Pilar Sánchez-Blázquez

Activation of I2‐imidazoline receptors enhances supraspinal morphine analgesia in mice: a model to detect agonist and antagonist activities at these receptors

This work investigates the receptor acted upon by imidazoline compounds in the modulation of morphine analgesia. The effects of highly selective imidazoline ligands on the supraspinal antinociception induced by morphine in mice were determined. Intracerebroventricular (i.c.v.) or subcutaneous (s.c.) administration of ligands selective for the I2‐imidazoline receptor, 2‐BFI, LSL 60101, LSL 61122 and aganodine, and the non selective ligand agmatine, increased morphine antinociception in a dose‐dependent manner. Neither moxonidine, a mixed I1‐imidazoline and α2‐adrenoceptor agonist, RX821002, a potent α2‐adrenoceptor antagonist that displays low affinity at I2‐imidazoline receptors, nor the selective non‐imidazoline α2‐adrenoceptor antagonist RS‐15385‐197, modified the analgesic responses to morphine. Administration of pertussis toxin (0.25 μg per mouse, i.c.v.) 6 days before the analgesic test blocked the ability of the I2‐imidazoline ligands to potentiate morphine antinociception. The increased effect of morphine induced by I2‐imidazoline ligands (agonists) was completely reversed by idazoxan and BU 224. Identical results were obtained with IBI, which alkylates I2‐imidazoline binding sites. Thus, both agonist and antagonist properties of imidazoline ligands at the I2‐imidazoline receptors were observed. Pre‐treatment (30 min) with deprenyl, an irreversible inhibitor of monoamine oxidase B (IMAO‐B), produced an increase of morphine antinociception. Clorgyline, an irreversible IMAO‐A, given 30 min before morphine did not alter the effect of the opioid. At longer intervals (24 h) a single dose of either clorgyline or deprenyl reduced the density of I2‐imidazoline receptors and prevented the I2‐mediated potentiation of morphine analgesia. These results demonstrate functional interaction between I2‐imidazoline and opioid receptors. The involvement of Gi‐Go transducer proteins in this modulatory effect is also suggested.

Agonists determine the pattern of G-protein activation in μ-opioid receptor-mediated supraspinal analgesia

The opioids heroin, methadone, buprenorphine, and morphine produce supraspinal antinociception in CD-1 mice that is antagonized by Cys(2), Tyr(3), Orn(5), Pen(7)-amide but not by naltrindole or nor-binaltorphimine. The patterns of GTP-binding regulatory proteins (G-proteins) activation exhibited by these agonists at mu-opioid receptors were characterized. The expression of alpha-subunits of Gi-protein classes, Gi1, Gi2, Gi3, Go1, Go2 and Gz, and those of the Gq-protein family, Gq and G11, was reduced by administration of antisense oligodeoxynucleotides (ODNs) complementary to sequences in their respective mRNAs. The ODN treatments demonstrated differences in the analgesic profiles of these opioids. Though the knock-down of G(i2)alpha or G(z)alpha subunits diminished the analgesic effects of the four opioids, impairment of G(i3)alpha did not modify the potency of morphine. In mice with reduced G(i1)alpha, G(o1)alpha or G(11)alpha levels, antinociception induced by heroin and methadone was diminished, but buprenorphine and morphine showed no change in their effects. Also, antinociception induced by heroin and buprenorphine, but neither morphine nor methadone, required intact G(o2)alpha or G(q)alpha levels. Thus, morphine, heroin, methadone, and buprenorphine showed different patterns of G-protein activation in evoking mu-opioid receptor-mediated supraspinal antinociception. Therefore, after binding identical receptors, each agonist determines the classes of GTP-binding regulatory transducer proteins to be activated.

