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Background Different epitopes of the granin family of proteins, chromogranin (Cg) A, CgB and secretogranin (Sg) II, have been demonstrated in normal human pancreas, gastrointestinal tract, adrenal medulla and in several neuroendocrine tumours (NETs). SgIII has been recently reported in endocrine pancreas. The aim of the present study was to examine the expression of SgIII in different NETs and compare it with the expression of CgA, CgB and SgII epitopes. Material and methods Tissue specimens from 47 NETs were analyzed. Antibodies to CgA 250–284, CgB 244–255, SgII 172–186 (C-terminal secretoneurin) and SgIII 348–361 were used for immunostaining. Results SgIII was expressed in 41 of 47 NETs. The expression of SgIII agreed well with that of CgA, CgB and SgII, with exceptions of phaeochromocytomas, where more CgB and SgII immunoreactive cells were observed and parathyroid adenomas, which were only stained by CgA. In rectal NETs more cells expressed SgIII than CgA. Conclusions This is the first report on SgIII expression in various NETs. A majority of tumours studied displayed SgIII immunostaining, which indicates a functional relationship with the other granins.

Chromogranin A-like immunoreactivity (CgA-LI) has been, and remains, the most widely used diagnostic and prognostic marker for endocrine tumors. The availability of assay kits combined with moderately high sensitivity and specificity has meant that there has been no great incentive to develop alternative markers. However, circulating concentrations of CgA-LI are elevated in several non-neoplastic diseases and in patients receiving acid-suppression therapy which may lead to false positive diagnosis. Additionally, certain endocrine tumors, such as rectal carcinoids, do not express the CgA gene so that there is a need for additional markers to complement CgA measurements. Plasma concentrations of the CgA-derived peptide, pancreastatin, measured with antisera of defined regional specificity, have a prognostic value in patients with metastatic midgut carcinoid tumors receiving somatostatin analog therapy or hepatic artery chemoembolization. Other CgA-derived peptides with potential as tumor markers are vasostatin-1, WE-14, catestatin, GE-25, and EL-35 but their value has yet to be fully assessed. Circulating concentrations of chromogranin B-like immunoreactivity (CgB-LI) are not elevated in non-neoplastic diseases and measurements of CCB, the COOH-terminal fragment of CgB, may be useful as a biochemical marker for neuroendocrine differentiation in lung tumors. Antisera to the secretogranin II-derived peptide, secretoneurin detects carcinoid tumors of the appendix with greater frequency than antisera to CgA and are of value in identifying therapy-resistant carcinoma of the prostate (clinical stage D3). Measurement of concentrations of a second secretogranin II-derived peptide, EM-66 in tumor tissue has been used to differentiate between benign and malignant pheochromocytoma. These examples point to a limited although potentially valuable role for granin-derived peptides as tumor markers.

Chromogranin A (CgA) and secretogranin II (SgII) of the granin family of uniquely acidic proteins secreted from elements of the diffuse neuroendocrine system are also produced by cells involved in inflammation. CgA and the CgA-derived peptides vasostatin-I and catestatin are products of polymorphonuclear neutrophils accumulating at sites of injury or infections while SgII and the Sg II-derived secretoneurin may contribute to neurogenic inflammation when released from sensory nerve terminals. This review is directed towards vasostatin-I, catestatin and secretoneurin as modulators of cells and tissues associated with inflammatory conditions. The accumulated literature indicates that concerted effects of vasostatin-I and catestatin may be relevant for the first-line host-defence against invading microorganisms, contrasting the apparent lack of antibacterial potencies in secretoneurin. Oppositely directed effects of vasostatin-I and secretoneurin on endothelial permeability and transendothelial extravasation are particularly striking. While vasostatin-I protects the integrity of the endothelial barrier against the disruptive effects of proinflammatory agents, secretoneurin activates transendothelial extravasation, chemotaxis and migration of leukocytes. Oppositely directed effects of vasostatin-I and secretoneurin on formation of blood vessels are also indicated, vasostatin-I inhibiting angiogenetic parameters while secretoneurin activates not only angiogenesis but also vascularization.

