Corticotropin-like Intermediate Peptide Research Articles

Short Communication: Identification of equine corticotropin-like intermediate lobe peptide (CLIP) binding to an adrenocortipcotrophic hormone (ACTH) assay capture antibody

A chemiluminescent immunoassay is commonly employed to measure adrenocorticotrophic hormone (ACTH) concentrations to assist pituitary pars intermedia dysfunction diagnosis. In a previous study, seasonally-dependent assay cross-reactivity to endogenous equine corticotropin-like intermediate lobe peptide (CLIP, ACTH 18–39) was suspected. The present study aimed to demonstrate binding of endogenous equine CLIP to the capture antibody of the ACTH chemiluminescent immunoassay. Liquid chromatography – mass spectrometry (LCMS) methods were optimised to identify selected ions from synthetic human ACTH, α-melanocyte stimulating hormone (α-MSH, ACTH 1–17) and CLIP. Synthetic ACTH and CLIP bound to the capture antibody of the chemiluminescent ACTH assay, but α-MSH did not. Equine endogenous CLIP was detected by LCMS in pony plasma taken in the autumn and could be eluted from the capture antibody of the ACTH chemiluminescent immunoassay. Further research is required to enable quantification of CLIP. Equine CLIP may alter measured ACTH concentrations in vivo.

Profiles of pro-opiomelanocortin and encoded peptides, and their processing enzymes in equine pituitary pars intermedia dysfunction.

Equine pituitary pars intermedia dysfunction (PPID) is characterized by hyperplasia of the pars intermedia (PI) melanotrophs of the pituitary gland (PG), and increased production of proopiomelanocortin (POMC). POMC is cleaved by prohormone convertase 1 (PC1) to produce adrenocorticotropic hormone (ACTH), and further processing of ACTH by PC2 to produce alpha-melanocyte stimulating hormone (α-MSH) and corticotropin-like intermediate peptide (CLIP). High plasma ACTH concentrations in horses with PPID might be related to reduced conversion of ACTH to α-MSH by PCs. The hypothesis of this study was that PC1 and PC2 expression in the pituitary gland are altered in PPID, resulting in an abnormal relative abundance of POMC derived proteins. The objectives of this study were to identify the partial sequences of equine POMC, PC1, and PC2 mRNAs; and to determine whether the expression of POMC, PC1, and PC2 mRNAs in whole pituitary extracts, and POMC-protein in the cavernous sinus blood of horses are altered in PPID. We confirmed (RT-PCR and sequencing) that the partial sequences obtained match the corresponding regions of predicted equine POMC, PC1 and PC2 sequences. The expression (quantification by RT-qPCR) of POMC, PC1 and PC2 mRNAs were found upregulated in the pituitary of horses with PPID. Plasma (measured using RIA/ELISA) ACTH and α-MSH were elevated in PPID horses. These results indicate distinct differences in gene and protein expression of POMC and its intermediates, and processing enzymes in PPID. It provides evidence to support the notion that local, pituitary-specific inadequacies in prohormone processing likely contribute to equine PPID.

Fatigue in Multiple Sclerosis: Is it related to cytokines and hypothalamic-pituitary-adrenal axis?

