steroid receptors had greater affinities for their appropriate hormones. High affinity receptors for estrogens and progestins were not detectable in these cells. Triiodothyronine receptors were demonstrable but at a very low binding capacity (1,100 sites/cell). The Kd of these receptors was 0.6 X 10(-10) M. Cytogenetic studies revealed 44 chromosomes/mitosis with several unique markers. These receptor and karyotypic features suggest that the 64-24 cells may be useful in studying androgen action on breast cancer independently of estrogen or progestin influence, as well as the effects of thyroid hormone and glucocorticoids on breast cancer cells.
Assuntos
Carcinoma/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Cariotipagem , Ratos , Receptores Androgênicos/metabolismo , Receptores de Glucocorticoides/metabolismo , Hormônios Tireóideos/metabolismoRESUMO
The effects of 1 alpha, 25-dihydroxyvitamin D3 (VD3) on proliferation, differentiation, and macromolecular synthesis in the new Philadelphia chromosome-positive chronic myelogenous leukemia cell line, RWLeu-4, were investigated. Binding of [3H]VD3 was saturable, with approximately 2000-3000 sites/cell, and half-maximal binding occurring at 0.21-0.33 nM. Treatment of RWLeu-4 cells with VD3 induced 24R-hydroxylase activity, a marker of vitamin D3 responsiveness in many tissues. Exposure of RWLeu-4 cells to VD3 also inhibited proliferation and DNA synthesis with a 50% effective dose of 3.5-10 nM within 72 h; in addition, protein and RNA synthesis were inhibited by VD3 treatment. Exposure of RWLeu-4 cells to 5 nM VD3 for 72 h caused 50% of the cells to differentiate into macrophage/monocyte type cells as judged by nitroblue tetrazolium staining and adherence to plastic. Progressive expression of cell surface maturation-specific antigens of the monocyte/macrophage lineage was induced by treatment of RWLeu-4 cells with VD3 for 24 to 72 h at doses that inhibited cellular proliferation. c-myc RNA, which is constitutively expressed in RWLeu-4 cells, increased after 0.5 h of treatment with 50 nM VD3 and then rapidly decreased to barely detectable levels after 4 h of treatment. Finally, the in vitro tyrosine kinase activity associated with the p210bcr-abl oncogene product was decreased approximately 50% by VD3 treatment. Because of the presence of a functional receptor-effector system for VD3 and multiple biological responses to the hormone, these cells provide a unique model system with which to probe the specific effects of VD3 on cell growth and differentiation in chronic myelogenous leukemia.
Assuntos
Calcitriol/farmacologia , Sistema Enzimático do Citocromo P-450 , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Macrófagos/citologia , Monócitos/citologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , DNA de Neoplasias/biossíntese , Indução Enzimática/efeitos dos fármacos , Proteínas de Fusão bcr-abl/metabolismo , Expressão Gênica , Humanos , Técnicas In Vitro , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-myc , RNA Mensageiro/genética , RNA Neoplásico/genética , Receptores de Calcitriol , Receptores de Esteroides/fisiologia , Esteroide Hidroxilases/biossíntese , Células Tumorais Cultivadas , Vitamina D3 24-HidroxilaseRESUMO
A 58-year-old man was initially seen with fatigue and weight loss. Laboratory examination detected hypercalcemia, elevated 1,25-dihydroxycholecalciferol levels, low parathyroid hormone (PTH) concentrations, and subperiosteal bone resorption. The patient underwent subtotal parathyroidectomy for presumed hyperparathyroidism, but serum calcium and 1,25-dihydroxycholecalciferol levels remained elevated following surgery. Search for another cause of the hypercalcemia disclosed enlarged para-aortic lymph nodes, biopsy specimens of which demonstrated Hodgkin's disease. After treatment of the patient with two cycles of chemotherapy with mechlorethamine hydrochloride, vincristine sulfate, procarbazine hydrochloride, and prednisone, serum calcium, 1,25-dihydroxycholecalciferol, and PTH levels normalized. We speculate that the humoral hypercalcemia in this patient resulted from tumor production of 1,25-dihydroxycholecalciferol.
