Br-DIF-1 accelerates dimethyl sulphoxide-induced differentiation of P19CL6 embryonic carcinoma cells into cardiomyocytes.

BACKGROUND AND PURPOSE: Stem cell transplantation therapy is a promising option for treatment of severe ischaemic heart disease. Dimethyl sulphoxide (DMSO) differentiates P19CL6 embryonic carcinoma cells into cardiomyocyte-like cells, but with low differentiation capacity. To improve the degree of this differentiation, we have assessed several derivatives of the differentiation-inducing factor-1 (DIF-1), originally found in the cellular slime mould Dictyostelium discoideum, on P19CL6 cells. EXPERIMENTAL APPROACH: P19CL6 cells were cultured with each derivative and 1% DMSO for up to 16 days. Differentiation was assessed by measuring the number of beating and non-beating aggregates, and the expression of genes relevant to cardiac tissue. The mechanism of action was investigated using a T-type Ca(2+) channel blocker. KEY RESULTS: Of all the DIF-1 derivatives tested only Br-DIF-1 showed any effects on cardiomyocyte differentiation. In the presence of 1% DMSO, Br-DIF-1 (0.3-3 µM) significantly and dose-dependently increased the number of spontaneously beating aggregates compared with 1% DMSO alone, by day 16. Expression of mRNA for T-type calcium channels was significantly increased by Br-DIF-1 + 1% DMSO compared with 1% DMSO alone. Mibefradil (a T-type Ca(2+) channel blocker; 100 nM) and a small interfering RNA for the T-type Ca(2+) channel both significantly decreased the beating rate of aggregates induced by Br-DIF-1 + 1% DMSO. CONCLUSIONS AND IMPLICATIONS: Br-DIF-1 accelerated the differentiation, induced by 1% DMSO, of P19CL6 cells into spontaneously beating cardiomyocyte-like cells, partly by enhancing the expression of the T-type Ca(2+) channel gene.

Tissue distribution of YM758, a novel If channel inhibitor, in pregnant and lactating rats.

In this study the tissue distribution of radioactivity in pregnant and lactating rats was investigated by quantitatively determining radioactivity concentrations and by whole-body autoradioluminograms after a single oral administration of 14C-YM758. In addition, the transfer of radioactivity into the reproductive tissues, foetus, and milk is discussed in terms of the localization of transporters in syncytiotrophoblast and mammary gland. The radioactivity concentrations in the liver were the highest of all the tissues and organs tested at all the sampling times. The radioactivity in main tissues (liver and kidney), including reproductive tissues (amniotic fluid, placenta, ovary, and uterus), was not retained for a long time, as in the plasma. The tissue/plasma (T/P) ratio of radioactivity in the foetus was below 1.0, which might be due to Mdr1-mediated export of YM758 into blood via the blood-placenta barrier since YM758 is a substrate for hMDR1, not for hBCRP/rBcrp. The T/P ratio of radioactivity in the maternal milk 1 and 4 h after oral administration of 14C-YM758 was 7.2 and 11.0, respectively. To understand better the distribution of new drugs into the reproductive tissues/milk, and to interpret further the results of reproductive safety studies for drug development, the contribution of transporters expressed in the blood-placenta barrier and mammary gland to the drug-transfer into placenta and milk should be considered.

Comparative evaluation of absorption, distribution, and excretion of YM758, a novel If channel inhibitor, between albino and non-albino rats.

1. YM758 is a novel If channel inhibitor for the treatment of stable angina and atrial fibrillation. The absorption, distribution, and excretion of YM758 have been investigated in albino and non-albino rats after a single oral administration of (14)C-YM758 monophosphate. 2. YM758 was well absorbed from all segments of the gastrointestinal tract except for the stomach. After oral administration, the ratio of AUC(0-1 h) between the plasma concentrations of radioactivity and the unchanged drug was estimated to be 17.7%, which suggests metabolism. 3. The distribution of the radioactivity derived from (14)C-YM758 in tissues was evaluated both in albino and non-albino rats. The radioactivity concentrations in most tissues were higher than those in plasma, which indicates that the radioactivity is well distributed to tissues. Extensive accumulation and slower elimination of radioactivity were noted in the thoracic aorta of albino and non-albino rats as well as in the eyeballs of non-albino rats. The recovery rates of radioactivity in urine and bile after oral dosing to bile duct-cannulated albino rats were 17.8% and 57.3%, respectively. 4. These results suggest that YM758 was extensively absorbed, subjected to metabolism, and excreted mainly into the bile after oral administration to rats, and extensive accumulation of the unchanged drug and/or metabolites into tissues such as the thoracic aorta and eyeballs was observed.

A chemiluminescent assay for hydroperoxide level of phosphatidylcholine hydroperoxide fraction purified by two Sep-Pak cartridges in biological samples.

