Mu-selective Agonist Research Articles

Effects of κ‐ and μ‐Selective Opiate Agonists on Colonic Temperature in Normoglycaemic and Streptozotocin‐Treated Hyperglycaemic Mice*

The thermic responses of highly selective k-(U-69593 and U-50488H) and mu-(sufentanil and fentanyl) agonists were assessed in normoglycaemic and hyperglycaemic mice. Hyperglycaemia was induced by streptozotocin injection. The opiates were injected subcutaneously and colonic temperatures monitored for 90 min. after injection. In normoglycaemic animals the k-agonists induced a significant hypothermic response and hyperglycaemia did not modify this effect. In normoglycaemic mice, low doses of mu-selective agonists predominantly produced hypothermia, while in high doses resulted in hyperthermia. In chronic hyperglycaemic mice, hyperthermia was the predominant effect of mu-opiates. The present results characterize the time course and dose response of some of the currently available highly selective kappa- and mu-opiate agonists for the first time in mice and suggest that hyperglycaemia differentially affects the opiate receptor subtypes that mediate thermoregulatory events.

μ‐Opioid Receptors and Not K‐Opioid Receptors Are Coupled to the Adenylate Cyclase in the Cerebellum

The putative regulatory effect of opioids on adenylate cyclase was investigated in two different preparations containing, respectively, two different populations of opioid receptors: the rabbit cerebellum (greater than 75% mu-opioid receptors) and the guinea pig cerebellum (greater than 80% kappa-opioid receptors). In the mu-preparation, but not in the kappa-preparation, opioids inhibited the basal and the forskolin-stimulated adenylate cyclase activity in a dose-dependent manner and stereospecifically. The inhibition was in the 20-30% range, required the presence in the assay medium of Mg2+ and of GTP, but was independent of the presence of Na+. Pharmacological characterization of the inhibitory response in the rabbit cerebellum clearly showed that it was under the control of a mu-opioid binding site, with the effect being elicited by non-selective (etorphine and morphine) and mu-selective (Tyr-D-Ala-Gly-Me-Phe-Gly-ol) agonists, whereas delta- and kappa-selective agonists were almost totally ineffective. ADP ribosylation of inhibitory GTP-binding protein by pertussis toxin failed to block the inhibitory effect of opioids, and data presented suggest that this failure is likely to be the consequence of a limited access of the toxin to its substrate in rabbit cerebellum membranes.

Stimulation of endogenous opioid release displaces mu receptor binding in rat hippocampus

Physiological release of endogenous opioids in the rat hippocampus was detected by an in vitro radioligand displacement assay using [ 3H][ d-Ala 2, N-methyl-Phe 4,glyol 5]enkephalin ([ 3H]DAGO), a mu selective opioid agonist. In this assay, radioligand binding to opioid receptors in the in vitro hippocampal slice was reduced by competition with endogenous opioids released following tissue depolarization. Veratridine-induced opioid release caused displacement of [ 3H]DAGO that could be blocked by either tetrodotoxin addition or calcium removal from the incubation buffer. Maximal displacement of [ 3H]DAGO also required the presence of peptidase inhibitors in the incubation buffer. None of the buffer composition changes directly affected [ 3H]DAGO binding to rat brain membranes. Calcium-dependent displacement of [ 3H]DAGO binding from mu receptor sites elicited by focal electrical stimulation depended on the intensity and frequency of stimulation and positioning of the electrode in the slice. Maximal displacement of [ 3H]DAGO binding was observed following high intensity (150–300μA), high frequency (10–50 Hz) stimulation of the perforant path, a major afferent fiber system to the hippocampus previously shown to contain proenkephalin-derived opioids. Low frequency stimulation (0.1–1 Hz) was ineffective. Stimulation of the mossy fibers (containing both dynorphins and enkephalins) also significantly reduced mu receptor binding, but to a lesser extent. Electrical stimulation of the hippocampal slice at sites not containing opioid peptides did not cause mu receptor displacement. These results demonstrate that under physiological conditions, the release of endogenous opioids from the major opioid containing pathways can be detected in a single hippocampal slice following high frequency stimulation.

