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Abstract: Mucosal inflammation in patients with inflammatory bowel disease (IBD) is characterized by an alteration of prohormone chromogranin A (CgA) production. The recent demonstration of an implication of CgA in collagenous colitis and immune regulation provides a potential link between CgA-derived peptides (catestatin, CTS) and gut inflammation. Colitis was induced by administration of dextran sulfate sodium or 2, 4 dinitrobenzenesulfonic acid to C57BL/6 mice. Treatment with human (h)CTS or its proximal or distal part was started one day before colitis induction and colonic inflammatory markers were determined. Pro-inflammatory cytokines were evaluated in peritoneal isolated and bone marrow derived macrophages (BMDMs); p-STAT3 level was studied. Serum levels of CgA and CTS were assessed in experimental colitis and in a separate study in IBD patients and healthy controls. We show that sera from IBD patients and that in experimental colitis conditions the colonic level of mouse (m)CgA and mCTS are significantly increased. Moreover, in vivo treatment with human (h)CTS reduces the disease onset and suppresses exacerbated inflammatory responses in preclinical settings of colitis associated with an increase of p-STAT3. In vitro, hCTS treatment decreases proinflammatory cytokine release by peritoneal macrophages and BMDMs and increases p-STAT3 levels. These results support the hypothesis that CTS is increased during colitis and that hCTS modulates intestinal inflammation via the macrophage population and through a STAT-3 dependent pathway in a murine model of colitis. Identification of the molecular mechanism underlying the protective role of this peptide may lead to a novel therapeutic option in IBD. [Copyright &y& Elsevier]

The nociceptive effects of i.p administration of a synthetic peptide (CgA4-16) derived from chromogranin A (CgA) were studied on a model of inflammatory (somato-visceral) pain. Inflammatory mediators participate in controlling the activity of enterochromaffin cells that store and release chromogranins. Adult male Wistar rats were injected i.p with diluted acetic acid (AA) to induce abdominal writhes. Pharmacological agents were injected prior to CgA4-16 and/or AA together. While i.p CgA4-16 alone did not produce any effect, the peptide increased the number of abdominal constrictions induced by i.p AA administration in a dose-related manner. To determine the possible mechanisms involved in CgA4-16 produced pronociceptive effect, i.p diltiazem or indomethacin were tested. The pronociceptive effect induced by CgA4-16 was blocked by pretreatment of either substance. I.p administration of CGRP, substance P (SP) or capsaicin evoked dose-related abdominal writhing. CgA4-16, 20 min prior to CGRP or capsaicin, potentiated the nociceptive effects induced by CGRP or capsaicin, but not those induced by SP. Taken together, these data suggest for the first time that a CgA-derived peptide may modulate inflammatory pain. [Copyright &y& Elsevier]

Chromogranin A (CgA) is an acidic protein identified within a large variety of endocrine cells. Colocalized with catecholamines in chromaffin cells, CgA is a prohormone precursor of small biologically active peptides. Vasostatin (CgA 1–76) is the most conserved fragment of CgA and chromogranin A 47–66 peptide (CgA 47–66) possesses potent antimicrobial activities. The aim of this study was to test the hypothesis that CgA 47–66 may be involved in mechanisms modulating nociception. Thus, we used acetic acid (AA) which produces a delayed inflammatory response and episodes of abdominal writhing, a marker of pain, when injected intraperitoneally (i.p.) to rats.Administration (i.p.) of CgA 47–66 induced specific opposite dose-dependent effects depending on concentration. That is, CgA 47–66 below 0.5 mg/kg produced antinociceptive effects, whereas at 2 mg/kg it produced a marked pronociceptive effect. The latter effect was blocked by diltiazem and indomethacin.CgA 47–66-induced antinociceptive effects on AA-induced responses were reversed when the corticotropin-releasing factor (CRF) antagonist alpha-helical CRF 9–41 was i.p. injected to animals prior to AA and CgA 47–66 administration.The administration of i.p. calcitonin gene-related peptide (CGRP) or substance P (SP) evoked dose-dependent abdominal writhing; this effect was abolished when CgA 47–66 was injected. The present data suggest, for the first time, that a fragment of CgA, CgA 47–66, possesses potent antinociceptive effects at low doses. Although the mechanism triggered by this peptide is unknown, CRF receptors are likely to be involved. [Copyright &y& Elsevier]

