Powerful analgesics relieve pain primarily through activating mu opioid receptor (MOR), but the long-term use of MOR agonists, such as morphine, is limited by the rapid development of tolerance. ...Recently, it has been observed that simultaneous stimulation of the delta opioid receptor (DOR) and MOR limits the incidence of tolerance induced by MOR agonists. 3-(2
R,6
R,11
R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2
H)-yl-
N-phenylpropanamide (LP1) is a centrally acting agent with antinociceptive activity comparable to morphine and is able to bind and activate MOR and DOR. The aim of this work was to evaluate and compare the induction of tolerance to antinociceptive effects from treatment with LP1 and morphine.
Here, we evaluated the pharmacological effects of LP1 administered at a dose of 4
mg/kg subcutaneously (s.c.) twice per day for 9
days to male Sprague–Dawley rats. In addition, the LP1 mechanism of action was assessed by measurement of LP1-induced
35SGTPγS binding to the MOR and DOR.
Data obtained from the radiant heat tail flick test showed that LP1 maintained its antinociceptive profile until the ninth day, while tolerance to morphine (10
mg/kg s.c. twice per day) was observed on day 3. Moreover, LP1 significantly enhanced
35SGTPγS binding in the membranes of HEK293 cells expressing either the MOR or the DOR.
LP1 is a novel analgesic agent for chronic pain treatment, and its low tolerance-inducing capability may be correlated with its ability to bind both the MOR and DOR.
Previous studies have shown that the intracellular domains of opioid receptors serve as platforms for the formation of a multi-component signaling complex consisting of various interacting partners ...(Leontiadis et al., 2009, Cell Signal. 21, 1218–1228; Georganta et al., 2010, Neuropharmacology, 59(3), 139–148). In the present study we demonstrate that spinophilin a dendritic-spine enriched scaffold protein associates with δ- and μ-opioid receptors (δ-ΟR, μ-OR) constitutively in HEK293 an interaction that is altered upon agonist administration and enhanced upon forskolin treatment for both μ-OR and δ-ΟR. Spinophilin association with the opioid receptors is mediated via the third intracellular loop and a conserved region of the C-terminal tails. The portion of spinophilin responsible for interaction with the δ-OR and μ-OR is narrowed to a region encompassing amino acids 151–444. Spinophilin, RGS4, Gα and Gβγ subunits of G proteins form a multi-protein complex using specific regions of spinophilin and a conserved amino acid stretch of the C-terminal tails of both δ-μ-ORs. Expression of spinophilin in HEK293 cells potentiated DPDPE-mediated adenylyl-cyclase inhibition of δ-OR leaving unaffected the levels of cAMP accumulation mediated by the μ-OR. Moreover, measurements of extracellular signal regulated kinase (ERK1,2) phosphorylation indicated that the presence of spinophilin attenuated agonist-driven ERK1,2 phosphorylation mediated upon activation of the δ-OR but not the μ-OR. Collectively, these findings suggest that spinophilin associates with both δ- and μ-ΟR and G protein subunits in HEK293 cells participating in a multimeric signaling complex that displays a differential regulatory role in opioid receptor signaling.
► Direct physical association of δ-opioid receptor with spinophilin in HEK293 cells ► δ-μ-opioid receptors differentially couple with spinophilin. ► The COOH tail of opioid receptors docking site of spinophilin ► Spinophilin Gα, Gβγ, RGS4 and opioid receptors form a protein complex. ► δ-μ-opioid receptor signaling is differentially altered by spinophilin.
6,7-Benzomorphan derivatives, exhibiting different μ, δ, and κ receptor selectivity profiles depending on the N-substituent, represent a useful skeleton for the synthesis of new and better analgesic ...agents. In this work, an aromatic ring and/or alkyl residues have been used with an N-propanamide or N-acetamide spacer for the synthesis of a new series of 5,9-dimethyl-2′-hydroxy-6,7-benzomorphan derivatives (12–22). Data obtained by competition binding assays showed that the μ opioid receptor seems to prefer an interaction with the 6,7-benzomorphan ligands having an N-substituent with a propanamide spacer and less hindered amide. Highly stringent features are required for δ receptor interaction, while an N-acetamide spacer and/or bulkier amide could preferentially lead to κ receptor selectivity. In the propanamide series, compound 12 (named LP1) displayed high μ affinity (Ki=0.83nM), good δ affinity (Ki=29nM) and low affinity for the κ receptor (Ki=110nM), with a selectivity ratio δ/μ and κ/μ of 35.1 and 132.5, respectively. Further, in the adenylyl cyclase assay, LP1 displayed a μ/δ agonist profile, with IC50 values of 4.8 and 12nM at the μ and δ receptors, respectively. The antinociceptive potency of LP1 in the tail-flick test after sc administration in rat was comparable with the potency of morphine (ED50=2.03 and 2.7mg/kg, respectively), and was totally reversed by naloxone. LP1, possessing a μ/δ agonist profile, could represent a lead in further developing benzomorphan-based ligands with potent in vivo analgesic activity and a reduced tendency to induce side effects.
Novel dermorphin tetrapeptides are described in which Tyr1 is replaced by Dmt1, where d-Ala2 and Gly4 are N-methylated, and where Phe3-Gly4 residue is substituted by the constrained Aba3-Gly4 ...peptidomimetic. Most of these peptidic ligands displayed binding affinities in the nanomolar range for both μ- and δ-opioid receptors but no detectable affinity for the κ-opioid receptor. Measurements of cAMP accumulation, phosphorylation of extracellular signal-regulated kinase (ERK1/2) in HEK293 cells stably expressing each of these receptors individually, and functional screening in primary neuronal cultures confirmed the potent agonistic properties of these peptides. The most potent ligand H-Dmt-NMe-d-Ala-Aba-Gly-NH2 (BVD03) displayed mixed μ/δ opioid agonist properties with picomolar functional potencies. Functional electrophysiological in vitro assays using primary cortical and spinal cord networks showed that this analogue possessed electrophysiological similarity toward gabapentin and sufentanil, which makes it an interesting candidate for further study as an analgesic for neuropathic pain.
