Lawrence Toll

Sub-anesthetic concentrations of (R,S)-ketamine metabolites inhibit acetylcholine-evoked currents in α7 nicotinic acetylcholine receptors

The effect of the (R,S)-ketamine metabolites (R,S)-norketamine, (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine on the activity of α7 and α3β4 neuronal nicotinic acetylcholine receptors was investigated using patch-clamp techniques. The data indicated that (R,S)-dehydronorketamine inhibited acetylcholine-evoked currents in α7-nicotinic acetylcholine receptor, IC(50) = 55 ± 6 nM, and that (2S,6S)-hydroxynorketamine, (2R,6R)-hydroxynorketamine and (R,S)-norketamine also inhibited α7-nicotinic acetylcholine receptor function at concentrations ≤ 1 μM, while (R,S)-ketamine was inactive at these concentrations. The inhibitory effect of (R,S)-dehydronorketamine was voltage-independent and the compound did not competitively displace selective α7-nicotinic acetylcholine receptor ligands [(125)I]-α-bungarotoxin and [(3)H]-epibatidine indicating that (R,S)-dehydronorketamine is a negative allosteric modulator of the α7-nicotinic acetylcholine receptor. (R,S)-Ketamine and (R,S)-norketamine inhibited (S)-nicotine-induced whole-cell currents in cells expressing α3β4-nicotinic acetylcholine receptor, IC(50) 3.1 and 9.1 μM, respectively, while (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine were weak inhibitors, IC(50) >100 μM. The binding affinities of (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine at the NMDA receptor were also determined using rat brain membranes and the selective NMDA receptor antagonist [(3)H]-MK-801. The calculated K(i) values were 38.95 μM for (S)-dehydronorketamine, 21.19 μM for (2S,6S)-hydroxynorketamine and>100 μM for (2R,6R)-hydroxynorketamine. The results suggest that the inhibitory activity of ketamine metabolites at the α7-nicotinic acetylcholine receptor may contribute to the clinical effect of the drug.

Peptidomics of Cpefat/fat mouse brain regions: implications for neuropeptide processing

Quantitative peptidomics was used to compare levels of peptides in wild type (WT) and Cpefat/fat mice, which lack carboxypeptidase E (CPE) activity because of a point mutation. Six different brain regions were analyzed: amygdala, hippocampus, hypothalamus, prefrontal cortex, striatum, and thalamus. Altogether, 111 neuropeptides or other peptides derived from secretory pathway proteins were identified in WT mouse brain extracts by tandem mass spectrometry, and another 47 peptides were tentatively identified based on mass and other criteria. Most secretory pathway peptides were much lower in Cpefat/fat mouse brain, relative to WT mouse brain, indicating that CPE plays a major role in their biosynthesis. Other peptides were only partially reduced in the Cpefat/fat mice, indicating that another enzyme (presumably carboxypeptidase D) contributes to their biosynthesis. Approximately 10% of the secretory pathway peptides were present in the Cpefat/fat mouse brain at levels similar to those in WT mouse brain. Many peptides were greatly elevated in the Cpefat/fat mice; these peptide processing intermediates with C‐terminal Lys and/or Arg were generally not detectable in WT mice. Taken together, these results indicate that CPE contributes, either directly or indirectly, to the production of the majority of neuropeptides.

Determination of activity for nociceptin in the mouse vas deferens

The recently discovered neuropeptide nociceptin was found to inhibit electrically induced contractions of the mouse vas deferens. Nociceptin and its 14-Tyr analog were each partial agonists, but with high affinity (ED50 of 20 nM). This activity was not opioid in nature, as it was not inhibited by either selective or non-selective opiate antagonists.

