Structure-function analyses reveal a triple ß-turn receptor-bound conformation of adrenomedullin 2/intermedin and enable peptide antagonist design.

The cardioprotective vasodilator peptide adrenomedullin 2/intermedin (AM2/IMD) and the related adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) signal through three heterodimeric receptors comprising the calcitonin receptor-like class B G protein-coupled receptor (CLR) and a variable receptor activity-modifying protein (RAMP1, -2, or -3) that determines ligand selectivity. The CGRP receptor (RAMP1:CLR) favors CGRP binding, whereas the AM1 (RAMP2:CLR) and AM2 (RAMP3:CLR) receptors favor AM binding. How AM2/IMD binds the receptors and how RAMPs modulate its binding is unknown. Here, we show that AM2/IMD binds the three purified RAMP-CLR extracellular domain (ECD) complexes with a selectivity profile that is distinct from those of CGRP and AM. AM2/IMD bound all three ECD complexes but preferred the CGRP and AM2 receptor complexes. A 2.05 Å resolution crystal structure of an AM2/IMD antagonist fragment-bound RAMP1-CLR ECD complex revealed that AM2/IMD binds the complex through a unique triple ß-turn conformation that was confirmed by peptide and receptor mutagenesis. Comparisons of the receptor-bound conformations of AM2/IMD, AM, and a high-affinity CGRP analog revealed differences that may have implications for biased signaling. Guided by the structure, enhanced-affinity AM2/IMD antagonist variants were developed, including one that discriminates the AM1 and AM2 receptors with ∼40-fold difference in affinities and one stabilized by an intramolecular disulfide bond. These results reveal differences in how the three peptides engage the receptors, inform development of AM2/IMD-based pharmacological tools and therapeutics, and provide insights into RAMP modulation of receptor pharmacology.

Impact of ultrafiltration and nanofiltration of an industrial fish protein hydrolysate on its bioactive properties.

BACKGROUND: Numerous studies have demonstrated that in vitro controlled enzymatic hydrolysis of fish and shellfish proteins leads to bioactive peptides. Ultrafiltration (UF) and/or nanofiltration (NF) can be used to refine hydrolysates and also to fractionate them in order to obtain a peptide population enriched in selected sizes. This study was designed to highlight the impact of controlled UF and NF on the stability of biological activities of an industrial fish protein hydrolysate (FPH) and to understand whether fractionation could improve its content in bioactive peptides. RESULTS: The starting fish protein hydrolysate exhibited a balanced amino acid composition, a reproducible molecular weight (MW) profile, and a low sodium chloride content, allowing the study of its biological activity. Successive fractionation on UF and NF membranes allowed concentration of peptides of selected sizes, without, however, carrying out sharp separations, some MW classes being found in several fractions. Peptides containing Pro, Hyp, Asp and Glu were concentrated in the UF and NF retentates compared to the unfractionated hydrolysate and UF permeate, respectively. Gastrin/cholecystokinin-like peptides were present in the starting FPH, UF and NF fractions, but fractionation did not increase their concentration. In contrast, quantification of calcitonin gene-related peptide (CGRP)-like peptides demonstrated an increase in CGRP-like activities in the UF permeate, relative to the starting FPH. The starting hydrolysate also showed a potent antioxidant and radical scavenging activity, and a moderate angiotensin-converting enzyme (ACE)-1 inhibitory activity, which were not increased by UF and NF fractionation. CONCLUSION: Fractionation of an FPH using membrane separation, with a molecular weight cut-off adapted to the peptide composition, may provide an effective means to concentrate CGRP-like peptides and peptides enriched in selected amino acids. The peptide size distribution observed after UF and NF fractionation demonstrates that it is misleading to characterize the fractions obtained by membrane filtration according to the MW cut-off of the membrane only, as is currently done in the literature.

Use of a commercial protease and yeasts to obtain CGRP-like molecules from saithe protein.

Different bioactive molecules, such as CGRP-like peptides, can be found in fish protein hydrolysates. Calcitonin gene-related peptide (CGRP) is a neuropeptide known to act as a potent arterial and venous vasodilator in humans. This study focuses on the industrial obtaining of CGRP-like molecules from saithe (Pollachius virens) byproduct, derived from the filleting process. Protein from P. virens was primarily hydrolyzed with Alcalase and later treated with Saccharomyces cerevisiae live cells. Treatment with Saccharomyces doubled the quantity of bioactive molecules obtained. The CGRP-like molecules were partially purified by chromatography, and the immunoreactive material was further analyzed for its CGRP-like bioactivity, using a specific radioreceptor assay. The concentration of CGRP-like molecules increased over 100-fold after purification. The bioactive molecules were able to induce cyclic AMP stimulation in rat liver membranes. Finally, partial sequencing of the bioactive peptide was performed, showing some homology with alpha-actin and myosin of several fish species.

