Rivka Levy

Anandamide, a brain endogenous compound, interacts specifically with cannabinoid receptors and inhibits adenylate cyclase.

Abstract: A putative endogenous cannabinoid ligand, arachidonylethanolamide (termed “anandamide”), was isolated recently from porcine brain. Here we demonstrate that this compound is a specific cannabinoid agonist and exerts its action directly via the cannabinoid receptors. Anandamide specifically binds to membranes from cells transiently (COS) or stably (Chinese hamster ovary) transfected with an expression plasmid carrying the cannabinoid receptor DNA but not to membranes from control non‐transfected cells. Moreover, anandamide inhibited the forskolin‐stimulated adenylate cyclase in the transfected cells and in cells that naturally express cannabinoid receptors (N18TG2 neuroblastoma) but not in control nontransfected cells. As with exogenous cannabinoids, the inhibition by anandamide of the forskolin‐stimulated adenylate cyclase was blocked by treatment with pertussis toxin. These data indicate that anandamide is an endogenous agonist that may serve as a genuine neurotransmitter for the cannabinoid receptor.

The peripheral cannabinoid receptor: adenylate cyclase inhibition and G protein coupling

Two cannabinoid receptors, designated neuronal (or CB1) and peripheral (or CB2), have recently been cloned. Activation of CB1 receptors leads to inhibition of adenylate cyclase and N‐type voltage‐dependent Ca2+ channels. Here we show, using a CB2 transfected Chinese hamster ovary cell line, that this receptor binds a variety of tricyclic cannabinoid ligands as well as the endogenous ligand anandamide. Activation of the CB2 receptor by various tricyclic cannabinoids inhibits adenylate cyclase activity and this inhibition is pertussis toxin sensitive indicating that this receptor is coupled to the Gi/G0 GTP‐binding proteins. Interestingly, contrary to results with CB1, anandamide did not inhibit the CB2 coupled adenylate cyclase activity and δ 9‐tetrahydrocannabinol had only marginal effects. These results characterize the CB2 receptor as a functional and distinctive member of the cannabinoid receptor family.

Cannabinoid receptor activation differentially regulates the various adenylyl cyclase isozymes.

Abstract: Two cannabinoid receptors belonging to the superfamily of G protein‐coupled membrane receptors have been identified and cloned: the neuronal cannabinoid receptor (CB1) and the peripheral cannabinoid receptor (CB2). They have been shown to couple directly to the Gi/o subclass of G proteins and to mediate inhibition of adenylyl cyclase upon binding of a cannabinoid agonist. In several cases, however, cannabinoids have been reported to stimulate adenylyl cyclase activity, although the mechanism by which they did so was unclear. With the cloning of nine adenylyl cyclase isozymes with various properties, including different sensitivities to αs, αi/o, and βγ subunits, it became important to assess the signaling pattern mediated by each cannabinoid receptor via the different adenylyl cyclase isozymes. In this work, we present the results of cotransfection experiments between the two types of cannabinoid receptors and the nine adenylyl cyclase isoforms. We found that independently of the method used to stimulate specific adenylyl cyclase isozymes (e.g., ionomycin, forskolin, constitutively active αs, thyroid‐stimulating hormone receptor activation), activation of the cannabinoid receptors CB1 and CB2 inhibited the activity of adenylyl cyclase types I, V, VI, and VIII, whereas types II, IV, and VII were stimulated by cannabinoid receptor activation. The inhibition of adenylyl cyclase type III by cannabinoids was observed only when forskolin was used as stimulant. The activity of adenylyl cyclase type IX was inhibited only marginally by cannabinoids.

Electron microscopic study on reassembly of plasma high density apoprotein with various lipids.

Products resulting from the sonification of mixtures of plasma high density lipoprotein apoprotein and specific lipids were studied by electron microscopy using negative staining. Sonicates of apoprotein plus lecithin produced disc-shaped structures which stacked in aggregates with a 50–55-A repeat; the discs were 100–200 A in diameter. Incorporation of unesterified cholesterol into the mixture produced structures morphologically similar to those observed in sonicates of apoprotein plus lecithin. Disc-shaped particles from sonified mixtures of apoprotein, lecithin and unesterified cholesterol were ultracentrifugally isolated in the d 1.063–1.21 g/ml fraction and were incubated with a plasma d > 1.21 g/ml fraction containing lecithin: cholesterol acyltransferase activity. Electron microscopy following the incubation procedure showed a transformation of the disc-like structures into approximately spherical particles (50–100 A diameter). Similar spherical particles were also obtained after sonification of apoprotein-lecithin-unesterified cholesterol-cholesteryl ester mixtures. Results indicate a requirement for the presence of cholesteryl esters to maintain normal morphology of plasma high density lipoproteins.

Cannabinoids Δ9-Tetrahydrocannabinol and Cannabidiol Differentially Inhibit the Lipopolysaccharide-activated NF-κB and Interferon-β/STAT Proinflammatory Pathways in BV-2 Microglial Cells

