Morphine-like activity of natural human IgG autoantibodies is because of binding to the first and third extracellular loops of the mu-opioid receptor.
(33/2005)
We have previously demonstrated that randomly selected healthy individuals express anti-human mu-opioid receptor antibodies which behave as agonist in vitro. In this study, we show that the activity of these antibodies was not affected by the deletion of the amino-terminal region of the receptor. Using agarose-bound peptide columns, we affinity-purified IgG specifically directed toward each extracellular loop. Whatever its specificity, each anti-human mu-opioid receptor (hMOR) extracellular loop peptide IgG preparation was unable, when examined individually, to reduce adenylate cyclase activity. Activation of the hMOR was, however, achieved by the simultaneous binding of IgG to the first and third extracellular loops of the receptor. Our results suggest that the simultaneous binding of IgG antibodies to these two loops mimics morphine-induced receptor activation by triggering a coordinated shift of the third and sixth transmembrane helices. (+info)
Naloxone blocks endomorphin-1 but not endomorphin-2 induced inhibition of tachykinergic contractions of guinea-pig isolated bronchus.
(34/2005)
The recently identified endogenous agonists on the mu-opioid-receptor (mu OR), endomorphin-1 (EM-1) and endomorphin-2 (EM-2), induce a concentration dependent inhibition of electrical field stimulation (EFS)-induced tachykinin-mediated contractions of the guinea-pig bronchus (ED50s < 10 nM for both compounds). Surprisingly, only endomorphin-1 effects could be blocked by naloxone (10 microM), whereas endomorphin-2 effects were not affected by specific antagonists for the mu-, kappa-, and delta-opioid-receptor. (+info)
Presynaptic regulation of glutamate release in the ventral tegmental area during morphine withdrawal.
(35/2005)
The regulation of glutamate (Glu) release from the excitatory input to dopamine cells in the ventral tegmental area (VTA) during acute withdrawal from morphine was studied in slices from animals treated for 6-7 d with morphine. EPSCs were inhibited by opioid agonists acting at micro-subtype receptors but not by selective delta- or kappa-subtype agonists. The opioid inhibition was reduced by 65% with the potassium channel blocker 4-aminopyridine (4-AP; 100 microM) and a 12-lipoxygenase inhibitor, baicalein (5 microM), suggesting that opioids acted via a transduction pathway involving activation of a voltage-dependent potassium conductance by lipoxygenase metabolites as has been shown in the periaqueductal gray (). During withdrawal, neither the potency nor the efficacy of D-Ala-Met-enkephalin-Gly-ol (DAMGO) were changed; however, the blockade of micro-opioid inhibition by both 4-AP and baicalein was reduced. In addition, the potency of baclofen to depress EPSCs by GABA-B receptors and the effects of the GABA-uptake inhibitor NO-711 (10 microM) were increased in withdrawn rats. Finally, group 2 (but not group 4 or 1) metabotropic glutamate receptor-mediated presynaptic inhibition was also enhanced in morphine-withdrawn rats. These results suggest that one of the consequences of withdrawal from chronic morphine is an enhanced presynaptic inhibition of the excitatory inputs to the dopamine cells of the VTA. Inhibition of glutamate release during acute withdrawal would add to the inhibition of dopamine cells that is mediated by an augmented release of GABA (). (+info)
Calmodulin binding to G protein-coupling domain of opioid receptors.
(36/2005)
The ubiquitous intracellular Ca(2+) sensor calmodulin (CaM) regulates numerous proteins involved in cellular signaling of G protein-coupled receptors, but most known interactions between GPCRs and CaM occur downstream of the receptor. Using a sequence-based motif search, we have identified the third intracellular loop of the opioid receptor family as a possible direct contact point for interaction with CaM, in addition to its established role in G protein activation. Peptides derived from the third intracellular loop of the mu-opioid (OP(3)) receptor strongly bound CaM and were able to reduce binding interactions observed between CaM and immunopurified OP(3) receptor. Functionally, CaM reduced basal and agonist-stimulated (35)S-labeled guanosine 5'-3-O-(thio)triphosphate incorporation, a measure of G protein activation, in membranes containing recombinant OP(3) receptor. Changes in CaM membrane levels as a result of overexpression or antisense CaM suppression inversely affected basal and agonist-induced G protein activation. The ability of CaM to abolish high affinity binding sites of an agonist at OP(3) further supports the hypothesis of a direct interaction between CaM and opioid receptors. An OP(3) receptor mutant with a Lys(273) --> Ala substitution (K273A-OP(3)), an amino acid predicted to play a critical role in CaM binding based on motif structure, was found to be unaffected by changes in CaM levels but coupled more efficiently to G proteins than the wild-type receptor. Stimulation of both the OP(1) (delta-opioid) and OP(3) wild-type receptors, but not the K273A-OP(3) mutant, induced release of CaM from the plasma membrane. These results suggest that CaM directly competes with G proteins for binding to opioid receptors and that CaM may itself serve as an independent second messenger molecule that is released upon receptor stimulation. (+info)
Identification and characterization of three new alternatively spliced mu-opioid receptor isoforms.
