Analysis of selected genes in neuroendocrine tumours: insulinomas and phaeochromocytomas

Insulinomas and phaeochromocytomas are neuroendocrine tumours that may be either sporadic or manifestation of a familial cancer syndromes and are both derived from the neural crest. In the present study, gene components of different signalling pathways were investigated in sporadic human insulinomas and phaeochromocytomas to identify the responsible candidates. Ret and k-ras were tested for activating point mutations, and NF1 , p53 , BRCA1 , nm23-H1 , SDHB and SDHD for loss of heterozygosity (LOH). Twenty-two sporadic insulinomas and 15 phaeochromocytomas were analysed by the polymerase chain reaction using restriction fragment length polymorphism or dinucleotide repeat polymorphism methods. The results of our analysis demonstrate that the most frequent changes were point mutations of k-ras : 23% of insulinomas and 62% of phaeochromocytomas harboured k-ras mutations. The analysis also showed two phaeochromocytomas with point mutations of the ret oncogene. Only one insulinoma showed LOH of NF1 , and another showed LOH of p53 . Allelic loss of BRCA1 was detected in two insulinomas, and of nm23-H1 in another insulinoma. Allelic losses of the SDHB gene were present in two phaeochromocytoma and one insulinoma cases and allelic losses of SDHD were present in one phaeochromocytoma case. The changes observed in phaeochromocytomas were more homogenous and confined to k-ras and ret oncogenes, whereas insulinomas showed more heterogenic situation. Our findings may contribute to a better understanding of the genetic profile of neuroendocrine tumours.     

Positive feedback by autoexcitatory neuropeptides in neuroendocrine bag cells of Aplysia

Neurohormones are often secreted in large amounts from neuroendocrine cells during episodes of synchronous, repetitive spike activity. We report evidence that this pattern of activity in the neuroendocrine bag cells of Aplysia involves positive feedback by autoexcitatory transmitters. Intracellular stimulation of individual bag cells caused slow depolarizing afterpotentials and synchronous afterdischarges in the entire population of bag cells. Application of the bathing medium collected during bag cell activity mimicked these responses. Application of alpha-, beta-, or gamma-bag cell peptides (BCPs), 3 structurally related neuropeptides released from bag cells, also mimicked these responses. These autoexcitatory BCPs fulfill most of the strict criteria necessary for classification as neurotransmitters in this system. This is the first biological activity reported for beta- and gamma-BCPs and brings to 4 the number of bag cell neuropeptides derived from the egg-laying hormone/BCP precursor that are putative cotransmitters. Positive feedback by autoexcitatory transmission may provide a general mechanism for the generation of episodic activity in neuroendocrine systems.     

Combinatorial code of growth factors and neuropeptides define neuroendocrine differentiation in PC12 cells

Adrenal chromaffin cells constitute one of the first cell types to have been defined as a neuroendocrine cell type. Since they produce dopamine, these cells have been proposed for the treatment of neuronal deficits in human Parkinson's disease. However, the factors involved in the development of chromaffin cells are still poorly understood. Based on recent insights from stem cell research, we decided to study the role of extracellular matrices, growth factors and neuropeptides on the neuroendocrine differentiation in a serum-free medium of PC12 cells. Employing immunohistochemistry, quantitative PCR and HPLC analysis, neuroendocrine differentiation was determined by evaluating neurite outgrowth, catecholamine biosynthesis and release as well as neuropeptide and vesicular protein mRNA expression. The combination of bFGF, NGF and PACAP could prevent the inhibition of neurite process development induced by dexamethasone in PC12 cells cultured on ECM. Whereas glucocorticoids were essential in the regulation of enzymes of catecholamine biosynthesis and metabolism, growth factors and PACAP were more efficient in inducing neuropeptide and chromogranin B expression as well as release of dopamine and 3-methoxytyramine. Therefore, in addition to glucocorticoids, chromaffin cells need a gradient of matrix, growth factors, and neuropeptides to develop the full functional phenotype of a neuroendocrine cell.     

