The effect of streptozotocin-diabetes on beta-endorphin level and proopiomelanocortin gene expression in the rat pituitary

Streptozotocin-induced diabetes for 4 weeks resulted in a decrease in proopiomelanocortin (POMC) mRNA in both the anterior lobe (AL) and the neuro-intermediate lobe (NIL) of the rat pituitary. The beta-endorphin levels decreased in the NIL but not in the AL. It is concluded that the synthesis of POMC in the pituitary is inhibited in diabetic rats and that there is a decrease in beta-endorphin release from the anterior pituitary.     

Lack of neuronal NOS has consequences for the expression of POMC and POMC-derived peptides in the mouse pituitary

The relevance of NO in neuroendocrine signalling has been investigated by analysis of cellular expression of pro-opiomelanocortin (POMC) and the POMC-derived peptides beta-endorphin, alpha-melanocyte stimulating hormone and adrenocorticotropin. Expression patterns were studied in the pituitary gland of 150-day old wild-type and neuronal-NOS (nNOS) knock-out mice by using immunohistochemistry, in situ hybridization and Northern blot analysis. Remaining NO-generating capacities in the knock-out mice were demonstrated by immunohistochemical localization of inducible, endothelial and neuronal NOS isoforms. Quantitative analysis revealed that cellular expression of POMC mRNA was drastically reduced in the pituitary of knock-out mice in comparison to controls. In situ hybridization studies demonstrated that this reduction was most pronounced in the intermediate lobe, while the anterior lobe was much less affected. Immunostaining for the proteolytic fragments of POMC was significantly reduced in the intermediate lobe cells of knock-out mice. A moderate reduction of immunostaining for these peptides was also observed in adenopituitary cells of nNOS knock-out mice. Our data demonstrate that the lack of nNOS substantially affects cellular levels of pituitary opioid peptides, which may have consequences for the response of these animals to stress and pain.     

Processing of proopiomelanocortin (POMC) approached by immunoelectron microscopy pre-embedding method.

Immunoelectron microscopy pre-embedding method was applied to study the processing of proopiomelanocortin (POMC). Premature pituitary cells and cancer cells showed POMC derived peptides in perinuclear spaces (PNS), rough endoplasmic reticula (RER) and in a few secretory granules (SG). These cells demonstrated immature processing of POMC and suggested constitutive pathway. Cultured mouse fibroblasts transfected with human POMC gene was a good model for this system. Mature pituitary cells and pituitary adenomas showed POMC derived peptides predominantly in SG which correlated with authentic processing of POMC. Cultured mouse AtT 20 cells transfected with human POMC gene showed similar secretory pathway and processing. These results emphasized that SG is mandatory for proper processing of POMC. SG is probably essential for the regulated system of secretion.     

Release of endogenous 5-hydroxytryptamine from the intermediate lobe of the rat pituitary gland is inhibited by endogenous dopamine

The release of endogenous 5-hydroxytryptamine (5-HT) from the in vitro incubated neurointermediate lobe (NIL) of the rat hypophysis was measured by HPLC with electrochemical detection. 5-HT release evoked by electrical stimulation of the pituitary stalk from the NIL, but not from the isolated neural lobe (NL) was enhanced in the presence of the dopamine receptor antagonist sulpiride (1 microM). The opiate receptor antagonist naloxone (1 microM) had no effect on the evoked release of 5-HT from the NIL or NL. In conclusion, the release of endogenous 5-HT from the intermediate lobe is under inhibitory control of endogenous dopamine.     

Ultrastructure of the pituitary intermediate lobe in aging rats

The pituitary intermediate lobe, a source of pro-opiomelanocortin (POMC) peptides, was examined with light and electron microscopic techniques in Sprague-Dawley rats aged 5-18 months and Zucker rats aged 18 months. Cysts were common in the intermediate lobe in the Zucker animals, a finding also noted in human pituitary glands by other investigations. The nuclear envelopes were often indented in cells of aging rats, while those of young animals were generally smooth. Lipid droplets and lysosomes, rarely seen in tissue from young animals, were frequently observed in endocrine cells of older rats. Most cells had an abundance of secretory granules, suggestive of enhanced storage of peptides in the cytoplasm. Nerve terminals which were present among endocrine cells contained myelin figures in some of the old rats, and may indicate degenerative changes, while other terminals appeared normal. These morphologic findings suggest that the aging phenomena in intermediate lobe tissue have characteristics in common with nervous tissue, and may also reflect a diminished inhibitory neuroregulation.     

