Decreased spinal morphine/clonidine antinociceptive synergism in morphine-tolerant mice

The antinociceptive interactions between spinally administered opioids and the alpha₂ agonist clonidine were examined in placebo and morphine pellet-implanted mice using the tail flick test. In placebo pellet-implanted animals, coadministered morphine and clonidine produced a synergistic antinociceptive effect. In mice implanted with morphine pellets, the synergism decreased to an additive interaction. The interactions between clonidine and the mu agonist Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO), the delta agonist D-Pen²-D-Pen⁵-Enkephalin (DPDPE), and the kappa agonist U50-488H were also synergistic in placebo animals. In morphine pellet treated mice the DPDPE/clonidine interaction decreased to an antagonistic interaction, the DAMGO/clonidine remained synergistic and the U50-488H/clonidine interaction decreased to additive. These results support the proposal that the morphine spinal/supraspinal synergism depends upon the interaction between spinal opioid and alpha₂ receptors and a decrease in this interaction is a mechanism involved in development of tolerance to morphine. In addition, delta and kappa receptors appeared to be more involved in the morphine/clonidine decreased interaction than did mu opioid receptors.     

Alpha interferon interaction with opiate receptors in the rat brain

The preferential interactions of alpha-interferon (alpha-IFN) with delta and mu opiate receptors were studied. alpha-IFN (specific antiviral activity 2 X 10(3) U/mg protein) was shown to inhibit in the competitive manner 3H-naloxone and 3H-D-ala2, D-leu5-enkephalin (3H-DADL) specific binding to opiate receptor subpopulations. alpha-IFN was much more effective in decreasing 3H-DADL than 3H-naloxone binding in opiate receptors: K1 values averaged 160 +/- 30 and 1150 +/- 80 U/ml, respectively. IFN effective concentrations inhibiting 50% of 3H-naloxone opiate receptor binding in the absence or presence of 100 mmol/l NaCl were similar, and the "sodium shift" value was equal to 1. The independence of alpha-IFN activity of the presence of NA+ cations suggests the antagonist character of alpha-IFN interaction with opiate receptors. Thus, alpha-IFN employed appears to be an alpha-selective ligand displaying the in vitro properties of "pure" morphine antagonists.     

Comparative effects of opioid peptides on respiration and analgesia in rats

Receptor mechanisms for opiate induced respiratory depression and analgesia (tail-flick) were studied by the ED50 ratios and the apparent pA2 values of the interactions of naloxone with the mu-agonists morphine and D-ala2-me-phe4-met (O)ol5-enkephalin (FK-33824), and the delta-agonists D-ala2-D-leu5-enkephalin (DADL) and tyr-D-ser-gly-phe-leu-thr. The apparent pA2 values of morphine, FK-33824 and DADL for analgesia were similar, whereas the apparent pA2 values of the mu-agonists for respiratory depression were significantly lower than those of the delta-agonists. The ratio between the ED50 of FK-33824 in analgesia and respiratory depression was much lower than that of DADL. It is concluded that different receptors mediate the opiate-induced respiratory depression. One difficulty with the delta-receptors being maximally involved in this action is the high degree of antagonism shown by naloxone on the respiratory effects of the delta-agonists.     

Environmental and intraperitoneal ethanol influences morphine antinociceptive effect in mice

The ability of acute environmental or intraperitoneal (i.p.) ethanol to influence morphine antinociceptive effect was studied in mice. In order to induce tolerance to morphine analgesia, mice received daily injections of 10 mg/Kg morphine over a period of 10 days. Mice were divided into three groups: i.p. ethanol (E), environmental ethanol (E*), and control saline (M). During the induction of tolerance these groups were treated identically except on days 1 and 11. On these days, 10 minutes prior to morphine injection, mice received either i.p. ethanol (1g/Kg), environmental ethanol (a bottle of 10% ethanol placed next to the animals cage during the experiments), or an equivalent volume of saline. Analgesia was assessed using a standard hot plate protocol and dose-response cumulative curves for morphine analgesia were obtained on days 1 and 11. On day 1, both the i.p. and environmental administration of ethanol showed similar morphine-potentiation effects [Mean Effective Dose: ED50 (M1)=4.5 mg/kg; ED50 (E1)=2.4 mg/kg; ED50 (E*1)=2.1 mg/kg]. On day 11, control group mice showed a reduction of morphine analgesia at test [ED50 (M11)=14.1 mg/kg]. Mice receiving i.p. and environmental ethanol again showed a leftward shift in dose-response cumulative curves for morphine antinociception with respect to controls [ED50 (E11)=9.1 mg/kg; ED50 (E*11)=4.7 mg/kg]. I.p. ethanol administration at non-antinociceptive doses enhances the morphine antinociception effect similarly in tolerant and non-tolerant (naive) mice. The presence of environmental ethanol can also induce a similar pattern of increase in morphine antinociception effect.     

