The GBeta5 subunit that associates with the R7 subfamily of RGS proteins regulates mu-opioid effects

The Gbeta5 protein, which is similar in sequence to other G-protein beta subunits, mainly associates with the G-protein gamma-like (GGL) domains of the R7 subfamily of regulators of G-protein signalling (RGS) proteins. This paper reports the presence of the Gbeta5 protein and its mRNA in all areas of mouse CNS, and also its involvement in the cellular signals initiated at mu- and delta-opioid receptors. The expression of Gbeta5 and RGS9-2 proteins (member of the R7 subfamily of RGS) was reduced by blocking their mRNAs with antisense oligodeoxynucleotides (ODN). Knock-down of these proteins enhanced the potency and duration of antinociception promoted by morphine and [D-Ala2, N-MePhe4,Gly-ol5]-enkephalin (DAMGO), agonists at mu opioid receptors. However, the activity of the selective agonist at delta opioid receptors, [D-Pen(2,5)]-encephalin (DPDPE), appeared to be reduced. A single intracerebroventricular (i.c.v.) ED80 analgesic dose of morphine gave rise to acute tolerance in control mice, but did not promote tolerance in Gbeta5 or RGS9-2 knock-down animals. In a model of sustained morphine treatment, the impairment of Gbeta5 proteins facilitated the development of tolerance. This treatment did not alter the incidence of jumping behaviour precipitated by naloxone 3 days after commencing with chronic morphine. These results show differences in the signalling regulation of G-proteins when activated by mu or delta opioid agonists. For mu opioid receptors, acute tolerance, but probably not long-term tolerance, appears to depend on the function of Gbeta5 subunits and associated RGS proteins.     

Morphine alters the selective association between mu-opioid receptors and specific RGS proteins in mouse periaqueductal gray matter

In the CNS, several regulators of G-protein signalling (RGS) modulate the activity of mu-opioid receptors. In pull-down assays performed on membranes from mouse periaqueductal gray matter (PAG), mu-opioid receptors co-precipitated with delta-opioid receptors, Gi/o/z/q proteins, and the regulators of G-protein signalling RGS4, RGS9-2, RGS14, RGSZ1 and RGSZ2. No RGS2, RGS7, RGS10 and RGS11 proteins were associated with the mu receptors in these PAG membranes. In mice, an intracerebroventricular dose of 10 nmol morphine produced acute tolerance at mu receptors but did not disrupt the co-precipitation of mu-delta receptor complexes. However, this opioid reduced by more than 50% the co-precipitation of G alpha i/o/z subunits with mu receptors, and altered their association with some of the RGS proteins at 30 min, 3 h and 24 h after its administration. The association of RGS9-2 with mu receptors diminished by 30-40% 24 h after the administration of morphine, while that of RGSZ2 and of RGSZ1 increased. Morphine treatment recruited RGS4 to the PAG membranes, and 30 min and 3 h after the opioid challenge its association with mu receptors had increased. However, 24 h after morphine administration, the co-precipitation of RGS4 had decreased by about 30%. The opioid produced no change in the membrane levels of RGS9-2, RGS14, RGSZ1 and RGSZ2. Thus, in PAG synaptosomal membranes, a dynamic and selective link exists between, mu-opioid receptors, Gi/o/z proteins and certain RGS proteins.     

Administration of myr(+)-G(i2)alpha subunits prevents acute tolerance (tachyphylaxis) to mu-opioid effects in mice

The administration of efficacious doses of morphine or beta-endorphin causes acute tolerance (tachyphylaxis) to the effects of additional administrations of these opioids. Mice intracerebroventricularly (icv)-injected with biologically active myristoylated (myr(+))-G(i2)alpha subunits developed no tachyphylaxis to morphine antinociception in the tail-flick test. This treatment increased the potency of opioid-induced analgesia during the declining phase. Moreover, animals showing tachyphylaxis to opioid effects exhibited normal responses to the agonists after icv-administration of myr(+)-G(i2)alpha subunits. In morphine tolerant/dependent mice, an icv dose of 12 pmol/mouse myr(+)-G(i2)alpha subunits facilitated complete restoration of morphine antinociception in only 4 or 5 days instead of the 10 to 11 days required for post-dependent mice. This was observed when myr(+)-G alpha subunits were injected within the first 24 h of chronic morphine administration -- but not later when long-term tolerance takes place. These results suggest that during the course of an opioid effect a progressive reduction of receptor-regulated G-proteins occurs, and hence tachyphylaxis develops. Exogenous administration of myr(+)-G alpha subunits may be of therapeutic potential in improving agonist activity and accelerating the recovery of post-dependent receptors.     