用非同位素原位杂交组化在光镜和电镜水平观察前阿黑皮素mRNA在垂体的分布

用地高辛标记反意前阿黑皮素(pro-opiomelanocortin,POMC)cRNA探针原位杂交组化在光镜和电镜水平观察了POMC mRNA在大鼠垂体的分布,并比较了碱性磷酸酶(AKP)显色系统和辣根过氧化酶(HRP)显色系统在光镜水平上的敏感性.结果:POMC mRNA广泛地分布于垂体的中间叶和前叶.中间叶全部细胞均为POMC mRNA阳性.前叶中除前叶的腹侧缘和前叶与中间叶交界处阳性细胞较少外,都有较多的POMC mRNA阳性细胞分布.AKP显色系统比HRP显色系统敏感.在电镜水平,POMC mRNA主要分布于粗面内质网,少数分泌颗粒可能呈阳性反应,胞核未见阳性反应沉淀.文内还就阳性分泌颗粒在调节细胞合成功能方面可能起的作用进行了讨论.     

Corticotropin-releasing factor and forskolin increase proopiomelanocortin messenger RNA levels in rat anterior and intermediate cells in vitro

The effect of ovine corticotropin-releasing factor (CRF) on messenger ribonucleic acid (mRNA) level encoding proopiomelanocortin (POMC) was studied in serum-free primary cultures of intermediate (IL) and anterior lobe (AL) cells of rat pituitary. Levels of POMC mRNA were quantitated by hybridization to a 32P-labeled, single-stranded POMC complementary deoxyribonucleic acid (cDNA) probe. This effect was time dependent and after 48 h of treatment, POMC mRNA levels in IL cells and AL corticotrophs were increased by 116 +/- 9% and 118 +/- 2% of control values, respectively. Forskolin (1 microM) induced a similar increase in POMC mRNA in both pituitary cell types. These data suggest that CRF might stimulate the gene expression of POMC in pituitary melanotrope and corticotrope cells. Moreover, our findings are consistent with the role of cAMP as a second messenger for CRF in IL and AL corticotrophic cells.     

Detection of the messenger RNAs coding for the opioid peptide precursors in pituitary and adrenal by "in situ' hybridization: study in several mammal species

The messenger RNAs coding for opioid peptide precursors have been detected and mapped in histological sections by "in situ' hybridization using specific DNA probes labelled with 32P. Using bovine preproenkephalin A (PPA) cDNA, PPA mRNA was detected in adrenal medulla of bull, hamster and guinea pig. No signal was detected in adrenal of man, rat and cat. The pro-opiomelanocortin (POMC) mRNA was detected in pituitary of man, bull, cat, rat and pig, in all cells of the intermediate lobe as well as in scattered cells of the anterior lobe producing POMC. Adequate controls demonstrated the specificity of the labelling. These results provide evidence of the expression of the gene coding for PPA in the adrenal and for POMC in the pituitary. They show cross-hybridization of one DNA probe with mRNAs of various mammals and then provide evidence that one single probe can be used to analyze expression of a given gene in tissues of several animal species by "in situ' hybridization.     

Release of immunoreactive beta-endorphin in vitro from pituitaries of young and old male rats

The release of immunoreactive beta-endorphin (IR-BE) in vitro from the anterior pituitary (AP) and the neurointermediate lobe of the pituitary (NIL) from old male rats was significantly greater than from the AP and NIL from young male rats. In addition, the content and concentration of IR-BE in the AP and NIL was significantly greater in old than in young male rats, as was the concentration of IR-BE in the plasma. Chromatographic analysis revealed that in old male rats, the increase in IR-BE contained in and released by the AP and NIL, and found in the plasma, represented an increase in a peptide which coeluted with beta-endorphin rather than beta-lipotropin. These data suggest that both the AP and the NIL contribute to the elevation in plasma levels of IR-BE observed in old male rats, and that the increase in pituitary and plasma IR-BE in old male rats represents an increase in beta-endorphin.     

Electrophysiological study with K+- and Ca2+-sensitive micropipettes of GABA receptors in the rat neurointermediate lobe in vitro

An efflux of K+ and a decrease in extracellular Ca2+ activity are to be expected when GABA markedly depolarizes the membrane of unmyelinated axons or secretory cells. Accordingly, we used extracellular recordings of ionic movements to specify GABA receptor presence in parts of the pituitary having GABAergic innervation: the neurohypophysis and intermediate lobe (NIL). We identified a site of action having the same desensitization characteristics and pharmacological criteria as the GABA A receptor which modulates Cl- -conductance. Baclofen, a GABA B agonist, was without effect. The possible distribution of receptors and role of GABAergic synapses in modulating neurotransmitter and hormone release by the NIL are discussed.     

Immunocytochemical study of 18 tumours causing ectopic Cushing''s syndrome.

