BackgroundMammalian cells synthesize morphine and the respective biosynthetic pathway has been elucidated. Human neutrophils release this alkaloid into the media after exposure to morphine precursors. However, the exact role of endogenous morphine in inflammatory processes remains unclear. We postulate that morphine is released during infection and can be determined in the serum of patients with severe infection such as sepsis.MethodologyThe presence and subcellular immunolocalization of endogenous morphine was investigated by ELISA, mass spectrometry analysis and laser confocal microscopy. Neutrophils were activated with Interleukin-8 (IL-8) or lipopolysaccharide (LPS). Morphine secretion was determined by a morphine-specific ELISA. μ opioid receptor expression was assessed with flow cytometry. Serum morphine concentrations of septic patients were determined with a morphine-specific ELISA and morphine identity was confirmed in human neutrophils and serum of septic patients by mass spectrometry analysis. The effects of the concentration of morphine found in serum of septic patients on LPS-induced release of IL-8 by human neutrophils were tested.Principal FindingsWe confirmed the presence of morphine in human neutrophil extracts and showed its colocalisation with lactoferrin within the secondary granules of neutrophils. Morphine secretion was quantified in the supernatant of activated human polymorphonuclear neutrophils in the presence and absence of Ca2+. LPS and IL-8 were able to induce a significant release of morphine only in presence of Ca2+. LPS treatment increased μ opioid receptor expression on neutrophils. Low concentration of morphine (8 nM) significantly inhibited the release of IL-8 from neutrophils when coincubated with LPS. This effect was reversed by naloxone. Patients with sepsis, severe sepsis and septic shock had significant higher circulating morphine levels compared to patients with systemic inflammatory response syndrome and healthy controls. Mass spectrometry analysis showed that endogenous morphine from serum of patient with sepsis was identical to poppy-derived morphine.ConclusionsOur results indicate that morphine concentrations are increased significantly in the serum of patients with systemic infection and that morphine is, at least in part, secreted from neutrophils during sepsis. Morphine concentrations equivalent to those found in the serum of septic patients significantly inhibited LPS-induced IL-8 secretion in neutrophils.

Magainins are antimicrobial peptides isolated from the African clawed frog Xenopus laevis. They interact with bacterial membranes where they cause channel formation and membrane disruption. When added as a cocktail magainin 2 and PGLa are considerably more efficient when compared to the corresponding amounts of individual components. In order to investigate this synergistic interaction of PGLa and magainin a number of magainin variants have been prepared and investigated in biological and biophysical assays. In particular we report on the antimicrobial activities and solid-state NMR investigations of magainins that have been extended by a carboxyterminal GGC tripeptide to form covalently linked dimers. Notably, when the formation of the covalent linkage is prevented by exchanging the cystein by serine or alanine no loss in efficiency was observed indicating that the covalent interaction is not necessary for synergistic interaction. In a next step peptides labelled with (15)N and (2)H were reconstituted into oriented membranes and their topology studied by solid-state NMR spectroscopy. The tendency of some of these peptides to adopt membrane-spanning alignments does not correlate with their synergistic activities in antimicrobial assays. In contrast, the stable alignment of PGLa parallel to the surface of membranes made of Escherichia coli lipid extracts is strongly suggestive that the peptides develop synergistic activities when in an in-planar configuration. Notably, the phospholipid head groups of these samples show a high degree of disturbance suggesting that the synergistic interactions between the magainin peptides could be mediated through the lipid phase.

