Impulsive choices in mice lacking imprinted Nesp55.

Genomic imprinting is the process whereby germline epigenetic events lead to parent-of-origin specific monallelic expression of a number of key mammalian genes. The imprinted gene Nesp is expressed from the maternal allele only and encodes for Nesp55 protein. In the brain, Nesp55 is found predominately in discrete areas of the hypothalamus and midbrain. Previously, we have shown that loss of Nesp55 gives rise to alterations in novelty-related behaviour. Here, we extend these findings and demonstrate, using the Nespm/+ mouse model, that loss of Nesp55 leads to impulsive choices as measured by a delayed-reinforcement task, whereby Nespm/+ mice were less willing to wait for a delayed, larger reward, preferring instead to choose an immediate, smaller reward. These effects were highly specific as performance in another component of impulsive behaviour, the ability to stop a response once started as assayed in the stop-signal reaction time task, was equivalent to controls. We also showed changes in the serotonin system, a key neurotransmitter pathway mediating impulsive behaviour. First, we demonstrated that Nesp55 is co-localized with serotonin and then went on to show that in midbrain regions there were reductions in mRNA expression of the serotonin-specific genes Tph2 and Slc6a4, but not the dopamine-specific gene Th in Nespm/+ mice; suggesting an altered serotonergic system could contribute, in part, to the changes in impulsive behaviour. These data provide a novel mode of action for genomic imprinting in the brain and may have implications for pathological conditions characterized by maladaptive response control.

First localization and biochemical identification of chromogranin B- and secretoneurin-like immunoreactivity in the fetal human vagal/nucleus solitary complex.

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 is released into human airways by topical histamine but not capsaicin.

BACKGROUND: The neuropeptide secretoneurin, with potential relevance to leukocyte trafficking, is present in nerves of the nasal mucosa in allergic rhinitis and may be released in response to allergen and histamine exposure. There is no information on the occurrence and mechanisms of release of secretoneurin in healthy human airways. METHODS: The presence of secretoneurin in nasal biopsies and its release in response to nasal capsaicin and histamine challenges were examined. Symptoms and lavage fluid levels of fucose were recorded as markers of effects in part produced by neural activity. Bronchial histamine challenges followed by sputum induction and analysis of secretoneurin were also carried out. RESULTS: Nerves displaying secretoneurin immunoreactivity abounded in the nasal mucosa. Nasal capsaicin challenge produced local pain (P <0.05) and increased the levels of fucose (P <0.05), but failed to affect the levels of secretoneurin. Nasal histamine challenge produced symptoms (P <0.05) and increased the mucosal output of secretoneurin (P <0.05) and fucose (P <0.05). Bronchial histamine challenge increased the sputum levels of secretoneurin (P <0.05). CONCLUSIONS: We conclude that secretoneurin is present in healthy human airways and that histamine evokes its release in both nasal and bronchial mucosae. The present observations support the possibility that secretoneurin is involved in histamine-dependent responses of the human airway mucosa.

NESP55, a novel chromogranin-like peptide, is expressed in endocrine tumours of the pancreas and adrenal medulla but not in ileal carcinoids.

