Endogenous opiates and behavior: 2014

This paper is the thirty-seventh consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2014 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (endogenous opioids and receptors), and the roles of these opioid peptides and receptors in pain and analgesia (pain and analgesia); stress and social status (human studies); tolerance and dependence (opioid mediation of other analgesic responses); learning and memory (stress and social status); eating and drinking (stress-induced analgesia); alcohol and drugs of abuse (emotional responses in opioid-mediated behaviors); sexual activity and hormones, pregnancy, development and endocrinology (opioid involvement in stress response regulation); mental illness and mood (tolerance and dependence); seizures and neurologic disorders (learning and memory); electrical-related activity and neurophysiology (opiates and conditioned place preferences (CPP)); general activity and locomotion (eating and drinking); gastrointestinal, renal and hepatic functions (alcohol and drugs of abuse); cardiovascular responses (opiates and ethanol); respiration and thermoregulation (opiates and THC); and immunological responses (opiates and stimulants).This paper is the thirty-seventh consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2014 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (endogenous opioids and receptors), and the roles of these opioid peptides and receptors in pain and analgesia (pain and analgesia); stress and social status (human studies); tolerance and dependence (opioid mediation of other analgesic responses); learning and memory (stress and social status); eating and drinking (stress-induced analgesia); alcohol and drugs of abuse (emotional responses in opioid-mediated behaviors); sexual activity and hormones, pregnancy, development and endocrinology (opioid involvement in stress response regulation); mental illness and mood (tolerance and dependence); seizures and neurologic disorders (learning and memory); electrical-related activity and neurophysiology (opiates and conditioned place preferences (CPP)); general activity and locomotion (eating and drinking); gastrointestinal, renal and hepatic functions (alcohol and drugs of abuse); cardiovascular responses (opiates and ethanol); respiration and thermoregulation (opiates and THC); and immunological responses (opiates and stimulants).     

Annelid Endocrine Disruptors and a Survey of Invertebrate FMRFamide-Related Peptides

There is a growing body of literature describing the actionsof endocrine disruptors on annelids. These pollutants causedecreases in growth and reproductive output, delay sexual maturation,and inhibit the immune system in annelids. More studies areneeded to determine the mechanisms that underlie these responses.Most invertebrate endocrine disruptor research focuses on steroids.In recent years many new invertebrate peptide hormones includingthose related to the molluscan peptide FMRFamide have been identified.Since the storage of these peptides can be inhibited by steroidsduring insect metamorphosis, they may be affected by endocrinedisruptors. Therefore, it is worthwhile to give a brief overviewof this peptide family to those studying endocrine disruptionin invertebrates with the hope that they may begin to considerthese peptides in their future research. In 1977 Price and Greenbergisolated FMRFamide from the cerebral ganglia of the clam, Macrocallistanimbosa. Since then researchers have used bioassays and immunoassaysto identify a large number of FMRFamide-related peptides (FaRPs)from many invertebrate phyla. Even more peptides are predictedby the FaRP genes that have been sequenced. FaRPs have a varietyof functions and act as neurotransmitters, neuromodulators,or neurohormones. Each function is species and tissue specific.Most FaRP receptors are linked to a second messenger system.However, at least one is a ligand gated sodium channel. On goingstudies are examining FaRPs from the molecular to organismallevel.     

The next frontier in the molecular biology of the opioid system

The analgesic and euphoric properties of some plant alkaloids such as morphine have been known and exploited for centuries. In contrast, only during the last twenty years have we begun to unravel the molecular basis by which opiates exert their effects, mechanisms important to our general understanding of the nervous system. The analgesic response to opiates is the result of a cascade of biochemical events that are triggered by the interaction of the opiate with specific macromolecular components found on the membranes of nervous system tissues, the opioid receptors. The endogenous ligands of these receptors are small peptides, the opioid peptides. Although much has been learned about the structures and the mode of synthesis of the opioid peptides, little is understood about the structure of their receptors. The application of molecular genetic techniques was of great importance to the studies of the opioid peptides. It is now expected that this same technology will unravel the physical mysteries of the opioid receptors.     

Opioid receptor agonists and Ca2+ modulation in human B cell lines.

