Lawrence H. Lazarus

Current Research on Opioid Receptor Function

The use of opioid analgesics has a long history in clinical settings, although the comprehensive action of opioid receptors is still less understood. Nonetheless, recent studies have generated fresh insights into opioid receptor-mediated functions and their underlying mechanisms. Three major opioid receptors (μ-opioid receptor, MOR; δ-opioid receptor, DOR; and κ-opioid receptor, KOR) have been cloned in many species. Each opioid receptor is functionally sub-classified into several pharmacological subtypes, although, specific gene corresponding each of these receptor subtypes is still unidentified as only a single gene has been isolated for each opioid receptor. In addition to pain modulation and addiction, opioid receptors are widely involved in various physiological and pathophysiological activities, including the regulation of membrane ionic homeostasis, cell proliferation, emotional response, epileptic seizures, immune function, feeding, obesity, respiratory and cardiovascular control as well as some neurodegenerative disorders. In some species, they play an essential role in hibernation. One of the most exciting findings of the past decade is the opioid-receptor, especially DOR, mediated neuroprotection and cardioprotection. The upregulation of DOR expression and DOR activation increase the neuronal tolerance to hypoxic/ischemic stress. The DOR signal triggers (depending on stress duration and severity) different mechanisms at multiple levels to preserve neuronal survival, including the stabilization of homeostasis and increased pro-survival signaling (e.g., PKC-ERK-Bcl 2) and antioxidative capacity. In the heart, PKC and KATP channels are involved in the opioid receptor-mediated cardioprotection. The DOR-mediated neuroprotection and cardioprotection have the potential to significantly alter the clinical pharmacology in terms of prevention and treatment of life-threatening conditions like stroke and myocardial infarction. The main purpose of this article is to review the recent work done on opioids and their receptor functions. It shall provide an informative reference for better understanding the opioid system and further elucidation of the opioid receptor function from a physiological and pharmacological point of view.

Dmt and opioid peptides: a potent alliance.

The introduction of the Dmt (2′,6′‐dimethyl‐L‐tyrosine)–Tic pharmacophore into the design of opioid ligands produced an extraordinary family of potent δ‐opioid receptor antagonists and heralded a new phase in opioid research. First reviewed extensively in 1998, the incorporation of Dmt into a diverse group of opioid molecules stimulated the opioid field leading to the development of unique analogues with remarkable properties. This overview will document the crucial role played by this residue in the proliferation of opioid peptides with high receptor affinity (Ki equal to or less than 1 nM) and potent bioactivity. The discussion will include the metamorphosis between δ‐opioid receptor antagonists to δ‐agonists based solely on subtle structural changes at the C‐terminal region of the Dmt–Tic pharmacophore as well as their behavior in vivo. Dmt may be considered promiscuous due to the acquisition of potent μ‐agonism by dermorphin and endomorphin derivatives as well as by a unique class of opioidmimetics containing two Dmt residues separated by alkyl or pyrazinone linkers. Structural studies on the Dmt–Tic compounds were enhanced tremendously by x‐ray diffraction data for three potent and biologically diverse Dmt–Tic opioidmimetics that led to the development of pharmacophores for both δ‐opioid receptor agonists and antagonists. Molecular modeling studies of other unique Dmt opioid analogues illuminated structural differences between δ‐ and μ‐receptor ligand interactions. The future of these compounds as therapeutic applications for various medical syndromes including the control of cancer‐associated pain is only a matter of time and perseverance.

Anxiolytic- and antidepressant-like activities of H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512), a novel selective delta opioid receptor agonist

Knockout and pharmacological studies have shown that delta opioid peptide (DOP) receptor signalling regulates emotional responses. In the present study, the in vitro and in vivo pharmacological profile of the DOP ligand, H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512) was investigated. In receptor binding experiments performed on membranes of CHO cells expressing the human recombinant opioid receptors, UFP-512 displayed very high affinity (pKi 10.20) and selectivity (>150-fold) for DOP sites. In functional studies ([35S]GTP gamma S binding in CHOhDOP membranes and electrically stimulated mouse vas deferens) UFP-512 behaved as a DOP selective full agonist showing potency values more than 100-fold higher than DPDPE. In vivo, in the mouse forced swimming test, UFP-512 reduced immobility time both after intracerebroventricular (i.c.v.) and intraperitoneal (i.p.) administration. Similar effects were recorded in rats. Moreover, UFP-512 evoked anxiolytic-like effects in the mouse elevated plus maze and light-dark aversion assays. All these in vivo actions of UFP-512 were fully prevented by the selective DOP antagonist naltrindole (3 mg/kg, s.c.). In conclusion, the present findings demonstrate that UFP-512 behaves as a highly potent and selective agonist at DOP receptors and corroborate the proposal that the selective activation of DOP receptors elicits robust anxiolytic- and antidepressant-like effects in rodents.

