58 A.C. Howlett
consistent with Gi mediation of the inhibition component and Gs mediation of the
stimulation component. The finding that overexpression of Gαi1 could overcome
the stimulatory component led these researchers to suggest that the D 2 dopamine
receptors could sequester Gi proteins, resembling the response to pertussis toxin
treatment, and thereby precludetheir coupling totheCB 1 receptors(Jarrahianetal.
2004). The CB 1 receptor-mediated stimulation of cyclic AMP production required
greater concentrations of CP55940 than did inhibition (Jarrahian et al. 2004). The
efficacies of cannabinoid receptor agonists for regulation of Gs were not as great as
for regulation of Gi (Bonhaus et al. 1998). HU210, CP55940, and WIN55212-2 were
full agonists to inhibit forskolin-stimulated cyclic AMP accumulation by Gi, and
∆^9 -THC and anandamide were partial agonists. Following pertussis toxin treat-
ment, WIN55212-2 was a full agonist to stimulate cyclic AMP accumulation, but
HU210, CP55940,∆^9 -THC, and anandamide behaved as partial agonists for this
response.
3
Cannabinoid Receptor-Mediated Ca2+Fluxes and Phospholipases C and A
Cannabinoid and endocannabinoid compounds increased intracellular free Ca2+
as determined by fura-2 fluorescence in undifferentiated N18TG2 neuroblastoma
and NG108-15 neuroblastoma-glioma hybrid cells (Sugiura et al. 1996, 1997a,
1999). The CB 1 receptor and Gi/o proteins were implicated because this response
was blocked by SR141716 and pertussis toxin (Sugiura et al. 1996, 1999). From
studies directly measuring isotopic Ca2+influx into N18TG2 neuroblastoma cells,
the evidence suggests that desacetyllevonantradol stimulated Ca2+uptake via CB 1
receptor coupling to Gs, cyclic AMP production, and PKA activation (Bash et al.
2003). Further evidence suggested that a second component of Ca2+influx was due
to CB 1 receptor coupling to Gi/o, leading to receptor Tyr kinase transactivation,
PKC phosphorylation, and regulation of MAPK (Rubovitch et al. 2004). Evidence
for a CB 1 receptor-mediated Tyr phosphorylation of N18TG2 cell proteins that can
be immunoprecipitated with the CB 1 receptor has been reported (Peterson et al.
2004).
Some controversy exists regarding the ability of cannabinoid receptors to signal
through the inositol 1,4,5-triphosphate (IP 3 )-Ca2+mobilization pathway. In studies
using Ca2+reporter fura-2 fluorometry, Ca2+mobilization in N18TG2 neuroblas-
toma cells was blocked by a phospholipase C (PLC) inhibitor, indicating that PLCβ
could be the effector (Sugiura et al. 1996, 1997a). CB 1 receptor activation in cul-
tured cerebellar granule cells resulted in an augmented Ca2+signal in response to
depolarization by glutamate receptors or high K+(Netzeband et al. 1999). In these
cells, Ca2+was mobilized from a caffeine-sensitive and IP 3 receptor-sensitive pool.
This Ca2+signal was attenuated by SR141716, pertussis toxin, and a PLC inhibitor
(Netzeband et al. 1999), indicative of a CB 1 receptor-mediated PLC mechanism for
Ca2+mobilization from endoplasmic reticulum stores.
TheprimaryevidenceagainstaPLC-mediatedpathwayisthatagonist-stimulated
CB 1 receptors that were heterologously expressed in competent CHO cells failed to