BackgroundFatigue is a common symptom of Multiple Sclerosis (MS) that diminishes the quality of life of patients, but its exact mechanism remains poorly understood. There is not a generally adopted scale to determine MS fatigue. Studies that investigated physiopathology of fatigue symptom have shown dysregulation of hypothalamic-pituitaryadrenal (HPA) axis. In the current study, we aimed to compare the results obtained with two separate scales, namely the Fatigue Severity Scale (FSS) and the Neurological Fatigue Index-Multiple Sclerosis (NFI-MS), and assess the relationship between fatigue and serum IL-1β, TNF-α, IL-35, IL-2, IL-10, ACTH, cortisol, α-MSH, β-MSH, γ-MSH and CLIP (Corticotropinlike intermediate lobe peptide) in MS patients categorized as fatigued and non-fatigued on the basis of FSS scores. MethodsFor the study, a total of 54 (29 females, 25 males) patients diagnosed with RRMS including 26 with fatigue symptom (48.1%), and 26 healthy controls (13 females, 13 males) were enrolled. A FSS score ≥36 was considered as cut-off score to separate fatigued patients from nonfatigued patients. ResultsA significant positive correlation was determined between FSS score and NFI-MS scale, NFI-MS 1, NFI-MS 2, NFI-MS 3 and NFI-MS 4 scores. IL-1β, IL-10 and TNF-α levels did not differ between patient and control groups. IL-35 and IL-2 levels were significantly higher among MS patients (p<0.01). However, no difference was observed between fatigued and nonfatigued patients in the cytokines and HPA parameters studied. ACTH, cortisol and α-MSH were significantly higher in MS group (p=0.02, p<0.01 and p<0.01, respectively). CLIP level was significantly low in MS patient group (p<0.01). ConclusionNFI-MS scale is equally sensitive as FSS scale for assessment of MS fatigue; thus, it may also be widely used to evaluate that symptom. Generally HPA axis is hyperactive in MS patients, but it is not correlated with fatigue in our study. For the first time, levels of CLIP (a type of melanocortin) are studied, and determined to be lower among MS patients. Elevated levels of IL-35 and IL-2 suggest that these cytokines may have a prominent role in MS pathophysiology and can be investigated as potential targets for development of novel therapies.

Erratum to: Expression patterns of c-Fos early gene and phosphorylated ERK in the rat brain following 1-h immobilization stress: concomitant changes induced in association with stress-related sleep rebound

An immobilization stress (IS) of 1 h applied at the beginning of the dark phase is followed by a sleep rebound. During the restraint, serotonin released by the dorsal raphe nucleus within the arcuate area stimulates the availability of corticotropin-like intermediate lobe peptide (CLIP or ACTH18-39). Three hours after the restraint, CLIP, through its hypnogenic properties, contributes to the sleep rebound that follows the IS. Here, we immunohistochemically evaluated protein expression of the immediate early gene, c-Fos and phosphorylated extracellular signal-regulated kinase (p-ERK) in hypothalamic (preoptic area [POA], paraventricular nucleus [PVN], arcuate nucleus [ARC]) and brain stem (dorsal raphe [DR], locus coeruleus [LC]) nuclei involved in the acute response to stress and the subsequent stress-related sleep rebound (recovery period). Immediately after the 1-h restraint, c-Fos and p-ERK expression increased in all structures studied, particularly in PVN and LC. Three hours later, the number of p-ERK- and c-Fos-positive neurons was reduced in PVN and LC (p < 0.001) as well as in DR (p < 0.01) compared to control animals. In contrast, both c-Fos and p-ERK expression in POA neurons (p < 0.01) and c-Fos expression in ARC neurons (p < 0.001) were increased 3 h after the IS. The marked activation observed in PVN and LC nucleus immediately after the IS confirms that these structures are clearly reactive to stress. However, the high activity observed in POA and ARC neurons during the recovery period, not described to date, highlights the particular part played by these structures in the stress-related sleep rebound. An unbalance in the above processes may contribute to pathological outcomes, such as anxiety and depression.

Expression patterns of c-Fos early gene and phosphorylated ERK in the rat brain following 1-h immobilization stress: concomitant changes induced in association with stress-related sleep rebound