Cannabinoids have been shown to exert anti-inflammatory activities in various in vivo and in vitro experimental models as well as ameliorate various inflammatory degenerative diseases. However, the mechanisms of these effects are not completely understood. Using the BV-2 mouse microglial cell line and lipopolysaccharide (LPS) to induce an inflammatory response, we studied the signaling pathways engaged in the anti-inflammatory effects of cannabinoids as well as their influence on the expression of several genes known to be involved in inflammation. We found that the two major cannabinoids present in marijuana, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), decrease the production and release of proinflammatory cytokines, including interleukin-1β, interleukin-6, and interferon (IFN)β, from LPS-activated microglial cells. The cannabinoid anti-inflammatory action does not seem to involve the CB1 and CB2 cannabinoid receptors or the abn-CBD-sensitive receptors. In addition, we found that THC and CBD act through different, although partially overlapping, mechanisms. CBD, but not THC, reduces the activity of the NF-κB pathway, a primary pathway regulating the expression of proinflammatory genes. Moreover, CBD, but not THC, up-regulates the activation of the STAT3 transcription factor, an element of homeostatic mechanism(s) inducing anti-inflammatory events. Following CBD treatment, but less so with THC, we observed a decreased level of mRNA for the Socs3 gene, a main negative regulator of STATs and particularly of STAT3. However, both CBD and THC decreased the activation of the LPS-induced STAT1 transcription factor, a key player in IFNβ-dependent proinflammatory processes. In summary, our observations show that CBD and THC vary in their effects on the anti-inflammatory pathways, including the NF-κB and IFNβ-dependent pathways.

Differential Modulation of Adenylyl Cyclases I and II by Various Gβ Subunits

The accepted dogma concerning the regulation of adenylyl cyclase (AC) activity by Gβγ dimers states that the various isoforms of AC respond differently to the presence of free Gβγ. It has been demonstrated that AC I activity is inhibited and AC II activity is stimulated by Gβγ subunits. This result does not address the possible differences in modulation that may exist among the different Gβγ heterodimers. Six isoforms of Gβ and 12 isoforms of Gγ have been cloned to date. We have established a cell transfection system in which Gβ and Gγ cDNAs were cotransfected with either AC isoform I or II and the activity of these isoforms was determined. We found that while AC I activity was inhibited by both Gβ1/γ2 and Gβ5/γ2 combinations, AC II responded differentially and was stimulated by Gβ1/γ2 and inhibited by Gβ5/γ2. This finding demonstrates differential modulatory activity by different combinations of Gβγ on the same AC isoform and demonstrates another level of complexity within the AC signaling system.

Inhibition of adenylyl cyclase isoforms V and VI by various Gβγ subunits

An intriguing development in the G‐protein signaling field has been the finding that not only the Gα subunit, but also Gβγ subunits, affect a number of downstream target molecules. One of the downstream targets of Gβγ is adenylyl cyclase, and it has been demonstrated that a number of isoforms of adenylyl cyclase can be either inhibited or stimulated by Gβγ subunits. Until now, adenylyl cyclase type I has been the only isoform reported to be inhibited by free Gβγ. Here we show by transient cotransfection into COS‐7 cells of either adenylyl cyclase V or VI, together with Gγ2 and various Gβ subunits, that these two adenylyl cyclase isozymes are markedly inhibited by Gβγ. In addition, we show that Gβ1 and Gβ5 subunits differ in their activity. Gβ1 transfected alone markedly inhibited adenylyl cylcase V and VI (probably by recruiting endogenous Gγ subunits). On the other hand, Gβ5 produced less inhibition of these isozymes, and its activity was enhanced by the addition of Gγ2. These results demonstrate that adenylyl cyclase types V and VI are inhibited by Gβγ dimers and that Gβ1 and Gβ5 subunits differ in their capacity to regulate these adenylyl cyclase isozymes.—Bayewitch, M. L., Avidor‐Reiss, T., Levy, R., Pfeuffer, T., Nevo, I., Simonds, W. F., Vogel, Z. Inhibition of adenylyl cyclase isoforms V and VI by various Gβγ subunits. FASEB J. 12, 1019–1025 (1998)

Biophysical constraints of optogenetic inhibition at presynaptic terminals

We investigated the efficacy of optogenetic inhibition at presynaptic terminals using halorhodopsin, archaerhodopsin and chloride-conducting channelrhodopsins. Precisely timed activation of both archaerhodopsin and halorhodpsin at presynaptic terminals attenuated evoked release. However, sustained archaerhodopsin activation was paradoxically associated with increased spontaneous release. Activation of chloride-conducting channelrhodopsins triggered neurotransmitter release upon light onset. Thus, the biophysical properties of presynaptic terminals dictate unique boundary conditions for optogenetic manipulation.

Cannabidiol inhibits pathogenic T cells, decreases spinal microglial activation and ameliorates multiple sclerosis-like disease in C57BL/6 mice

BACKGROUND AND PURPOSE Cannabis extracts and several cannabinoids have been shown to exert broad anti‐inflammatory activities in experimental models of inflammatory CNS degenerative diseases. Clinical use of many cannabinoids is limited by their psychotropic effects. However, phytocannabinoids like cannabidiol (CBD), devoid of psychoactive activity, are, potentially, safe and effective alternatives for alleviating neuroinflammation and neurodegeneration.

Differential changes in GPR55 during microglial cell activation

We examined how lipopolysaccharide (LPS) and interferon gamma (IFN‐γ), known to differentially activate microglia, affect the expression of G protein‐coupled receptor 55 (GPR55), a novel cannabinoid receptor. We found that GPR55 mRNA is significantly expressed in both primary mouse microglia and the BV‐2 mouse microglial cell line, and that LPS down‐regulates this message. Conversely, IFN‐γ slightly decreases GPR55 mRNA in primary microglia, while it upregulates this message in BV‐2 cells. Moreover, the GPR55 agonist, lysophosphatidylinositol, increases ERK phosphorylation in BV‐2 stimulated with IFN‐γ, in correlation with the increased amount of GPR55 mRNA. Remarkably, these stimuli‐induced changes in GPR55 expression are similar to those observed with CB2‐R, suggesting that both receptors might be involved in neuroinflammation and that their expression is concomitantly controlled by the state of microglial activation.