Cloning of prepronociceptin has led to the discovery of other biologically active peptides

Among the opioid receptors family, the cloning of the mu, kappa and delta receptors was followed by that of another member, named ORL1 (Opiate Receptor Like 1). In spite of obvious homologies with the mu, kappa and delta receptors, ORL1 does not display a relevant affinity for the endogenous ligands of these former receptors (beta endorphin, enkephalins, dynorphin A...). This observation has prompted to search for an endogenous ligand of ORL1. A heptadecapeptide which fulfils this function, with a nanomolar affinity, has been found. It was named either nociceptin or orphanin FQ. It demonstrates, according either to the dose or to the route of administration, hyperalgesic, allodynic, antiopioidergic or even analgesic effects. It displays also many behavioural effects, modifying especially locomotion, exploratory behaviour, motivation, anxiety, memory, food intake. Nociceptin results from the cleavage of a large precursor protein, prepronociceptin (PPNOC). In this latter, nociceptin is flanked on its C-terminal region by another peptide which may be regarded either as a heptadecapeptide (NocII), or a bidecapeptide (NocIII) according to the inclusion or not of a fragment constituted by 3 arginine residues. Investigating the functions modulated by NocII, we observed that it stimulates locomotor activity of mice and shortens the forepaws licking latency in the hot plate test (55 degrees C); these effects are not shared by NocIII. The simultaneous administration of NocII and nociceptin resulted in animals put on the hot plate to the appearance of their respective effects, not modified by the presence of the other. A 41 amino acid peptide flanks nociceptin on its N-terminal region in PPNOC. It may be cleaved to generate a heptadecapeptide, named nocistatin on account of its antagonist effect on the hyperalgesia/allodynia induced by nociceptin. Thus, the discovery of ORL1 has led to that of nociceptin, that of its precursor PPNOC, and thereby to that of NocII/NocIII and nocistatin. The functions modulated by these peptides are being investigated whereas their receptors are yet unknown. These multiple targets allow to expect new strategies to modulate their functions.     

Human neutrophils as a source of nociceptin: a novel link between pain and inflammation

Nociceptin is a neuropeptide sharing sequence homology with classical opioid peptides but with a distinct pharmacological profile. Through activation of its receptor, NociR, nociceptin has been linked with several physiological functions in the central nervous system including memory, locomotion, and processing of pain signals. Recently, peripheral blood neutrophils (PMNs) were demonstrated to express a functional NociR, a result suggesting that additional functions of the neuropeptide remain to be elucidated. The present study investigated the possibility that PMNs may be a source of nociceptin and whether the neuropeptide elicits PMN early responses. We observed the presence of nociceptin in the synovial fluids from arthritic patients, an inflammatory milieu typically containing high numbers of PMNs. In addition, freshly isolated PMNs were found to express and secrete nociceptin following degranulation, identifying these inflammatory cells as a novel source of the neuropeptide. Incubation of PMNs with nociceptin elicited a specific pattern of cellular protein phosphorylation on tyrosine residues in a rapid and transient fashion. Moreover, nociceptin prevented intracellular accumulation of cAMP in fMLP-stimulated PMNs, an effect mimicked by the specific NociR synthetic agonist, Ro 64-6198. Taken together, these results show that nociceptin/NociR is present and functional in human neutrophils, and the results identify a novel dialogue pathway between neural and immune tissues.     

Solution structure of nociceptin peptides.

Peptides embedded in the sequence of pre-pro-nociceptin, i.e. nociceptin, nocistatin and orphanin FQ2, have shed light on the complexity of the mechanisms involving the peptide hormones related to pain and have opened up new perspectives for the clinical treatment of pain. The design of new ligands with high selectivity and bioavailability, in particular for ORL1, is important both for the elucidation and control of the physiological role of the receptor and for their therapeutic importance. The failure to obtain agonists and antagonists when using, for nociceptin, the same substitutions that are successful for opioids, and the conformational flexibility of them all, justify systematic efforts to study the solution conformation under conditions as close as possible to their natural environment. Structural studies of linear peptides in solution are hampered by their high flexibility. A direct structural study of the complex between a peptide and its receptor would overcome this difficulty, but such a study is not easy since opioid receptors are membrane proteins. Thus, conformational studies of lead peptides in solution are still important for drug design. This review deals with conformational studies of natural pre-nociceptin peptides in several solvents that mimic in part the different environments in which the peptides exert their action. None of the structural investigations yielded a completely reliable bioactive conformation, but the global conformation of the peptides in biomimetic environments can shed light on their interaction with receptors.     