Assuntos
Doença de Hodgkin/complicações , Hipercalcemia/complicações , Reabsorção Óssea/complicações , Calcitriol/sangue , Doença de Hodgkin/metabolismo , Humanos , Hipercalcemia/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
A patient with a left lower lung mass had muscle weakness, generalized hyperpigmentation, metabolic alkalosis, and profound hypokalemia. His elevated serum cortisol, corticosterone, and adrenocorticotropic hormone (ACTH) concentrations were not suppressed after midnight dexamethasone administration. Light and electron microscopic sections of the lung mass fitted the pathological criteria for adenocarcinoma. Immunocytochemical analysis of the tumor demonstrated specific staining with antibody to beta-endorphin, suggesting that the tumor cells made the common precursor molecule of ACTH, beta-lipotropin, and endorphin. This is, to the best of our knowledge, only the second case report of pulmonary adenocarcinoma associated with the syndrome of ectopic ACTH.
Assuntos
Síndrome de ACTH Ectópico/diagnóstico , Adenocarcinoma/diagnóstico , Neoplasias Pulmonares/diagnóstico , Síndromes Endócrinas Paraneoplásicas/diagnóstico , Síndrome de ACTH Ectópico/etiologia , Adenocarcinoma/complicações , Adenocarcinoma/ultraestrutura , Humanos , Neoplasias Pulmonares/complicações , Neoplasias Pulmonares/ultraestrutura , Masculino , Pessoa de Meia-IdadeRESUMO
Hormonal measurements in some women with hirsutism often reveal little or no elevation in androgen levels to explain the disorder. Thus, it has been postulated that increased sensitivity of the hair follicle to androgen may contribute to the development of hirsutism in such patients. We, therefore, sought androgen receptor abnormalities in skin fibroblasts cultured from 10 hirsute women (ages 17-43) and normal or mildly elevated plasma testosterone levels (28-82 ng/dl). Androgen receptor content (Ro) and binding affinity (Kd) in cultured pubic skin fibroblasts were measured using a dispersed, whole cell assay. Ten such cell lines from these women were compared with 19 pubic skin cell lines from 9 normal volunteers (6 males and 3 females) and from 10 other subjects (males with gynecomastia or hypospadias). There was no statistically significant difference in the mean androgen receptor content (11,600 +/- 2700 (SE) sites/cell fibroblasts vs 7900 +/- 700 sites/cell or binding affinity (2.0 +/- 0.3 (SE) X 10(-9) M vs 1.5 +/- 0.2 X 10(-9) M, respectively) between the patients' fibroblasts and those of the controls. We conclude that hirsutism cannot be explained by abnormalities in fibroblast androgen receptor number or affinity. These observations do not exclude the possibility that other mechanisms might lead to increased peripheral androgen sensitivity in such patients.
Assuntos
Hirsutismo/metabolismo , Receptores Androgênicos/análise , Receptores de Esteroides/análise , Pele/análise , Adolescente , Adulto , Feminino , Fibroblastos/análise , Ginecomastia/metabolismo , Humanos , Hipospadia/metabolismo , Técnicas In Vitro , MasculinoRESUMO
The antiandrogenic properties of delta 1-testolactone (17 alpha-oxa-D-homo-1,4-androstane-3,17-dione; Teslac) were investigated in vivo and in vitro. Teslac (75 mg/day for 7 days) inhibited the rise in ventral prostate weight induced by testosterone (T) (P less than 0.001), dihydrotestosterone (DHT) (P less than 0.05), and a combination of T plus 17 beta-estradiol (E2) (P less than 0.01) in immature castrate rats. Similar effects were seen on the seminal vesicles after T and T plus E2 (P less than 0.001). Teslac also decreased prostate and seminal vesicle weights in intact immature rats. The effects of Teslac were dose and time dependent. Teslac did not change the concentration of serum T or DHT. However, Teslac inhibited DHT binding to the androgen receptor (Ki = 2.5 +/- 0.8 X 10(-7) M) in cytosol of the rat prostate. Teslac also inhibited DHT binding to the androgen receptor in cultured human prepuce fibroblasts and cultured rat mammary tumor cells (Ki = 1.9 +/- 0.3 X 10(-5) M). The results indicate that Teslac, in addition to its antiaromatase activity, is an antiandrogen by virtue of its interaction with the androgen receptor.
