Interaction of calcium with3H-morphine binding to synaptosomes of the guinea-pig ileum

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Abstract
Specific binding sites for3H-morphine were assayed in subcellular fractions of a crude mitochondrial P2 pellet from the guinea-pig ileum longitudinal muscle-myenteric plexus, prepared by discontinuous sucrose-gradient fractionation. The highest specific binding (ca. 100 fmol/mg protein) was obtained in the fraction containing synaptosomes, as examined by electron microscopy. A synaptosomal fraction prepared from guinea-pig brain had comparable specific binding (ca. 130 fmol/mg) to that of the ileal synaptosomal fraction. Addition of calcium (10 mM) to the binding assay medium resulted in a marked decrease in particular of the specific3H-morphine binding. Detailed analysis of the specific3H-morphine binding in the synaptosomal fraction of the ileum revealed that 1) a saturable component of specific opiate binding was present between 0.34 and 21.74 nM of3H-morphine; 2) in the presence of 3 and 10 nM of calcium similar decreases of specific3H-morphine binding were obtained, indicating that this binding was maximally inhibited already by 3 mM of calcium; 3) both in the absence and presence of calcium theK D of specific3H-morphine binding was about 38 nM, indicating a non-competitive nature of the calcium inhibition; 4) addition of magnesium exhibited a similar effect as that of calcium, although magnesium appeared to be less potent than calcium in this respect. The data are discussed in the context of previously observed calcium effects on opioid actions in the electrically stimulated guinea-pig ileum bioassay and may contribute to the evidence that the interaction of calcium and opioids on the stimulus-release coupling mechanism at the neuromuscular junction of the guinea-pig ileum is occurring beyond opioid receptor activation as well.