Nociceptin/Orphanin FQ Receptor Structure, Signaling, Ligands, Functions, and Interactions with Opioid Systems

The NOP receptor (nociceptin/orphanin FQ opioid peptide receptor) is the most recently discovered member of the opioid receptor family and, together with its endogenous ligand, N/OFQ, make up the fourth members of the opioid receptor and opioid peptide family. Because of its more recent discovery, an understanding of the cellular and behavioral actions induced by NOP receptor activation are less well developed than for the other members of the opioid receptor family. All of these factors are important because NOP receptor activation has a clear modulatory role on mu opioid receptor-mediated actions and thereby affects opioid analgesia, tolerance development, and reward. In addition to opioid modulatory actions, NOP receptor activation has important effects on motor function and other physiologic processes. This review discusses how NOP pharmacology intersects, contrasts, and interacts with the mu opioid receptor in terms of tertiary structure and mechanism of receptor activation; location of receptors in the central nervous system; mechanisms of desensitization and downregulation; cellular actions; intracellular signal transduction pathways; and behavioral actions with respect to analgesia, tolerance, dependence, and reward. This is followed by a discussion of the agonists and antagonists that have most contributed to our current knowledge. Because NOP receptors are highly expressed in brain and spinal cord and NOP receptor activation sometimes synergizes with mu receptor-mediated actions and sometimes opposes them, an understanding of NOP receptor pharmacology in the context of these interactions with the opioid receptors will be crucial to the development of novel therapeutics that engage the NOP receptor.

Evidence for an NMDA receptor subunit in human keratinocytes and rat cardiocytes

Receptor binding studies have demonstrated the presence of an [3H]MK-801 ([3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine maleate) binding site in human keratinocytes. The affinity found in keratinocytes was lower than that found in brain membranes. Northern blots identified mRNA in human keratinocytes and rat cardiocytes, as well as rat brain, that hybridized with high stringency to a probe for NMDAR1, an NMDA receptor subunit. In each tissue, mRNA that hybridized to another glutamate binding protein that might be part of an NMDA receptor complex, was also present. The presence of NMDA or NMDA-like receptors in keratinocytes and rat cardiocytes together with the low affinity [3H]MK-801 binding suggests that this protein may be a general channel forming protein that is present in many tissues, and forms specific receptors by interacting with additional subunits.

Activities of mixed NOP and μ‐opioid receptor ligands

Compounds that activate both NOP and μ‐opioid receptors might be useful as analgesics and drug abuse medications. Studies were carried out to better understand the biological activity of such compounds.

Modulation of enkephalin release by nociceptin (orphanin FQ)

Nociceptin (orphanin FQ) is an endogenous peptide agonist for the newly discovered receptor (opioid receptor-like 1 receptor, ORL1) that bears striking homology to opioid receptors. Initial reports claimed that this peptide had hypoalgesic effects following i.c.v. or i.t. administration. The present study demonstrates that, in the presence of opioid receptor blockade, nociceptin can substantially alter the magnitude of the stimulated release of methionine-enkephalin from the guinea pig myenteric plexus. This effect is concentration dependent. Low doses (1 or 10 nM) inhibit whereas higher concentrations (100 or 1000 nM) enhance evoked enkephalin release. In contrast, in the absence of opioid receptor blockade, a statistically significant inhibition of stimulated enkephalin release is observed in response to 1, 100 or 1000 nM nociceptin. However, the magnitude of this effect did not differ among these concentrations. Furthermore, at 10 nM nociceptin, either an inhibition or enhancement of stimulated enkephalin release is manifest. The ability of naloxone to alter the nociceptin modulation of enkephalin release suggests that a component of the nociceptin modulation of enkephalin release is mediated via opioid receptors. This is consistent with the observation that this peptide has modest affinity for opioid receptors (L > K > 8) which, under appropriate conditions, should be sufficient to permit interactions with multiple opioid receptor types. This complicates dose responsiveness for nociceptin since both the naloxone-resistant (ORL1-mediated) and naloxone-sensitive (opioid receptor-mediated) component exhibit a concentration-dependent bimodality (albeit in opposite directions). Determination of i.c.v. or i.t. nociceptin dose responsiveness over several orders of magnitude is suggested before concluding the physiological effects of this peptide.

Small-molecule agonists and antagonists of the opioid receptor-like receptor (ORL1, NOP): ligand-based analysis of structural factors influencing intrinsic activity at NOP.