Opioid inhibition of kainic acid-induced scratching: Mediation by mu and sigma but not delta and kappa receptors

Scratching induced by intrathecal (IT) administration of kainic acid (0.5 nmol) to rats was inhibited by IT pretreatment with the selective mu agonists levorphanol (30 and 90 nmol), [D-Ala 2,N-Met-Phe 4, Gly 5-ol]-enkaphalin (DAGO, 0.4 and 1.1 nmol), or morphine (90 nmol), the mixed mu-delta agonist [D-Ala 2,D-Leu 5]-enkaphalinamide (DADLE, 10 and 30 nmol), or the sigma/phencyclidine (PCP) agonists dextrorphan (90 nmol) or (+)-N-allyl-N-normetazocine ([+]-NAM, 90 nmol). The kappa agonists dynorphin (1.1 nmol) and ethylketocyclazocine (EKC, 90 nmol) had no significant effect, nor did the selective delta agonist [D-Pen 2,D-Pen 5]-enkaphalinamide (DPDPE, 90 nmol). The nonopioids (+)-3(3-hydroxyphenyl)-N-(1-propyl)piperidine([+])-3-PPP, 90 nmol) and PCP (90 nmol), selective for sigma and PCP sites, respectively, both antagonized kainic-induced scratching. Levorphanol- and DADLE-induced attenuation of scratching was partially antagonized by naltrexone. These findings suggest that opioid inhibition of kainic acid-induced scratching is mediated by classical mu receptors as well as sigma and PCP sites.

Opioid ligands related to tifluadom

In an effort to prepare ligands with kappa selective affinity suitable for use in affinity chromatography, we synthesized the haloacetamido derivatives 17 and 18 of tifluadom. These compounds showed modest opioid binding affinity but were more active at the mu than at the kappa site. The nitro compounds 7 and 16 were prepared as potential intermediates. The former, a weak mu agonist, was devoid of kappa affinity (IC 50 > 10 −6 M), whereas the latter was a mu selective agonist.

Comparative effects of the opioids fentanyl and buprenorphine on ventricular vulnerability during acute coronary artery occlusion

Fentanyl, a mu selective opioid agonist in wide clinical use, raises the ventricular fibrillation threshold in the normal canine myocardium. We have previously shown that this effect is amplified by haemorrhagic stress. In order to determine if mu receptor activation is antifibrillatory during acute myocardial ischaemia, we compared the effects of two mu selective agents, fentanyl and buprenorphine, in open chest chloralose anaesthetised dogs. Each drug was administered intravenously in two doses 1 h apart (fentanyl 30 micrograms.kg-1.dose; buprenorphine 0.3 mg.kg-1.dose). Ventricular fibrillation threshold was measured during right ventricular pacing using the single stimulus technique. The threshold was determined before and during a 10 min left anterior descending coronary artery occlusion. Prior to fentanyl administration, ventricular fibrillation threshold decreased from a control value of 19(SEM 2) mA to 12(1) mA during coronary artery occlusion. After the first dose of this drug an attenuation in the ischaemia induced fall in fibrillation threshold from 23(4) mA to 15(2) mA was observed. After the second dose of fentanyl the decline in fibrillation threshold was significantly blunted at 22(4) mA during control and 18(3) mA during occlusion, p less than 0.05 compared to no drug. In an additional series of experiments atropine sulphate abolished the antifibrillatory action of fentanyl, indicating that vagal efferent activation is responsible for the protective effect of the drug during acute myocardial ischaemia. This is in contrast with its mode of action during haemorrhage, when it enhances vagal afferent inhibition of sympathetic tone, and atropine pretreatment is without effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Mu-Opioid peptide modulation of cardiovascular and sympathoadrenal responses to stress