Neuroendocrine secretory protein 55, NESP55, is an acidic protein belonging to the chromogranin family. The distribution of NESP55 in human tumours is not known. The aim of the present study was to study the expression of NESP55 in human gastrointestinal, pancreatic and adrenal tumours. A total of 118 human endocrine and nonendocrine tumours were examined by immunocytochemistry, and compared to the expression of chromogranin A (CgA) in the same tumours. Pancreatic endocrine tumours (14 out of 25), pheochromocytomas (19 out of 19), and neuroblastomas (seven out of 14) expressed NESP55, with the same strong labelling pattern in both benign and malignant tumours. Expression of NESP55 in pancreatic endocrine tumours and pheochromocytomas was confirmed by Western and Northern blot analysis. Immunocytochemical analysis demonstrated no labelling in ileal carcinoids (zero out of 15), and adrenocortical adenomas (zero out of 15). The majority of gastrointestinal and pancreatic carcinomas were negative for NESP55, with focal staining observed in two out of 30 tumours. In contrast, CgA was present in all neuroendocrine tumours examined (25 out of 25 pancreatic endocrine tumours, 19 out of 19 pheochromocytomas, 14 out of 14 neuroblastomas and 15 out of 15 ileal carcinoids). Thus, the expression of NESP55 in endocrine tumours of the gastrointestinal tract, pancreas and adrenals differs from that of CgA. Neuroendocrine secretory protein 55 is found in a subset of neuroendocrine tumours showing differentiation towards adrenal chromaffin cells and pancreatic islets cells.

Secretoneurin promotes pertussis toxin-sensitive neurite outgrowth in cerebellar granule cells.

The neuropeptide secretoneurin (SN) is an endoproteolytic product of the chromogranin secretogranin II. We investigated the effects of SN on the differentiation of immature cerebellar granule cells derived from the external granular layer (EGL). Secretoneurin caused concentration-dependent increases in neurite outgrowth, reflecting differentiation. The maximum effect was reached at a concentration of 100 nm SN. Secretoneurin immunoneutralization using specific antiserum significantly decreased neurite outgrowth; however, neurite morphology was altered. An affinity chromatography-purified antibody significantly inhibited the outgrowth response to SN (p < 0.001) without altering the morphology. Binding studies suggest the existence of specific G-protein-coupled receptors on the surface of monocytes that recognize SN. Assuming that SN promotes neurite outgrowth in EGL cells by acting through a similar G-protein-coupled mechanism, we treated SN-stimulated EGL cultures with pertussis toxin. Exposure to pertussis toxin (0.1 micro g/mL) showed a significant inhibition of the SN-induced outgrowth. To establish a second messenger pathway we used the protein kinase C inhibitor staurosporine. We found that EGL cell viability was not enhanced following chronic SN treatment for 24 h. These data indicate that SN is a novel trophic substance that can affect cerebellar maturation, primarily by accelerating granule cell differentiation through a signalling mechanism that is coupled to pertussis toxin-sensitive G-proteins.

Distribution and intraneuronal trafficking of a novel member of the chromogranin family, NESP55, in the rat peripheral nervous system.

NESP55 (neuroendocrine secretory protein of M(r) 55000) is a novel member of the chromogranin family. In the present study, we have investigated the distribution, axonal transport and proteolytic processing of NESP55 in the peripheral nervous system. The amount of NESP55 immunoreactivity in adrenal gland was more than 240 times higher than that in the vas deferens. Double or triple immunostaining demonstrated that NESP55 immunoreactivity was highly co-localized with tyrosine hydroxylase immunoreactivity in bundles of thin axons and postganglionic sympathetic neurons; that NESP55 immunoreactivity also co-existed with vesicular acetylcholine transporter immunoreactivity in large-sized axons in sciatic nerves, and that NESP55 immunoreactivity overlapped with calcitonin gene-related peptide immunoreactivity in some large-sized axons, but NESP55 immunoreactivity was not detected in sensory neurons. Strong NESP55 immunoreactivity was found in cell bodies and axons, but it was not detectable in any terminal region by immunohistochemistry. In crush-operated sciatic nerves, NESP55 immunoreactivity could be found as early as 1 h after operation, and accumulated amounts increased substantially with time. However, NESP55 immunoreactivity was only observed in axons proximal to the crush, but none or very little distal to the crush, which was consistent with the data from radioimmunoassay. Finally, extracts of the normal and crushed sciatic nerve and vas deferens were subjected to high-performance liquid chromatography followed by radioimmunoassay. The results indicate that NESP55 is processed slowly to small peptides (GAIPIRRH) during axonal transport. NESP55 immunoreactivity was only detected in axons proximal to the crush. The data in the present study indicate that NESP55 immunoreactivity is widely distributed in adrenergic, cholinergic, and peptidergic neurons, but not in sensory neurons, and that this peptide is anterogradely, but not retrogradely, transported with fast axonal transport and slowly processed to smaller peptides during axonal transport in the peripheral nervous system.