A chemiluminescent assay for hydroperoxide level of phosphatidylcholine hydroperoxide (PCOOH) fraction purified from biological samples was presented. This method utilized of two Sep-Pak cartridges. A lipid soluble fraction was isolated from each homogenized tissue or blood by Folch's method. The mixture of phosphatidylcholine (PC) and PCOOH was separated from the lipid soluble fraction by a Sep-Pak silica cartridge. A Sep-Pak tC18 cartridge made complete separation of both PCOOH and PC possible. The hydroperoxide level of PCOOH fraction was quantified by the reaction with ferrous ion using 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin++ +-3-one as a chemiluminescent dye. The mixture of positional isomers, 1-hexadecanoyl-2-[9, or 10-hydroperoxyl octadecanoyl]-sn-glycero-3-phosphocholine was used as an authentic standard. The good recovery rate for authentic PCOOH of 87.1 +/- 11.6% (mean +/- S.E., n = 4) was obtained by using two Sep-Pak cartridges. Linear calibration curve was obtained in the range from 2.5 to 20 nmol, and the detection limit of the standard was 10 pmol (signal-to-noise ratio > 3). This method was applied to the investigation of the lipid peroxidation induced by reperfusion of the liver with cold preservation, mimicking liver transplantation in rats. The effect of liposome-encapsulated dichloromethylene diphosphonate (LEDD), which eliminate of Kupffer cells to prevent the generation of oxygen radicals on the lipid peroxidation, was compared with the untreated group as a control. After 1 h reperfusion at 37 degrees C the hydroperoxide level obtained the liver without preservation in the untreated group was 12.4 +/- 2.4 nmol/100 mg lipid (n = 4) and levels increased significantly by prolongation of the preservation time. On the other hand, the hydroperoxide level in the LEDD treated group did not change up to 24 h preservation. These results suggest that this improved assay for hydroperoxide level of PCOOH fraction in biological samples can be applied to investigations involving lipid peroxidation because of its simplicity and accuracy.

Novel acyl alpha-pyronoids, dictyopyrone A, B, and C, from Dictyostelium cellular slime molds.

For the elucidation of the diversity of secondary metabolites of Dictyostelium cellular slime molds, we investigate the constituent of three species of slime molds. From the methanol extract of their fruit bodies, we obtained three novel compounds, dictyopyrone A (1) and B (2) from D. discoideum and D. rhizoposium and dictyopyrone C (3) from D. longosporum. They possess a unique alpha-pyrone moiety with a side chain at the C-3 position. Their relative structures were elucidated by spectral means, and the absolute configuration was confirmed by asymmetric synthesis of 1. Since these compounds were obtained from different species of Dictyostelium slime molds, they may be a type of compound common to this genus.

A fluorometric assay for cyclic guanosine 3',5'-monophosphate incorporating a Sep-Pak cartridge and enzymatic cycling.

We developed a sensitive and nonradioactive fluorometric assay for cyclic guanosine 3',5'-monophosphate (cGMP). Guanine nucleotides except cGMP were enzymatically phosphorylated to GTP. cGMP, absorbed into a Sep-Pak amino propyl cartridge, was eluted separately from GTP. Purified cGMP was enzymatically converted to GTP, which was applied to the GTP-GDP cycle using succinic thiokinase and pyruvate kinase. When pyruvic acid produced by the GTP-GDP cycle was reduced by lactate dehydrogenase, a reduced form of nicotinamide adenine dinucleotide (NADH) was equivalently oxidized to NAD(+). NAD(+) was further converted into fluorescent compound, which was excited at 370 nm and emitted fluorescence at 460 nm, by a strong alkali. When 20 nmol NADH was used for this assay, the calibration curve over 50 to 500 fmol cGMP became sufficiently linear. The detection limit for cGMP was ca. 5 fmol (signal to noise ratio >3). Using this assay, we confirmed that the cGMP content in the left atrial strip of dog was changed from 11.4 +/- 3.8 to 19.3 +/- 2.6 fmol/mg wet wt of tissue (mean +/- SE, n = 6) by electrical driving at 1 Hz. Carbachol (1 microM) further increased the cGMP to 45.6 +/- 9.2 fmol/mg wet wt of tissue. From these results, it is suggested that this novel assay for cGMP is highly sensitive and can be applied to various biological samples.

Fragility of the electron transport chain and superoxide generation in mitochondria of the liver graft after cold ischemia.