An immobilization stress (IS) of 1 h applied at the beginning of the dark phase is followed by a sleep rebound. During the restraint, serotonin released by the dorsal raphe nucleus within the arcuate area stimulates the availability of corticotropin-like intermediate lobe peptide (CLIP or ACTH18-39). Three hours after the restraint, CLIP, through its hypnogenic properties, contributes to the sleep rebound that follows the IS. Here, we immunohistochemically evaluated protein expression of the immediate early gene, c-Fos and phosphorylated extracellular signal-regulated kinase (p-ERK) in hypothalamic (preoptic area [POA], paraventricular nucleus [PVN], arcuate nucleus [ARC]) and brain stem (dorsal raphe [DR], locus coeruleus [LC]) nuclei involved in the acute response to stress and the subsequent stress-related sleep rebound (recovery period). Immediately after the 1-h restraint, c-Fos and p-ERK expression increased in all structures studied, particularly in PVN and LC. Three hours later, the number of p-ERK- and c-Fos-positive neurons was reduced in PVN and LC (p < 0.001) as well as in DR (p < 0.01) compared to control animals. In contrast, both c-Fos and p-ERK expression in POA neurons (p < 0.01) and c-Fos expression in ARC neurons (p < 0.001) were increased 3 h after the IS. The marked activation observed in PVN and LC nucleus immediately after the IS confirms that these structures are clearly reactive to stress. However, the high activity observed in POA and ARC neurons during the recovery period, not described to date, highlights the particular part played by these structures in the stress-related sleep rebound. An unbalance in the above processes may contribute to pathological outcomes, such as anxiety and depression.

Molecular cloning of propiomelanocortin cDNA and multi-tissue mRNA expression in Parasilurus asotus

We investigated the structure,evolution,and tissue specific expression of Parasilurus asotus proopiomelanocortin(POMC) cDNA using RT-PCR and RACE.The POMC cDNA was 1 099 bp in length and contained a 129 bp 5′UTR,a 322 bp 3′UTR,and a 648 bp open reading frame encoding a protein of 215 amino acids.The catfish POMC protein contained a signal peptide(SP,Met1-Ala28),an N-terminal peptide(Gln29-Asp102),adrenocorticotropic hormone(ACTH,Ser105-Met144),α-melanocyte stimulating hormone(α-MSH,Ser105-Val117),corticotropin-like intermediate lobe peptide(CLIP,Arg123–Met144),β-lipotropin(β-LPH,Glu145-Ser158),β-MSH(Asp161-Ser177),and β-Endorphin(β-EP,Tyr178-Leu215).Catfish POMC protein does not contain a γ-MSH region and the majority of the joining peptide and part of the γ-LPH were deleted.The protein shared the highest similarity(88%) with channel catfish(Parasilurus asotus) POMC.The most highly conserved active peptide was α-MSH,followed by β-MSH,β-EP,and CLIP whereas γ-MSH,joining peptide(JP),and γ-LPH were located in the most divergent region.The protein had a calculated molecular weight of 24.66 kD,a theoreti-cal isoelectric point of 7.25,was hydrophilic in most regions,and was rich in B cell antigenicity positions.Catfish POMC mRNA was primarily expressed in the pituitary and the concentration of mRNA was much higher in brain than in other tissues.

The Multiplicity of Post-Translational Modifications in Pro-Opiomelanocortin-Derived Peptides

The precursor protein, pro-opiomelanocortin (POMC) undergoes extensive post-translational processing in a tissue-specific manner to yield various biologically active peptides involved in diverse cellular functions. The recently developed method of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for direct tissue analysis has proved to be a powerful tool for investigating the distribution of peptides and proteins. In particular, topological mass spectrometry analysis using MALDI-MS can selectively provide a mass profile of the hormones included in cell secretory granules. An advantage of this technology is that it is possible to analyze a frozen thin slice section, avoiding an extraction procedure. Subsequently, tandem mass spectrometry (MS/MS) has a profound impact on addressing the modified residues in the hormone molecules. Based on these strategies with mass spectrometry, several interesting molecular forms of POMC-derived peptides have been found in the fish pituitary, such as novel sites of acetylation in α-melanocyte-stimulating hormone (MSH), hydroxylation of a proline residue in β-MSH, and the phosphorylated form of corticotropin-like intermediate lobe peptide.