Monocytes appear to play a central role in inflammatory processes like atherogenesis or lung inflammation both as the progenitors of foam cells and as a potential source of factors mediating further inflammatory processes. However, signals mediating the influx of monocytes into the inflammatory focus remain partly unknown. Secretoneurin (SN) is a more recently characterised 33-amino acid neuropeptide that is co-released from afferent nerve endings together with substance P (SP) and calcitonin gene-related peptide (CGRP). Furthermore, SN has been shown to affect human fibroblast, endothelial, smooth muscle, eosinophil and monocyte functions in vitro. An activity of SN on monocyte adhesion to the vascular wall has not yet been reported. The aim of this study was to investigate whether the adhesion properties of human monocytes (U937 and Mono Mac-6) to endothelial cells could be influenced by SN. In an in vitro model of the vascular wall, incubation of arterial (rat aortic endothelial cells) and venous endothelial cells (immortalised human umbilical vein endothelial line: EA.hy 926) with SN resulted in a time- and concentration-dependent increase in monocyte adhesion with a maximal effect seen after 4-6 h at a concentration of 10(-8) M SN. Increased monocyte adhesion seems not to be tissue-specific as SN-induced adhesion was observed on both arterial and venous endothelial cells. A specific antibody preparation against SN completely abolished increased monocyte adhesion toward SN-stimulated endothelium. Since adhesion was enhanced to a similar degree and with similar time kinetics as responses evoked by interleukin-1 (IL-1, 1 ng/ml) or lipopolysaccharide (LPS, 100 ng/ml), involvement of identical adhesion molecules can be suggested. Our observations provide substantial evidence that in inflammatory processes, SN might play a role in recruitment of monocytes to inflamed tissue.

Secretoneurin (SN) is a 33-34 amino acid peptide derived from the most conserved sequence of the secretogranin (SgII) precursor. SgII is a granin protein found in the secretory granules of neuroendocrine tissues. There are two paralogs of teleost SgII that we name here SgIIa and SgIIb. Processing of these proteins would yield SNa and SNb in fish. Secretoneurin immunoreactivity is found within all the major pituitary cell types in mammals. In goldfish, it appears to be mainly expressed in the prolactin cells of the rostral pars distalis. We have investigated the paracrine role of goldfish SN (SNa) to stimulate luteinizing hormone from gonadotrophs in the neighboring proximal pars distalis. Another source of SN is the hypophysiotropic neurons that may deliver SN to target cells by direct pituitary innervation. Little else is known about the neuroendocrine role of SN. We also discuss the evolution, distribution and production of SN in the pituitary.

This review focus on neuroendocrine tumours (NETs), with special reference to the immunohistochemical analysis of granins and granin-related peptides and their usefulness in identifying and characterizing the great diversity of NET types. Granins, their derived peptides, and complex protein-processing enzyme systems that cleave granins and prohormones, have to some extent cell-specific expression patterns in normal and neoplastic NE cells. The marker most commonly used in routine histopathology to differentiate between non-NETs and NETs is chromogranin (Cg) A, to some extent CgB. Other members of the granin family may also be of diagnostic value by identifying special NET types, e.g. secretogranin (Sg) VI was only found in pancreatic NETs and phaeochromocytomas. SgIII has recently arisen as an important NET marker; it was strongly expressed in NETs, with some exceptions--phaeochromocytomas expressed few cells and parathyroid adenomas none. Some expression patterns of granin-related peptides seem valuable in differentiating between some benign and malignant NETs, some may also provide prognostic information, among which: well-differentiated NET types expressed more CgA epitopes than the poorly differentiated ones, except insulinomas, where the opposite was noted; medullary thyroid carcinomas containing few cells immunoreactive to a CgB antibody were related to a bad prognosis; C-terminal secretoneurin visualized a cell type related to malignancy in phaeochromocytomas. Further research will probably establish new staining patterns with marker functions for granins in NETs which may be of histopathological diagnostic value.

