Cloning and characterization of RGS9-2: a striatal-enriched alternatively spliced product of the RGS9 gene.
Regulators of G-protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) for alpha subunits of heterotrimeric G-proteins. Previous in situ hybridization analysis of mRNAs encoding RGS3-RGS11 revealed region-specific expression patterns in rat brain. RGS9 showed a particularly striking pattern of almost exclusive enrichment in striatum. In a parallel study, RGS9 cDNA, here referred to as RGS9-1, was cloned from retinal cDNA libraries, and the encoded protein was identified as a GAP for transducin (Galphat) in rod outer segments. In the present study we identify a novel splice variant of RGS9, RGS9-2, cloned from a mouse forebrain cDNA library, which encodes a striatal-specific isoform of the protein. RGS9-2 is 191 amino acids longer than the retinal isoform, has a unique 3' untranslated region, and is highly enriched in striatum, with much lower levels seen in other brain regions and no expression detectable in retina. Immunohistochemistry showed that RGS9-2 protein is restricted to striatal neuropil and absent in striatal terminal fields. The functional activity of RGS9-2 is supported by the finding that it, but not RGS9-1, dampens the Gi/o-coupled mu-opioid receptor response in vitro. Characterization of a bacterial artificial chromosome genomic clone of approximately 200 kb indicates that these isoforms represent alternatively spliced mRNAs from a single gene and that the RGS domain, conserved among all known RGS members, is encoded over three distinct exons. The distinct C-terminal domains of RGS9-2 and RGS9-1 presumably contribute to unique regulatory properties in the neural and retinal cells in which these proteins are selectively expressed. (+info)
Presynaptic inhibition of GABA(B)-mediated synaptic potentials in the ventral tegmental area during morphine withdrawal.
Opioids increase the firing of dopamine cells in the ventral tegmental area by presynaptic inhibition of GABA release. This report describes an acute presynaptic inhibition of GABAB-mediated IPSPs by mu- and kappa-opioid receptors and the effects of withdrawal from chronic morphine treatment on the release of GABA at this synapse. In slices taken from morphine-treated guinea pigs after washing out the morphine (withdrawn slices), a low concentration of a mu receptor agonist increased, rather than decreased, the amplitude of the GABAB IPSP. In withdrawn slices, after blocking A1-adenosine receptors with 8-cyclopentyl-1, 3-dipropylxantine, mu-opioid receptor activation inhibited the IPSP at all concentrations and increased the maximal inhibition. In addition, during withdrawal, there was a tonic increase in adenosine tone that was further increased by forskolin or D1-dopamine receptor activation, suggesting that metabolism of cAMP was the source of adenosine. The results indicate that during acute morphine withdrawal, there was an upregulation of the basal level of an opioid-sensitive adenylyl cyclase. Inhibition of this basal activity by opioids had two effects. First, a decrease in the formation of cAMP that decreased adenosine tone. This effect predominated at low mu receptor occupancy and increased the amplitude of the IPSP. Higher agonist concentrations inhibited transmitter release by both kinase-dependent and -independent pathways. This study indicates that the consequences of the morphine-induced upregulation of the cAMP cascade on synaptic transmission are dependent on the makeup of receptors and second messenger pathways present on any given terminal. (+info)
Molecular modeling of mu opioid receptor and receptor-ligand interaction.
AIM: To construct the 3D structural model of mu opioid receptor (mu OR) and study the interaction between mu OR and fentanyl derivatives. METHODS: The 3D structure of mu OR was modeled using the bacteriorhodopsin (bRh) as a template, in which the alignments of transmembrane (TM) of bRh and mu OR were achieved by scoring the alignment between the amino acid sequence of mu OR and the structure of bRh. The fentanyl derivatives were docked into the 7 helices of mu OR and the binding energies were calculated. RESULTS: (1) The receptor-ligand interaction models were obtained for fentanyl derivatives. (2) In these models, the fundamental binding sites were possibly Asp147 and His297. The negatively charged oxygen of Asp147 and the positively charged ammonium group of ligand formed the potent electrostatic and hydrogen-binding interactions. Whereas the interactions between the positively charged nitrogen of His297 and the carbonyl oxygen of ligand were weak. In addition, there were some pi-pi interactions between the receptor and the ligand. (3) The binding energies of the receptor-ligand complexes had a good correlation with the analgesic activities (-lg ED50) of the fentanyl derivatives. CONCLUSION: This model is helpful for understanding the receptor-ligand interaction and for designing novel mu OR selective ligands. (+info)
Binding properties of C-truncated delta opioid receptors.