RGS9 proteins facilitate acute tolerance to mu‐opioid effects

This paper reports that regulators of G‐protein signalling (RGS) proteins modulate the timing and amplitude of opioid signals by a push–pull mechanism. This is achieved without noticeable changes in the binding properties of opioids, e.g. β‐endorphin to mu‐opioid receptors. The expression of RGS proteins was reduced by blocking their mRNA with antisense oligodeoxynucleotides (ODN). Knock down of RGS2 or RGS3 diminished morphine and β‐endorphin analgesia, whereas that of RGS9 or RGS12 enhanced this activity. In mice with impaired RGS9, but not impaired RGS2, the potency and, in particular, the duration of opioid antinociception increased. Further, the animals did not exhibit acute tolerance generated by a single and efficacious dose of morphine, nor did they develop tolerance after a daily i.c.v. injection of the opioid for 4 days. In a model of sustained morphine treatment, the impairment of RGS9 proteins facilitated increases in the response to the delivered opioid. This was only effective for 2–3 h after the subcutaneous implantation of an oily morphine pellet; later, tolerance developed. To reduce the impact of the chronic morphine acting on opioid receptors, other RGS proteins presumably substitute the GTPase‐activating function of RGS9 on morphine‐activated Gα‐GTP subunits. The desensitization of mu‐opioid receptors appears to be a cell membrane‐limited process facilitated by RGS9′s sequestering of agonist‐segregated Gα subunits.

The Mu-Opioid Receptor and the NMDA Receptor Associate in PAG Neurons: Implications in Pain Control

The capacity of opioids to alleviate inflammatory pain is negatively regulated by the glutamate-binding N-methyl-D-aspartate receptor (NMDAR). Increased activity of this receptor complicates the clinical use of opioids to treat persistent neuropathic pain. Immunohistochemical and ultrastructural studies have demonstrated the coexistence of both receptors within single neurons of the CNS, including those in the mesencephalic periaqueductal gray (PAG), a region that is implicated in the opioid control of nociception. We now report that mu-opioid receptors (MOR) and NMDAR NR1 subunits associate in the postsynaptic structures of PAG neurons. Morphine disrupts this complex by protein kinase-C (PKC)-mediated phosphorylation of the NR1 C1 segment and potentiates the NMDAR–CaMKII, pathway that is implicated in morphine tolerance. Inhibition of PKC, but not PKA or GRK2, restored the MOR–NR1 association and rescued the analgesic effect of morphine as well. The administration of N-methyl-D-aspartic acid separated the MOR–NR1 complex, increased MOR Ser phosphorylation, reduced the association of the MOR with G-proteins, and diminished the antinociceptive capacity of morphine. Inhibition of PKA, but not PKC, CaMKII, or GRK2, blocked these effects and preserved morphine antinociception. Thus, the opposing activities of the MOR and NMDAR in pain control affect their relation within neurons of structures such as the PAG. This finding could be exploited in developing bifunctional drugs that would act exclusively on those NMDARs associated with MORs.

Opiates as antidepressants

The pathophysiology of mood disorders involves several genetic and social predisposing factors, as well as a dysregulated response to a chronic stressor, i.e. chronic pain. Our present view that depression involves a dysfunction of the monoaminergic system is a result of important clinical and preclinical observations over the past 40 years. In fact, current pharmacological treatment for depression is based on the use of drugs that act mainly by enhancing brain serotonin and noradrenaline neurotransmission by the blockade of the active reuptake mechanism for these neurotransmitters. However, a substantial number of patients do not respond adequately to antidepressant drugs. In view of this, there is an intense search to identify novel targets (receptors) for antidepressant therapy. Opioid peptides and their receptors are potential candidates for the development of novel antidepressant treatment. In this context, endogenous opioid peptides are co-expressed in brain areas known to play a major role in affective disorders and in the action of antidepressant drugs. The actions of endogenous opioids and opiates are mediated by three receptor subtypes (mu, delta and kappa), which are coupled to different intracellular effector systems. Also, antidepressants which increase the availability of noradrenaline and serotonin through the inhibition of the reuptake of both monoamines lead to the enhancement of the opioid pathway. Tricyclic antidepressants show an analgesic effect in neuropathic and inflammatory pain that is blocked by the opioid antagonist naloxone. A compilation of the most significant studies will illustrate the actual and potential value of the opioid system for clinical research and drug development.