RGSZ1 and GAIP regulate mu- but not delta-opioid receptors in mouse CNS: role in tachyphylaxis and acute tolerance.

In the CNS, the regulators of G-protein signaling (RGS) proteins belonging to the Rz subfamily, RGS19 (G(alpha) interacting protein (GAIP)) and RGS20 (Z1), control the activity of opioid agonists at mu but not at delta receptors. Rz proteins show high selectivity in deactivating G(alpha)z-GTP subunits. After reducing the expression of RGSZ1 with antisense oligodeoxynucleotides (ODN), the supraspinal antinociception produced by morphine, heroin, DAMGO ([D-Ala2, N-MePhe4,Gly-ol5]-enkephalin), and endomorphin-1 was notably increased. No change was observed in the effect of endomorphin-2. This agrees with the proposed existence of different mu receptors for the endomorphins. The activities of DPDPE ([D-Pen2,5]-enkephalin) and [D-Ala2] deltorphin II, agonists at delta receptors, were also unchanged. Knockdown of GAIP and of the GAIP interacting protein C-terminus (GIPC) led to changes in agonist effects at mu but not at delta receptors. The impairment of RGSZ1 extended the duration of morphine analgesia by at least 1 h beyond that observed in control animals. CTOP (Cys2, Tyr3, Orn5, Pen7-amide) antagonized morphine analgesia when given during the period in which the effect of morphine was enhanced by RGSZ1 knockdown. Thus, in naive mice, morphine tachyphylaxis originated in the presence of the opioid agonist and during the analgesia time course. The knockdown of RGSZ1 facilitated the development of tolerance to a single dose of morphine and accelerated tolerance to continuous delivery of the opioid. These results indicate that mu but not delta receptors are linked to Rz regulation. The mu receptor-mediated activation of Gz proteins is effective at recruiting the adaptive mechanisms leading to the development of opioid desensitization.     

Effector antagonism by the regulators of G protein signalling (RGS) proteins causes desensitization of mu-opioid receptors in the CNS

Rationale In cell culture systems, agonists can promote the phosphorylation and internalization of receptors coupled to G proteins (GPCR), leading to their desensitization. However, in the CNS opioid agonists promote a profound desensitization of their analgesic effects without diminishing the presence of their receptors in the neuronal membrane. Recent studies have indicated that CNS proteins of the RGS family, specific regulators of G protein signalling, may be involved in mu-opioid receptor desensitization in vivo.Objective In this work we review the role played by RGS proteins in the intensity and duration of the effects of mu-opioid receptor agonists, and how they influence the delayed tolerance that develops in response to specific doses of opioids.Results RGS proteins are GTPase-activating proteins (GAP) that accelerate the hydrolysis of GGTP to terminate signalling at effectors. The GAP activity of RGS-R4 and RGS-Rz proteins restricts the amplitude of opioid analgesia, and the efficient deactivation of GzGTP subunits by RGS-Rz proteins prevents mu receptor desensitization. However, RGS-R7 proteins antagonize effectors by binding to and sequestering mu receptor-activated Gi/o/z subunits. Thus, they reduce the pool of receptor-regulated G proteins and hence, the effects of agonists. The delayed tolerance observed following morphine administration correlates with the transfer of G subunits from mu receptors to RGS-R7 proteins and the subsequent stabilization of this association.Conclusion In the CNS, the RGS proteins control the activity of mu opioid receptors through GAP-dependent (RGS-R4 and RGS-Rz) as well as by GAP-independent mechanisms (RGS-R7). As a result, they can both antagonize effectors and desensitize receptors under certain circumstances.     

