RESUMEN
The effects of corticotropin-releasing hormone (CRH) and dexamethasone on proopiomelanocortin (POMC) mRNA levels in cultured pituitary adenoma cells were studied in 10 patients with Cushing's disease. As a control, POMC mRNA levels in cells from nonadenomatous tissues were examined in four patients. Human POMC mRNA in the cells was analyzed by Northern blot hybridization. Human POMC DNA probe hybridized with only a single size class of RNA (approximately 1,200 nucleotides) from the adenoma and nonadenoma cells of each patient. The size of POMC mRNA did not change through the culture or after incubation with CRH or dexamethasone. CRH increased POMC mRNA levels in these cells in a dose- and time-dependent manner. The minimum concentration of CRH required to elevate POMC mRNA levels in these cells exposed for 15 h was 0.1 nM. The minimum duration of 1 nM CRH treatment required to increase these levels was 3 h under our conditions. Inhibitory effects of 1 and 10 micrograms/dl dexamethasone on ACTH release and POMC mRNA levels in nonadenoma cells were greater than those in adenoma cells. These results suggest the following: (a) that the mRNA in cultured pituitary adenoma cells is qualitatively the same as that in vivo; (b) that responses of mRNA levels to CRH are time- and dose-dependent; and (c) that adenoma cells resist the inhibitory effect of dexamethasone on POMC mRNA levels and ACTH release.
Asunto(s)
Adenoma/metabolismo , Hormona Liberadora de Corticotropina/farmacología , Dexametasona/farmacología , Neoplasias Hipofisarias/metabolismo , Proopiomelanocortina/metabolismo , ARN Mensajero/metabolismo , Hormona Adrenocorticotrópica/metabolismo , Humanos , Hibridación de Ácido Nucleico , Adenohipófisis , ARN Mensajero/aislamiento & purificación , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Plasma immunoreactive corticotropin-releasing factor (I-CRF) levels were determined by using a human CRF radioimmunoassay and an immunoaffinity procedure. The basal plasma I-CRF level in normal subjects was 6 +/- 0.5 pg/ml (mean +/- SD). We found that most plasma I-CRF levels were affected by stress, negative feedback, and circadian rhythm. Basal I-CRF levels were high in patients with Addison's disease, Nelson's syndrome, hypopituitarism stemming from pituitary macroadenoma, and CRF- and adrenocorticotropic hormone-producing tumors. A very low, but significant, amount of I-CRF was detected (1-3 pg/ml) in patients with Cushing's syndrome, in corticosteroid-treated patients, and in a patient with hypothalamic hypopituitarism. These results suggest that a major component of plasma I-CRF is of hypothalamic origin, however, other extrahypothalamic tissues cannot be ruled out as a minor source of plasma I-CRF.
Asunto(s)
Enfermedades de las Glándulas Suprarrenales/sangre , Hormona Liberadora de Corticotropina/sangre , Enfermedades Hipotalámicas/sangre , Enfermedades de la Hipófisis/sangre , Hormona Adrenocorticotrópica/sangre , Adulto , Cromatografía Líquida de Alta Presión , Síndrome de Cushing/sangre , Humanos , Hidrocortisona/sangre , Insulina/farmacología , Metirapona , RadioinmunoensayoRESUMEN
Effects of ACTH and ACTH fragments on immunoreactive corticotropin-releasing factor (I-CRF) release were examined by utilizing rat hypothalamic perifusion system and a rat CRF RIA. ACTH-(1-39) had a dose-related inhibitory effect on I-CRF release. Mean percent inhibition of I-CRF release was 52, 55, 49, 30 and less than 5 percent by ACTH-(1-39), ACTH-(1-24), alpha-MSH and ACTH-(18-39) at 2.2 nM concentrations, respectively. These results suggest the presence of a negative short-loop feedback mechanism, and also that the active core is contained within the ACTH-(1-17) structure.