Eighteen cases of Cushing's syndrome caused by ectopic production of peptide hormones were investigated by histological and immunocytochemical methods and the findings correlated with clinical and biochemical observations. Immunocytochemistry showed immunoreactive adrenocorticotrophic hormone (ACTH) or peptides derived from the ACTH precursor (pro-opiomelanocortin (POMC], or both, in a total of 10 cases: five of these also contained immunoreactive-alpha-melanocyte stimulating hormone, indicating more extensive translational processing of POMC than normally occurs in healthy corticotrophs of the anterior pituitary; in two further cases peptides capable of stimulating ACTH release from the anterior pituitary were present. In the remaining six cases immunocytochemistry failed to show the presence of ACTH, other POMC derived peptides, or peptides with ACTH releasing properties. These findings correlate well with the histological and clinical observations, in that the six tumours had been clinically overt, caused rapid death, and histologically seemed to be highly malignant. In contrast, the 12 other tumours were occult to radiological examination, patients had a much improved survival rate, and histologically the tumours seemed to be less aggressive. All but one of the tumours in this series showed a degree of neuroendocrine differentiation, indicated by the presence of neuron specific enolase. These results suggest that one feature of highly malignant tumours, which cause an ectopic endocrine syndrome, is a high secretion of peptide hormones, leaving amounts that are too small to be shown by immunocytochemistry.     

Distribution of beta-endorphin and related peptides in the hypothalamus and pituitary.

β-Endorphin and its precursors, β-lipotropin and pro-opiocortin, in the pituitary and hypothalamus of the rat, were studied by means of radioimmunoassays with antisera to β-endorphin, β-melanocyte stimulating hormone and adrenocorticotropin. Gel filtration chromatography in combination with radioimmunoassay revealed the existence of β-endorphin, β-lipotropin and pro-opiocortin in all the tissues studied, with the highest concentration in the posterior-intermediate lobe of the pituitary gland. Great variation was observed in the relative amounts of these three β-endorphin-like immuno-reactive peptides in different regions. The ratio of β-endorphin to β-lipotropin is highest in the hypothalamus, is less in the posterior intermediate pituitary lobe and lowest in the anterior pituitary lobe. A β-melanocyte-stimulating hormone-like immunoreactive peptide, which is larger than porcine β-melanocyte-stimlating hormone and smaller than β-lipotropin, was detected in the posterior-intermediate lobe but not in the anterior lobe of the pituitary.The results suggest that the pro-opiocortin may act mainly as a precursor of adrenocorticotropin in the anterior pituitary and that β-endorphin may act mainly in the intermediate lobe of the pituitary where there is conversion of β-lipotropin to β-endorphin and the β-melanocyte-stimulating hormone-like immunoreactive peptide.     

The elevation of immunoreactive beta-endorphin in old male rats is related to alterations in dopamine and serotonin

The concentration of immunoreactive beta-endorphin (IR-BE) in the anterior pituitary (AP) and the neurointermediate lobe of the pituitary (NIL) was elevated in old as compared to young male rats. Treatment of old male rats with the dopamine precursor, L-DOPA, did not affect the concentration of IR-BE in the AP and produced a significant reduction in the concentration of IR-BE in the NIL. By contrast, administration of the serotonergic neurotoxin, p-CPA, significantly diminished the concentration of IR-BE in the AP of old male rats, while the concentration of IR-BE in the NIL remained unchanged. Hypothalamic IR-BE was decreased in old male rats and was not influenced by administration of L-DOPA or p-CPA. Chromatographic analysis indicated that in the AP of old animals the amount of beta-endorphin relative to beta-lipotropin was increased and was diminished slightly by the treatments. Alterations in IR-BE in the NIL and hypothalamus were represented solely by beta-endorphin. These data suggest that in old male rats, a decrease in dopaminergic activity contributes to the increase in IR-BE levels in the NIL, and an increase in serotonergic function, at least in part, is responsible for the elevation in the level of IR-BE in the AP.     

Presence of arginine-vasopressin in bovine pituitary intraglandular colloid of intermediate lobe origin

In order to determine whether pituitary intraglandular colloid might contain enough hormonal material to be considered a transport medium for intermediate lobe peptides, arginine-vasopressin was assayed in three pools of colloid collected from nearly 100 steers. Colloid samples taken from the pituitary glands of freshly slaughtered cattle were pooled, freed of extraneous tissue, and lyophilized. Three such pools were further extracted on octadecylsilyl-silica and assayed. The radioimmunoassay showed that arginine-vasopressin was indeed present in the bovine intraglandular colloid at levels ranging from 0.18 to 6.95 ng/mg dry weight. These results indicate that the intraglandular colloid contains a sufficient amount of arginine-vasopressin to be considered as a transport medium for this and other intermediate lobe peptides.     

