Manserin, a secretogranin II-derived peptide, distributes in the rat endocrine pancreas colocalized with islet-cell specific manner

Manserin is a recently characterized 40-amino acid neuropeptide derived from secretogranin II, a protein belonging to the chromogranin family. Although the physiological roles of manserin have not been elucidated to date, manserin has been shown to distribute in not only the brain but also the endocrine system such as the pituitary and adrenal glands, suggesting its role in the endocrine system. The present study aimed to explore the occurrence and distribution of manserin in the rat pancreas using an immunohistochemical technique with a polyclonal antibody against rat manserin. Immunoreactivity for manserin was readily detected in almost whole islets of Langerhans whereas not at all in the exocrine pancreas. Manserin-expressing cells were not colocalized with the glucagon-secreting cells (α cells), whereas they colocalized with insulin-secreting cells (β cells) and somatostatin-secreting cells (δ cells), although their intracellular distribution was different. These results indicate that manserin, occurring in the endocrine pancreas, may have a potential role in the endocrine system.     

Secretogranin II and its Derivative Peptide,Manserin, are Differentially Localized in Purkinje Cells and Unipolar Brush Cells in the Rat Cerebellum

The cerebellum has long been recognized as the primary center of motor coordination in the central nervous system. Cerebellar neuropeptides have been postulated to be involved in such motor coordination, though this role is not fully understood. We herein investigated the localization of novel neuropeptide, “manserin” in the adult rat cerebellum. Punctate signals of manserin immunoreactivity were observed in the granular layer of the rat cerebellum. Manserin signals were also observed in the fibers and fiber terminals in the granular layer as well as the molecular layer. Manserin did not localize in Purkinje cells. Interestingly, cerebellar manserin was preferentially colocalized with unipolar brush cells, a class of excitatory granular layer interneuron, which are known to be involved in vestibullocerebellar functions. These results indicate that manserin plays pivotal roles in the cerebellar functions.     

Existence of manserin, a secretogranin II-derived neuropeptide, in the rat inner ear: relevance to modulation of auditory and vestibular system

Manserin is a 40-amino acid neuropeptide derived from rat brain. Manserin has been shown to distribute in the neuroendocrine system, such as the pituitary and adrenal glands, but it has been little studied in other organs. In this study, the authors examined localization of manserin in the inner ear of the adult Wistar rat using immunohistochemical analyses. Manserin immunoreactivity was detected in the neuronal terminals of the organ of Corti and type II spiral ganglion cells. In addition to being identified in the auditory system, manserin was detected at the synapses of the vestibular system, such as saccule, utricle, and semicircular canal. These results suggest that inner ear manserin may be involved in the function of peripheral auditory and vestibular systems.     

Recently identified a novel neuropeptide manserin colocalize with the TUNEL-positive cells in the top villi of the rat duodenum.

We recently isolated a novel 40 amino acid neuropeptide designated manserin from the rat brain. Manserin is derived from secretogranin II, a member of granin acidic secretory protein family by proteolytic processing, as previously reported secretoneurin and EM66. Manserin peptide are localized in the endocrine cells of the pituitary. In this study, we further investigated the manserin localization in the digestive system by immunohistochemical analysis using antimanserin antibody. In the duodenum, manserin immunostaining was exclusively observed in the nuclei of top villi instead of cytosol as observed in neurons in our previous study. Interestingly, manserin-positive cells in the duodenum are colocalized with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) positive cells, the cells whose DNA was damaged. Since the top villi of duodenum epithelial cells are known to undergo spontaneous apoptosis during epithelial cell turn over, and since other peptides such as secretoneurin and EM66 derived from SgII have been reported to be cancer-related, these results indicated that manserin peptide may have a role in apoptosis and/or cancer pathogenesis in the digestive organ.     

Characterization of Hypothalamic Neurons Expressing a Neuropeptide Receptor, GALR2, Using Combined in Situ Hybridization–Immunohistochemistry

Our laboratory is interested in characterizing the neurotransmitter and hormonal phenotype of neurons in the rat hypothalamus expressing novel neuropeptide receptors of the neuropeptide Y and galanin families. In this review, we describe a technique combining nonradioactive in situ hybridization to detect mRNA and fluorescence immunohistochemistry to detect protein antigens. We examined paraffin sections of rat hypothalamus using confocal microscopy to determine whether mRNA for the galanin receptor, GALR2, was colocalized at the cellular level of resolution with somatostatin or tyrosine hydroxylase immunoreactivity. We found that many neurons in the hypothalamus expressed both GALR2 mRNA and either somatostatin or tyrosine hydroxylase immunoreactivity. The simultaneous detection of mRNA and protein immunoreactivity in individual neurons using the confocal microscope for visualization is an excellent tool for the analysis of newly characterized genes in the central nervous system.     

