Activation of G protein by opioid receptors: role of receptor number and G-protein concentration.

The collision-coupling model for receptor-G-protein interaction predicts that the rate of G-protein activation is dependent on receptor density, but not G-protein levels. C6 cells expressing mu- or delta-opioid receptors, or SH-SY5Y cells, were treated with beta-funaltrexamine (mu) or naltrindole-5'-isothiocyanate (delta) to decrease receptor number. The time course of full or partial agonist-stimulated ¿35SGTPgammaS binding did not vary in C6 cell membranes containing <1-25 pmol/mg mu-opioid receptor, or 1.

Membrane microviscosity modulates mu-opioid receptor conformational transitions and agonist efficacy.

The influence of membrane microviscosity on mu-opioid agonist and antagonist binding, as well as agonist efficacy, was examined in membranes prepared from SH-SY5Y cells and from a C6 glioma cell line stably expressing the rat mu-opioid receptor (C6mu). Addition of cholesteryl hemisuccinate (CHS) to cell membranes increased membrane microviscosity and reduced the inhibitory effect of sodium and guanine nucleotides on the affinity of the full agonists sufentanil and [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO) for the mu-opioid receptor. Binding of the antagonists [3H]naltrexone and [3H]diprenorphine and the partial agonist nalbuphine was unaffected by CHS.

Opioid efficacy in a C6 glioma cell line stably expressing the human kappa opioid receptor.

A C6 glioma cell line stably transfected with the human kappa opioid receptor (kappaOR) was used to characterize receptor binding and G protein activation via the kappaOR by a comprehensive series of opioid ligands. The ligand-binding affinity for [3H]5alpha,7alpha, 8beta(-)-N-methyl-N-(7-Cl-pyrrolidinyl)-1-oxaspiro(4, 5)dec-8-yl)benzene acetamide (U69593) was similar to that observed in monkey brain membranes and was 10-fold lower in the presence of sodium and GDP. Both peptide and nonpeptide agonists maximally stimulated [35S]GTPgammaS binding.

Delta opioid receptor down-regulation is independent of functional G protein yet is dependent on agonist efficacy.

Chronic treatment of C6 glioma cells stably expressing the rat delta opioid receptor (C6delta) with full agonists resulted in receptor down-regulation. Chronic [D-Ser2,L-Leu5]enkephalyl-Thr treatment caused a decrease in cell surface as well as a decrease in agonist-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate binding. Treatment with full agonists for 12 hr resulted in a 90% decrease in receptor number that was paralleled by a decrease in the ability of agonist to stimulate [35S]guanosine-5'-O-(3-thio)triphosphate binding and inhibit forskolin-stimulated adenylyl cyclase.

Stimulation of guanosine-5'-O-(3-[35S]thio)triphosphate binding by endogenous opioids acting at a cloned mu receptor.

The ability of endogenous opioids to activate G proteins was measured in membranes from C6 rat glioma cells stably expressing a cloned rat mu receptor. Peptides representing each of the three known families of endogenous opioids (enkephalins, endorphins and dynorphins) were studied, as well as two recently discovered endogenous opioids, endomorphin-1 and -2, which are thought to represent a fourth family of endogenous opioid peptides. Stimulation of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding to membranes was used as a measure of G protein activation.

Synthesis and characterization of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-labeled fluorescent ligands for the mu opioid receptor.

A series of opioid ligands utilizing the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophores 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene++ +-3-propionic acid or 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza- s-indacene-3-propionic acid were synthesized and characterized for their ability to act as a suitable fluorescent label for the mu opioid receptor. All compounds displaced the mu opioid receptor binding of [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol in monkey brain membranes with high affinity. The binding of fluorescent ligands to delta and kappa receptors was highly variable.

Opioid efficacy in a C6 glioma cell line stably expressing the delta opioid receptor.