Immunohistochemical and chromatographic studies of peptides with tachykinin-like immunoreactivity in the central nervous system of the lamprey

The distribution and chemical properties of compounds with tachykinin-like immunoreactivity (TK-LI) in the spinal cord and brain of lampreys (Lampetra fluviatilis and Ichthyomyzon unicuspis) were investigated by means of immunohistochemistry and various chromatographic methods combined with radioimmunoassay. The distribution of TK immunoreactive fibers in the lamprey spinal cord was investigated with 13 different TK antisera which gave positive staining in pilot experiments. The antisera were raised against substance P (SP) (n = 6), physalaemin (PHY) (n = 1), neurokinin A (NKA) (n = 2), kassinin (KAS) (n = 2) or eledoisin (ELE) (n = 2). Pre-incubation of these antisera with their corresponding TKs abolished or reduced the immunostaining. Four different patterns of distribution were found with the 13 antisera, and they did not seem to be related to the TKs against which the antisera were raised. The different patterns could be explained by assuming the presence of the three different TKs. Six different antisera, raised against SP (n = 2), KAS (n = 2) or ELE (n = 2), were used for radioimmunoassay. The TK-LI material eluted as several separate components in various chromatographic systems. The central nervous system (CNS) of the lamprey did not contain measurable amounts of SP, NKA, neurokinin B (NKB), KAS or ELE. The present data imply that the lamprey CNS contains at least three different TKs probably different from SP, PHY, NKA, NKB, KAS or ELE; these are possibly new, not earlier described TKs. The three hypothetical TKs differ in their distribution.

New δ-opioid antagonists as pharmacological probes

The δ-opioid receptor belongs to the G protein- coupled family of receptors containing seven putative transmembrane (TM) α-helical regions, distinguished by a disulphide bond between extracellular loops (EL) 1 and 2, and an extracellular N-terminal domain[56xEvans, C.J. et al. Science. 1992; 258: 1952–1955Crossref | PubMedSee all References, 57xKieffer, B.L., Befort, K., Gaveriaux-Ruff, C., and Hirth, G.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12048–12052Crossref | PubMedSee all References]. The intracellular loop 3 couples G proteins[58xMerkouris, M. et al. Mol. Pharmacol. 1996; 50: 985–992PubMedSee all References[58] and the intracellular C-terminal sequence influences receptor turnover[59xSegredo, V., Burford, N.T., Lameh, J., and Sadee, W. J. Neurochem. 1997; 68: 2395–2404Crossref | PubMedSee all References[59]. Site-directed mutagenesis and formation of μ/δ- and κ/δ-chimeric receptors identified some residues and regions within the 372 amino acid δ-receptor that are involved in ligand recognition. Although the extracellular N-terminal region does not participate in ligand binding, specific residues in both the EL and the TM regions are important. Amino acids, Arg291 ([60xPepin, M-C. et al. J. Biol. Chem. 1997; 272: 9260–9267Crossref | PubMed | Scopus (50)See all References[60]) and Arg292 ([61xWang, W., Shahrestanifar, M., Jin, J., and Howells, R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12436–12440Crossref | PubMed | Scopus (83)See all References, 62xMeng, F. et al. Eur. J. Pharmacol. 1996; 311: 285–292Crossref | PubMed | Scopus (56)See all References]) in EL-3 and Lys108 ([63xFukuda, K., Terasko, K., Kato, S., and Mori, K. FEBS Lett. 1995; 373: 171–181Abstract | Full Text PDF | Scopus (41)See all References, 64xMinami, M. et al. Mol. Pharmacol. 1996; 50: 1413–1422PubMedSee all References]) in EL-1 may associate with an anionic residue in an opioid ligand. The hydroxyl group of Tyr is a candidate for hydrogen bonding with Asp95 ([65xKong, H. et al. J. Biol. Chem. 1993; 268: 23055–23058PubMedSee all References[65]) in TM2, Asp128 in TM3 ([66xBefort, K. et al. Mol. Pharmacol. 1996; 49: 216–223PubMedSee all References[66]) or with Ser177 in TM4 ([67xClaude, P.A. et al. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5715–5719Crossref | PubMed | Scopus (57)See all References[67]). The aromatic residues Trp268 ([68xValiquette, M. et al. J. Biol. Chem. 1996; 271: 18789–18796Crossref | PubMed | Scopus (99)See all References[68]), Tyr129, Trp173, Trp274 and Tyr308 spanning TM3–TM7 play a role in general ligand recognition; modifications at Tyr129 in TM3 drastically altered ligand–receptor interaction[69xBefort, K. et al. J. Biol. Chem. 1996; 271: 10161–10168Crossref | PubMed | Scopus (148)See all References[69].References

Interaction of deltorphin with opioid receptors: molecular determinants for affinity and selectivity.