AIM: To study the role of C-terminal delta opioid receptor involved in ligand binding affinity and selectivity. METHODS: The 31 amino acid residues of C-terminal truncated delta opioid receptors and the wild-type were expressed stably in Chinese hamster ovary (CHO) cells, respectively. Then the ligand binding properties of the products were studied by receptor binding assay. RESULTS: A typical mutated receptor clone CHO-T and a wild-type receptor clone CHO-W were obtained. The Kd values of [3H] diprenorphine (Dip) and [3H]leucine-2-alanine enkephalin (DADLE) bound to CHO-T were similar to CHO-W. Both the specific [3H]Dip bindings of CHO-T and CHO-W were strongly inhibited by delta selective agonists with similar Ki, but neither by mu nor kappa selective agonists. CONCLUSION: The C-terminal of the delta opioid receptor is not involved in the ligands binding affinity and selectivity. (+info)
Interaction models of 3-methylfentanyl derivatives with mu opioid receptors.
AIM: To study the interaction model of 3-methylfentanyl derivatives with mu opioid receptor. METHODS: After a systematic conformational search, a three-dimensional quantitative structure-activity relationship study was carried out with comparative molecular field analysis (CoMFA). RESULTS: 1) The 6 CoMFA models had good predictive values and each model corresponded to the minimum-energy conformations of 13 compounds studied; 2) The important geometric parameters of mu pharmacophore d1 (A), d2 (A), d3 (A), d4 (A), d5 (A), and d6 (A) were 5.2, 5.4, 4.9, 10.6, 10.2, and 5.8 in Model A; 5.2, 6.5, 3.6, 10.6, 11.6, and 5.8 in Model B; 5.2, 4.6, 4.9, 11.6, 9.2, and 6.5 in Model C; 5.2, 5.4, 4.9, 10.5, 10.3, and 5.8 in Model D; 3.6, 5.4, 4.9, 5.7, 7.5, and 5.7 in Model E; 5.2, 4.7, 4.9, 11.2, 9.5, and 6.4 in Model F, respectively. CONCLUSIONS: The several bioactive conformations of fentanyl analogs possibly existed and did not need to be the absolute minimum-energy conformation, each of which was involved in the interaction with mu opioid receptor. (+info)
Absence of G-protein activation by mu-opioid receptor agonists in the spinal cord of mu-opioid receptor knockout mice.
1. The ability of mu-opioid receptor agonists to activate G-proteins in the spinal cord of mu-opioid receptor knockout mice was examined by monitoring the binding to membranes of the non-hydrolyzable analogue of GTP, guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS). 2. In the receptor binding study, Scatchard analysis of [3H][D-Ala2,NHPhe4,Gly-ol]enkephalin ([3H]DAMGO; mu-opioid receptor ligand) binding revealed that the heterozygous mu-knockout mice displayed approximately 40% reduction in the number of mu-receptors as compared to the wild-type mice. The homozygous mu-knockout mice showed no detectable mu-binding sites. 3. The newly isolated mu-opioid peptides endomorphin-1 and -2, the synthetic selective mu-opioid receptor agonist DAMGO and the prototype of mu-opioid receptor agonist morphine each produced concentration-dependent increases in [35S]GTPgammaS binding in wild-type mice. This stimulation was reduced by 55-70% of the wild-type level in heterozygous, and virtually eliminated in homozygous knockout mice. 4. No differences in the [35S]GTPgammaS binding stimulated by specific delta1- ([D-Pen2,5]enkephalin), delta2-([D-Ala2]deltorphin II) or kappa1-(U50,488H) opioid receptor agonists were noted in mice of any of the three genotypes. 5. The data clearly indicate that mu-opioid receptor gene products play a key role in G-protein activation by endomorphins, DAMGO and morphine in the mouse spinal cord. They support the idea that mu-opioid receptor densities could be rate-limiting steps in the G-protein activation by mu-opioid receptor agonists in the spinal cord. These thus indicate a limited physiological mu-receptor reserve. Furthermore, little change in delta1-, delta2- or kappa1-opioid receptor-G-protein complex appears to accompany mu-opioid receptor gene deletions in this region. (+info)
Nitrocinnamoyl and chlorocinnamoyl derivatives of dihydrocodeinone: in vivo and in vitro characterization of mu-selective agonist and antagonist activity.