A C6 glioma cell line stably transfected with the rat delta opioid receptor (C6delta) was used to characterize receptor binding and G protein activation by both peptide and nonpeptide delta opioid ligands. The ligand binding affinities for [3H]naltrindole and [3H]pCl-[D-Pen2,D-Pen5]enkephalin (DPDPE) were similar to those observed in monkey brain membranes. The nonpeptide agonists, BW373U86 and SNC80, as well as peptide agonist [D-Ser2, L-Leu5]enkephalyl-Thr maximally stimulated [35S]GTPgammaS binding by 640, 654 and 576%, respectively, over basal.

Down-regulation of mu-opioid receptor by full but not partial agonists is independent of G protein coupling.

In C6 glial cells stably expressing rat mu-opioid receptor, opioid agonist activation is negatively coupled to adenylyl cyclase through pertussis toxin-sensitive G proteins. In membranes, [D-Ala2, N-MePhe4,Gly-ol5]enkephalin (DAMGO) increases guanosine-5'-O-(3-[35S]thio)triphosphate (GTP[gamma-35S]) binding by 367% with an EC50 value of 28 nM. Prolonged exposure to agonists induced desensitization of the receptor as estimated by a reduction in the maximal stimulation of GTP[gamma-35S] binding by DAMGO and rightward shifts in the dose-response curves.

Regulation of mu-opioid receptor in neural cells by extracellular sodium.

SH-SY5Y neural cells expressing mu- and delta-opioid receptors were maintained viable in isotonic, sodium-free buffer in vitro. Intracellular sodium levels were manipulated by various methods, and ligand binding to intact cells was studied. In physiological buffer containing 118 mM sodium, [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol ([3H]-DAMGO) and [3H]naltrexone bound to mu receptor with KD values of 3.

Zipeprol: preclinical assessment of abuse potential.

Zipeprol was evaluated in a number of in vitro and in vivo assays predictive of stimulant, depressant, or opioid abuse potential. Zipeprol had affinity for mu and kappa opioid binding sites as well as sigma binding sites. However, it failed to exert opioid-like agonist actions in rodents, and did not attenuate withdrawal signs in morphine- or pentobarbital-dependent rats.

Characterization of opioid agonist efficacy in a C6 glioma cell line expressing the mu opioid receptor.

In C6 glioma cells stably expressing a homogeneous population of the cloned rat mu opioid receptor, the binding affinities of opioid agonists and subsequent activation of G protein were examined. Opioid receptor number in membranes of these cells was high (10-30 pmol/mg protein [3H]diprenorphine binding sites). Opioids were found to bind to the receptor with high affinity [Tyr-D-Ala-Gly-(Me)Phe-Gly-ol (DAMGO) 0.

Reversible modulation of opioid receptor binding in intact neural cells by endogenous guanosine triphosphate.

Incubation of SH-SY5Y neural cells with mycophenolic acid (MPA), an inhibitor of inosine monophosphate dehydrogenase (the key enzyme in purine nucleotide biosynthesis), reduced the cellular content of GTP by 94% relative to its concentration in control cells (43 nmol/mg protein) without altering the level of GDP. Although in GTP-depleted intact cells the receptor binding parameters (Kd and Bmax) of the opioid antagonist [3H]naltrexone were unchanged from those in untreated cells, the binding affinity of the mu-selective opioid agonist [3H]Tyr-D-Ala-Gly-(Me)- Phe-Gly-ol ([3H]DAMGO) was enhanced 2-fold. Furthermore, the kinetics of ligand/receptor interaction revealed that in the nucleotide-depleted cells, the dissociation rate constant for [3H]DAMGO was reduced by 44%.

Behavioral effects and receptor binding affinities of fentanyl derivatives in rhesus monkeys.

