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.2013 Nov 20;19(15):1766-82.
doi: 10.1089/ars.2012.5100. Epub 2013 Jun 1.

Cannabinoid receptors couple to NMDA receptors to reduce the production of NO and the mobilization of zinc induced by glutamate

Affiliations

Cannabinoid receptors couple to NMDA receptors to reduce the production of NO and the mobilization of zinc induced by glutamate

Pilar Sánchez-Blázquez et al. Antioxid Redox Signal..

Abstract

Aims: Overactivation of glutamate N-methyl-D-aspartate receptor (NMDAR) increases the cytosolic concentrations of calcium and zinc, which significantly contributes to neural death. Since cannabinoids prevent the NMDAR-mediated increase in cytosolic calcium, we investigated whether they also control the rise of potentially toxic free zinc ions, as well as the processes implicated in this phenomenon.

Results: The cannabinoid receptors type 1 (CNR1) and NMDARs are cross-regulated in different regions of the nervous system. Cannabinoids abrogated the stimulation of the nitric oxide-zinc pathway by NMDAR, an effect that required the histidine triad nucleotide-binding protein 1 (HINT1). Conversely, NMDAR antagonism reduced the analgesia promoted by the CNR1 agonist WIN55,212-2 and impaired its capacity to internalize CNR1s. At the cell surface, CNR1s co-immunoprecipitated with the NR1 subunits of NMDARs, an association that diminished after the administration of NMDA in vivo or as a consequence of neuropathic overactivation of NMDARs, both situations in which cannabinoids do not control NMDAR activity. Under these circumstances, inhibition of protein kinase A (PKA) restored the association between CNR1s and NR1 subunits, and cannabinoids regained control over NMDAR activity. Notably, CNR1 and NR1 associated poorly in HINT1(-/-) mice, in which there was little cross-regulation between these receptors.

Innovation: The CNR1 can regulate NMDAR function when the receptor is coupled to HINT1. Thus, internalization of CNR1s drives the co-internalization of the NR1 subunits, neutralizing the overactivation of NMDARs.

Conclusion: Cannabinoids require the HINT1 protein to counteract the toxic effects of NMDAR-mediated NO production and zinc release. This study situates the HINT1 protein at the forefront of cannabinoid protection against NMDAR-mediated brain damage.