Role of TrkB expression in rat adrenal gland during acute immobilization stress

Expression of tyrosine receptor kinase B (TrkB), a receptor for brain‐derived neurotrophic factor (BDNF), is markedly elevated in the adrenal medulla during immobilization stress. Catecholamine release was confirmed in vitro by stimulating chromaffin cells with recombinant BDNF. We investigated the role of TrkB and the localization of BDNF in the adrenal gland during immobilization stress for 60 min. Blood catecholamine levels increased after stimulation with TrkB expressed in the adrenal medulla during 60‐min stress; however, blood catecholamine levels did not increase in adrenalectomized rats. Furthermore, expression of BDNF mRNA and protein was detected in the adrenal medulla during 60‐min stress. Similarly, in rats undergoing sympathetic nerve block with propranolol, BDNF mRNA and protein were detected in the adrenal medulla during 60‐min stress. These results suggest that signal transduction of TrkB in the adrenal medulla evokes catecholamine release. In addition, catecholamine release was evoked by both the hypothalamic–pituitary–adrenal axis and autocrine signaling by BDNF in the adrenal gland. BDNF–TrkB interaction may play a role in a positive feedback loop in the adrenal medulla during immobilization stress.     

Human napsin A: expression, immunochemical detection, and tissue localization

PCR-coupled cDNA subtraction hybridization was adapted to identify the genes expressed in the adrenocortical tissues from high salt diet-treated rat. A novel cDNA clone, termed salt-inducible kinase (SIK), encoding a polypeptide (776 amino acids) with significant similarity to protein serine/ threonine kinases in the SNF1/AMPK family was isolated. An in vitro kinase assay demonstrated that SIK protein had autophosphorylation activity. Northern blot revealed that SIK mRNA levels were markedly augmented by ACTH treatment both in rat adrenal glands and in Y1 cells. SIK may play an important role in the regulation of adrenocortical functions in response to high plasma salt and ACTH stimulation.     

Induction of Adrenal Tyrosine Hydroxylase mRNA by Single Immobilization Stress Occurs Even After Splanchnic Transection and in the Presence of Cholinergic Antagonists

Abstract: Immobilization (IMO) stress elevates plasma catecholamines and increases tyrosine hydroxylase (TH) gene expression in rat adrenals. This study examined the mechanism(s) of IMO-induced changes in adrenal TH mRNA levels. Innervation of the adrenal medulla is predominantly cholinergic and splanchnicotomy as well as nicotinic receptor antagonists prevent the cold-induced rise in TH mRNA levels. In this study, the IMO-induced rise in plasma catecholamines, but not TH mRNA levels, was reduced by the antagonist chlorisondamine. Muscarinic antagonist atropine also did not prevent the IMO stress-elicited rise in TH mRNA. Furthermore, denervation of the adrenals by unilateral splanchnicotomy did not block the IMO-induced rise in TH mRNA but completely prevented the induction of neuropeptide Y mRNA. These results suggest that (1) the large increase in adrenal TH gene expression elicited by a single IMO stress is not regulated via cholinergic receptors or splanchnic innervation, and (2) there is a dissociation between regulatory mechanisms of catecholamine secretion and elevation of TH gene expression in the adrenal medulla of rats during IMO stress.     

Effects of ethanol on the concentration of neuropeptides, ACTH and corticosterone during immobilization stress

It was shown, that stress increased the level of ACTH, beta-endorphin and corticosterone in the blood plasma of the rat. Injection of ethanol (1 g/kg) decreased the level of ACTH, but increased the levels of beta-endorphin in the rat subjected to immobilization stress. The immobilization lowered the levels of met-enkephalin in the striatum and medulla oblongata, but increased the content of neuropeptide in the adrenal glands. The concentration of leu-enkephalin and DSIP remained unchanged following the stress. Ethanol reversed the action of immobilization on the level of met-enkephalin in the striatum, but increased the content of DSIP in the thalamus. These results indicate that ethanol modified the activity of pituitary-adrenal-axis during stress and probably the stress-protective action of ethanol partly performed with the involvement of DSIP.     

Biochemical characterization and immunocytochemical localization of EM66, a novel peptide derived from secretogranin II, in the rat pituitary and adrenal glands.