Molecular characterization of calcitonin gene-related peptide (CGRP) related peptides (CGRP, amylin, adrenomedullin and adrenomedullin-2/intermedin) in goldfish (Carassius auratus): cloning and distribution.

To further characterize the structure and function of calcitonin gene-related peptide (CGRP) related peptides in fish, we have cloned cDNA sequences for CGRP, amylin, adrenomedullin (AM) and adrenomedullin-2/intermedin (IMD) in goldfish (Carassius auratus) and examined their tissue distribution. CGRP, amylin, AM and IMD cDNAs were isolated by reverse transcription (RT) and rapid amplification of cDNA ends (RACE). The cloned sequences contain the complete four mature peptides, which present a high degree of identity with mature peptide sequences from other fish. Phylogenetic analyses show that goldfish AM and IMD form a sub-family within the CGRP-related peptides that is distinct from the CGRP/amylin sub-family. The distribution of goldfish CGRP-like peptides mRNA expression in different tissues and within the brain was studied by RT-PCR. CGRP, IMD and AM are detected throughout the brain, in pituitary and in most peripheral tissues examined. Amylin mRNA is mostly expressed in the brain, in particular posterior brain, optic tectum and hypothalamus, but is also present in pituitary, gonad, kidney and muscle. Our results suggest that goldfish CGRP, amylin, AM and IMD are conserved peptides that show the typical structure characteristics present in their mammalian counterparts. The widespread distributions of CGRP, AM and IMD suggest that these peptides could be involved in the regulation of many diverse physiological functions in fish. Amylin mRNA distribution suggests possible new roles for this peptide in teleosts, including the control of reproduction.

Occurrence of a CGRP-like molecule in siki (Centroscymnus coelolepsis) hydrolysate of industrial origin.

Fish protein hydrolysates (FPH) may have potential as bioactive components in functional foods as nutraceuticals. This study focused on the identification of calcitonin gene-related peptide (CGRP) molecules in FPH. CGRP is a neuropeptide belonging to the calcitonin/CGRP family and is known as potent arterial and venous vasodilator in humans. Hydrolysates of industrial origin were prepared from siki (Centroscymnus coelolepsis) heads and were analyzed for the presence of CGRP-like molecules using specific radioimmunoassays and radioreceptorassays. The biological activity of the CGRP-related molecules was assessed by their ability to stimulate the adenylate cyclase activity in rat liver membranes. They were finally purified using gel exclusion chromatography and high-performance liquid chromatography (HPLC). These molecules presented a molecular weight around 1500-2500 Da and were obtained with a purification factor of 79. The incorporation of FPH with CGRP-like molecules in functional foods could lead to the development of new useful products for health and nutrition markets.

Expresión de neuropéptidos en la articulación temporomandibular, 1: GGRP y articulación temporomandibular / Neuropeptides expression in the temporomandibular joint, 1: CGRP and temporomandibular joint

Antecedentes: neuropéptidos como el péptido relacionado con el gen de la calcitonina (CGRP), sustancia P (SP) y neurokinina A (NKA) se relacionan con desarrollo y progresión de enfermedad degenerativa articular. Estudios previos mostraron su rol en respueta vascular y nociceptiva en artritis y su papel modulardor en hiperalgesia y dolor de tipo artrítico, comprobando su presencia en líquido sinovial de articulación temporomandibular. Propósito: evaluar presencia y contenido de neuropéptidos en tejidos retrodiscal hiperplásico de la articulación temporomandibular en pacientes con enfermedad degenerativa articular mediante radioinmunoensayo. Métodos: ocho pacientes de sexo femenino (15 articulaciones), premenopáusicas, no embarazadas ni lactando por un año, fueron diagnosticadas con enfermedad articular degenerativa (osteoartrosis). Se registraron niveles de dolor en escala visual análoga, donde 0 es ausencia de dolor y 16 dolor agudo; se clasificó la degeneración ósea entre leve, moderada y severa, de acuerdo con hallazgos de resonanacia nuclear magnética. Las pacientes fueron sometidas a cirugía abierta de ATM donde se tomaron las muestras. Los especímenes se colocaron en bloques plásticos con medio congelante y se almacenaron a -700C hasta la extracción de los neuropéptidos por radioinmunoensayo con el estuche específico para cada uno. Resultados: se estableció una relacion directamente proporcional entre grado de degeneración ósea y expresión de CGRP, y entre clasificación de osteoartrosis con escala visual análoga. Los hallazgos mostraron correlación definitiva entre niveles de dolor y expresión del péptido relacionado con el gen de la calcitonina. Conclusiones: CGRP, SP y NKA, si se expresan en tejido retrodiscal de ATM en humanos con enfermedad degenerativa articular, relacionándose CGRP directamente con niveles de osteoartrosis y dolor

