Overview and Historical Perspective of Four Papers Presented on Research Related to the Endogenous Opioid System

The endogenous opioid system is now known to play a major role in many aspects of normal physiology and neurobiology, as well as a central role in many of the addictive disease disorders. There are many areas of ongoing exciting research regarding this diverse system, including some new approaches to understanding both the brain localization and specific molecular mechanisms underlying behaviors. These include the use of small interfering RNA directed against the mu opioid receptor system, which when instilled in the substantia nigra-ventral tegmental area has been shown to result in both an ablation of heroin-induced locomotor activity and conditioned place preference. Further, a wide variety of human molecular genetics studies identify the mu opioid receptor as an important contributor to the vulnerability to develop an addiction, as well as a gene whose functional variants may alter physiological and pathological states in a wide variety of ways (e.g., Levran et al., 2008; Nielsen et al., 2008; Zhang et al., 2009). When research first began at The Rockefeller University in 1964 to attempt to develop a pharmacological treatment for opiate addiction, it was conceptualized that a therapeutic agent should be targeted to a specific site of action of opiate drugs like heroin and its active metabolites, primarily morphine (Dole et al., 1966). Further, the probable existence of specific “opiate (later opioid) receptors” was postulated, a concept that had been introduced by a few different groups, including that of the late Martin in Kentucky, and Collier and colleagues in England. Thus, in the selection of a pharmacological agent, criteria were set that it should be orally effective, long-acting, and targeted at what was then simply called “the opiate receptor,” and, of course, methadone was the agent chosen (Dole et al., 1966). In 1970, Dole and Ingolia published the first paper that gave the scientific methodological approach that ultimately led to the discovery of specific opiate receptors, that is the approach of using stereoselective binding of an opiate-like compound, in this case, the binding to brain membranes of active l (or R) enantiomer of methadone and the inactive d (or more correctly S) enantiomer (Ingolia and Dole, 1970). The labeled enantiomers of methadone had too low specific activity to allow precise differentiation between binding of the active and inactive enantiomers in brain membranes (Ingolia and Dole, 1970). Goldstein built on this technology; using the Dole protocol, he also looked for stereoselective binding of a compound theoretically targeted at the mu opioid receptor. This work also suffered from the lack of availability of a high enough specific radioactive ligand (Goldstein et al., 1971). Success was achieved in early 1973 when, in a very short period of time, three groups working independently, but all using the technique of steroeselective binding of a presumably opiate-directed agonist or antagonist (but using adequately high specific activity compounds to detect a significant difference) were reported by the groups of Snyder, Simon, and Terenius, respectively (Pert and Snyder, 1973; Simon et al., 1973; Terenius, 1973). Over the next years, several groups using a variety of different chemical probes delineated the existence of not one single opiate receptor, but rather three classes, which were named mu, delta, and kappa. Over the next several years, the genes for each of the three classes of endogenous opioids, proopiomelanocortin (POMC) gene (yielding beta-endorphin and many other biologically important peptides), the enkephalin gene, and the dynorphin gene were cloned. In 1992, two independent scientific groups, each using the technique of expression cloning and the same cell line, working in Los Angeles and Strasbourg, France, respectively, successfully cloned the gene of the delta opioid receptor (Evans et al., 1992; Kieffer et al., 1992). In rapid succession, the mu and kappa opioid receptors were subsequently cloned, first in rats and mice, and then in humans. In 1994, research began in collaboration with Lei Yu, who headed one of the two groups that first cloned the mu opioid receptor in rodents and in humans, to do extensive sequencing of this coding region of the gene (using the then-available laborious gel sequencing and running the gels both forward and in reverse directions for appropriate identification of any variants) (Bond et al., 1998). In the early sequencing work (1995–1996) studying subjects from three different ethnic-cultural groups, two quite common variants were found, “the C17T” variant and “the A118G” variant (Bond et al., 1998). Functional studies were performed on the A118G variant because it was at a site of putative glycosylation, and, like the C17T variant, resulted in an amino acid change in the N-terminus of the mu opioid receptor. But in the case of the A118G variant, the change of amino acid was from asparagine to aspartic acid, and thus undoubtedly changing the physical properties of the receptor terminus (Bond et al., 1998). While functionality of the A118G variant was being studied, the existence of each of these two variants was reported with no studies of their possible altered functionality. The group of Goldman described the A118G variant but found in their populations that there was no association of this variant with alcoholism (Bergen et al., 1997). In another independent study, reported by the groups of Berrettini in the U.S. in collaboration with Hoehe in Germany, the C17T variant was identified in a primarily African-American population, but no association was identified with substance abuse (Berrettini et al., 1997). What was found in the next two years of study was that the receptor resulting from the A118G variant bound beta-endorphin, the longest (31 amino acids) and longest half-life of the endogenous opioids, three times more tightly than the prototype receptor, but binding of no other peptide nor natural or synthetic mu opioid receptor ligand was altered (Bond et al, 1998). It was further found that signal transduction through the G protein inwardly rectifying potassium channel was increased three-fold with the A118G variant-related receptor. Again, this happened only when beta-endorphin was the ligand. It has been demonstrated that this A118G variant alters objectively measured stress responsivity as well as response to pain and other physiological events modulated by the mu opioid receptor (Bond et al., 1998; LaForge et al., 2000; Kreek et al., 2005; Kroslak et al., 2007; Kreek and LaForge, 2007; Kreek, 2008). Further, as was predicted initially, it was found in a central Swedish population with only modest admixture that the A118G variant was very significantly associated with opiate addiction and also with alcoholism, with high attributable risk to this one variant in each of these complex disorders (Bart et al., 2004; Bart et al., 2005).