Endogenous chromogranin A (CgA)-derived peptides are secreted by nervous, endocrine and immune cells. Chromofungin (Chr: CgA47-66) is one of these peptides that display antimicrobial activities and activate neutrophils, with important implications in inflammation and innate immunity. The aim of the present study is to examine the effects of Chr on isolated and Langendorff perfused rat hearts. The study was performed by using the isolated and Langendorff perfused rat hearts, Elisa assay and real-time PCR. We found that, under basal conditions, increasing doses (11–165 nM) of Chr induced negative inotropic effects without changing coronary pressure. This action was mediated by the AKT/eNOS/cGMP/PKG pathway. We also found that Chr acted as a postconditioning (PostC) agent against ischemia/reperfusion (I/R) damages, reducing infarct size and LDH level. Cardioprotection involved PI3K, RISK pathway, MitoKATP and miRNA-21. We suggest that Chr directly affects heart performance, protects against I/R myocardial injuries through the activation of prosurvival kinases. Results may propose Chr as a new physiological neuroendocrine modulator able to prevent heart dysfunctions, also encouraging the clarification of its clinical potential. [ABSTRACT FROM AUTHOR]

Abstract: Cytoskeleton scaffold in cardiac myocytes provides structural support and compartmentalization of intracellular components. It is implicated in cardiac pathologies including hypertrophy and failure, playing a key role in the determinism of contractile and diastolic dysfunctions. Chromogranin A (CgA) and its derived peptides have revealed themselves as novel cardiovascular modulators. In humans, normal CgA levels considerably increase in several pathologies, including heart failure. Recent data have shown on the unstimulated rat heart that human recombinant Vasostatin-1 (hrVS-1) and rat chromogranin A 1-64 (rCgA1-64) induce negative inotropic and lusitropic effects counteracting the β-adrenergic-dependent positive inotropism with a functional non-competitive antagonism. This study investigates, on the isolated Langendorff perfused rat heart, whether cardiac cytoskeleton is involved in the modulation of contractility and relaxation exerted by hrVS-1 and rCgA1-64. Cytoskeleton impairment by either cytochalasin-D (actin polymerization inhibitor), BDM (myosin ATP-ase antagonist) or wortmannin (inhibitor of PI3-K/Akt transduction cascade), or W-7 (calcium–calmodulin antagonist) abolished hrVS-1 and rCgA1-64-mediated inotropism and lusitropism. Using fluorescent phalloidin, we showed on rat cardiac H9C2 cells that hrVS-1 (10nM÷10µM) stimulates actin polymerization. Taken together these data indicate that in the rat heart, the actin cytoskeletal network strongly contributes to the cardiotropic action of CgA-derived peptides. [ABSTRACT FROM AUTHOR]

Abstract: Chromogranins/secretogranins are members of the granin family present in secretory vesicles of nervous, endocrine and immune cells. In chromaffin cells, activation of nicotinic cholinergic receptors induces the release, with catecholamines, of bioactive peptides resulting from a natural processing. During the past decade, our laboratory has characterized new antimicrobial chromogranin-derived peptides in the secretions of stimulated bovine chromaffin cells. They act at the micromolar range against bacteria, fungi, yeasts, and are non-toxic for the mammalian cells. They are recovered in several biological fluids involved in defence mechanisms (human serum, neutrophil secretions and saliva). These new antimicrobial peptides demonstrate the major role of the adrenal medulla in innate immunity. In this review we focus on the antimicrobial peptides derived from human and bovine chromogranin A (CGA), chromogranin B (CGB) and secretogranin II (SGII) emphasizing their direct action against pathogens and their effects on immune cells. [ABSTRACT FROM AUTHOR]

Designed histidine-rich amphipathic cationic peptides, such as LAH4, have enhanced membrane disruption and antibiotic properties when the peptide adopts an alignment parallel to the membrane surface. Although this was previously achieved by lowering the pH, here we have designed a new generation of histidine-rich peptides that adopt a surface alignment at neutral pH. In vitro, this new generation of peptides are powerful antibiotics in terms of the concentrations required for antibiotic activity; the spectrum of target bacteria,fungi,and parasites;and the speed with which they kill. Further modifications to the peptides, including the addition of more hydrophobic residues at the N terminus, the inclusion of a helix-breaking proline residue or using D-amino acids as building blocks, modulated the biophysical properties of the peptides and led to substantial changes in toxicity to human and parasite cells but had only a minimal effect on the antibacterial and antifungal activity. Using a range of biophysical methods, in particular solid-state NMR, we show that the peptides are highly efficient at disrupting the anionic lipid component of model membranes. However, we also show that effective pore formation in such model membranes may be related to, but is not essential for, high antimicrobial activity by cationic amphipathic helical peptides. The information in this study comprises a new layer of detail in the understanding of the action of cationic helical antimicrobial peptides and shows that rational design is capable of producing potentially therapeutic membrane active peptides with properties tailored to their function. [ABSTRACT FROM AUTHOR]