Itch-scratch responses induced by opioids through central Mu opioid receptors in mice

We examined scratch-inducing effects of intracisternal, intrathecal and intradermal injections of morphine and some opioid agonists in mice. Intracisternal injection of morphine (3 nmol/animal) and the mu-receptor agonist [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]enkephalin (DAMGO; 0.2 nmol/animal) elicited scratching of the face, with little effect on scratching of the trunk. Intracisternal injection of the delta-receptor agonist [D-Pen(2,5)]enkephalin (DPDPE) and the kappa-receptor agonist U50488 were without effects. Intrathecal injection of morphine (0.1-3 nmol/animal) produced a dose-dependent increase in body scratching, with little effects on face scratching. Face scratching induced by intrathecal morphine (3 nmol/animal) was almost abolished by subcutaneous pretreatment with naloxone (1 mg/kg). Intradermal injections of morphine (3-100 nmol/site), DAMGO (1-100 nmol/site), DPDPE (10 and 100 nmol/site) and U50488 (10-100 nmol/site) did not elicit scratching of the site of injection. Intradermal injection of histamine (100 nmol/site) induced the scratching in ICR, but not ddY, mice and serotonin (30 and 50 nmol/site) elicited the scratching in either strain of mice. The results suggest that opioids induce scratching, and probably itching, through central mu-opioid receptors in the mouse.     

Dorsal noradrenergic bundle lesions fail to alter opiate withdrawal or suppression of opiate withdrawal by clonidine

Previous work has shown that clonidine effectively supresses many of the signs of opiate withdrawal. The present study was designed to test the hypothesis that the supression of opiate withdrawal by clonidine is mediated by forebrain noradrenergic projections of the locus coeruleus. Two groups of 24 rats each were subjected to either a 6-hydroxydopamine lesion of the dorsal noradrenergic bundle (Lesion group) or a sham, vehicle injection (Sham group). All rats were made dependent on morphine by subcutaneous implantation of one 75 mg silastic morphine pellet for three days followed by 3 more days with two additional 75 mg pellets. Following removal of the morphine pellet, withdrawal was precipitated in all rats by subcutaneous injection of 4 mg/kg of naloxone. Pretreatment 10 min. before withdrawal with clonidine (0.1 or 0.2 mg/kg) produced a significant attenuation of withdrawal signs as compared to saline injected rats; this effect was equally significant in both sham and lesion groups. Lesions of the locus coeruleus had no effect on withdrawal, nor did they affect the ameliorating action of clonidine. These results substantiate the observation that clonidine can effectively attenuate signs of opiate withdrawal in the rat, but fail to support the hypothesis that these effects are mediated by the forebrain projections of the locus coeruleus.     

Effect of clonidine on the chronic morphine tolerance and on the sensitivity of the smooth muscles in mice

Clonidine, when administered for prolonged period showed no tolerance to its analgesic activity. Prior exposure to clonidine attenuated the tolerance development to morphine-induced analgesia and the supersensitivity to acetylcholine (ACh) in ileum during chronic morphine treatment. Further, acute administration of lower doses of clonidine (upto 1 mg) produced supersensitivity in ileum to ACh while the higher dose (10 mg) induced subsensitivity. In vas deferens, clonidine in all the concentrations tested induced dose and time dependent supersensitivity to norepinephrine (NE) similar to that produced by morphine. Chronic clonidine treatment failed to alter the ACh responses in ileum while it produced supersensitivity to NE in vas deferens. The results suggest that clonidine and morphine possess comparable properties and the antagonism of chronic morphine tolerance by clonidine may be the therapeutic basis for its clinical application in the treatment of opiate addicts.     

