Neuroendocrine Secretory Protein Research Articles

Peripheral projections of NESP55 containing neurons in the rat sympathetic ganglia

The peripheral projections of neurons expressing neuroendocrine secretory protein 55 (NESP55), a novel member of the chromogranin family, were studied by retrograde tracing technique. It was found that NESP55 positive neurons in the rat superior cervical ganglion projected to a number of targets including the submandibular gland, the cervical lymph nodes, the forehead skin, the iris, but not to the thyroid. Among these NESP55 positive target-projecting neurons, a subpopulation contained neuropeptide Y (NPY), a vasoconstrictor. Forepaw pad projecting neurons were found exclusively in the stellate ganglion, almost all of which (approximately 90%) were immunoreactive to NESP55. Colocalization of NESP55 and calcitonin gene-related peptide (CGRP), a peptide involved in sudomotor effects, was observed in a subpopulation of these paw pad projecting neurons, as was colocalization of NESP55 and NPY. The data suggest that NESP55 may have a functional role in some populations of sympathetic neurons.

Physiological manipulation of cellular activity tunes protein and ultrastructural profiles in a neuroendocrine cell

To study in vivo the dynamics of the biosynthetic and secretory processes in a neuroendocrine cell, we use the proopiomelanocortin-producing intermediate pituitary melanotrope cells of Xenopus laevis. The activity of these cells can be simply manipulated by adapting the animal to a white or a black background, resulting in inactive and hyperactive cells respectively. Here, we applied differential display proteomics and field emission scanning electron microscopy (FESEM) to examine the changes in architecture accompanying the gradual transition of the inactive to the hyperactive melanotrope cells. The proteomic analysis showed differential expression of neuroendocrine secretory proteins, endoplasmic reticulum (ER)-resident chaperones, and housekeeping and metabolic proteins. The FESEM study revealed changes in the ultrastructure of the ER and Golgi and the number of secretory granules. We conclude that activation of neuroendocrine cells tunes their molecular machineries and organelles to become professional secretors.

Sorting of the Neuroendocrine Secretory Protein Secretogranin II into the Regulated Secretory Pathway: ROLE OF N- AND C-TERMINAL α-HELICAL DOMAINS

Secretogranin II (SgII) belongs to the granin family of prohormones widely distributed in dense-core secretory granules (DCGs) of endocrine, neuroendocrine, and neuronal cells, including sympathoadrenal chromaffin cells. The mechanisms by which secretory proteins, and granins in particular, are sorted into the regulated secretory pathway are unsettled. We designed a strategy based on novel chimeric forms of human SgII fused to fluorescent (green fluorescent protein) or chemiluminescent (embryonic alkaline phosphatase) reporters to identify trafficking determinants mediating DCG targeting of SgII in sympathoadrenal cells. Three-dimensional deconvolution fluorescence microscopy and secretagogue-stimulated release studies demonstrate that SgII chimeras are correctly targeted to DCGs and released by exocytosis in PC12 and primary chromaffin cells. Results from a Golgi-retained mutant form of SgII suggest that sorting of SgII into DCGs depends on a saturable sorting machinery at the trans-Golgi/trans-Golgi network. Truncation analyses reveal the presence of DCG-targeting signals within both the N- and C-terminal regions of SgII, with the putative alpha-helix-containing SgII-(25-41) and SgII-(334-348) acting as sufficient, independent sorting domains. This study defines sequence features of SgII mediating vesicular targeting in sympathoadrenal cells and suggests a mechanism by which discrete domains of the molecule function in sorting, perhaps by virtue of a particular arrangement in tertiary structure and/or interaction with a specific component of the DCG membrane.

Chromogranin A, a Member of Neuroendocrine Secretory Proteins as a Selective Marker for Laboratory Diagnosis of Pheochromocytoma.

