Demonstration of two classes of opiate binding sites in the nervous tissue of the marine mollusc Mytilus edulis. Positive homotropic cooperativity of lower affinity binding sites

[³H]Etorphine, ¹²⁵I-FK 33-824 ( a highly potent enkephalin derivative of enhanced stability), and ¹²⁵I-levallorphan bound stereospecifically, with high affinity, and reversibly to pedal ganglia membranes of the marine mollusc Mytilus edulis. Each ligand exhibited (by Scatchard analysis) noncooperative binding to a class of high affinity sites (Kd = 1 to 3 nM) and cooperative binding to a class of lower affinity sites (Kd = 6 to 11 nM). Hill analyses of the cooperative site revealed Hill coefficients n = 2.6 to 3.7, values which indicate markedly positive homotropic cooperativity. Opiate binding was undetectable in non-neuronal tissues of M. edulis such as muscle and mantle. Many of the properties of the noncooperative sites are similar to those of brain opiate receptors. Opiate binding to pedal ganglia was of high affinity and was markedly inhibited by trypsin. Very similar dissociation and association rates were observed in both tissues. In addition, opiate agonist and antagonist binding was affected differentially by sodium and manganese. Finally, the relative potencies of a series of opiate narcotic agonists and antagonists and opioid peptides in displacing 1 nM ¹²⁵I-FK 33-824 binding to pedal ganglia were very similar to those determined for rat brain homogenates. Similar relative potencies were also observed for the abilities of these drugs to increase endogenous dopamine levels in intact M. edulis. These findings support the pharmacological relevance of the noncooperative opiate binding sites in the pedal ganglia. Possible assignments for the two classes of opiate binding sites in M. edulis pedal ganglia include 1) pre- and post-synaptic receptor sites in analogy to the α-adrenergic system of rat heart membranes; 2) different pharmacological subclasses of opiate receptors; or 3) a receptor and a metabolic enzyme. This study represents the first biochemical analysis of specific opiate binding in an invertebrate system. The findings of pharmacological effects of opiates in this and other systems, and the recent finding of enkephalin in the neuronal tissue of earthworms suggest that opiate systems may be widespread throughout the invertebrate phyla. The potential value of such a system for the study of the detailed mechanism of opiate action is discussed.