We report for the first time that morphine-6-glucuronide, a highly analgesic morphine-derived molecule, is present in adrenal chromaffin granules and secreted from chromaffin cells upon stimulation. We also demonstrate that phosphatidylethanolamine-binding protein (alternatively named Raf-1 kinase inhibitor protein or RKIP) acts as an endogenous morphine-6-glucuronide-binding protein. An UDP-glucuronosyltransferase 2B-like enzyme, described to transform morphine into morphine-6-glucuronide, has been immunodetected in the chromaffin granule matrix, and morphine-6-glucurohide de novo synthesis has been characterized, demonstrating the possible involvement of intragranular UDP-glucuronosyltransferase 2B-like enzyme in morphine-6-glucuronide metabolism. Once secreted into the circulation, morphine-6-glucuronide may mediate several systemic actions (e.g. on immune cells) based on its affinity for μ-opioid receptors. These activities could be facilitated by phosphatidylethanolamine-binding protein (PEBP), acting as a molecular shield and preventing morphine-6-glucuronide from rapid clearance. Taken together, our data represent an important observation on the role of morphine-6-glucuronide as a new endocrine factor. [ABSTRACT FROM AUTHOR]

Abstract: Vasostatin-I (CgA1-76) is a naturally occurring and biologically active N-terminal peptide derived from chromogranin A (CgA), produced and secreted at high concentrations by neuroendocrine tissues and also from a range of neuroendocrine tumors. This study aims to examine the hypothesis that in the absence of classical protein receptors CgA1-76 may, like its two derived peptides CgA1-40 and CgA47-66, perturb the lipid microenvironment of other membrane receptors, as a basis for the largely inhibitory activities of these CgA peptides. The nature of the interactions between phospholipids and vasostatin-derived fragments was studied in the Langmuir film balance apparatus at 37 °C. The synthetic peptides CgA1-40 and CgA47-66 and a recombinant fragment (VS-I) containing vasostatin-I (Ser-Thr-Ala-CgA1-78) were compared for their effects on monolayers of phosphatidylcholine and phosphatidylethanolamine from pig brain and defined species of phosphatidylserine. Marked differences in surface pressure–area isotherms and phase-transition plateaus were apparent with the three classes of phospholipids on VS-I, CgA1-40 and CgA47-66 in physiological buffer or pure water. The results indicate that VS-I and CgA47-66 at 5–10 nM concentrations may engage in electrostatic as well as hydrophobic interactions with membrane-relevant phospholipids at physiological conditions, VS-I in particular enhancing the fluidity of saturated species of phosphatidylserine. [Copyright &y& Elsevier]

Abstract: Chromogranin A (CgA), an acidic granule protein of the regulated secretory pathway in the diffuse neuroendocrine system, is postulated to serve as a prohormone for regulatory peptides. Betagranin (rCgA1–128), the first N-terminal cleavage product of rat CgA, is 87% homologous to the bovine vasostatin I (bCgA1–76), previously shown to be vasoinhibitory in bovine resistance arteries. In this study the vasoactivity of homologous rat and bovine peptides was investigated in the rat posterior cerebral artery. Firstly, we examined the interaction of rhodamine (Rh)-labelled bCgA7–40 and bCgA47–70 with elements of the arterial wall by fluorescence microscopy. Secondly, rCgA7–57, bCgA1–40, bCgA7–40 and bCgA47–66 (chromofungin) were studied for effects on arterial tone and intracellular calcium as function of pressure in an arteriograph. Although without dilator or constrictor responses at 60–150 mm Hg, the rat peptide (rCgA7–57) evoked a significant delay in the onset of forced dilatation at 170 mm Hg, in contrast to the bovine peptides bCgA1–40, bCgA7–40 and bCgA47–66 (chromofungin). Neither Rh-bCgA7–40 nor Rh-bCgA47–70 stained the endothelial layer, while Rh-bCgA47–70 but not Rh-bCgA7–40 stained the smooth muscle compartment. Analogously, bCgA47–66 but not bCgA7–40 reduced intracellular calcium, however without modifying the myogenic response. Thus, the betagranin peptide rCgA7–57 and the two bovine chromofungin-containing peptides, highly homologous to the corresponding sequence (rCgA47–66), affected the rat cerebral artery without vasodilator effects, indicating significant species differences in vasoactivity of the N-terminal domain of CgA. [Copyright &y& Elsevier]