Expression of oligodendroglia and Schwann cell markers in human nervous system tumors

Summary Sixty tumors of the central and peripheral nervous system and seven brain metastases of extracranial carcinomas were examined using the peroxidase-antiperoxidase method to study the expression of myelin basic protein (MBP), myelin-associated glycoprotein (MAG) and the HNK-1/Leu-7 epitope. The immunocytochemical findings were compared with and correlated to Western blot results. None of the tumor types, including oligodendrogliomas, neurinomas and neurofibromas, expressed MAG and MBP, whereas myelin sheaths and their remnants within the tumors yielded specific immunoreactions. In contrast, the HNK-1/Leu-7 antibodies labelled the majority of the tumors tested including oligodendrogliomas and Schwann cell tumors. As demonstrated by Western blot experiments the HNK-1/anti-Leu-7 antibodies exhibited positive reactions with diverse polypeptides both in tumors and in non-neoplastic brain tissue at positions not corresponding to MAG. This suggests that the epitope recognized by HNK-1/Leu-7 antibodies is shared by a variety of unrelated proteins in normal and neoplastic tissues. Our results strongly indicate the absence of detectable amounts of MBP and MAG in oligodendrogliomas and Schwann cell tumors. The immunomorphological and immunochemical findings clearly showed the wide distribution of the HNK-1 epitope within different tumor types of the central and peripheral nervous system. In conclusion, the data demonstrate that specific cell markers for human oligodendrogliomas and Schwann cell tumors are still lacking.     

Disturbances in the hypothalamo-pituitary-adrenal and other neuroendocrine axes in bulimia

Disturbances in the hypothalamo-pituitary-adrenal (HPA) and other endocrine axes were assessed in 24 women with bulimia and healthy controls. Overnight blood samples for measuring nocturnal plasma cortisol, prolactin (PRL), growth hormone (GH), luteinizing hormone (LH), and follicle stimulating hormone (FSH) were obtained at 30-min intervals. A 1.5 mg dexamethasone suppression test (DST) and a TRH-test were performed. Patients were monitored closely while their nutritional intake was recorded over 21 days. Compared with healthy controls, nocturnal cortisol plasma levels were not elevated in the bulimics. There was a trend toward insufficient cortisol suppression in the DST in patients with bulimia, which was most pronounced in patients with signs of restricted caloric intake. Plasma dexamethasone levels were significantly reduced in bulimics compared with healthy controls. There was a trend for blunted thyrotropin stimulating hormone (TSH) responses to thyrotropin releasing hormone (TRH) in bulimia. The prolactin response to TRH was significantly reduced in bulimics with a history of anorexia nervosa. Plasma LH and plasma FSH were significantly reduced in bulimics with signs of reduced caloric intake [low T3, high levels of beta-hydroxy-butyric acid (BHBA), reduced daily caloric intake, high number of fasting days] as compared with healthy controls. Bulimics with high BHBA levels had significantly reduced nocturnal prolactin plasma levels. Results show that multiple neuroendocrine disturbances exist in bulimia in a milder form than in anorexia nervosa. Evidence for the impact of caloric intake on endocrine functions is presented. Endocrine dysfunctions in our bulimic sample did not show a positive association with the presence of depressive symptoms.     

Expression of cell division markers in the hippocampus in Alzheimer’s disease and other neurodegenerative conditions