Recruitment of RGS2 and RGS4 to the plasma membrane by G proteins and receptors reflects functional interactions

N-terminally green fluorescent protein (GFP)-tagged regulator of G protein signaling (RGS) 2 and RGS4 fusion proteins expressed in human embryonic kidney 293 cells localized to the nucleus and cytosol, respectively. They were selectively recruited to the plasma membrane by G proteins and correspondingly by receptors that activate those G proteins: GFP-RGS2 when coexpressed with Galphas, beta2-adrenergic receptor, Galphaq, or AT1A angiotensin II receptor, and GFP-RGS4 when coexpressed with Galphai2 or M2 muscarinic receptor. G protein mutants with reduced RGS affinity did not produce this effect, implying that the recruitment involves direct binding to G proteins and is independent of downstream signaling events. Neither agonists nor inverse agonists altered receptor-promoted RGS association with the plasma membrane, and expressing either constitutively activated or poorly activated G protein mutants produced effects similar to those of their wild-type counterparts. Thus, intracellular interactions between these proteins seem to be relatively stable and insensitive to the activation state of the G protein, in contrast to the transient increases in RGS-G protein association known to be caused by G protein activation in solution-based assays. G protein effects on RGS localization were mirrored by RGS effects on G protein function. RGS4 was more potent than RGS2 in promoting steady-state Gi GTPase activity, whereas RGS2 inhibited Gs-dependent increases in intracellular cAMP, suggesting that G protein signaling in cells is regulated by the selective recruitment of RGS proteins to the plasma membrane.     

Bremazocine is an agonist at kappa-opioid receptors and an antagonist at mu-opioid receptors in the guinea-pig myenteric plexus.

The agonist and antagonist activity of bremazocine at opioid receptors in the guinea-pig myenteric plexus preparation was determined in untreated tissues and in tissues in which either mu-9 or kappa-opioid receptors were blocked preferentially. After pretreatment of the tissue with beta-funaltrexamine for 90 min followed by washing out, the IC50 value of the selective mu-ligand [D-Ala2,MePhe4,Gly-ol5]enkephalin was increased 67 fold whereas the IC50 values of the selective kappa-ligand U-69,593 and of the non-selective kappa-ligand bremazocine were not significantly changed. In this experimental design bremazocine acted only on kappa-receptors. After pretreatment of the tissue with beta-chlornaltrexamine and 10 microM of the mu-ligand for 30 min followed by washout, the IC50 value of the mu-ligand was increased 2 fold whereas the IC50 value of the selective kappa-ligand was increased 32 fold and that of bremazocine 62 fold. Under these experimental conditions, it was shown that bremazocine is an antagonist against [D-Ala2,MePhe4,Gly-ol5]enkephalin at the mu-receptor (Ke = 1.6 nM). The residual agonist activity of bremazocine is at the kappa-receptor. In naive myenteric plexus preparations the mu-antagonist activity of bremazocine cannot be demonstrated because its potency at the kappa-receptor is very high. This dual action may be of importance for the responses of bremazocine in other peripheral and central tissues.     

Roles of mu-opioid receptors in development of tolerance to diisopropylfluorophosphate (DFP)

Anatomical evidence indicates that cholinergic and opioidergic systems are co-localized and acting on the same neuron. However, the regulatory mechanisms between cholinergic and opioidergic system have not been well characterized. In the present study, the potential involvement of mu-opioid receptors in mediating the changes of toxic signs and muscarinic receptor binding after administration of irreversible anti-acetylcholinesterase diisopropylfluorophosphate (DFP) was investigated. DFP (1 mg/kg/day, subcutaneous injection, s.c.)-induced tremors and chewing movements were monitored during the 28-day treatment period in mu-opioid receptor knockout and wild type mice. Autoradiographic studies of total, M1, and M2 muscarinic receptors were conducted using [(3)H]-quinuclidinyl benzilate, [(3)H]-pirenzepine, and [(3)H]-AF-DX384 as ligands, respectively. DFP-induced tremors in both mu-opioid receptor knockout and wild type mice showed tolerance development. However, DFP-induced tremors in mu-opioid receptor knockout mice showed delayed tolerance development than that of DFP-treated wild type controls. DFP-induced chewing movements in both mu-opioid receptor knockout and wild type mice failed to show development of tolerance after four weeks of treatment. M2 muscarinic receptor binding of DFP-treated mu-opioid receptor knockout mice was significantly decreased than that of the DFP-treated wild type controls in the striatum, but not in the cortex and hippocampus. However, there were no significant differences in total and M1 muscarinic receptor binding between DFP-treated mu-opioid receptor knockout and wild type mice in the cortex, striatum and hippocampus. These studies indicate that mu-opioid receptors play an important role through the striatal M2 muscarinic receptors to regulate the development of tolerance to DFP-induced tremors.     

The RGSZ2 protein exists in a complex with mu-opioid receptors and regulates the desensitizing capacity of Gz proteins.