The function of chromogranin A (CGA) is reviewed, and the radioimmunometric determination of plasma CGA was evaluated as a marker of pheochromocytoma using a comparison of pheochromocytoma patients immediately before surgery (group P, n=25, 635+/-451 ng/ml) with other groups of patients, i.e. pheochromocytoma patients approximately 1 year after removal of tumor (group PP, n=13, 69+/-33 ng/ml), medullary thyroid carcinoma patients (group M, n= 22, 106+/-59 ng/ml), congenital adrenal hyperplasy patients (n=33, 65+/-40 ng/ml), and controls (n=31, 66+/-29 ng/ml). A CGA level above cut off value 130 ng/ml was found in 24 of 25 patients in group P, 1 (relapse) of 13 patients in group PP, and 4 of 22 patients in group M. In the group P we found a significant association between the size of the tumors removed and plasma CGA concentrations (p=0.0016), and also a significant (p=0.0016) relationship between plasma CGA concentrations and PASS score rating the malignity of pheochromocytoma. We can conclude that plasma CGA concentration as determined by radioimmunometric assay (which is simple without the necessity of special laboratory equipment) is an effective marker of pheochromocytoma with association to malignity and tumor mass.

Neuroendocrine secretory protein 55 (NESP55) in the spinal cord of rat: An immunocytochemical study

The immunohistochemical expression of a novel chromogranin-like protein, neuroendocrine secretory protein 55 (NESP55), in the rat spinal cord was investigated. NESP55-immunoreactive cells were detected in the ventral horn, intermediate laminae, and deep dorsal horn, comprising motoneurons, autonomic neurons, and interneurons throughout all spinal segments. Within laminae I-II of the dorsal horn, one or two NESP55-positive cells were often seen. Nerve fibers also contained NESP55 immunoreactivity (IR) and were particularly prominent in the ventral horn. No nerve terminals/varicosities appeared to contain NESP55 in any spinal lamina. Double-staining experiments revealed that a high proportion of the NESP55-positive neurons were cholinergic. Moreover, NESP55-IR in the motoneurons was evenly distributed in the whole cytoplasm with a finely granular appearance. In contrast, the fluorescent material in the preganglionic neurons was concentrated in the perinuclear region and largely overlapped with the trans-Golgi network marker TGN38. Our data provide detailed morphological information on the distribution of NESP55-IR in the rat spinal cord. Also, the differential intracellular expression of NESP55-IR in the spinal motoneurons and autonomic neurons suggests that NESP55 may be processed into different secretory granules and may be involved in both constitutive and regulated pathways in these neurons.

The GNAS Locus: Quintessential Complex Gene Encoding Gsα, XLαs, and other Imprinted Transcripts

The currently estimated number of genes in the human genome is much smaller than previously predicted. As an explanation for this disparity, most individual genes have multiple transcriptional units that represent a variety of biologically important gene products. GNAS exemplifies a gene of such complexity. One of its products is the α-subunit of the stimulatory heterotrimeric G protein (Gsα), a ubiquitous signaling protein essential for numerous different cellular responses. Loss-of-function and gain-of-function mutations within Gsα-coding GNAS exons are found in various human disorders, including Albright’s hereditary osteodystrophy, pseudohypoparathyroidism, fibrous dysplasia of bone, and some tumors of different origin. While Gsα expression in most tissues is biallelic, paternal Gsα expression is silenced in a small number of tissues, playing an important role in the development of phenotypes associated with GNAS mutations. Additional products derived exclusively from the paternal GNAS allele include XLαs, a protein partially identical to Gsα, and two non-coding RNA molecules, the A/B transcript and the antisense transcript. The maternal GNAS allele leads to NESP55, a chromogranin-like neuroendocrine secretory protein. In vivo animal models have demonstrated the importance of each of the exclusively imprinted GNAS products in normal mammalian physiology. However, although one or more of these products are also disrupted by most naturally occurring GNAS mutations, their roles in disease pathogenesis remain unknown. To further our understanding of the significance of this gene in physiology and pathophysiology, it will be important to elucidate the cellular roles and the mechanisms regulating the expression of each GNAS product.

Exon expression profiling reveals stimulus-mediated exon use in neural cells

Exon centric microarrays were used to resolve the calcium-modulated gene expression response into transcript-level an exon-level regulation.

