Dopamine and endogenous cannabinoids display complex interactions in the basal ganglia. One possible level of interaction is between CB1 cannabinoid and D2 dopamine receptors. Here, we demonstrate that a regulated association of CB1 and D2 receptors profoundly alters CB1 signaling. This provides the first evidence that CB1/D2 receptor complexes exist, are dynamic, and are agonist-regulated with highest complex levels detected when both receptors are stimulated with subsaturating concentrations of agonist. The consequence of this interaction is a differential preference for signaling through a "nonpreferred" G protein. In this case, D2 receptor activation, simultaneously with CB1 receptor stimulation, results in the receptor complex coupling to G␣s protein in preference to the expected G␣i/o proteins. The result of this interaction is an increase in the second messenger cAMP, reversing an initial synergistic inhibition of adenylyl cyclase activity seen at subthreshold concentrations of cannabinoid agonist. Additionally, a pertussis toxin insensitive component in the activation of extracellular signal-regulated kinase (ERK) 1/2 kinases by the cannabinoid agonist CP 55,940 [(1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol] is revealed in cells stably expressing both CB1 and D2 receptors. Thus, concurrent receptor stimulation promotes a heterooligomeric receptor complex and an apparent shift of CB1 signaling from a pertussis toxin-sensitive inhibition to a partly pertussis toxin-insensitive stimulation of adenylyl cyclase and ERK 1/2 phosphorylation.
Microglia, as phagocytes and antigen-presenting cells in the central nervous system, are activated in such disease processes as stroke and multiple sclerosis. Because peripheral macrophages are capable of producing endocannabinoids, we have examined endocannabinoid production in a macrophage-colony stimulating factor (M-CSF)-dependent rat microglial cell line (RTMGL1) using reversed phase high-pressure liquid chromatography and liquid chromatography-mass spectroscopy. We determined that cultured microglial cells produce the endocannabinoid 2-arachidonylglycerol (2-AG) as well as anandamide in smaller quantities. When 2-AG, but not anandamide, is added exogenously, RT-MGL1 microglia increase their proliferation. This increased proliferation is blocked by an antagonist of the CB 2 receptor N-[(1S)-endo-1,3,3-trimethyl bicyclo heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) and mimicked by the CB 2 receptor-specific agonist 1,1-dimethylbutyl-1-deoxy-⌬ 9 -tetrahydrocannabinol (JWH133). Accompanying the increase in proliferation seen with 2-AG is an increase in active ERK1 that is also blocked with SR144528. The RTMGL1 microglial cells, which exist in a primed state, express the CB 1 and CB 2 receptors as demonstrated by reverse transcription-polymerase chain reaction and immunostaining. The CB 2 receptor in untreated cells is expressed both at the cell surface and internally, and exposure of the cells to 2-AG significantly increases receptor internalization. These data suggest that 2-AG activation of CB 2 receptors may contribute to the proliferative response of microglial cells, as occurs in neurodegenerative disorders.
SummaryMolecular mechanisms regulating the development of physiological and behavioral tolerance to cannabinoids are not well understood. Two cellular correlates implicated in the development and maintenance of tolerance are CB 1 cannabinoid receptor internalization and uncoupling of receptor signal transduction. Both processes have been proposed as mediators of tolerance because of observations that chronic Δ 9 -tetrahydrocannabinol (THC) treatment causes both region-specific decreases in CB 1 receptors and G-protein coupling in the brain. To determine the balance of these two processes in regulating CB 1 receptor signaling during sustained receptor stimulation, we evaluated the parameters affecting ERK1/2 MAP kinase activity in HEK293 cells stably expressing CB 1 receptors. CB 1 receptor stimulation by the potent CB 1 receptor agonist, CP 55,940 transiently activated ERK1/2. To determine if CB 1 receptor desensitization or internalization was responsible for the transient nature of ERK1/2 activation, we evaluated ERK1/2 phosphorylation in HEK293 cells expressing a desensitization-deficient CB 1 receptor (S426A/S430A CB 1 ). Here, the duration of S426A/S430A CB 1 receptor-mediated activation of ERK1/2 was markedly prolonged relative to wild-type receptors, and was dynamically reversed by SR141716A. Interestingly, the S426A/S430A CB 1 receptor was still able to recruit βarrestin-2, a key mediator of receptor desensitization, to the cell surface following agonist activation. In contrast to a central role for desensitization, pharmacological and genetic approaches suggested CB 1 receptor internalization is dispensable in the transient activation of ERK1/2. This study indicates that the duration of ERK1/2 activation by CB 1 receptors is regulated by receptor desensitization and underscores the importance of G-protein uncoupling in the regulation of CB 1 receptor signaling.
The nucleus accumbens (Acb) shell and core are essential components of neural circuitry mediating the reward and motor effects produced by activation of dopamine D2 or cannabinoid-1 (CB1) receptors. D2 receptors can form heterodimeric complexes with cannabinoid-1 (CB1) receptors and are also involved in control of the availability of both dopamine and endocannabinoids. Thus, the subcellular locations of D2 and CB1 receptors with respect to each other are implicit to their physiological actions in the Acb. We used electron microscopic immunocytochemistry to determine these locations in the Acb shell and core of rat brain. In each region, many neuronal profiles showed endomembrane and plasmalemmal distributions of one or both receptors. Approximately one-third of the labeled profiles were somata and dendrites, some of which showed overlapping subcellular distributions of D2 and CB1 immunoreactivities. The remaining labeled profiles were small axons and axon terminals containing CB1 and/or D2 receptors. Of the labeled terminals forming recognizable synapses, ~20% of those containing CB1 receptors contacted D2-labeled dendrites, while conversely, almost 15% of those containing D2 receptors contacted CB1-labeled dendrites. These results provide the first ultrastructural evidence that D2 and CB1 receptors in the Acb shell and core have subcellular distributions supporting both intracellular associations and local involvement of D2 receptors in making available endocannabinoids that are active on CB1 receptors in synaptic neurons. These distributions have direct relevance to the rewarding and euphoric as well as motor effects produced by marijuana and by addictive drugs enhancing dopamine levels in the Acb.
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