Recent studies, showing that cell cycle-related nuclear proteins p105 and Ki-67 are associated with Alzheimer’s disease (AD)-related cytoskeletal pathology, suggested that these proteins, in addition to their functions in regulating the cell cycle, may have more specialised functions in the adult nervous system. In order to test this hypothesis we studied the expression of the cell cycle-related proteins Ki-67, pCNA and p53 in the hippocampi of 33 subjects, including some with AD or other neurodegenerative disorders and some with no neurological disease. By immunohistochemistry we found nuclear expression of Ki-67 in all subregions of the hippocampus, with the highest levels in the dentate gyrus. Both neurons and glial cells expressed this protein. The proportion of cells positive for Ki-67 and the distribution pattern varied considerably depending on the pathological diagnosis. Neuronal nuclear expression of Ki-67 was increased in AD but was also elevated in young Down’s syndrome subjects and in those with Pick’s disease. Expression of this protein was therefore not AD-specific. We did not find nuclear pCNA or p53 expressed in our patient groups. Contrary to previous studies AD-type neurofibrillary tangles were not labelled with any of the cell cycle markers used. The presence of nuclear Ki-67 expression indicates that some hippocampal neurons are not in the quiescent G₀ phase but have re-entered the cell cycle. The absence of nuclear pCNA or p53 suggests that the cycle is arrested in G₁. The significance of our findings and their relationship to the production of neurodegenerative cell death via an apoptotic mechanism are discussed. Received: 10 June 1996 / Revised, accepted: 31 July 1996     

Evidence of conserved neuroendocrine interactions in the thymus: intrathymic expression of neuropeptides in mammalian and non-mammalian vertebrates

The function of lymphoid organs and immune cells is often modulated by hormones, steroids and neuropeptides produced by the neuroendocrine and immune systems. The thymus intrinsically produces these factors and a comparative analysis of the expression of neuropeptides in the thymus of different species would highlight the evolutionary importance of neuroendocrine interaction in T cell development. In this review, we highlight the evidence which describes the intrathymic expression and function of various neuropeptides and their receptors, in particular somatostatin, substance P, vasointestinal polypeptide, calcitonin gene-related peptide and neuropeptide Y, in mammals (human, rodent) and non-mammals (avian, amphibian and teleost), and conclude that neuropeptides play a conserved role in vertebrate thymocyte development.     

Psychosocial dwarfism: psychopathological aspects and putative neuroendocrine markers

There exists an extensive terminology for defining the situation of children who, in varying circumstances, suffer from affective deprivation (AD), within an unsatisfactory family situation or in institutions. Nevertheless, the neuroendocrine mechanisms (if they exist) determining it have yet to be identified. Our objective was to determine if specific neuroendocrine markers, all of them previously implicated in affective disorders, could be modified, and in which sense, in affective deprivation syndrome of the child. For this purpose, we studied three separate groups of children: (1) control group (CG); (2) children suffering from AD; and (3) children with non-organic failure to thrive (NOFT). In every case, we studied the serum levels of melatonin, serotonin, β-endorphins and adrenocorticotropic hormone (ACTH); and kynurenine pathway tryptophan metabolites (both during the day and at night). Significantly, there was a conspicuous reduction in the levels of each of the neuroendocrine markers (melatonin, serotonin, β-endorphins and ACTH) in the group suffering from affective deficiency, a diminution which was even more noticeable in the group of patients presenting delayed growth. Furthermore, as also occurs in other affective disorders, there were corresponding modifications in the metabolisation of tryptophan. We report the existence of neuroendocrine mechanisms that are associated with the above-mentioned clinical manifestations in these patients, mechanisms that may underlie the close connection existing between AD syndrome and the cause of NOFT. These data suggest that the AD syndrome and NOFT comprise a single process, but one with a different evolutionary continuum of psychosocial dwarfism.     

Immunocytochemical localization and development of multiple kinds of neuropeptides and neuroendocrine proteins in the chick ultimobranchial gland.

