Induction of morphine-6-glucuronide synthesis by heroin self-administration in the rat

Department of Physiology and Pharmacology Vittorio Erspamer, Sapienza University of Rome, Rome, Italy.
Psychopharmacology (Impact Factor: 3.88). 10/2011; 221(2):195-203. DOI: 10.1007/s00213-011-2534-7
Source: PubMed


Heroin is rapidly metabolized to morphine that in turn is transformed into morphine-3-glucuronide (M3G), an inactive metabolite at mu-opioid receptor (MOR), and morphine-6-glucuronide (M6G), a potent MOR agonist. We have found that rats that had received repeated intraperitoneal injections of heroin exhibit measurable levels of M6G (which is usually undetectable in this species).
The goal of the present study was to investigate whether M6G synthesis can be induced by intravenous (i.v.) heroin self-administration (SA).
Rats were trained to self-administer either heroin (50 μg/kg per infusion) or saline for 20 consecutive 6-h sessions and then challenged with an intraperitoneal challenge of 10 mg/kg of heroin. Plasma levels of heroin, morphine, 6-mono-acetyl morphine, M3G, and M6G were quantified 2 h after the challenge. In vitro morphine glucuronidation was studied in microsomal preparations obtained from the liver of the same rats.
Heroin SA induced the synthesis of M6G, as indicated by detectable plasma levels of M6G (89.7 ± 37.0 ng/ml vs. 7.35 ± 7.35 ng/ml after saline SA). Most important, the in vitro V (max) for M6G synthesis was correlated with plasma levels of M6G (r (2) = 0.78). Microsomal preparations from saline SA rats produced negligible amounts of M6G.
Both in vivo and in vitro data indicate that i.v. heroin SA induces the synthesis of M6G. These data are discussed in the light of previous studies conducted in heroin addicts indicating that in humans heroin enhances the synthesis of the active metabolite of heroin and morphine.

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    • "We have previously observed that both in humans (Antonilli et al., 2003a) and rats (Meringolo et al., 2012) diacetylmorphine (heroin) self-administration leads to a reduced blood M3G/M6G ratio. Liver is the primary site of this heroin action as demonstrated by the converging findings that, when incubated with morphine, both liver microsomes obtained from rats repeatedly exposed to heroin and primary cultures of rat hepatocytes, likewise exposed to heroin, yield less M3G than controls but measurable quantities of M6G, which is usually not synthesized in the rat (Antonilli et al., 2003b, 2005; Graziani et al., 2008). "
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    ABSTRACT: We have previously found that phenantrenic opioids, such as heroin or naltrexone, modulate morphine glucuronidation in the rat. Here we further investigated the effects of phenantrenic opioids on morphine glucuronidation comparing the effects of codeine and heroin. In particular, we measured the synthesis of morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) from morphine: in the liver microsomal preparations obtained from rats repeatedly treated with two different doses of codeine (ex vivo study); in primary cultures of rat hepatocytes previously incubated for 72h with codeine, or heroin (in vitro study); in the latter conditions, the levels of expression of genes coding for uridine-5'-diphosphate-glucuronosyltransferases (UGTs) A1, A6, A7 and 2B1 were also determined; finally, the levels of glucuronic acid in rat hepatocytes previously incubated for 72h with codeine or heroin were assessed. The ex vivo study shows that codeine exposure in vivo stimulated liver microsomal M3G formation and de novo synthesis of M6G. Differently, in primary hepatocyte cultures both codeine and heroin inhibited M3G formation, whereas heroin only stimulated de novo synthesis of M6G; moreover, codeine significantly reduced UGT2B1 expression at 6h and caused a trend toward inhibition of UGT1A1 expression at 72h; heroin enhanced UGT2B1 expression and inhibited that of UGT1A1 at 72h; finally, both codeine and heroin depleted UDPGA content of hepatocytes. In conclusion, codeine affects liver glucuronidation of morphine enlightening the possible contribution of changes in the spectrum of UGT gene expression and co-factor synthesis in this phenomenon.
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    ABSTRACT: Morphine is converted to morphine 3-β-D-glucuronide (M3G) by the UDP-glucuronosyltransferase Ugt2b1 in the endoplasmic reticulum (ER) of rat liver. Because of its luminal localization, UGT activity requires UDP-glucuronate import and glucuronide export across the ER membrane. The former transport is generally considered to be rate limiting and to explain the latency of UGT activities in intact microsomal vesicles. However, some observations indicate that the release of bulky glucuronides, such as M3G, might also be rate limiting for glucuronidation. This assumption was tested by characterizing the transport of M3G and its distribution between the intra- and extravesicular spaces during synthesis in rat liver microsomes. The amount of vesicle-associated M3G was measured using rapid filtration and LC-MS measurement. Our results reveal a remarkable accumulation of newly synthesized M3G in the microsomal lumen above the equilibrium. The transport showed a linear concentration-dependence in a wide range (5-200 μM). Therefore, the build-up of high (about 20 μM) luminal M3G concentration could adjust the rate of release to that of synthesis (44.85 ± 4.08 pmol/min/mg protein) during the conjugation of 100 μM morphine. These data can explain earlier findings indicative of separate intracellular pools of M3G in rat liver. Accumulation of bulky glucuronides in the ER lumen might also play an important role in their targeting and in the control of biliary excretion. © 2012 BioFactors, 2013.
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