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.     

The R7 subfamily of RGS proteins assists tachyphylaxis and acute tolerance at mu-opioid receptors.

Members of the R7 subfamily of regulators of G-protein signaling (RGS) proteins (RGS6, RGS7, RGS9-2, and RGS11) are found in the mouse CNS. The expression of these proteins was effectively reduced in different neural structures by blocking their mRNA with antisense oligodeoxynucleotides (ODNs). This was achieved without noticeable changes in the binding characteristics of labeled beta-endorphin to opioid receptors. Knockdown of R7 proteins enhanced the potency of antinociception promoted by morphine and [D-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO)-both agonists at mu-opioid receptors. The duration of morphine analgesia was greatly increased in RGS9-2 and in RGS11 knockdown mice. The impairment of R7 proteins brought about different changes in the analgesic activity of selective delta agonists. Knockdown of RGS11 reduced [D-Ala(2)]deltorphin II analgesic effects. Those of RGS6 and RGS9-2 proteins caused [D-Ala(2)]deltorphin II to produce a smoothened time-course curve-the peak effect blunted and analgesia extended during the declining phase. RGS9-2 impairment also promoted a similar pattern of change for [D-Pen(2,5)]-enkephalin (DPDPE). RGS7-deficient mice showed an increased response to both [D-Ala(2)]deltorphin II and DPDPE analgesic effects. A single intracerebroventricular (i.c.v.) ED(80) analgesic dose of morphine gave rise to acute tolerance in control mice, but did not promote tolerance in RGS6, RGS7, RGS9-2, or RGS11 knockdown animals. Thus, R7 proteins play a critical role in agonist tachyphylaxis and acute tolerance at mu-opioid receptors, and show differences in their modulation of delta-opioid receptors.     

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.     

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.     

Acute and chronic morphine treatments and morphine withdrawal differentially regulate GRK2 and GRK5 gene expression in rat brain

Opioid agonist stimulates activation of G protein-coupled receptor kinase (GRK) and causes desensitization of opioid signaling, which plays an important role in opioid tolerance. The current study investigated the potential regulatory effects of acute and chronic morphine administration and withdrawal on GRK2 and GRK5 gene expression in rat brain. Our results showed that the initial morphine treatment (10 mg/kg) significantly increased GRK mRNA levels in cerebral cortex, hippocampus, and lateral thalamic nuclei. A significant decrease in GRK5 mRNA levels was observed in periaqueductal gray. In strong contrast, repeated administration of morphine for 9 days failed to cause any significant increase in GRK5 mRNA in any of these brain regions. Chronic morphine treatment resulted in 30-70% down-regulation of GRK2 expression in cerebral cortex, hippocampus, thalamus, and locus coeruleus, opposite to what observed with the single morphine administration. Moreover, spontaneous and naloxone-precipitated morphine withdrawal resulted in aberrant increases in GRK2 and GRK5 mRNA levels in these brain regions. Taken together, our study suggests that opioid not only induces rapid negative feedback regulation on opioid signals through activation of GRK but also exerts its impact, via controlling levels of GRK gene expression, on the regulatory machinery itself over a longer period of time in brain.     

Up-regulation of RGS4 mRNA by opioid receptor agonists in PC12 cells expressing cloned mu- or kappa-opioid receptors.

The regulators of G-protein signaling (RGS) proteins have been shown to modulate the function of some heterotrimeric G-proteins by stimulating the GTPase activity of G-protein alpha subunits. In this study, by northern blotting analysis, we investigated the regulation of RGS4 mRNA by opioid receptor agonists in PC12 cells stably expressing either cloned mu- or kappa-opioid receptors. Treatment with respective opioid receptor agonists (mu: morphine) and [D-Ala(2), MePhe(4), Gly(ol)(5)] enkephalin (DAMGO), kappa: (+)-(5 alpha,7 alpha,8 beta)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro-(4,5)dec-8-y1]benzeneacetamide (U69,593)) for 0.5-24 h significantly and transiently increased the expression of RGS4 mRNA by 140-170% of the control level in a concentration-dependent manner which peaked when treated for 2 h, while treatment of non-transfected PC12 cells with opioid receptor agonists did not. The up-regulation of RGS4 mRNA was significantly blocked by co-treatment with respective opioid antagonists (mu: naloxone, kappa: norbinaltorphimine) or pretreatment with pertussis toxin. These results suggest that the activation of mu- or kappa-opioid receptors increases RGS4 mRNA level, which might contribute to opioid desentilization.     