Chromogranins as markers of altered hippocampal circuitry in temporal lobe epilepsy.

Chromogranins are polypeptides which are widely expressed in the central nervous system. They are stored in dense core vesicles of nerve terminals, from where they are released upon stimulation. Using immunocytochemistry, we investigated the distribution of chromogranin A, chromogranin B, secretoneurin, and, for comparison, dynorphin in hippocampal specimens removed at routine surgery from patients with drug-resistant mesial temporal lobe epilepsy and in autopsy tissues from nonneurologically deceased subjects. In post mortem controls (n = 21), immunoreactivity for all 4 peptides (most prominently for chromogranin B and dynorphin) was observed in the terminal field of mossy fibers. For chromogranins, staining was observed also in sectors CA1 to CA3 and in the subiculum. Chromogranin B immunoreactivity was found in the inner molecular layer of the dentate gyrus, the area of terminating associational-commissural fibers. Secretoneurin and dynorphin immunoreactivity labeled the outer molecular layer and the stratum lacunosum moleculare of sectors CA1 to CA3, where projections from the entorhinal cortex terminate. In specimens with Ammon's horn sclerosis (n = 25), staining for all 3 chromogranins and for dynorphin was reduced in the hilus of the dentate gyrus. Instead, intense staining was observed in the inner molecular layer, presumably delineating terminals of sprouted mossy fibers. Specimens obtained from temporal lobe epilepsy patients without Ammon's horn sclerosis (n = 4) lacked this pronounced rearrangement of mossy fibers. In the stratum lacunosum moleculare of sector CA1, secretoneurin and dynorphin immunoreactivity was reduced in sclerotic, but not in nonsclerotic, specimens, paralleling the partial loss of fibers arising from the entorhinal cortex. Instead, presumably sprouted secretoneurin-immunoreactive fibers were found in the outer dentate molecular layer in sclerotic specimens. These changes in staining patterns for chromogranins and dynorphin mark profound plastic and functional rearrangement of hippocampal circuitry in temporal lobe epilepsy.

Differential regulation of chromogranin A, chromogranin B and secretogranin II in rat brain by phencyclidine treatment.

Chromogranin A, chromogranin B and secretogranin II belong to the chromogranin family which consists of large protein molecules that are found in large dense core vesicles. Chromogranins are endoproteolytically processed to smaller peptides. This study was designed to elucidate the regulation of chromgranin expression by acute and subchronic phencyclidine administration. The behavioral syndrome produced by phencyclidine represents a pharmacological model for some aspects of schizophrenia [Jentsch and Roth (1999) Neuropsychopharmacology 20, 201-225]. Tissue concentrations of chromogranins were measured with specific radioimmunoassays. Alterations in secretogranin II gene expression were investigated by in situ hybridization. A single dose of phencyclidine (10mg/kg) led to a transient decrease in secretoneurin tissue levels in the prefrontal cortex after 4h followed by an increase in secretoneurin tissue levels after 12h. Repeated phencyclidine treatment (10mg/kg/day) for five days resulted in elevated secretoneurin levels in cortical areas whereas chromogranin A and chromogranin B tissue levels were unchanged. After the same treatment, a significant increase in the number of secretoneurin containing neurons was found in cortical layers II-III, and V-VI as revealed by immunocytochemistry. The increases in secretoneurin levels were paralleled by an increased number of secretogranin II messenger RNA containing neurons as well as by an increased expression of secretogranin II by individual neurons. The present study shows that secretoneurin II tissue concentration and secretogranin II messenger RNA expression is distinctly altered after acute and subchronic phencyclidine application. From these results we suggest that phencyclidine may induce synaptic alterations in specific brain areas and may contribute to a better understanding of synaptic dysfunction which may also occur in schizophrenia.

