Alain Puget

Evidence for a new type of opioid binding site in the brain of the frog Rana ridibunda

The crude membrane fraction from the brain of the frog Rana ridibunda was shown to contain 0.7-0.8 pmol/mg protein for a site with high (KD = 0.1 nM) and about 3.2 pmol/mg protein for a site with lower (KD = 10-15 nM) affinity for the opiate agonist [3H]etorphine and for the opiate antagonist [3H]diprenorphine. In addition to its very high affinity for the two tritiated oripavine derivatives, the high affinity site displayed (i) a considerably reduced ability to bind the agonist but not the antagonist in the presence of Na+ ions and (ii) pronounced stereospecificity. These properties are all typical of an opioid receptor site. The lower affinity site, which was about four times as abundant as the other exhibited none of the aforementioned characteristics and is therefore probably not opioid in nature. Detailed testing of the potency of various unlabelled opioid ligands to inhibit the binding of [3H]etorphine at the high affinity site showed that the latter consists of a mixture of several types of opioid sites, including a major type with an apparent binding profile clearly different from those of mammalian brain mu, delta- and kappa-opioid sites. In particular, this major type of site, which accounted for about 70% of the opioid binding in frog brain membranes, bound mu ([D-Ala2,MePhe4,Glyol5]enkephalin), delta ([D-Thr2,Leu5]enkephalyl-Thr) and kappa (U50,488) selective ligands with much lower affinity than did mu-, delta- and kappa-opioid receptor sites, respectively.

Species differences in the localization of neuropeptide FF receptors in rodent and lagomorph brain and spinal cord

Quantitative in vitro receptor autoradiography of [125I][D-Tyr1,(NMe)Phe3]NPFF was used to study the regional distribution of neuropeptide FF receptors in rodent and lagomorph brain. In rat, mouse, rabbit, and Afghan pika [125I][D-Tyr1,(NMe)Phe3]NPFF binding sites were enriched in the superficial layers of dorsal horn of the spinal cord and in parabrachial nucleus, central gray matter, hypothalamus, and reunions thalamic nucleus. In other neuroanatomical regions, important species differences in NPFF receptor patterns are observed. In marked contrast, the brain and the spinal cord of the Octodon degus are devoid of NPFF receptors. The present study shows that in different species regional variations in brain NPFF receptor binding occur.

The natural tolerance of the afghan pika (Ochotona rufescens) to morphine

A lagomorph, the afghan pika, Ochotona rufescens showed no effect whatever following the subcutaneous injection of morphine in doses up to 50 mg per 100 g of body weight, i.e. 250 times the ED50 for the rat. Higher doses were toxic and induced convulsions. However, the pika is responsive to synthetic opiates such as etorphine, pentazocine and phenoperidine. Interestingly enough, morphine antagonized the opiate response elicited by those narcotics to which the animal is sensitive. Pharmacokinetic analysis demonstrated that morphine enters the pikas brain as readily as it does the rats. In addition, opiate receptor sites, which are present in normal amounts in pika brain retained their high affinity for 3H-etorphine (KD = 0.3 nM), 3H-naloxone (KD = 1.2 nM) and morphine. Moreover, binding of morphine to pika brain homogenates was inhibited in the presence of sodium ions (agonist response). Therefore, the antagonism of phenoperidine action by morphine appeared not to occur at the opiate receptor site; the mechanism of the pikas natural tolerance to morphine may reside in molecular events that normally preceed (metabolism?) or follow (enzyme activation?) the interaction between the drug and its specific recognition sites.