The sensory neuropeptides secretoneurin (SN) and substance P (SP) are involved in “neurogenic” inflammatory processes as they occur in bronchial asthma or allergic rhinitis. A possible interaction with basophils has not been reported to date. Basophils were isolated from healthy donors by magnetic cell sorting technique and migration was explored using Boyden microchemotaxis chambers. SN [10 − 8 M] and SP [10 − 6 to 10 − 8 M] proved to be chemoattractants equally potent to FMLP [10 − 8 M] or LPS [10 pg/ml]. Specific anti-SN antibodies and a trypsinization preparation of SN were used to determine the specificity of the SN effect on basophils. The preincubation of basophils with neurokinin-1 (NK-1) or -2 (NK-2) receptor antagonists revealed the SP effect to act via NK-1 receptors in basophils. In addition, we were able to show phosphodiesterases and phosphoinositide-3 kinases to be engaged in the downstream signalling pathway. Our observations reveal for the first time a link between basophils, which are engaged in allergic processes, and the neuropeptides SN and SP. Furthermore, our data might suggest phosphodiesterases or phosphoinositide-3 kinases to be new therapeutic targets for the treatment of allergic diseases such as asthma or allergic rhinitis.

Introduction Chromogranin (Cg) and secretogranin (Sg) are members of the granin family of proteins, which are expressed in neuroendocrine and nervous tissue. In recent publications we have presented generation of region-specific antibodies against CgA and CgB and also development of several region-specific radioimmunoassays for measurements of specific parts of the Cgs. In this study we describe generation of antibodies against SgII, SgIII, SgV and the proconvertases PC1/3 and PC2 and development of radioimmunoassays for measurements of these proteins. Materials and methods Peptides homologous to defined parts of the secretogranin and proconvertase molecules were selected and synthesised. Antibodies were raised, radioimmunoassays were developed and circulating levels of the proteins in plasma samples from 22 patients with neuroendocrine tumours were measured in the assays. Results Increased plasma concentrations were recorded in 11, 4 and 3 of the patients with the SgII 154–165 (N-terminal secretoneurin), the SgII 172–186 (C-terminal Secretoneurin) and the SgII 225–242 assays respectively. The SgIII, SgV, PC1/3 and PC2 assays failed to detect increased concentrations in any of the patients. Conclusion Increased concentrations of SgII, especially the N-terminal part of secretoneurin could be measured in plasma from patients with endocrine pancreatic tumours and in this case this assay was quite comparable to measurements of CgA and CgB. Even though secretoneurin was not as frequently increased as CgA and CgB in patients with carcinoid tumours or pheochromocytoma it may be a useful marker for endocrine pancreatic tumours.

The human vagal/nucleus solitary complex is a primary visceral relay station and an integrative brain stem area which displays a high density of chromogranin B- and secretoneurin-like immunoreactivity. In this study, we localized and biochemically identified these proteins during prenatal development. At prenatal week 11, 15, 20 and 37, we performed a chromatographic analysis to identify the molecular forms of PE-11, a peptide within the chromogranin B sequence, and secretoneurin, a peptide within secretogranin II. Their localization was studied with immunocytochemistry, and was compared to that of substance P which is well established as a functional neuropeptide in the vagal/nucleus solitary complex. At prenatal week 11, chromogranin B-, secretoneurin- and substance P-like immunoreactivities were detected consisting of varicosities, varicose fibers and single cells. At the same time, PE-11 and secretoneurin appeared as a single peak in chromatographic analysis. Prohormone convertases PC1- and PC2-like immunoreactivities were also present at week 11. In general, the density for each peptide increased during later fetal stages with the highest density at week 37. These results demonstrate that each chromogranin peptide is expressed during human fetal life in neurons of the vagal/nucleus solitary complex indicating that these peptides could be important during prenatal development.