Asunto(s)
Hormona Adrenocorticotrópica/farmacología , Hormona Liberadora de Corticotropina/metabolismo , Hipotálamo/metabolismo , Animales , Péptido de la Porción Intermedia de la Adenohipófisis Similar a la Corticotropina , Cosintropina/farmacología , Hipotálamo/efectos de los fármacos , Técnicas In Vitro , Cinética , Masculino , Hormonas Estimuladoras de los Melanocitos/farmacología , Fragmentos de Péptidos/farmacología , RatasRESUMEN
Immunoreactive ACTH (I-ACTH) levels in the rat anterior pituitary and plasma, and immunoreactive corticotropin-releasing factor (I-CRF) concentrations in the median eminence (ME) and plasma were determined after adrenalectomy and in insulin-induced hypoglycemia. I-CRF was detected in plasma from normal rats (mean +/- SD, 5.6 +/- 0.9 pg/ml; n = 6). Gel filtration chromatography of I-CRF from pooled plasma of these rats revealed a single peak which eluted in the position of authentic rat CRF. I-CRF levels in ME and I-ACTH levels in anterior pituitary decreased immediately after adrenalectomy, then gradually increased to high levels 14 days after surgery. Plasma I-CRF and I-ACTH concentrations increased immediately after surgery, slightly decreased to near the control levels at 24 h, and then increased to high concentrations 14 days after surgery. Plasma and ME I-CRF levels 14 days after adrenalectomy, followed by daily dexamethasone replacement, were almost the same as control levels. In insulin-induced hypoglycemia, plasma I-ACTH and I-CRF concentrations increased and ME I-CRF content decreased at 30 and 60 min. These results suggest that plasma I-CRF levels reflect changes in hypothalamic CRF levels.
Asunto(s)
Hormona Liberadora de Corticotropina/sangre , Adrenalectomía , Hormona Adrenocorticotrópica/sangre , Hormona Adrenocorticotrópica/metabolismo , Animales , Cromatografía en Gel , Hormona Liberadora de Corticotropina/metabolismo , Hipoglucemia/inducido químicamente , Hipoglucemia/metabolismo , Hipotálamo/metabolismo , Insulina , Masculino , Eminencia Media/metabolismo , Adenohipófisis/metabolismo , Radioinmunoensayo , Ratas , Ratas EndogámicasRESUMEN
Immunoreactive ACTH (I-ACTH) concentrations in the anterior pituitary (AP), intermediate-posterior pituitary (IP) and plasma, and immunoreactive corticotropin-releasing factor (I-CRF) concentrations in the median eminence (ME) and IP, were determined in adrenalectomized rats from 3 h till 14 days after surgery. Plasma I-ACTH concentrations showed the typical triphasic response over time. AP I-ACTH concentrations decreased immediately after surgery, then increased to high concentrations 3 days after surgery. I-ACTH concentrations in IP did not change through these periods. I-CRF concentrations in ME and IP decreased immediately after surgery, then gradually increased to high concentrations (ME) or to control levels (IP) 14 days after surgery. These results raise the possibility that the I-CRF in IP is of hypothalamic origin.
Asunto(s)
Adrenalectomía , Hormona Adrenocorticotrópica/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Eminencia Media/metabolismo , Neurohipófisis/metabolismo , Hormona Adrenocorticotrópica/sangre , Animales , Cinética , Masculino , Adenohipófisis/metabolismo , Ratas , Ratas EndogámicasRESUMEN
To investigate whether an increase in the intracellular glutathione disulfide (GSSG) concentration actually regulates T4-5'-deiodination in intact cells, rat hepatocytes in primary culture were exposed to glutathione-oxidizing agents (diamide and tertiary butylhydroperoxide) or vinblastine, and their effects on 5'-deiodination of T4 were studied. Deiodinating activity was determined from the 125I- fraction released from [3',5'-125I]T4 added to the serum-free culture medium. Total glutathione (T-GSH) and GSSG levels were determined enzymatically. Diamide (1 mM) and tertiary butylhydroperoxide (0.5 mM) increased the GSSG fraction to approximately 40% of the T-GSH at 5 min, followed by a rapid decrease in GSSG. Glucose deprivation of the medium caused a greater GSSG level at 5 min, followed by a delayed normalization of the increased GSSG level. T4-5'-deiodinating activity was minimally decreased in hepatocytes exposed to 1 mM diamide in the presence of glucose in the medium, but was significantly inhibited in the absence of glucose. Vinblastine, in contrast, gradually and steadily increased the GSSG fraction, and by 3 h, GSSG exceeded 20% of T-GSH (at 10(-4) M vinblastine). This was accompanied by a significant inhibition of 5'-deiodinating activity. When the enzyme activity was inhibited, the T-GSH level was decreased to 40-80% of the control level, which per se cannot account for the decreased T4-5'-deiodinating activity, as reported previously. These data suggest that the increased GSSG level, but not the T-GSH concentration, modulates T4-5'-deiodination in intact cells, and that glucose stimulates the enzyme activity by maintaining glutathione in the reduced form, probably through supplying NADPH, a cofactor for GSSG reductase.