Short-term tolerance to morphine: effects of diazepam, phenobarbital, and amphetamine

Short-term tolerance to morphine, which can be demonstrated in as little as 3 hours after a single administration of the opiate, was examined in animals chronically pretreated with diazepam, phenobarbital, or amphetamine. Tail-flick latency in mice and changes in plasma corticosterone in rats were the parameters tested in these experiments. Rats primed with either saline or morphine, 10 mg/kg, were injected 3 hours subsequently with morphine, 5 mg/kg. Those primed with saline showed the characteristic plasma corticosterone elevation following morphine, when serial blood samples were examined, whereas those previously treated with morphine did not. Mice were primed with saline or either of two doses of morphine, 30 or 100 mg/kg, 3.5 hours prior to estimation of tail-flick latency and ED50 determinations. Mice primed with either dose of morphine had significantly higher ED50's than those primed with saline. Chronic treatment with diazepam or amphetamine in either species did not significantly alter short-term tolerance development by either parameter. However, with phenobarbital pretreatment, the plasma corticosterone response was attenuated and short-term tolerance to morphine's analgesic effects did not occur. Further studies in morphine-pelleted mice showed that analgesic tolerance occurred similarly in all groups. This suggests that barbiturates may delay the process.     

Effects of epinephrine, clonidine, L-phenylephrine, and morphine on intestinal secretion mediated by Escherichia coli heat-stable enterotoxin in pig jejunum

Perfusion of pig jejunum with Escherichia coli heat-stable enterotoxin (strain 1261) reversed net absorption of water and electrolytes to net secretion. Addition of the alpha-adrenergic agonists clonidine (5 X 10(-7) M) or L-phenylephrine (5 X 10(-6) M), or the opiate agonist morphine (3.6 X 10(-6) M) to the perfusate reduced the secretory response to enterotoxin and stimulated absorption in normal jejunum. Epinephrine (5 X 10(-5) M) did not stimulate absorption in controls but reduced chloride loss in the presence of enterotoxin. Mucosal sodium--potassium adenosine triphosphatase was unchanged but disaccharidase activity was decreased in the presence of enterotoxin. The results suggest that alpha-adrenergic agonists and opiate agonists may exert an antidiarrheal action by increasing net transport across intestinal epithelium.     

Comparison of the amounts of hCG and PMSG to induce ovulation in 50% of the animals, mice, Syrian hamsters and rats]

On the day of diestrus, female mice, syrian hamsters and rats, showing regular 4-day estrous cycles, were injected with hCG or PMSG and were inspected for the presence of ovulation the following day. The dose level expected to cause an effect in 50% of the animals (ED50) was calculated using the Van der Waerden method. When hCG was injected into i. v., i. p. and s. c., the ED50 values per animal and per body weight (kg) in parenthesis were as follows; 0.2 (7.7), 0.3 (11.5) and 0.7 (26.9) I. U. for mice, 1.0 (9.5), 1.8 (17.1) and 2.6 (24.8) I. U. for syrian hamsters and 1.3 (4.6), 3.5 (12.3) and 7.5 (26.3) I. U. for rats, respectively. In PMSG study, the ED50 values per animal and per body weight (kg) in parenthesis were as follows: i. v., 0.8 (30.8); i. p., 2.0 (76.9); s. c., 2.8 (107.7) I. U. for mice, i. v., 3.6 (34.3); i. p., 8.0 (76.2); s. c., 13.2 (125.7) I. U. for syrian hamsters and i. v., 6.0 (76.8); i. p., 20.8 (73.0); s. c., 76.8 (269.5) I. U. for rats, respectively. From these results, the intravenous ED50 value was lower than other routes in three rodents with hCG or PMSG. In all injection routes, the ED50 value for mouse was lower than others. However, there were not significant differences in the ED50 values per body weight (kg) among three rodents. In particular, subcutaneous ED50 of hCG and intraperitoneal ED50 of PMSG were almost same values among three rodents, respectively. Given that the ED50 value per body weight (kg) in one of three rodents is determined, its value may be possible to be extrapolated to remaining two rodents.     

Studies on the effects of glucose in vitro and of the glycaemic state in vivo on the binding characteristics of mu, delta and kappa opiate receptors in mouse brain.

