Regulation of the cAMP cascade, gene expression and immune function by cannabinoid receptors
Introduction
Historically, the mechanism by which cannabinoids produce their broad array of physiological effects has been attributed to nonspecific intercalation of these lipophilic compounds into the lipid bilayer of the cell membrane resulting in the disruption of membrane processes. Although the specific mechanism(s) of action for cannabinoids still remains to be fully elucidated, a number of distinct lines of evidence have supported the involvement of receptors. Many of the early observations were made by using the central nervous system (CNS) and CNS-derived cell-lines as models. One of the most compelling is the general lack of correlation between the degree of lipophilicity of specific cannabinoid congeners and their biologic activity (Thomas et al., 1990). Moreover, early studies demonstrated negative regulation of adenylate cyclase following exposure to cannabinoids, an enzyme almost exclusively associated in mammalian systems with membrane bound receptors (Howlett, 1985; Howlett and Fleming, 1984). The above also correlates well with binding studies which demonstrated specific and saturable binding by cannabinoids in brain synaptosome preparations (Harris et al., 1978). Lastly, a novel guanine-nucleotide-binding protein (G-protein) coupled receptor was cloned from a rat brain cDNA library which exhibited stereospecific cannabinoid binding and negative regulation of adenylate cyclase when transfected into chinese hamster ovary cells (CHO) cells in the presence of cannabinoid compounds (Matsuda et al., 1990). Since the identification of a cannabinoid receptor in neuronal tissues (cannabinoid receptor type 1 (CB1)), a second major form of the receptor (cannabinoid receptor type 2 (CB2)) has been isolated and cloned from the promyelocytic line HL60 (Munro et al., 1993). The two receptors share approximately 68% identity within their transmembrane regions, that portion of the receptor believed to possess the ligand binding domain (Munro et al., 1993). In spite of the marked differences in identity, most cannabinoid compounds bind with similar affinity to both CB1 and CB2. One exception to this rule is the plant derived cannabinoid, cannabinol, which possesses significantly greater binding affinity for CB2 than for CB1 (Munro et al., 1993); exhibits good binding affinity to mouse spleen cells (Schatz et al., 1997); and has immunomodulatory activity in a number of leukocyte preparations (Condie et al., 1996).
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CB1 and CB2 receptor distribution
Shortly following the identification of CB1 in rat brain, CB1 was also identified in human brain (Gerard et al., 1990) and human testis (Gerard et al., 1991) and found to be highly conserved between these two as well as other species. The first identification of cannabinoid receptors within the immune system was in mouse spleen cells (Kaminski et al., 1992). This cellular preparation exhibited: (a) saturable specific binding of the high affinity cannabinoid receptor radioligand, [3H]-CP-55940
Regulation of adenylate cyclase by cannabinoid compounds
As initially suggested by Howlett et al. (1986)from studies in neuronal cell lines prior to the identification of cannabinoid receptors, both CB1 and CB2 negatively regulate adenylate cyclase activity through a pertussis toxin sensitive GTP-binding protein (Kaminski et al., 1992). Modulation of adenylate cyclase by cannabinoid compounds has been compellingly demonstrated in virtually every tissue and cell-line shown to express functional cannabinoid receptors as well as in cell-lines devoid of
Does the inhibition of adenylate cyclase by cannabinoids have any relevance to immune function?
As discussed above, one of the earliest signalling events initiated by ligand binding to cannabinoid receptors is the inhibition of adenylate cyclase which leads to a decrease in the production and accumulation of intracellular cyclic adenosine 3′:5′-monophosphate (cAMP). That this, in turn, leads to an inhibition of immune function is contrary to a long held immunologic axiom, that the primary role of the cAMP signalling cascade in immune function is to serve as a negative regulatory pathway.
The role of the cAMP signalling cascade in T-helper cell function
In the remainder of this chapter, the discussion will primarily focus on the role of the cAMP signalling cascade in cannabinoid-mediated disruption of two immunological responses: (a) interleukin-2 (IL-2) expression by T-cells; and (b) inducible nitric oxide synthase (iNOS) expression by macrophages. We will first focus on the disregulation of interleukin-2 (IL-2) by cannabinoids in helper T-cells.
Recently, studies have focused on the effects exerted by cannabinoids, through an inhibition of
The role of the cAMP signaling cascade in the regulation of iNOS in macrophages
Recently, it has been demonstrated that cannabinoids inhibit the induction of iNOS gene expression in LPS-stimulated macrophages (Jeon et al., 1996). The iNOS catalyzes the production of large amounts of NO from l-arginine and molecular oxygen (Palmer et al., 1988). Expression of iNOS is rapidly induced in macrophages by LPS which is a major constituent of gram negative bacterial cell membranes. The production of NO in turn contributes to the cytolytic function of macrophages and is believed to
Conclusions
Cannabinoids are well established as being immune modulators. Elucidation of the specific mechanisms responsible from this biologic effect has a number of significant implications. First, from a basic science standpoint, cannabinoids are a very useful class of pharmacologic probes that can be utilized to characterize the signaling events within the cAMP cascade and its role in a variety of biological processes. As discussed above, much has already been gleaned from the use of cannabinoids as
Acknowledgements
This work was supported by funds from NIDA Grants DA07908 and DA09789.
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