Opiates and opioid peptides have been shown to modulate lymphocyte functions; however, little attention has been given to the type of receptors or receptor signaling mechanisms that are involved. Receptor-mediated signaling via ionized free Ca2+ is an event thought to be important in the triggering of lymphocyte activities. We report use of the calcium indicator dye, indo-1, and flow cytometry to identify B lymphocyte calcium responses to physiologic concentrations of opioid peptides. The human B cell lines Nalm 6 and JY responded to the naturally occurring opioid pentapeptide methionine-enkephalin or other opiate receptor agonists with a rapid, dose-dependent rise in free cytoplasmic Ca2+. This opioid peptide effect on Ca2+ modulation was inhibited by the opiate receptor antagonist naloxone. The synthetic enkephalin analogue DAMGO with specificity for mu-type opiate receptors and the synthetic opiate receptor agonists U50,488H and U69,593 with selectivity for kappa-type sites also stimulated calcium responses when applied to the B cell lines. These studies provide evidence that human B cell lines express functional opiate receptors of the mu- and kappa-types and suggest that such receptors, coupled with Ca2+ modulation, are instrumental in the B cell response to opiates and endogenous opioid neuropeptides.     

Localisation of Globodera pallida FMRFamide-related peptide encoding genes using in situ hybridisation

The present study employed an in situ hybridisation technique to detect the expression of a number of FMRFamide-like peptide encoding (flp) genes, previously identified from Globodera pallida, in whole-mount preparations of the J(2) stage of this worm. gpflp-1, encoding the FMRFamide-related peptide (FaRP) KSAYMRFamide, was expressed in neurones associated with the circumpharyngeal nerve ring and specifically in a number of cell bodies in the lumbar ganglia of the perianal nerve ring. The lumbar ganglia and pre-anal ganglia along with the BDU neurones and a number of cells in the retrovesicular ganglion were observed to express gpflp-2, encoding KNKFEFIRFamide. gpflp-3 (encoding KHEYLRFamide) expression was localised to the anterior ganglion and a number of paired cells posterior to the circumpharyngeal nerve ring whilst expression of gpflp-4, encoding a number of -P(G/Q)VLRFamides, was localised to the retrovesicular ganglion. No expression of gpflp-5 was observed. Identification of the reactive cells has implicated distinct roles for the FaRPs encoded on these genes in regulation of both dorsal and ventral body wall muscles, the musculature of the vulva and in the function of a number of sensory structures in both the head and tail of G. pallida. Comparison with the expression patterns of analogous genes in Caenorhabditis elegans suggests that, whilst some of the encoded peptides are conserved between nematode species, their functions therein are distinct. Furthermore, the expression of some of these genes in a number of interneurones supports the idea that FaRPs fulfil neuromodulatory as well as neurotransmitter roles.     

FaRP cell distribution in the developing CNS suggests the involvement of FaRPs in all parts of the chromatophore control pathway in Sepia officinalis (Cephalopoda)

The FMRFamide-related peptide (FaRP) family includes a wide range of neuropeptides that have a role in many biological functions. In cephalopods, these peptides intervene in the peculiar body patterning system used for communication and camouflage. This system is particularly well developed in the cuttlefish and is functional immediately after hatching (stage 30). In this study, we investigate when and how the neural structures involved in the control of body patterning emerge and combine during Sepia embryogenesis, by studying the expression or the production of FaRPs. We detected FaRP expression and production in the nervous system of embryos from the beginning of organogenesis (stage 16). The wider FaRP expression was observed concomitantly with brain differentiation (around stage 22). Until hatching, FaRP-positive cells were located in specific areas of the central and peripheral nervous system (CNS and PNS). Most of these areas were implicated in the control of body patterns, suggesting that FaRPs are involved in all parts of the neural body pattern control system, from the ‘receptive areas’ via the CNS to the chromatophore effectors.     

Opiate modulating properties of nociceptin/orphanin FQ

The recently discovered peptide nociceptin/orphanin FQ (N/OFQ) and its receptor NOR share many structural similarities with the opioid peptides and their receptors. The anatomical distributions of N/OFQ and NOR are similar to those of opioid peptides and receptors. In addition, NOR and opiate receptors couple via the same G-proteins to similar effectors, such as Ca(2+) channels, K(+) channels, adenylyl cyclase, and several protein kinases. Thus, the behavioral effects of N/OFQ have been investigated in the context of known opiate effects, and a possible connection has been sought between the effects of these two homologous signaling systems. Originally characterized as a nociception-producing peptide, N/OFQ has now been shown to have diverse effects on nociception, as well as effects on many other behaviors. With regard to nociception, the peptide has been reported to produce hyperalgesia, reversal of opioid-mediated analgesia, analgesia, and allodynia. N/OFQ also has effects on other behaviors, such as locomotion, feeding, anxiety, spatial attention, reproductive behaviors, and opiate tolerance. The relationship between opiates and N/OFQ is strengthened by the fact that opiates also affect these behaviors. However, the exact nature of the relationship of N/OFQ with opiates-opiate-like versus antiopiate-remains controversial. This review will detail the diverse effects of N/OFQ and suggest that this peptide, like other putative antiopiate peptides, can be described as 'opiate modulating. '     

