Signal transduction and receptor modulation

Binding of agonist at cannabinoid receptors leads to activation of predominantly pertussis toxin-sensitive G proteins. These, in turn, cause inhibition of adenylyl cyclase and voltage-activated calcium channels or activation of potassium channels. The most common molecular/cellular assays of CB1 receptor function, therefore, involve enhancement of [35S]-GTPyS binding to preparations from brain tissue or cells and the inhibition of cyclic AMP accumulation in intact brain slice or cell preparations. In isolated tissues, CB1 receptor function can be studied by examining inhibition of electrically-evoked neurotransmitter release in the guinea-pig ileum or mouse vas deferens through measurement of alterations in contractile responses (see Table 13.4). From such studies in a variety of preparations, it is apparent that CB1 receptor inhibition of transmitter release is associated with a direct inhibition of calcium currents, while the enhancement of potassium fluxes maybe secondary to inhibition of adenylyl cyclase activity (Pertwee 1997).

Turning to enzymatic pathways, heterologous expression of CB1 receptors in CHO or L cells revealed a lack of coupling to the phospholipase C pathway (Felder et al. 1992).

Table 13.4 Cannabinoid receptor regulation of transmitter/hormone release in vitro

Neurotransmitter

Effect of

Effect of CB1

Source

agonist

antagonist

5HT

1

Reversal

Mouse cerebral cortical slicesa

ACh

1 (THC)

Human platelets (from migraineurs)b

1

Enhancement

Rat hippocampal slicesc

1

Enhancement

Mouse urinary bladderd

1

Enhancement

Guinea-pig ileume

1

No enhancement

Rat striatal slicesf

1

Human ileumg

1

Enhancement

Rat cortical synaptosomesh

1

Enhancement

Rat hippocampal synaptosomesh

=

Rat striatal synaptosomesh

1

No effect

Guinea-pig tracheai

1

Enhancement

Mouse hippocampal slices'

=

Mouse striatal slicesk

ACTH

t

Not reversed

Rat anterior pituitary cellsl

CCK

1

Rat hippocampal slicesm

=

Rat cortical slices"

CGRP

t (AEA)

Not reversed

Rat mesenteric arteryo

CRF

t

Reversal

Rat median eminence fragmentsp

DA

1

Enhancement

Guinea-pig retinaq

1

Reversal

Rat striatal slicesr

1

Reversal

Rat striatal slicess

=

No effect

Rat striatal slices1

t (THC)

Rat nucleus accumbens slicesu

GABA

1

Reversal

Cultured rat hippocampal neuronesv

1

Reversal

Rat nucleus accumbensw

=

Rat substantia nigrax

1

Rat hippocampal slicesy

1

Reversal

Rat hippocampal slices2

1

Reversal

Rat striatal slicesaa

1

Reversal

Human hippocampal slicesab

1

Reversal

Rat nucleus accumbens (shell)ac

GH

t

Not reversed

Rat anterior pituitary cellsah

Glu

1

Reversal

Rat substantia nigra pars reticulataad

1

Reversal

Rat striatal slicesae

1

Reversal

Mouse nucleus accumbensaf

GnRH

t

Reversal

Rat median eminence fragmentsag

Table 13.4 (Contd.)

Neurotransmitter

Effect of

Effect of CB1

Source

Agonist

Antagonist

LH

1

Reversal

Rat anterior pituitary cellsal

1

Reversal

Mouse vas deferensaj

=

No effect

Rat hippocampal slicesak

1

Reversal

Guinea-pig hippocampal slicesal

NA

1

Reversal

Human hippocampal slicesam

1

Reversal

Human atrial segmentsan

=

No effect

Rat hippocampal slicesao

=

No effect

Mouse hippocampal slicesap

PRL

1

Reversal

Rat anterior pituitary cellsaq

Test

1

Mouse testesar

a (Nakazi etal. 2000); b(Volfeetal. 1985); c(Gifford and Ashby 1996); d(Pertweeetal. 1996); e(Pertweeand Fernando 1996); f(Gifford etal. 1997b); g(Croci etal. 1998); h(Gifford etal. 2000); i(Spicuzza etal. 2000); j(Kathmann etal. 2001); k(Kathmann etal. 2001); '(Wenger etal. 2000); m(Beinfeld and Connolly 2001); n(Beinfeld and Connolly 2001); o(Zygmunt etal. 1999); p(Prevot etal. 1998); q(Schlickeretal. 1996); r(Cadogan etal. 1997); s(Kathmann etal. 1999); ^Szabo etal. 1999); u(Szabo etal. 1999); v(Ohno-Shosaku etal. 2001; Irving etal. 2000); w(Hoffman and Lupica 2001); x(Romeroetal. 1997); y(Wilson and Nicoll 2001); z(Katona etal. 1999; Hajos etal. 2000); aa(Szabo etal. 1998); ab(Katona etal. 2000); ac(Hoffman and Lupica 2001); ad(Szabo etal. 2000); ae(Gerdeman and Lovinger 2001; Huang etal. 2001); af(Robbeetal. 2001); ag(Prevotetal. 1998); ah(Wenger etal. 2000); ai(Wenger etal. 2000); aJ(Ishac etal. 1996); ak(Gifford etal. 1997a); al(Schlicker etal. 1997); am(Schlicker etal. 1997); an(Molderings etal. 1999); ao(Schlicker etal. 1997); ap(Schlicker etal. 1997); aq (Wenger etal. 2000); ar(Dalterio etal. 1977).

Key: 5HT serotonin; ACh acetylcholine; AEA anandamide; ACTH adrenocorticotrophic hormone; CCK cholecystokinin; CGRP calcitonin gene-related peptide; CRF corticotropin-releasing factor; DA dopamine; GABA y-aminobutyric acid; GH growth hormone; Glu glutamate; GnRH gonadotropin-releasing hormone; LH luteinizing hormone; NA noradrenaline; PRL prolactin; Test testosterone; THC tetrahydrocannabinol; f increase/stimulation; | decrease/inhibition; = no effect.

Indeed, in rat hippocampal slices, THC appears to inhibit phosphoinositide turnover, albeit through a pertussis toxin-insensitive mechanism (Nah etal. 1993).

Given that arachidonic acid derivatives are putative endogenous ligands of the cannabinoid receptors, it is of note that cannabinoid receptor activation has been reported to couple to an increase in arachidonic acid release (Burstein et al. 1994) and eicosanoid biosynthesis, as well as synthesis of the endocannabinoid anandamide (Hunter and Burstein 1997). Furthermore, it is apparent that activation of cannabinoid receptors by either anandamide or THC in WI-38 fibroblasts leads to stimulation of the ERK/MAP kinase signal transduction pathway which, in turn, leads to increased phosphorylation and activation of the arachidonate-specific cytoplasmic phospholipase A2 (Wartmann et al. 1995). ERK/MAP kinase activation has also been reported to couple cannabinoid receptor activation to other effectors, including the NHE-1 isoform of the Na+ /H+ exchanger (Bouaboula etal. 1999) and glucose metabolism in primary astrocytes (Sanchez et al. 1998). Other studies suggest, however, that cannabinoids can inhibit the activation of ERK/MAP kinases via CB2 receptor activation (Faubert and Kaminski 2000).

In cultured macrophage-like cells, it is apparent that CB2 receptors couple to the inhibition of nitric oxide production stimulated by bacterial lipopolysaccharide (Ross et al. 2000). However, whether this phenomenon is involved in the putative immunosuppressive actions of cannabinoids in vivo remains to be elucidated.

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