The effect of glucose on the binding characteristics of opiate receptor subtypes was investigated in brain membranes from normoglycaemic lean Aston (C57BL/6J) mice using [3H][D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO), [3H][D-Pen2,D-Pen5]enkephalin (DPDPE) and [3H]U69,593 as selective ligands for mu, delta and kappa opiate receptors respectively. The equilibrium dissociation constants (Kd) and maximal binding capacities (Bmax) of [3H]DAMGO and [3H]DPDPE were unaltered by 20mM glucose in vitro. Similarly, [3H]U69,593 binding was not modified by increasing the concentration of glucose from 0 to 20mM (P between 0.10 and 0.05), or by the presence of 20mM fructose and of 20mM 3-O-me-glucose, a non-metabolisable sugar, in the incubation medium. The nonselective opiate ligand, [3H]diprenorphine, bound with similar affinity and binding capacity to brain membranes prepared from control and streptozotocin-diabetic Swiss (CD1) mice. The addition of 20mM glucose or of 20mM fructose in vitro induced no changes in their binding parameters. The affinity and binding capacity of [3H]U69,593 to STZ-diabetic Swiss mouse brain membranes was not significantly different to that of normoglycaemic controls; 20mM glucose in vitro had no effect on ligand binding to kappa sites in STZ-diabetic mouse brain membranes. We conclude that glucose does not interact directly with the opiate receptor to modfy it in such as way as could explain the altered sensitivity to different opioid agonists seen in obese and hyperglycaemic animal models in vivo.     

Yohimbine reduces morphine tolerance in guinea-pig ileum

Opiates are known to inhibit electrically-evoked twitches of longitudinal muscle-myenteric plexus strips from guinea-pig ileum. When this preparation was incubated with morphine for 1 h tolerance developed to the inhibitory effect, since dose-response curves were shifted to the right. In the present study, the effects of alpha-2 adrenergic agents on the tolerance induced by morphine in this preparation was investigated. Addition of yohimbine 10 microM (but not 0.1 or 1 microM) to the incubating medium reduced the magnitude of opiate tolerance. This effect did not appear in the presence of the alpha-2 agonists clonidine or guanfacine (10 microM). Our results provide evidence of the longitudinal muscle-myenteric plexus as a useful model for the study of the relationship between morphine tolerance and alpha-2 adrenergic mechanisms.     

The possible involvement of endogenous ligands for mu-, delta- and kappa-opioid receptors in modulating morphine-induced CPP expression in rats

Previous studies suggested that electroacupuncture (EA) can suppress opioid dependence by the release of endogenous opioid peptides. To explore the site of action and the receptors involved, we tried to inject highly specific agonists for μ-, δ- and κ-opioid receptors into the CNS to test whether it can suppress morphine-induced conditioned place preference (CPP) in the rat. Male Sprague–Dawley rats were trained with 4 mg/kg morphine, i.p. for 4 days to establish the CPP model. This CPP can be prevented by (a) i.p. injection of 3 mg/kg dose of morphine, (b) intracerebroventricular (i.c.v.) injection of micrograms doses of the selective μ-opioid receptor agonist DAMGO, δ-agonist DPDPE or κ-agonist U-50,488H or (c) microinjection of DAMGO, DPDPE or U50488H into the shell of the nucleus accumbens (NAc). The results suggest that the release of endogenous μ-, δ- and κ-opioid agonists in the NAc shell may play a role for EA suppression of opiate addiction.     

Restraint stress enhances morphine-induced analgesia in the rat without changing apparent affinity of receptor

Antagonism of morphine analgesia (tail-flick assay) by naloxone was assessed quantitatively by in vivo "apparent" pA2 determination in unstressed rats and in rats subjected to restraint stress. Restrained rats had a higher baseline tail-flick latency than did unstressed (unrestrained) animals, and were more sensitive to the analgesic effect of morphine, as reflected in lower morphine ED50s. There was no significant difference between apparent pA2 values of unstressed and restrained rats using pA2 regression line analysis. This suggests that while stress enhances the analgesic effect of morphine, it does not appreciably alter opiate receptor affinity for naloxone under the conditions of this study.     