Neuroendocrine secretory protein 55 (NESP55) immunoreactivity in male and female rat superior cervical ganglion and other sympathetic ganglia

Neuroendocrine secretory protein 55 (NESP55) is a soluble, acidic and heat-stable protein, belonging to the class of chromogranins. It is expressed specifically in endocrine cells and the nervous system, and is probably involved in both constitutive and regulated secretion. In the present study, we investigated the distribution of NESP55 in various rat sympathetic ganglia by immunohistochemistry . The expression of NESP55-IR was detected in a subpopulation of principal neurons in the rat SCG, which was also TH positive, and, thus, adrenergic. In the rat stellate ganglion, more than two thirds of NESP55 positive neurons were adrenergic. Colocalization of NESP55 and calcitonin gene-related peptide (CGRP) in cholinergic neurons was also observed. In the rat thoracic chain, however, the majority of NESP55 positive neurons appeared to lack TH. No detectable NESP55-IR was found in the mouse SCG. Furthermore, in the sexually dimorphic SCG, it was demonstrated that, 80% of the NESP55 positive principal neurons were also NPY positive in the male rat, while a slightly higher, but statistically significant proportion, 87%, was found in the female. Whether or not this small difference is physiologically significant is unknown. The present data provide basic knowledge about the expression of NESP55 in the sympathetic autonomic nervous system of rat, which may further our understanding of the functional significance of NESP55.

Differential Expression and Processing of Chromogranin A and Secretogranin II in Relation to the Secretory Status of Endocrine Cells

Chromogranin A (CgA) and secretogranin II (SgII) are neuroendocrine secretory proteins that participate in regulation of the secretory pathway and also serve as precursors of biologically active peptides. To investigate whether there is a relationship between the expression, distribution, and processing of CgA and SgII and the degree of secretory activity, we employed two melanotrope subpopulations of the pituitary intermediate lobe that exhibit opposite secretory phenotypes. Thus, although one of the melanotrope subtypes shows high secretory activity, the other exhibits characteristics of a hormone storage phenotype. Our data show that SgII expression levels were higher in secretory melanotropes, whereas CgA expression showed similar rates in both cell subsets. The use of various antibodies revealed the presence of the unprocessed proteins as well as three CgA-derived peptides (67, 45, and 30 kDa) and six SgII-derived peptides (81, 66, 55, 37, 32, and 30 kDa) in both subpopulations. However, the smallest molecular forms of both granins predominated in secretory melanotropes, whereas the largest SgII- and CgA-immunoreactive peptides were more abundant in storage melanotropes, which is suggestive of a more extensive processing of granins in the secretory subset. Confocal microscopy studies showed that CgA immunoreactivity was higher in storage cells, but SgII immunoreactivity was higher in secretory melanotropes. Taken together, our results indicate that SgII and CgA are differentially regulated in melanotrope subpopulations. Thus, SgII expression is strongly related to the secretory activity of melanotrope cells, whereas CgA expression may not be related to secretory rate, but, rather, to hormone storage in this endocrine cell type.

Neuroendocrine tumors of the gastrointestinal tract: recent advances in molecular genetics, diagnosis, and treatment

Neuroendocrine tumors of the gastrointestinal tract represent a small area within oncology, but many new data have been reported during the past year. This paper updates the recent findings. An N-terminally truncated variant of heat shock protein 70 (Hsp70) has been identified in neuroendocrine tumors but not in normal pancreatic islets. CDX-2 is a homeobox gene product essential for intestinal development and differentiation that is expressed at high levels in intestinal neuroendocrine tumors, but not in other types. A new marker has been identified, neuroendocrine secretory protein-55, a member of the chromogranin family, specific for endocrine pancreatic tumors but not for intestinal neuroendocrine tumors. Positron emission tomography has significantly improved imaging of neuroendocrine tumors of the gastroenteropancreatic tract. Tracers such as C-5-hydroxy-L-tryptophan show very high sensitivity for detection of tumors, higher than for somatostatin receptor scintigraphy. A new somatostatin analogue SOM230 binding to four of five somatostatin receptors has recently come into clinical trials. Increased knowledge of the molecular background for the development of neuroendocrine tumors may improve the management of these tumors in the future. New tumor markers have been developed and the localization techniques have been significantly improved. That will of course lead to earlier diagnosis and improved possibilities for surgical cure of these patients.

Imprinted Nesp55 influences behavioral reactivity to novel environments.