The effects of mu- and delta-opioid receptor activation on sympathoadrenal and cardiovascular responses to stress were examined in conscious rats. The mu-selective agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAGO) or the delta-selective agonist [D-Pen2,D-Pen5]enkephalin (DPDPE) was injected into a lateral cerebral ventricle, then rats were stressed by restraint. Plasma catecholamines were measured, and arterial blood pressure and heart rate were recorded continuously. Restraint stress evoked increases in plasma catecholamines and heart rate in saline-pretreated rats. Both DAGO and DPDPE increased basal plasma levels of catecholamines and blood pressure, and DAGO, 5 nmol, produced bradycardia. DAGO, 5 nmol, but not DPDPE, potentiated the plasma catecholamine responses to restraint. However, the presence of DAGO or DPDPE during restraint resulted in decreases in heart rate and blood pressure. The effects of DAGO and DPDPE on plasma catecholamines, heart rate, and blood pressure were blocked by a mu-selective dose of naloxone but were not reversed by the delta-selective antagonist ICI 174864. These results indicate that mu-receptor stimulation during restraint stress facilitates sympathoadrenal and parasympathetic outflow and results in vasodilatation of some peripheral vascular beds.

Centrally administered opioid peptides stimulate saccharin intake in nondeprived rats

Endogenous opioid peptides are thought to play a role in mediating the pleasurable or rewarding aspects of the ingestion of certain foods and liquids. We therefore measured the effects of central administration of selective opioid agonists and naloxone on the intake of two concentrations of saccharin solution. All tests were performed on nondeprived rats, such that the taste of the solutions provided the primary incentive to consume. Intracerebroventricular (ICV) administration of the selective mu agonist [D-Ala 2,MePhe 4,Gly-ol 5]enkephalin (DAGO) and the selective delta agonist Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET) (3 nmol) increased intake of a 0.15% saccharin solution by approximately 10 ml over 3 hr. Water was available simultaneously, but intake was minimal. The selective kappa agonist U-50,488H did not increase intake of the saccharin solution. Naloxone (30 and 100 μg, ICV) caused a 44% reduction in saccharin solution intake in the first hour; two- and three-hour cumulative intakes were not different from control. DAGO and DTLET were also tested when rats were given a weaker saccharin solution (0.006%) along with water. Both agonists caused small increases in saccharin and water intake, but the increases above baseline were much smaller than those observed with the more palatable 0.15% saccharin solution. These results are consistent with reports by others which suggest that endogenous opioids influence taste preferences or palatability. Further, they indicate a role for central mu and delta opioid receptors in the mediation of this influence.

Effects of pertussis toxin on opioid regulation of catecholamine release from rat and guinea pig brain slices

Opioid agonists selective for mu-, delta-, and kappa-receptors are all capable of regulating the stimulated release of noradrenaline from three terminal fields (cortex, hippocampus, and cerebellum) of the noradrenergic projections from locus coeruleus in the guinea pig brain. Intracerebroventricular injections of pertussis toxin abolished the ability of a mu-selective agonist and of a delta-selective agonist to inhibit stimulated noradrenaline release, but left unaffected the concentration-related inhibition of NE release by a kappa agonist. Thus, mu- and delta-receptors have been shown to be coupled to their effector system in these noradrenergic neurons via guanyl nucleotide binding proteins (G proteins) which are sensitive to pertussis toxin, while kappa-receptors in the same neurons appear to be coupled through a different mechanism which is significantly less sensitive to pertussis toxin. In contrast to opioid receptor regulation of noradrenaline release in guinea pig hippocampus, mu-, but not delta- or kappa-agonists are capable of regulation of stimulated noradrenaline release from rat hippocampus and cortex, and kappa-, but not mu- or delta-agonists are capable of inhibiting the stimulated release of dopamine from rat striatum and cortex. Pertussis toxin injections significantly attenuated mu-agonist inhibition of noradrenaline release, but had no effect on the ability of a kappa-selective agonist to regulated dopamine release, confirming the insensitivity of the kappa-receptor-effector coupling system to pertussis toxin.