Assuntos
Antagonistas de Androgênios/farmacologia , Neoplasias Mamárias Experimentais/metabolismo , Próstata/fisiologia , Receptores Androgênicos/metabolismo , Receptores de Esteroides/metabolismo , Pele/metabolismo , Testolactona/farmacologia , Animais , Castração , Células Cultivadas , Citosol/metabolismo , Di-Hidrotestosterona/metabolismo , Feminino , Fibroblastos/metabolismo , Cinética , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Receptores Androgênicos/efeitos dos fármacos , Glândulas Seminais/fisiologiaRESUMO
Administration of the antifungal drug ketoconazole reduces serum 1,25-dihydroxyvitamin D (1,25-D) levels in normal subjects. To determine whether a similar effect occurs in hypercalcemic patients, ketoconazole (200 mg every 8 h for 7 days) was given to nine patients with confirmed primary hyperparathyroidism, three patients with probable primary hyperparathyroidism who were awaiting surgery, and three patients with mild hypercalcemia of uncertain etiology who were being followed. Ketoconazole administration led to a significant reduction in mean serum 1,25-D levels in the hypercalcemic patients [basal, 64 +/- 7 (+/- SEM) pg/mL (154 +/- 17 pmol/L) vs. 36 +/- 5 pg/mL (86 +/- 12 pmol/L) after ketoconazole; P less than 0.001]. Serum total calcium fell slightly but significantly [basal, 11.05 +/- 0.17 mg/dL (2.76 +/- 0.04 mmol/L) vs. 10.77 +/- 0.16 (2.69 +/- 0.04 mmol/L) after ketoconazole; P less than 0.02], but the falls in total serum calcium and serum 1,25-D after ketoconazole treatment were not correlated with one another. Ketoconazole administration did not alter serum ionized calcium, 25-hydroxyvitamin D, phosphate, alkaline phosphatase, or PTH concentrations or urinary cAMP excretion. The responses to ketoconazole were similar in all three patient subgroups. We conclude that short term administration of ketoconazole to hypercalcemic patients causes a substantial fall in serum 1,25-D and a small fall in total serum calcium. These effects render ketoconazole a potentially useful agent for investigation of the importance of 1,25-D in patients with hypercalcemic disorders and for their treatment.
Assuntos
Calcitriol/sangue , Cálcio/sangue , Hipercalcemia/sangue , Cetoconazol/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The antimycotic agent ketoconazole is known to inhibit several cytochrome P450-dependent enzymes involved in the biosynthesis of steroid hormones from cholesterol. Since 1,25-dihydroxyvitamin D is also a sterol synthesized by cytochrome P450-dependent enzymes, we assessed whether ketoconazole would lower serum 1,25-dihydroxyvitamin D levels. In nine normal men, administration of ketoconazole for 1 week in doses of 300-1200 mg/day led to a dose-dependent reduction in serum 1,25-dihydroxyvitamin D levels (r = -0.64; P less than 0.001). At the highest dose taken by each man (1200 mg/day in six, 900 mg/day in one, and 600 mg/day in two), serum levels of 1,25-dihydroxyvitamin D fell significantly compared to baseline [14 +/- 1 (+/- SEM) vs. 39 +/- 3 pg/ml; P less than 0.001), but there was no change in serum levels of 25-hydroxyvitamin D, PTH, calcium, phosphate, or alkaline phosphatase. Ketoconazole may be potentially useful in exploring the pathogenetic role of 1,25-dihydroxyvitamin D in disorders of calcium metabolism and in treatment of patients with hypercalcemic disorders or renal stone disease.
