Stimulatory Effect Of Corticotropin-releasing Hormone Research Articles

Testosterone and dihydrotestosterone modulate the redox homeostasis of endothelium.

The predominance of cardiovascular diseases among men compared to premenopausal women has been attributed to testosterone, which is implicated in vascular remodeling. Molecular mechanisms underlying its role have not been clarified but oxidative stress-induced inflammation may be important. We therefore investigated in vitro the effects of testosterone and dihydrotestosterone, (a nonaromatized androgen), on redox homeostasis in absence (basal conditions) and after corticotropin-releasing hormone-induced pro-oxidant action in macroendothelial cells. More specifically, we explored their role on well-established antioxidant enzymes activity, namely endothelial nitric oxide synthase, superoxide dismutase, catalase, and glutathione. We observed that both androgens significantly increased the intracellular reactive oxygen species levels, endothelial nitric oxide synthase activity, nitric oxide concentration as well as superoxide dismutase activity and decreased catalase activity. These effects of Testosterone and DHT were reversed in the presence of the androgen receptor antagonist, flutamide. Moreover, testosterone and dihydrotestosterone similarly enhanced the stimulatory effect of corticotropin-releasing hormone on intracellular reactive oxygen species levels and superoxide dismutase activity but did not influence the inhibitory effect on endothelial nitric oxide synthase activity, nitric oxide release and catalase activity. Finally, androgens did not have a detectable effect on glutathione levels or the glutathione/glutathione plus glutathione disulfide ratio. Our results reveal that testosterone and DHT rise the intracellular redox threshold of the endothelial cell and increases NO synthesis. These findings suggest that the action of testosterone is affected by the redox status of the endothelium and help to explain its controversial effects on the cardiovascular system.

17-β estradiol attenuates the pro-oxidant activity of corticotropin-releasing hormone in macroendothelial cells.

Corticotropin-releasing hormone, which is the predominant regulator of neuroendocrine responses to stress, attenuates inflammation through stimulation of glucocorticoid release. Enhanced corticotropin-releasing hormone expression has been detected in inflammatory cells of the vascular endothelium, where it acts as a local regulator of endothelial redox homeostasis. Estrogens have beneficial effects on endothelial integrity and function, though the mechanism underlying their antioxidative effect remains as yet largely unknown. We therefore investigated the effect of 17β-estradiol on pro-oxidant action of corticotropin-releasing hormone in vitro in macroendothelial cells, and, more specifically, the role of 17β-estradiol on corticotropin-releasing hormone-induced activities/release of the antioxidant enzymes namely, endothelial nitric oxide synthase, superoxide dismutase, catalase, and glutathione. We observed that 17β-estradiol abolished the stimulatory effect of corticotropin-releasing hormone on intracellular reactive oxygen species levels and counteracted its inhibitory effect on endothelial nitric oxide synthase activity and nitric oxide release. In addition, 17β-estradiol significantly induced superoxide dismutase and catalase activity, an effect that was not significantly influenced by corticotropin-releasing hormone. Finally, 17β-estradiol significantly increased glutathione levels and the glutathione/glutathione + glutathione disulfide ratio, an action that was partially blocked by corticotropin-releasing hormone. Our results reveal that 17β-estradiol counterbalances corticotropin-releasing hormone-mediated pro-inflammatory action and thereby maintains the physiological threshold of the endothelial cell redox environment. These observations may be of importance, considering the protective role of estrogen in the development of atherosclerosis.

Corticotropin-releasing hormone (CRH) stimulates cocaine- and amphetamine-regulated transcript gene (CART1) expression through CRH type 1 receptor (CRHR1) in chicken anterior pituitary