Post-translational modifications of pro-opiomelanocrtin related hormones in medaka pituitary based on mass spectrometric analyses

Direct tissue matrix-assisted laser desorption ionization with time-of-flight mass spectrometry analysis provides a selective detection of mass profile for the peptides contained into cell secretory granules. By this mass spectrometry with slice of pituitary, two novel molecular forms of pro-opimelanocrtin related hormone were found in the orange-red strain medaka (Oryzias latipes var.). The structures of [N,O-diacetyl Serine1, O-acetyl Serine3]-α-melanocyte-stimulating hormone (MSH) and [hydroxyproline15]-β-MSH, together with [phosphoserine15]-corticotropin-like intermediate lobe peptide, were determined for the first time using a collision-induced dissociation with electrospray ionization mass spectrometry. A combination of mass spectrometry analyses is thus a powerful tool to lead to the elucidation of the post-translational processing from the pre-prohormone.

Effect of Corticotropin-Like Intermediate Lobe Peptide on Presynaptic and Postsynaptic Glutamate Receptors and Postsynaptic GABA Receptors in Rat Brain

We studied the effect of corticotropin-like intermediate lobe peptide (CLIP) on presynaptic NMDA receptors and postsynaptic GABA, NMDA, and AMPA receptors in rat brain. CLIP inhibited presynaptic and postsynaptic NMDA receptors, but potentiated postsynaptic GABA and AMPA receptors. Our results indicate that CLIP modulates function of ionotropic receptors for glutamate and GABA.

Mass spectrometric map of neuropeptide expression and analysis of the γ-prepro-tachykinin gene expression in the medaka ( Oryzias latipes) brain

Neuropeptides have important roles in modulating behavioral patterns such as social interaction. With the aim to determine the presence of neuropeptides known to be involved in social interaction as well as novel peptides, we used MALDI-TOF/MS to analyze neuropeptide profiles in some medaka brain regions. In the telencephalon, hypothalamus, and pituitary gland, 3, 6, and 10 peaks, respectively, were identified as neuropeptides (Arg-vasotocin [AVT], growth hormone-releasing hormone [GHRH], neuropeptide FF, substance P [SP], somatostatin-1 and -2, melanin-concentrating hormone [MCH], MCH gene-related peptide [Mgrp], melanocyte-stimulating hormone [MSH], corticotropin-like intermediate lobe peptide [CLIP], and β-endorphin). The neuropeptide profile of telencephalon similar to that of the hypothalamus, but completely different from that of pituitary gland. For the future genetic analysis, we identified cDNAs encoding precursor proteins for the identified peptides. We also detect its expression of γ-prepro-tachykinin gene encoding a SP precursor protein in both the telencephalon and hypothalamus. Our results indicated that the medaka brain contains some neuropeptides (AVT, SP, and somatostatins) that may be involved in modulating medaka behaviors such as social interaction.

Identification of Glioma Neovascularization-related Proteins by Using MALDI-FTMS and Nano-LC Fractionation to Microdissected Tumor Vessels

The identification of angiogenesis-related proteins is important for the development of new antiangiogenic therapies, and such proteins are potential new biomarkers for gliomas. The aim of this study was to identify proteins that are exclusively present in glioma neovasculature and not in the vasculature of normal brain. We combined advanced proteomics techniques to compare the expression profiles of microdissected blood vessels from glioma with blood vessels of normal control brain samples. We measured the enzymatic generated peptide profiles from these microdissected samples by MALDI-FTMS. Subsequently, the samples were fractionated by nano-LC prior to MALDI-TOF/TOF. This combined approach enabled us to identify four proteins that appeared to be exclusively expressed in the glioma blood vessels. Two of these proteins, fibronectin and colligin 2, were validated on tissue sections using specific antibodies. We found that both proteins are present in active angiogenesis in glioma, other neoplasms, and reactive conditions in which neoangiogenesis takes place. This work proves that gel-free mass spectrometric techniques can be used on relatively small numbers of cells generated by microdissection procedures to successfully identify differentially expressed proteins.