Assuntos
Calcitriol/sangue , Cetoconazol/farmacologia , Adolescente , Adulto , Depressão Química , Relação Dose-Resposta a Droga , Humanos , Cetoconazol/sangue , MasculinoRESUMO
Using a dispersed intact cell assay system, we screened a number of compounds for their ability to compete for nuclear binding of L-[125I]T3 in cultured human skin fibroblasts incubated for 90 min at 37 degrees C. T3 inhibited nuclear [125I]T3 binding by 50% at a concentration of 3.4 +/- 0.3 (+/- SE) X 10(-10) M. 3,5-Dimethyl-3'-isopropyl-thyronine, a nonhalogenated thyroid hormone agonist, had an affinity for the nuclear thyroid hormone receptor (4.4 X 10(-9) M, as judged by 50% inhibition of nuclear [125I]T3 binding) that correlates well with its thyromimetic potency. Of several radiographic contrast agents and other compounds tested (iodipamide, iopanoic acid, sodium ipodate, sodium diatrizoate, sodium tyropanoate, diphenylhydantoin, carbamazepine, amiodarone hydrochloride, propylthiouracil, propranolol, and potassium iodide), only sodium ipodate (Oragrafin) and iopanoic acid (Telepaque) interfered with nuclear [125I]T3 binding, with 50% inhibition at 5 X 10(-5) and 1.8 X 10(-4) M, respectively. Interestingly, diphenylhydantoin and amiodarone, two compounds previously thought to interact with thyroid hormone receptors, did not impair fibroblast nuclear [125I]T3 binding at concentrations up to 10(-3) M. We conclude that this in vitro assay system with intact human cells is useful in evaluating the nuclear T3 receptor affinity of compounds that affect thyroid hormone action or metabolism. These studies more closely approximate in vivo conditions and, therefore, provide information not obtainable by studies with isolated nuclei or nuclear extracts.
Assuntos
Núcleo Celular/metabolismo , Meios de Contraste/farmacologia , Receptores de Superfície Celular/efeitos dos fármacos , Ligação Competitiva , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Receptores dos Hormônios Tireóideos , Pele/metabolismoRESUMO
Although several drugs with antiandrogenic properties have been used to treat such conditions as prostatic carcinoma, precocious puberty, acne, and hirsutism, their relative strengths in human tissues are not known. Most of the compounds that are effective clinically in opposing androgen action interact with the androgen receptor in various assay systems. To determine in human cells the relative potencies of these agents as well as others with androgenic properties, we measured the abilities of various compounds to compete with [3H]dihydrotestosterone [( 3H]DHT) for androgen-binding sites in dispersed human genital skin fibroblasts at 22 degrees C. The concentrations of unlabeled DHT, methyltrienolone (a synthetic non- metabolizeable androgen), and testosterone required for 50% inhibition of [3H]DHT binding were similar, approximately 1 nM [0.87 +/- 0.12 (+/- SE), 1.18 +/- 0.18, and 1.01 +/- 0.20 nM, respectively]. The relative binding activities, defined by the ratio of the concentration of methyltrienolone to the concentration of competitor required for 50% displacement of [3H]DHT, were as follows: spironolactone greater than R2956 (a synthetic antiandrogen) greater than megestrol acetate greater than cyproterone acetate greater than estradiol greater than flutamide much greater than testolactone greater than cimetidine. Danazol, an androgen agonist that causes hirsutism, was nearly as effective as spironolactone in its ability to compete for the fibroblast androgen receptor, 50% inhibition of fibroblast [3H]DHT binding was achieved by 1.76 +/- 0.31 nM spironolactone and 2.85 +/- 0.50 nM danazol. Two other compounds that induce hirsutism, diphenylhydantoin and diazoxide, did not displace [3H]DHT. We conclude that 1) of the compounds tested, spironolactone, which is rapidly metabolized in vivo to a much less potent competitor, is the most potent antiandrogen in its ability to interact in vitro with human skin fibroblast androgen receptors; 2) estradiol is a relatively potent androgen receptor binder; and 3) this receptor assay, combined with metabolic clearance and pharmacokinetic considerations, should be useful in selecting drugs for androgen and antiandrogen therapy.
