The effects of endomorphin-1 and endomorphin-2 in CHO cells expressing recombinant mu-opioid receptors and SH-SY5Y cells.
(49/2005)
1 Endomorphin-1 and -2 (E-1/E-2) have been proposed as endogenous ligands for the mu-opioid receptor. The aims of this study are to characterize the binding of E-1/E-2 and the subsequent effects on cyclic AMP formation and [Ca2+]i levels in SH-SY5Y and Chinese hamster ovary (CHO) cells expressing endogenous and recombinant mu-opioid receptors. 2 E-1 displaced [3H]-diprenorphine ([3H]-DPN) binding in CHO micro and SH-SY5Y membranes with pKi values of 8.02+/-0.09 and 8.54+/-0.13 respectively. E-2 displaced [3H]-DPN binding in CHOmu and SH-SY5Y cells with pKi values of 7.82+/-0.11 and 8.43+/-0.13 respectively. E-1/E-2 bound weakly to CHOdelta and CHOkappa membranes, with IC50 values of greater than 10 microM. 3 In CHOmu cells, E-1/E-2 inhibited forskolin (1 microM) stimulated cyclic AMP formation with pIC50 values of 8.03+/-0.16 (Imax = 53.0+/-9. 3%) and 8.15+/-0.24 (Imax = 56.3+/-3.8%) respectively. In SH-SY5Y cells E1/E2 inhibited forskolin stimulated cyclic AMP formation with pIC50 values of 7.72+/-0.13 (Imax=46.9+/-5.6%) and 8.11+/-0.31 (Imax = 40.2+/-2.8%) respectively. 4 E-1/E-2 (1 microM) increased [Ca2+]i in fura-2 loaded CHOmu cell suspensions in a thapsigargin sensitive and naloxone reversible manner. Mean increases observed were 106+/-28 and 69+/-6.7 nM respectively. In single adherent cells E-1/E-2 (1 microM) increased [Ca2+]i with a mean 340/380 ratio change of 0.81+/-0.09 and 0.40+/-0.08 ratio units respectively. E-1/E-2 failed to increase intracellular calcium in CHOdelta, CHOkappa and SH-SY5Y cells. 5 These data show that E-1/E-2 bind with high affinity and selectivity to mu-opioid receptors and modulate signal transduction pathways typical of opioids. This provides further evidence that these two peptides may be endogenous ligands at the mu-opioid receptor. (+info)
Effects of regulators of G protein-signaling proteins on the functional response of the mu-opioid receptor in a melanophore-based assay.
(50/2005)
The goal of the present study was to investigate a possible role for regulators of G protein-signaling (RGS) proteins in opioid receptor (OR) desensitization using cultured Xenopus laevis dermal melanophores. Morphine-induced pigment aggregation in a melanophore cell line stably expressing the murine mu OR (muOR) was quantified over time. Responses of the muOR (a G(i)-linked receptor) exhibited a time-dependent desensitization, which varied with the concentration of morphine used. In contrast, much less desensitization was observed in response to melatonin, effects mediated through the cells' endogenous melatonin receptor (which is also G(i)-linked). To further study OR desensitization, melanophores lacking a muOR were transiently transfected with plasmids encoding the muOR alone or in combination with plasmids encoding one of several RGS subtypes (RGS1, RGS2, RGS3, or RGS4). Overexpression of RGS2, but not the other RGS subtypes, produced a rightward shift in the morphine concentration-response curve. RGS protein overexpression also decreased the magnitude of morphine-induced responses. Finally, the effect of a mutant form of Galpha(i1), which is insensitive to RGS action, was investigated with respect to its ability to alter the response of the muOR to morphine. Expression of the mutant Galpha(i1) prolonged morphine-induced pigment aggregation and produced leftward shifts in concentration-response curves, compared with expression of wild-type Galpha(i1). These results demonstrate that specific RGS proteins can dampen signals initiated by agonist activation of the muOR, and support a possible role for RGS proteins in OR desensitization. (+info)
Ring-constrained orvinols as analogs of buprenorphine: differences in opioid activity related to configuration of C(20) hydroxyl group.