Two 14beta-p-nitrocinnamoyl derivatives of dihydrocodeinone, 14beta-(p-nitrocinnamoylamino)-7,8-dihydrocodeinone (CACO) and N-cyclopropylmethylnor-14beta-(p-nitrocinnamoylamino)- 7, 8-dihydrocodeinone (N-CPM-CACO), and the corresponding chlorocinnamoylamino analogs, 14beta-(p-chlorocinnamoylamino)-7, 8-dihydrocodeinone (CAM) and N-cyclopropylmethylnor-14beta-(p-chlorocinnamoylamino) -7, 8-dihydrocodeinone (MC-CAM), were tested in opioid receptor binding assays and the mouse tail-flick test to characterize the opioid affinity, selectivity, and antinociceptive properties of these compounds. In competition binding assays, all four compounds bound to the mu opioid receptor with high affinity. When bovine striatal membranes were incubated with any of the four dihydrocodeinones, binding to the mu receptor was inhibited in a concentration-dependent, wash-resistant manner. Saturation binding experiments demonstrated that the wash-resistant inhibition of mu binding was due to a decrease in the Bmax value for the binding of the mu-selective peptide [3H][D-Ala2, MePhe4,Gly(ol)5] enkephalin and not a change in the Kd value, suggesting an irreversible interaction of the compounds with the mu receptor. In the mouse 55 degrees C warm water tail-flick test, both CACO and N-CPM-CACO acted as short-term mu-selective agonists when administered by i. c.v. injection, whereas CAM and MC-CAM produced no measurable antinociception at doses up to 30 nmol. Pretreatment of mice for 24 h with any of the four dihydrocodeinone derivatives produced a dose-dependent antagonism of antinociception mediated by the mu but not the delta or kappa receptors. Long-term antagonism of morphine-induced antinociception lasted for at least 48 h after i.c. v. administration. Finally, shifts in the morphine dose-response lines after 24-h pretreatment with the four dihydrocodeinone compounds suggest that the nitrocinnamoylamino derivatives may produce a greater magnitude long-term antagonism of morphine-induced antinociception than the chlorocinnamoylamino analogs. (+info)
kappa- and mu-Opioid inhibition of N-type calcium currents is attenuated by 4beta-phorbol 12-myristate 13-acetate and protein kinase C in rat dorsal root ganglion neurons.
In rat dorsal root ganglion neurons, activation of kappa- and mu-opioid receptors decreases N-type calcium current, whereas a constitutively active form of protein kinase C (PKC; i.e., PKM, a PKC catalytic subunit fragment) increases N-type calcium current. PKC also attenuates inhibition of calcium current by several G protein-linked neurotransmitter systems. We examined the effects of activation of endogenous PKC by 4beta-phorbol 12-myristate 13-acetate (PMA) and dialysis of cells with PKM and a pseudosubstrate inhibitor PKC(19-31) (PKC-I) on kappa- and mu-opioid-mediated inhibition of calcium current, calcium current amplitude, and rundown. PMA modestly increased peak calcium current and substantially reduced calcium current "rundown," effects blocked by PKC-I. In contrast, PKC-I decreased calcium current and increased current rundown. PMA attenuated morphine-, dynorphin A-, and U50, 488- but not pentobarbitol-related inhibition of calcium current. Similar effects were seen with intracellular dialysis of PKM. Intracellular PKC-I did not block opioid inhibition of calcium current but did reverse PMA and PKM effects on opioid receptor coupling to calcium channels. Because neither PMA nor PKM changed the proportion of omega-CgTX-inhibited current, their effects were not due to a decrease in the proportion of N-type current. After omega-CgTX treatment, there were no differences in the dynorphin A effects on control and PMA- or PKM-treated neurons, suggesting that PKC primarily affected coupling to N-type calcium channels. These data suggest that in acutely dissociated rat dorsal root ganglion neurons, endogenous PKC is required for maintenance of calcium current, may play a role in regulation of neuronal calcium channels, and could be involved in tolerance and/or cross-talk inhibition of opioid responsiveness. (+info)