Assuntos
Receptores Androgênicos/metabolismo , Receptores de Esteroides/metabolismo , Pele/metabolismo , Ligação Competitiva , Células Cultivadas , Di-Hidrotestosterona/metabolismo , Fibroblastos/metabolismo , Humanos , Recém-Nascido , Masculino , PênisRESUMO
We used cultured skin fibroblasts from patients with hereditary resistance to 1,25-dihydroxyvitamin D [1,25-(OH)2D] and normal hormone binding to soluble extract from cells [i.e. receptor-positive resistance to 1,25-(OH)2D] to characterize DNA binding of the receptor for 1,25-(OH)2D. Occupied receptor was generated by incubating soluble extracts from cells with [3H]1,25-(OH)2D3; occupied receptor was applied to columns of DNA-cellulose and then eluted with linear gradients of KCl. Occupied receptors of cells from other sources eluted as a single peak at 0.20-0.26 M KCl; this elution pattern was independent of tissue (skin, breast cancer, or osteosarcoma) or species (human or rat) of origin of the receptors. With cells from two kindreds in whom there was mildly decreased localization of the hormone-receptor complex to the nucleus in vitro, occupied receptor interacted abnormally with DNA-cellulose (elution at 0.09-0.13 M KCl vs. normal at 0.20-0.26 M KCl); this suggested mutation(s) that affected a DNA-binding domain of the receptor in these two kindreds. With receptor-positive cells from two other kindreds in whom there was unmeasurable hormone localization to the nucleus, the elution pattern of occupied receptors from DNA-cellulose was normal; this suggested mutation(s) which did not affect the same DNA-binding site. We conclude that our demonstration of two distinct elution profiles from DNA-cellulose reflects two independent classes of mutation, either of which can cause receptor-positive resistance to 1,25-(OH)2D.
Assuntos
Erros Inatos do Metabolismo/genética , Mutação , Receptores de Esteroides/genética , Animais , Calcitriol/metabolismo , Linhagem Celular , Células Cultivadas , Celulose/análogos & derivados , Cromatografia de Afinidade , DNA/análogos & derivados , Resistência a Medicamentos , Fibroblastos/metabolismo , Humanos , Erros Inatos do Metabolismo/metabolismo , Ratos , Receptores de Calcitriol , Receptores de Esteroides/isolamento & purificação , Receptores de Esteroides/metabolismo , Raquitismo/genética , Raquitismo/metabolismo , Pele/metabolismoRESUMO
The cellular mechanisms that cause the syndrome of generalized, peripheral, and pituitary resistance to thyroid hormones are unclear. In order to investigate the possibility of a defect at the nuclear receptor for T3, we examined: 1) equilibrium binding of [125I]T3 (64 analyses) to the nuclei of intact cultured fibroblasts from 12 patients with both sporadic and familial generalized or selective pituitary resistance, 17 normal subjects, and two apparently normal siblings from affected families; and 2) kinetics of [125I]T3 nuclear uptake (22 analyses) in intact fibroblasts from 4 generalized resistant patients from two different kindreds, compared to one apparently normal sister of one affected patient and 6 normal subjects. Statistical analysis of the equilibrium binding parameters showed no differences in nuclear binding capacity for T3 among all groups. The equilibrium dissociation constants (Kd) also were not significantly different in fibroblasts from the selective pituitary resistant patients, the normal siblings, and the normal subjects. In contrast, the KdS from the general resistant patients were significantly increased (median = 1.94 X 10(-10) M, 5-95% confidence interval = 1.18-2.93 X 10(-10) M 1.11, 0.77-1.25 X 10(-10) M; P less than 0.008). Since there was considerable overlap of the equilibrium binding values among groups, we studied the kinetics of [125I]T3 nuclear uptake in selected cell lines from normal subjects and familial generalized resistant patients. Kinetic analysis of the association curve revealed that, compared to the normal subjects, maximum binding at 10(-10) M [125I]T3 was significantly reduced to 50% in two resistant patients, to 10% in another two patients, and to 65% in one apparently-normal sibling. The empiric parameter lambda that corresponds to the slope at the origin of the curve was not significantly different in any patient compared to normal. The calculated apparent association constant, derived from lambda, and the binding capacity obtained at equilibrium, showed differences that were significant for only one of the patients. We conclude that cultured fibroblasts from patients with familial generalized resistance to thyroid hormone display abnormal kinetics of T3 nuclear uptake which provide a convenient in vitro method for the recognition of thyroid hormone resistance at the cell level. Furthermore, because cultured fibroblasts can be grown under controlled experimental conditions, they are a suitable tissue for further study of the molecular basis of these disorders.