Hypophysectomy and neurointermediate pituitary lobectomy reduce serum immunoglobulin M (IgM) and IgG and intestinal IgA responses to Salmonella enterica serovar Typhimurium infection in rats

The influence of anterior pituitary hormones on the gastrointestinal tract of humans and animals has been reported. Hypophysectomy (HYPOX) in the rat causes atrophy of the intestinal mucosa, reduction of gastric secretion and intestinal absorption, and increased susceptibility to infections. To our knowledge, there are no studies on the humoral immune response of the gut-associated lymphoid tissue after HYPOX. We have reported that decreased secretion of vasopressin and oxytocin due to neurointermediate pituitary lobectomy (NIL) diminishes humoral and cell-mediated immune responses. However, no data have been published on whether NIL can affect intestinal immune responses. We analyzed the effects of HYPOX and NIL on bacterial colonization of the intestinal lumen, Peyer's patches, and spleen as well as the serum immunoglobulin G (IgG) and IgM and specific intestinal IgA levels in response to Salmonella enterica serovar Typhimurium oral infection. Results showed the following: (i) Salmonella serovar Typhimurium was eliminated from the intestinal lumen at the same rate in rats that underwent a sham operation, HYPOX, and NIL; (ii) Salmonella serovar Typhimurium colonization of Peyer's patches and spleen was significantly higher in both HYPOX and NIL rats than in sham-operated rats; (iii) serum IgG and IgM and intestinal IgA against surface proteins of Salmonella serovar Typhimurium were significantly lower in HYPOX and NIL rats than in sham-operated rats; and (iv) compared to NIL rats, higher Peyer's patch and spleen bacterial colonization and decreased IgG, IgM, and IgA production were observed in HYPOX rats. We conclude that hormones from each pituitary lobe affect the systemic and gastrointestinal humoral immune responses through different mechanisms.     

Relative enrichment of the lighter 59 kDa form of glutamic acid decarboxylase in nerve endings: an immunoblotting study in pituitary neurointermediate lobe.

Previous studies established that glutamate decarboxylase (GAD) is present in the brain of higher vertebrates in two forms composed of the homodimeric association of two subunits 59 and 63 kDa, respectively, that were found to be structurally related. We have performed quantitative comparative immunoblotting of whole brain and pituitary neurointermediate lobe (NIL) extracts. While GAD in the brain is present in cell bodies and nerve endings, it is known to be contained in the NIL exclusively in nerve endings that belong to a relatively long gamma-aminobutyric acid (GABA)ergic pathway of central origin. The relative immunolabelling ratio of the 59 kDa to the 63 kDa subunit was at least 10-fold higher in the NIL when compared to whole brain extracts. Accordingly we suggest that the GAD composed of the 59 kDa subunits, which appears to be greatly enriched in GABAergic nerve endings, might represent the form of GAD on which short term activity regulation is exerted in relation to neuronal activity. It might result from the post-translational processing of the GAD formed from the 63 kDa subunits during the axonal transport.     

Corticotropin releasing hormone and proopiomelanocortin involvement in the cutaneous response to stress

The skin is a known target organ for the proopiomelanocortin (POMC)-derived neuropeptides alpha-melanocyte stimulating hormone (alpha-MSH), beta-endorphin, and ACTH and also a source of these peptides. Skin expression levels of the POMC gene and POMC/corticotropin releasing hormone (CRH) peptides are not static but are determined by such factors as the physiological changes associated with hair cycle (highest in anagen phase), ultraviolet radiation (UVR) exposure, immune cytokine release, or the presence of cutaneous pathology. Among the cytokines, the proinflammatory interleukin-1 produces important upregulation of cutaneous levels of POMC mRNA, POMC peptides, and MSH receptors; UVR also stimulates expression of all the components of the CRH/POMC system including expression of the corresponding receptors. Molecular characterization of the cutaneous POMC gene shows mRNA forms similar to those found in the pituitary, which are expressed together with shorter variants. The receptors for POMC peptides expressed in the skin are functional and include MC1, MC5 and mu-opiate, although most predominant are those of the MC1 class recognizing MSH and ACTH. Receptors for CRH are also present in the skin. Because expression of, for example, the MC1 receptor is stimulated in a similar dose-dependent manner by UVR, cytokines, MSH peptides or melanin precursors, actions of the ligand peptides represent a stochastic (predictable) nonspecific response to environmental/endogenous stresses. The powerful effects of POMC peptides and probably CRH on the skin pigmentary, immune, and adnexal systems are consistent with stress-neutralizing activity addressed at maintaining skin integrity to restrict disruptions of internal homeostasis. Hence, cutaneous expression of the CRH/POMC system is highly organized, encoding mediators and receptors similar to the hypothalamic-pituitary-adrenal (HPA) axis. This CRH/POMC skin system appears to generate a function analogous to the HPA axis, that in the skin is expressed as a highly localized response which neutralizes noxious stimuli and attendant immune reactions.