Neuroendocrine secretory protein 55 (NESP55): alternative splicing onto transcripts of the GNAS gene and posttranslational processing of a maternally expressed protein.

Recent studies established a novel genomically imprinted gene located 45 kb upstream of the human GNAS1 locus. This locus encoded for the Neuroendocrine Secretory Protein with an apparent molecular weight of 55,000 (NESP55), which is transcribed exclusively from the maternal allele. We sequenced rat and human NESP55 and investigated tissue-specific splicing of its mRNA and posttranslational modifications of the protein in various tissues. Alternative mRNA splicing of NESP55 was analyzed by sequencing of cDNA clones, RT-PCR and Northern blotting. Two main splice variants, which were generated in a tissue-specific manner, were identified: The open reading frame encoding NESP55 was spliced onto exons 2-13 of Gsalpha in the adrenal medulla, pituitary and the brain. In addition, in the pituitary a second shorter, prominent mRNA transcript was generated by splicing of NESP55 onto exons 2, 3 and N1 of Gsalpha. Several of the cDNA clones isolated contained inverted repeats of 50-150 bp at their 5' or 3' termini, which might form hairpin stems and thus alter mRNA stability. The NESP55 open reading frame encoded a hydrophilic protein of 28,018 Da (human) and 29,218 Da (rat), respectively, which resembled the class of acidic, neuroendocrine secretory proteins collectively called chromogranins. NESP55 is highly conserved among mammalian species. It is posttranslationally acidified by the addition of keratan sulfate glycosaminoglycan chains and differentially processed by endopeptidases in various endocrine and neuronal tissues.

Elevated levels of serum secretoneurin in patients with therapy resistant carcinoma of the prostate.

PURPOSE: The majority of prostate cancers show some degree of neuroendocrine differentiation. It was previously demonstrated that chromogranin A, a constituent of large dense core vesicles of neuroendocrine cells, is frequently elevated in patients with metastatic prostate cancer. We evaluate the expression of secretoneurin, which is generated by proteolytic processing of secretogranin II (chromogranin C), in patients with prostate disease. MATERIALS AND METHODS: Secretoneurin was measured in sera of 16 healthy men whose blood was drawn for prostate cancer screening (controls), and in 9 patients with prostatitis, 19 with benign prostate hyperplasia and 54 with prostate cancer detected by radioimmunoassay. Therapy resistant disease (clinical stage D3) was noted in 20 prostate cancer cases. Serum prostate specific antigen was measured in all patients and controls. In addition, chromogranin A, prostate acid phosphatase and interleukin-6 were determined in patients with D3 prostate cancer. Molecular properties of secretoneurin immunoreactivity were analyzed by gel filtration chromatography followed by radioimmunoassay. RESULTS: Mean secretoneurin was 58.9+/-8 fmol./ml. in patients with therapy resistant prostate cancer. Levels were significantly higher than those measured in sera from controls and patients with prostatitis, benign prostatic hyperplasia and pT2 or pT3 prostate cancer. There was a statistically significant correlation between secretoneurin and chromogranin A in patients with endocrine therapy failure (r = 0.543, p<0.05). There was no correlation between serum secretoneurin and prostate specific antigen, prostate acid phosphatase or interleukin-6. Gel filtration chromatography analysis of sera of 3 patients with D3 prostate cancer revealed a peak of secretoneurin immunoreactivity where intact secretoneurin elutes, thus showing that the processed peptide is circulating in the blood. CONCLUSIONS: Secretoneurin is elevated in sera of patients with endocrine therapy refractory prostate cancer. Our results support the concept that neuroendocrine differentiation is associated with prostate cancer progression.

