Immunocytochemical demonstration of melanin-concentrating hormone and proopiomelanocortin-like products in the brain of the trout and carp

Immunocytochemistry on frozen sections revealed that in both the trout and the carp, parvocellular neurones located in the medial basal hypothalamus (medial nucleus lateralis tuberis) were immunostained by antisera against three molecules known to be derived from the proopiomelanocortin (POMC) molecule, viz: alpha-melanocyte-stimulating hormone (alpha MSH), ACTH, and salmonid NPP--the whole N-terminal sequence preceding ACTH in the POMC precursor. Axons from these neurones extended into various regions of the brain but did not appear to project into the pituitary gland. Antiserum against salmonid melanin-concentrating hormone (MCH) immunostained magnocellular neurones in the lateral basal hypothalamus (lateral nucleus lateralis tuberis). Axons from some of these neurones projected into the brain while other axons extended into the pituitary gland. In the carp, but not in the trout, some MCH neurones were also immunostained by antisera against alpha MSH but not by antisera against the other POMC molecules.     

Mimecan in pituitary corticotroph cells may regulate ACTH secretion and the HPAA

Mimecan is a protein of unknown function that is expressed in the pituitary tissues of mouse and human. In this study, we observed the function of mimecan on the proopiomelanocortin (POMC) gene in the pituitary and the hypothalamo-pituitary-adrenal axis (HPAA). Incubating pituitary corticotroph AtT-20 cells with recombinant mimecan protein stimulated adrenocorticotrophic hormone (ACTH) secretion without significantly up-regulating POMC gene expression. In addition, pituitary corticotroph AtT-20 cell corticotropin-releasing hormone receptor 1 (CRHR1) gene expression was induced by mimecan. Interestingly, long-term mimecan overexpression in corticotroph cells increased CRHR1 mRNA levels while slightly decreasing POMC mRNA expression and ACTH secretion. Using mimecan knockout mice, we found that, although the serum ACTH concentration was not significantly different between wild type and mimecan knockout mice under basal conditions, the serum ACTH level was relatively lower in mimecan knockout mice after treatment with corticotropin-releasing hormone (CRH). Meanwhile, we observed that POMC and CRHR1 gene expression decreased in primary cultured knockout mouse pituitary cells compared with wild type cells. Taken together, these data suggest that mimecan expressed in pituitary corticotroph cells mainly regulates ACTH secretion in the pituitary and coordinates the HPAA.     

Defective glucocorticoid regulation of proopiomelanocortin gene expression and peptide secretion in a small cell lung cancer cell line

A human small cell lung cancer cell line (COR L103) that actively expresses the proopiomelanocortin (POMC) gene has been used as a model of extrapituitary ACTH-secreting tumors to investigate the phenomenon of resistence of ACTH production to glucocorticoids. After both short term (24 h) and long term (10 days) exposure to hydrocortisone at concentrations of 500 and 1000 nM, the accumulation of intracellular POMC mRNA, ACTH, and ACTH precursor peptides in the culture medium was not suppressed. These finding contrast with those in the pituitary corticotroph cell line AtT20, in which POMC mRNA, ACTH, and ACTH precursors were suppressed under the same conditions. Two other genes that are regulated by glucocorticoids in other cell types, the tyrosine amino transferase gene and the glucocorticoid receptor gene, were expressed in COR L103 cells. However, neither gene appeared to be regulated by hydrocortisone in this small cell lung cancer cell line. Further studies demonstrated that glucocorticoid receptor binding could be detected in the nucleus and cytoplasm, with a Kd of 5 X 10(-9) M. It is concluded that nonsuppression of POMC by glucocorticoids is probably part of a more global defect of glucocorticoid signaling in these cells, but that this defect lies distal to steroid binding in the nucleus.     

Comparison of ACTH and ACTH precursor peptides secreted by human pituitary and lung tumour cells in vitro

The molecular forms of ACTH secreted by established human small cell lung cancer (SCLC) cells and primary cultures derived from a bronchial carcinoid tumour, a pituitary adenoma and hyperplastic pituitary tissue have been characterized by Sephadex G-75 chromatography and quantified with two novel immunoradiometric assays for ACTH and ACTH precursor peptides. Pro-opiomelanocortin (POMC; Mr 31,000) and pro-ACTH (Mr 22,000) were secreted by all cell types. No smaller peptides were identified in the culture media from SCLC and bronchial carcinoid cells, implying a deficiency in the enzymes and/or intracellular organelles required for extensive POMC processing. A more heterogeneous profile of ACTH-containing peptides was produced by cells of pituitary origin, indicating more extensive proteolytic processing of POMC. However, the major peptide secreted by cells from a large aggressive pituitary adenoma was unprocessed POMC (Mr 31,000). These results suggest that both lung and pituitary cells in vitro retain their in-vivo pattern of POMC processing and provide valuable models in which to study the regulation of ACTH synthesis and secretion.     

Effects of protein kinase-C-related adrenocorticotropin secretagogues and interleukin-1 on proopiomelanocortin gene expression in rat anterior pituitary cells

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.     

Localization, expression and control of adrenocorticotropic hormone in the nucleus preopticus and pituitary gland of common carp (Cyprinus carpio L.)

Adrenocorticotropic hormone (ACTH) takes a central role in the hypothalamo-pituitary-interrenal axis (HPI axis), which is activated during stress. ACTH is produced by the corticotrope cells of the pituitary pars distalis (PD) and is under control of factors from the nucleus preopticus (NPO). The distribution of ACTH in the hypothalamo-pituitary system in common carp (Cyprinus carpio L.) was assessed by immunohistochemistry. ACTH and beta-endorphin immunoreactivity was observed in the ACTH cells in the PD and in the NPO. Nerve fibers, originating from the NPO and projecting to the pituitary gland, contain beta-endorphin, but not ACTH, and these fibers either control the pituitary pars intermedia (PI) through beta-endorphin or release it to the blood. The release of pituitary ACTH (studied in a superfusion setup) must in vivo be under predominant inhibitory control of dopamine. Release of ACTH is stimulated by corticotropin-releasing hormone, but only when ACTH cells experience dopaminergic inhibition. The expression of the precursor pro-opiomelanocortin in (POMC) NPO, PD and PI was studied in an acute restraint stress paradigm by real-time quantitative polymerase chain reaction (RQ-PCR). POMC gene expression is upregulated in these three key tissues of the hypothalamo-pituitary complex, revealing a hitherto unforeseen complex role for POMC-derived peptides in the regulation of responses to stress.