Chronic pain leads to concomitant noradrenergic impairment and mood disorders

BACKGROUND Patients suffering chronic pain are at high risk of suffering long-lasting emotional disturbances characterized by persistent low mood and anxiety. We propose that this might be the result of a functional impairment in noradrenergic circuits associated with locus coeruleus (LC) and prefrontal cortex, where emotional and sensorial pain processes overlap. METHODS We used a chronic constriction injury of sciatic nerve as a model of neuropathic pain in male Sprague-Dawley rats to assess the time-dependent changes that might potentially precipitate mood disorders (2, 7, 14, and 28 days after injury). This was measured through a combination of behavioral, electrophysiological, microdialysis, immunohistochemical, and Western blot assays. RESULTS As expected, nerve injury produced an early and stable decrease in sensorial pain threshold over the testing period. By contrast, long-term neuropathic pain (28 days after injury) resulted in an inability to cope with stressful situations, provoking depressive and anxiogenic-like behaviors, even more intense than the aversiveness associated with pain perception. The onset of these behavioral changes coincided with irruption of noradrenergic dysfunction, evident as: an increase in LC bursting activity; in tyrosine hydroxylase expression and that of the noradrenaline transporter; and enhanced expression and sensitivity of α2-adrenoceptors in the LC. CONCLUSIONS Long-term neuropathic pain leads to anxio-depressive-like behaviors that are more predominant than the aversion of a painful experience. These changes are consistent with the impairment of noradrenergic system described in depressive disorders.

RGSZ1 and GAIP regulate μ- but not δ-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 (Gα interacting protein (GAIP)) and RGS20 (Z1), control the activity of opioid agonists at μ but not at δ receptors. Rz proteins show high selectivity in deactivating Gαz-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 μ receptors for the endomorphins. The activities of DPDPE ([D-Pen2,5]-enkephalin) and [D-Ala2] deltorphin II, agonists at δ receptors, were also unchanged. Knockdown of GAIP and of the GAIP interacting protein C-terminus (GIPC) led to changes in agonist effects at μ but not at δ 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 μ but not δ receptors are linked to Rz regulation. The μ receptor-mediated activation of Gz proteins is effective at recruiting the adaptive mechanisms leading to the development of opioid desensitization.

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 RGSZ2 protein exists in a complex with μ-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 GαzGTP 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 μ-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 δ 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 μ-opioid receptors. While a morphine challenge reduced the association of Gi/o/z with μ receptors, it increased their association with the RGSZ2 and RGSZ1 proteins. However, only Gαz subunits co-precipitated with RGSZ2. Doses of morphine that produced acute tolerance maintained the association of Gα subunits with RGSZ proteins even after the analgesic effects had ceased. These results indicate that both RGSZ1 and RGSZ2 proteins influence μ receptor signaling by sequestering Gα subunits, therefore behaving as effector antagonists.

The R7 Subfamily of RGS Proteins Assists Tachyphylaxis and Acute Tolerance at μ -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 β-endorphin to opioid receptors. Knockdown of R7 proteins enhanced the potency of antinociception promoted by morphine and [D-Ala2, N-MePhe4, Gly-ol5]-enkephalin (DAMGO)—both agonists at μ-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 δ agonists. Knockdown of RGS11 reduced [D-Ala2]deltorphin II analgesic effects. Those of RGS6 and RGS9-2 proteins caused [D-Ala2]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-Pen2,5]-enkephalin (DPDPE). RGS7-deficient mice showed an increased response to both [D-Ala2]deltorphin II and DPDPE analgesic effects. 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 RGS6, RGS7, RGS9-2, or RGS11 knockdown animals. Thus, R7 proteins play a critical role in agonist tachyphylaxis and acute tolerance at μ-opioid receptors, and show differences in their modulation of δ-opioid receptors.