Phosphoinositide 3-kinase accelerates necrotic cell death during hypoxia.

Using H9c2 cells derived from rat cardiomyocytes, we investigated the mechanism of cell death during hypoxia in the presence of serum and glucose. Hypoxic cell death is by necrosis and is accompanied by metabolic acidosis. Moreover, hypoxic cell death is inhibited by Hepes buffer as well as by 2-deoxyglucose, an inhibitor of glycolysis, indicating that metabolic acidosis should play an essential role in hypoxic injury. The involvement of phosphoinositide 3-kinase (PI 3-kinase), which is known to activate glucose metabolism, was examined using its inhibitor, LY290042, or adenovirus-mediated gene transfer. Hypoxic cell death was inhibited by LY294002 in a dose-dependent manner. Overexpression of dominant negative PI 3-kinase was found to reduce cell death, whereas wild-type PI 3-kinase enhanced it. Dominant negative PI 3-kinase also reduced glucose consumption and acidosis, but this was stimulated by wild-type PI 3-kinase. The data indicate that PI 3-kinase stimulates cell death by enhancing metabolic acidosis. LY294002 significantly reduced glucose uptake, showing that PI 3-kinase regulates glycolysis at the step of glucose transport. These findings indicate the pivotal role of glucose metabolism in hypoxic cell death, and reveal a novel death-promoting effect of PI 3-kinase during hypoxia, despite this enzyme being considered to be a survival-promoting factor.

Michaelis-Menten elimination kinetics of acetate during ethanol oxidation.

BACKGROUND: The redistribution of acetate cannot be explained using a linear kinetic model. We studied the pharmacokinetics of acetate during ethanol oxidation in the rabbit. METHODS: An ethanol saline solution (0.5, 1.5, and 2.5 g/kg) was injected bolus intravenously. We measured blood ethanol, acetaldehyde, and acetate concentrations by using head-space gas chromatography. RESULTS: Blood acetate concentration changed in three phases: an ascending, a plateau, and a declining phase. The first-order rate constant of the declining phase was smaller than that of the ascending phase and decreased dose dependently. Statistical moment analysis of the blood acetate profiles showed that the normalized area under the curve (AUC/Dose) and the mean residence time (MRT) increased with increasing dose amount. These increases suggest a capacity-limited elimination of acetate. We attempted simultaneous multiline fitting, using the three blood acetate disappearance curves, to determine the pharmacokinetic model. Consequently, the blood acetate profile was best described by a Michaelis-Menten elimination kinetic model. The Vmax and Km values of acetate elimination were 40.80 +/- 14.10 mM/hr and 0.47 +/- 0.19 mM, respectively. The fraction of a dose of ethanol converted to acetate (f(AcA)) was calculated to be 0.54. The estimated values are average parameter values of three different doses. Fitted curves suggest smaller f(AcA) at a low dose and larger f(AcA) at a higher dose, which indicate increases of accumulation and redistribution of acetate at higher doses. CONCLUSIONS: Acetate elimination during ethanol oxidation obeys capacity-limited kinetics.

Sevoflurane does not inhibit human platelet aggregation induced by thrombin.