BackgroundMorphine, the principal active agent in opium, is not restricted to plants, but is also present in different animal tissues and cell types, including the mammalian brain. In fact, its biosynthetic pathway has been elucidated in a human neural cell line. These data suggest a role for morphine in brain physiology (e.g., neurotransmission), but this hypothesis remains a matter of debate. Recently, using the adrenal neuroendocrine chromaffin cell model, we have shown the presence of morphine-6-glucuronide (M6G) in secretory granules and their secretion products, leading us to propose that these endogenous alkaloids might represent new neuroendocrine factors. Here, we investigate the potential function of endogenous alkaloids in the central nervous system.Methodology and Principal FindingsMicroscopy, molecular biology, electrophysiology, and proteomic tools were applied to human neuroblastoma SH-SY5Y cells (i) to characterize morphine and M6G, and (ii) to demonstrate the presence of the UDP-glucuronyltransferase 2B7 enzyme, which is responsible for the formation of M6G from morphine. We show that morphine is secreted in response to nicotine stimulation via a Ca2+-dependent mechanism involving specific storage and release mechanisms. We also show that morphine and M6G at concentrations as low as 10−10 M are able to evoke specific naloxone-reversible membrane currents, indicating possible autocrine/paracrine regulation in SH-SY5Y cells. Microscopy and proteomic approaches were employed to detect and quantify endogenous morphine in the mouse brain. Morphine is present in the hippocampus, cortex, olfactory bulb, and cerebellum at concentration ranging from 1.45 to 7.5 pmol/g. In the cerebellum, morphine immunoreactivity is localized to GABA basket cells and their termini, which form close contacts on Purkinje cell bodies.Conclusions/SignificanceThe presence of morphine in the brain and its localization in particular areas lead us to conclude that it has a specific function in neuromodulation and/or neurotransmission. Furthermore, its presence in cerebellar basket cell termini suggests that morphine has signaling functions in Purkinje cells that remain to be discovered.

The structural requirements for the synergistic enhancement of antimicrobial activities of the cationic linear peptides PGLa and magainin 2 were investigated. In a first step the antimicrobial activities were evaluated for a number of modifications of the sequences and equimolar mixtures thereof. In particular fluorophore labelled peptides maintain a high degree of antimicrobial activity and considerable synergism when tested conjointly. Thereafter, circular dichroism spectroscopy indicated that these extended sequences adopt helical conformations in the presence of model membranes similar to the unmodified sequences. Energy transfer between the fluorophores suggested that the peptides reside in close proximity to each other when bound to the membrane surface at high concentrations. The fluorophore interactions quickly diminish at lower peptide-to-lipid ratios indicating that the peptide-peptide interactions are weak. Furthermore, (15)N solid-state NMR measurements of the membrane topology of [(15)N-Ala14]-PGLa and of its fluorophorecarrying analogue reconstituted into supported 1, 2-didecanoyl-sn-glycero-3-phosphocholine bilayers were performed. These experiments revealed no correlation between the topological state of PGLa and the observed synergistic enhancement of antimicrobial activities due to the presence of magainins. These results suggest that lipid mediated interactions rather than the formation of tight peptide-peptide complexes in the membrane are responsible for synergistic activities of the mixtures.

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 UDPglucuronosyltransferase 2B-like enzyme, described to transform morphine into morphine-6-glucuronide, has been immunodetected in the chromaffin granule matrix, and morphine-6-glucuronide 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 -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.For 20 years, cerebral endogenous morphine has been isolated and characterized, and its structure has been shown to be identical to morphine from poppy (1, 2). In the 1990s, few groups succeeded in characterizing endogenous morphine from the organs and fluids of vertebrates, including bovine brain and adrenal gland, rat heart and adrenal gland, and human heart and urine (for review, Refs. 1-3); invertebrates (4 -6); and parasites (7,8). In mammals, the endogenous morphine synthetic pathway is associated with the dopamine and catecholamine biosynthetic pathway (for review, see Refs. 1, 3, and 9). Very recently, several crucial steps were reached when Zenk and co-workers (10, 11) demonstrated that morphine can be formed using a multistep biosynthetic route, and Zhu et al. (12) showed that human primary polymorphonuclear cells are able to synthesize morphine. These authors have shown morphine de novo synthesis in human and animal primary and cancer cell cultures.Chromaffin cells are neuroendocrine cells originating from the neural crest and are the predominant cell type in the adrenal medulla (for review, see Ref. 13). These cells possess the catecholamine biosynthetic pathway, leading to dopamine and adrenaline/noradrenaline synthesis (13). Chromaffin secretory granules contain a complex mixture of peptides and proteins that are co-released with catecholamines into the circulation in response to splanchnic nerve stimulation (13). Based on the morphine biosynthetic pathway and on the presence of morphine in bovine and rat adrenal glands (14, 15), rat pheochromocytoma PC-12 cells (10, 16), and eel chromaffin cells (17), we hypothesized that mammalian chromaffin cells might have the capacity to synthesize morphine and that their secretory granules could potentially release this alkaloid into the blood.Several mole...