Expression of three proopiomelanocortin subtype genes and mass spectrometric identification of POMC-derived peptides in pars distalis and pars intermedia of barfin flounder pituitary

Proopiomelanocortin (POMC) is a common precursor of adrenocorticotropic hormone (ACTH), melanophore-stimulating hormone (MSH), and endorphin (END). In pituitary gland, POMC receives posttranslational processing by which different peptides are generated in the pars distalis (PD) and pars intermedia (PI). Recently, we cloned three subtypes of the POMC gene in pituitary gland of barfin flounder. The present study was undertaken to elucidate whether the three POMC genes are expressed in both the PD and PI of barfin flounder pituitary, and to identify peptides derived from POMCs in these lobes. We amplified the transcripts of POMC-A, -B and -C in both the PD and PI by the reverse transcription-polymerase chain reaction. In situ hybridization also detected signals for these three subtypes in the PD and PI. These results demonstrated that all three POMC genes are expressed in both the PD and PI of barfin flounder pituitary. By mass spectrometric analyses, ACTH-A, Des-acetyl (Ac)-α-MSH-A/B (amino acid sequence of α-MSH-A is identical to that of α-MSH-B), β-MSH-A, corticotropin-like intermediate lobe peptide (CLIP)-A, and N-terminal peptide ( N-POMC)-A were identified in the PD. Moreover, Des-Ac-α-MSH-A/B, α-MSH-A/B, β-MSH-A and -B, N-β-lipotropin-A, CLIP-A, N-Ac-β-END-A 1–41 (C-terminally truncated form of N-Ac-β-END-A), and N-POMC-A were identified in the PI. Predominant detection of POMC-A-derived peptides indicates the greatest production of POMC-A and no detection of POMC-C-derived peptides indicates the lowest production of POMC-C in both the PD and PI. ACTH-A is specifically produced in the PD, however, the occurrence of Des-Ac-α-MSH-A, CLIP-A, and β-MSH-A shows that the entire POMC-A is further cleaved into small peptides as in the PI. In the PI, some peptides receive modification or truncation as shown by the occurrence of α-MSH-A/B and N-Ac-β-END-A 1–41. These results show differential posttranslational processing of POMC between the PD and PI in barfin flounder pituitary.

Trends in the evolution of the proopiomelanocortin gene

The POMC gene is perhaps the most extensively studied member of the opioid/orphanin gene family. In Phylum Chordata this gene has been characterized in representatives of every class within the Gnathostomata, as well as in one representative agnathan vertebrate, the marine lamprey. This review provides a systematic overview of trends in the evolution of the melanocortins (ACTH/α-MSH, β-MSH, γ-MSH, and δ-MSH) and β-endorphin in gnathostomes, and advances the hypothesis that the appearance of γ-MSH occurred early in the radiation of the gnathostomes. A summary of the extensive work on POMC genes in the marine lamprey is also provided, as well as a reevaluation of the conserved regions in the sequence of CLIP (corticotropin-like-intermediate lobe peptide) in the POMC sequences of the various groups of gnathostomes.

Molecular cloning of proopiomelanocortin cDNA and multi-tissue mRNA expression in channel catfish

Channel catfish ( Ictalurus punctatus) proopiomelanocortin ( POMC) cDNA was cloned to investigate its structure, evolution, and expression in different tissues. POMC is an important gene in the hypothalamus–pituitary–adrenal axis, the main mediator of the stress response. POMC gene was isolated from a pituitary cDNA library and nucleotide sequence was determined. POMC cDNA is composed of 1164 nucleotides with a 639 nucleotide open reading frame encoding a protein of 212 amino acids. Catfish POMC protein contains a signal peptide (SP, Met 1–Ala 28), N-terminal peptide (Gln 29–Glu 101), adrenocorticotropic hormone (ACTH, Ser 104–Met 142), α-melanocyte stimulating hormone (α-MSH, Ser 104–Val 116), corticotropin-like intermediate lobe peptide (CLIP, Arg 121–Met 142), β-lipotropin (β-LPH, Glu 145–His 212), γ-lipotropin (γ-LPH, Glu 145– Ser 177), β-MSH (Asp 161–Ser 177), and β-endorphin (β-EP, Tyr 180–His 212). Catfish POMC protein does not contain a γ-MSH region and most of the joining peptide and part of the γ-LPH are deleted. Protein sequence alignment showed the highest similarity with the carp ( Cyprinus carpio) POMC I (66.5%) and POMC II (67%), while the sea lamprey ( Petromyzon marinus) POC (17.9%) and POM (18.8%) were the most divergent. The average similarity was 46.95% among the 44 POMC proteins from 36 different species analyzed. Compared to the POMC mRNA levels in the pituitary, the concentration of the POMC mRNA was 0.0594% in the anterior kidney and 0.0012–0.0045% in all the other tissues except in the skin where the lowest expression (0.0005%) was observed. Overall architecture of channel catfish POMC is highly similar to those from other teleosts.