BACKGROUND: Sevoflurane reportedly inhibits adenosine diphosphate-induced platelet aggregation by suppressing thromboxane A2 formation. The increase in intracellular calcium concentration that fosters platelet aggregation, however, is also induced by other cell signaling pathways, such as activation of the production of inositol 1,4,5-triphosphate by thrombin. The current study aimed to clarify the net influence of sevoflurane on thrombin-induced platelet aggregation. METHODS: Washed platelets were stimulated by thrombin after incubation with 0.5, 1.0, or 1.5 mM sevoflurane, halothane, or isoflurane. Aggregation curves were measured by an aggregometer. Intracellular calcium concentration was measured fluorometrically using fura-2. Calcium mobilization via plasma membrane calcium channels and the dense tubular system was assessed differentially. Intracellular inositol 1,4,5-triphosphate was measured by radioimmunoassay. RESULTS: Halothane significantly suppressed aggregation ratios at 5 min compared with those in controls (89 +/- 7%) to 71 +/- 10% (1.0 mM) and 60 +/- 11% (1.5 mM) and the increase in intracellular calcium concentration (controls, 821 +/- 95 nM vs. 440 +/- 124 nM [1.0 mM] or 410 +/- 74 nM [1.5 mM]). Halothane also significantly inhibited release of calcium from the dense tubular system (controls, 220 +/- 48 nM vs. 142 +/- 31 nM [1.0 mM]). Neither sevoflurane nor isoflurane produced a net change in aggregation ratios, intracellular calcium concentration, or calcium mobilization. Halothane (1 mM) significantly suppressed inositol 1,4,5-triphosphate concentrations, whereas neither 1 mM isoflurane nor 1 mM sevoflurane had any effect. CONCLUSIONS: Although sevoflurane has been reported to inhibit human platelet aggregation induced by weak agonists such as adenosine diphosphate, it does not inhibit human platelet aggregation induced by strong agonists such as thrombin.

Michaelis-Menten elimination kinetics of acetate in rabbit.

BACKGROUND: Acetate redistribution from hepatic to peripheral tissues was reported during ethanol metabolism when saturating conditions were reached for acetate metabolism. Because this redistribution cannot be clarified by linear kinetics, elimination kinetics of acetate was studied in the rabbit. METHODS: A sodium-acetate solution in physiological saline (0.5 and 1.0 g/kg of body weight) was injected as an intravenous bolus. The blood acetate profile was measured by headspace gas chromatography. RESULTS: Blood acetate disappeared rapidly. Statistical moment analysis of the blood acetate profiles showed that the normalized area under the curve and the mean residence time increased with an increasing dose amount. These increases suggested a capacity-limited elimination of acetate. Simultaneous multilines fitting after two acetate doses was used to estimate the pharmacokinetic model by the application of minimum Akaike's information criterion estimation. As a result, the blood acetate concentration-time curve was best described by a two-compartment open model with Michaelis-Menten elimination kinetics. The Vmax value was approximately two times larger than that of ethanol obtained by using the same compartment model. The Km value (1.5 mM) was almost the same as that of ethanol and corresponded to blood acetate levels during ethanol oxidation that had been reported to be approximately 2 mM. CONCLUSION: The elimination of acetate obeys nonlinear kinetics, which can clarify the saturation of acetate metabolism.

Effects of repeated cocaine administration on alcohol consumption.

OBJECTIVE: Alcohol consumption (alcohol preference or alcohol intake) in animals is an index of human drinking behavior. Cocaine is the most frequently abused drug at present. Therefore, an increasing number of cases demonstrating concurrent use of alcohol and cocaine is being noted. We examined whether cocaine affects alcohol consumption and studied the mechanism of change in alcohol consumption following cocaine administration. METHOD: We measured alcohol consumption in inbred mice, C57BL/6J and C3H/HeJ, when 10 or 50 mg/kg cocaine was administered intraperitoneally once a day for 1 week. Then the rate of blood ethanol disappearance from C57BL/6J mice in vivo was measured. Also, liver alcohol dehydrogenase(ADH) and aldehyde dehydrogenase(ALDH) activity in vitro were measured in the C57BL/6J mice. RESULTS: Following 50 mg/kg cocaine administration, alcohol consumption was reduced in C57BL/6J mice, but there was no clear change in C3H/HeJ mice. The rate of blood ethanol disappearance was not changed by pretreatment with cocaine. Neither liver ADH nor ALDH activity was changed by repeated cocaine administration. CONCLUSIONS: The present study showed that repeated cocaine administration decreased alcohol consumption in C57BL/6J mice without altering the metabolism of ethanol.

Noncompetitive-like inhibition of ethanol elimination by cyanamide treatment: pharmacokinetic study.