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Figures

FIG. 1.
FIG. 1.
Effect of GPCR agonists on zinc mobilization from endogenous stores. Coronal mouse frontal cortex slices from WT and HINT1−/− mice were oxygenated and preloaded for 1 h with 50 μM of the cell-permeable Newport Green diacetate, assaying the preparations after its removal. The evolution of spontaneous endogenous zinc release was determined in Control (no treatment) sections, obtaining fluorescent images at various intervals. Images were obtained by confocal microscopy through a 10×0.4 HC PL APO objective on a Leica DMIII 6000 CS confocal fluorescence microscope equipped with a TCS SP5 scanning laser (excitation, 488 nm; emission, 498–520 nm). The cortical regions studied are indicated as A, B, or C, and the data shown were obtained 30 min post-treatment (for each treatment, the images on the left are the controls obtained before adding the compounds under study). The agonists used were: cannabinoids at CNR1s WIN55,212-2, CP55,940, and methanandamide; morphine (MOR); quinpirole (D2R); apomorphine (D1R and D2R); clonidine (α2A); and DPAT (5HT1A). The CNR2 antagonist JTE907 (up to 10 μM; Tocris, 2479) did not alter the capacity of cannabinoids to promote zinc release (not shown); thus, their effects were mostly mediated through CNR1. The NMDAR antagonist MK801 was co-administered with WIN55,212-2. For each agonist, the assays on WT and HINT1−/− mice were performed during the same run. The assays were done in triplicate, and the data were analyzed to determine the means of their luminosity (grayscale of 256 channels; 0 black and 255 white) and 95% confidence intervals (Sigmaplot/Sigmastat v12). For details, see Materials and Methods. Representative images were color indexed and presented in pseudocolor. Scale bar=500 μm. CNR1, cannabinoid receptors type 1; DPAT, (±)-8-Hydroxy-2-dipropylaminotetralin hydrobromide; GPCR, G protein coupled receptor; HINT1, histidine triad nucleotide-binding protein 1; NMDAR,N-methyl-D-aspartate receptor; WT, wild type. To see this illustration in color, the reader is referred to the web version of this article atwww.liebertpub.com/ars
FIG. 2.
FIG. 2.
Cannabinoids produce HINT1-dependent hypofunction of NMDARs: their effect on NMDA-mediated nNOS/NO activation and zinc release. NMDAR-mediated production of NO and the subsequent mobilization of zinc ions. The data shown were obtained at 30 min post-treatment. Control: baseline vehicle; (A): agonist 3 μM NMDA; MK801+A: 3 μM MK801+3 μM NMDA; L-NNA+A: 10 μM L-NNA+3 μM NMDA. Influence of cannabinoids and of agonists at other GPCRs on the release of zinc promoted by NMDA. Different sections were used to analyze the effect of NMDA when administered alone or in combination with the GPCR agonists. The CNR1 cannabinoid antagonist Ly320135 (3 μM) was used to diminish the effects of WIN55,212-2 (see Fig. 1 for details). L-NNA, NG-Nitro-L-arginine; nNOS, neural nitric oxide synthase; NO, nitric oxide. To see this illustration in color, the reader is referred to the web version of this article atwww.liebertpub.com/ars
FIG. 3.
FIG. 3.
CNR1 and NMDAR associate in the nervous system and interact physicallyin vitro. (A) Solubilized cortical synaptosomes were incubated with biotinylated IgGs directed against the first extracellular loop of CNR1. After recovery with streptavidin-sepharose, the CNR1-containing complexes were processed to remove the IgGs before detecting any associated proteins (see Materials and Methods). The antibodies used to detect NR subunits were NR1, ab1880; NR2A, ab14596; NR2B, ab14400; NR3AB, ab2639. The antibody directed against the NR1 extracellular peptide sequence (Sigma-Genosys; 482–496) was used to co-immunoprecipitate CNR1. IP stands for immunoprecipitation.(B) BiFC analysis of the interaction between the CNR1 and NR1 subunits. CHO cells were transiently co-transfected with cDNAs encoding NR1VN173 and CNR1VC155 (0.3 μg), and confocal fluorescent signals were obtained when NR1VN173 and CNR1VC155 associated. For each pair of images:Left, Phase field and fluorescent images are combined;Right, Fluorescent image of cells in the field. SPR analysis of the CNR1-NR1 interaction. The CNR1 C-terminal sequence (401–473) interacts with the NR1 C-terminus containing the C1 region. Sensorgrams were constructed with CNR1 Ct in fluid phase at the concentrations indicated (μg/ml), and no signal was detected with NR1 C0-C2. NR1 C0-C1-C2 or NR1 C0-C2 C-terminus variants were incubated with the GST-CNR1 C-terminus (401–473) or GST alone (control), and (P) the GST complex was captured with glutathione sepharose and probed in western blots (WB) with an antibody against the NR1 C2 region (ab6485). The CNR1 C-terminal sequence co-immunoprecipitated with NR1 C0-C1-C2 but not with NR1 C0-C2, and GST did not bind to the NR1 C1 segment. BiFC, bimolecular fluorescence complementation; SPR, surface plasmon resonance.