The regulator of G-protein signaling RGS17(Z2) is a member of the RGS-Rz subfamily of GTPase-activating proteins (GAP) that efficiently deactivate GalphazGTP subunits. We have found that in the central nervous system (CNS), the levels of RGSZ2 mRNA and protein are elevated in the hypothalamus, midbrain, and pons-medulla, and that RGSZ2 is glycosylated in synaptosomal membranes isolated from CNS tissue. In analyzing the function of RGSZ2 in the CNS, we found that when the expression of RGSZ2 was impaired, the antinociceptive response to morphine and [D-Ala2, N-MePhe4, Gly-ol5]-enkephalin (DAMGO) augmented. This potentiation involved mu-opioid receptors and increased tolerance to further doses of these agonists administered 24 h later. High doses of morphine promoted agonist desensitization even within the analgesia time-course, a phenomenon that appears to be related to the great capacity of morphine to activate Gz proteins. In contrast, the knockdown of RGSZ2 proteins did not affect the activity of delta receptor agonists, [D-Pen2,5]-enkephalin (DPDPE), and [D-Ala2] deltorphin II. In membranes from periaqueductal gray matter (PAG), both RGSZ2 and the related RGS20(Z1) co-precipitated with mu-opioid receptors. While a morphine challenge reduced the association of Gi/o/z with mu receptors, it increased their association with the RGSZ2 and RGSZ1 proteins. However, only Galphaz subunits co-precipitated with RGSZ2. Doses of morphine that produced acute tolerance maintained the association of Galpha subunits with RGSZ proteins even after the analgesic effects had ceased. These results indicate that both RGSZ1 and RGSZ2 proteins influence mu receptor signaling by sequestering Galpha subunits, therefore behaving as effector antagonists.     

Chronic opioid antagonist treatment dose-dependently regulates mu-opioid receptors and trafficking proteins in vivo

Chronic opioid antagonist treatment increases the density of mu-opioid receptors (muOR) in many model systems. In previous studies, naltrexone treatment produced an increase in muOR density accompanied by decreases in GRK-2 and DYN-2 protein abundance. To examine the relationship between changes in receptor density and proteins involved in receptor trafficking, the dose-dependent effect of chronic naloxone infusion was determined. Dose-dependent antagonism of morphine analgesia was also examined. Mice were infused with naloxone (0.1, 1.0, 5.0 mg/kg/day sc) for 7 days via osmotic pump. Controls were treated with placebo pellets. On the 7th day, morphine dose-response studies were determined using the tail flick. Other mice were sacrificed at the end of the treatment and spinal cords were collected for determination of muOR density and GRK-2 and DYN-2 protein abundance. Naloxone infusion dose-dependently increased spinal muOR density with no change in affinity. The increases in mu-receptor density were proportional to dose-dependent decreases in GRK-2 and DYN-2 protein levels. Furthermore, naloxone dose-dependently antagonized morphine. These data suggest that opioid antagonist-induced muOR up-regulation in mouse spinal cord is associated with regulation of proteins involved in receptor trafficking and support suggestions that opioid antagonist-induced receptor up-regulation is due to reduced constitutive internalization of opioid receptors.     

R7BP Complexes With RGS9-2 and RGS7 in the Striatum Differentially Control Motor Learning and Locomotor Responses to Cocaine

In the striatum, signaling through G protein-coupled dopamine receptors mediates motor and reward behavior, and underlies the effects of addictive drugs. The extent of receptor responses is determined by RGS9-2/Gβ5 complexes, a striatally enriched regulator that limits the lifetime of activated G proteins. Recent studies suggest that the function of RGS9-2/Gβ5 is controlled by the association with an additional subunit, R7BP, making elucidation of its contribution to striatal signaling essential for understanding molecular mechanisms of behaviors mediated by the striatum. In this study, we report that elimination of R7BP in mice results in motor coordination deficits and greater locomotor response to morphine administration, consistent with the essential role of R7BP in maintaining RGS9-2 expression in the striatum. However, in contrast to previously reported observations with RGS9-2 knockouts, mice lacking R7BP do not show higher sensitivity to locomotor-stimulating effects of cocaine. Using a striatum-specific knockdown approach, we show that the sensitivity of motor stimulation to cocaine is instead dependent on RGS7, whose complex formation with R7BP is dictated by RGS9-2 expression. These results indicate that dopamine signaling in the striatum is controlled by concerted interplay between two RGS proteins, RGS7 and RGS9-2, which are balanced by a common subunit, R7BP.     