Asunto(s)
Glucosa/farmacología , Glutatión/análogos & derivados , Yodo/metabolismo , Hígado/metabolismo , Tiroxina/metabolismo , Animales , Células Cultivadas , Diamida/farmacología , Glutatión/metabolismo , Glutatión/farmacología , Disulfuro de Glutatión , Hígado/efectos de los fármacos , Peróxidos/farmacología , Ratas , Vinblastina/farmacología , terc-ButilhidroperóxidoRESUMEN
Immunoreactive ACTH (I-ACTH) concentrations in the anterior pituitary, intermediate-posterior pituitary (IP), and plasma and immunoreactive corticotropin-releasing factor (I-CRF) concentrations in the median eminence and IP were determined in rats receiving dexamethasone for various periods from 16 h to 10 days. Plasma I-ACTH concentrations were decreased 16 h after a single injection of dexamethasone. Anterior pituitary I-ACTH concentrations did not decrease until 4 days after the start of dexamethasone medication. IP I-ACTH concentrations did not change throughout these periods. I-CRF concentrations in median eminence and IP rapidly decreased after dexamethasone administration. These results raise the possibility that the source of I-CRF in the IP is hypothalamic.
Asunto(s)
Hormona Liberadora de Corticotropina/metabolismo , Dexametasona/farmacología , Eminencia Media/fisiología , Neurohipófisis/fisiología , Hipófisis/fisiología , Hormona Adrenocorticotrópica/sangre , Hormona Adrenocorticotrópica/metabolismo , Animales , Técnicas In Vitro , Cinética , Masculino , Eminencia Media/efectos de los fármacos , Hipófisis/efectos de los fármacos , Neurohipófisis/efectos de los fármacos , Ratas , Ratas EndogámicasRESUMEN
To study the effect of acute stress on ACTH secretion and synthesis in rat pituitary and hypothalamus, ACTH content and POMC mRNA levels (measured by use of Northern blot analysis) in these tissues as well as the levels of ACTH in plasma and those of CRF in the hypothalamus were determined after insulin-induced hypoglycemia. Plasma ACTH levels increased at 30 and 60 min. ACTH levels in the anterior pituitary lobe (AP) decreased at 30 min, and then returned to control levels at 60 min. No change was seen in the intermediate-posterior pituitary (IP) or the hypothalamus after insulin injection. CRF levels decreased at 30 and 60 min, then returned to control levels at 90 min in the medial basal hypothalamus, including the median eminence. Hybridization with a cDNA probe revealed a single size class of POMC mRNA in AP, IP, and hypothalamus, and the size of POMC mRNA in these tissues did not change during the experimental period. POMC mRNA levels in AP increased at 60 min and reached a peak at 120 min, but those in IP and hypothalamus did not change. These results suggest that 1) insulin-induced hypoglycemia stimulates both secretion and synthesis of ACTH (at least by increasing POMC mRNA levels) in the AP, and 2) the levels of ACTH and POMC mRNA in the IP and hypothalamus are not affected by insulin-induced hypoglycemia.
Asunto(s)
Hipoglucemia/metabolismo , Insulina/farmacología , Adenohipófisis/análisis , Proopiomelanocortina/genética , ARN Mensajero/análisis , Hormona Adrenocorticotrópica/sangre , Animales , Glucemia/análisis , Hormona Liberadora de Corticotropina/análisis , Hipotálamo/análisis , Masculino , Ratas , Ratas EndogámicasRESUMEN
To study the effect of acute stress on CRF release and synthesis in rat hypothalamus, ACTH levels in plasma, CRF contents in the median eminence (ME), and CRF mRNA levels in the hypothalamus without ME and cerebral cortex were determined after insulin-induced hypoglycemia. Plasma ACTH levels increased at 30 and 60 min, while ME CRF content decreased at 30 and 60 min, then returned to the control level at 90 min. Hybridization with a cRNA probe revealed a single size class of CRF mRNA in the hypothalamus and cerebral cortex (approximately 1300 nucleotides), and the size of CRF mRNA in these tissues did not change during the experimental period. CRF mRNA levels in the hypothalamus increased to 130% of the control value at 30 min and reached a peak (186% of the control value) at 120 min, but these levels in the cerebral cortex did not change. These results suggest that insulin-induced hypoglycemia stimulates CRF synthesis by increasing CRF mRNA levels in the hypothalamus as well as CRF release, and that release and synthesis of CRF in the cerebral cortex are independent of those in the hypothalamus.