(51/2005)
The relative positions of the C(20) substituents in buprenorphine, particularly the hydroxyl group, have been implicated in its actions as a partial mu-agonist and a kappa-antagonist. This hypothesis has been examined by the synthesis and pharmacological characterization of five orvinols in which the C(20) carbon atom of buprenorphine is constrained in a five-membered ring, fixing the hydroxyl group above (beta) or below (alpha) the plane of the ring. All five compounds were nonselective in binding assays with similar, low nanomolar affinities. The compounds acted as delta-agonists in the mouse vas deferens and kappa-agonists in the myenteric plexus-longitudinal muscle of the guinea pig ileum and in Chinese hamster ovary (CHO) cells expressing the human kappa-opioid receptor (CHO-hkor). All were lower efficacy mu-agonists than buprenorphine as measured by the [(35)S]guanosine-5'-O-(3-thio)triphosphate assay in SH-SY5Y cells. The major difference between the isomers was an 11- to 12-fold higher potency of the beta-OH isomer (BU46) compared with the alpha-OH isomer (BU47) at the kappa-receptor in the guinea pig ileum and CHO-hkor cells and a somewhat higher efficacy of BU46 in CHO-hkor cells. BU46 and BU47 were evaluated in vivo. BU46 was a full agonist in the mouse writhing assay and antinociception was prevented by norbinaltorphimine, showing a kappa-mechanism of action. In contrast, BU47 acted as an antagonist of mu-, delta-, and kappa-mediated antinociception in the writhing assay. The results show that the configuration of the hydroxyl group is not important in binding affinity at mu-, delta-, or kappa-receptors but does influence kappa-potency and kappa-efficacy, particularly in vivo. (+info)
L-glutamate and gamma-aminobutyric acid efflux from rat cerebrocortical synaptosomes: modulation by kappa- and mu- but not delta- and opioid receptor like-1 receptors.
(52/2005)
The modulation by delta-, kappa-, mu-, and opioid receptor like-1 (ORL(1)) agonists and antagonists of L-glutamate (L-Glu) and gamma-aminobutyric acid (GABA) efflux from superfused rat cerebrocortical synaptosomes was studied. Tetrodotoxin (0.5 microM) inhibited the spontaneous efflux of both transmitters by 20%. Ca(2+) omission decreased GABA and facilitated L-Glu efflux. The neurotransmitter overflow evoked by K(+) concentrations in the 7.5- to 10-mM range was largely Ca(2+) dependent and tetrodotoxin sensitive. Neither the delta-receptor agonist deltorphin (up to 0.3 microM) nor the ORL(1) receptor agonist nociceptin (up to 1 microM) significantly affected either spontaneous or K(+)-evoked neurotransmitter efflux. Conversely, the ORL(1) ligand [Phe(1)(CH(2)-NH)Gly(2)]nociceptin(1-13)NH(2) (0.3 microM) caused a naloxone-sensitive inhibition of both L-Glu- and GABA-stimulated overflow. The kappa-receptor agonist (-)-U50,488 failed to modulate spontaneous L-Glu and GABA efflux. However, it similarly inhibited the K(+)-evoked overflow of both neurotransmitters (EC(50) approximately 100 nM; E(max) approximately 25-30% inhibition) in a norbinaltorphimine-sensitive manner. The selective mu-receptor agonist endomorphin 1 inhibited both spontaneous (EC(50) approximately 50 nM) and K(+)-evoked (EC(50) approximately 10 nM; E(max) approximately 50% inhibition) L-Glu efflux in a naloxone-sensitive manner. Conversely, it significantly inhibited only K(+)-evoked GABA efflux (EC(50) approximately 10 nM), although with a lower maximal effect (E(max) approximately 25-30% inhibition). It is concluded that, in the rat cerebral cortex, L-Glu and GABA efflux from nerve terminals is under the direct inhibitory control of kappa- and mu- (but not delta- or ORL(1)) receptors. Because glutamatergic terminals emerged as a preferential target of mu-receptor agonists, the activation of this receptor may advocate both relevant analgesic and neuroprotective effects. (+info)
Modulation of respiratory frequency by peptidergic input to rhythmogenic neurons in the preBotzinger complex.
(53/2005)
Neurokinin-1 receptor (NK1R) and mu-opioid receptor (muOR) agonists affected respiratory rhythm when injected directly into the preBotzinger Complex (preBotC), the hypothesized site for respiratory rhythmogenesis in mammals. These effects were mediated by actions on preBotC rhythmogenic neurons. The distribution of NK1R+ neurons anatomically defined the preBotC. Type 1 neurons in the preBotC, which have rhythmogenic properties, expressed both NK1Rs and muORs, whereas type 2 neurons expressed only NK1Rs. These findings suggest that the preBotC is a definable anatomic structure with unique physiological function and that a subpopulation of neurons expressing both NK1Rs and muORs generate respiratory rhythm and modulate respiratory frequency. (+info)
3D modeling, ligand binding and activation studies of the cloned mouse delta, mu; and kappa opioid receptors.
