Reticuline exposure to invertebrate ganglia increases endogenous morphine levels.

OBJECTIVES: Given the presence of morphine, its metabolites and precursors in mammalian and invertebrate tissues, it became important to determine if exposing tissues to an opiate alkaloid precursor, reticuline, would result in increasing endogenous morphine levels.

METHOD: Endogenous morphine levels were determined by high pressure liquid chromatography coupled to electrochemical detection and radioimmunoassay following incubation of Mytilus edulis pedal ganglia with reticuline. Nitric oxide (NO) release was determined in real-time via an amperometric probe. Mu opiate receptor affinity for opiate alkaloid precursors was determined by a receptor displacement assay.

RESULTS: Morphine is present in the pedal ganglia of Mytilus edulis (1.43 +/- 0.41 ng/mg +/- SEM ganglionic wet weight). Ganglia incubated with 50 ng of reticuline, a morphine precursor in plants, for 1 hour exhibited a statistical increase in their endogenous morphine levels (6.7 +/- 0.7 ng/mg tissue wet weight; P<0.01). This phenomenon is concentration dependent. The increase in ganglionic morphine levels occurs gradually over the 60 min incubation period, beginning 10 minutes post reticuline addition. We show that reticuline (10(-6) M) does not stimulate ganglionic NO release in a manner resembling that of morphine (10(-6) M), which releases NO seconds after its exposure to the ganglia and lasts for 5 minutes. With reticuline, there is a 3 minute delay, which is followed by an extended release period. Furthermore, in binding displacement experiments both reticuline and salutaridine (another morphine precursor) exhibit no binding affinity for the pedal ganglia mu opiate receptor subtype. This finding is further substantiated using the positive control of human monocytes where the mu3 opiate receptor subtype has been cloned.

CONCLUSION: Taken together, we surmise that the morphine's precursors are being converted to morphine. The experiments strongly indicate that pedal ganglia can synthesize morphine from reticuline.