Chromogranin A (CGA) is a protein that is stored and released together with neurotransmitters and hormones in the nervous, endocrine and diffuse neuroendocrine systems. As human vasostatins I and II [CGA(1–76) and CGA(1–113), respectively] have been reported to affect vessel motility and exert concentration‐dependent cardiosuppressive effects on isolated whole heart preparations of eel, frog and rat (i.e. negative inotropism and antiadrenergic activity), we investigated the presence of vasostatin‐containing peptides in rat heart. Rat heart extracts were purified by RP‐HPLC, and the resulting fractions analyzed for the presence of CGA N‐terminal fragments using dot‐blot analysis. CGA‐immunoreactive fractions were submitted to western blot and MS analysis using the TOF/TOF technique. Four endogenous N‐terminal CGA‐derived peptides [CGA(4–113), CGA(1–124), CGA(1–135) and CGA(1–199)] containing the vasostatin sequence were characterized. The following post‐translational modifications of these fragments were identified: phosphorylation at Ser96, O‐glycosylation (trisaccharide, NAcGal‐Gal‐NeuAc) at Thr126, and oxidation at three methionine residues. This first identification of CGA‐derived peptides containing the vasostatin motif in rat heart supports their role in cardiac physiology by an autocrine/paracrine mechanism.

Alzheimer’s disease is the most common form of dementia that affects about 50 million of sufferers worldwide. A major role for the initiation and progression of Alzheimer’s disease has been associated with the amyloid β-peptide (Aβ), which is a protease cleavage product of the amyloid precursor protein. The amyloid precursor protein is an integral membrane protein with a single transmembrane domain. Here, we assessed the structural integrity of the transmembrane domain within oriented phosphatidylcholine lipid bilayers and determined the tilt angle distribution and dynamics of various subdomains using solid-state NMR and attenuated total reflectance Fourier transform infrared spectroscopies. Although the overall secondary structure of the transmembrane domain is α-helical, pronounced conformational and topological heterogeneities were observed for the γ- and, to a lesser extent, the ζ-cleavage site, with pronounced implications for the production of Aβ and related peptides, the development of the disease, and pharmaceutical innovation.

The study of peptide-lipid and peptide-peptide interactions as well as their topology and dynamics using biophysical and structural approaches have changed our view how antimicrobial peptides work and function. It has become obvious that both the peptides and the lipids arrange in soft supramolecular arrangements which are highly dynamic and able to change and mutually adapt their conformation, membrane penetration, and detailed morphology. This can occur on a local and a global level. This review focuses on cationic amphipathic peptides of the magainin family which were studied extensively by biophysical approaches. They are found intercalated at the membrane interface where they cause membrane thinning and ultimately lysis. Interestingly, mixtures of two of those peptides namely magainin 2 and PGLa which occur naturally as a cocktail in the frog skin exhibit synergistic enhancement of antimicrobial activities when investigated together in antimicrobial assays but also in biophysical experiments with model membranes. Detailed dose-response curves, presented here for the first time, show a cooperative behavior for the individual peptides which is much increased when PGLa and magainin are added as equimolar mixture. This has important consequences for their bacterial killing activities and resistance development. In membranes that carry unsaturations both peptides align parallel to the membrane surface where they have been shown to arrange into mesophases involving the peptides and the lipids. This supramolecular structuration comes along with much-increased membrane affinities for the peptide mixture. Because this synergism is most pronounced in membranes representing the bacterial lipid composition it can potentially be used to increase the therapeutic window of pharmaceutical formulations.