Organotypic cultures of fetal mouse spinal cord-
ganglion explants (2-4 weeks in vitro) contain
forskolin-stimulated
adenylate cyclase (AC) activity that is inhibited by
levorphanol and other
opioid agonists in a dose-dependent manner. Inhibition by
levorphanol no longer occurs if
sodium is omitted from the incubation and the
levorphanol inhibition is blocked by the
opioid antagonist,
naloxone. These findings together with the ineffectiveness of
dextrorphan indicate that the
opioid inhibition of
forskolin-stimulated AC is receptor mediated. Both the delta- and
kappa-receptor subtypes appear to be involved since the selective delta-
opioid agonist, [
D-Pen2, D-Pen5]enkephalin, and the selective kappa-
opioid agonist, t-3,4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)cyclohexyl]-
benzene acetamide (U-50,488H) are both effective at nanomolar concentrations. In contrast, the selective mu-
opioid agonist, Tyr-D-
Ala-Gly-N-MePhe-Gly-ol, has no significant effect even at micromolar concentrations. Both cord and
ganglion components of the explants contain
opioid-sensitive AC.
Forskolin-stimulated AC of the explants is also inhibited by
serotonin and
carbachol. The
serotonin effect appears to be mediated by 5-HT1A receptors, based on relative agonist and antagonist selectivity. Chronic exposure of cultures to
morphine results in enhanced basal and
forskolin-stimulated AC as well as attenuation of
opioid-inhibition of AC assayed in the presence of
forskolin; treatment of explants with
pertussis toxin causes similar changes in the AC system. The inhibitory effect of
serotonin is also attenuated by the
pertussis toxin treatment. Basal AC activity of the explants (assayed without
forskolin present) is stimulated to a small but significant extent by
opioids and by
serotonin. The
opioid stimulatory effect is markedly enhanced following either
morphine or
pertussis toxin treatment of the explants. The attenuation of
opioid- and
serotonin-inhibition of AC produced by chronic exposure to
pertussis toxin and the attenuation of
opioid inhibition produced by exposure to
morphine are consonant with the attenuation of
opioid and monoaminergic depression of sensory evoked dorsal horn network responses after similar chronic treatments. It is proposed that the inhibitory effects of
opioids and
serotonin on these neurons are mediated by receptors that are negatively coupled via a
pertussis toxin sensitive Gi
protein to AC. Furthermore, alterations of AC with chronic
morphine treatment may be involved in the development of physiologic tolerance to
opioids.