Asunto(s)
Hormona Adrenocorticotrópica/sangre , Hormona Liberadora de Corticotropina/genética , Hipoglucemia/metabolismo , Hipotálamo/metabolismo , Insulina/farmacología , ARN Mensajero/metabolismo , Animales , Hipoglucemia/inducido químicamente , Hipotálamo/efectos de los fármacos , Masculino , Eminencia Media/efectos de los fármacos , Eminencia Media/metabolismo , Plásmidos , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Ratas , Ratas EndogámicasRESUMEN
To examine the effects of the cAMP-independent protein kinase-C system and interleukin-1 (IL-1) on secretion of ACTH and POMC gene expression in cultured rat anterior pituitary (AP) cells, AP cells were incubated with CRF, 8-bromo-cAMP, arginine vasopressin, angiotensin II, norepinephrine, and phorbol 12-myristate 13-acetate. After 15 h of incubation, CRF and 8-bromo-cAMP increased both ACTH release and the POMC mRNA level. Arginine vasopressin, angiotensin II, norepinephrine, or phorbol 12-myristate 13-acetate stimulated ACTH release but failed to increase basal or CRF-stimulated POMC mRNA levels. Human recombinant IL-1 alpha and -beta increased neither ACTH release nor POMC mRNA levels after 3 h of incubation. After 15 h of incubation, 100 pM to 10 nM IL-1 alpha and -beta increased ACTH release. However, POMC mRNA levels were significantly elevated only by 10 pM IL-1 beta. These results suggest that the CRF-cAMP system plays a major role in both ACTH release and expression of the POMC gene in AP cells, but the cAMP-independent protein kinase-C system contributes only to ACTH release; that acute stimulation of ACTH release from AP with IL-1 administration is not due to direct action of IL-1 at the pituitary level; that chronic exposure of AP cells to IL-1 alpha or -beta can stimulate ACTH release; and that the direct effects of IL-1 alpha and -beta on POMC gene expression, if any, seem to be minimal.
Asunto(s)
Hormona Adrenocorticotrópica/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-1/farmacología , Adenohipófisis/metabolismo , Proopiomelanocortina/genética , Proteína Quinasa C/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Angiotensina II/farmacología , Animales , Arginina Vasopresina/farmacología , Hormona Liberadora de Corticotropina/farmacología , Norepinefrina/farmacología , Adenohipófisis/efectos de los fármacos , ARN Mensajero/biosíntesis , Ratas , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
ACTH responsiveness in vitro to synthetic corticotropin-releasing factor (CRF), lysine-8-vasopressin, and cAMP was examined using superfusion of pituitary adenoma tissue and the nonadenomatous tissue from 16 patients with Cushing's disease. Sensitivity of adenomas to lysine-8-vasopressin and cAMP was similar to that of nonadenomatous tissues; however, sensitivity of adenomas to CRF was lower than that of nonadenomatous tissues in 7 of 16 patients. CRF-induced ACTH secretion from adenomas was inhibited by Ca2+-free medium in all instances and by dexamethasone and somatostatin in some. Angiotensins I and II stimulated ACTH secretion from both adenomas and nonadenomatous tissues, while angiotensin I-induced ACTH secretion was inhibited by angiotensin-converting enzyme inhibitor. These results suggest that the sensitivity of the pituitary corticotroph adenomas to CRF in some patients is low. This may be due to an abnormality of the step(s) before cAMP formation, such as the CRF receptor.