BACKGROUND: After cold ischemia, electrons transferred in the electron transport chain may leak out of the mitochondria in proportion to the deterioration of mitochondrial oxidative phosphorylation. This seems to be one major cause of the lipid peroxidation that occurs mainly in the hepatocytes at reperfusion in liver transplantation. To examine this hypothesis, we investigated superoxide generation and the amount of oxidative phosphorylation in the mitochondria isolated from rat livers after cold preservation. METHODS: Rat liver was preserved in University of Wisconsin solution at 4 degrees C for 24 hr. The mitochondrial fraction was prepared, and the amount of ATP synthesis and superoxide generation was investigated. Superoxide generation in the electron transport chain of submitochondrial particles was also measured by a chemiluminescence recorder. RESULTS: The amount of ATP synthesis was significantly decreased after 12 hr of cold preservation. In the whole mitochondria, superoxide production in the presence of succinate was approximately 1/2000 to 1/3000 less than that observed in the submitochondrial particles at any determination point, and superoxide production was not affected by cold preservation. In the presence of antimycin A, superoxide production in the mitochondria after 18 hr of preservation increased significantly. CONCLUSION: These results indicate that the electron transfer in the complex III of the mitochondrial membrane becomes leaky after long periods of cold ischemia, but that leakage of superoxide anion did not increase, although the mitochondrial respiratory phosphorylation was deteriorated. We conclude that superoxide through the mitochondrial membrane cannot cause lipid peroxidation in hepatocytes at reperfusion even after a long period of cold ischemia.

Norepinephrine measurement in the isolated, blood-perfused papillary muscle of the dog by using ex vivo microdialysis.

Subendocardial interstitial norepinephrine (NE) was measured in the isolated, blood-perfused papillary muscle (PM) preparation of the dog by using ex vivo microdialysis. A microdialysis fiber was inserted into the base of the PM and perfused with Ringer's solution at a rate of 1 microl/min. Dialyzed NE concentrations were measured by high-performance liquid chromatography with an electrical detector. The basal dialyzed NE concentration from the subendocardial interstitium was 1.74+/-0.24 nM (n = 12, mean +/- SE). When the anterior septal artery was occluded for 5 min, the dialyzed NE concentration from the subendocardial interstitium 0-5 min after occlusion increased to 2.90 +/-0.61 nM (n = 12, p<0.05 versus before occlusion). When desmethylimipramine, a neuronal uptake 1 inhibitor, was administered into the anterior septal artery at a rate of 100 nmol/ml for 30 min, dialyzed NE concentration substantially increased from 1.55+/-0.33 to 2.63+/-0.34 nM (n = 3, p<0.05). Likewise, the occlusion-induced increase in dialyzed NE was augmented to 3.75+/-0.90 nM by desmethylimipramine infusion into the anterior septal artery. These observations suggested that the ex vivo microdialysis of the isolated, blood-perfused PM preparation of the dog is a sensitive method for measuring the subendocardial interstitial NE and that coronary artery occlusion increases the subendocardial interstitial NE as early as within 5 min.

Pharmacological properties of pteleprenine, a quinoline alkaloid extracted from Orixa japonica, on guinea-pig ileum and canine left atrium.

We have investigated the pharmacological properties of pteleprenine, a quinoline alkaloid, on contractile responses of the guinea-pig ileum and on inotropic responses of the canine left atrium. Although pteleprenine (0.1-1 microM) had no effect on the contraction of the ileum induced by acetylcholine at 10 microM it significantly inhibited acetylcholine-induced contraction of the ileum. Pteleprenine (0.1-10 microM) reduced nicotine induced-contraction of the ileum in a concentration-dependent manner yet had no maximum relaxant effect even at a concentration of 10 microM. From Schild analysis the pA2 of pteleprenine on the guinea-pig ileum was found to be 6.6. The contraction of the ileum induced by 10 microM 1,1-dimethyl-4-phenylpiperazinium, a specific agonist of nicotinic acetylcholine receptors, was concentration-dependently suppressed by 10 nM-10 microM pteleprenine. In contrast, 0.1-10 microM pteleprenine did not antagonize the acetylcholine- and nicotine-induced negative inotropic contractile responses of the canine left atrium. These results show that pteleprenine has inhibitory action against nicotinic acetylcholine receptors in the guinea-pig ileum but not in the canine left atrium. Our findings also suggest that pteleprenine might be a novel lead compound as a nicotinic receptor antagonist.

Kupffer cells generate superoxide anions and modulate reperfusion injury in rat livers after cold preservation.