The ultimobranchial gland is an endocrine organ consisting of C cell groups. In chickens, the glands are richly supplied by nerve fibers immunoreactive for neurofilaments. It was found by immunocytochemical staining that C cells of chick ultimobranchial glands showed immunoreactivities for multiple kinds of neuropeptides and neuroendocrine proteins in addition to calcitonin, i.e., calcitonin gene-related peptide (CGRP), somatostatin, neurotensin, chromogranin A, and tyrosine hydroxylase. Furthermore, enkephalin-immunoreactive cells that showed long cytoplasmic processes and large cell bodies, being distinct from the C cell feature, were detected. The densities of these cells per unit area of ultimobranchial gland were assessed using computer-assisted image analysis system; calcitonin cells were 42.9 +/- 10.0%; CGRP cells 26.9 +/- 5.6%; neurotensin cells 8.6 +/- 6.9%; somatostatin cells 3.1 +/- 1.4%; chromogranin A cells 11.8 +/- 1.8%; tyrosine hydroxylase cells 10.0 +/- 5.2%; enkephalin cells 2.9 +/- 1.3%. Dense distributions of peptidergic nerve fibers were also detected in chick ultimobranchial glands. Numerous varicose fibers immunoreactive for substance P were distributed in the close vicinity to C cell clusters and blood vessels. Enkephalin-immunoreactive fibers were also prominent around C cell clusters. Galanin-, vasoactive intestinal peptide (VIP)-, and tyrosine hydroxylase-immunoreactive fibers were distributed around blood vessels only. Subsequently, the ontogeny of these neuropeptides, neuroendocrine proteins, and peptidergic innervations was examined in chickens at various developmental stages. In 10-day-old embryos, weak to moderately intense immunoreactivity for calcitonin was already present in almost all C cells. Immunoreactivities for somatostatin, CGRP, and tyrosine hydroxylase began to appear at this age. At 12 days of incubation, substance P-immunoreactive fibers were first detected in the parenchyma of ultimobranchial glands. Considerable numbers of enkephalin-immunoreactive fibers and cells were also observed. At 14 days of incubation, the largest populations of somatostatin- and enkephalin-immunoreactive cells were attained; the densities of somatostatin- and enkephalin-immunoreactive cells per unit area were 21.2 +/- 3.2% and 12.9 +/- 3.1%, respectively. Substance P-immunoreactive fibers became numerous throughout the gland at this age. Thereafter, calcitonin-, CGRP-, tyrosine hydroxylase-immunoreactive cells progressively increased in number with embryonic age, whereas somatostatin- and enkephalin-immunoreactive cells started to decrease. Chromogranin A- and neurotensin-immunoreactive cells began to appear at 16 days and 18 days of incubation, respectively. Galanin-, VIP-, and tyrosine hydroxylase-immunoreactive fibers were inconspicuous during embryonic life.     

Distribution of neuropeptide FF in porcine spinal cord in comparison with other neuropeptides and serotonin

A large number of neurotransmitters and neuropeptides are concentrated in the dorsal horn of the spinal cord, Where they interact in a complex manner and modulate sensory mechanisms. Most studies are carried out in the rat, and little is known of other species. It is relevant to study mammals with a more complex central nervous system, because pain mechanisms are central in both human and veterinary medicine. Immunoreactivity for neuropeptide FF, an amidated octapeptide originally isolated from bovine brain, was found immunocytochemically at all levels of porcine spinal cord. In contrast to other species studied so far, the peptide immunoreactivity in porcine spinal cord was confined to the intermediolateral gray matter, especially to the intermediolateral cell column and lamina X of the gray matter. This distribution was remarkably different from that of substance P, proenkephalin A-derived peptides, thyrotropin-releasing hormone, serotonin, and neuropeptide Y. Pharmacologic administration of neuropeptide FF alters behavior in assays for analgesia. The distribution of neuropeptide FF immunoreactivity as revealed by this study suggests that there may be marked species differences in the distribution and function of the peptide. © 1994 Wiley-Liss, Inc.     