Asunto(s)
Hormona Adrenocorticotrópica/metabolismo , Hormona Liberadora de Corticotropina/farmacología , Síndrome de Cushing/metabolismo , Hipófisis/metabolismo , Adenoma/metabolismo , Angiotensina I/farmacología , Bromocriptina/farmacología , Calcio/fisiología , AMP Cíclico/farmacología , Dexametasona/farmacología , Hormona Liberadora de Gonadotropina/farmacología , Humanos , Técnicas In Vitro , Lipresina/farmacología , Hipófisis/efectos de los fármacos , Neoplasias Hipofisarias/metabolismo , Radioinmunoensayo , Somatostatina/farmacología , Hormona Liberadora de Tirotropina/farmacologíaRESUMEN
The effect of ACTH administration on plasma CRH levels was studied. In five patients with Addison's disease and three patients with hypopituitarism, bolus iv injection of 0.25 and 0.5 mg ACTH-(1-24) reduced plasma CRH levels (that had become elevated 48 h after discontinuation of corticosteroid replacement) to near-normal levels at 30-60 min in a dose-dependent manner. Plasma immunoreactive beta-endorphin levels were similarly decreased in patients with Addison's disease. ACTH-(1-24) (0.25 and 0.5 mg) injection failed to inhibit plasma CRH levels in five normal subjects. Basal CRH release from the rat hypothalamic median eminence in vitro was inhibited by 0.22 and 2.2 nM ACTH-(1-24) and ACTH-(1-39) in a dose-dependent manner. These results suggest that in the absence of negative feedback control of ACTH secretion by glucocorticoids, ACTH can regulate its secretion by inhibition of hypothalamic CRH release.
Asunto(s)
Enfermedad de Addison/fisiopatología , Hormona Adrenocorticotrópica/fisiología , Hormona Liberadora de Corticotropina/metabolismo , Hipopituitarismo/fisiopatología , Hormona Adrenocorticotrópica/farmacología , Adulto , Anciano , Animales , Cosintropina/sangre , Cosintropina/farmacología , Retroalimentación , Femenino , Humanos , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Cinética , Masculino , Eminencia Media/efectos de los fármacos , Eminencia Media/metabolismo , Persona de Mediana Edad , RatasRESUMEN
To study possible adrenergic modulation of pituitary-adrenal responses to insulin-induced hypoglycemia and CRH we examined the effect of nonselective alpha-blockade (phentolamine) and nonselective beta-blockade (propranolol) on plasma ACTH, cortisol, and vasopressin (AVP) responses to hypoglycemia and CRH in five normal men. Infusion of propranolol or phentolamine did not alter basal plasma ACTH or cortisol levels. The propranolol infusion enhanced the stimulatory effect of hypoglycemia on ACTH, cortisol, and AVP secretion and also enhanced the stimulatory effect of CRH on ACTH and cortisol secretion. Infusion of phentolamine inhibited hypoglycemia-induced ACTH and AVP secretion, but had no effect on the stimulatory effect of CRH on ACTH and cortisol secretion. The increments of plasma ACTH and cortisol induced by an almost maximal dose of CRH (1 microgram/kg) were smaller than those induced by hypoglycemia. The propranolol-induced enhancement of the ACTH response to hypoglycemia was almost the same as the ACTH response to CRH alone. From these results we conclude that propranolol may act at the pituitary level to enhance CRH action, rather than AVP action, and that the ACTH response to hypoglycemia may be mediated by hypothalamic alpha-adrenergic activation.
Asunto(s)
Hormona Adrenocorticotrópica/sangre , Hormona Liberadora de Corticotropina/farmacología , Hipoglucemia/fisiopatología , Insulina/farmacología , Fentolamina/farmacología , Propranolol/farmacología , Glucemia/análisis , Femenino , Hemodinámica/efectos de los fármacos , Humanos , Hidrocortisona/sangre , Insulina/sangre , Vasopresinas/sangreRESUMEN
The concentrations of immunoreactive corticotropin-releasing factor (I-CRF) in human cerebrospinal fluid (CSF) were measured utilizing immunoaffinity chromatography and RIA in patients with no endocrine disease, patients with Cushing's disease, Nelson's syndrome, Sheehan's syndrome, Addison's disease and steroid treated patients. On high performance liquid chromatography, the elution profile and retention time of I-CRF in CSF were not identical with ovine CRF. I-CRF concentrations in CSF from patients with Cushing's disease and Sheehan's syndrome were lower than those from normal subjects, however those from patients with Nelson's syndrome and Addison's disease were within the normal range. I-CRF concentrations in CSF from patients with Cushing's disease returned to normal levels 2-9 months after pituitary adenomectomy. These results suggest that CSF I-CRF concentrations are reduced by increased plasma corticosteroid levels.