Cocaine- and amphetamine-regulated transcript (CART) peptide(s) is generally viewed as neuropeptide(s) and can control food intake in vertebrates, however, our recent study revealed that CART1 peptide is predominantly expressed in chicken anterior pituitary, suggesting that cCART1 peptide is a novel pituitary hormone in chickens and its expression is likely controlled by hypothalamic factor(s). To test this hypothesis, in this study, we examined the spatial expression of CART1 in chicken anterior pituitary and investigated the effect of hypothalamic corticotropin-releasing hormone (CRH) on pituitary cCART1 expression. The results showed that: 1) CART1 is expressed in both caudal and cephalic lobes of chicken anterior pituitary, revealed by quantitative real-time PCR (qPCR), western blot and immuno-histochemical staining; 2) CRH potently stimulates cCART1 mRNA expression in cultured chick pituitary cells, as examined by qPCR, and this effect is blocked by CP154526 (and not K41498), an antagonist specific for chicken CRH type I receptor (cCRHR1), suggesting that cCRHR1 expressed on corticotrophs mediates this action; 3) the stimulatory effect of CRH on pituitary cCART1 expression is inhibited by pharmacological drugs targeting the intracellular AC/cAMP/PKA, PLC/IP3/Ca2+, and MEK/ERK signaling pathways. This finding, together with the functional coupling of these signaling pathways to cCRHR1 expressed in CHO cells demonstrated by luciferase reporter assay systems, indicates that these intracellular signaling pathways coupled to cCRHR1 can mediate CRH action. Collectively, our present study offers the first substantial evidence that hypothalamic CRH can stimulate pituitary CART1 expression via activation of CRHR1 in a vertebrate species.

Mitotane reduces human and mouse ACTH-secreting pituitary cell viability and function

Medical therapy for Cushing's disease (CD) is currently based on agents mainly targeting adrenocortical function. Lately, pituitary-directed drugs have been developed, with limited efficacy. Mitotane, a potent adrenolytic drug, has been recently investigated for the treatment of CD, but the direct pituitary effects have not been clarified so far. The aim of our study was to investigate whether mitotane may affect corticotroph function and cell survival in the mouse pituitary cell line AtT20/D16v-F2 and in the primary cultures of human ACTH-secreting pituitary adenomas, as an in vitro model of pituitary corticotrophs. We found that in the AtT20/D16v-F2 cell line and in primary cultures, mitotane reduces cell viability by inducing caspase-mediated apoptosis and reduces ACTH secretion. In the AtT20/D16v-F2 cell line, mitotane reduces Pomc expression and blocks the stimulatory effects of corticotropin-releasing hormone on cell viability, ACTH secretion, and Pomc expression. These effects were apparent at mitotane doses greater than those usually necessary for reducing cortisol secretion in Cushing's syndrome, but still in the therapeutic window for adrenocortical carcinoma treatment. In conclusion, our results demonstrate that mitotane affects cell viability and function of human and mouse ACTH-secreting pituitary adenoma cells. These data indicate that mitotane could have direct pituitary effects on corticotroph cells.

Evidence that corticotropin-releasing hormone acts as a growth hormone-releasing factor in a primitive teleost, the European eel (Anguilla anguilla).

The inhibitory control of growth hormone (GH) release by somatostatin (SRIH) has been conserved throughout vertebrate evolution. In contrast, the neuropeptides involved in the stimulatory control of GH vary according to species and/or physiological situations. We investigated the direct pituitary regulation of GH release in a primitive teleost, the European eel (Anguilla anguilla L.) at the juvenile stage. Short-term serum-free primary cultures of dispersed pituitary cells were used, and GH release was measured by an homologous radioimmunoassay. Whereas growth hormone-releasing hormone (GHRH), gonadotropin-releasing hormone (GnRH), thyrotropin-releasing hormone (TRH), neuropeptide Y (NPY) and cholecystokinin (CCK) failed to induce any change in GH release, corticotropin-releasing hormone (CRH) dose-dependently stimulated GH release with a significant effect at 1 nM and a maximal effect (> or =400% of controls at 24 h) at 100 nM. In agreement with our previous studies, PACAP also stimulated GH release but its maximal effect was lower than that of CRH. Proopiomelanocortin (POMC)-peptides, corticotropin (ACTH), melanotropin (alpha-MSH), beta-endorphin) had no effect on GH release, at any dose tested (0.1-1000 nM), indicating that the stimulatory effect of CRH on GH release by somatotrophs was not mediated by CRH-induced release of POMC-peptides from corticotrophs and melanotrophs. The CRH antagonist, alpha-helical CRH(9-41), significantly inhibited the stimulatory effect of CRH on GH release, suggesting the implication of specific CRH receptors related to mammalian ones. The stimulatory effect of CRH on GH release was reduced after 24 h of incubation, indicating a desensitization. In contrast, no desensitization to the inhibitory effect of SRIH was observed. SRIH inhibited CRH action in a dose-dependent manner. The effect of SRIH was overriding, 1 nM SRIH being able to abolish the effect of 1000 nM CRH. In conclusion, in the eel, CRH stimulates GH release directly at the pituitary cell level. GH and cortisol secretions could interact in controlling several physiological functions such as metabolism and ion exchange. This study suggests that CRH may have played an important early role in vertebrates co-ordinating the activation of various endocrine axes involved in metamorphosis, osmoregulation, stress and fasting. The stimulatory role of CRH on GH release may have been partially conserved during evolution, as it is found in some human physio-pathological situations such as stress, fasting and depression.