Differential regulation of RGS proteins in the prefrontal cortex of short- and long-term human opiate abusers

Opiate addiction is characterized by drug tolerance and dependence which involve adaptive changes in μ-opioid receptors (MORs) signaling. Regulators of G-protein signaling RGS9, RGS4 and RGS10 proteins negatively regulate G(αi/o) protein activity modulating MOR function. An important role of RGS proteins in drug addiction has been described but the status of RGS proteins in human brain of opiate addicts remains unknown. The present study evaluated the immunoreactivity levels of RGS4, RGS9 and RGS10 proteins in prefrontal cortex of short- (n = 15) and long-term (n = 21) opiate abusers and in matched control subjects. RGS4 protein was not altered in short-term opiate abusers but, in long-term abusers it was significantly up-regulated (Δ = 29 ± 6%). RGS10 protein expression was significantly decreased in short-term (Δ = -42 ± 7%) but remained unaltered in long-term opiate abusers. RGS9 protein levels in opiate abusers did not differ from matched controls either in the short-term or in the long-term opiate abuser groups. RGS4, RGS9 and RGS10 levels were also studied in brains (frontal cortex) of rats submitted to acute and chronic morphine treatment and to spontaneous and naloxone-precipitated opiate withdrawal. Chronic morphine treatment in rats was associated with an increase in RGS4 protein immunoreactivity (Δ = 28 ± 7%), which persisted in spontaneous (Δ = 35 ± 8%) and naloxone-precipitated withdrawal (Δ = 30 ± 9%) without significant changes in RGS9 and RGS10 proteins. The specific modulation of RGS4 and RGS10 protein expression observed in the prefrontal cortex of opiate abusers might be relevant in the neurobiology of opiate tolerance, dependence and withdrawal. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.     

Nucleus Accumbens-Specific Interventions in RGS9-2 Activity Modulate Responses to Morphine

Regulator of G protein signalling 9-2 (Rgs9-2) modulates the actions of a wide range of CNS-acting drugs by controlling signal transduction of several GPCRs in the striatum. RGS9-2 acts via a complex mechanism that involves interactions with Gα subunits, the Gβ5 protein, and the adaptor protein R7BP. Our recent work identified Rgs9-2 complexes in the striatum associated with acute or chronic exposures to mu opioid receptor (MOR) agonists. In this study we use several new genetic tools that allow manipulations of Rgs9-2 activity in particular brain regions of adult mice in order to better understand the mechanism via which this protein modulates opiate addiction and analgesia. We used adeno-associated viruses (AAVs) to express forms of Rgs9-2 in the dorsal and ventral striatum (nucleus accumbens, NAc) in order to examine the influence of this protein in morphine actions. Consistent with earlier behavioural findings from constitutive Rgs9 knockout mice, we show that Rgs9-2 actions in the NAc modulate morphine reward and dependence. Notably, Rgs9-2 in the NAc affects the analgesic actions of morphine as well as the development of analgesic tolerance. Using optogenetics we demonstrate that activation of Channelrhodopsin2 in Rgs9-2-expressing neurons, or in D1 dopamine receptor (Drd1)-enriched medium spiny neurons, accelerates the development of morphine tolerance, whereas activation of D2 dopamine receptor (Drd2)-enriched neurons does not significantly affect the development of tolerance. Together, these data provide new information on the signal transduction mechanisms underlying opiate actions in the NAc.     

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.     

Differential desensitization of mu- and delta- opioid receptors in selected neural pathways following chronic morphine treatment.

