Evidence for distinct subcellular sites of opiate receptors. Demonstration of opiate receptors in smooth microsomal fractions isolated from rat brain.

Abstract

We have found opiate receptors enriched in two distinct subcellular fractions obtained from rat brain cellfree homogenates. By several criteria, one fraction contains synaptic plasma membranes, while the other represents predominantly smooth microsomes. Using marker enzyme analysis, we determined that the occurrence of opiate receptors in the smooth microsomal fraction cannot be attributed to synaptic plasma membrane cross-contamination. Electron microscopic examination revealed that the two fractions enriched in opiate receptors differed markedly in morphology. In particular, smooth microsomes appeared to lack synaptic junctional complexes, postsynaptic densities, or membranes identifiable as presynaptic. Also, opiate receptors in microsomes were localized on membranes which were distinctly lighter ( p = 1.08-1.14 g/ml) than synaptic plasma membrane receptors (p = 1.13-1.18 g/ ml) when either agonist or antagonist binding was measured. We also studied the characteristics of binding of opiate agonists to both receptor populations. AIthough both microsomal and synaptic membrane opiate receptors were sensitive to NaCl when agonist binding was assessed, microsomal receptors were considerably less sensitive to guanine nucleotides than were synaptic membrane receptors. The subsensitivity appeared due to the inability of relatively low concentrations of guanine nucleotides (10 w) to accelerate the dissociation rate of bound peptide ligand. Finally, microsomal opiate receptors displayed faster rates of dissociation (K-l = 0.077 min”) than did synaptic membrane receptors (ZL1 = 0.017 min”). The data are discussed in relation to the concepts that microsomal opiate receptors either represent a distinct population of synaptic membranes or newly synthesized receptors in transit to synaptic membrane sites.