Levels of neuropeptides nocistatin, nociceptin/orphanin FQ and their precursor protein in a rat neuropathic pain model

Neuropeptides nociceptin/orphanin FQ (N/OFQ) and nocistatin (NST) are related to pain modulation. The amounts of these peptides and their precursor protein, prepronociceptin (ppN/OFQ) in the brain, spinal cord and serum samples of rats with partial sciatic nerve ligation (PSNL) were compared with those in na?ve rats using radioimmunoassay (RIA). There was a significant rise in the levels of ppN/OFQ, N/OFQ and NST in the brains of PSNL rats. Their spinal cords showed significantly increased ppN/OFQ and NST levels but no change in N/OFQ levels. The PSNL rats also had increased serum NST (statistically significant) and N/OFQ (statistically insignificant) with decreased ppN/OFQ suggesting important roles of these peptides in neuropathic pain mechanism.     

Ultrastructural localization of Hsp-72 examined with a new polyclonal antibody raised against the truncated variable domain of the heat shock protein

In spite of the intensive search for the determination of the continuously widening physiological and pathological roles of different stress proteins, their ultrastructural localization at the electron microscopic (EM) level has hardly been examined. As it becomes increasingly evident that the function and physiological effectiveness of stress proteins are highly dependent on their spatial location and their associations with diverse regulator proteins, the demand for morphological studies which can identify their detailed distribution within the cells is evident. The reason for the practical lack of studies carried out at the EM level, lies in the shortage of reagents with suitable specificity and avidity necessary for this type of examination. To create such a reagent, a polyclonal antibody was raised using a recombinant truncated form of the inducible Hsp-72 protein. The antibody was extensively characterized, using different immunochemical methods to determine and verify its specificity, and then it was tried in ultrastructural examinations. Using the new antibody, it was possible to analyze the intracellular distribution of Hsp-72 with the immunogold technique. The localization of Hsp-72 was demonstrated directly at the ultrastructural level in the cytoplasm (especially at the cisterns of the RER), in the nucleus (mainly around the heterochromatic regions) and at both sides of the nuclear envelope close to the membrane pores. Apart from these localizations, Hsp-72 was found in several membrane bordered intracellular structures, which mainly belong to the endosomal-lysosomal system. We provide the first morphological verification of the appearance of Hsp-72 on the surface of the cells. Also novel is the indication, that the stress protein may recycle from the cell surface using a common route which includes coated pits and the endosomal system.     

Pain peptides. Solution structure of orphanin FQ2

Orphanin FQ2 (OFQ2) is a novel heptadecapeptide generated from prepronociceptin (PPNOC), the same precursor of nociceptin/orphanin FQ and nocistatin. OFQ2 is a potent analgesic when administered both supraspinally and spinally. In order to clarify the structural relationship with all peptides generated from PPNOC, we have undertaken the conformational study of OFQ2 in water and in structure-promoting solvent media. Nuclear magnetic resonance data and theoretical calculations are consistent with a well defined helical structure from Met(5) to Ser(16). The uniform distribution of hydrophobic residues along the helix suggests that OFQ2 may interact with the transmembrane helices of a receptor akin to those of nociceptin and opioids.     

Nocistatin: a novel neuropeptide encoded by the gene for the nociceptin/orphanin FQ precursor