Genomic imprinting results in parent-of-origin-dependent monoallelic expression of selected genes. Although their importance in development and physiology is recognized, few imprinted genes have been investigated for their effects on brain function. Gnas is a complex imprinted locus whose gene products are involved in early postnatal adaptations and neuroendocrine functions. Gnas encodes the stimulatory G-protein subunit Gsalpha and two other imprinted protein-coding transcripts. Of these, the Nesp transcript, expressed exclusively from the maternal allele, codes for neuroendocrine secretory protein 55 (Nesp55), a chromogranin-like polypeptide associated with the constitutive secretory pathway but with an unknown function. Nesp is expressed in restricted brain nuclei, suggesting an involvement in specific behaviors. We have generated a knockout of Nesp55 in mice. Nesp55-deficient mice develop normally, excluding a role of this protein in the severe postnatal effects associated with imprinting of the Gnas cluster. Behavioral analysis of adult Nesp55 mutants revealed, in three separate tasks, abnormal reactivity to novel environments independent of general locomotor activity and anxiety. This phenotype may be related to prominent Nesp55 expression in the noradrenergic locus coeruleus. These results indicate a role of maternally expressed Nesp55 in controlling exploratory behavior and are the first demonstration that imprinted genes affect such a fundamental behavior.

Neuroendocrine secretory protein-55 (NESP-55) expression discriminates pancreatic endocrine tumors and pheochromocytomas from gastrointestinal and pulmonary carcinoids.

Neuroendocrine secretory protein-55 (NESP-55), the latest addition to the chromogranin family, is a product of a genomically imprinted gene transcribed exclusively from the maternal allele. Initial studies have shown it to have a less widespread distribution than that of chromogranin A in normal tissues. It has also been suggested that NESP-55 may be a marker of neuroendocrine tumors differentiating toward the adrenal chromaffin and pancreatic islet cells. Metastatic gastrointestinal and pulmonary carcinoids may occasionally be difficult to distinguish from pancreatic endocrine tumors (PETs) and pheochromocytomas on morphologic grounds alone. We studied neuroendocrine tumors from these sites to see if NESP-55 expression could reliably discriminate pulmonary and gastrointestinal carcinoids from neuroendocrine tumors arising in the pancreas or the adrenal medulla. Sixty-three neuroendocrine tumors positive for one or more immunohistochemical marker of neuroendocrine differentiation (chromogranin A, chromogranin B, synaptophysin, secretogranin II, neuron-specific enolase) were selected for the study and consisted of 34 typical carcinoids (15 pulmonary, 11 ileal, 4 gastric, and 4 rectal), 19 PETs, and 10 pheochromocytomas (4 sporadic, 3 MEN-2, 2 neurofibromatosis type 1, and 1 VHL). All cases were stained for NESP-55 after microwave antigen retrieval using a rabbit polyclonal antibody at a dilution of 1:1000. Sections of normal adrenal medulla were used as positive controls for NESP-55 staining. Negative controls consisted of omission of primary antibody and replacement with normal rabbit serum at an equivalent concentration. NESP-55 immunoreactivity was seen as brown finely granular cytoplasmic staining with prominent perinuclear accentuation. All gastric and ileal carcinoids studied were completely negative for NESP-55. One of four rectal and 1 of 15 pulmonary carcinoids showed focal positivity for it in less than 5% of tumor cells. In contrast, all 10 pheochromocytomas and 14 of 19 PETs showed strong immunohistochemical staining in a variable proportion of tumor cells. Diffuse positivity (>75% of tumor cells) was seen in 6 of 14 PETs and 8 of 10 pheochromocytomas. Our results indicate that, in contrast to the other granins, NESP-55 reactivity is restricted to endocrine tumors of the pancreas and the adrenal medulla. Immunohistochemical expression of NESP-55 may thus be useful in assigning a pancreatic or adrenal origin to metastatic endocrine tumors of unknown origin.

Imprinting of Nesp55 gene in cattle.

