Effects on the Immune System 387cannabinoid receptor—and which impacted studies on effects of cannabinoids on
immunity—was conducted by Howlett and coworkers. Using a pharmacological
approach, it was demonstrated that THC,∆^8 -tetrahydrocannabinol, levonantradol,
and desacetyllevonantradol inhibited adenylate cyclase activity in plasma mem-
branes of neuroblastoma cells (Howlett and Fleming 1984). This inhibition was
not blocked by atropine, yohimbine, or naloxone, suggesting that muscarinic,α-2-
adrenergic and opiate receptors were not required for the response. Furthermore,
the inhibition of adenylate cyclase appeared to be specific for cannabinoids that
were psychoactive, since CBD and CBN produced minimal effects. In addition, the
inhibition of adenylate cyclase activity was stereoselective, since dextronantradol
as compared to its stereoisomer levonantradol did not produce the response. Fi-
nally, inhibition was observed as concentration-dependent over a nanomolar range
for both THC and its synthetic analog, desacetyllevonantradol. In a subsequent
set of experiments, it was shown that the inhibitory effect of these psychoactive
compounds was related to the ability of adenylate cyclase to be regulated by di-
valent cations and guanine nucleotides (Howlett 1985). Howlett et al. (1986) also
demonstrated that pertussis toxin treatment abolished the cannabimimetic inhi-
bition of adenylate cyclase activity, but only for intact neuroblastoma cells, neu-
roblastoma/glioma hybrid cells, or their derivative membranes. These results were
consistent with the existence of a “cannabinoid” receptor, since receptor-mediated
inhibition of adenylate cyclase requires the presence of a guanine nucleotide-
binding protein complex, Gi, which can be functionally inactivated as a result of
an adenosine diphosphate (ADP)-ribosylation modification catalyzed by pertussis
toxin. Devane et al. (1988) used a tritium-labeled biologically active synthetic bi-
cyclic cannabinoid (CP 55,940) to identify and characterize specific ligand binding
in rat brain. Collectively, the data fulfilled the criteria for the existence of a high-
affinity and stereoselective, pharmacologically distinct cannabinoid receptor in
brain tissue.
Matsuda and colleagues (Matsuda et al. 1990) reported on the cloning and
expression of a complementary DNA (cDNA) from rat brain that encoded a G
protein-coupled receptor that exhibited all of the properties described by Howlett
and colleagues. Its messenger RNA (mRNA) was found in cell lines and regions
of the brain that contained cannabinoid receptors based on radioligand binding
analysis. Thus, by the early 1990s, a framework of rigorous cellular, molecular,
pharmacological, and physiological methodology had been established that al-
lowed for the systematic characterization and definition of a neuronal or central
cannabinoid receptor, currently referred to as the CB 1 receptor. It was within this
framework of definable criteria for assessing effects of cannabinoids that a sec-
ond cannabinoid receptor was discovered that was associated in distribution and
functional relevance with the immune system
In 1992, Kaminski and colleagues (Kaminski et al. 1992) demonstrated through
an equilibrium binding assay that membranes from mouse spleen had specific
binding sites for cannabinoids. In addition, using specific primers for the cannabi-
noid receptor identified in brain, they amplified from splenic RNA using RNA
transcriptase-polymerase chain reaction (RT-PCR), an 854-kb product that hy-
bridized with brain cannabinoid receptor cDNA. These studies demonstrated that