A diagram summarizing the proposed connection between MOR and NMDAR and the role of the Akt-nNOS pathway in cross-regulation.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011278#s4" target="_blank">Discussion</a> for details.</p

Control of Akt-nNOS pathway by the action of morphine on MORs.

<p>10 nmol morphine was icv-injected into CD1 mice. At various intervals post-injection, the mice were sacrificed and the PAG structures were collected. The presence of Akt and nNOS in the PAG synaptosomal membrane was determined. Also, the association of these proteins with the MOR was evaluated. At each time point studied, the PAG structures from four to six mice were pooled. MOR proteins were immunoprecipitated and the co-precipitated Akt and nNOS were evaluated. Immunosignals (average optical density of the pixels within the object area/mm²; Quantity One Software, BioRad) were expressed as the change relative to the control animals (attributed an arbitrary value of 1). Each data point represents the mean of three assays performed on PAG samples obtained from independent groups of mice. Data are presented as the area below the curve (Sigmaplot v11), the upper curve indicates the +SEM. For comparison, we have superimposed the control levels of the pair of curves being compared. <i>Upper panel:</i> The effect of morphine on the presence of Akt in the synaptosomal PAG membrane and the MOR environment. The presence of activating Thr308 and Ser473 phosphorylations in the membrane-associated Akt. Insets: Representative western blots of these data. An absence of phosphorylation in Akt recruited to the MOR was shown but not plotted. MOR-rcted stands for MOR-recruited. <i>Lower panel</i>: A similar study of nNOS was carried out. Enzyme levels associated with PAG synaptosomal membranes and MORs were determined. The presence of Akt-activating Ser1417 phosphorylation and CaMKII-inactivating Ser847 phosphorylation were also determined in membrane-associated nNOS. Insets: Representative nNOS western blots are shown. nNOS that was recruited to the MOR environment lacked Ser1417 and Ser847 phosphorylation.</p

The Influence of the Akt-nNOS pathway on morphine-induced NMDAR potentiation.

<p><i>Upper panel</i>: The influence of 10 nmol icv morphine on NMDAR activity was evaluated. The levels of NMDAR1 and of NMDAR2A subunits and activating phosphorylations at NR1 Ser890 (PKC site) and NR2A Tyr1325 (Src site) were monitored in the CD1 PAG synaptosomal membranes during the post-morphine intervals indicated. The presence of the Thr286 autophosphorylation of NMDAR-activated CaMKII was also determined. Administration of LNNA or naltrindole before morphine greatly reduced NR1, NR2A and CaMKII phosphorylation. <i>Lower panel</i>: Naltrindole decreased Akt/PKB activating phosphorylation. Please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011278#s2" target="_blank">Materials and Methods</a> for further details.</p

The Influence of Akt and nNOS in the production of supraspinal analgesia and changes in MOR-associated transduction as a result of morphine exposure.

<p><i>Upper panel</i>: CD1 mice were icv-injected with increasing doses of morphine and antinociception was monitored with the warm water (52°C) tail-flick test. Data correspond to the peak-effect interval at 30 min post-injection and are presented as dose-response curves. Individual values represent the mean ± SEM from a group of eight mice. The development of morphine-induced single-dose tolerance and its pharmacological rescue were studied 24 h after receiving a priming dose of 3 nmol or 10 nmol morphine. The analgesic effect of increasing test doses of morphine was then evaluated at 30 min post-injection. The nNOS inhibitor, LNNA (7 nmol), and the mixed Delta-opioid receptor antagonist/Akt inhibitor, naltrindole (1 nmol), were icv-injected prior to the 10 nmol morphine priming dose. LNNA was injected 30 min before the priming dose, whereas naltrindole was administered 90 min before the priming dose. <i>Lower panel</i>: LNNA and naltrindole were administered to the CD1 mice prior to the 10 nmol morphine priming dose. Mice were sacrificed at the indicated time points. For each time point studied, PAG structures from four to six mice were pooled. The MOR was immunoprecipitated and serine phosphorylation was evaluated (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011278#s2" target="_blank">Materials and Methods</a>). Co-precipitation of Gαi2 subunits with MORs and RGSZ2 proteins was also evaluated. Further data and details are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011278#pone-0011278-g001" target="_blank">Fig. 1</a>.</p

Chronology of morphine-triggered changes in MOR-regulated signaling proteins.

<p>10 nmol morphine was icv-injected into the CD1 mice at time 0. The data, the mean of three assays performed on PAG samples obtained from independent groups of mice, are presented as the area below the curve (Sigmaplot v11) and the upper curve indicates +SEM. In instances where a pair of curves is plotted together, the control levels have been superimposed. Details are provided in the legends for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011278#pone-0011278-g001" target="_blank">Figs 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011278#pone-0011278-g003" target="_blank">3</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011278#pone-0011278-g004" target="_blank">4</a>.</p

HINT1 protein binds to a series of signaling proteins at the C terminus.