Characterization of secretogranin II (SgII) mRNA in various vertebrates has revealed selective conservation of the amino acid sequences of two regions of the protein, i.e., the bioactive peptide secretoneurin and a flanking novel peptide that we named EM66. To help elucidate the possible role of EM66, we examined the occurrence as well as the cellular and subcellular distribution of EM66 in rat pituitary and adrenal glands by using a polyclonal antibody raised against the recombinant human EM66 peptide. High-performance liquid chromatography (HPLC) analysis of rat pituitary and adrenal extracts combined with a radioimmunoassay resolved EM66-immunoreactive material exhibiting the same retention time as recombinant EM66. In the rat pituitary, double-labeling immunohistochemical (IHC) studies showed that EM66 immunoreactivity (IR) was present in gonadotrophs, lactotrophs, thyrotrophs, and melanotrophs, whereas corticotrophs were devoid of labeling. EM66-IR was also observed in nerve endings in the neural lobe. Immunocytochemical staining at the electron microscopic level revealed that EM66-IR is sequestered in the secretory granules within gonadotrophs and lactotrophs. In the adrenal medulla, double IHC labeling showed that EM66-IR occurs exclusively in epinephrine-synthesizing cells. At the ultrastructural level, EM66-IR was seen in chromaffin vesicles of adrenomedullary cells. These results demonstrate that post-translational processing of SgII generates a novel peptide that exhibits a cell-specific distribution in the rat pituitary and adrenal glands where it is stored in secretory granules, supporting the notion that EM66 may play a role in the endocrine system.     

Age-Related Bidirectional Changes in Neuropeptide Y Peptides in Rat Adrenal Glands, Brain, and Blood

Age-related changes in neuropeptide Y (NPY) regulation were studied in rat adrenal glands, brains, and blood by radioimmunoassay and biochemical characterization using reversed phase HPLC and gel filtration chromatography. NPY immunoreactivity (pmol/g tissue +/- SEM) in rat adrenal glands increased from 7 +/- 1 (6 weeks old) to 1,500 +/- 580 (69 weeks old). Biochemical characterization by HPLC showed that this increase was due to those of NPY and methionine sulfoxide NPY. In contrast, in rat brain, NPY content decreased in an age-dependent manner specifically in striatum, hippocampus, medulla oblongata, and spinal cord and the sulfoxide form was not detected. In rat blood, the circulating level of NPY was high (3-5 pmol/ml plasma +/- SEM) but did not change significantly with age or by adrenal demedullation. Only a small increase of the sulfoxide form of NPY was observed in aged rat plasma. The age-dependent changes in regulation and modification of NPY in adrenal glands and in specific brain areas may have physiological relevance in the regulation of catecholamine release from adrenal glands and some brain functions during aging.     

Splanchnic Nerve Transection Abolishes the Age-Dependent Increase of Neuropeptide Y-Like Immunoreactivity in Rat Adrenal Gland

Using an antiserum directed against porcine neuropeptide Y (NPY), a high concentration of NPY immunoreactivity (NPY-IR) was detected in rat adrenal gland. The level of NPY-IR in the adrenal gland was found to increase considerably with age. Biochemical characterization by reverse-phase HPLC indicated that this increase was due to accumulations of NPY and another molecular form of NPY-like immunoreactive peptide. Chronic denervation of the splanchnic nerve abolished this age-dependent increase of NPY-IR rat adrenal gland.     

Generation of self-antigen reactive, anti-urocortin specific antibodies by immunization of recombinantly expressed urocortin fusion proteins

Urocortin is a recently described 40-meric neuropeptide, which was originally detected in the rat mid-brain and is believed to play a key role in response to stress situations. While its function in the central nervous system is rather well established, the biological role in the periphery is still to be determined. To investigate its distribution and effect on peripheral cells and tissues, in the present study, urocortin was recombinantly expressed and specific antibodies were generated. So far, the immunological detection of urocortin in the rat was largely dependent on antisera generated in rabbits. However, the polyclonal nature of the serum and the remote species origin tend to show cross-reactivities and higher backgrounds. On the other hand, generation of mouse antibodies to rat urocortin was hampered since mouse and rat urocortin sequences are identical, and such antibodies would represent auto-reactive antibodies. Despite such restrictions, the immunization with a combination of various recombinantly expressed urocortin fusion proteins resulted in the successful generation of mouse antiurocortin antisera, whose specificities were confirmed by ELISA and Western blot analysis. To produce the recombinant proteins for immunization, a cDNA encoding the mature urocortin sequence was cloned and expressed in fusion either with the glutathione-S-transferase, the maltose-binding protein, thioredoxin, or a 6X His tag. Depending on the expression system, the solubility and yield of the recombinant proteins greatly varied. Together with the newly generated antibodies, these recombinantly expressed urocortin proteins will serve as valuable tools in further investigations of the biological function of urocortin.     

