Akira Takanaka

Effects of ATP antagonists on purinoceptor-operated inward currents in rat phaeochromocytoma cells

The effects of suramin, reactive blue 2 (RB2) and d-tubocurarine (d-TC) were investigated electrophysiologically to elucidate the mechanisms underlying their antagonism of P2 purinoceptor-mediated responses. All three compounds inhibited an adenosine triphosphate (ATP)-activated inward current in rat phaeochromocytoma PC12 cells in a concentration-dependent manner. The order of potency was RB2 > suramin > d-TC. The inhibition induced by suramin or RB2 was reversible, whereas that induced by d-TC was not reversed after a 5-min rinse. The inactivation of the ATP-activated current was accelerated by d-TC but not by suramin or RB2. RB2 administered simultaneously with ATP exerted much weaker inhibition compared to that induced by prior administration, suggesting that RB2 is a slowly acting antagonist. This was not observed for suramin or d-TC. Suramin and RB2 caused a parallel shift in the concentration/response curve for the ATP-activated current. With d-TC the maximal response of ATP was decreased but the concentration producing half-maximal response was unchanged. The voltage dependency of the ATP-activated current showed less inward rectification in the presence of d-TC. Suramin or RB2 did not affect the voltage dependency. These results suggest that suramin and RB2 reversibly block binding of ATP to receptors, whereas d-TC blocks ion permeability through the ATP-activated channel.

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.

Reversible and selective antagonism by suramin of ATP-activated inward current in PC12 phaeochromocytoma cells.

1 The effects of suramin on an adenosine 5′‐triphosphate (ATP)‐ activated inward current were investigated in PC12 phaeochromocytoma cells with whole‐cell voltage‐clamp techniques. 2 Suramin (30 to 300 μm) inhibited the ATP‐activated current in a dose‐dependent manner. The inhibitory effects were reversible and competitive. 3 Suramin also suppressed the current activated by adenosine 5′‐0‐(3‐thio triphosphate) but did not affect the current activated by nicotine. Suramin did not affect the suppression of a K current induced by methacholine. 4 The results suggest that suramin antagonizes the ATP‐receptor‐operated membrane current reversibly and selectively.

Selective and competitive antagonism by suramin of ATP‐stimulated catecholamine‐secretion from PC12 phaeochromocytoma cells

1 Suramin, a putative P2‐antagonist, (10 to 300 μm) inhibited the adenosine 5′‐triphosphate (ATP)‐stimulated secretion of [3H]‐noradrenaline or endogenous dopamine from phaeochromocytoma PC12 cells in a concentration‐dependent manner. Suramin (300 μm) did not affect the dopamine‐secretion stimulated by high K+ or nicotine. 2 Suramin shifted the concentration‐response curve for ATP to the right. The antagonism was competitive with a pA2 value of 4.52. 3 ATP also stimulated an increase in intracellular Ca2+ concentration as determined by fura‐2 methods. Suramin antagonized this effect over the same concentration range that antagonized the ATP‐stimulated catecholamine secretion. 4 These results suggest that suramin can be used as a selective and competitive antagonist of ATP in experiments concerning mechanisms of catecholamine‐secretion.

Complete cDNA sequence of a major 3-methylcholanthrene-inducible cytochrome P-450 isozyme (P-450AFB) of Syrian hamsters with high activity toward aflatoxin B1

Cytochrome P-450AFB is major isozyme inducible by 3-methylcholanthrene in Syrian golden hamsters and shows high potency toward aflatoxin B1 activation. We have isolated and sequenced cDNA clones to P-450AFB by immunoscreening a hamster liver cDNA library in lambda gt11. The longest clone contains an open reading frame of 1482 nucleotides and encodes a protein of 494 amino acids with a molecular weight of 57,420. The sequence of P-450AFB shares a 73% and 65% homology with that of mouse P-450 15 alpha (IIA3) and rat P-450a (IIA1), respectively, indicating that P-450AFB is a unique gene of the P-450IIA subfamily. The apparent concentration of a mRNA species hybridizable to the clone as well as the concentration of a protein immunoreactive to P-450AFB was increased significantly by the treatment with 3-methyl-cholanthrene, which indicates that the increase in P-450AFB protein is due mainly to an elevation of the mRNA.

Effect of adrenalectomy or alloxan diabetes on the substrate interaction with cytochrome p-450 in the oxidation of drugs by liver microsomes☆

Abstract Adrenalectomy or alloxan diabetes markedly decreased the magnitude of cytochrome P-450 spectral change induced by hexobarbital or aminopyrine in male rat liver microsomes. The decrease in the binding capacity of cytochrome P-450 for hexobarbital or aminopyrine was assumed to be responsible. On the other hand, the binding capacity of cytochrome P-450 for aniline or zoxazolamine was not significantly affected by adrenalectomy or alloxan diabetes. In contrast to male rats, the binding capacity by the liver of female rats to cytochrome P-450 was not affected by adrenalectomy or alloxan diabetes. The K m (Michaelis constant) value for hexobarbital hydroxylation and the K s (spectral dissociation constant) value for the hexobarbital-induced spectral change were increased in liver microsomes from the adrenalectomized or diabetic male rats, whereas the K m and K s , values for aniline were not affected. These results suggest that the decrease in the binding capacity of cytochrome P-450 for hexobarbital or aminopyrine is a factor responsible for the decrease in the oxidation of drugs by liver microsomes from the adrenalectomized or diabetic male rats. The impairment of the androgen-dependent regulatory mechanism for the binding capacity of cytochrome P-450 was assumed to be a factor responsible for the decrease in the binding capacity in liver microsomes from the adrenalectomized or diabetic rats.