We identified a novel neuropeptide and named it "nocistatin." Its presence was expected by analysis of the precursor for the neuropeptide nociceptin or orphanin FQ (Noc/OFQ), previously identified as an endogenous ligand for the orphan opioid receptor-like receptor. The precursor prepronociceptin/orphanin FQ (ppNoc/OFQ) comprises at least two bioactive peptides, nocistatin and Noc/OFQ. Noc/OFQ is involved in a broad range of pharmacological actions in various tissues from the central nervous system to the periphery. In pain transmission, Noc/OFQ is reported to have different effects including nociception, no effect, and analgesia, depending on the animal species tested, doses, route of administration, and so on. We found that intrathecal administration of Noc/OFQ induced pain responses including allodynia and hyperalgesia. Simultaneous administration of nocistatin blocked the allodynia and hyperalgesia induced by Noc/OFQ, whereas anti-nocistatin antibody decreased the threshold for the Noc/OFQ-induced allodynia. The endogenous heptadecapeptide nocistatin was isolated from bovine brains and recently identified in mouse, rat, and human brain and in human cerebrospinal fluid. Although human, rat and mouse ppNoc/OFQ produced larger respective counterparts with 30, 35, and 41 amino acid residues, all peptides showed the antinociceptive activity. This activity was ascribed to the carboxyl-terminal hexapeptide of nocistatin, Glu-Gln-Lys-Gln-Leu-Gln, which is conserved beyond species. Nocistatin also attenuated the allodynia and hyperalgesia evoked by prostaglandin E(2) and the inflammatory hyperalgesia induced by formalin or carrageenan/kaolin, and reversed the Noc/OFQ-induced inhibition of morphine analgesia at picogram doses. Furthermore, nocistatin counteracted the impairment of learning and memory induced by Noc/OFQ or scopolamine. Nocistatin is widely present in the spinal cord and brain. Although nocistatin did not bind to the Noc/OFQ receptor, it bound to the membrane of mouse brain and spinal cord with a high affinity. Nocistatin is a novel bioactive peptide produced from the same precursor as Noc/OFQ, and it plays important roles in the regulation of pain transmission and learning and memory processes in the central nervous system.     

The nociceptin (ORL1) receptor: molecular cloning and functional architecture

Nociceptin and the ORL1 receptor share high sequence similarity with opioid peptides, particularly dynorphin A, and their receptors. However, nociceptin and dynorphin A may use distinct molecular pathways to bind and activate their cognate receptors. Activation of the kappa-opioid receptor by dynorphin A is thought to require interactions of its N-terminal hydrophobic domain (Y(1)GGF) with the receptor opioid binding pocket, located within the transmembrane helix bundle, while activation of the ORL1 receptor appears to require interactions of the positively charged core (R(8)KSARK) of nociceptin with the negatively charged second extracellular receptor loop.     

Structure of complex viruses and virus-infected cells by electron cryo tomography

In microbiology, and in particular in virus research, electron microscopy (EM) is an important tool, offering a broad approach for investigating viral structure throughout their intracellular and extracellular life cycles. Currently, molecular tools and rapid developments in advanced light microscopy dominate the field and supply an enormous amount of information concerning virus biology. In recent years, numerous fascinating high-resolution EM structures obtained by single-particle electron cryo microscopy (cryo-EM) were revealed for viral particles that possess icosahedral symmetry. However, no comprehensive three-dimensional analysis of complex viruses or viruses within cells has yet been achieved using EM. Recent developments in electron cryo-tomography render this a proficient tool for the analysis of complex viruses and viruses within cells in greater detail.     

Synthesis and biological studies of nociceptin derivatives containing the DTPA chelating group for further labeling with therapeutic radionuclides

Nociceptin is an endogenous anti-opiate heptadecapeptide primarily interacting with the nociceptin (NOP) receptor. This neuropeptide-receptor system is involved in pain regulation, tolerance to and dependence on opiates as well as many other physiological and pathophysiological events. The role and mechanisms of nociceptin in pathological conditions is not clearly known yet. In an attempt to have a radiopharmaceutical labeled either with 99mTc or (111)In, we incorporated diethylenetriaminepentaacetic acid (DTPA) as chelator into the structure of [Arg14,Lys15]nociceptin(1-17)-NH2 at the epsilon-amino group of Lys15. Such a radiopeptide may be useful in imaging for diagnostical purposes. Preparation of the peptide ligands was carried out by solid phase synthesis. Two peptides containing DTPA were obtained and purified. The products were [Arg14,Lys(DTPA)15]nociceptin(1-17)-NH2 and its cross-linked dimer on the basis of mass spectrometric analysis. In (115)In3+ binding experiments the conjugates exhibited preserved indium ion chelating properties, indicating the potential use of radiolabeled DTPA-nociceptin derivatives as radiopharmaceutical. Biological properties of these compounds were studied in rat brain membrane preparations by radioligand binding, functional biochemical [35S]GTPgammaS binding assays and mouse vas deferens (MVD) bioassay. Besides the similar in vitro binding characteristics to nociceptin receptor, both of the DTPA-chelated compounds were more potent and efficient than nociceptin in functional biochemical and mouse vas deferens bioassays. Our further aim is to radiolabel these compounds in order to get a radiopharmaceutical which can be used diagnostically.     