Apparent precoupling of κ- but not μ-opioid receptors with a G protein in the absence of agonist

Abstract Rabbit and guinea-pig cerebellum membranes contain a very high (> 80%) proportion of μ- and κ-opioid receptors, respectively. Rabbit (μ) and guinea-pig (κ) cerebellum membranes wre (i) labeled either with the opiate agonist. [ 3 H]etorphine (K d = 0.1–0.2 nM), or with the opiate antagonist. [ 3 diprenorphine (K d = 0.1 nM), in the absence or presence of Na + and/or 5′-guanylylimidodiphosphate (GppNHp), (ii) solubilized with digitonin (1%, w : v) and (iii) the radioactivity in the soluble extracts analyzed by ultracentrifugation in sucrose gradients. In the soluble extracts from rabbit cerebellum (μ) membranes, bound [ 3 H]etorphine sedimented faster (s 20 ≅ 12S) that bound [ 3 H]diprenorphine (10s), while is those from guinea-pig cerebellum (κ) membranes, bound [ 3 H]etorphine and bound [ 3 H]diprenorphine sedimented at the same position (12S). Na + selectively decreased recovery of the bound tritiated agonist in the two soluble preparations. When they had been generated in the presence of GppNHp but in the absence of Na + , the [ 3 etorphine complexes of the μ- and κ-opioid receptors as well as the [ 3 Hdiprenorphine complex of the κ-opioid receptor were all recovered at position 10S, indicating that GppNHp had induced a decrease of the apparent molecular size of the two types of opioid receptors. these data are interpreted in terms of μ- and κ-opioid receptors being capable of physically interacting with a G protein (GTP binding regulatory protein) yet, unlike the μ-opioid receptor which does so only in the presence of an agonist, the κ-opioid receptor appears to be precoupled with a G protein.

Opioid receptor types in the brain of the afghan pika (Ochotona rufescens), a species which is naturally tolerant to morphine

The rabbit is normally sensitive to morphine while another lagomorph, the Afghan pika Ochotona rufescens is naturally tolerant to the analgesic effects elicited by the opium alkaloid. In spite of the different responsiveness of the two species to morphine we find that the pika brain and the rabbit brain both contain a mixture of mu-, delta- and kappa-opioid sites in nearly the same proportions: 46-47% mu, 23% delta and 28-30% kappa. Moreover, apparent binding of morphine in pika and rabbit brain membranes is inhibited in the presence of Na+ ions and/or of 5-guanylylimidodiphosphate indicating that morphine should behave as an opiate agonist (analgesic) not only in rabbits, which it does but also in pikas, which it does not. Taken together these results suggest that the natural tolerance of the Afghan pika to morphine may not reside in modified opioid receptor types and that its origin should be sought elsewhere.

Morphine metabolism in the naturally morphine-tolerant afghan pika: a preliminary study.

The afghan pika (Ochotona rufescens), a lagomorph which is naturally tolerant to the analgesic action of morphine, metabolizes morphine into morphine 3-glucuronide apparently faster than does the rabbit, another lagomorph which is however normally responsive to morphine. In the two species, following morphine administration, another unidentified component appears very soon (5 min) in pika blood plasma and much later (60 min) in rabbit blood plasma. This unknown component which appears not to be morphine derived might be involved in the natural resistance of the Afghan pika to morphine.

Regulation by Na+ ions and GppNHp of the interaction between a G protein and the amphibian type of opioid receptor

Digitonin treatment of frog brain membranes in 50 mM Tris-HCl yields a soluble extract that contains nearly equal amounts of free and G protein-bound opioid receptor molecules (Mollereau et al., 1988, J. Biol. Chem. 263, 18003). We report here that the balance of the two forms of the opioid receptor in digitonin solution is dependent on the environment of the membrane suspension at the time of solubilization with the detergent. Preincubating the membrane suspension with 50 microM GppNHp or with 120 mM NaCl results, in the two cases, in a digitonin extract that no longer displays the G protein-bound form of the receptor, i.e., the form of the receptor which exhibits high affinity for the opiate agonist etorphine in binding studies, as well as large apparent molecular size in sucrose gradients. Assuming that the situation in soluble extracts faithfully reflects the one in the membrane, these results would exclude the possibility that in a physiological environment the opioid receptor is in part precoupled with a G protein in the absence of an opioid agonist.