N-demethylation of morphine in rat brain is localised in sites with high opiate receptor content

Abstract

N-DEMETHYLATION of morphine, a principal biotransformation of this opiate alkaloid, has been demonstrated in several species including man^1–4. The primary site of the reaction is the liver^5,6 but its existence in the central nervous system (CNS) has been inferred from experiments both in vitro with rat brain slices⁷, and in vivo with hepatectomised rats⁸. The enzymatic N-demethylation of morphine and other biologically active tertiary amines has been studied extensively and both Axelrod^5,6 and Beckett et al.⁹ have proposed that N-demethylation of narcotics is intimately involved in the mechanism of analgesia and the development of tolerance to and dependence on these agents. Subsequently, other investigators have questioned the validity of these hypotheses^10,11 and critical reviews of the experimental support of these theories have been published^12,13. Belleau and Morgan¹⁴ have revived interest in the biological significance of the N-demethylation of narcotics by proposing a provocative concept of clastic binding to opiate receptors in which the process of N-demethylation participates in conjunction with the opiate receptor in the mechanism of opiate action. This concept requires that the location of the sites of N-demethylation within the CNS is proximal to or coincident with that of the opiate receptors. Previous reports^7,8,15 have described the N-demethylation of morphine by the whole brain, and our study was initiated to locate the reaction at specific sites. To do this in vivo, we used a new, highly sensitive assay. A mixture of 6-³H-morphine¹⁶, and N–¹⁴CH₃-morphine (Amersham/Searle), of known isotope ratio, was injected into each rat and, after killing, the isotope ratio of selected tissues was determined accurately by combustion in a tissue oxidiser. N-demethylation of this mixture generates ¹⁴C-containing fragments originating from the N-methyl group, but the N-demethylated products of the reaction contain only ³H. The former are dissipated from the site of reaction much more rapidly than the latter, so that an increase in the ratio of ³H to ¹⁴C in a specific tissue would serve to indicate the presence, and also the extent, of any in situ N-demethylation.