FMRFamide-related peptides and serotonin regulate Drosophila melanogaster heart rate: mechanisms and structure requirements

Drosophila melanogaster FMRFamide-related peptides (FaRPs) include SDNFMRFamide, PDNFMRFamide, and TDVDHVFLRFamide (dromyosuppressin, DMS); each peptide contains a C-terminal FMRFamide but a different N-terminal extension. FaRPs and serotonin (5-HT) each affect the frequency of D. melanogaster heart contractions in vivo. We examined the cellular expression of FaRPs and 5-HT, and the activities of FMRFamide, SDNFMRFamide, PDNFMRFamide, or DMS and 5-HT on heart rate. FaRPs and 5-HT were not co-localized; FaRP-and 5-HT-immunoreactive fibers extended from different brain cells and innervated the anterior D. melanogaster dorsal vessel. However, no neuron expressed both a FaRP and 5-HT. The effect of FMRFamide and 5-HT was not different from the effect of 5-HT alone on heart rate. The effect of PDNFMRFamide and 5-HT showed an additive effect on heart rate. SDNFMRFamide and 5-HT or DMS and 5-HT resulted in non-additive effects on heart rate. Our data provide evidence for the complexity of FaRP and 5-HT interactions to regulate frequency of heart contractions in vivo. Our results also confirm the biological importance of FaRP N-terminal amino acid extensions.     

On the specificity of naloxone as an opiate antagonist.

Since the discovery of endogenous opioid peptides in brain (68,69,97,113, 128) and the pituitary gland (26,81,105,125) there has been considerable interest in their possible roles in a variety of physiological and pharmacological processes. Many studies have used antagonism by naloxone as a criterion for implicating endogenous opiates in a process, assuming that naloxene has no pharmacological actions other than those related to blockade of opiate receptors. The doses of naloxene used are often higher than those required to antagonize the analgesic and other effects of morphine. However, multiple forms of opiate receptors are present in nervous tissue and higher concentrations of naloxene are required to antagonize effects mediated by some of these receptors (83). Although the earlier literature supports the assumption that the effects of naloxene are due to the blockade of opiate receptors (87), there are an increasing number of reports which indicate that naloxene may have pharmacological actions unrelated to opiate receptor blockade. The subsequent review serves to emphasize that antagonism by naloxene is a necessary but not sufficient criterion for invoking the mediation of a response by an endogenous opiate (61). Additional lines of evidence which serve to strengthen the conclusion that endogenous opiates mediate a process will be considered.     

The endoplasmic reticulum Ca2+-pump SERCA2b interacts with G protein-coupled receptors and enhances their expression at the cell surface

Calcium (Ca(2+)) plays a pivotal role in both cellular signaling and protein synthesis. However, it is not well understood how calcium metabolism and synthesis of secreted and membrane-bound proteins are related. Here we demonstrate that the sarco(endo)plasmic reticulum Ca(2+) ATPase 2b (SERCA2b), which maintains high Ca(2+) concentration in the lumen of the endoplasmic reticulum, interacts specifically with the human delta opioid receptor during early steps of receptor biogenesis in human embryonic kidney 293 cells. The interaction involves newly synthesized incompletely folded receptor precursors, because the association between the delta opioid receptor and SERCA2b (i) was short-lived and took place soon after receptor translation, (ii) was not affected by misfolding of the receptor, and (iii) decreased if receptor folding was enhanced by opioid receptor pharmacological chaperone. The physical association with SERCA2b was found to be a universal feature among G protein-coupled receptors within family A and was shown to occur also between the endogenously expressed luteinizing hormone receptor and SERCA2b in rat ovaries. Importantly, active SERCA2b rather than undisturbed Ca(2+) homeostasis was found to be essential for delta opioid receptor biogenesis, as inhibition of its Ca(2+) pumping activity by thapsigargin reduced the interaction and impaired the efficiency of receptor maturation, two phenomena that were not affected by a Ca(2+) ionophore A23187. Nevertheless, inhibition of SERCA2b did not compromise the functionality of receptors that were able to mature. Thus, we propose that the association with SERCA2b is required for efficient folding and/or membrane integration of G protein-coupled receptors.     