Regulation of adrenocorticotropin secretion in vitro by anterior pituitary corticotrophs from fallow deer ( Dama Dama)

The actions of corticotropin-releasing hormone (CRH), vasopressin (VP), the synthetic glucocorticoid dexamethasone (DEX), and mifepristone (RU 486), a glucocorticoid antagonist, on the secretion of adrenocorticotropin (ACTH) by cultured fallow deer corticotrophs were studied in vitro. On Day 5 of primary culture, corticotrophs were challenged for up to 4 hr with medium alone (Contrpl), CRH, VP, DEX, forskolin (FSK), phorbol ester (TPA), cyclic AMP (cAMP), and/or RU 486 at various concentrations and combinations. CRH, VP, FSK and TPA each stimulated (P < 0.01) the secretion of ACTH in dose- and time-related manners. Relative to Control, CRH at 0.001 and 0.1 μM and VP at 0.01 and 1 μM increased (P < 0.01) medium concentration of ACTH by 7.3-, 13.5-, 3.7- and 9.0-fold, respectively. There was a treatment × incubation time interaction (P < 0.01) such that at 30-min posttreatment, CRH-induced ACTH secretion tended (P < 0.10) to be less than that obtained via VP treatment, whereas at 1, 3, and 4 hr posttreatment, medium concentration of ACTH from cells treated with 0.1 μM CRH was greater (P < 0.05) than that in cells treated with 1 μM VP. At equimolar doses of 0.01 and 0.1 μM, CRH was 3.4- and 3.0-fold more potent (treatment × dose, P < 0.05) than VP. Cotreatment with 1 μM DEX reduced (P < 0.001) the stimulatory effects of CRH (0.1 μM), VP (1 μM), FSK (10 μM), TPA (0.1 μM), and cAMP (0.001 M). However, the coaddition of RU 486 (1 μM) to the CRH plus DEX- and the FSK plus DEX-treated wells partially negated the inhibitory effects of DEX. RU 486 completely negated the inhibitory effects of DEX on the VP-, TPA-, and cAMP-stimulated secretion of ACTH. These data indicate that CRH is a more potent stimulator of ACTH secretion than is VP in primary culture of fallow deer pituitary cells. This study also demonstrates the utility of an in vitro culture system to investigate stress-related hormonal interactions in cervids.

Pituitary-adrenal function and hypothalamic beta-endorphin release in vitro following food deprivation

Basal and dexamethasone-suppressed adrenal gluco- corticoid secretion and hypothalamic β-endorphin (BE) release in vitro were investigated in rats deprived of food for 24, 48, 72, and 96 h. Fasting for up to 48 h neither caused significant changes in serum corticosterone levels nor in the suppressive effect of dexamethasone. Food deprivation for 72–96 h resulted in increased basal serum corticosterone, diminished suppression by dexa- methasone, and a significant involution of the thymus. Basal in vitro BE release from hypothalamic expiants was significantly increased after the first day of food deprivation, and in vitro perifusion with corticotropin-releasing hormone (CRH) failed to enhance BE release further. With continuing food deprivation, basal BE release remained significantly greater than that from hypothalami originating from normally fed control rats. The stimulatory effect of CRH on BE release was only partially restored after 2 days of fasting. The results suggest that food deprivation for more than 2 days increases basal glucocorticoid secretion, and signs of impairment in hypothalamic-pituitary-adrenal regulation become apparent. These findings might be implicated in the pathogenetic mechanisms of endocrine dysregulation in diseases related to caloric reduction.