(37/2005)
We have identified four new mu-opiod receptor (MOR)-1 exons, indicating that the gene now contains at least nine exons spanning more than 200 kilobases. Replacement of exon 4 by combinations of the new exons yields three new receptors. When expressed in Chinese hamster ovary cells, all three variants displayed high affinity for mu-opioid ligands, but kappa and delta drugs were inactive. However, there were subtle, but significant, differences in the binding profiles of the three variants among themselves and from MOR-1. Immunohistochemically, the major variant, MOR-1C, displayed a regional distribution quite distinct from that of MOR-1. Region-specific processing also was seen at the mRNA level. Antisense mapping revealed that the four new exons were all involved in morphine analgesia. Together with two other variants generated from alternative splicing of exon 4, there are now six distinct MOR-1 receptors. (+info)
Actions of opioids on excitatory and inhibitory transmission in substantia gelatinosa of adult rat spinal cord.
(38/2005)
1. The actions of opioid receptor agonists on synaptic transmission in substantia gelatinosa (SG) neurones in adult (6- to 10-week-old) rat spinal cord slices were examined by use of the blind whole-cell patch-clamp technique. 2. Both the mu-receptor agonist DAMGO (1 microM) and the delta-receptor agonist DPDPE (1 microM) reduced the amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) which were monosynaptically evoked by stimulating Adelta afferent fibres. Both also decreased the frequency of miniature EPSCs without affecting their amplitude. 3. In contrast, the kappa-receptor agonist U-69593 (1 microM) had little effect on the evoked and miniature EPSCs. 4. The effects of DAMGO and DPDPE were not seen in the presence of the mu-receptor antagonist CTAP (1 microM) and the delta-receptor antagonist naltrindole (1 microM), respectively. 5. Neither DAMGO nor DPDPE at 1 microM affected the responses of SG neurones to bath-applied AMPA (10 microM). 6. Evoked and miniature inhibitory postsynaptic currents (IPSCs), mediated by either the GABAA or the glycine receptor, were unaffected by the mu-, delta- and kappa-receptor agonists. Similar results were also obtained in SG neurones in young adult (3- to 4-week-old) rat spinal cord slices. 7. These results indicate that opioids suppress excitatory but not inhibitory synaptic transmission, possibly through the activation of mu- and delta- but not kappa-receptors in adult rat spinal cord SG neurones; these actions are presynaptic in origin. Such an action of opioids may be a possible mechanism for the antinociception produced by their intrathecal administration. (+info)
Opioid modulation of calcium current in cultured sensory neurons: mu-modulation of baroreceptor input.
(39/2005)
We used the whole cell open-patch or perforated-patch technique to characterize mu-opioid modulation of Ca(2+) current (I(Ca)) in nodose sensory neurons and in a specific subpopulation of nodose cells, aortic baroreceptor neurons. The mu-opiate receptor agonist Tyr-D-Ala-Gly-MePhe-Gly-ol enkephalin (DAGO) inhibited I(Ca) in 95% of neonatal [postnatal day (P)1-P3] nodose neurons. To the contrary, only 64% of juvenile cells (P20-P35) and 61% of adult cells (P60-P110) responded to DAGO. DAGO-mediated inhibition of I(Ca) was naloxone sensitive, irreversible in the presence of guanosine 5'-O-(3-thiotriphosphate), absent with guanosine 5'-O-(2-thiodiphosphate), and eliminated with pertussis toxin; DAGO's inhibition of I(Ca) was G protein mediated. Incubation of neurons with omega-conotoxin GVIA eliminated the effect of DAGO in neonatal but not in juvenile cells. In the latter, DAGO reduced 37% of the current remaining in the presence of omega-conotoxin. In the subset of nodose neurons, aortic baroafferents, the effect of DAGO was concentration dependent, with an IC(50) of 1.82 x 10(-8) M. DAGO slowed activation of I(Ca), but activation curves constructed from tail currents were the same with and without DAGO (100 nM). In summary, mu-opiate modulation of I(Ca) in nodose neurons was demonstrated in three age groups, including specifically labeled baroafferents. The demonstration of a mechanism of action of mu-opioids on baroreceptor afferents provides a basis for the attenuation of the baroreflex that occurs at the level of the nucleus tractus solitarii. (+info)
MAP kinase activation by mu opioid receptor involves phosphatidylinositol 3-kinase but not the cAMP/PKA pathway.
(40/2005)
The involvement of protein kinases was studied in mu opioid receptor activation of mitogen-activated protein (MAP) kinase using cells transfected with the receptor clone. The cAMP/protein kinase A (PKA) pathway is known to be the major biochemical pathway for mu opioid receptor signaling. However, our data showed that stimulating adenylyl cyclase or activating PKA had no effect on mu receptor enhancement of MAP kinase activity, suggesting that the cAMP/PKA pathway is not involved in mediating the mu receptor activation of MAP kinase. Inhibition of phosphatidylinositol (PI) 3-kinase reduced mu receptor enhancement of MAP kinase activity, suggesting PI 3-kinase involvement. Together, these results show that cross-talk between the mu opioid receptor and the MAP kinase cascade is not mediated by the cAMP/PKA pathway, but involves PI 3-kinase. (+info)