Species differences in the effect of morphine administration or adrenalectomy on the substrate interactions with cytochrome P-450 and drug oxidations by liver microsomes.

Abstract The hexobarbital hydroxylation and the magnitude of hexobarbital interaction with cytochrome P-450 were markedly decreased in liver microsomes from morphine-treated male rats, whereas they were not decreased in liver microsomes from morphine-treated female rats, male and female mice and rabbits. The binding capacity of cytochrome P-450 for hexobarbital calculated from the magnitude of hexobarbital interaction with cytochrome P-450 in liver microsomes was also decreased only in the morphine-treated male rats. Similarly, the hexobarbital hydroxylation, the magnitude of hexobarbital interaction with cytochrome P-450 and the binding capacity of cytochrome P-450 in liver microsomes were decreased only in adrenalectomized male rats, but not in adrenalectomized female rats, male and female mice. In contrast to the results obtained with hexobarbital, the aniline hydroxylation, the magnitude of aniline interaction with cytochrome P-450 and the binding capacity of cytochrome P-450 for aniline were not decreased in liver microsomes from male and female rats, mice and rabbits by the morphine treatment or adrenalectomy. These results therefore support the previous suggestion that the decrease in the hexobarbital hydroxylation and in the magnitude of hexobarbital interaction with cytochrome P-450 is mainly due to the decrease in the binding capacity of cytochrome P-450 for hexobarbital, probably through an impairment of the action of androgen to increase the binding capacity of cytochrome P-450 for hexobarbital.

Characterization of benzo[a]pyrene metabolism and related cytochrome P‐450 isozymes in Syrian hamster livers

Cytochrome P-450 monooxygenases of golden Syrian hamsters were characterized with respect to benzo[a]pyrene metabolism. Male hamsters were treated with phenobarbital, 3-methylcholanthrene, dexamethasone, benzoflavone, or ethanol, and the activity of aryl hydrocarbon hydroxylase and benzo[a]pyrene activation was determined by mutagenicity testing in hepatic microsomes. Aryl hydrocarbon hydroxylase activity was induced markedly by treatment with phenobarbital but not with 3-methylcholanthrene, nor with other chemicals. The degree of benzo[a]pyrene activation on mutagenicity testing was significantly elevated by treatment with 3-methylcholanthrene and phenobarbital but was reduced with dexamethasone. Immunoinhibition of these activities and Western blotting of hepatic microsomes using antibodies against cytochrome P-450 isozymes suggested that the isozymes responsible for benzo[a]pyrene metabolism in Syrian hamsters belong to the CYP1A, CYP2A, and CYP3A families, a result that differs from observations in rats.

Purification and characterization of a form of cytochrome P-450 with high specificity for aflatoxin B1 from 3-methylcholanthrene treated hamster liver

A form of cytochrome P-450 highly active in inducing mutagenicity of aflatoxin B1 was purified to a specific content of 15.1 nmol/mg of protein from 3-methylcholanthrene-treated hamster liver. This species of cytochrome P-450, having its absorption maximum at 448.5 nm in carbon monoxide-complex of reduced form and low spin ferric ion, is of molecular weight of 56,000 and distinctly different in physicochemical and catalytic properties from major forms of cytochrome P-450 purified from phenobarbital- or 3-methylcholanthrene-treated rat liver. In the induction of aflatoxin B1 mutagenicity, this hamster cytochrome P-450 is 50 times more potent than those from rat liver.

N-Nitrosodialkylamine dealkylation in reconstituted systems containing cytochrome P-450 purified from phenobarbitaland β-naphthoflavone-treated rats

Five cytochrome P-450 forms were purified from livers of rats pretreated with phenobarbital (PB) or β-naphthoflavone (BNF), and the oxidative dealkylation of N-nitrosodialkylamines by the reconstituted cytochrome P-450 systems was measured. PB-II (P450IIB1) showed very high N-nitrosomethybutylamine (NMBA) debutylase activity, high NMBA demethylase activity and high N-nitrosomethyl-benzylamine (NMBeA) debenzylase activity, suggesting that the increase following PB treatment in hepatic microsomal NMBA debutylation and NMBeA debenzylation was due to the induction of PB-II. BNF-H (P450IA2) showed very high NMBA debutylase and high NMBeA debenzylase activities, and BNF-L (P450IA1) showed NMBA debutylase and high NMBeA debenzylase activities. These results suggested that the increase by BNF pretreatment in hepatic microsomal NMBA debutylation was due mainly to the induction of BNF-H and in some part to that of BNF-L. PB-II also showed very high dealkylation activity of lipophilic N-nitrosodialkylamines with long alkyl moieties. On the other hand, BNF-H dealkylated N-nitrosodipropylamine (NDPA), N-nitrosomethylbutylamine (NMBA) and N-nitrosoethylbutylamine (NEBA) at higher rates than N-nitrosodibutylamine (NDBA). BNF-L dealkylated NEBA at higher rates than NMBeA and NDBA. These results reveal that substrate specificity of each cytochrome P-450 form in N-nitrosodialkylamine metabolism is different from each other and several forms of cytochrome P-450 support each N-nitrosamine dealkylase activity in mammalians.