Chromatographic characterization of neuropeptides in post mortem human brain

Reverse phase high performance liquid chromatography was used to establish the immunoreactive species of five neuropeptides (thyrotropin-releasing hormone, luteinising hormone-releasing hormone, neurotensin, substance-P and somatostatin) in three areas of post mortem human brain--the hypothalamus, amygdala and cortex. In the majority of cases the major immunoreactive peak corresponded to the authentic peptide, although other peaks of immunoreactivity were observed in several instances. It was established that somatostatin-14 was present as the major immunoreactive form and that somatostatin-28 did not occur in any of the three brain areas, although other somatostatin-immunoreactive peaks of unknown structure were detected. In addition to authentic neurotensin in the cortex, a substantial peak of immunoreactivity corresponding to the elution time of neurotensin (1-11) was observed. LH-RH was not detected in the amygdala, but was present in the cortex as a minor component of overall immunoreactivity. The major peak of substance-P immunoreactivity in all three brain areas corresponded to authentic substance-P; in addition immunoreactive material eluting in the region of [Met-O] substance-P, substance-P (5-11) and substance-P (6-11) were detected. TRH occurred as the major peak in all three areas, although minor peaks of immunoreactivity were seen in the amygdala.     

Effects of vasopressin and other neuropeptides on rostral medullary sympathoexcitatory neurons 'in vitro'

Neurons with intrinsic pacemaker activity and presumed sympathoexcitatory function were recorded in rat tissue slices within the confines of the rostroventrolateral reticular nucleus (RVL). These cells were excited in dose-dependent fashion by arginine vasopressin (AVP, 10(8)-10(6) M) but not by oxytocin (up to 10(7) M). The effect of AVP was mimicked by the V1-selective agonist [Phe2,Orn8]vasotocin (VT) (1 microM) but not by the V2-agonist [Val4,D-Arg8]vasopressin (VP) (1.9 microM). The effect of AVP (10(-7) M) was completely blocked by SKF 101926 (10(7) M), a non-selective antagonist and by d(CH2)5[Tyr(Me)2]AVP, a V1-selective antagonist but was unaffected by the V2-selective antagonist d(CH2)5[D-Ile2,Ile4,Ala-NH2 9]AVP. These cells were also activated by thyrotropin-releasing hormone (TRH) (10(-7)-10(-6) M), calcitonin gene-related peptide (CGRP) (4 X 10(-8) M), substance P, (10(-6) M), neuropeptide Y (NPY) (10(-8) M) and inhibited by Met-enkephalin (10(-6) M) and morphine (2 mM). Corticotropin-releasing factor (CRF) (10(-7) M) and angiotensin II (10(-6) M) were ineffective. In conclusion, RVL pacemaker neurons have vasopressin receptors reminiscent of the V1 (vascular and pressor) subtype. Their pacemaking activity is modulated by low doses of several other peptides also known to produce large vasomotor effects after introduction into the cerebroventricular space.     

Effects of substance P and selected other neuropeptides on the synthesis of interleukin-1 β and interleukin-6 in human monocytes: a re-examination

Recent studies have suggested that substance P (SP) and some other neuropeptides are able to induce the synthesis of the proinflammatory cytokines interleukin-1 (IL-1) and interleukin-6 (IL-6) in peripheral blood mononuclear cells. In the present study, we re-examined these findings by using a completely endotoxin-free monocyte cultivation system. We demonstrate that the neuropeptides SP, vasoactive intestinal peptide, substance K, cholecystokinine, a-endorphin, and β-endorphin are consistently unable to induce the synthesis of IL-1 and IL-6 in human peripheral blood monocytes. However, low amounts of LPS ( 1 pg/ml) synergized with SP to induce IL-6 mRNA expression. In contrast to its lack of effect in monocytes, we were able to confirm the ability of SP to induce cytokine synthesis in astrocytic cells. Our results raise questions about previous results claiming a neuropeptide-induced synthesis of proinflammatory cytokines in human monocytes. In conjunction with other studies, we suggest that undetected levels of endotoxin/LPS in the culture medium may have been primarily responsible for results suggesting an inductive effect of neuropeptides on cytokine synthesis in monocytes.     