Advanced Correlative Light/Electron Microscopy: Current Methods and New Developments Using Tokuyasu Cryosections

Microscopy is an essential tool for analysis of cellular structures and function. With the advent of new fluorescent probes and super-resolution light microscopy techniques, the study of dynamic processes in living cells has been greatly facilitated. Fluorescence light microscopy provides analytical, quantitative, and three-dimensional (3D) data with emphasis on analysis of live cells using fluorescent markers. Sample preparation is easy and relatively inexpensive, and the use of appropriate tags provides the ability to track specific proteins of interest. Of course, only electron microscopy (EM) achieves the highest definition in terms of ultrastructure and protein labeling. To fill the gap between light microscopy and EM, correlative light and electron microscopy (CLEM) strategies have been developed. In particular, hybrid techniques based upon immuno-EM provide sensitive protein detection combined with high-resolution information on cell structures and protein localization. By adding the third dimension to EM with electron tomography (ET) combined with rapid freezing, CLEM techniques now provide additional tools for quantitative 3D analysis. Here, we overview the major methods applied and highlight the latest advances in the field of CLEM. We then focus on two selected techniques that use cryosections as substrate for combined biomolecular imaging. Finally, we provide a perspective of future developments in the field. (J Histochem Cytochem 57:1103–1112, 2009)     

Correlative light and electron microscopic immunocytochemistry on the same section with colloidal gold

Ultrastructural localization of growth hormone in rat anterior pituitary and of muscle-specific actin in rabbit arterial smooth muscle cells was accomplished with a post-embedment procedure using colloidal gold. Plastic sections (2 microns) were mounted on slides, deplasticized, immunostained with immunoglobulin-colloidal gold particles, re-embedded in Epon, and sectioned for electron microscopy. This procedure enabled light and electron microscopic localization of these intracellular antigens on the same section. Positive immunostaining was demonstrated with this procedure with a muscle-specific actin antibody which previously failed to localize antigenic sites by EM. The procedure described yielded staining of high specificity, with minimal background and well-preserved ultrastructure. This re-embedding technique is useful in situations where problems with post-embedding EM immunostaining exist and where correlative LM and EM immunostaining is essential.     

Role of nociceptin systems in learning and memory

This article summarizes our recent finding that the nociceptin system is involved in the regulation of learning and memory. The nociceptin-knockout mice show greater learning ability in the water maze task, an enhanced latent learning in the water finding task, better memory in the passive avoidance task, and further, larger long-term potentiation in the hippocampal CA1 region than wild-type mice. Nociceptin itself induces an impairment of passive avoidance task in wild-type mice, which is reversed by naloxone benzoylhydrazone (NalBzoH). Thus, the nociceptin system seems to play negative roles in learning and memory, and NalBzoH may act as a potent antagonist for the nociceptin receptor.     

Integrated Electron Microscopy: Super-Duper Resolution

Since its inception, electron microscopy (EM) has revealed that cellular membranes are organized into structurally distinct subdomains, created by localized protein and lipid assemblies to perform specific complex cellular functions. Caveolae are membrane subdomains that function as signaling platforms, endocytic carriers, sensors of membrane tension, and mechanical stress, as well as in lipid homeostasis. They were first discovered almost 60 years ago by pioneering electron microscopists. While new and exciting developments in SUPER-resolution fluorescent light microscopy facilitate studies of the spatial organization of fluorescently labeled protein components, these techniques cannot reveal the underlying cellular structures. Thus, equally exciting are developments in EM: genetically encoded probes for protein localization at sub-10 nm resolution, more powerful instruments that allow imaging of larger cell volumes, and computational methods for reconstructing three-dimensional images. Used in combination, as done by Ludwig et al. in the current issue of PLOS Biology, these tools reveal high-resolution insights into the composition and organization of the caveolae coat and the formation of these specialized structures. Together, these advances are contributing to a resurgence in EM.     