Asunto(s)
Enfermedades de las Glándulas Suprarrenales/líquido cefalorraquídeo , Hormona Liberadora de Corticotropina/líquido cefalorraquídeo , Enfermedades de la Hipófisis/líquido cefalorraquídeo , Enfermedad de Addison/líquido cefalorraquídeo , Corticoesteroides/uso terapéutico , Síndrome de Cushing/líquido cefalorraquídeo , Síndrome de Cushing/cirugía , Enfermedades del Sistema Endocrino/tratamiento farmacológico , Femenino , Humanos , Hipopituitarismo/líquido cefalorraquídeo , Cinética , Masculino , Síndrome de Nelson/líquido cefalorraquídeoRESUMEN
Immunoreactive corticotropin-releasing factor (I-CRF) and ACTH (I-ACTH) were examined using RIA, immunoaffinity chromatography, and gel filtration chromatography in human hypothalamus, adrenal (cortex and medulla), lung cancer, and pheochromocytoma. I-CRF and I-ACTH were present in these tissues. Gel filtration of I-ACTH in the adrenal, pheochromocytoma, and lung cancer showed the presence of larger amounts of I-ACTH with large molecular weight forms in contrast to the hypothalamus. Gel filtration of I-CRF in these tissues showed the main peak eluted at the position of synthetic rat CRF. High performance liquid chromatography of this main peak showed two components which eluted in the positions of synthetic rat CRF and oxidized CRF. These elution positions were the same in all tissues and identical with those in the hypothalamus. These results suggest the presence of I-ACTH and I-CRF in these tissues and that CRF outside the brain is identical to hypothalamic CRF.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/análisis , Glándulas Suprarrenales/análisis , Hormona Adrenocorticotrópica/aislamiento & purificación , Hormona Liberadora de Corticotropina/aislamiento & purificación , Hipotálamo/análisis , Neoplasias Pulmonares/análisis , Feocromocitoma/análisis , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Humanos , Inmunoquímica , RadioinmunoensayoRESUMEN
The distribution and characterization of immunoreactive corticotropin-releasing factor (I-CRF) in human tissues were examined using a rat CRF RIA, immunoaffinity chromatography, gel filtration chromatography, and high performance liquid chromatography. High concentrations of I-CRF were found in the hypothalamus and pituitary stalk. In addition, I-CRF was found in the posterior pituitary, thalamus, cerebral cortex, cerebellum, pons, medulla oblongata, spinal cord, and outside the brain and in the adrenal, lung, liver, stomach, duodenum, and pancreas. The major component of I-CRF from these tissues eluted in the position of rat CRF on gel filtration chromatography. High performance liquid chromatography of this major component showed two main peaks which eluted in the positions of CRF and oxidized CRF. These elution positions were the same in all tissues. These results indicate the presence of I-CRF outside the brain and suggest that this CRF is identical to hypothalamic CRF.
Asunto(s)
Hormona Liberadora de Corticotropina/análisis , Glándulas Suprarrenales/análisis , Química Encefálica , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Humanos , Inmunoquímica , Pulmón/análisis , Páncreas/análisis , Radioinmunoensayo , Estómago/análisisRESUMEN
A human plasma CRH-binding protein (CRH-BP) was identified and characterized by chemical cross-linking of 125I-Tyr-hCRH to human plasma using disuccinimidyl suberate. The apparent mol wt of the cross-linked complex determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography was approximately 43,000. The mol wt was slightly lower in the nonreduced state, suggesting the presence of intramolecular disulfide bonds. Subtracting the mol wt of 125I-Tyr-CRH, the BP appeared to have a mol wt of approximately 38,000. Binding was specific since the appearance of the 43,000 dalton band was not affected by unlabeled ACTH, vasopressin, serum albumin, or gamma-globulin, but was inhibited by unlabeled hCRH dose dependently. Pretreatment of plasma with 0.1 mol/L HCl, 0.01 mol/L NaOH, 10 mmol/L dithiothreitol, or trypsin before cross-linking abolished its ability to bind 125I-Tyr-hCRH. Rat, rabbit, or goat plasma or human cerebrospinal fluid did not bind 125I-Tyr-CRH. It is unlikely that CRH-BP is a CRH receptor, because the estimated mol wt of the CRH-BP is smaller than the reported size of CRH receptors, and the CRH-BP did not bind to ovine CRH. The binding of 125I-Tyr-CRH to CRH-BP decreased in the third trimester of pregnancy, when plasma CRH levels were markedly elevated. However, after dissociating endogenous CRH from the CRH-BP, the binding was almost the same as in nonpregnant subjects. In addition, CRH-BP inhibited CRH-induced ACTH secretion from cultured rat anterior pituitary cells. We conclude that most of the increased plasma CRH found in pregnant women is bound to CRH-BP, and so is inactive, therefore plasma ACTH levels do not increase to above the normal range.