These studies examined the opioid receptor binding affinities and behavioral effects of several fentanyl derivatives in rhesus monkeys. OHM3295, OHM3296, OHM3326 and OHM3463 displayed high affinity for mu (IC50 = 7-66 nM) as compared to kappa (IC50 = 263-3255 nM) or delta (IC50 = 480-4500 nM) receptors as measured by their ability to displace [3H](D-Ala2-Me-Phe4,Glyol5)enkephalin, [3H](5,7,8[beta])-N-[2- (1-pyrrolidinyl)1-oxaspiro[4,5]dec-8-yl]benzeneacetamide and [3H](D-Pen2-D-Pen5)enkephalin, respectively. All four compounds maintained i.

Binding affinity and selectivity of opioids at mu, delta and kappa receptors in monkey brain membranes.

The binding parameters of radiolabeled DAMGO (mu), DPDPE and pCl-DPDPE (delta) and 5 alpha, 7 alpha, 8 beta-N-methyl-N-[7-(1- pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl]benzeneacetamide (also known as U69593, kappa) and the affinity and selectivity profiles of various opioid agonists and antagonists at the three opioid receptor types were determined in membranes from brain cortex of rhesus monkey. Among the 10 opioids with established mu-selective actions, etonitazene inhibited the binding of [3H]DAMGO with a Ki of 0.02 nM (0.

Irreversible opioid antagonist effects of clocinnamox on opioid analgesia and mu receptor binding in mice.

The effects of the systemically active irreversible opioid receptor antagonist clocinnamox (C-CAM; 14 beta-(p-chlorocinnamoylamino)-7,8-dihydro-N- cyclopropylmethyl normorphinone mesylate) on mu receptor binding to cerebral membranes and on mu opioid analgesia were assessed using mice. After systemic administration, C-CAM produced a dose-dependent decrease in the Bmax values of both [3H]DAMGO ([D-Ala2, N-MePhe4, Gly5-ol][tyrosyl-3,5-3H]enkephalin) and [3H]naltrexone without affecting the Kd value of either ligand. After administration of 3.

Antipodal alpha-N-(methyl through decyl)-N-normetazocines (5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphans): in vitro and in vivo properties.

The enantiomeric (-)- and (+)-N-(methyl through decyl) normetazocines (5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphans) were synthesized and their in vitro and in vivo activities determined. Increasingly bulky enantiomeric N-alkyl homologs were prepared until their interaction with the sigma 1 receptor decreased and their insolubility became a hindrance to their evaluation in vivo and/or in vitro. The (-)-methyl, -pentyl, -hexyl, and -heptyl homologs were essentially as potent as, or more potent than, morphine in the tail-flick, phenylquinone, and hot-plate assays for antinociceptive activity; the (-)-propyl homolog had narcotic antagonist activity between that of nalorphine and naloxone in the tail-flick vs morphine assay, and it also displayed antagonist properties in the single-dose suppression assay in the rhesus monkey.

Comparison of kappa opioids in rhesus monkeys: behavioral effects and receptor binding affinities.

Bremazocine, [5R-(5,7,8 beta)]-N-methyl-N-[7-(1-pyrrolidinyl)1-oxaspiro [4,5]dec-8-yl]-4-benzofuranacetamide (Cl-977), (+-)-trans-3,4-dichloro-N- methyl-(2-(pyrrolidin-1-yl)-5-methoxy-1,2,3,4-tetrahydronapth++ +-1-yl benzeneacetamide methanesulfonate (DUP 747), ethylketocyclazocine (EKC), nalorphine, (+/-)-trans-N-methyl-N-[2-(1- pyrrolidnyl)-cyclohexyl]benzo[b]thiophene-4-acetamide (PD117302), trans-(+/-)-3,4-dichloro-N-methyl-[2-(1-pyrrolidinyl)- cyclohexyl]benzeneacetamide (U-50,488), (5,7,8 beta)-N-methyl-N[2-(1- pyrrolidinyl), 1-oxaspiro[4,5]dec-8-yl benzeneacetamide (U-69,593) and spiradoline were compared in rhesus monkeys for their discriminative stimulus, analgesic and respiratory effects. Selected compounds also were studied for their binding affinities at mu [[3H](D-Ala2-Me-Phe4,Glyol5)enkephalin], kappa ([3H]U-69,593) and delta [[3H](D-Pen2-D-Pen5) enkephalin], opioid receptors in monkey brain membranes. All compounds substituted completely (> or = 90%) for EKC in monkeys discriminating between EKC and saline, with the exception that DUP 747 produced a maximum of 74% EKC responding.