Clonidine Inhibition of Norepinephrine Release from Normal and Morphine-Tolerant Guinea Pig Cortical Slices

Endogenous norepinephrine (NE) release in cerebral cortex slices taken from normal and morphine-tolerant guinea pigs was measured by HPLC. In normal slices, a linear relationship was found between electrically evoked NE release and the log of the frequency of stimulation in the range of 1-20 Hz. The efficiency of the alpha 2-mediated autofeedback was tested by adding the alpha 2-agonist clonidine and the alpha 2 agonist idazoxan. NE release was dose-dependently reduced by clonidine (1 nmol/L-1 mumol/L) and increased by idazoxan (10-100 nmol/L). The inhibition by clonidine was significantly greater at 1 Hz than at 3 Hz, whereas the absolute increase in NE release induced by idazoxan was greater at 3 Hz than at 1 Hz. Morphine at 1 mumol/L (a concentration per se ineffective) shifted to the left the clonidine concentrations able to inhibit NE release at 3 and 1 Hz (1-10 nmol/L), but at both frequencies, the opiate reduced the maximal inhibition induced by clonidine at 1 mumol/L. In slices taken from morphine-tolerant guinea pigs (in the presence of morphine at 1 mumol/L), clonidine (1 nmol/L-1 mumol/L) was ineffective at the stimulation rate of 3 Hz, but it was more active than in normal slices at 1 Hz. Such a response pattern suggests a reduced availability of alpha 2 receptors and an increase in their sensitivity to clonidine. However, chronic morphine treatment did not influence the physiological autoinhibition because the increase in NE release elicited by idazoxan (10-100 nmol/L) at 1 and 3 Hz was the same in normal and in "morphine-tolerant" slices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mu-delta opioid interactions. II: Beta-FNA inhibits DPDPE-induced increases in morphine EEG and EEG spectral power.

In the present study, the effects of beta-FNA on DPDPE-induced increases in morphine EEG and EEG power spectra were assessed. Adult female Sprague-Dawley rats were implanted with cortical EEG electrodes and permanent indwelling ICV and IV cannulae. Rats were administered ICV beta-FNA at 20 nmol or ICV sterile water. Then 18-24 h later, rats were administered ICV DPDPE at 2.5 nmol or ICV sterile water followed, 10 min later, by IV morphine at 3 mg/kg. Morphine-induced changes in EEG global (1-50 Hz) spectral parameters, the duration of morphine-induced high voltage EEG bursts, the period of EEG and behavioral excitation, and the latency to onset of slow-wave sleep were statistically analyzed using a one-way analysis of variance. beta-FNA pretreatment significantly decreased morphine-induced total spectral power seen in the DPDPE + morphine group. beta-FNA pretreatment also significantly decreased the duration of morphine-induced EEG bursts, the period of EEG and behavioral excitation, and the latency to onset of slow-wave sleep in the DPDPE + morphine group. These data, therefore, suggest that DPDPE may be increasing the effects of morphine on EEG through delta opioid receptors associated with the mu-delta opioid receptor complex.     

Antinociceptive interactions of opioid delta receptor agonists with morphine in mice: supra- and sub-additivity.

In this study, the antinociceptive interactions of fixed ratio combinations of intracerebroventricularly (i.c.v.) given morphine and subantinociceptive doses of the delta agonists, [D-Pen2, D-Pen5]enkephalin (DPDPE), [D-Ala2, Glu4]deltorphin (DELT) or [Met5]enkephalin (MET) were examined using the mouse warm water tail flick test. When morphine was coadministered with DPDPE or DELT in a 4:1 and 9:1 mixture, respectively, a synergistic antinociceptive effect was observed. In contrast, when morphine was coadministered with MET in a 1:2 fixed ratio mixture, a subadditive interaction occurred. These results demonstrate both positive and negative modulatory interactions of delta agonists with morphine in an antinociceptive endpoint and that these interactions can be either supra- or subadditive. The data support the concept of a functional interaction between opioid mu and delta receptors and a potential regulatory role for the endogenous ligands of the opioid delta receptor.     

Alpha-2 adrenergic and opioid receptor-mediated gastroprotection.