Asunto(s)
Proteínas Portadoras/sangre , Hormona Liberadora de Corticotropina/sangre , Embarazo/sangre , Hormona Adrenocorticotrópica/metabolismo , Adulto , Animales , Unión Competitiva , Proteínas Sanguíneas/metabolismo , Proteínas Portadoras/fisiología , Células Cultivadas , Reactivos de Enlaces Cruzados , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Masculino , Adenohipófisis/metabolismo , Unión Proteica , Radioinmunoensayo , RatasRESUMEN
ACTH responses to corticotropin-releasing hormone (CRH) were studied in three patients with the ectopic ACTH syndrome caused by lung cancer. Plasma ACTH responded to synthetic CRH in two of three patients. Tumor tissues obtained from these two patients contained CRH and ACTH. In one patient, tumor ACTH secretion was stimulated by CRH in vitro. Tumor CRH was immunologically, chromatographically, and biologically similar to hypothalamic CRH. In addition, multiple forms of immunoreactive beta-endorphin were present in plasma and the tumor extracts. From these results, we conclude that some patients with the ectopic ACTH syndrome have tumors that produce both ACTH and CRH and that CRH can stimulate ACTH secretion by such tumors. Other patients with the ectopic ACTH syndrome do not have ACTH responses to CRH. Therefore, procedures other than CRH testing are needed to differentiate patients with Cushing's syndrome due to ectopic ACTH/CRH production from those with Cushing's disease, since the latter also usually have ACTH responses to CRH.
Asunto(s)
Síndrome de ACTH Ectópico/etiología , Hormona Liberadora de Corticotropina/metabolismo , Neoplasias Pulmonares/metabolismo , Síndromes Paraneoplásicos Endocrinos/etiología , Anciano , Carcinoma de Células Pequeñas/metabolismo , Hormona Liberadora de Corticotropina/fisiología , Endorfinas/sangre , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Radioinmunoensayo , betaendorfinaRESUMEN
We investigated the effects of serotonin, cyproheptadine and reserpine on corticotropin-releasing factor (CRF) release from the rat hypothalamus, and the effect of cyproheptadine on CRF-induced adrenocorticotropic hormone (ACTH) secretion from the anterior pituitary (AP) in vitro using a perifusion system for rat hypothalami and AP, and a rat CRF radioimmunoassay. Cyproheptadine, 10(-8) M, had a direct inhibitory effect on both basal and 10(-9) M CRF-induced ACTH secretion from the rat AP in vitro. In addition, 10(-9)-10(-7) M cyproheptadine inhibited basal CRF release in a dose-dependent fashion, and also suppressed serotonin- and KCl-induced CRF release. Conversely, 10(-9)-10(-7) M reserpine failed to influence CRF release from the rat hypothalamus. These results indicate that a serotonergic mechanism may be involved in the CRF-releasing mechanism, and inhibition of depolarization-dependent calcium entry into cells and/or nerve endings. In addition an anti-serotonergic mechanism is involved in the inhibitory action of cyproheptadine.
Asunto(s)
Hormona Liberadora de Corticotropina/metabolismo , Ciproheptadina/farmacología , Hipotálamo/metabolismo , Reserpina/farmacología , Serotonina/farmacología , Hormona Adrenocorticotrópica/metabolismo , Animales , Interacciones Farmacológicas , Masculino , Adenohipófisis/metabolismo , Potasio/farmacología , Ratas , Ratas EndogámicasRESUMEN
A time course study on the changes of rat hypothalamic corticotropin-releasing factor (CRF) levels and ACTH levels in plasma, pituitary and hypothalamus after an acute treatment with reserpine was examined using a rat CRF RIA. The massive and prolonged depletion of hypothalamic norepinephrine and dopamine levels provoked by a single injection of reserpine (2 and 8 mg/kg, i.p.) caused a transient decrease of hypothalamic CRF levels and ACTH levels in the anterior pituitary glands, and an increase in plasma ACTH levels. There was a strong correlation between the depletion of hypothalamic CRF and norepinephrine levels. These results suggest that: acute depletion of hypothalamic norepinephrine levels cause the initial release of CRF that stimulates pituitary ACTH secretion, and the depletion of CRF and ACTH stores at the early stage; and noradrenergic pathways may be involved in the inhibitory mechanism of CRF release.