Pro-melanin-concentrating hormone gene (PMCH) is localized on human chromosome 12q and rat chromosome 7

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide that may be involved in regulation of the stress response and food intake behavior in mammals. MCH and two other putative neuropeptides, NEI and NGE, are encoded by the same precursor, designated pro-melanin-concentrating hormone (PMCH). A panel of somatic cell hybrids segregating either human or rat chromosomes was used to determine the chromosomal localization of the PMCH locus. It was assigned to human chromosome 12q and to rat chromosome 7. This is the first neuropeptide-encoding gene found in this new synteny group conserved in rat and human.     

Characterization of hypothalamic neurons expressing a neuropeptide receptor, GALR2, using combined in situ hybridization-immunohistochemistry.

Our laboratory is interested in characterizing the neurotransmitter and hormonal phenotype of neurons in the rat hypothalamus expressing novel neuropeptide receptors of the neuropeptide Y and galanin families. In this review, we describe a technique combining nonradioactive in situ hybridization to detect mRNA and fluorescence immunohistochemistry to detect protein antigens. We examined paraffin sections of rat hypothalamus using confocal microscopy to determine whether mRNA for the galanin receptor, GALR2, was colocalized at the cellular level of resolution with somatostatin or tyrosine hydroxylase immunoreactivity. We found that many neurons in the hypothalamus expressed both GALR2 mRNA and either somatostatin or tyrosine hydroxylase immunoreactivity. The simultaneous detection of mRNA and protein immunoreactivity in individual neurons using the confocal microscope for visualization is an excellent tool for the analysis of newly characterized genes in the central nervous system.     

Differential expression of a stress-modulating gene, BRE, in the adrenal gland, in adrenal neoplasia, and in abnormal adrenal tissues.

Genes that modulate the action of hormones and cytokines play a critical role in stress response, survival, and in growth and differentiation of cells. Many of these biological response modifiers are responsible for various pathological conditions, including inflammation, infection, cachexia, aging, genetic disorders, and cancer. We have previously identified a new gene, BRE, that is responsive to DNA damage and retinoic acid. Using multiple-tissue dot-blotting and Northern blotting, BRE was recently found to be strongly expressed in adrenal cortex and medulla, in testis, and in pancreas, whereas low expression was found in the thyroid, thymus, small intestine and stomach. In situ hybridization and immunohistochemical staining indicated that BRE was strongly expressed in the zona glomerulosa of the adrenal cortex, which synthesizes and secretes the mineralocorticoid hormones. It is also highly expressed in the glial and neuronal cells of the brain and in the round spermatids, Sertoli cells, and Leydig cells of the testis, all of which are associated with steroid hormones and/or TNF synthesis. However, BRE expression was downregulated in human adrenal adenoma and pheochromocytoma, whereas its expression was enhanced in abnormal adrenal tissues of rats chronically treated with nitrate or nitrite. These data, taken together, indicate that the expression of BRE is apparently associated with steroids and/or TNF production and the regulation of endocrine functions. BRE may play an important role in the endocrine and immune system, such as the cytokine-endocrine interaction of the adrenal gland.     

人肾上腺基因表达谱的建立及其功能的新认识

为深入理解人类肾上腺（AD）的功能,构建了正常人肾上腺cDNA文库,并利用大规模表达序列标签（ESTs）测序和生物信息学技术,研究显示参与基因/蛋白表达的基因类型表达最多,其次为能量代谢类.肾上腺中表达丰度最高的3个基因均为参与类固醇合成的酶类和蛋白.一些重要的基因首次显示在肾上腺表达,包括神经激素和神经肽,如促肾上腺皮质激素释放激素（CRH）,黑色素浓激素（MCH）,urocortin,可卡因和安非他明调节肽（CART）和垂体腺苷酸环化酶激活肽（PACAP）;许多重要介质的受体,如细胞因子、神经肽及神经递质受体;参与胆固醇代谢的基因,如LDL受体、HDL结合蛋白和胆固醇合成酶.研究结果表明在肾上腺表达丰度最高的基因与该器官的功能特异性有关,除类固醇激素外,许多神经肽、细胞因子在肾上腺产生,肾上腺与体内其他重要的系统间存在广泛的应答,而且在人肾上腺局部可能存在一个CRH-ACTH-皮质醇调节网络.