Distribution and origin of secretoneurin-immunoreactive nerves in the female rat uterus.

Secretoneurin is a 33-amino acid peptide derived from secretogranin II. Secretoneurin immunoreactivity has been localized in the peripheral nervous system where it exerts potent chemotactic activity for monocytes and may play a role in inflammation. Secretoneurin could play a role in this process, although the presence and distribution of secretoneurin-immunoreactive neurons in the female reproductive system has not been documented. Thus, this study was undertaken to examine secretoneurin immunoreactivity in nerves of the rat uterus and uterine cervix. A moderate plexus of secretoneurin-immunoreactive nerve fibers was present in the myometrium and endometrium of the uterus as well as in the smooth muscle and endocervix of the cervix. Many of these fibers were associated with the vasculature as well as the myometrium. Secretoneurin immunoreactivity was present in small- to medium-sized neurons of dorsal root and nodose ganglia. Retrograde tracing with FluoroGold indicated that some of these sensory neurons project axons to the cervix and uterine horns. Secretoneurin-immunoreactive terminal-like structures were associated with neurons in the sacral parasympathetic nucleus of the lumbosacral spinal cord. In addition, some secretoneurin terminals were apposed to pelvic parasympathetic neurons in the paracervical ganglia that projected axons to the uterus and cervix. Double-immunostaining indicated co-existence of calcitonin gene-related peptide or substance P with secretoneurin in some sensory neurons, in some terminals of the pelvic ganglia, as well as nerve fibers in the uterine horn and cervix. Finally, fibers in the uterus and cervix were depleted of secretoneurin by capsaicin treatment. This study indicates that secretoneurin is present in the uterus in C-afferent nerve fibers whose cell bodies are located in sensory ganglia. Some of these fibers contain both secretoneurin and calcitonin gene-related peptide or substance P. These substances have functions in inflammatory reactions. Further, secretoneurin could influence postganglionic parasympathetic "uterine-related" neurons in the pelvic ganglia and preganglionic parasympathetic neurons in the lumbosacral spinal cord.

Levels and molecular properties of secretoneurin-immunoreactivity in the serum and urine of control and neuroendocrine tumor patients.

We have determined the levels of secretoneurin (SN), a novel 33-amino acid neuropeptide belonging to the class of chromogranins, in the serum and urine of healthy subjects and patients suffering from various tumors. SN serum levels averaged 22.1+/-1.1 fmol/mL. They were 5-fold higher in younger children and then declined continuously. SN levels were positively correlated with serum creatinine, suggesting an influence of renal function on the clearance of SN from the serum. In the urine 80.0 fmol/mL SN was present. In patients with endocrine tumors like gut carcinoids, endocrine pancreatic tumors, oat cell lung carcinomas, and pheochromocytomas, SN serum levels were elevated up to 45-fold. Patients suffering from neuroblastomas, insulinomas, pituitary adenomas including acromegaly, and solid nonendocrine tumors had concentrations in the normal range. In human serum, SN-immunoreactivity was confined to the free peptide SN; neither larger intermediate-sized forms nor the precursor secretogranin II were detected. An efficient removal of the small molecule SN from the serum by the kidney explains why SN serum levels are lower when compared to chromogranin A, which is present as large molecule in serum.

Localization of neuroendocrine secretory protein 55 messenger RNA in the rat brain.

Neuroendocrine secretory protein 55 (NESP55) is a recently characterized secretory protein localized to large dense-core vesicles resembling the class of chromogranins. We investigated the distribution of the messenger RNA encoding for NESP55 in the rat brain by in situ hybridization with specific 35S-labelled oligonucleotides. NESP55 messenger RNA was detected only on neuronal but not glial cells. In the brain, expression of NESP55 messenger RNA was most prominent in several areas throughout the midbrain and brainstem, including the locus coeruleus, the raphe complex and the reticular formation. NESP55 messenger RNA-expressing cells were also found in many areas and nuclei throughout the hypothalamus. Neocortical areas, the hippocampus and the cerebellum were devoid of NESP55 messenger RNA-containing neurons. From this distribution pattern, a significant overlap of NESP55 expression with the noradrenergic, adrenergic and serotonergic transmitter systems was evident. The present study defines, for the first time, the cellular localizaton of NESP55 messenger RNA in the rat brain. The present results provide the basis for future studies defining the as yet obscure function of NESP55.