Identification of proopiomelanocortin-related peptides in the rostral pars distalis of the pituitary in coelacanth: evolutional implications

The coelacanth fish, genus Latimeria, flourished during the Devonian Period and is considered among the closest living relatives of tetrapods. It may therefore provide important information on the evolution of fishes into tetrapods. However, little is known about the components of the endocrine system in this fish. Here we describe the structural characterization of pituitary hormones derived from proopiomelanocortin (POMC) in Latimeria chalumnae. We identified α-melanocyte-stimulating hormone (MSH), N–Des-acetyl–α-MSH, β-MSH, N-terminal peptide containing γ-MSH, corticotropin-like intermediate lobe peptide (CLIP), and N–acetyl–β-endorpin (END) in an extract from the rostral pars distalis of the pituitary by reversed-phase high-performance liquid chromatography, amino acid sequence analysis, and mass spectrometry. The occurrence of three different MSHs and one β-END indicates that the structural organization of coelacanth POMC is the same as that of lungfish, tetrapods, and primitive ray-finned fish. The coelacanth α-MSH is identical to its mammalian counterpart. The coelacanth β-MSH shows the highest sequence identity with the amphibian counterpart, and γ-MSH and CLIP show the highest sequence identity with their amphibian and bird counterparts, whereas coelacanth β-END is most similar to the sturgeon peptide. The coexistence of tetrapod-type and fish-type characteristics in the putative coelacanth POMC molecule reflects the phylogenetic position of this fish. When each hormonal segment was compared between coelacanth, lungfish, and tetrapod, MSH and CLIP of coelacanth were closer to their tetrapod counterparts than those of lungfish, whereas β-MSH and β-END of coelacanth are less closely related to their tetrapod counterparts than those of lungfish. γ-MSH and CLIP may have evolved at a different rate from β-MSH and β-END in both the coelacanth and lungfish.

Identification of carp proopiomelanocortin-related peptides and their effects on phagocytes

We report the immunomodulating effects of proopiomelanocortin (POMC)-related peptides on phagocytic cells in carp. The complete amino acid sequences of two carp POMCs (I and II) were deduced from the nucleotide sequences after cDNA cloning. Both POMCs consist of 194 amino acids (91% sequence identity) including identical α-melanotropin (MSH) and β-endorphin (EP). All hormonal peptides derived from two POMCs were identified by mass spectrometry after separation by high-performance liquid chromatography of an acid-acetone extract from a single pituitary. These peptides were α-MSH,N -Des-Ac-α-MSH, di-Ac-α-MSH, β-MSH I, β-MSH-II, N -Ac-β-EP(1–29), corticotropin-like intermediate lobe peptide I and II and N -terminal peptide of POMC I and II. The immunomodulating effects of synthetic MSHs and EPs on phagocytic cells from carp head kidney were examined. Di-Ac-α-MSH, β-MSH I,N -Ac-β-EP(1–29) and β-EP(1–29) increased the production of superoxide anion at 0·1–100ng ml−1for these MSHs and 1–100ng ml−1for EPs in RPMI 1640 medium.