Opioids induce convulsions and wet dog shakes in rats: mediation by hippocampal mu, but not delta or kappa opioid receptors

The opioid receptor subtypes and brain regions involved in eliciting convulsions and wet dog shakes (WDS) were studied by testing different opioid receptor selective agonists in unanesthetized rats. Selective mu agonists, [NMe-Phe3-D-Pro4]-morphiceptin (PL017) and [D-Ala2-N-methyl-Phe3-Gly5-ol]-enkephalin, induced convulsions and WDS when unilaterally injected into the ventral hippocampus. [D-Ala2,D-Leu5]-enkephalin (DADLE), a mixed mu and delta agonist, also elicited such behavioral changes, but its effect was less potent than the selective mu agonists. DADLE-induced WDS were dose dependent, and both convulsions and WDS were antagonized by the irreversible mu receptor antagonist, beta-funaltrexamine, but not by the selective delta receptor antagonist, ICI-174,864. Treatment with the selective delta agonist [D-Pen2,5]-enkephalin or the selective kappa agonists U-50,488H, dynorphin-A amide, or dynorphin-A(1-8) did not produce convulsions or WDS. The injection of a high dose of PL017 intraventricularly or into other brain regions such as the dorsal hippocampus, frontal cortex, striatum, and amygdala did not produce convulsions or WDS, therefore suggesting the ventral hippocampus is an important site for the expression of opioid-induced convulsions and WDS. These results suggest that opioid-induced convulsions and WDS are mediated exclusively by mu but not delta or kappa opioid receptors in the ventral hippocampus.

Antagonist-induced opioid receptor up-regulation. I. Characterization of supersensitivity to selective mu and kappa agonists.

Antagonist-induced opioid receptor up-regulation. I. Characterization of supersensitivity to selective mu and kappa agonists.

Mu and delta opiate receptors coupled negatively to adenylate cyclase on embryonic neurons from the mouse striatum in primary cultures

Primary cultures of pure populations of neuronal or glial cells from the striatum, the cerebral cortex, and the mesencephalon of the mouse embryo were used to look for the presence of opiate receptors coupled to adenylate cyclase. Leu-enkephalin inhibited cAMP production in membranes of embryonic striatal neurons but not in those of other cell types examined. Mu and delta opiate receptors seemed to be coupled negatively to adenylate cyclase in embryonic striatal neurons. It was found that DTLET (a selective delta agonist), as well as DAGO (a selective mu agonist), inhibited cAMP production on these cells. DTLET but not, however, DAGO produced a similar effect on homogenates from the adult rat striatum and on membranes from the neuroblastoma x glioma hybrid cell line NG 108-15, two preparations known to possess only delta receptors negatively coupled to adenylate cyclase. The selective kappa agonist U 50.488 was ineffective on all types of membrane preparations used. The inhibitory effects of both DTLET and DAGO on basal adenylate cyclase activity in striatal neurons were reversed by naloxone with a similar efficacy. Two other selective mu agonists, trimu 5 and morphiceptin, inhibited cAMP production in membranes of striatal neurons as well. The nonadditivity of the inhibitory effects of DTLET and DAGO on basal or forskolin-induced activation of adenylate cyclase suggested that mu and delta receptors were colocalized on a similar subpopulation of striatal cells in primary culture. These cells possess dopaminergic receptors of the D1 subtype as well since the amplitude of the inhibitory effects of DTLET and DAGO on cAMP production was increased in the presence of dopamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Anticonvulsant effects of mu (DAGO) and delta (DPDPE) enkephalins in rats.