Relative amounts and molecular forms of NESP55 in various bovine tissues.

NESP55 (neuroendocrine secretory protein with Mr 55,000) comprises a novel chromogranin-like protein, which is paternally imprinted at the genomic level. We used antisera raised against GAIPIRRH, a peptide present at the C-terminus of this protein, and against TC-14, a peptide located in the N-terminal half of NESP55. Radioimmunoassay, gel-filtration chromatography and immunoblotting were used to determine the levels and the molecular forms of NESP55 in different bovine organs. The tissues with the highest levels of GAIPIRRH immunoreactivity were, in decreasing order: the adrenal medulla, the anterior pituitary, the posterior pituitary, various brain regions, and the intestine. The degree of proteolytic processing revealed differences among the tissues analyzed. The lowest processing was detected in the anterior pituitary and in the brain where only a peak corresponding to the intact precursor was present. This was also true for cerebrospinal fluid (CSF). In the posterior pituitary and in the intestine, the free peptide GAIPIRRH was the predominant molecular form. GAIPIRRH-IR, as in the CSF, is present in serum mainly as an intact precursor. A relatively high concentration of GAIPIRRH-IR was found in the kidney medulla, probably due to an endocytotic re-uptake of this molecule from the tubuli after filtration in the glomeruli. The present study is consistent with the concept that NESP55, like the other chromogranins, becomes proteolytically processed. The function of this new chromogranin-like protein, therefore, might be to represent a precursor of biologically active peptides.

The new chromogranin-like protein NESP55 is preferentially localized in adrenaline-synthesizing cells of the bovine and rat adrenal medulla.

The protein NESP55, a new member of the chromogranin family, is present in large dense-core secretory granules of neuroendocrine tissues. We investigated its cellular distribution in adrenal medulla with immunohistochemistry and in situ hybridization. A preferential co-localization of NESP55 with phenylethanolamine-N-methyltransferase in the adrenergic cell population was found by immunolabelling of consecutive sections. Noradrenergic cells also contained small amounts of NESP55, but the levels as measured by radioimmunoassay were five times lower. The distribution of NESP55 mRNA was similar to preproenkephalin mRNA which previously was shown to be confined to adrenaline-producing cells of the adrenal medulla. The present study indicates that stimulation of adrenergic cells will release significantly higher amounts of NESP55. The functional implications of this preferential secretion, however, have yet to be discovered.

A soluble gradient of the neuropeptide secretoneurin promotes the transendothelial migration of monocytes in vitro.

Secretoneurin, derived from the chromogranin secretogranin II, triggers the selective migration of human monocytes, eosinophils, fibroblasts, endothelial and smooth muscle cells. More recently, we located specific binding sites on the human monocytic cell line MonoMac-6. Differentiated U937 transendothelial diapedesis was evaluated using an in vitro model of the vascular wall and specific monoclonal antibodies against CD11/CD18 and the alpha-chains of the very late activation antigen (VLA)-4 were used to evaluate involved adhesion molecules. Results showed a significant migratory response to secretoneurin between 10(-8) to 10(-10) M. Migration was comparable to a maximal effect induced by the monocyte chemotactic agent N-formyl-Met-Leu-Phe (fMLP, 10(-8) M). Rabbit anti-secretoneurin antibodies were able to block the neuropeptide effect but not of fMLP and a trypsinized secretoneurin preparation as well as the secretogranin II-fragment EL-17 were ineffective in eliciting migration. Transmigration of U937 across endothelial-layers toward secretoneurin is inhibited by antibodies to CD11/CD18 adhesion molecules. The novel neuropeptide secretoneurin may play a role in regulating migration of monocytes into the subendothelial space, supposing a role in inflammatory responses.