Assuntos
Núcleo Celular/metabolismo , Receptores de Superfície Celular/metabolismo , Hormônios Tireóideos/farmacologia , Adolescente , Adulto , Células Cultivadas , Criança , Pré-Escolar , Resistência a Medicamentos , Feminino , Fibroblastos/metabolismo , Humanos , Lactente , Cinética , Masculino , Pessoa de Meia-Idade , Receptores dos Hormônios Tireóideos , Doenças da Glândula Tireoide/genética , Doenças da Glândula Tireoide/metabolismo , Hormônios Tireóideos/fisiologiaRESUMO
Patients with Graves' disease lacking eye symptoms frequently have abnormal intraocular pressure (IOP) increases on upward gaze (greater than or equal to 3 mm Hg) indicative of apparent subclinical ophthalmopathy. Because of the close relationship between Graves' disease (GD) and Hashimoto's thyroiditis (HT), we examined 30 patients with a history of HT as well as 26 patients with a history of GD, 4 patients with a history of subacute thyroiditis, 1 patient with a history of silent thyroiditis, and 25 normal subjects for the presence of IOP abnormalities at 15 degrees and 25 degrees upgaze. While all of the patients were asymptomatic, had no exophthalmos, and were euthyroid at the time of the exam, Hertel exophthalmometer readings (mean +/- SD) for the patients with GD were significantly higher (P less than 0.005) than those for either the HT patients or normal subjects (17.1 +/- 2.4 vs. 14.5 +/- 2.3 vs. 14.4 +/- 4.2 mm, respectively). At 15 degrees upgaze, IOP abnormalities occurred in 25% and 13% of patients with GD and HT, respectively. At 25 degrees upgaze, these figures rose to 54% for the GD patients and 37% in HT patients. Only 1 of 25 normal subjects had elevated IOP changes on upgaze, as did the 1 patient with silent thyroiditis, but the patients with subacute thyroiditis did not. These data suggest the frequent presence of extraocular muscle restriction in patients with a history of HT as well as in patients with a history of GD. Maximal detection of these IOP abnormalities requires that patients be examined at 25 degrees upgaze. These data support the belief that the autoimmune bases of both GD and HT are closely linked, at least as manifested by eye muscle involvement.
Assuntos
Doenças Autoimunes/complicações , Oftalmopatias/fisiopatologia , Pressão Intraocular , Doenças da Glândula Tireoide/complicações , Adulto , Idoso , Oftalmopatias/etiologia , Feminino , Doença de Graves/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Músculos/fisiopatologia , Tireoidite/complicações , Tireoidite Autoimune/complicaçõesRESUMO
UNLABELLED: The syndrome of rickets, alopecia, hypocalcemia, and high circulating levels of 1,25-dihydroxyvitamin D (1,25-(OH)2D) apparently is caused by resistance of target tissues to 1,25-(OH)2D. To evaluate this, we cultured cells from explants of long bone of one patient with this syndrome and from a control without any preexisting disorder of mineral metabolism. The cultured cells showed morphological features of fibroblasts but contained alkaline phosphatase activity without detectable acid phosphatase activity, indicating an osteoblastic origin for some or all of the cultured cells. Receptors for 1,25-(OH)2D were assessed by three methods: high affinity uptake of hormone in nuclei of dispersed cells, high affinity binding in hypertonic extracts (herein termed cytosol) from cells, and sedimentation velocity of bound [3H]1,25-(OH)2D3 in extracts of cell nuclei. With cells cultured from bone of the normal control, receptors for 1,25-(OH)2D exhibited properties indistinguishable from those found with cultured skin fibroblasts. With cells cultured from bone of the patient with resistance to 1,25-(OH)2D, high affinity uptake of 1,25-(OH)2D into nuclei was unmeasurable, but high affinity binding of hormone with cytosol was normal; these abnormal findings also were indistinguishable from abnormal findings obtained with fibroblasts cultured from skin of that patient. IN CONCLUSION: 1) Cells cultured from explants of human bone showed morphological features of fibroblasts but retained a marker enzyme characteristic of osteoblasts. Significant admixture of osteoblast-like cells with fibroblasts was possible. 2) Cells cultured from bone of a patient with familial resistance to 1,25-(OH)2D exhibit a defect in vitamin D metabolism, indistinguishable from the defect observed with cells cultured from skin of the same patient.