Kunisuke Nagamatsu

Morphine metabolism in isolated rat hepatocytes and its implications for hepatotoxicity.

Isolated rat hepatocytes metabolized morphine to its glucuronide conjugate, morphinone-glutathione conjugate, normorphine and morphinone. Addition of morphine to the isolated hepatocytes induced a marked decrease in the level of glutathione in the cells and resulted in cell death. The formation of glutathione conjugate was correlated well with the loss of intracellular glutathione. The cytotoxicity of morphinone was higher than that of morphine. Naloxone and normorphine showed no cytotoxic effect on the cells. Naloxone inhibited the formation of morphinone-glutathione conjugate and prevented the morphine-induced cytotoxicity. Naloxone also blocked morphine-induced liver damage in vivo. In contrast, the morphinone-induced hepatotoxicity was not prevented by naloxone. It is concluded that morphine has a hepatotoxic effect, that the morphine-induced hepatotoxicity is due to its metabolic activation, and that naloxone acts as an inhibitor of an enzyme converting morphine to morphinone.

In vitro formation of codeinone from codeine by rat or guinea pig liver homogenate and its acute toxicity in mice.

In vitro metabolism of codeine was investigated by using a 9000 g supernatant fraction of rat or guinea pig liver homogenate. When a mixture of [N-14CH3] and [C-6-3H]codeine was incubated with the rat liver 9000 g supernatant fraction in the presence of NAD, formation of codeinone, morphine and norcodeine was detected. Replacement of NAD with NADP abolished only the formation of codeinone. On the other hand, when the guinea pig liver homogenate was used in the presence of NAD, codeinone was the main metabolite of codeine. NADP was also ineffective in forming codeinone with the guinea pig liver homogenate. The acute toxicity of codeinone was thirty times higher than that of codeine. The roles of codeinone as a metabolic intermediate and in the acute toxicity of codeine are discussed.

Effects of glutathione and phenobarbital on the toxicity of codeinone.

The ability of sulfhydryl compounds to provide protection against the acute toxicity of codeinone, a toxic metabolite of codeine, was investigated in mice. Subcutaneous administration of codeinone produced a slight reduction in hepatic glutathione concentration. Pretreatment of the mice with glutathione or cysteine significantly increased the survival rate for mice given a lethal dose of codeinone (10 mg/kg). The lethality of codeine was lowered by naloxone, whereas that of codeinone was not blocked by naloxone. The strychnine-like convulsant action of codeinone could be prevented by phenobarbital pretreatment. Glutathione pretreatment reduced the amounts of radioactivity in tissues of mice injected with [N-methyl-3-H]codeinone. A possible explanation for these observations is that glutathione reacts in vivo with codeinone and plays a role as a scavenger of this compound. This assumption is supported by the observation that codeinone reacts non-enzymatically with glutathione under physiological conditions.

Accentuation by pertussis toxin of the 5-hydroxytryptamine-induced potentiation of ATP-evoked responses in rat pheochromocytoma cells

We previously demonstrated that 5-hydroxytryptamine (5-HT) enhances the cationic current activated by extracellular ATP in rat pheochromocytoma PC12 cells. We report here that pertussis toxin (PTX) modulates this 5-HT-dependent enhancement in these cells. 5-HT potentiated ATP-evoked intracellular Ca2+ concentration ([Ca]i) rise and dopamine release over a concentration range from 1 to 100 microM. When cells were pre-treated with PTX, this potentiation was accentuated. Pretreatment with PTX also accentuated the 5-HT-dependent enhancement of the ATP-activated current. These results suggest that the enhancement by 5-HT of the ATP-evoked responses is negatively regulated by a mechanism mediated through PTX-sensitive GTP-binding protein.

Effect of sodium selenite on morphine-induced hepatotoxicity in mice.

The effects of selenite on morphine hepatotoxicity and metabolism were examined. Pretreatment of male mice with sodium selenite (2 mg/kg, ip) 24 hr before morphine administration significantly protected them against the hepatotoxic effects of morphine as evidenced by decreased plasma alanine and aspartate transaminase values. This treatment inhibited the decrease in hepatic glutathione content that occurs in animals receiving hepatotoxic doses of morphine. Selenium pretreatment decreased the in vivo covalent bonding of morphine to hepatic proteins. Selenium also lowered the in vitro covalent bonding of morphine to hepatic microsomal proteins.

Mouse brain opioid receptor identification by direct ultraviolet photoaffinity labeling

[3H]Morphine, PL-017[prolyl-3,4-3H,D-prolyl,3,4-3H] ([3H]PL-017) and enkephalin-(2-D-penicillamine,5-D-penicillamine)[tyrosyl-2,6-3H] ([3H]DPDPE) were directly cross-linked to mouse brain opiate receptors by an ultraviolet (254 nm) irradiation procedure. [3H]Morphine preferentially and specifically labeled a 58 kDa protein. The labeling of this protein was suppressed by the addition of excess naloxone. Dithiothreitol reduced the irreversible binding of [3H]morphine and [3H]PL-017 to the receptor protein. In the acid hydrolysate of [3H]DPDPE-labeled opiate receptors, dityrosine, was detected. These results suggest that the [3H]tyrosine residue of [3H]DPDPE covalently bound the tyrosine residue of delta-opioid receptors. The direct UV-photoaffinity labeling method using commercially available radioactive opiates described here should be a useful tool for characterization and purification of the opiate receptors.