A major focus of work has been to study the use of site-directed alkylating agents to define opiate receptor subtypes. Previous work demonstrated that pretreatment of membranes with the fentanyl derivative FIT eliminates the higher affinity 3H-D-ala2-D-leu5-enkephalin binding site. Additional work has shown that this delta-selective irreversible ligand in addition alters the properties of the lower affinity 3H-D-ala2-D-leu5-enkephalin (3H-DADL) binding site. Additional experiments have shown that pretreatment of membranes with both FIT and the mu-receptor directed irreversible ligand, BIT, produces a membrane preparation highly enriched with kappa opiate receptors. Using this protocol, the distribution of kappa receptors in the hippocampus of several species were determined using autoradiographic techniques. Of some interest is the finding that opiate receptors in the rat pituitary are confined to the neural lobe and are exclusively kappa receptors. A second line of investigation has been to identify in vivo manipulations of rats that will produce selective alterations in opiate receptors assayed in vitro. Thus using the site-directed alkylating agents to define 3H-DADL binding sites, we have shown that whereas chronic morphine causes an up regulation of the lower affinity 3H-DADL binding site, chronic electroconvulsive shock causes a down-regulation of this binding site. It is likely that these observations may lead to a greater understanding of the mechanisms underlying the development of tolerance and dependence to opiates.