Clara cell secretory protein-expressing cells of the airway neuroepithelial body microenvironment include a label-retaining subset and are critical for epithelial renewal after progenitor cell depletion.

Stem cells with potential to contribute to the re-establishment of the normal bronchiolar epithelium have not been definitively demonstrated. We previously established that neuroepithelial bodies (NEBs) sequester regenerative cells that contribute to bronchiolar regeneration after selective chemical depletion of Clara cells, a major progenitor cell population. Two candidate stem cells were identified on the basis of proliferative potential after chemical ablation: a pollutant-resistant subpopulation of Clara cells that retain their expression of Clara cell secretory protein (CCSP) (variant CCSP-expressing [CE] cells or vCE cells) and calcitonin gene-related peptide (CGRP)-expressing pulmonary neuroendocrine cells (PNECs). In the present study, two populations of label-retaining cells were identified within the NEB: CGRP-expressing cells and a subpopulation of CE cells. To investigate contributions made by CE and CGRP-expressing cells to epithelial renewal, CE cells were ablated through acute administration of ganciclovir to transgenic mice expressing herpes simplex virus thymidine kinase under the regulatory control of the mouse CCSP promoter. CGRP-immunoreactive PNECs proliferated after depletion of CE cells, yet were unable to repopulate CE cell-depleted airways. These results support the notion that vCE cells represent either an airway stem cell or are critical for stem cell maintenance, and suggest that PNECs are not sufficient for epithelial renewal.

Isolation, structure, synthesis, and activity of a new member of the calcitonin gene-related peptide family from frog skin and molecular cloning of its precursor.

Calcitonin gene-related peptide has been extracted from the skin exudate of a single living specimen of the frog Phyllomedusa bicolor and purified to homogeneity by a two-step protocol. A total volume of 250 microl of exudate yielded 380 microg of purified peptide. Mass spectrometric analysis and gas phase sequencing of the purified peptide as well as chemical synthesis and cDNA analysis were consistent with the structure SCDTSTCATQRLADFLSRSGGIGSPDFVPTDVSANSF amide and the presence of a disulfide bridge linking Cys(2) and Cys(7). The skin peptide, named skin calcitonin gene-related peptide, differs significantly from all other members of the calcitonin gene-related peptide family of peptides at nine positions but binds with high affinity to calcitonin gene-related peptide receptors in the rat brain and acts as an agonist in the rat vas deferens bioassay with potencies equal to those of human CGRP. Reverse transcriptase-polymerase chain reaction coupled with cDNA cloning and sequencing demonstrated that skin calcitonin gene-related peptide isolated in the skin is identical to that present in the frog's central and enteric nervous systems. These data, which indicate for the first time the existence of calcitonin gene-related peptide in the frog skin, add further support to the brain-skin-gut triangle hypothesis as a useful tool in the identification and/or isolation of mammalian peptides that are present in the brain and other tissues in only minute quantities.

Trifluoroacetate, a contaminant in purified proteins, inhibits proliferation of osteoblasts and chondrocytes.

Peptides purified by HPLC are often in the form of a trifluoroacetate (TFA) salt, because trifluoroacetic acid is used as a solvent in reversed-phase HPLC separation. However, the potential effects of this contaminant in culture systems have not been addressed previously. TFA (10(-8) to 10(-7) M) reduced cell numbers and thymidine incorporation into fetal rat osteoblast cultures after 24 h. Similar effects were found in cultures of articular chondrocytes and neonatal mouse calvariae, indicating that the effect is not specific to one cell type or to one species of origin. When the activities of the TFA and hydrochloride salts of amylin, amylin-(1-8), and calcitonin were compared in osteoblasts, cell proliferation was consistently less with the TFA salts of these peptides, resulting in failure to detect a proliferative effect or wrongly attributing an antiproliferative effect. This finding is likely to be relevant to all studies of purified peptides in concentrations above 10(-9) M in whatever cell or tissue type. Such peptides should be converted to a hydrochloride or biologically equivalent salt before assessment of their biological effects is undertaken.