The effects of highly selective mu and delta opioid peptide agonists were determined in two rat models of experimentally-induced convulsions, the flurothyl threshold test and the maximal electroshock test. Intracerebroventricular injections of the mu selective enkephalin DAGO (0.3-2.2 nmol) resulted in a dose-related protection in both seizure models. Pretreatment with a low dose of naloxone (29 nmol) or the irreversible mu antagonist beta-FNA (21 nmol), but not the delta opioid antagonist ICI 154,129 (50 nmol), antagonized the anticonvulsant actions of DAGO. Intracerebroventricular injections of the delta selective enkephalin DPDPE (70-140 nmol) also resulted in seizure protection. These effects were selectively antagonized by the delta antagonist ICI 174,864 (2.8 nmol), but not by pretreatment with beta-FNA. Thus, using agonists and antagonists highly selective for mu and delta opioid receptors, anticonvulsant actions of enkephalin have been described against chemically- and electrically-induced convulsions in rats.

MU but not delta opioid receptor stimulation intensifies kainic acid-induced neurotoxicity in rat hippocampus

In the present study, we compared the effects of the selective mu agonist, [D-Ala 2-N-methyl-pHe 4-Gly-ol]-enkephalon (DAGO), and the selective delta agonist, [D-Pen 2,5]-enkephalin (DPDPE), on kainic acid-induced neurotoxicity in rats. Infusion of kainic acid ( 0.5 ug 1.5 ul , i.c.v. ) alone caused pyramidal cell loss predominantly in hippocampal field CA3 with minimal involvement of the CA1 field. Coadministration of DAGO plus kainic acid into the lateral ventricle intensified the extent of degeneration of hippocampal pyramidal cells in the CA1 field. The potentiating effect of DAGO was completely blocked by naltrexone. In contrast, DPDPE had no significant effect on kainic acid-induced neurotoxicity. Thus, activation of mu but not delta receptors intensifies the neurotoxic effects of kainic acid in the hippocampus.

Antifibrillatory action of the narcotic agonist fentanyl

Morphine, an opiate alkaloid with mixed mu- and delta-agonist properties, raises the ventricular fibrillation threshold in anesthetized dogs by altering autonomic tone. To elucidate further underlying structure-activity relationships, the effect of fentanyl, a nonalkaloid, mu-selective agonist in wide clinical use, was studied. Fentanyl (30 μg/kg) was given intravenously to 27 chloralose-anesthetized dogs, and ventricular fibrillation threshold was measured by means of the single-stimulus technique. In the baseline state fentanyl raised the ventricular fibrillation threshold by 14%. When the dogs were subjected to hemorrhagic stress, this effect was amplified to 29% ( p < 0.0001). Bilateral cervical vagotomy abolished fentanyl's antifibrillatory effect, but neither atropine sulfate (0.4 mg/kg/hr) nor atropine methylnitrate (0.5 mg/kg/hr) did so. Fentanyl's influence on the fibrillation threshold during hemorrhage was significantly reduced by bilateral stellate ganglionectomy ( p < 0.005). It is concluded that fentanyl raises the ventricular fibrillation threshold by its known sympathoinhibitory action rather than by its vagal efferent activating effect. The facts that an intact vagus is required and that hemorrhage amplifies the effect suggest that the antifibrillatory effect of fentanyl is mediated through the afferent component of the baroreflex arc.

In-vivo studies with the opioid antagonist, 16-methylcyprenorphine

The effect of the opioid antagonist, 16-methylcyprenorphine (RX8008M), on the antinociceptive action of the mu-selective agonist, morphine, and the kappa-selective agonist, U50488H, has been investigated in the mouse abdominal constriction test. RX8008M produced a dose-dependent antagonism of the antinociceptive effect of morphine, but did not antagonize the response to U50488H. RX8008M should prove a useful probe for the in-vivo characterization of the receptor selectivity of opioid drugs.