Modulation of sodium-sensitive GTPase by partial opiate agonists. An explanation for the dual requirement for Na+ and GTP in inhibitory regulation of adenylate cyclase

Opiates and opioid peptides inhibit adenylate cyclase and stimulate specific low Km GTPase activity in membranes from neuroblastoma x glioma NG108-15 hybrid cells. The effects of opiate agonists on both enzymes are mediated by high affinity stereospecific receptors and require Mg2+, GTP, and Na+. In the presence of Mg2+, Na+ inhibits basal GTPase activity; opiates stimulate GTP hydrolysis by antagonizing the Na+-induced inhibition. Activation of GTPase leads, in turn, to inactivation of GTP-stimulated adenylate cyclase activity. The intrinsic activities (or efficacies) of a series of opiates are identical for stimulation of GTPase and inhibition of adenylate cyclase. These results provide a mechanism for the dual requirement for Na+ and GTP in the inhibitory coupling of opiate receptors to the adenylate cyclase system in these cells and may be of general significance to the action of other inhibitory hormones.     

Sigma1 binding in a human neuroblastoma cell line

Behaviorally, sigma₁ agents modulate opioid analgesia. To examine possible mechanisms responsible for these interactions, we have identified a cell line containing both sigma₁ and opioid receptors. [³H](+)-pentazocine binding in BE(2)-C human neuroblastoma cells is high affinity (K_D 3.4±0.7 nM) and high density (B_max 2.98±0.14 pmol/mg protein). Competition studies reveal a selectivity profile similar to that of sigma₁ sites in guinea pig brain. (+)-Pentazocine has no effect upon either basal or forskolin-stimulated cyclase in the BE(2)-C cells, but cAMP accumulation is inhibited by the morphine, DPDPE and naloxone benzoylhydrazone. (+)-Pentazocine at concentrations as high as 10 μM does not affect this opioid effect, implying that sigma₁/opioid interactions are not mediated at the level of the cell. This suggests that their behavioral interactions result from interacting neural circuits. Although (+)-pentazocine is without effect in the cyclase system, it does block carbachol-stimulated phosphoinositol turnover (IC₅₀ 6.5±1.14 μM). The specificity of the effect is confirmed by the ability of haloperidol (1 μM) to shift the IC₅₀ value of (+)-pentazocine 2-fold to the right. Special issue dedicated to Dr. Eric J. Simon.     

Endogenous opiates and behavior: 2004

This paper is the 27th consecutive installment of the annual review of research concerning the endogenous opioid system, now spanning over 30 years of research. It summarizes papers published during 2004 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior, and the roles of these opioid peptides and receptors in pain and analgesia; stress and social status; tolerance and dependence; learning and memory; eating and drinking; alcohol and drugs of abuse; sexual activity and hormones, pregnancy, development and endocrinology; mental illness and mood; seizures and neurologic disorders; electrical-related activity and neurophysiology; general activity and locomotion; gastrointestinal, renal and hepatic functions; cardiovascular responses; respiration and thermoregulation; and immunological responses.     

Sex differences in opioid analgesia, hyperalgesia, tolerance and withdrawal: Central mechanisms of action and roles of gonadal hormones

This article reviews sex differences in opiate analgesic and related processes as part of a Special Issue in Hormones and Behavior. The research findings on sex differences are organized in the following manner: (a) systemic opioid analgesia across mu, delta and kappa opioid receptor subtypes and drug efficacy at their respective receptors, (b) effects of the activational and organizational roles of gonadal steroid hormones and estrus phase on systemic analgesic responses, (c) sex differences in spinal opioid analgesia, (d) sex differences in supraspinal opioid analgesia and gonadal hormone effects, (e) the contribution of genetic variance to analgesic sex differences, (f) sex differences in opioid-induced hyperalgesia, (g) sex differences in tolerance and withdrawal-dependence effects, and (h) implications for clinical therapies.     

Guanine nucleotides inhibit binding of agonists and antagonists to soluble opiate receptors

The guanine nucleotides GDP, GTP, and guanosine-5'-(beta, gamma-imido)triphosphate inhibit binding of opiates and opioid peptides to receptors solubilized from membranes of neuroblastoma X glioma NG108-15 hybrid cells. The inhibition reflects decreased affinity of receptors for opioid ligands. Whereas in membranes, only opioid agonist binding is sensitive to guanine nucleotide inhibition, both agonist and antagonist binding is reduced in the case of soluble receptors. Furthermore, soluble receptors are more sensitive to the effects of guanine nucleotides than are membrane-bound receptors. These observations are consistent with the suggestion that solubilized receptors may be complexes of an opiate binding protein and a guanine nucleotide-sensitive regulatory component.     