Levels and molecular forms of chromogranins in human childhood neuroblastomas and ganglioneuromas.

The chromogranins are a class of acidic proteins found in large secretory granules of neuroendocrine tissues and tumors derived from them. We measured the relative amounts and characterized the molecular forms of two members of this family, i.e. chromogranin A and secretogranin II, in 14 neuroblastomas and five ganglioneuromas. In all the tumors investigated significant amounts of chromogranin A and secretogranin II were found. Neuroblastomas contained two times and ganglioneuromas 45 times more secretogranin II compared to chromogranin A. Both proteins were processed in these tumors to a great extent to smaller peptides, only limited amounts of intact chromogranin A or secretogranin II were present. In general, proteolytic processing of secretogranin II to the small neuropeptide secretoneurin was more complete than that of chromogranin A to the peptide GE-25. Proteolytic processing of both chromogranins as well as the total amounts of these proteins were unrelated to tumor staging.

Presence of chromogranins and regulation of their synthesis and processing in a neuroendocrine prostate tumor cell line.

BACKGROUND: Small-cell carcinoma and carcinoid tumors of the prostate display a neuroendocrine phenotype. To some extent, adenocarcinomas of the prostate also express neuroendocrine properties. Prostatic neuroendocrine tumors do not respond to androgen ablation therapy. The regulation of synthesis of chromogranins and their processing into neuropeptides have not yet been studied in neuroendocrine cells of the prostate. We used CRL-5813 cells which were derived from a metastasis from small-cell prostate cancer for studies on steroid receptor expression and chromogranin processing. METHODS: The expression of steroid receptor mRNA in CRL-5813 cells was examined by polymerase chain reaction. The synthesis and secretion of chromogranin- and secretogranin II-derived peptides were investigated by radioimmunoassays and high-performance liquid chromatography in untreated cells and in cells treated with the protein kinase A activator forskolin or basic fibroblast growth factor (bFGF). RESULTS: cDNA fragments for alpha-estrogen receptor and androgen receptor but not for beta-estrogen receptor, progesterone receptor, and glucocorticoid receptor were amplified from CRL-5813 cells. These cells were found to contain typical markers of large dense-core vesicles, i.e., chromogranins A and B and secretogranin II. Forskolin significantly stimulated the synthesis and secretion of the chromogranin B-derived peptide PE-11 and the secretogranin II-derived secretoneurin. bFGF significantly induced PE-11 protein levels in cell extracts. CONCLUSIONS. Our results demonstrate the expression of typical large dense-core vesicle proteins, i.e., chromogranins, in a small-cell prostate cancer cell line and their upregulation by a protein kinase A activator and, in part, by bFGF.

Formation and sequence analysis of secretoneurin, a neuropeptide derived from secretogranin II, in mammalian, bird, reptile, amphibian and fish brains.

Secretoneurin is a recently-characterized neuropeptide derived from secretogranin II, a protein belonging to the class of chromogranins. We investigated the phylogeny of this peptide by immunoblotting and gel-filtration high performance liquid chromatography followed by radioimmunoassay of brain extracts of various species including chicken, lizard, frog and fish. In addition the amino acid sequence of secretoneurin from pig, hamster, rabbit, guinea-pig and chicken was established by reverse transcriptase polymerase chain reaction. Secretoneurin is strongly conserved during evolution, it is not only expressed in various mammalian species but found also in the brain of birds, reptiles, amphibians and fish. In all these species a significant or near complete processing of secretogranin II to secretoneurin was observed. These data provide significant evidence for the neuropeptide nature of the novel functional peptide.