RGS-Rz and RGS9-2 proteins control mu-opioid receptor desensitisation in CNS: the role of activated Galphaz subunits

Two consecutive i.c.v. administrations of analgesic doses of mu-opioid receptor agonists lead to a profound desensitisation of the latter receptors; a third dose produced less than 20% of the effect obtained with the first administration. Desensitisation was still effective 24h later. Impairing the activity of Galphaz but not Galphai2 subunits prevented tolerance developing after the administration of three consecutive doses of morphine. Further, the i.c.v. injection of Galphai2 subunits potentiated morphine analgesia and abolished acute tolerance, whereas i.c.v.-administered Galphaz subunits produced a rapid and robust loss of the response to morphine. The RGSZ1 and RGSZ2 proteins selectively deactivate GalphazGTP subunits, and their knockdown increased the effects produced by the first dose of morphine. However, impairing their activity also accelerated tachyphylaxis following successive doses of morphine, and facilitated the development of acute morphine tolerance. In contrast, inhibiting the RGS9-2 proteins, which bind to GalphaoGTP and GalphaiGTP but only weakly deactivates them, preserved the effects of consecutive morphine doses and abolished the generation of acute tolerance. Therefore, desensitisation of mu-opioid receptors can be achieved by reducing the responsiveness of post-receptor elements (via the possible action of activated Galphaz subunits) and/or by depleting the pool of receptor-regulated G proteins that agonists need to propagate their effects, e.g., through the activity of RGS9-2 proteins.     

The interaction of mu-opioid receptors with ion channels and G-proteins

The state-of-the-art in investigations of the mu-opioid receptors (ORs) is reviewed. Published information on the interaction between mu-ORs and various G-proteins is summarized and data on the neutral antagonists and inverse agonists of mu-opioid receptor are generalized. The notions about the spontaneous activation of mu-ORs are considered and numerous reports on the interaction between mu-ORs and GIRK, KATP, and Kv channels are analyzed. The role of L, N, and P/Q types of Ca(2+)-channels in mu-OR-mediated intracellular signaling is discussed.     

Opioid agonist efficacy predicts the magnitude of tolerance and the regulation of mu-opioid receptors and dynamin-2

It has been proposed that opioid agonist efficacy may play a role in tolerance and the regulation of opioid receptor density. To address this issue, the present studies estimated the in vivo efficacy of three opioid agonists and then examined changes in spinal mu-opioid receptor density following chronic treatment in the mouse. In addition, tolerance and regulation of the trafficking protein dynamin-2 were determined. To evaluate efficacy, the method of irreversible receptor alkylation was employed and the efficacy parameter tau estimated. Mice were injected with the irreversible mu-opioid receptor antagonist clocinnamox (0.32-25.6 mg/kg, i.p), and 24 h later, the analgesic potency of s.c. morphine, oxycodone and etorphine were determined. Clocinnamox dose-dependently antagonized the analgesic effects of morphine, etorphine and oxycodone. The shift to the right of the dose-response curves was greater for morphine and oxycodone compared to etorphine and the highest dose of clocinnamox reduced the maximal effect of morphine and oxycodone, but not etorphine. The order of efficacy calculated from these results was etorphine>morphine>oxycodone. Other mice were infused for 7 days with oxycodone (10-150 mg/kg/day, s.c.) or etorphine (50-250 microg/kg/day, s.c.) and the analgesic potency of s.c. morphine determined. The low efficacy agonist (oxycodone) produced more tolerance than the high efficacy agonist (etorphine) at equi-effective infusion doses. In saturation binding experiments, the low efficacy opioid agonists (morphine, oxycodone) did not regulate the density of spinal mu-opioid receptors, while etorphine produced approximately 40% reduction in mu-opioid receptor density. Furthermore, etorphine increased spinal dynamin-2 abundance, while oxycodone did not produce any significant change in dynamin-2 abundance. Overall, these data indicate that high efficacy agonists produce less tolerance at equi-effective doses. Furthermore, increased efficacy was associated with mu-opioid receptor downregulation and dynamin-2 upregulation. Conversely, lower efficacy agonists produced more tolerance at equi-effective doses, but did not regulate mu-opioid receptor density or dynamin-2 abundance. Taken together, these studies indicate that agonist efficacy plays an important role in tolerance and regulation of receptors and trafficking proteins.     

