SEASONAL VARIATION OF STEREOSPECIFIC ENKEPHALIN BINDING AND PHARMACOLOGICAL ACTIVITY IN MARINE MOLLUSCS NERVOUS TISSUE

SUMMARY The ability of various pentapeptides to alter basal dopamine levels was found to be seasonal in nature. In the spring and fall DALA and methionine enkephalin caused dopamine levels to increase while in the summer they did not. Enkephalin binding followed a similar pattern, that is binding was highest in the spring and fall and lowest in the summer. Recently, exogenous opioid peptides were shown to increase dopamine levels in the pedal ganglia of the marine mollusc Mytilus edulis / Stefano and Catapane 1979 /. This effect occurred at relatively low concentrations of the peptides and was reversed by naloxone, findings which suggested an opiate receptor mechanism. Subsequently, stereospecific, high affinity opiate binding sites were demonstrated in the neuronal tissue of this organism / Stefano and Hiripi 1980 /. Other reports now substantiate the widespread occurrence of opiate systems throughout the invertebrate phyla. For example, morphine and methionine and leucine enkephalins have been shown to increase both dopamine and cyclic GMP levels and to decrease cyclic AMP levels in the freshwater bivalve Anodonta cygnea / Stefano and Catapane 1979 /. Stereospecific binding of / 3 H/ Diprenorphine to Drosophila heads has been reported / Pert and Taylor 1979 /. Furthermore, the neuronal localization of immunoreactive enkephalin and B-endorphin have been demonstrated in the earthworm / Aluments et al. 1979 /. In a study aimed at elucidating the physiological role of enkephalinergic systems in invertebrates, we now report that the actions of the enkephalins on the metabolism of dopamine and their stereo-specific binding are seasonally dependent. The ability of methionine enkephalin and of D-Ala 2 , met 5 -enkephalinamide /DALA/ to alter basal dopamine levels in M. edulis was examined during various months of the year / Table 1 /. In April, May and September, methionine enkephalin /4×10 −5 M/ and DALA /10 −5 M/ each produced approximately a 40% increase in endogenous dopamine levels within 60 min of the last drug application. Half-maximal stimulation occurred at 1×10 −6 M met-enkephalin and at 5×10 −6 M DALA /data not shown/. In contrast, in June, July and August, application of the same concentrations of these peptides resulted in no detectable alteration in the basal dopamine levels / Table 1 /. In addition, higher doses /1.2×10 −4 M met-enkephalin or 8×10 −5 M DALA/ were also ineffective in stimulating dopamine during the summer months. Stereospecific binding of 125 I-labelled FK 33-824, the highly potent methionine sulfoxide-carbinol derivative of met-enkephalin, to the nervous tissue of M.edulis was also observed to vary seasonally / Table 2 /. In all months during which the study was carried out, binding of 125 I-FK 33–824 was stereospecific, saturable, and reversible /data not shown/. In April, maximal binding occurred at 10 −8 M peptide to approximately 2.5×10 −13 mol sites/mg protein; half-maximal binding occurred at 3×10 −9 M ligand to 72 mol sites/mg protein. In May the stereospecific binding of 125 I-FK 33–824 /3×10 −8 M/ was decreased by 30% relative to the binding of this ligand under the same conditions in April. In the summer months /June, July and August/ stereospecific binding of the peptide was reduced to approximately 33% of that observed in April. Thus, enkephalin binding to the nervous tissue of M.edulis would appear to vary in a cyclic manner; binding is lower in the summer months and higher in the Spring and Fall. It has been demonstrated previously / Stefano and Catapane 1977 / that monoamine levels in the ganglia of M.edulis undergo seasonal changes; these were shown to be higher in the warmer months and lower in the colder months. This phenomenon was also observed in the present study / Table 1 /. It is interesting that in those months in which the dopamine levels of M.edulis ganglia are high, stereospecific enkephalin binding and enkephalin stimulation of dopamine in this tissue are minimal. At the end of the summer, stereospecific opiate binding increases with time, while dopamine basal levels decrease toward those values observed in the Spring. In the latter part of September and in October M.edulis endogenous dopamine is 28.1 ± 1.9 μg/gm /mean ±SEM/ / Stefano and Catapane 1977 /; the dopamine level continues to decrease monotonically until a minimal level is observed in the month of February / Stefano and Catapane 1977 /. This seasonal variation in opiate activity has also been observed in another invertebrate organism. In September and October, morphine produced a stimulation in cyclic GMP levels in the bivalve Anodonta cygnea , while in July morphine application under the same conditions reduced cyclic GMP levels / Stefano and Hiripi 1979 /. This seasonal variation in the sensitivity of the bivalves to applied opiates suggests a modulatory role for the pentapeptides which varies quantitatively in the different seasons of the year. It is of interest to speculate that during the summer months endogenous opioid ligand levels and therefore opiate receptor occupancy increase relative to those present in the Fall or Spring. This increase in endogenous opiate peptides would account for the higher levels of basal dopamine observed in the summer months. In addition, the observed reduction in stereospecific 125 I-labelled FK 33–824 binding during the summer might thus reflect occupation of opiate receptor sites by endogenous ligand which is not readily displaced by the radiolabelled peptide. Other possibilities include altered opiate receptor affinity or number during the summer months. The present study demonstrating the seasonal dependence of enkephalin binding and pharmacological activity in the bivalve M.edulis provides a possible explanation for the inability of previous investigators to detect stereospecific binding of opiates in invertebrates / Pert et al. 1974 /. This study also raises important questions concerning the physiological role of the endogenous opioid ligands throughout invertebrate nervous systems.