Immunohistochemical mapping of enkephalins, NPY, CGRP, and GRP in the cat amygdala.

This immunohistochemical study shows a wide distribution of neuropeptides in the cat amygdala. Neuropeptide Y is present along the whole amygdaloid complex, and fibers and cell bodies containing neuropeptide Y are observed in all the nuclei studied. Leucine-enkephalin-, gastrin-releasing peptide/bombesin-, and calcitonin gene-related peptide-immunoreactive fibers and perikarya are observed only in discrete nuclei of the amygdaloid complex, whereas only fibers -but no cell bodies- containing methionine-enkephalin-Arg6-Gly7-Leu8 have been observed. No immunoreactivity has been found for gamma-melanocyte-stimulating hormone, dynorphin A (1-17), or galanin. These data are compared with those reported in the amygdala of other mammals.

The peptide content of colonic afferents decreases following colonic inflammation.

Peripheral injury produces long term changes in peptide content in dorsal root ganglion (DRG) cells that contribute to the inflammatory process in the periphery and neuronal plasticity in the spinal cord. We report here the proportion of colonic afferents labeled for calcitonin gene-related peptide (CGRP), substance P (SP) or somatostatin (Som) in the T13-L2 and L6-S2 DRG and changes in the percentage of SP or CGRP labeled afferents 6, 24, and 72 h following induction of experimental colitis. Following injection of fluorogold (FG) into the descending colon, significantly more FG labeled DRG cells were observed in the T13-L2 than L6-S2 DRG. In noninflamed rats, in both spinal regions, 60-70% of the colonic afferents that were labeled with FG were double labeled for SP. Similar results were obtained when double labeling for CGRP. Only 20-30% of the FG labeled afferents were double labeled for Som. Following experimental colitis induced by intracolonic zymosan, there was a significant decrease in the percentage of cells double labeled for SP in the T13-L2 and L6-S2 DRG at 6, 24, and 72 h. The percentage of CGRP double labeled cells was decreased in the T13-L2 DRG at all time points, but only at 24 h in the L6-S2 DRG. The cell bodies of CGRP labeled colonic afferents were significantly larger than SP or Som in control rats. Inflammation did not affect the mean size of the double labeled cells. These results suggest that colonic inflammation increases SP and CGRP release in the spinal cord and the colon that is manifest as a decrease in peptide content in the cell bodies of the colonic afferents during the first 72 h following injury.

A sensitive sandwich enzyme immunoassay for calcitonin gene-related peptide (CGRP): characterization and application.

Thirty mouse monoclonal antibodies (mAbs) directed against rat calcitonin gene-related peptide-alpha (CGRP-alpha) have been obtained. These mAbs are classified in 2 groups, one recognizing the peptide N-terminus and the other binding the C-terminus. A two-site immunometric assay was developed using mAb CGRP-83 as capture antibody, whereas mAb CGRP-72 acts as tracer, covalently labeled with enzyme acetylcholinesterase. This assay appeared sensitive (limit of detection: 2 pg/ml) and precise, allowing quantitative measurement of all human and murine CGRP isoforms. The assay was used to determine specific concentrations of CGRP in different rat, mice and guinea pig samples. The validity of the test was demonstrated by HPLC fractionation experiments.

Isolation of an acid fraction from a fish protein hydrolysate with a calcitonin-gene-related-peptide-like biological activity.

The possibility of obtaining calcitonin and/or calcitonin-gene-related peptide (CGRP) immunorelated molecules from partly digested proteins was investigated with fish and shrimp hydrolysates. These two peptides were quantified by both radioimmunoassay and radioreceptor assay; the positive extracts were partly purified. Different hydrolysates were analysed: cod head, stomach and viscera hydrolysates, a shrimp hydrolysate and two sardine hydrolysates. Although each cod extract interacted in the CGRP radioimmunoassay, none of these extracts was able to displace the CT binding to its antibody. In contrast, shrimp and sardine hydrolysates interacted with both radioimmunoassays. Radioreceptor assays performed on the same extracts demonstrated that only three extracts contained the structural determinants that allowed them to interact in the CGRP radioreceptor assay. No interaction with the calcitonin radioreceptor assay could be demonstrated. Molecular sieving of the two sardine extracts showed that the immunoreactivity was resolved into two main fractions. The higher-molecular-mass fraction interacted only in the CGRP radioreceptor assay. The results obtained suggest the presence of a biologically related CGRP molecule in peptone hydrolysates and requires further investigation into the role of these peptide fragments in the regulation of intestinal function by partly digested proteins.

