Kouji Ohno

Metabolic dehydration of prostaglandin E2 and cellular uptake of the dehydration product: Correlation with prostaglandin E2-induced growth inhibition

When L-1210 murine leukemia cells were incubated with 60 microM PGE2 in culture medium containing fetal calf serum for various time, cell proliferation was inhibited dependent on incubation time. However, when the medium containing PGE2 was changed every 6 h during 24 h exposure to cells, growth inhibition became much weaker. Moreover, when the medium containing PGE2 was aged by preincubating without cells at 37 degrees C, growth inhibitory effect of the medium was enhanced with preincubation time, suggesting that active growth inhibitory compound(s) accumulated during preincubation. In culture medium containing fetal calf serum, PGE2 degraded time-dependently and the major product was identified as PGA2 by HPLC. Furthermore, when cells were incubated with the medium containing 60 microM[3H]PGE2 or the same medium aged by preincubation, we observed that the radioactivity was taken up by the cells time-dependently, and identified the incorporated radioactivity as PGA2. This uptake was closely correlated with decrease in viable cell number during incubation. These results suggested that growth inhibitory effect of PGE2 was due to the metabolic dehydration of PGE2 to PGA2, and PGA2, after taken up by cells, exerted cell growth inhibition.

Induction of γ-glutamylcysteine synthetase by prostaglandin A2 in L-1210 cells

Abstract Effects of prostaglandin A2 (PGA2) on glutathione (GSH) status in L-1210 cells were examined. When the cells were cultured in the presence of PGA2, a persistent rise of cellular GSH concentration was observed 6 h after the addition of PGA2. This stimulatory effect of PGA2 was abolished if the cells were pretreated with an enzyme inhibitor of GSH synthesis, buthionine sulfoximine. Subsequent study with cell free extract of cultured L-1210 has revealed that PGA2 stimulated the biosynthesis of γ-glutamylcysteine synthetase (EC 6.3.2.2). Actinomycin D inhibited this stimulatory effect of PGA2 on cultured cells. The optimal pH, Km value for glutamic acid and sensitivity to inhibitors of γ-glutamylcysteine synthetase from PGA2 treated and nontreated cells were virtually the same. Thus, our findings suggest that PGA2 induced γ-glutamylcysteine synthetase in cultured L-1210 cells which is responsible for the elevated level of GSH in these cells.

Induction of intestinal cytochrome P450 (CYP3A) by rifampicin in beagle dogs.

Both male and female beagle dogs (four dogs/sex) were orally treated with rifampicin (Rif) at the dose of 10 mg/kg/day for 7 days and an additional eight dogs (four dogs/sex) were used as a control. The inducible effect of Rif on intestinal cytochrome P450, especially CYP3A enzyme, was investigated by measuring microsomal testosterone 6beta-hydroxylation (6beta-OHT) activity, immunoblot and ELISA analysis. In male dogs, microsomal 6beta-OHT activity in the duodenum, upper, middle and lower part of the jejunum and the ileum of the control was 229, 204, 194, 129 and 57 pmol/min/mg protein, while the activity of the Rif-treated dogs significantly increased to 456, 486, 430, 192 and 138 pmol/min/mg protein, respectively. The activity of intestinal 6beta-OHT in the control and Rif-treated female dogs showed almost similar levels to those observed in the corresponding male dogs. The activity of intestinal 6beta-OHT in both control and Rif-treated dogs was specifically inhibited by anti-CYP3A12 antiserum. The apparent K(m) value for 6beta-OHT activity in all sections of the small intestine was comparable with that in the liver, and no significant changes were observed in between control and Rif-treated dogs. In both control and Rif-treated dogs, immunoblotting of intestinal microsomes with anti-CYP3A12 antiserum produced a band indistinguishable from that of purified CYP3A12 or of immunoreactive CYP3A12 in liver microsomes. A significant increase in intestinal CYP3A content by Rif treatment was quantitatively verified by the ELISA analysis and the magnitude of its increase correlated well with that of 6beta-OHT activity elevation. Furthermore, the results of immunohistochemistry using the anti-CYP3A12 antiserum indicated that CYP3A protein was specifically distributed in epithelial cells throughout the small intestine and appeared to be predominant at the apical side of villus cells. These results demonstrate that Rif induces not only hepatic CYP3A12 but also intestinal CYP3A in dogs.

Characterization of the transport system of prostaglandin A2 in L-1210 murine leukemia cells.