Previous studies have shown that the intracellular domains of opioid receptors serve as platforms for the formation of a multi-component signaling complex consisting of various interacting partners ...(Leontiadis et al., 2009, Cell Signal. 21, 1218-1228; Georganta et al., 2010, Neuropharmacology, 59(3), 139-148). In the present study we demonstrate that spinophilin a dendritic-spine enriched scaffold protein associates with d- and mu -opioid receptors (d-OR, mu -OR) constitutively in HEK293 an interaction that is altered upon agonist administration and enhanced upon forskolin treatment for both mu -OR and d-OR. Spinophilin association with the opioid receptors is mediated via the third intracellular loop and a conserved region of the C-terminal tails. The portion of spinophilin responsible for interaction with the d-OR and mu -OR is narrowed to a region encompassing amino acids 151-444. Spinophilin, RGS4, Galpha and Gbetagamma subunits of G proteins form a multi-protein complex using specific regions of spinophilin and a conserved amino acid stretch of the C-terminal tails of both d- mu -ORs. Expression of spinophilin in HEK293 cells potentiated DPDPE-mediated adenylyl-cyclase inhibition of d-OR leaving unaffected the levels of cAMP accumulation mediated by the mu -OR. Moreover, measurements of extracellular signal regulated kinase (ERK1,2) phosphorylation indicated that the presence of spinophilin attenuated agonist-driven ERK1,2 phosphorylation mediated upon activation of the d-OR but not the mu -OR. Collectively, these findings suggest that spinophilin associates with both d- and mu -OR and G protein subunits in HEK293 cells participating in a multimeric signaling complex that displays a differential regulatory role in opioid receptor signaling.
This thesis focuses on the use of animal caps to address questions concerning neural induction, patterning and neurogenesis in Xenopus embryos. Animal caps can be neuralised by the inhibition of BMPs ...through the action of inhibitors, such as noggin, but they do not form neurons. Instead, primary neurogenesis requires additional signalling by retinoic acid (RA), acting through the retinoid receptors, RARIRXR. In the presence of RA, retinoid signalling (RS) activates gene expression. Key target genes of RS during development are the Hox genes that are believed to be important for both posterior patterning and neurogenesis. In contrast, in the absence of RA, RS promotes active repression; a process that is required for normal head formation. However, much less is known of the genes that are affected by active repression. Although RS is sufficient to promote neurogenesis in noggin-neuralised animal caps, it is likely to be working in conjunction with other, endogenous, signalling pathways, mediated for example by FGF and Wnt. In this study, animal caps were analysed for signalling molecule expression after neuralisation by noggin and treatment with RARlRXR (±RA) and it was shown that the expression of some, but not all, FGFs and Wnts respond to RS in the animal cap. This may be significant for neural induction, patterning and neurogenesis; related processes in the animal cap. In addition, the receptor isotypes RARa and RARy seem to elicit different responses from some genes. Positive RS (RARlRXR+RA) was shown to promote posterior markers, such as Hox genes and the expression of Wnt3A. However, negative RS (RARlRXR-RA) was shown to inhibit Wnt3A and activate xSaiF. Consequently, RARs promote Wnt signalling posteriorly via positive signalling and inhibit Wnt signalling anteriorly by negative signalling. In conclusion, this thesis shows that animal caps can be used to investigate the effects of RS in Xenopus neural development and indicates a major role for RS along the length of the AlP axis of the embryo.
This thesis focuses on the use of animal caps to address questions concerning neural induction, patterning and neurogenesis in Xenopus embryos. Animal caps can be neuralised by the inhibition of BMPs ...through the action of inhibitors, such as noggin, but they do not form neurons. Instead, primary neurogenesis requires additional signalling by retinoic acid (RA), acting through the retinoid receptors, RARIRXR. In the presence of RA, retinoid signalling (RS) activates gene expression. Key target genes of RS during development are the Hox genes that are believed to be important for both posterior patterning and neurogenesis. In contrast, in the absence of RA, RS promotes active repression; a process that is required for normal head formation. However, much less is known of the genes that are affected by active repression. Although RS is sufficient to promote neurogenesis in noggin-neuralised animal caps, it is likely to be working in conjunction with other, endogenous, signalling pathways, mediated for example by FGF and Wnt. In this study, animal caps were analysed for signalling molecule expression after neuralisation by noggin and treatment with RARlRXR (±RA) and it was shown that the expression of some, but not all, FGFs and Wnts respond to RS in the animal cap. This may be significant for neural induction, patterning and neurogenesis; related processes in the animal cap. In addition, the receptor isotypes RARa and RARy seem to elicit different responses from some genes. Positive RS (RARlRXR+RA) was shown to promote posterior markers, such as Hox genes and the expression of Wnt3A. However, negative RS (RARlRXR-RA) was shown to inhibit Wnt3A and activate xSaiF. Consequently, RARs promote Wnt signalling posteriorly via positive signalling and inhibit Wnt signalling anteriorly by negative signalling. In conclusion, this thesis shows that animal caps can be used to investigate the effects of RS in Xenopus neural development and indicates a major role for RS along the length of the AlP axis of the embryo.
![Superpotent [Dmt1]Dermorphi...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/0022-2623/small "Superpotent [Dmt1]Dermorphin Tetrapeptides Containing the 4‑Aminotetrahydro-2-benzazepin-3-one Scaffold with Mixed μ/δ Opioid Receptor Agonistic Properties")