Studies in mouse and human have established that the exon encoding the neuroendocrine secretory protein Nesp55 is transcribed exclusively from the maternal allele. To study the imprinting status of Nesp55 in cattle, we examined transcripts of this exon in a wide range of tissues from four fetuses and the dam of one of the fetuses (not all tissues were examined in all subjects). A polymorphic site was used to determine transcript status as monoallelic or biallelic. The expression analysis was by direct sequencing of RT-PCR products amplified from the tissues. Nesp55 transcripts were detected in many tissues, in contrast to previous studies in which Nesp55 was detected by immunoassays in endocrine and nervous tissues only. In all five individuals, Nesp55 showed monoallelic expression. In one of the fetuses, parental origin of the expressed allele could not be determined; in the others monoallelic expression was clearly maternal. Thus, bovine Nesp55 is maternally expressed like its counterparts in mouse and human.

Importance of vesicle proteins in the diagnosis and treatment of neuroendocrine tumors.

We have analyzed the expression of synaptic vesicle proteins in human neuroendocrine tumors and the potential use of vesicle proteins in the diagnosis and treatment of neuroendocrine tumors. Biopsies from endocrine and nonendocrine tumors of the gastrointestinal tract, pancreas, and adrenals were examined by immunocytochemistry using antibodies against synaptic vesicle protein 2 (SV2), vesicular monoamine transporter 1 and 2 (VMAT1 and 2), and neuroendocrine secretory protein 55 (NESP55). SV2 was expressed in all endocrine tumors of the gastrointestinal tract and pancreas as well as in gastrointestinal stromal tumors (GISTs). None of the adenocarcinomas expressed SV2. VMAT1 and 2 were expressed in amine-producing tumors of the gastrointestinal tract (ECL cell and EC cell carcinoids) and in a small number of peptide-producing pancreatic endocrine tumors. NESP55 was expressed in neuroblastomas and adrenal pheochromocytomas as well as in a subgroup of pancreatic endocrine tumors. The importance of VMAT1 and 2 for the uptake of 123I-MIBG in tumor cells was demonstrated. It was concluded that neuroendocrine tumors express multiple synaptic vesicle proteins that are useful in the histopathological diagnosis and classification of tumors. Vesicle proteins may prove to be useful for targeting tumor therapy.

Identification of calcium-inducible genes in primary keratinocytes using suppression-subtractive hybridization.

Terminal differentiation in epidermal keratinocytes involves major biochemical changes including the expression of many new differentiation-specific genes. To further understand this process, we performed suppression-subtractive hybridization of keratinocytes cultured under high-calcium condition, known to induce differentiation in vitro. We randomly isolated 300 clones representing 90 different genes. By reverse Northern blot analyses, 20 different genes were found to be overexpressed, of which 13 were confirmed as differentially expressed genes during keratinocyte differentiation by Northern blot analysis. Of those, five genes, transglutaminase 1, keratin 6, interleukin-1 receptor antagonist, kallikrein 7, and heat shock protein 27, are known to be up-regulated during epidermal differentiation. Six genes, ferritin-L chain, ribosomal protein S6, tumor-associated calcium signal transducer 2, neuroendocrine secretory protein 55, phosphoserine aminotransferase, and neutrophil gelatinase-associated lipocalin, heretofore were not known to be up-regulated during keratinocyte differentiation. We also identified two novel genes. One of these maps to chromosome 1q21 of the epidermal differentiation complex, and its expression level was strongly increased in differentiating keratinocytes. These differentially expressed genes may provide significant opportunities for further understanding of the epidermal keratinocyte differentiation.

Expression Analysis of Genes identified by Molecular Profiling of VGP Melanomas and MGP Melanoma-Positive Lymph Nodes

Microarray profiling is a powerful approach to establish gene expression patterns for different histopathological stages of a malignancy, and at the same time, to identify individual genes that may have important functions in the early and/or advanced stages of a neoplasm. To identify genes that hitherto have not been shown to be expressed or play a role in advanced-stage melanomas, we conducted microarray analyses with RNAs from primary melanoma and melanoma-positive lymph node specimens. Using RT-PCR, quantitative, real-time RT-PCR, and fluorochrome oligonucleotide-based optical imaging, we established the level and pattern of expression of five of the identified known genes [Suppression of Tumorigenicity 13 (ST13), Cystatin 8 (CST-8), Dyskeratosis Congentia 1 (DKC1), Neuroendocrine Secretory Protein 55 (NESP55), Niemann-Pick Disease, type C2 (NP-C2)], and a gene with unknown function (16.7 kD Hypothetical Protein) in benign and atypical nevocytic lesions, advanced-stage melanomas, and melanoma-positive lymph nodes.