FIG. 4.
FIG. 4.
The HINT1 protein binds to the CNR1 C-terminal sequence and the C1 segment of the NMDAR NR 1 subunit. (A) The HINT1 protein interacted with the CNR1 C-terminal sequence, as evident by SPR and co-precipitationin vitro.(B) Direct physical association between HINT1 and NMDAR NR1 subunit. For BiFC analysis of the interaction between CNR1/NR1 and HINT1, CHO cells were transiently co-transfected with cDNAs encoding CNR1VC155/HINT1VN173 and NR1VN173/HINT1VC155 (0.3 μg). Details as in Figure 3.
FIG. 5.
FIG. 5.
The antagonism of NMDARs impairs the capacity of Win55,212-2 to internalize CNR1s. (A) Mice received an icv dose of WIN55,212-2 (20 nmol). Groups of mice were then sacrificed at the time points indicated, and the synaptosomes obtained from the frontal cortex were processed into membrane and soluble fractions (see Materials and Methods). CNR1 was immunoprecipitated, and the membrane and internalized CNR1 and its co-immunoprecipitated proteins were assessed in western blots. */ΦSignificantly different from the control group (C membrane/C′ internalized) that did not receive the cannabinoid.(B) Mice that received icv WIN55,212-2 (20 nmol) were processed as described in(A). The membrane and internalized NR1 subunits were immunodetected directly in western blots. */ΦSignificantly different from the corresponding membrane or internalized control (0 min) that did not receive the cannabinoid.(C, D) Mice received an icv dose of WIN55,212-2 (20 nmol) along with MK801 (1 nmol) and were processed as in(A) and(B).
FIG. 6.
FIG. 6.
HINT1 protein determines the control of the NMDAR by CNR1 internalization. (A) MK801 stabilizes the association between NR subunits at the NMDAR. The NMDAR antagonist MK801 (1 nmol, icv) was administered 30 min before sacrificing the mice. The CNR1 was immunoprecipitated from brain synaptosomes, and the co-immunoprecipitation of NR1 and NR2 subunits was then assessed in western blots.(B, C) WT and HINT1−/− mice received three consecutive icv doses of WIN55,212-2 (20 nmol) spaced 90 min apart, alone or along with MK801 (1 nmol). The mice were then sacrificed 3 h after the last injection, CNR1 was immunoprecipitated from the membrane and soluble fraction of cortical synaptosomes, and the content of CNR1 and NR1 subunits was determined. *Significantly different from the corresponding control value that did not receive the cannabinoid.
FIG. 7.
FIG. 7.
Antagonism of WIN55,212-2-induced supraspinal analgesia by MK801: the influence of the HINT1 protein. (A) The effects of various icv doses of WIN55,212-2 were studied in mice lacking the HINT1 protein and in their WT littermates. Each point is the mean±SEM from groups of at least six mice. Both WT and HINT1-deficient mice showed comparable responses to increasing doses of WIN55,212-2. The NMDAR antagonist, MK801 (1 nmol), was icv-injected to the mice 30 min before WIN55,212-2. *Significantly different from the group that received saline instead of MK801:p<0.05.(B)Left panel, Cortical cell cultures from wild-type and HINT1−/− mice were exposed to increasing concentrations of NMDA for 24 h. Cell death was measured by LDH efflux into the medium. The data shown are the mean±SEM from 20 wells per group. *Significant difference between wild-type and HINT1−/− cultured neurons,p<0.05.Right panel, cultures were exposed to a fixed concentration of 50 μM NMDA for 24 h in the presence or absence (–) of increasing concentrations of the cannabinoid agonist WIN55,212-2. To rule out the possible participation of CNR2, the assay was conducted in the presence of 3 μM of the CNR2 antagonist JTE907. *Significant difference with regard to NMDA alone,p<0.05. (1–4) Fluorescence photomicrographs of cortical cell cultures immunolabeled with an anti-MAP2ab. (1) WT: 50 μM NMDA; (2) WT: 50 μM NMDA plus 100 nM WIN55,212-2; (3) HINT1−/−: 50 μM NMDA; and (4) HINT1−/−; 50 μM NMDA plus 100 nM WIN55,212-2.(C) NMDA-induced changes in [Ca2+]i concentration within the cells. Bar graph indicates the mean+S.E.M fluorescence ratio (494/523 nm) averaged across 10–15 cells per condition. HINT1+/+ and HINT1−/− cultures were exposed during 2 min perfusion to NMDA (15 μM) alone or in combination with MK801 (10 μM) and WIN55,212-2 (100 nM). Cells were incubated in artificial cerebrospinal fluid (ACSF). *Significant difference relative to NMDA, Φbetween wild-type and HINT1−/− cultured neurons,p<0.01.(D) The absence of HINT1 reduces thein vivo association between the CNR1 and NR1 subunits. The CNR1 was immunoprecipitated from solubilized cortical synaptosomes or cortical cell cultures obtained from WT and HINT1−/− mice, and the CNR1 and co-immunoprecipitated NR1 was assessed. The NR1 content is shown.