Cutting edge: nociceptin stimulates neutrophil chemotaxis and recruitment: inhibition by aspirin-triggered-15-epi-lipoxin A4

The nociceptin receptor (Noci-R) is a G protein-coupled receptor present in neural tissues and its activation by nociceptin is involved in the processing of pain signals. Here, we report that Noci-R is present and functional on peripheral blood polymorphonuclear leukocytes (PMN). Human PMN express mRNA for Noci-R, its nucleotide sequence determined, and specific binding with [(125)I]-labeled nociceptin gave an apparent K(d) approximately 1.5 nM for this PMN opioid receptor. Nociceptin evoked PMN chemotaxis with maximal activity at 100 pM, without intracellular Ca(2+) mobilization. When injected in murine air pouches, nociceptin elicited leukocyte infiltration in a concentration-dependent fashion. Nociceptin-stimulated PMN infiltration was inhibited by treating mice with a synthetic analog of the aspirin-triggered lipid mediator 15-epi-lipoxin A(4). The present results identify nociceptin as a potent chemoattractant and provide a novel link between the neural and immune systems that are blocked by aspirin-triggered lipid mediators and may be relevant in neurogenic inflammation.     

Organellar Na+/H+ exchangers: novel players in organelle pH regulation and their emerging functions

Mammalian Na+/H+ exchangers (NHEs) play a fundamental role in cellular ion homeostasis. NHEs exhibit an appreciable variation in expression, regulation, and physiological function, dictated by their dynamics in subcellular localization and/or interaction with regulatory proteins. In recent years, a subgroup of NHEs consisting of four isoforms has been identified, and its members predominantly localize to the membranes of the Golgi apparatus and endosomes. These organellar NHEs constitute a family of transporters with an emerging function in the regulation of luminal pH and in intracellular membrane trafficking as expressed, for example, in cell polarity development. Moreover, specific roles of a variety of cofactors, regulating the intracellular dynamics of these transporters, are also becoming apparent, thereby providing further insight into their mechanism of action and overall functioning. Interestingly, organellar NHEs have been related to mental disorders, implying a potential role in the brain, thus expanding the physiological significance of these transporters.     

Blockade effects of (Nphe1)Nociceptin(1-13)-NH(2) on anti-nociception induced by intrathecal administration of nociceptin in rats

The present study investigated the roles of the opioid-receptor-like (ORL1) receptor and its endogenous ligand nociceptin on nociception in the spinal cord of rats. Intrathecal administration of 10 nmol of nociceptin produced significant increases in hindpaw withdrawal latencies (HWLs) to thermal and mechanical stimulation. There were no significant changes of average maximum angles in inclined plane tests after intrathecal injection of 10 nmol of nociceptin in rats. The intrathecal nociceptin-induced increases in HWL were antagonized by intrathecal administration of (Nphe1)Nociceptin(1-13)-NH(2), a selective antagonist of ORL1 receptor, in a dose-dependent manner. The results demonstrated that ORL1 receptor is involved in the nociceptin-induced anti-nociceptive effect in the spinal cord of rats.     

Nociceptin/orphanin FQ in spinal nociceptive mechanisms under normal and pathological conditions

Nociceptin and its receptor are present in dorsal spinal cord, indicating a possible role for this peptide in pain transmission. The majority of functional studies using behavioral and electrophysiological studies have shown that nociceptin applied at spinal level produces antinociception through pre- and post-synaptic mechanisms. The spinal inhibitory effect of nociceptin is not sensitive to antagonists of opioid receptors such as naloxone. Thus, nociceptin-induced antinociception is mediated by a novel mechanism independent of activation of classic opioid receptors. This has raised the possibility that agonists of the nociceptin receptor may represent a novel class of analgesics. Supporting this hypothesis, several groups have shown that intrathecal nociceptin alleviated hyperalgesic and allodynic responses in rats after inflammation or partial peripheral nerve injury. Electrophysiological studies have also indicated that the antinociceptive potency of spinal nociceptin is maintained or enhanced after nerve injury. It is concluded that the predominant action of nociceptin in the spinal cord appears to be inhibitory. The physiological role of nociceptin in spinal nociceptive mechanisms remains to be defined. Moreover, further evaluation of nociceptin as a new analgesic calls the development of non-peptide brain penetrating agents.