Influence of a 1-h immobilization stress on sleep and CLIP (ACTH 18–39) brain contents in adrenalectomized rats

Basal sleep amounts in adrenalectomized rats (AdX), as compared to intact animals, exhibit a significant increase in slow-wave sleep (SWS), a tendency towards an increase in paradoxical sleep (PS), and circadian rhythms (SWS and PS) flattened in amplitude. An immobilization stress (IS) of 1 h, imposed on AdX rats at the beginning of the dark period, is accompanied by an intense polygraphic waking. Just after the IS, SWS amount become significantly higher than in control rats (+44%/11 h of darkness) whereas significant increases of PS occur only 5–10 h after the IS (+24%/11 h of darkness). A specific radioimmunoassay for CLIP (corticotropin-like intermediate lobe peptide or ACTH 18–39) was performed in biopsies taken either from the nucleus raphe dorsalis (nRD) or the arcuate nucleus (AN). In the nRD, just after the IS, phosphorylated CLIP (Ph-CLIP) concentration exhibits a decreasing tendency, but 4 h later, it increases significantly (+22%, p<0.05). In the AN, Ph-CLIP concentration remains unchanged after the IS as well as 4 h later. These results differ from those previously reported in intact animals also submitted to a 1-h IS, that is, a SWS rebound less marked (+27%/11 h of darkness), a PS rebound more important starting immediately after the IS (+46%/11 h of darkness) and a significant increase in Ph-CLIP occurring just after the end of the restraint. In conclusion, data obtained after a restraint stress either in AdX or in control rats point out the dependence of the PS rebound on the nRD Ph-CLIP concentration.

Immunocytochemical and ultrastructural characterization of melanotropin and adrenocorticotropin cells from the Mediterranean yellowtail (Seriola dumerilii, Risso 1810).

Melanotropin (MSH) and adrenocorticotropin (ACTH) are pituitary hormones derived from a common precursor: the proopiomelanocortin (POMC), which is processed differently in the melanotropic and corticotropic cells of several vertebrates. While ACTH is a major final product in corticotropes, it is further processed into alpha-MSH and corticotropin-like intermediate lobe peptide (CLIP) in melanotropes. Cells which are immunoreactive to ACTH (ACTH cells) and to both alpha-MSH and ACTH (MSH cells) have been described in a number of teleosts, including the Mediterranean yellowtail, by light microscopic immunocytochemistry. However, these cells have been ultrastructurally characterized only in a few species. In this paper, we use electron microscopy to identify and characterize the cells producing MSH and ACTH in M. yellowtail (Seriola dumerilii). Pituitaries from adult specimens were dissected and processed for conventional and immunocytochemical electron microscopy. An immunogold technique was performed using anti-synthetic alpha-MSH and anti-human (h) ACTH (1-24) sera. MSH cells had round secretory granules with a granular content of varying electron density and compactness, which were immunogold-labeled with anti-alpha-MSH. Homogeneous and electron-dense secretory granules found in the Golgi area of these cells reacted with both anti-alpha-MSH and anti-hACTH (1-24). ACTH cells had round secretory granules with a homogeneous and medium or high electron-dense core and narrow clear halo, which were grouped in the cell area near the neurohypophysis (NH). Some granules showed an osmiophilic semicore in the medium electron-dense content, which has not been described in other teleost pituitary cells. Immunogold-labeling over the secretory granules only was obtained with all the antisera used. Some ACTH cells showed involutive features. MSH and ACTH are respective final products of the POMC in two ultrastructurally different cells of the pituitary of M. yellowtail, MSH and ACTH cells. The immature granules in the Golgi area of MSH cells seem to be the site of proteolitic cleavage of ACTH into alpha-MSH and CLIP.