Phosphatidylethanolamine-binding protein (PEBP), alternatively named Raf-1 kinase inhibitor protein, is the precursor of the hippocampal cholinergic neurostimulating peptide (HCNP) corresponding to its natural N-terminal fragment, previously described to be released by hippocampal neurons. PEBP is a soluble cytoplasmic protein, also associated with plasma and reticulum membranes of numerous cell types. In the present report, using biochemistry and cell biology techniques, we report for the first time the presence of PEBP in bovine chromaffin cell, a well described secretion model. We have examined its presence at the subcellular level and characterized this protein on both secretory granule membranes and intragranular matrix. In addition, its presence in bovine chromaffin cell and platelet exocytotic medium, as well as in serum, was reported showing that it is secreted. Like many other proteins that lack signal sequence, PEBP may be secreted through non-classic signal secretory mechanisms, which could be due to interactions with granule membrane lipids and lipid rafts. By two-dimensional liquid chromatography-tandem mass spectrometry, HCNP was detected among the intragranular matrix components. The observation that PEBP and HCNP were secreted with catecholamines into the circulation prompted us to investigate endocrine effects of this peptide on cardiovascular system. By using as bioassay an isolated and perfused frog (Rana esculenta) heart preparation, we show here that HCNP acts on the cardiac mechanical performance exerting a negative inotropism and counteracting the adrenergic stimulation of isoproterenol. All together, these data suggest that PEBP and HCNP might be considered as new endocrine factors involved in cardiac physiology. [ABSTRACT FROM AUTHOR]

The negative inotropic effects of synthetic peptides derived from the N-terminus of chromogranin A (CgA) were studied in an avascular model of the vertebrate myocardium, the isolated working frog heart (Rana esculenta). The peptides were frog and bovine CgA4–16 and CgA47–66, and bovine CgA1–40 with (CgA1–40SS) and without an intact disulfide bridge (CgA1–40SH). Under basal cardiac conditions, four of the peptides caused a concentration-dependent negative inotropism that was comparable to the negative inotropy reported for human recombinant vasostatin I (CgA1–78) and bovine CgA7–57. By comparison of the structural characteristics of the bovine and frog sequences with their minimally effective concentrations ranging from 68 to 125 nM of peptide, the results were consistent with the natural structure (CgA17–38SS) being essential for the negative inotropism. In addition, the partial sequences of the frog and bovine vasostatin I were effective in counteracting the characteristic positive inotropism exerted by isoproterenol (1 nM) at minimally effective concentrations ranging from 45 to 272 nM. Taken together, these results extend the first evidence for a cardiosuppressive role of the N-terminal domain of chromogranin A known for its co-storage with catecholamines in the sympathoadrenal system of vertebrates. [Copyright &y& Elsevier]

[Display omitted] • Ctl mitigated the LPS-induced cell injury in H9c2 cardiomyocytes. • Ctl modulated TLR4 through a direct binding to the partner protein MD-2. • Ctl suppressed the LPS-dependent inflammation and oxidative stress in H9c2 cells. Global public health is threatened by new pathogens, antimicrobial resistant microorganisms and a rapid decline of conventional antimicrobials efficacy. Thus, numerous medical procedures become life-threating. Sepsis can lead to tissue damage such as myocardium inflammation, associated with reduction of contractility and diastolic dysfunction, which may cause death. In this perspective, growing interest and attention are paid on host defence peptides considered as new potential antimicrobials. In the present study, we investigated the physiological and biochemical properties of Cateslytin (Ctl), an endogenous antimicrobial chromogranin A-derived peptide, in H9c2 cardiomyocytes exposed to lipopolysaccharide (LPS) infection. We showed that both Ctl (L and D) enantiomers, but not their scrambled counterparts, significantly increased cardiomyocytes viability following LPS, even if L -Ctl was effective at lower concentration (1 nM) compared to D -Ctl (10 nM). L -Ctl mitigated LPS-induced LDH release and oxidative stress, as visible by a reduction of MDA and protein carbonyl groups content, and by an increase of SOD activity. Molecular docking simulations strongly suggested that L -Ctl modulates TLR4 through a direct binding to the partner protein MD-2. Molecular analyses indicated that the protection mediated by L -Ctl against LPS-evoked sepsis targeted the TLR4/ERK/JNK/p38-MAPK pathway, regulating NFkB p65, NFkB p52 and COX2 expression and repressing the mRNA expression levels of the LPS-induced proinflammatory factors IL‐1β, IL‐6, TNF‐α and NOS2. These findings indicate that Ctl could be considered as a possible candidate for the development of new antimicrobials strategies in the treatment of myocarditis. Interestingly, L -enantiomeric Ctl showed remarkable properties in strengthening the anti-inflammatory and anti-oxidant effects on cardiomyocytes. [ABSTRACT FROM AUTHOR]