1. Morphine produces a plethora of pharmacological effects and its chronic administration induces several side-effects. The cellular mechanisms by which opiates induce these side-effects are not fully understood. Several studies suggest that regulation of adenylyl cyclase activity by opioids and other transmitters plays an important role in the control of neural function. 2. The aim of this study was to evaluate desensitization of mu- and delta- opioid receptors, defined as a reduced ability of opioid agonists to inhibit adenylyl cyclase activity, in four different brain structures known to be involved in opiate drug actions: caudate putamen, nucleus accumbens, thalamus and periaqueductal gray (PAG). Opiate regulation of adenylyl cyclase in these regions has been studied in control and morphine-dependent rats. 3. The chronic morphine treatment used in the present study (subcutaneous administration of 15.4 mg morphine/rat/day for 6 days via osmotic pump) induced significant physical dependence as indicated by naloxone-precipitated withdrawal symptoms. 4. Basal adenylyl cyclase in the four brain regions was not modified by this chronic morphine treatment. In the PAG and the thalamus, a desensitization of mu- and delta-opioid receptors was observed, characterized by a reduced ability of Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO; mu), Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE; delta) and [D-Ala2]-deltorphin-II (DT-II; delta) to inhibit adenylyl cyclase, activity following chronic morphine treatment. 5. The opioid receptor desensitization in PAG and thalamus appeared to be heterologous since the metabotropic glutamate receptor agonists, L-AP4 and glutamate, and the 5-hydroxytryptamine (5-HT)1A receptor agonist, R(+)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), also showed reduced inhibition of adenylyl cyclase activity following chronic morphine treatment. 6. In the nucleus accumbens and the caudate putamen, desensitization of delta-opioid receptor-mediated inhibition without modification of mu-opioid receptor-mediated inhibition was observed. An indirect mechanism probably involving dopaminergic systems is proposed to explain the desensitization of delta-mediated responses and the lack of mu-opioid receptor desensitization after chronic morphine treatment in caudate putamen and nucleus accumbens. 7. These results suggest that adaptive responses occurring during chronic morphine administration are not identical in all opiate-sensitive neural populations.     

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.     

R7BP Modulates Opiate Analgesia and Tolerance but not Withdrawal

The adaptor protein R7 family binding protein (R7BP) modulates G protein coupled receptor (GPCR) signaling and desensitization by controlling the function of regulator of G protein signaling (RGS) proteins. R7BP is expressed throughout the brain and appears to modulate the membrane localization and stability of three proteins that belong to R7 RGS family: RGS6, RGS7, and RGS9-2. RGS9-2 is a potent negative modulator of opiate and psychostimulant addiction and promotes the development of analgesic tolerance to morphine, whereas the role of RGS6 and RGS7 in addiction remains unknown. Recent studies revealed that functional deletion of R7BP reduces R7 protein activity by preventing their anchoring to the cell membrane and enhances GPCR responsiveness in the basal ganglia. Here, we take advantage of R7BP knockout mice in order to examine the way interventions in R7 proteins function throughout the brain affect opiate actions. Our results suggest that R7BP is a negative modulator of the analgesic and locomotor activating actions of morphine. We also report that R7BP contributes to the development of morphine tolerance. Finally, our data suggest that although prevention of R7BP actions enhances the analgesic responses to morphine, it does not affect the severity of somatic withdrawal signs. Our data suggest that interventions in R7BP actions enhance the analgesic effect of morphine and prevent tolerance, without affecting withdrawal, pointing to R7BP complexes as potential new targets for analgesic drugs.     

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.     

Effects of single and repeated administration of methamphetamine or morphine on neuroglycan C gene expression in the rat brain

The rearrangement of neural networks associated with the behavioural sensitization and tolerance induced by psychostimulants is poorly understood. We have investigated the effects of repeated administration of methamphetamine (chronic MAP), which induces behavioural sensitization, or morphine (chronic morphine), which induces tolerance to its antinociceptive effect, on the mRNA levels of neural network-related genes in the rat brain. A gene of special interest was that for neuroglycan C (NGC), a neural tissue-specific transmembrane chondroitin sulphate proteoglycan. Single MAP (acute MAP) administration significantly decreased NGC mRNA levels in the frontal cortex, ventral tegmental area (VTA), and amygdala compared to vehicle-treated groups. Repeated MAP (chronic MAP) administration significantly increased NGC mRNA levels in the frontal cortex, nucleus accumbens (NAc), striatum, hippocampus, VTA, and amygdala compared to acute MAP treatment. Single morphine (acute morphine) administration significantly increased NGC mRNA levels in the NAc, striatum, hippocampus, VTA, and amygdala compared to vehicle-treated groups. Chronic morphine administration significantly decreased NGC mRNA levels in the NAc, striatum, VTA, and amygdala compared to acute treatment. In addition, the NGC protein level in the NAc was increased after chronic MAP and acute morphine treatment. Dopamine and opioid receptor antagonists attenuated the effect of MAP and morphine respectively on NGC mRNA levels. These results suggest that the sensitization to MAP is associated with up-regulation of NGC gene expression, while the tolerance to the morphine-induced analgesic effect is associated with the down-regulation of NGC gene expression.     