The effect of acetaldehyde accumulation of ethanol elimination is of interest in medico-legal practice in Japan. We examined the pharmacokinetic mechanism of the inhibition of ethanol metabolism by cyanamide, an inhibitor of mitochondrial aldehyde dehydrogenase. An ethanol solution (0.25-2.0 g/kg body weight) was injected intravenously into male rabbits with or without administration of cyanamide. Cyanamide was injected intraperitoneally (25 mg/kg body weight) to the cyanamide-treated group 2 hr before ethanol injection. Blood ethanol and acetaldehyde concentrations were measured periodically by head-space gas chromatography. The MULTI(RUNGE) computer program was applied for the pharmacokinetic analysis. One- or two-compartment open models with Michaelis-Menten elimination kinetics were used for simultaneous multi-line fitting. The ethanol elimination rate decreased by cyanamide treatment. The border-point concentration between pseudolinear and curvilinear phases was not affected by cyanamide treatment. The estimated Vmax value decreased by cyanamide treatment, whereas the K(m) value did not change. Our results correspond to a noncompetitive-like inhibition of ethanol metabolism. K(m) is related to the border point between pseudolinear and curvilinear phases. Thus, our findings in the blood ethanol concentration-time curve suggest adequate curve-fitting. The product, or competitive, inhibition of alcohol dehydrogenase by acetaldehyde had been reported in enzymological study. The pharmacokinetic manner of inhibition in vivo was different from the enzymologic mechanism in vitro. Other metabolic factors related to ethanol metabolism are thought to be more important than acetaldehyde accumulation itself.

Fundamental studies on alcohol dependence and disposition.

This article reviews some recent studies on alcohol preference, dependence, metabolism and pharmacokinetics which were mainly carried out in our department. The inbred strains of mice with genetically different alcohol drinking behavior and alcohol animal model treated with the neurotoxins, 6-hydroxydopamine and 5,7-dihydroxytryptamine, are useful for a behavioral and pharmacological approach to evaluate the contribution of specific neural systems to alcohol, drug dependence mechanism and alcohol drinking behavior. The relations between alcohol preference and some physiological conditions are reviewed. On the drug-alcohol interaction, some drugs containing the chemical group = CHONO2, antimony and methamphetamine are addressed. This article also deals with recent topics in the pharmacokinetics and pharmacodynamics of alcohol. The dose-dependency of the alcohol elimination rate, the first-pass metabolism during alcohol drinking, and the pharmacodynamic model for describing pulse rate reaction to plasma acetaldehyde are discussed.

Problems in pharmacokinetic analysis of alcohol disposition: a trial of the Bayesian least-squares method.

The technical problems of the pharmacokinetic analysis of alcohol disposition were studied using the Michaelis-Menten elimination kinetic model. This model was defined by two forms of equations: differential and integrated, with the latter being derived by integration of the differential equation. We compared the parameter values, estimated by one-line curve-fitting, using these two equation forms. We concluded that, for the kinetic analysis of alcohol disposition, curve-fitting with the differential equation was superior to that with the integrated equation. We also studied the methodological problems involved in one-line fitting. The ordinary least-squares (OLS) method was compared with the Bayesian least-squares (BLS) method. Correlation between the Vmax and beta (ethanol elimination rate) values, and between the Vmax and K(m) values was seen when the parameter values were estimated by the OLS method. These results suggested that one-line fitting by the OLS method was not adequate for Michaelis-Menten-type elimination kinetic analysis. BLS analysis resulted in no correlation between the estimated parameter values that did not change with the level of the dose. The BLS method seemed to be more useful than the OLS method for the estimation of individual pharmacokinetic parameter values.

Fluorinated analogues as mechanistic probes in valproic acid hepatotoxicity: hepatic microvesicular steatosis and glutathione status.