A comparison of four extraction methods for substance P, neurokinin A and calcitonin gene-related peptide from human dental pulp tissue.

Measuring neuropeptides in biological tissues by radioimmunoassay requires efficient extraction that maintains their immunoreactivity. Many different methods for extraction have been described, but there is little information on optimal extraction methods for individual neuropeptides from human dental pulp tissue. The aim was therefore to identify an effective extraction procedure for three pulpal neuropeptides; substance P, neurokinin A and calcitonin gene-related peptide. Tissue was obtained from 20 pulps taken from teeth freshly extracted for orthodontic reasons. The pulp samples were divided into four equal groups and different extraction methods were used for each group. Boiling whole pulp in acetic acid gave the highest overall yield and, in addition, offered an easy and rapid means of pulp tissue processing. The use of protease inhibitors did not increase the recovery of the immunoreactive neuropeptides but did provide the best combination of maximal recoveries and minimal variability. These results should be useful for planning the extraction of these neuropeptides from human pulp tissue in future studies.

Primary structure, distribution, and effects on motility of CGRP in the intestine of the cod Gadus morhua.

Calcitonin gene-related peptide (CGRP) was isolated from an extract of the intestine of the cod Gadus morhua. The primary structure of this 37-amino acid peptide was established as follows: ACNTA TCVTH RLADF LSRSG GIGNS NFVPT NVGSK AF-NH2. The peptide shows close structural similarities to other nonmammalian (3-4 amino acid substitutions) and mammalian (5-8 amino acid substitutions) CGRPs, and it contains the two residues Asp14 and Phe15 that seem to be characteristic for CGRP in nonmammalian vertebrates. Cod CGRP (10(-9)-10(-7) M) inhibited the motility of spontaneously active ring preparations from the cod intestine and was significantly (P < 0.05) more potent than rat alpha-CGRP. Neither prostaglandins nor nitric oxide is involved in the inhibitory response produced by cod CGRP, and the lack of effect of tetrodotoxin suggests an action of CGRP on receptors on the intestinal smooth muscle cells. The competitive CGRP antagonist human alpha-CGRP-(8-37) significantly (P < 0.05) reduced the response to cod CGRP. Immunohistochemistry demonstrated CGRP-immunoreactive neurons intrinsic to the intestine, and a dense innervation with immunoreactive nerve fibers was observed in the myenteric plexus and the circular muscle layer. Myotomy studies show that CGRP-containing nerves project orally and anally in the myenteric plexus, whereas nerve fibers in the circular muscle layer project mainly anally, indicating a role for CGRP in descending inhibitory pathways of the cod intestine.

Isolation and molecular characterization of porcine calcitonin gene-related peptide (CGRP) and its endocrine effects in the porcine pancreas.

The aim of this study was to investigate the possible role of porcine calcitonin gene-related peptide (CGRP) in the regulation of the endocrine porcine pancreas. Initially, we isolated and purified CGRP from extracts of porcine adrenal glands and pancreases. A single molecular form of the peptide was found in the two tissues. The adrenal peptide was sequenced and found to differ from human alpha-CGRP at six positions and from human beta-CGRP at three positions. By immunohistochemistry, CGRP was found in nerve fibers in the pancreatic ganglia. A synthetic replica of the porcine peptide was infused at different dose levels (10(-10), 10(-9), and 10(-8) M) into isolated perfused porcine pancreata. With 5 mmol/L glucose in the perfusate. CGRP at 10(-10) and 10(-9) M increased insulin and glucagon secretion, whereas significant decreases were observed with 10(-8) M. Somatostatin secretion was increased significantly by 10(-8) M CGRP. In immunoneutralization studies (n = 6) using a high-affinity somatostatin antibody, the inhibitory effect of CGRP at 10(-8) M was reversed to a significant stimulation of insulin and glucagon secretion. Insulin secretion in response to square-wave increases in glucose concentration to 11 mM was inhibited dose dependently by CGRP; at 10(-8) M the insulin output decreased by 72+/-9% (n = 6). The present results indicate that CGRP may be involved in the regulation of insulin and glucagon secretion from the porcine pancreas.