Effects of cyclosporine A on the hypothalamic-pituitary-adrenal axis and anterior pituitary interleukin-6 mRNA expression during chronic inflammatory stress in the rat

In the rat, adjuvant arthritis (AA) is an inflammatory joint disease associated with chronic stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. We have investigated the effects of the immunosuppressive agent cyclosporine A (CsA) on plasma levels of adrenocorticotropin (ACTH) and corticosterone (B), as well as on anterior pituitary proopiomelanocortin (POMC) and interleukin (IL)-6 mRNA accumulation in control and adjuvant-injected animals. In control animals, CsA reduced basal anterior pituitary POMC and IL-6 mRNA and decreased plasma levels of ACTH and B. Adjuvant-injected animals that were treated with CsA showed no clinical signs of AA. Moreover, CsA inhibited the arthritis-induced increases in pituitary POMC and IL-6 mRNA levels and in circulating ACTH and B. In vitro, CsA reduced the POMC mRNA content of cultured anterior pituitary cells and diminished the stimulatory effects of corticotropin-releasing hormone (CRH) on POMC mRNA expression and ACTH secretion from these cells. These data indicate that CsA has a direct action on the HPA axis and also reduces the activation of the HPA axis seen in chronic inflammatory arthritis.

Effects of corticotropin-releasing hormone on the postoperative course of elderly patients under long-term artificial respiration.

In both human and animal studies a stimulatory effect of corticotropin-releasing hormone (CRH) on respiration and on cognitive parameters has been demonstrated. Our own studies employing human CRH (hCRH) iv in healthy volunteers and different groups of patients have shown hCRH to be a safe drug. We prospectively studied the clinical effects of a standardized dose of 100 micrograms hCRH iv in 12 elderly patients following major abdominal surgery who remained comatose and were under prolonged respirator therapy over a mean period of 37 days. Cardio-respiratory parameters, blood gas values, plasma cortisol and catecholamines were evaluated before and 30 min following hCRH injection. Furthermore, vigilance was tested using a score system. Ventilation was markedly enhanced following hCRH injection while the cardiovascular parameters were only moderately affected. Vigilance was augmented in all subjects and improved impressively in five patients. The changes were of great benefit for the patients treated and supported their respirator weaning procedures and mobilization training.

Effects of Corticotropin-Releasing Hormone and Adrenocorticotropin on Prostaglandin Output by Human Placenta and Fetal Membranes

We have examined the possibility that corticotropin-releasing hormone (CRH) and adrenocorticotropin (ACTH) might affect prostaglandin (PG) E2 and PGF2 alpha output by term placenta, amnion and decidua during short-term (48-hour) culture, and that effects of CRH might be mediated by ACTH. In all three tissues PG output was stimulated by both CRH and ACTH. These effects were inhibited in the presence of antisera to CRH and to ACTH. Moreover, in placenta, but not in amnion or decidua, the stimulatory effects of CRH on PGE2 and PGF2 alpha output were attenuated in the presence of an antibody to ACTH. Our results support the possibility of paracrine stimulation by CRH and ACTH of PG production in intrauterine tissues, and suggest that in part the effects of CRH on placental PG output might be mediated through ACTH.

Growth and Secretory Responses of Enriched Populations of Corticotropes*

The purpose of this study was to learn whether enriched populations of corticotropes could be grown without the other pituitary cell types. Corticotropes populations were enriched to 80-90% by counterflow centrifugation in an elutriator with the Sanderson chamber. After initial separation into small, medium, and large fractions, the cells were stimulated for 3 h with 0.5 nM corticotropin-releasing hormone (CRH) and then re-eluted to remove the enlarged corticotropes. More ACTH (6- to 10-fold) was released in media with 10% fetal bovine serum (FBS) than was released in media with no serum. The effects of FBS could not be mimicked by 0.3% BSA. Corticotropes grew in serum-containing media as long as they were plated at a density of at least 2500 cells per well. The corticotropes expanded in size and assumed two major morphological subtypes. Both stored ACTH and beta-endorphin. One subtype was flattened and pleomorphic. The other subtype was stellate with multiple processes. Cell counts showed a 2.5- to 3.8-fold increase in the number of labeled corticotropes during the first 21 days of culture. Then the numbers of cells declined rapidly. Basal secretion of ACTH rose 1.6-fold during the first week, plateaued after 14 days and then declined to less than 30% of first week levels. CRH stimulation produced dose-dependent increases in media ACTH. In 7 day cultures, both basal and stimulated levels of ACTH were similar to those in 7 day cultures of mixed pituitary cells (containing equivalent numbers of corticotropes). Stimulatory effects of CRH were evident for up to 42 days of culture. Arginine vasopressin enhanced CRH-mediated secretion in most cultures in the first week. Pretreatment with glucocorticoids (100 nM corticosterone) for 15 h blocked CRH-mediated secretion in all cultures. The studies showed that corticotropes do not need the other pituitary cell types for basic plating and basal and CRH-mediated secretory responses. Further tests of specific growth factors are needed to learn whether they will maintain function for longer periods.