(54/2005)
Refined 3D models of the transmembrane domains of the cloned delta, mu and kappa opioid receptors belonging to the superfamily of G-protein coupled receptors (GPCRs) were constructed from a multiple sequence alignment using the alpha carbon template of rhodopsin recently reported. Other key steps in the procedure were relaxation of the 3D helix bundle by unconstrained energy optimization and assessment of the stability of the structure by performing unconstrained molecular dynamics simulations of the energy optimized structure. The results were stable ligand-free models of the TM domains of the three opioid receptors. The ligand-free delta receptor was then used to develop a systematic and reliable procedure to identify and assess putative binding sites that would be suitable for similar investigation of the other two receptors and GPCRs in general. To this end, a non-selective, 'universal' antagonist, naltrexone, and agonist, etorphine, were used as probes. These ligands were first docked in all sites of the model delta opioid receptor which were sterically accessible and to which the protonated amine of the ligands could be anchored to a complementary proton-accepting residue. Using these criteria, nine ligand-receptor complexes with different binding pockets were identified and refined by energy minimization. The properties of all these possible ligand-substrate complexes were then examined for consistency with known experimental results of mutations in both opioid and other GPCRs. Using this procedure, the lowest energy agonist-receptor and antagonist-receptor complexes consistent with these experimental results were identified. These complexes were then used to probe the mechanism of receptor activation by identifying differences in receptor conformation between the agonist and the antagonist complex during unconstrained dynamics simulation. The results lent support to a possible activation mechanism of the mouse delta opioid receptor similar to that recently proposed for several other GPCRs. They also allowed the selection of candidate sites for future mutagenesis experiments. (+info)
Time course of changes in mu-opioid receptor mRNA levels in the periaqueductal gray of rat brain by a single or repeated injections of antisense oligodeoxynucleotides.
(55/2005)
The effect of phosphorothioated antisense oligodeoxynucleotide (AS ODN) against the mu-opioid receptor (MOR) on MOR mRNA level in the periaqueductal gray (PAG) of rat brain was investigated. The MOR mRNA levels at 3, 6, 12, 24, 48 and 72 h after MOR AS ODN microinjection into the PAG were determined by reverse transcriptase-polymerase chain reaction. The MOR mRNA level was significantly decreased only at 12 h after the injection of 10 microg MOR AS ODN. When 10 microg MOR AS ODN was given three times at the interval of 48 h, MOR mRNA levels were significantly decreased at 6, 12 and 24 h after the last injection of the AS ODN. However, MOR mRNA levels were not significantly changed by three injections at 48-h interval of MOR sense ODN or AS ODNs against delta- and kappa-opioid receptors, although the two latter AS ODNs significantly reduced the respective targeted mRNA levels. In conclusion, the present results show that the selective decrease in MOR mRNA is at least one reason why the reported diminished effects of MOR agonists are produced in animals pretreated with MOR AS ODN, although they could be produced through several mechanisms in which MOR mRNA level does not change. (+info)
Selective modulation of excitatory transmission by mu-opioid receptor activation in rat supraoptic neurons.
(56/2005)
Opioid peptides have profound inhibitory effects on the production of oxytocin and vasopressin, but their direct effects on magnocellular neuroendocrine neurons appear to be relatively weak. We tested whether a presynaptic mechanism is involved in this inhibition. The effects of mu-opioid receptor agonist D-Ala(2), N-CH(3)-Phe(4), Gly(5)-ol-enkephalin (DAGO) on excitatory and inhibitory transmission were studied in supraoptic nucleus (SON) neurons from rat hypothalamic slices using whole cell recording. DAGO reduced the amplitude of evoked glutamatergic excitatory postsynaptic currents (EPSCs) in a dose-dependent manner. In the presence of tetrodotoxin (TTX) to block spike activity, DAGO also reduced the frequency of spontaneous miniature EPSCs without altering their amplitude distribution, rising time, or decaying time constant. The above effects of DAGO were reversed by wash out, or by addition of opioid receptor antagonist naloxone or selective mu-antagonist Cys(2)-Tyr(3)-Orn(5)-Pen(7)-NH(2) (CTOP). In contrast, DAGO had no significant effect on the evoked and spontaneous miniature GABAergic inhibitory postsynaptic currents (IPSCs) in most SON neurons. A direct membrane hyperpolarization of SON neurons was not detected in the presence of DAGO. These results indicate that mu-opioid receptor activation selectively inhibits excitatory activity in SON neurons via a presynaptic mechanism. (+info)