Effects of opioid agonists selective for mu, kappa and delta opioid receptors on schedule-controlled responding in rhesus monkeys: antagonism by quadazocine.

Rhesus monkeys were trained to respond under a fixed-ratio 30 schedule of food reinforcement. The mu opioid agonists alfentanil and fentanyl, the kappa opioid agonists ethylketocyclazocine (EKC) and U69,593, the delta opioid agonist BW373U86 [(+-)-4-((R*)-a-((2S*5R*)-4-allyl-2,5-dimethyl-1-piperazinal)-3-h ydroxy- benzyl)-N,N-diethylbenzamide dihydrochloride] and the nonopioid, noncompetitive N-methyl-D-aspartate antagonist ketamine all produced a dose-dependent decrease in rates of responding. Quadazocine (0.

Altered transition between agonist- and antagonist-favoring states of mu-opioid receptor in brain membranes with modified microviscosity.

In unmodified synaptosomal brain membranes the presence of NaCl inhibited the binding to mu receptors of the tritiated opioid agonists etorphine, Tyr-D-Ala-Gly-(Me)Phe-Gly-ol, and sufentanil by 53, 43, and 37%, respectively, and increased that of the antagonist [3H]naltrexone by 54%. On the other hand, in membranes whose microviscosity was increased by incorporation of cholesteryl hemisuccinate (CHS) the effects of sodium on opioid agonist and antagonist binding were abolished and strongly reduced, respectively. Furthermore, in the modified membranes the ability of sodium to protect the opioid receptor from inactivation by the sulfhydryl-reactive agent N-ethylmaleimide (NEM) was diminished.

Go mediates the coupling of the mu opioid receptor to adenylyl cyclase in cloned neural cells and brain.

In membranes from SH-SY5Y human neuroblastoma cells differentiated with retinoic acid, the mu-selective agonist Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) inhibited cAMP formation with an IC50 of 26 nM. Two separate antibodies raised against distinct regions of the Go alpha sequence attenuated the effect of DAMGO by 50-60%, whereas antibodies to Gi alpha 1,2 or Gi alpha 3 reduced the mu-opioid signal insignificantly or to a lesser extent. In contrast, inhibition of adenylyl cyclase by the delta-opioid agonist Tyr-D-Pen-Gly-Phe-D-Pen-OH (DPDPE; Pen = penicillamine) was very sensitive to the Gi alpha 1,2 antibody.

Receptor mechanisms of opioid tolerance in SH-SY5Y human neural cells.

In differentiated SH-SY5Y human neuroblastoma cells, various opioids exhibited a wide range of potencies (Ki) in acutely inhibiting adenylate cyclase to different extents (Imax). After exposure of the cells to opioids for 24 hr, the initially reduced cAMP content of the cells recovered toward pre-exposure levels. Withdrawal of agonist from, or addition of antagonist to, the tolerant cells rapidly increased the cAMP content to 1.

Opioid and non-opioid effects of novel butyrophenone analogues.

Haloperidol, haloperidol propionate, and a haloperidol analogue N-3-(p-fluorobenzoyl) propyl-4-phenyl-4-propionyl-oxypiperidine (NIH 10495) were evaluated in several in vitro and in vivo tests of opioid effects. Haloperidol bound to opioid receptors with very low affinity and had no opioid agonist effects in the other test systems. Haloperidol propionate was 10 times less potent than NIH 10495 in the binding assay and in the smooth-muscle assay.