Prostaglandin (PG) A2 has been shown to be actively incorporated into mammalian cells and transferred to the cell nucleus. To characterize the properties of the PGA2 transfer system, we examined the status of PGA2 in L-1210 cells with modified cellular glutathione (GSH) levels. PGA2 in the cytosol fraction of the cells existed in its free-form, the GSH conjugate-form and a macromolecule associate-form (protein bound-form). When the GSH level was lowered under culture conditions, the amount of free-form increased while that of the protein bound-form was unchanged. When PGA2-loaded cells were incubated in a salt solution, free- and conjugate-forms were emitted from the cells. A concomitant decrease and increase of protein bound PGA2 in cytosol and nuclei, respectively, were observed. Subsequent studies with isolated cellular fractions revealed that PGA2 bound to cytosolic protein was transported into the nuclear interior in a temperature-dependent manner. The binding of PGA2 to the protein and subsequent transport to the nuclei were inhibited by PGJ2 and 4-hydroxy-cyclopentenone, but not by PGB2, PGD2, PGE2, PGF2 alpha, arachidonic acid or oleic acid. N-Ethylmaleimide (NEM) and p-chloromercuribenzoate (PCMB) strongly interfered with these transfer processes, suggesting that sulfhydryl components are involved in the transport of PGA2. Subfractionation of cytosol by gel chromatography proved the presence of two proteins (100-150 kDa and 25-35 kDa) that are capable of transporting PGA2 to cell nuclei. Though 40-45 kDa proteins, which correspond to GSH S-transferases, bound to PGA2, they lacked the nuclear transport activities.

Protective effect of prostaglandin A2 against menadione-induced cell injury in cultured porcine aorta endothelial cells

Prostaglandin A2 (PGA2) stimulates the biosynthesis of gamma-glutamylcysteine synthetase and elevates glutathione (GSH) contents in cultured mammalian cells. To clarify the importance of gamma-glutamylcysteine synthetase induction in the defence of endothelial cells against oxidative stress, the effect of PGA2 on menadione (2-methyl-1,4-naphthoquinone)-induced cell injury was examined. Incubation of porcine aorta endothelial cells with menadione produced marked loss of cellular GSH and protein sulfhydryl groups, followed by leakage of lactic dehydrogenase (LDH) into the culture medium. The LDH leakage and modification of protein thiol was, however, completely prevented by pretreatment of the cells with PGA2. The protective effect of PGA2 was more potent than that of cysteine delivery agents such as methionine, N-acetylcysteine or 2-oxo-4-thiazolidine carboxylic acid (OTC). The results suggest that cellular GSH plays an important role in the defence against oxidative stress, and induction of gamma-glutamylcysteine synthetase is effective for protecting vascular endothelial cells.

Subcellular distribution and isoelectric heterogeneity of the substrate for ADP-ribosyl transferase from Clostridium botulinum

When the homogenate of bovine adrenal gland was subjected to subcellular fractionation, an Mr 21,000 substrate for botulinum ADP-ribosyl transferase was found not only in the membrane fractions but also in the cytosol; the amounts in the 10,000 x g precipitates and the 100,000 x g supernatant were about 21 and 56% of the total amount, respectively. Each fraction gave a single ADP-ribosylated protein band on SDS-polyacrylamide gel electrophoresis, but yielded on isoelectric focusing at least three bands between pH 5.5 and 6.0, suggesting the presence of multiple forms of the substrate of a similar molecular weight but different isoelectric points. ADP-ribosylated protein bands from the membrane and cytosol overlapped each other on both electrophoreses. After ammonium sulfate fractionation, the substrate from the cytosol showed requirement of divalent cations or guanine nucleotides for the reaction. Among cations tested, calcium, magnesium and manganese stimulated, whereas cadmium and lanthanum inhibited the reaction. Guanine nucleotides such as GTP, GDP and GTP-gamma-S also stimulated the substrate activity in the cytosol as that in the membrane fraction. However, no additive effects were observed when the nucleotides and cations were added together.

Cellular Uptake and Nuclear Accumulation of Prostaglandin A and J, a Mechanism of Prostaglandin-Induced Growth Inhibition

Prostaglandins (PGs) of the E, A and D series have been shown to cause inhibition of growth in cultured cells and to induce differentiation in several cases (Jaffe and Santoro 1977; Honn et al. 1981; Narumiya et al. 1986a). Since some types of PGs, particularly PGE and D, stimulate adenylate cyclase in various systems, it has been suggested that PG-induced growth inhibition is mediated by intracellular cyclic AMP (cAMP) (Johnson and Pastan 1971). This hypothesis, however, has been questioned recently. Bregman et al. (1982) could not find any correlation between the intracellular level of cAMP and growth inhibition in experiments using PGA and E-derivatives. Wiley et al. (1983) used BHK-21 cells, the cAMP response of which had been desensitized to PGE1, and showed that PGE1 still caused inhibition of DNA synthesis in these cells. The same authors further used S-49 cyc - cells which lacked the N-component of adenylate cyclase and did not increase cAMP in response to PG, and found that inhibition of DNA synthesis by PGA1 still occurred (Hughes-Fulford et al. 1985). Thus, the mechanism of PG-induced growth inhibition has not been elucidated as yet. During our study on PGD2 -induced growth inhibition we found that this PG underwent enzymatic dehydration in serum to 9-deoxy-Δ9,12-13,14-dihydro-PGD2 (Δ12-PGJ2) (Fig. 1) (Kikawa et al. 1984). This dehydration product is devoid of most conventional PGD2 activities including stimulation of adenylate cyclase, but has several times stronger growth inhibitory activity than PGD2, suggesting that growth inhibition is mediated via a mechanism completely different from other PG actions (Kikawa et al. 1984; Narumiya and Toda 1985).