The interaction of mu-opioid receptors with intracellular signal systems

Review is devoted a modem state of researches of micro-opioid receptor. It is discussed literature data on the integration of micro-opioid receptor with adenylyl cyclase, phospholipase A2, C and D. It is analyzed reports on the interaction of micro-opioid receptor with NO-synthase and guanylyl cyclase.     

The analgesic efficacy of fentanyl: relationship to tolerance and mu-opioid receptor regulation

This study determined if fentanyl analgesic efficacy predicts the magnitude of tolerance and mu-opioid receptor regulation. To estimate efficacy, mice were injected i.p. with saline or clocinnamox (CCAM), an irreversible mu-opioid receptor antagonist, (0.32-25.6 mg/kg) and 24 h later fentanyl cumulative dose-response studies were conducted. CCAM dose dependently shifted the fentanyl dose-response function to the right. The apparent efficacy (tau) of fentanyl, based on the operational model of agonism, was estimated as 58, indicating that fentanyl is a high analgesic efficacy agonist. Next, mice were infused with fentanyl (1, 2 or 4 mg/kg/day) for 7 days. Controls were implanted with placebo pellets. At the end of 7 days, morphine cumulative dose-response studies or mu-opioid receptor saturation binding studies were conducted. Fentanyl infusions dose dependently decreased morphine potency with the highest fentanyl dose reducing morphine potency by approximately 6 fold. Chronic infusion with fentanyl (4 mg/kg/day) significantly reduced mu-opioid receptor density by 28% without altering affinity, whereas lower infusion doses had no effect. Taken together, the present results strengthen the proposal that opioid analgesic efficacy predicts mu-opioid receptor regulation and the magnitude of tolerance.     

Unexpected prosecretory action component of loperamide at mu-opioid receptors in the guinea-pig colonic mucosa in vitro.

1. In a voltage clamp setting (Ussing chamber), the antidiarrhoeal drug, loperamide (Lop) slightly augmented prostaglandin E1 (PGE1) plus theophylline-stimulated net chloride secretion above control values at low concentrations (10(-10) and 10(-9) M) but inhibited it at higher concentrations (10(-6) and 10(-5) M). The apparently weak prosecretory action component of Lop was turned into a clear cut antisecretory effect by pretreatment with 2 x 10(-7) M naloxonazine plus 10(-7) M CTOP-NH2 (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2), two selective mu opioid receptor antagonists. This indicates a prosecretory effect of loperamide at mu opioid receptors. The antisecretory effect of low Lop concentrations, uncovered by mu opioid receptor blockade, was prevented by additional blockade of kappa opioid receptors by 5 x 10(-9) M nor-binaltorphimine (nor-BNI). 2. The nonselective opioid antagonist, naloxone, at 10(-6) M did not significantly reduce either PGE1 plus theophylline-stimulated net chloride secretion in Lop-free controls or the antisecretory action of Lop. By contrast, the partial agonist ethylketocyclazocine (EKC), which activates kappa but blocks mu opioid receptors, concentration-dependently inhibited PGE1 plus theophylline-stimulated net chloride secretion without any consistent prosecretory action component. Nor-BNI at 5 x 10(-8) M significantly blocked the antisecretory action of EKC. 3. It is concluded that, in the guinea-pig colonic mucosa under the present conditions, mu opioid receptors mediate enhancement and kappa opioid receptors inhibition of PGE1-stimulated net chloride secretion by low Lop concentrations. The two opposite actions are largely masked by superimposition. An opioid receptor-independent mechanism of action contributes to the antisecretory effect of Lop at high concentrations.     

Similarity between mu-opioid receptors in mouse vas deferens and guinea-pig ileum.

The effects of the opioid receptor agonist RX783006 and of the opioid receptor partial agonist (+)-meptazinol have been examined on electrically-induced twitch responses of the guinea-pig isolated ileum and of the mouse isolated vas deferens. Log10 concentration-tissue state curves were determined for (+)-meptazinol and for RX783006, alone, in combination and, when appropriate, in the presence of naloxone (30 nM). Analysis of these log10 concentration-tissue state curves using the null equations derived and verified in the previous paper allows quantitation of the characteristics of the interaction of (+)-meptazinol with the opioid receptors in these tissues. The results indicate that the apparent differences in the actions of (+)-meptazinol on isolated electrically-stimulated guinea-pig ileum and mouse vas deferens can be accounted for without the need to postulate differences between mu-opioid receptors in these two tissues.