FIG. 8.
FIG. 8.
PKC, PKA, and CaMKII regulate WIN55,212-2 analgesia and the CNR1-NR1 association. (A) Effect of PKC and CaMKII inhibition on WIN55,212-2 analgesia. Mice received the PKC inhibitor Gö7874 (1 nmol) or the CaMKII inhibitor KN93 (7 nmol) icv, 30 min before the cannabinoid agonist (6 nmol). In one set of assays, MK801 (1 nmol) was given 10 min after the kinase inhibitors; then, WIN55,212-2 was injected icv 20 min after the NMDAR antagonist, and the time course of analgesia was determined. *Significantly different from the group that received WIN55,212-2 and saline instead of the kinase inhibitors (left panel); or from the groups not receiving MK801 (right panel),p<0.05. The efficacy of the doses and intervals used were previously determined for NMDA, MK801, and kinase inhibitors (59).(B) Association of CNR1 with the NMDAR. Kinase inhibitors were icv injected into the animals 30 min before sacrifice (1 nmol Gö7874; 5 nmol PKA inhibitor; and 7 nmol KN93); CNR1 was immunoprecipitated from solubilized synaptosomal cortical membranes, and its association with NR1 subunits was determined in western blots. *Significantly different from the control group (C) that received saline instead of the kinase inhibitors,p<0.05. The effect of inhibiting PKC, PKA, or CaMKII on MK801; the impairment of WIN55,212-2-induced co-internalization of CNR1 and NR1 subunits. Kinase inhibitors were icv-injected 30 min after MK801 (1 nmol) and 30 min before WIN55,212-2. The mice were sacrificed 30 min later, and the presence of internalized CN1R and NR1 subunits was determined. *Significantly different from the control group (C) that received WIN55,212-2 and MK801 but saline instead of the kinase inhibitors,p<0.05. CaMKII, Ca2+/calmodulin-dependent protein kinase II; PKA, protein kinase A; PKC, protein kinase C.
FIG. 9.
FIG. 9.
Plasticity of the CNR1-NR1 association. (A) Influence of chronic constriction injury (CCI) and direct NMDA administration to mice on NR1 co-immunoprecipitation with CNR1s. Mice suffering from CCI were sacrificed on day 7, and the CNR1-NR1 association was determined in membranes isolated from cerebral cortex synaptosomes. A group of mice also received 5 nmol of the PKA inhibitor 6–22 amide 30 min before they were sacrificed. A similar study was performed after icv injecting NMDA (15 pmol per mouse) and assessing the CNR1-NR1 association in synaptosomes 24 h later. The presence of CaMKII and of its P-Thr286 form is shown. For each assay (column), the cortical structures from six mice were pooled for theex vivo determinations. C and IP stand for control mice and immunoprecipitation, respectively. *Significantly different from the Sham-operated control group or mice not receiving NMDA (C),p<0.05.(B) The sequence of WIN55,212-2 and NMDA icv injection influences the internalization of CNR1 and NR1 subunits. The mice received 20 nmol WIN55,212-2 and 15 pmol NMDA using the protocols indicated The mice were sacrificed 90 min after NMDA or 30 after WIN55,212-2, and the levels of CNR1 and NR1 subunits were determined. *Significantly different from the indicated control group,p<0.05. PKA model: NMDAR activates PKA: Calcium fluxes recruit Calmodulin (CaM) to form Ca2+-CaM, which is necessary to regulate CaMKII (1) and activate adenylyl cyclase types I and VIII. (2) Increased cAMP production activates PKA, which, in turn, inhibits protein phosphatase 1 (PP1: 3) that opposes CaMKII activity. (4) CaMKII consolidates its autophosphorylation on Thr286, which is now being activated independently of Ca2+-CaM levels and ready to regulate CNR1 activity or to remove RGSZ2.Gαi/oGTP from HINT1 proteins. Moreover, when PKA acts on certain serines in the NR1 C1, the regulatory segment promotes the separation of CNR1s from NMDARs. Inset: Diagram of the mechanism proposed for WIN55,212-2 induced NMDAR hypofunction. CNR1 and the NMDAR associate in the postsynapse, with PKC and CaMKII enhancing this association, and PKA diminishing it. (1) The agonist binds to CNR1 and (2) promotes co-internalization of CNR1 and NR1 subunits. These proteins separate in the cytosol, and (3) CNR1 returns to the plasma membrane. (4) The re-sensitized CNR1 re-associates with new NR1 subunits, and (5) the cycle starts again, while the agonist continues in the receptor environment. RGSZ2, regulator of G protein signaling 17 (Z2).
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