Opioid receptor analyses of deltorphin A (H-Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) analogues indicated the following: (a) increased negativity differentially affected affinities (Ki) and selectivity (Ki mu/Ki delta); (b) shifted sequence heptapeptides, [Asp5,Leu6,Met-NH2(7)] and [Asp4,His5,Leu6,Met-NH2(7)], reversed selectivity (delta-->mu); (c) substitutions at positions 4, 5, and 6 diminished selectivity, with changes in residue 5 being the most detrimental; (d) C-terminal deletions differentially effected Ki. These are the first data to demonstrate a reversal of delta selectivity in heptapeptides containing a negative charge and indicate that modifications in affinity occur through changes in both anionic and hydrophobic properties of residues at specific positions in the peptide. Deltorphin analogues might also be applied to differentiate between opioid receptor subsites.

Na+ mechanism of δ-opioid receptor induced protection from anoxic K+ leakage in the cortex

Abstract.Activation of δ-opioid receptors (DOR) attenuates anoxic K+ leakage and protects cortical neurons from anoxic insults by inhibiting Na+ influx. It is unknown, however, which pathway(s) that mediates the Na+ influx is the target of DOR signal. In the present work, we found that, in the cortex, (1) DOR protection was largely dependent on the inhibition of anoxic Na+ influxes mediated by voltage-gated Na+ channels; (2) DOR activation inhibited Na+ influx mediated by ionotropic glutamate N-methyl-D-aspartate (NMDA) receptors, but not that by non-NMDA receptors, although both played a role in anoxic K+ derangement; and (3) DOR activation had little effect on Na+/Ca2+ exchanger-based response to anoxia. We conclude that DOR activation attenuates anoxic K+ derangement by restricting Na+ influx mediated by Na+ channels and NMDA receptors, and that non-NMDA receptors and Na+/Ca2+ exchangers, although involved in anoxic K+ derangement in certain degrees, are less likely the targets of DOR signal.

δ-Opioid receptors protect from anoxic disruption of Na+ homeostasis via Na+ channel regulation

Hypoxic/ischemic disruption of ionic homeostasis is a critical trigger of neuronal injury/death in the brain. There is, however, no promising strategy against such pathophysiologic change to protect the brain from hypoxic/ischemic injury. Here, we present a novel finding that activation of δ-opioid receptors (DOR) reduced anoxic Na+ influx in the mouse cortex, which was completely blocked by DOR antagonism with naltrindole. Furthermore, we co-expressed DOR and Na+ channels in Xenopus oocytes and showed that DOR expression and activation indeed play an inhibitory role in Na+ channel regulation by decreasing the amplitude of sodium currents and increasing activation threshold of Na+ channels. Our results suggest that DOR protects from anoxic disruption of Na+ homeostasis via Na+ channel regulation. These data may potentially have significant impacts on understanding the intrinsic mechanism of neuronal responses to stress and provide clues for better solutions of hypoxic/ischemic encephalopathy, and for the exploration of acupuncture mechanism since acupuncture activates opioid system.

Opioid infidelity: novel opioid peptides with dual high affinity for δ- and μ-receptors

Deltorphins represent the paragon of delta-opioid-receptor ligands of natural origin, since they exceed the affinity and selectivity of the endogenous enkephalins by orders of magnitude. A series of opioid peptides have been developed in which the change in a single amino acid causes an extraordinary increase in mu-receptor binding while maintaining high affinity for the delta-receptor. The peptides appear to have a similar extended conformation in solution with a type-I beta-turn in the N-terminus region, suggesting that tertiary architecture plays a pivotal role in enabling the peptide to bind indiscriminately to mu- and delta-receptors. These dual-affinity peptide ligands can serve to mask delta- and mu-receptors while mapping kappa-receptors in the nervous system, to provide an understanding of the differences and similarities in the structure of the binding domains of delta- and mu-receptors, and might lead to a comprehensive new regime for the clinical management of acute and chronic pain.

Radioimmunoassay for the tachykinin peptide physalaemin: detection of a physalaemin-like substance in rabbit stomach.

An antiserum against the amphibian tachykinin physalaemin was specific for the NH2-terminal region of this peptide. The cross-reactivity of antiserum PS-1 with uperolein was 2.03%, phyllomedusin, 0.36%, and kassinin, 0.013%; negligible (<0.0001%) recognition was shown for substance P as well as with numerous other polypeptide hormones. The assay readily detected 1 pg (0.79 fmol) physalaemin with 50% displacement at 6.79 ± 1.98 pg (5.29 ± 1.54 fmol). Rabbit stomach, extracted by boiling followed by homogenization in 1.0 n formic acid, contained physalaemin-like immunoreactivity. The concentration detected in the antrum was 43.0 ± 7.3 ng/g dry weight with less in the fundus, pylorus, corpus, and duodenum. Chromatography of the extracts on Bio-Gel P4 columns in 0.1 n formic acid gave a single, symmetrical peak of immunoreactivity which eluted with an apparent molecular weight of approximately 1700. These data are the first to demonstrate the presence of a peptide with physalaemin-like immunoreactivity in mammalian tissues.