Proteolytic processing of chromogranin A by the prohormone convertase PC2

The neuroendocrine secretory protein chromogranin A (CgA) is a precursor for various biologically active peptides. Several single and paired basic residues are present within its primary amino acid sequence comprising cleavage sites for prohormone convertases. In this study, SH-SY5Y human neuroblastoma cells were stably transfected with the prohormone convertase PC2 to analyse the proteolytic processing of endogenous chromogranin A and, in particular, the formation of the chromogranin-A-derived peptide GE-25. Our analyses revealed a significant change in the pattern of proteolytic conversion of chromogranin A in cells expressing PC2. Mock-transfected control cells contained mainly the intact chromogranin A molecule and hardly any shorter products were found. On the other hand, PC2-transfected cells showed extensive processing of chromogranin A, resulting in significantly lower amounts of the intact precursor and especially high levels of the free peptide GE-25.

Neuroendocrine secretory protein 55: a novel marker for the constitutive secretory pathway.

The chromogranins constitute a class of acidic proteins comprising the structurally related chromogranins A and B and secretogranin II. These proteins are widely distributed in endocrine and nervous tissues; they are localized to the large dense core vesicles and released from them after stimulation of cells. In all the tissues examined chromogranins are proteolytically processed into small peptides, some of which have defined physiological activities. Chromogranin A plays a key role in large dense core vesicle biogenesis and can induce the formation of the regulated pathway. We have recently cloned neuroendocrine secretory protein 55 (NESP55), a protein that shares several features with the class of chromogranins. NESP55 is a soluble, acidic, heat-stable secretory protein that is expressed exclusively in endocrine and nervous tissues, although less widely than chromogranins. NESP55 is genomically imprinted and transcribed only from the maternal allele. It is proteolytically processed in some tissues into the small octapeptide GAIPIRRH located at the C terminus of NESP55. In the brain NESP55 is found in cell bodies and axons but not in terminals. At the subcellular level NESP55 is localized to a large vesicle, which is anterogradely transported by the fast axonal flow in neurons. From this vesicle NESP55 is constitutively released. However, in some tissues like the adrenal, medulla, and bovine splenic nerve, NESP55 is also found in the large dense transmitter storage organelles. Thus, NESP55 represents a novel peptidergic marker for a large constitutively secreting vesicle pool found in the central and peripheral nervous system.

The Gsα Gene: Predominant Maternal Origin of Transcription in Human Thyroid Gland and Gonads

Mutations in the guanine nucleotide binding alpha-subunit 1 gene (GNAS1) cause Albright's hereditary osteodistrophy, and the parent of transmission determines variable phenotypic expression of the disease. This has suggested that GNAS1 may be under tissue-specific imprinting control, although studies so far available have failed to clearly define the pattern of GNAS1 expression in humans. To establish if GNAS1 is imprinted in human endocrine tissues, we selected 14 thyroid, 10 granulosa cell, 13 pituitary (3 normal glands, 7 GH-secreting adenomas, and 3 nonfunctioning adenomas), 3 adrenal, and 11 lymphocyte samples shown to be heterozygous for a known polymorphism in exon 5. RNA from these tissues was analyzed by RT-PCR, and expression from both parental alleles was evaluated by enzymatic digestion and subsequent quantification of the resulting fragments. The parental origin of Gs alpha was assessed by evaluating neuroendocrine secretory protein 55 and extra large alphas-like protein transcripts, which have been shown to be monoallelically and parent-specifically expressed from the maternal and paternal allele, respectively. By this approach, the great majority of thyroid (n = 12), ovarian (n = 7), and pituitary (n = 11) samples showed an almost exclusive or significantly predominant expression of the maternal allele over the paternal one, whereas in lymphocyte and adrenal samples both alleles were equally expressed. Our results provide evidence for a predominant maternal origin of GNAS1 transcripts in different human adult endocrine tissues, particularly thyroid, ovary, and pituitary, and strongly suggest that this mechanism may play a crucial role in the determination of the phenotypic expression of Albright's hereditary osteodistrophy.

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 55 000) 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.