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1.
Mol Pain ; 7: 24, 2011 Apr 12.
Article in English | MEDLINE | ID: mdl-21486473

ABSTRACT

Hedonic reward, dependence and addiction are unwanted effects of opioid analgesics, linked to the phasic cycle of µ opioid receptor activation, tolerance and withdrawal. In vitro studies of recombinant G protein coupled receptors (GPCRs) over expressed in cell lines reveal an alternative tonic signaling mechanism that is independent of agonist. Such studies demonstrate that constitutive GPCR signaling can be inhibited by inverse agonists but not by neutral antagonists. However, ligand-independent activity has been difficult to examine in vivo, at the systems level, due to relatively low levels of constitutive activity of most GPCRs including µ receptors, often necessitating mutagenesis or pharmacological manipulation to enhance basal signaling. We previously demonstrated that the absence of ß-arrestin 2 (ß-arr2) augments the constitutive coupling of µ receptors to voltage-activated Ca²+ channels in primary afferent dorsal root ganglion neurons from ß-arr2⁻/⁻ mice. We used this in vitro approach to characterize neutral competitive antagonists and inverse agonists of the constitutively active wild type µ receptors in neurons. We administered these agents to ß-arr2⁻/⁻ mice to explore the role of constitutive µ receptor activity in nociception and hedonic tone. This study demonstrates that the induction of constitutive µ receptor activity in vivo in ß-arr2⁻/⁻ mice prolongs tail withdrawal from noxious heat, a phenomenon that was reversed by inverse agonists, but not by antagonists that lack negative efficacy. By contrast, the aversive effects of inverse agonists were similar in ß-arr2⁻/⁻ and ß-arr2+/+ mice, suggesting that hedonic tone was unaffected.


Subject(s)
Arrestins/genetics , Receptors, Opioid, mu/metabolism , Animals , Electrophysiology , Female , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , Male , Mice , Mice, Mutant Strains , Naloxone/pharmacology , Naltrexone/pharmacology , Pain Measurement , Peptide Fragments/pharmacology , Receptors, Opioid, mu/agonists , Receptors, Opioid, mu/antagonists & inhibitors , Somatostatin/pharmacology , beta-Arrestin 2 , beta-Arrestins
2.
Mol Pharmacol ; 76(1): 134-43, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19357247

ABSTRACT

Recombinant micro and delta opioid receptors expressed in cell lines can form heterodimers with distinctive properties and trafficking. However, a role for opioid receptor heterodimerization in neurons has yet to be identified. The inhibitory coupling of opioid receptors to voltage-dependent Ca(2+) channels (VDCCs) is a relatively inefficient process and therefore provides a sensitive assay of altered opioid receptor function and expression. We examined micro-receptor coupling to VDCCs in dorsal root ganglion neurons of delta(+/+), delta(+/-), and delta(-/-) mice. Neurons deficient in delta receptors exhibited reduced inhibition of VDCCs by morphine and [D-Ala(2),Phe(4),Gly(5)-ol]-enkephalin (DAMGO). An absence of delta receptors caused reduced efficacy of DAMGO without affecting potency. An absence of delta receptors reduced neither the density of VDCCs nor their inhibition by either the GABA(B) receptor agonist baclofen or intracellular guanosine 5'-O-(3-thio)triphosphate. Flow cytometry revealed a reduction in micro-receptor surface expression in delta(-/-) neurons without altered DAMGO-induced internalization. There was no change in micro-receptor mRNA levels. D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)-sensitive mu-receptor-coupling efficacy was fully restored to delta(+/+) levels in delta(-/-) neurons by expression of recombinant delta receptors. However, the dimerization-deficient delta-15 construct expressed in delta(-/-) neurons failed to fully restore the inhibitory coupling of micro-receptors compared with that seen in delta(+/+) neurons, suggesting that, although not essential for micro-receptor function, micro-delta receptor dimerization contributes to full micro-agonist efficacy. Because DAMGO exhibited a similar potency in delta(+/+) and delta(-/-) neurons and caused similar levels of internalization, the role for heterodimerization is probably at the level of receptor biosynthesis.


Subject(s)
Calcium Channels/physiology , Ganglia, Spinal/metabolism , Receptors, Opioid, delta/physiology , Receptors, Opioid, mu/physiology , Animals , Dimerization , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Mice , Mice, Inbred C57BL , Receptors, Opioid, delta/chemistry , Receptors, Opioid, mu/chemistry
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