Opiate Receptor Revisited

Abstract

OUR identification of opiate receptors 34 yr ago1seems distant. Today, receptors for drugs and neurotransmitters are taken for granted. Almost all receptors have been cloned, providing intimate understanding of signal transduction. Modern drug development uses robotics to screen millions of chemicals at dozens of receptors and their subtypes. By contrast, when we began our work, how drugs and neurotransmitters signaled was a mystery, with receptors and second messengers essentially black boxes. Drug development usually required screening in intact rodents with no way to discriminate whether differential drug potency stemmed from receptor affinity, metabolism, or target penetration.Identification in 1970 of the nicotinic acetylcholine receptor in the electric organ of the electric eel by the binding of radiolabeled α-bungarotoxin was an important advance. Paradoxically, this success convinced most workers in the field that it would never be possible to monitor receptors for drugs and most neurotransmitters in mammalian brain. Here is the reasoning. The acetylcholine receptors comprise 20% of the protein in the electric organ, yet detecting these receptors required the extraordinarily potent and specific α-bungarotoxin labeled with 125I to high specific radioactivity. By contrast, the opiate pharmacologist Vincent Dole, M.D. (Rockefeller University, New York, New York; 1913–2006), had calculated that opiate receptors would likely constitute no more than one millionth by weight of mammalian brain, and no magic toxins existed for the receptors. A few laboratories had attempted to monitor the binding of [3H]opiates and other drugs to brain membranes, but nonspecific binding to tissue lipid, carbohydrates, and proteins was great, and no specific receptor interactions had ever been demonstrated.I became interested in receptors at a time that my faculty colleague Pedro Cuatrecasas, M.D. (Professor, Johns Hopkins University, Baltimore, Maryland), had demonstrated insulin receptors by monitoring the binding of 125I-insulin to tissue membranes. Pedro used a custom-made vacuum manifold in which he could trap tissue membranes with receptor-bound insulin on glass fiber filters and wash away nonspecific binding vigorously but so rapidly that receptor-bound insulin was not also removed. I read a publication describing the sequencing of nerve growth factor, an impressive technical feat in those days, which reported a close similarity in amino acid sequence to insulin. I suggested a collaboration in which my postdoctoral fellow Shailesh Banerjee, Ph.D. (Department of Pharmacology, Johns Hopkins University), would use Pedro's equipment and attempt to identify nerve growth factor receptors, a project in which we were eminently successful.2In 1971 President Nixon had declared “War on Heroin” and appointed my friend, Jerome Jaffe, M.D. (Professor of Psychiatry, University of Maryland, Baltimore, Maryland), as his czar of drug abuse. After importuning by myself and some colleagues, Jaffe established a series of Drug Abuse Research Centers, and Johns Hopkins received one. Although I had never previously worked with opiates, I could see that finding their receptors would be the most effective way to learn how they work. Accordingly, I was willing to spend $3,000, which seemed an astronomic amount of money, to obtain a custom preparation of [3H]naloxone, the potent, pure opiate antagonist. My graduate student Candace Pert, Ph.D. (Johns Hopkins University), had been studying the uptake of choline as an acetylcholine precursor into preparations of the guinea pig ileum. Accordingly, she monitored binding of [3H]naloxone in these preparations and at the same time examined binding in rat brain membranes using Pedro's vacuum manifold. After a relatively few manipulations of washing conditions, we obtained what seemed to be specific receptor interactions which were approximately twofold to threefold greater than nonspecific binding, assessed in the presence o