Secretoneurin (SN), a newly discovered neuropeptide, may be implicated in inflammatory responses as it was shown to modulate leukocyte, endothelial and mesenchymal cell functions. Neutrophils placed above pulmonary arterial or venous endothelial monolayers migrated through this cellular barrier in response to apical or basal stimulation with SN in a dose-dependent manner. At optimal concentrations of 10 −6 to 10 −8 M, SN was nearly equally effective in stimulating neutrophil transmigration as was tumor necrosis factor-α at 10 ng/ml or a chemotactic gradient of formyl-Met-Leu-Phe (10 −8 M). Stimulation of transendothelial migration appears to be specific, since a trypsin digest of SN was ineffective and excess concentrations of anti-SN antibodies completely abolished the effect. Inhibition of cyclooxygenase or nitric oxide synthase did not affect the action of SN. Preincubation of endothelial cells with pertussistoxin (PTx) or choleratoxin (CTx), and the presence of staurosporine significantly inhibited transmigration, suggesting that SN uses a signalling pathway that is coupled to G-proteins and protein kinase C in endothelium. Moreover, SN treatment resulted in transient elevation of cytoplasmatic calcium concentration in endothelial cells. These data support the hypothesis that SN might contribute to neurogenic inflammation in vivo and reveal signalling mechanisms of SN in endothelial cells.

Chromogranins are neuropeptide precursors stored in large dense core vesicles in which they are processed to smaller peptides. Although these peptides are widespread in the CNS, it is still unknown if they are behaviourally active. For example, even though secretoneurin, a 33-amino acid peptide derived from secretogranin II, was shown to induce release of dopamine from rat striatal neurons, work on the functional significance of this result is still missing. In order to investigate the behavioural effects of chromogranin-derived peptides, we studied the total locomotor activity and rearing behaviour of male albino Sprague–Dawley rats in the open field experimental paradigm. Measurements were performed every 5 min during half an hour before and 2 h after an intracerebroventricular injection of GE-19, GAIPIRRH, secretoneurin or vehicle. None of the tested chromogranin-derived peptides (at a concentration of 20 μM) affected locomotion and rearing behaviour. However, the administration of secretoneurin and GAIPIRRH increased the thigmotaxis, suggesting a possible anxiolytic action. In male Swiss albino mice, which were tested in the black-and-white box paradigm, only GE-19 produced sedation at a dose of 0.72 nmol in 41% of the mice. Overall, there is only little evidence that any of the examined chromogranin-derived peptides produces a behaviourally significant effect, even when given intracerebroventricularly.

Secretoneurin (SN), a novel neuropeptide, specifically induces the selective chemotactic migration of monocytes in vitro and in vivo. Mediation of the migratory effects by yet unidentified receptors for secretoneurin is likely. Whether phagocytes other than monocytes are targets of SN's action, is currently unknown. Since previous data suggested that SN does not induce chemotaxis of neutrophils, we tested SN priming for respiratory burst activity, adherence to endothelial cell (EC) monolayers and effects on chemoattractant-stimulated migration of neutrophils. We found that priming of neutrophils with SN fails to enhance triggered respiratory burst activity or adherence to EC as compared to tumor necrosis factor-alpha (TNF), a well-known neutrophil-priming cytokine. Pretreatment, however, revealed an increase in spontaneous locomotion, and significant antagonism of formylpeptide (FMLP)-stimulated migration of neutrophils in modified Boyden chamber assays. The latter effect was similar to inhibition of FMLP-stimulated chemotaxis by TNF. The effects of SN on neutrophil migration could be abolished by an antiserum to SN, indicating its specificity. Data show that in vitro neutrophils are specifically affected by SN possibly by activation of a priming-type receptor. Since neutrophils represent a valuable tool for receptor research, further studies on SN receptors of leukocytes may be performed on these cells.