Clonidine inhibited the development of gastric mucosal lesions induced by either acidified ethanol or indomethacin. The ED50 values were: 7.1 and 5.2 microg x kg(-1) orally, respectively. The gastroprotective effect was antagonised by the pre-synaptic alpha-2 antagonist yohimbine, the more selective alpha-2 antagonist Ch-38083 and the pre-synaptic alpha-2B antagonist prazosin. Moreover, the non-selective opioid receptor antagonist naloxone, the delta receptor selective naltrindole also reversed the clonidine-induced mucosal protective action. Clonidine was also effective following intracerebroventricular administration with the ED50 of 37 ng/rat against ethanol-induced mucosal damage. Our results suggest that: 1) the gastroprotective effect of clonidine is likely to be mediated by alpha-2B adrenoceptor subtype; 2) there is an interaction between pre-synaptic alpha-2 adrenoceptors and opioid system; and 3) clonidine can induce gastroprotection by central mechanism.     

Differential knockdown of delta-opioid receptor subtypes in the rat brain by antisense oligodeoxynucleotides targeting mRNA.

Two antisense oligodeoxynucleotides (A-ODN), targeting delta-opioid receptor mRNA (DOR) and two mismatch ODN sequences (mODN) were continuously infused for 24 days into the lateral brain ventricles of Wistar rats. The density of delta-opioid receptors in rat brain homogenates was measured by saturation binding experiments using four selective ligands, two agonists ([D-Ala2, Glu4]-deltorphin and DPDPE) and two antagonists (Dmt-Tic-OH and naltrindole), and by immunoblotting SDS solubilized receptor protein. In brain membranes of mODN or saline-infused rats, the rank order of delta-opioid receptor density, calculated by Bmax values of the four delta-opioid receptor ligands, was: [D-Ala2, Glu4]deltorphin approximately Dmt-Tic-OH approximately naltrindole (86-118 fmo/mg protein) > DPDPE (73.6+/-6.3 fmol/mg protein). At the end of the 24 day infusion of A-ODN targeting DOR nucleotide sequence 280299 (A-ODN280-299), the Bmax of DPDPE (62.4+/-3.2 fmol/mg protein) was significantly higher than that of Dmt-Tic-OH (31.5+/-3.9 fmol/mg protein). Moreover, both the Kd value for DPDPE saturation binding and the Ki value for Dmt-Tic-OH displacement by DPDPE were halved. In contrast, an A-ODN treatment targeting exon 3 (A-ODN741-760) decreased the specific binding of [D-Ala2, Glu4]deltorphin and Dmt-Tic-OH significantly less (67%-81%) than the binding of DPDPE (53%), without changes in DPDPE Ki and KD values. No A-ODN treatment modified the specific binding of the micro-opioid agonist DAMGO and of the k-selective opioid receptor ligand U69593. On the Western blot of solubilized striatum proteins, A-ODN(280-299) and A-ODN(741-760) downregulated the levels of the DOR protein, whereas the corresponding mODN were inactive. The 24-day infusion of A-ODN(280-299) inhibited the rat locomotor response to [D-Ala2, Glu4]deltorphin but not to DPDPE. Intracerebroventricular (i.c.v.) infusion of A-ODN(741-760) reduced the locomotor responses to both delta-opioid receptor agonists, whereas mODN infusion never affected agonist potencies. In conclusion, these results demonstrate that 24-day continuous i.c.v. infusion of A-ODN targeting the nucleotide sequence 280-299 of DOR can differentially knockdown delta1 and delta2 binding sites in the rat brain.     

Intracerebroventricular injection of antibodies directed against Gs alpha enhances the supraspinal antinociception induced by morphine, beta-endorphin and clonidine in mice.

The intracerebroventricular (i.c.v.) injection of antisera directed against different sequences of Gs alpha to mice enhanced the antinociceptive potency of the opioids morphine, beta h-endorphin-(1-31) and of the alpha 2-agonist clonidine when studied 24 h later in the tail-flick test. The activity of DAGO, DADLE, DPDPE and [D-Ala2]-Deltorphin II remained unchanged after that treatment. Cholera toxin (0.5 microgram/mouse, i.c.v.), agent that impairs the receptor regulation of Gs transducer proteins promoted comparable changes in the supraspinal analgesia induced by these substances. Six days after a single i.c.v. injection (0.5 microgram/mouse) of pertussis toxin the antinociceptive activity of all the opioids and clonidine appeared diminished. It is concluded that opioids and clonidine promote analgesia after binding to receptors functionally coupled to Gi/G(o) proteins, moreover, the activity of morphine, beta-endorphin and clonidine in this test seems to be counteracted by a process involving activation of Gs alpha transducer proteins.