Glycosylated phosducin-like protein long regulates opioid receptor function in mouse brain

Phosducin (Phd), a protein that in retina regulates rhodopsin desensitization by controlling the activity of Gt beta gamma-dependent G-protein-coupled receptor kinases (GRKs), is present in very low levels in the CNS of mammals. However, this tissue contains proteins of related sequence and function. This paper reports the presence of N-glycosylated phosducin-like protein long (PhLP(L)) in all structures of mouse CNS, mainly in synaptic plasma membranes and associated with G beta subunits and 14-3-3 proteins. To analyze the role PhLP(L) in opioid receptor desensitization, its expression was reduced by the use of antisense oligodeoxynucleotides (ODNs). The antinociception induced by morphine, [D-Ala(2), N-MePhe(4),Gly-ol(5)]-enkephalin (DAMGO), beta-endorphin, [D-Ala(2)]deltorphin II, [D-Pen(2,5)]-enkephalin (DPDPE) or clonidine in the tail-flick test was reduced in PhLP(L)-knock-down mice. A single intracerebroventricular (icv)-ED(80) analgesic dose of morphine gave rise to acute tolerance that lasted for 4 days, but which was prevented or reversed by icv-injection of myristoylated (myr(+)) G(i2)alpha subunits. PhLP(L) knock-down brought about a myr(+)-G(i2)alpha subunit-insensitive acute tolerance to morphine that was still present after 8 days. It also diminished the specific binding of (125)I-Tyr(27)-beta-endorphin-(1-31) (human) to mouse periaqueductal gray matter membranes. After being exposed to chronic morphine treatment, post-dependent mice required about 10 days for complete recovery of morphine antinociception. The impairment of PhLP(L) extended this period beyond 17 days. It is concluded that PhLP(L) knock-down facilitates desensitization and uncoupling of opioid receptors.     

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.     

吗啡镇痛耐受大鼠中脑导水管周围灰质μ受体和δ受体协同表达下调

目的:测定吗啡镇痛耐受大鼠中脑导水管周围灰质(PAG)中μ受体和δ受体mRNA和蛋白表达的变化,以进一步探索吗啡耐受的发生机制。方法:健康成年♂Wistar大鼠32只,随机平均分为生理盐水对照组(NS组,n=16)和吗啡组(MS组,n=16)。采用盲法进行给药和疼痛行为测定。每天早、晚两次皮下注射(sc)药物并于上午注射前、后30 min测定大鼠的热甩尾潜伏期(TFL)作为痛阈指标。NS组和MS组分别sc生理盐水1 ml·kg-1和吗啡10 mg·kg-1,连续注射7 d,d8早晨各组分别取8只处死,剩余大鼠(分别记为NSN组和MSN组)d8早、晚两次sc纳洛酮0.4 mg·kg-1,并在d9处死。以TFL恢复至基础水平作为出现吗啡耐受的标准。采用免疫组织化学染色方法和实时定量PCR法分别检测PAG中μ和δ受体的蛋白表达和mRNA表达。结果:观察期两组大鼠的体重增加量在组间差异无显著性。两组大鼠每天注射前测定的TFL差异无显著性。MS组在d1 sc吗啡后TFL明显升高(P<0.05);在d2应用吗啡后,TFL升高达到最高值,且明显高于d1用药后水平(P<0.05),随后该组应用吗啡后的TFL逐渐降低,直至d7用药后TFL降至基础水平(P>0.05)。应用纳洛酮后,MSN亚组的TFL与NS组、NSN亚组以及基础值相比差异无显著性。连续应用吗啡7 d后,MS组PAG中μ和δ受体的蛋白表达和mRNA表达均较NS组显著降低(P<0.05)。结论:PAG中μ受体和δ受体共同参与了吗啡耐受机制,而且吗啡耐受大鼠PAG中μ受体和δ受体发生了协同表达下调。