<p>PAG synaptosomal membranes prepared from HINT1 (−/−) 129 SvJ mice were used to immunoprecipitate MORs and the association of the signaling proteins with MORs was studied. Signals corresponding to the MOR in HINT1 (−/−) and (+/+) mice were paired and co-precipitated proteins were directly compared. These assays were repeated twice on samples obtained from different mice. The results were consistent across replicates.</p

Morphine induces endocytosis of neuronal μ-opioid receptors through the sustained transfer of Gα subunits to RGSZ2 proteins-4

<p><b>Copyright information:</b></p><p>Taken from "Morphine induces endocytosis of neuronal μ-opioid receptors through the sustained transfer of Gα subunits to RGSZ2 proteins"</p><p>http://www.molecularpain.com/content/3/1/19</p><p>Molecular Pain 2007;3():19-19.</p><p>Published online 17 Jul 2007</p><p>PMCID:PMC1947952.</p><p></p>olerance was monitored at various intervals post-opioid administration by measuring the analgesia produced by the release of the opioid. Groups of 10 mice were sacrificed at different intervals and the dorsal horns of the cervical-dorsal spinal cords were removed. To analyze the phosphorylation and presence of MORs in the plasma membrane, the immunoprecipitation was performed under denaturing conditions. For every post-opioid interval analyzed, densitometric signals associated with 55–65, 70–75, and 90–100 kDa were pooled and normalized to those obtained probing the anti-MOR IgGs (heavy chain). The assay was repeated twice and the results were comparable. Further details as in Fig. 1

Morphine induces endocytosis of neuronal μ-opioid receptors through the sustained transfer of Gα subunits to RGSZ2 proteins-6

<p><b>Copyright information:</b></p><p>Taken from "Morphine induces endocytosis of neuronal μ-opioid receptors through the sustained transfer of Gα subunits to RGSZ2 proteins"</p><p>http://www.molecularpain.com/content/3/1/19</p><p>Molecular Pain 2007;3():19-19.</p><p>Published online 17 Jul 2007</p><p>PMCID:PMC1947952.</p><p></p>00 pmol DAMGO. Groups of mice that had received the same opioid treatment were sacrificed at various intervals post-opioid administration. The control mice received icv saline instead of the opioid treatment. The PAG synaptosomes (P2) and supernatant (S3) were obtained and the variations in the surface and internalized MORs (representative data in Figs 1-3), and in the association of surface MORs with Gαi2 subunits, was analyzed (see data in Fig. 6). The densitometric signals corresponding to MORs and the associated Gαi2 subunits that were observed in PAG from control mice injected with saline alone were attributed an arbitrary value of 1. The MOR and Gαi2 values corresponding to mice killed at the post-opioid intervals studied were then normalized to the levels observed for the controls. After normalization of the data, the levels of surface MORs observed at the different post-opioid intervals were correlated with the co-precipitation with Gαi2 subunits, as well as with the levels of internalized MORs. Regression lines, regression coefficients and their confidence intervals of 95% are shown (Sigmaplot v10/Sigmastat v 3.5). The data were pooled from two independent assays

Morphine induces endocytosis of neuronal μ-opioid receptors through the sustained transfer of Gα subunits to RGSZ2 proteins-0

<p><b>Copyright information:</b></p><p>Taken from "Morphine induces endocytosis of neuronal μ-opioid receptors through the sustained transfer of Gα subunits to RGSZ2 proteins"</p><p>http://www.molecularpain.com/content/3/1/19</p><p>Molecular Pain 2007;3():19-19.</p><p>Published online 17 Jul 2007</p><p>PMCID:PMC1947952.</p><p></p>ck test at various time intervals post-injection. Antinociception was expressed as a percentage of the maximum possible effect after setting a cut-off time of 10 seconds. The values shown are the mean ± SEM from groups of 10–15 mice. Effect of morphine treatment on internalization and phosphorylation of the C terminal Ser375 of MOR. The PAG synaptosomes (P2) and supernatant (S3) were obtained at various intervals post-morphine administration. For each time point studied the PAG structures from 6 to 8 mice were pooled. To reduce the risk of interference with signals from proteins other than the MORs, the study of these receptors and their Ser375 phosphorylation was performed by immunoprecipitation after releasing the associated proteins by SDS solubilization (denaturing conditions, see Methods). In order to detect additional protein bands, the areas inside the rectangles were overexposed. The densitometric immunosignals associated with the 55–65 kDa band (average optical density of the pixels within the object area/mm2; Quantity One Software, BioRad) were normalized to those obtained probing the anti-MOR IgGs hc (heavy chain) with the appropriate secondary antibody. These IgGs were detached from the immunoprecipitated MORs and processed in parallel gels/blots (see Methods). Each bar is the mean ± SEM of three assays performed on PAG samples obtained from independent groups of mice. The data are expressed relative to the levels observed for the control group (attributed an arbitrary value of 1)

Morphine induces endocytosis of neuronal μ-opioid receptors through the sustained transfer of Gα subunits to RGSZ2 proteins-8

<p><b>Copyright information:</b></p><p>Taken from "Morphine induces endocytosis of neuronal μ-opioid receptors through the sustained transfer of Gα subunits to RGSZ2 proteins"</p><p>http://www.molecularpain.com/content/3/1/19</p><p>Molecular Pain 2007;3():19-19.</p><p>Published online 17 Jul 2007</p><p>PMCID:PMC1947952.</p><p></p>uated by icv-injection of a second and identical dose of this opioid at different time intervals. , the animals received several icv-injections of 10 nmol morphine spaced 6 h, 24 h, 48 h and 72 h from the first, and antinociception was determined in the tail-flick test at its peak effect 30 min after each injection. Values shown are the mean ± SEM from groups of 8–12 mice. *Statistically significant with respect to the control (First dose) group; ANOVA, Student-Newman-Keuls test (SigmaStat, SPSS Science Software, Erkrath, Germany). Significance was set at < 0.05. The internalization and Ser375 phosphorylation of the MORs was studied after administration of a second dose of 10 nmol morphine 6 h after the first. For further details see Fig. 1 and Results