脑损伤并胫骨骨折大鼠骨痂中神经肽的分布及意义*☆

目的: 研究证明，含有P物质、降钙素基因相关肽、血管活性肠肽、神经肽Y和酪氨酸羟化酶等神经肽的肽能神经共同存在于骨组织中，主要分布于骨代谢活跃的区域，表明这些肽能神经与骨的生长、发育密切相关。观察脑损伤后大鼠胫骨骨痂中神经肽的表达。 方法：实验于2007-02/05在广西医科大学实验动物中心完成。①实验分组：雄性Wistar大鼠130只，体质量450~550 g，随机数字表法分为单纯骨折组（n =60），脑损伤合并骨折组（n =60），正常对照组（n =10）。②实验方法：麻醉后显露大鼠右颅顶骨，中线旁2 mm 处开直径5 mm 骨窗，液压打击致中度脑损伤，并制备大鼠胫骨骨折模型，其中单纯骨折组头部只做颅骨开窗，正常对照组不做任何处理。③实验评估：术后3，7，14，21，28，35 d 苏木精-伊红染色和神经肽免疫组织化学染色观察神经肽在大鼠胫骨中的分布及胫骨骨折骨痂的连续性及骨折愈合情况。计算机X射线摄像仪（CR）摄片测定术后14，21，28 d 脑损伤合并骨折组及单纯骨折组骨痂面积大小。 结果：纳入大鼠130只，均进入结果分析。①脑损伤合并骨折组早期形成大量纤维骨痂和软骨骨痂，骨痂中神经肽免疫阳性神经纤维较多，明显增厚的骨膜内层骨祖细胞、幼稚的软骨细胞胞质内降钙素基因相关肽、P物质、血管活性肠肽、酪氨酸羟化酶、神经肽Y强阳性表达。②脑损伤合并骨折组14 d 纤维骨痂中的软骨细胞团增大，骨膜下软骨细胞层增厚；21 d 小梁骨明显增厚，软骨岛增大；28 d 仍可见大量的纤维骨痂和软骨骨痂，软骨细胞团周边有少量结构稀疏的编织骨形成。单纯骨折组骨膜反应轻，纤维骨痂量少，骨内成骨和软骨内成骨并存，以前者为主，骨折愈合过程明显晚于脑损伤合并骨折组。③14，21 d 脑损伤合并骨折组骨痂面积较单纯骨折组大（P < 0.01）；21，28 d 脑损伤合并骨折组骨痂面积变化明显快于单纯骨折组，提示骨痂塑性快（P < 0.01）。CR摄片发现，各骨折组大鼠骨折端均未发现不愈合现象，单纯骨折组骨折线清晰，骨痂量较少；脑损伤合并骨折组骨性愈合较好，骨痂量较多，骨折线模糊。 结论：正常大鼠骨生长活跃区有丰富的肽能神经支配。脑损伤后骨痂中神经肽有显著改变，并引起骨痂量和质的改变，骨折愈合加速。     

Immunohistochemical markers in rat cortex: co-localization of calretinin and calbindin-D28k with neuropeptides and GABA.

Calretinin and calbindin-D28k are two calcium-binding proteins which are present in separate populations of interneurons in cerebral cortex and hippocampus. To identify these cells with the populations expressing different transmitters, two-colour immunofluorescence was done with antibodies against the calcium-binding proteins plus antibodies against vasoactive intestinal peptide (VIP), somatostatin (SRIF), or gamma-aminobutyric acid (GABA). In neocortex, calretinin is partially co-localized with VIP (especially in the deeper layers) and is not co-localized with SRIF. Calbindin is largely co-localized with SRIF, and not with VIP. Both calretinin and calbindin are partially co-localized with GABA. In piriform and entorhinal cortex, the patterns resemble those in neocortex. In hippocampus, preliminary data indicate greater heterogeneity, especially in the ventral part; at least a few double-positive cells are present for every combination of calcium-binding protein and neuropeptide. These results expand the known diversity of local-circuit neurons in cortical regions.