Multiple opiate receptors: emerging concepts

Increasing biochemical evidence indicates that the wide spectrum of opiate pharmacological actions are mediated via heterogeneous classes of receptors. μ receptors have been identified as the high affinity sites where morphine-like opiates exert their analgesic effects. δ receptors have a somewhat different CNS distribution and have been identified as sites relatively selective for the naturally occuring enkephalins. Recent biochemical studies provide evidence for two additional classes of opiate receptor sites which were originally proposed on the basis of physiological studies. Ketocyclazocine-like opiates produce their unique ataxic and sedative effects via interaction with K receptors, and SKF-10,047 (N-allylnorcyclazocine) and related opiates produce stimulant and psychotomimetic effects via interactions with σ receptors.Many opiate drugs interact at multiple receptor sites. Thus, the constellation of neuropharmacological actions of a particular opioid ligand may reflect its various potencies at a combination of μ, δ, K, and σ receptors.     

Calcium influx into phospholipid vesicles caused by dynorphin neuropeptides

Dynorphins, endogeneous opioid peptides, function as ligands to the opioid kappa receptors but also induce non-opioid excitotoxic effects. Dynorphin A can increase the intra-neuronal calcium concentration through a non-opioid and non-NMDA mechanism. In this investigation, we show that big dynorphin, dynorphin A and to some extent dynorphin A (1-13), but not dynorphin B, allow calcium to enter into large unilamellar phospholipid vesicles with partly negative headgroups. The effects parallel the previously studied potency of dynorphins to translocate through biological membranes and to cause calcein leakage from large unilamellar phospholipid vesicles. There is no calcium ion influx into vesicles with zwitterionic headgroups. We have also investigated if the dynorphins can translocate through the vesicle membranes and estimated the relative strength of interaction of the peptides with the vesicles by fluorescence resonance energy transfer. The results show that dynorphins do not translocate in this membrane model system. There is a strong electrostatic contribution to the interaction of the peptides with the membrane model system.     

N-terminal extended conjugates of the agonists and antagonists of both bradykinin receptor subtypes: structure-activity relationship, cell imaging using ligands conjugated with fluorophores and prospect for functionally active cargoes

Peptide agonists and antagonists of both bradykinin (BK) B(1) and B(2) receptors (B(1)R, B(2)R) are known to tolerate to a certain level N-terminal sequence extensions. Using this strategy, we produced and characterized the full set of fluorescent ligands by extending both agonists and antagonist peptides at both receptor subtypes with 5(6)-carboxyfluorescein (CF) and the ε-aminocaproyl (ε-ACA) optional spacer. Alternatively, kinin receptor ligands were extended with another carboxylic acid cargo (chlorambucil, biotinyl, pentafluorocinnamoyl, AlexaFluor-350 (AF350), ferrocenoyl, cetirizine) or with fluorescein isothiocyanate. N-terminal extension always reduced receptor affinity, more importantly for bulkier substituents and more so for the agonist version compared to the antagonist. This loss was generally alleviated by the presence of the spacer and modulated by the species of origin for the receptor. We report and review the pharmacological properties of these N-terminally extended peptides and the use of fluorophore-conjugated ligands in imaging of cell receptors and of angiotensin converting enzyme (ACE) in intact cells. Antagonists (B(1)R: B-10376: CF-ε-ACA-Lys-Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-BK; B(2)R: B-10380: CF-ε-ACA-D-Arg-[Hyp(3), Igl(5), D-Igl(7), Oic(8)]-BK and fluorescein-5-thiocarbamoyl (FTC)-B-9430) label the plasma membrane of cells expressing the cognate receptors. The B(2)R agonists CF-ε-ACA-BK, AF350-ε-ACA-BK and FTC-B-9972 are found in endosomes and model the endosomal degradation of BK in a complementary manner. The uneven surface fluorescence associated to the B(1)R agonist B-10378 (CF-ε-ACA-Lys-des-Arg(9)-BK) is compatible with a particular form of agonist-induced receptor translocation. CF-ε-ACA-BK binds to the carboxydipeptidase ACE with an affinity identical to that of BK. Metal- or drug-containing cargoes further show the prospect of ligands that confer special signaling to kinin receptors.