This study was designed to determine the source of reactive oxygen species (ROS) and whether Kupffer cells modulate the injury of perfused rat liver after cold preservation. The livers of male Lewis rats pretreated with schizophyllan glucan (SPG) (10 mg/kg, SPG group) or gadolinium chloride (5 mg/kg; Gd group) and untreated rats (control group) were preserved in University of Wisconsin solution for 0, 12, and 24 hours at 4 degrees C and then perfused for 60 minutes with oxygenated Krebs-Henseleit bicarbonate buffer. Real-time chemiluminescence (CL) of the liver during perfusion was measured using a sensitive photomultiplier, and reperfusion injury was assessed by measuring lipid peroxidation and lactate dehydrogenase release. CL intensity reached a peak within 5 minutes of reperfusion, and superoxide dismutase completely inhibited this CL in all groups. In the control group, the total CL intensity was high after 24 hours of preservation. It significantly (P < .05 vs. control group) increased in the SPG group, while it decreased in the Gd group after 12 and 24 hours of preservation. The total CL intensity decreased by 70% (when diphenyliodonium chloride (100 micromol/L; an NADPH oxidase inhibitor) was added to the perfusate before preservation of untreated rats. Lipid peroxidation and lactate dehydrogenase release significantly (P < .05) deteriorated in the SPG group, while they ameliorated in the Gd group after 24 hours of preservation. These results demonstrate that Kupffer cells primarily generate superoxide anions and modulate the organ injury in the initial phase of reperfusion after cold preservation.

Analysis of bilirubin fraction in the bile for early diagnosis of acute rejection in living related liver transplantation.

The diagnosis of acute rejection in liver transplantation usually needs hepatic biopsy, but hepatic biopsy sometimes involves severe complications. We analyzed biliary bilirubin fraction after living related liver transplantation by using high performance liquid chromatography (HPLC) and investigated availability for the early diagnosis of acute rejection retrospectively. Nine children with liver cirrhosis due to biliary atresia were included in this study, who underwent living related liver transplantation at The Second Department of Surgery, Tohoku University School of Medicine. Bile was collected daily from a biliary canulae inserted into the hepatic duct of the graft under aseptic and without exposure to the light. We measured the proportion of bilirubin diglucuronide (BDG), bilirubin monoglucuronide (BMG) and unconjugated bilirubin (UCB) of bile pigments in the bile by HPLC. In three of four patients with acute rejection, BDG + BMG (= Bc) was above 85% and BDG/Bc ratio was below 0.6 at the time of hepatic biopsy. After rejection therapy, BDG/Bc ratio increased in their bile. The remaining one case with acute rejection as well as bile duct injury due to arterial thrombosis of S2, Bc was below 85%, and BDG/Bc ratio was below 0.6. In four of the other five patients who had several severe complications, i.e., arterial or portal vein thrombosis, bile stasis due to cholangitis and sepsis due to necrotizing myofascitis, Bc was below 85% and BDG/Bc ratio was below 0.6. We concluded that analysis of biliary bilirubin fraction after liver transplantation could be reliable as a noninvasive maker and valuable for the early diagnosis of acute rejection.

[Pharmacological properties of pteleprenine, a quinoline alkaloid from Orixa japonica (Rutaceae), in guinea pig ileum and canine left atrium].

We investigated the pharmacological properties of pteleprenine, a quinoline alkaloid contained in the Rutaceous plant, Orixa japonica, on the contractile responses of the guinea pig ileum and on the inotropic responses of the canine left atrium. Contractile responses of the ileum to acetylcholine and histamine were not inhibited by less than 10(-6) M of pteleprenine. Meanwhile, the nicotine induced-contraction of the ileum was dose-dependently diminished by pteleprenine, but the contractile response to nicotine did not reach the maximum value in the presence of 10(-5) M of pteleprenine. The pA2 value of pteleprenine was 6.6 as determined from the Schild plot, which slope was nearly unity. Furthermore, the contraction of the ileum by 10(-5) M of 1,1-dimethyl-4-phenylpiperazinium (DMPP), a specific agonist of nicotinic acetylcholine receptors, was also dose-dependently suppressed by pteleprenine. On the other hand, 10(-5) M of pteleprenine did not have considerable inhibitory effects on acetylcholine- and nicotine-induced negative inotropic effects in the canine left atrium. From these results, it is suggested that pteleprenine has a specific inhibitory effect on nicotinic acetylcholine receptors in the guinea pig ileum.

Changes in the ability of ATP synthesis in the mitochondrial membrane in the rat liver injured by carbon tetrachloride.

We investigated the changes in the ability of adenosine triphosphate (ATP) synthesis in the inner mitochondrial membrane with the acute and chronic liver injuries by carbon tetrachloride (CCl4) in rats. The ability of ATP synthesis was evaluated by measuring the activity of proton ATPase and the ability of oxygen consumption using isolated mitochondria. In the acute liver injury, the ability of ATP synthesis at 6 hr after injection was preserved relatively well in spite of hepatocyte injuries. On the other hand, at 12 hr after CCl4 injection, the necrosis of hepatocyte was widely recognized. In the chronic liver injury, severe fibrosis was induced but the ability of ATP synthesis was kept as well as that in the normal liver. These results suggest that the mitochondria have some resistivity against radicals produced by CCl4.