Immunocytochemical and ultrastructural characterization of melanotropin and adrenocorticotropin cells from the mediterranean yellowtail (Seriola dumerilii, Risso 1810)

Background Melanotropin (MSH) and adrenocorticotropin (ACTH) are pituitary hormones derived from a common precursor: the proopiomelanocortin (POMC), which is processed differently in the melanotropic and corticotropic cells of several vertebrates. While ACTH is a major final product in corticotropes, it is further processed into α-MSH and corticotropin-like intermediate lobe peptide (CLIP) in melanotropes. Cells which are immunoreactive to ACTH (ACTH cells) and to both α-MSH and ACTH (MSH cells) have been described in a number of teleosts, including the Mediterranean yellowtail, by light microscopic immunocytochemistry. However, these cells have been ultrastructurally characterized only in a few species. In this paper, we use electron microscopy to identify and characterize the cells producing MSH and ACTH in M. yellowtail (Seriola dumerilii). Methods Pituitaries from adult specimens were dissected and processed for conventional and immunocytochemical electron microscopy. An immunogold technique was performed using anti-synthetic α-MSH and anti-human (h) ACTH (1-24) sera. Results MSH cells had round secretory granules with a granular content of varying electron density and compactness, which were immunogold-labeled with anti-α-MSH. Homogeneous and electron-dense secretory granules found in the Golgi area of these cells reacted with both anti-α-MSH and anti-hACTH (1-24). ACTH cells had round secretory granules with a homogeneous and medium or high electron-dense core and narrow clear halo, which were grouped in the cell area near the neurohypophysis (NH). Some granules showed an osmiophilic semicore in the medium electron-dense content, which has not been described in other teleost pituitary cells. Immunogold-labeling over the secretory granules only was obtained with all the antisera used. Some ACTH cells showed involutive features. Conclusions MSH and ACTH are respective final products of the POMC in two ultrastructurally different cells of the pituitary of M. yellowtail, MSH and ACTH cells. The immature granules in the Golgi area of MSH cells seem to be the site of proteolitic cleavage of ACTH into α-MSH and CLIP. Anat. Rec. 249:74–80, 1997. © 1997 Wiley-Liss, Inc.

Influence of a 1 h immobilization stress on sleep states and corticotropin-like intermediate lobe peptide (CLIP or ACTH 18–39, Ph-ACTH 18–39) brain contents in the rat

A 1 h immobilization stress (IS) was imposed to rats at the beginning of the dark period, i.e., when the animals start to be active. The IS was accompanied by an intense polygraphic waking and followed, over 12 h of the dark period, by a significant rebound of slow-wave sleep (SWS, +17%) and paradoxical sleep (PS, +57%). In order to estimate the IS-related changes in the endogenous concentrations of corticotropin-like intermediate lobe peptide (CLIP, ACTH 18–39) and related compounds, a specific radioimmunoassay (RIA) was used. Assays performed in cerebral biopsies taken from arcuate (AN) and raphe dorsalis (nRD) nuclei led to the obtention of 2 main immunoreactive peaks, corresponding to CLIP and its phosphorylated form Ph-CLIP. Just after the end of the IS and within the nRD, Ph-CLIP immunoreactivity increased by about 95%. Four hours later, i.e., when PS rebound was maximal, a 37% increase in Ph-CLIP immunoreactivity was measured in the AN. These observations have never been described before. In the blood, at the end of the restraint, CLIP/ACTH 1–39 total immunoreactivity was increased by 330%. It returned to baseline level 4 h later. Blood concentration of corticosterone was also increased by 56% at the end of the IS and was close to baseline level 4 h later. Data reported here indicate that the IS first triggers an increase in Ph–CLIP within the nRD. Since the nRD contains sleep permissive components, this increase might be determinant for the SWS and PS rebound induction. The changes observed in the blood as regards CLIP/ACTH 1–39 total immunoreactivity and corticosterone concentration testify to the efficacy of the IS and are part of the conventional picture accompanying such a situation. Finally, the increase in Ph-CLIP, occurring in the AN 4 h after the end of the restraint, might be part of the restorative processes necessary to compensate the stress overshoot. © 1997 Elsevier Science B.V. All rights reserved.