Effects of β-chlornaltrexamine on food intake, body weight and opioid-induced feeding

β-Chlornaltrexamine (β-CNA) is a non-equilibrium opioid receptor antagonist which alkylates and inactivates opioid receptors. Because opioid peptides are thought to contribute to the regulation of food intake, we examined the effects of intracerebroventricular (icv) injections of β-CNA on the food intake and body weight of male rats. We also tested the ability of β-CNA to block food intake stimulated by selective agonists of kappa, mu and delta opioid receptors: dynorphin A (DYN), Tyr-D-Ala-Gly-(Me)Phe-Gly-ol (DAGO), and [(D-Ser 2, Leu 5]-enkephalin-Thr 6 (DSLET). Treatment with β-CNA caused a long-term (2–4 days) reduction in daily food intake and a concomitant reduction in body weight. An additional experiment indicated that the weight loss after β-CNA treatment could be completely accounted for by the reduction in intake. β-CNA treatment also abolished or greatly attenuated the feeding effects of DAGO, DSLET and DYN, even when these peptides were tested 26 hours after β-CNA administration. The long duration of the effects of β-CNA suggests that this compound will be a useful pharmacological tool in further study of the opioid feeding system.

The choice of opioid receptor subtype in isolated preparations by ohmefentanyl.

Ohmefentanyl has significant inhibitory actions on the electrically evoked contractions of guinea pig ileum and mouse vas deferens. Their IC50 values are 0.15 nM and 0.89 nM, respectively. In the guinea pig ileum and the mouse vas deferens, both mu-antagonist naloxone and (mu + kappa)-antagonist Mr. 2266 antagonize readily the agonist action of ohmefentanyl, indicating that ohmefentanyl acts on mu-receptors in the guinea pig ileum and the mouse vas deferens. Like other mu-agonists, ohmefentanyl has agonist activity but no antagonist action in the rat vas deferens. In the rabbit vas deferens, the activity of ohmefentanyl is very weak. The IC50 value is 149 nM. These results further indicate that ohmefentanyl is a potent and selective mu-agonist.

Subclass specificity of anti-idiotypic anti-opiate receptor antibodies in rat brain, guinea pig cerebellum, & neuroblastoma x glioma (NG 108-15)

Receptor subclass recognition properties of affinity-purified rabbit polyclonal anti-idiotypic anti-opiate receptor antibodies in various membrane preparations have been determined. The anti-receptor immunoglobulins significantly decrease binding of 3H-[D-Ala 2,-MePhe 4,Gly-ol 5]enkephalin, a highly selective mu agonist, in rat neural membrane. In the presence of a concentration of the unlabeled ligand sufficient to block existing mu sites, the antibodies compete, to a lesser degree with 3H-[D-Ala 2,D-Leu 5]enkephalin for delta site occupancy in both rat neural membrane, and neuroblastoma x glioma membrane preparations. The antibodies do not displace 3H-ethylketocyclazocine from rat brain or guinea pig cerebellum.

The stimulation of food intake by selective agonists of mu, kappa and delta opioid receptors

It is known that under some conditions the administration of opioid agonists will stimulate food intake. However, the lack of receptor selectivity of some of the agonists which produce this effect leaves open the question of which receptor types are actually involved. In the experiments presented here, rats were given intracerebroventricular injections of Dynorphin 1–17 (DYN), [D-ala 2 6MePhe 4, GlGly-ol 5] enkephalin (DAGO), and [D-ser 2, leu 5]enkephalin-thr (DSLET); these peptides are thought to be selective agonists at kappa, mu and delta opioid receptors, respectively. All three peptides stimulated food intake in non-deprived rats at doses in the 3 – 10 nmol range; water intake was also increased in some cases. Generally, DYN stimulated feeding at a lowet dose than DAGO or DSLET and the magnitude of the effect tended to be greater. On the other hand, DAGO more consistently increased water intake. In some cases, DYN also caused episodes of “barrel-rolling” and postural abnormalities, whereas DAGO had sedative and/or cataleptic effects. These results are interpreted as an involvement of more than one opioid receptor types in the regulation of appetite, possibly with separate opioid systems contributing to food and water intake.