The recently discovered fourth member of the opioid receptor family, the nociceptin receptor (NOP) and its endogenous ligand, the heptadecaptide nociceptin, are involved in several central nervous system pathways, such as nociception, reward, tolerance, and feeding. The discovery of small-molecule ligands for NOP is being actively pursued for several therapeutic applications. This review presents a brief overview of the several recently reported NOP ligands, classified as NOP agonists and antagonists, with an emphasis on the analysis of the structural features that may be important for modulating the agonist/antagonist profile (intrinsic activity) of these ligands. Structure-activity relationships in our own series of dihydroindolinone-based NOP ligands and those of the various reported ligands indicate that the lipophilic substituent on the common basic nitrogen present in all NOP ligands plays a role in determining the agonist/antagonist profile of the NOP ligand. This analysis provides a basis for the rational drug design of NOP ligands of desired intrinsic activity and provides a framework for developing pharmacophore models for high affinity binding and intrinsic activity at the NOP receptor. Since NOP agonists and antagonists both have therapeutic value, rational approaches for obtaining both within a high-affinity binding class of compounds are very useful for designing potent and selective NOP ligands with the desired profile of intrinsic efficacy.

Challenges for opioid receptor nomenclature: IUPHAR Review 9

Recent developments in the study of the structure and function of opioid receptors raise significant challenges for the definition of individual receptor types and the development of a nomenclature that precisely describes isoforms that may subserve different functions in vivo. Presentations at the 2013 meeting of the International Narcotics Research Conference in Cairns, Australia, considered some of the new discoveries that are now unravelling the complexities of opioid receptor signalling. Variable processing of opioid receptor messenger RNAs may lead to the presence of several isoforms of the μ receptor. Each opioid receptor type can function either as a monomer or as part of a homo‐ or heterodimer or higher multimer. Additionally, recent evidence points to the existence of agonist bias in the signal transduction pathways activated through μ receptors, and to the presence of regulatory allosteric sites on the receptors. This brief review summarizes the recent discoveries that raise challenges for receptor definition and the characterization of signal transduction pathways activated by specific receptor forms.

Comparison of the Antinociceptive and Antirewarding Profiles of Novel Bifunctional Nociceptin Receptor/μ-Opioid Receptor Ligands: Implications for Therapeutic Applications

The nociceptin receptor (NOPr), a member of the opioid receptor family, is a target for the treatment of pain and drug abuse. Nociceptin/orphanin FQ (N/OFQ), the endogenous peptide for NOPr, not only modulates opioid antinociception, but also blocks the rewarding effects of several abused drugs, such as morphine, cocaine, and amphetamine. We hypothesized that NOPr agonists, with bifunctional activity at the μ-opioid receptor (MOPr), may function as nonaddicting analgesics or as drug abuse medications. Bifunctional small-molecule NOPr agonists possessing different selectivities and efficacies at MOPr were evaluated in an acute thermal antinociception assay, and for their ability to induce conditioned place preference (CPP) and their effect on morphine-induced CPP. 1-(1-Cyclooctylpiperidin-4-yl)-indolin-2-one) (SR14150), a high-affinity NOPr partial agonist, with low MOPr affinity and efficacy, produced analgesia that was naloxone-reversible. SR14150 did not induce CPP alone, nor did it attenuate morphine-induced CPP. 3-Ethyl-1-(1-(4-isopropylcyclohexyl)piperidin-4-yl)-indolin-2-one (SR16507), which has high affinity for both NOPr and MOPr, full agonist activity at NOPr, and partial agonist activity at MOPr, was also a potent analgesic and produced CPP alone, but also modestly attenuated morphine CPP. 1-(1-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)piperidinl-4-yl)-indolin-2-one (SR16835), a NOPr full agonist and low-affinity MOPr partial agonist, was not antinociceptive, did not produce CPP alone, but attenuated morphine CPP. Our results suggest that NOPr full-agonist activity is required to modulate opioid-induced reward, whereas a bifunctional NOPr/MOPr partial agonist profile may be suitable as a nonaddicting analgesic. The opioid-modulating effects of the NOPr ligands may be used effectively to produce better medications for treatment of drug abuse and pain.