Continuous elevation of intracellular
cyclic AMP (cAMP) by culturing
neuroblastoma x
glioma NG108-15 hybrid cells in the presence of
forskolin and isobutylmethylxanthine (
IBMX) in a chemically defined medium resulted in differentiation of the hybrid cells, as indicated by extension of neurite-like structures and induction of a subclass of
G-protein, Go, as monitored by Western analysis. This cellular differentiation also resulted in an initial 25 to 30% increase in [3H]
diprenorphine binding 3 hr after
forskolin and
IBMX treatment, followed by a decrease in
opioid receptor density to the maximal level of 35% of control 4 days later. However, the potencies and maximal inhibitory levels of various
opioid agonists to inhibit
adenylate cyclase activity was not altered during differentiation. When the differentiated hybrid cells were treated with
DADLE chronically, an apparent decrease in the ability of the agonist to desensitize and to down-regulate the
delta-opioid receptor was observed. It is unlikely that this observed attenuation was due to activation of
cAMP-dependent protein kinase A, because (1) attenuation of
DADLE desensitization was time-dependent, reaching maximal effects 48 hr after the initiation of treatment; and (2) pretreatment of NG108-15 cells with
forskolin and
IBMX resulted in attenuation of
forskolin's ability to stimulate
adenylate cyclase activity and parallel decrease in the ability of
forskolin to activate the
cAMP-dependent protein kinases in these cells was also observed. Thus, it is unlikely that the activation of
protein kinase A by
forskolin and
IBMX is the cause for the attenuation of
DADLE-induced
delta-opioid receptor desensitization in differentiated NG108-15 cells.