Analytical extraction of regulatory peptides from rat lung tissue.

We evaluated protocols for the extraction of calcitonin gene-related peptide, neuropeptide Y, substance P, peptide YY and beta-endorphin from rat lung tissue for subsequent radioimmunoassay. The effects of varying acidity of the extraction solution and repeating extraction on the recovery of peptide immunoreactivity and non-specific tracer-binding were compared by analysis of variance. Moreover, variability of immunoreactivity was quantified for comparison. Considering all three criteria, the optimal acidity for extraction was: 0.1 M or 1 M acetic acid for CGRP and beta-endorphin, 0.1 M acetic acid for NPY, 1 M acetic acid for substance P and phosphate buffer for peptide YY. Double or combined extraction unambiguously improved assay results only for substance P. Reversed-phase high-performance liquid chromatography of CGRP-, NPY- and SP-immunoreactivity obtained from selected extracts suggested that differences in recovery of these peptides are not explainable by differential peptide fragmentation during extraction.

A lobster cysteine protease with immunoreactivity and activities of calcitonin and CGRP.

The high concentrations of molecules immunologically related to salmon calcitonin (CT) and/or to human calcitonin gene-related peptide (CGRP) in the oesophagus of the norway lobster Nephrops norvegicus have been examined. In the present study. We report the purification of these molecules by means of a specific radioimmunoassay for calcitonin and calcitonin gene related peptide. The immunoreactive molecules were tested for their functional similarities with CT and CGRP. This was investigated by measuring their ability to interact with CGRP and CT radioreceptor assays and to stimulate the adenylate cyclase activity in rat liver and kidney membranes, respectively. In addition, the purified product was injected in young rats in order to check for a CT-like biological activity of these molecules. The combination of these tests led us to purify a molecular form of 33 kDa. N-terminal sequence analysis of this protein revealed a considerable homology with the lobster cysteine proteases and the human cathepsin L. Control experiments performed with the highly purified American lobster cysteine protease I showed that crustacean cysteine proteases given in vivo to rats induce a fall in the plasma calcium and phosphate levels. This study therefore adds further documentation for a common ancestral origin of CT, CGRP and the much large cysteine proteases from invertebrates.

Substance P- and calcitonin gene-related peptide-immunofluorescent nerves in the repair of experimental bone defects.

Healing of an experimental bony defect in the rat's tibia was studied with an immunofluorescent technique to clarify when and where substance P (SP) and calcitonin gene-related peptide (CGRP) would develop. The normal tibia showed a few SP- and CGRP-immunofluorescent nerve fibres. In the experimental tibia, the number of these fibres increased on the 6th day after operation, reached a peak of proliferation on the 15th day and reverted to normal after the 24th day. The changes were associated with the development and decay of callus tissue suggesting that harmful stimuli from the injured site in a bone could be mediated by sensory nerves throughout the repair period. Most of the SP- and CGRP-immunofluorescence was seen near the vessels, frequently in the same nerve fibres. The SP- and CGRP-immunofluorescent nerves seemed to take part jointly in callus formation through the enhancement of local blood flow.

Extraction and quantitation of neuropeptides in bone by radioimmunoassay.

The feasibility of extracting and quantifying neuropeptides in bone by radioimmunoassay was investigated in a study including 60 diaphyseal rat femora. Substance P, calcitonin gene-related peptide, neuropeptide Y and vasoactive intestinal polypeptide, previously identified in bone by immunohistochemistry, were extracted from separate homogenates of bone, periosteum and bone marrow in a solution of 4% EDTA and 2 M acetic acid. Measurable amounts of all four neuropeptides in bone, periosteum and bone marrow were obtained by radioimmunoassay in a reproducible manner. The neuropeptide immunoreactivities were characterized by reverse-phase high performance liquid chromatography. Among the four neuropeptides analyzed, neuropeptide Y consistently exhibited the highest concentrations in the different tissues. Overall, cortical bone showed the lowest neuropeptide concentrations. The concentration of vasoactive intestinal polypeptide was higher in periosteum than in bone marrow, whereas that of calcitonin gene-related peptide was uniform in these tissues. The distributional differences observed in bone tissue may be explained by a variety of physiological roles attributed to neuropeptides such as regulation of nociception, vasoactivity, immune function and local bone metabolism. The described methodology offers a new means of investigating a neuropeptidergic involvement in various disorders of the skeleton.