It is postulated that the hepatotoxicity of valproic acid (VPA) results from the mitochondrial beta-oxidation of its cytochrome P450 metabolite, 2-propyl-4-pentenoic acid (4-ene VPA), to 2-propyl-(E)-2,4-pentadienoic acid ((E)-2,4-diene VPA) which, in the CoA thioester form, either depletes GSH or produces a putative inhibitor of beta-oxidation enzymes. In order to test this hypothesis, 2-fluoro-2-propyl-4-pentenoic acid (alpha-fluoro-4-ene VPA) which was expected to be inert to beta-oxidative metabolism was synthesized and its effect on rat liver studied in comparison with that of 4-ene VPA. Similarly, the known hepatotoxicant 4-pentenoic acid (4-PA) and 2,2-difluoro-4-pentenoic acid (F2-4-PA) were compared. Male Sprague-Dawley rats (150-180 g, 4 rats per group) were dosed ip with 4-ene VPA (0.7 mmol/kg per day), 4-PA (1.0 mmol/kg per day), or equivalent amounts of their alpha-fluorinated analogues for 5 days. Both 4-ene VPA and 4-PA induced severe hepatic microvesicular steatosis ( > 85% affected hepatocytes), and 4-ene VPA produced mitochondrial alterations. By contrast, alpha-fluoro-4-ene VPA and F2-4-PA were not observed to cause morphological changes in the liver. The major metabolite of 4-ene VPA in the rat urine and serum was the beta-oxidation product (E)-2,4-diene VPA. The N-acetylcysteine (NAC) conjugate of (E)-2,4-diene VPA was also found in the urine. Neither (E)-2,4-diene VPA nor the NAC conjugate could be detected in the rats administered alpha-fluoro-4-ene VPA. In a second set of rats (3 rats per group), total liver GSH levels were determined to be depleted to 56% and 72% of control following doses of 4-ene VPA (1.4 mmol/kg) and equivalent alpha-fluoro-4-ene VPA, respectively. Mitochondrial GSH remained unchanged in the alpha-fluoro-4-ene VPA treated group but was reduced to 68% of control in the rats administered 4-ene VPA. These results strongly support the theory that hepatotoxicity of 4-ene VPA, and possibly VPA itself, is mediated largely through beta-oxidation of 4-ene VPA to reactive intermediates that are capable of depleting mitochondrial GSH.

Pharmacokinetics of ethanol drinking--absorption kinetics and first-pass effect.

The pharmacokinetics of alcohol drinking was studied in male rabbits. Various doses (D) of ethanol (0.25, 0.5, 1, 2 and 3 g/kg BW) were administered perorally to the animals and blood alcohol (ethanol) concentration (BAC) was measured. Simultaneous multiline fitting was attempted using the BAC-time curves of 5 peroral doses and of 5 intravenous doses. The two-compartment open model with parallel first-order and Michaelis-Menten elimination and first-order absorption kinetics was concluded to be the best model. To estimate the value of the absorption rate constant (ka) and the bioavailability (F) at each dose, curve fitting was also attempted by using a single BAC-time curve. The F value decreased with a low dose (0.25 g/kg), but complete systemic bioavailability was noted in higher doses. A first-pass effect was suggested to occur with the low dose. With low doses, the peak BAC might be too low to saturate the ethanol metabolism. The conventional method using area under the BAC-time curve (AUC) underestimated the F value. The other conventional method using theoretical BAC at time zero (C0) [F = C0oral x D(i.v.)/C0i.v. x Doral)] was compared to the AUC method. The estimated F value by this C0 equation method was closer to it by curve fitting than that by the AUC method. The effects of various ka, F and D values were also studied using a computer simulation. Consequently, the conventional AUC method was concluded to overestimate the first-pass metabolism of ethanol. The C0 equation method is thought to be more useful.

The effects of neurotoxins 6-hydroxydopamine and 5,7-dihydroxytryptamine into the rat nucleus accumbens on the alcohol drinking behavior.

To elucidate the relationship between drinking behavior and brain monoamines in the brain rewarding system, the effects of injection of 6-hydroxydopamine (6-OHDA) and 5,7-dihydroxytryptamine (5,7-DHT) into the rat nucleus accumbens (ACC), which would affect dopamine- and indoleamine-containing neurons, respectively, on the rat alcohol preference score were examined. The 6-OHDA (4 micrograms/side)-treated rats showed a higher alcohol preference score for 2 weeks following the treatment, and a lowered dopamine (DA) level in the ACC and midbrain after ACC injection of 6-OHDA. On the other hand, although the serotonin (5-HT) and DA levels in the midbrain and 5-HT level in ACC were lowered after the injection of 5,7-DHT, the alcohol preference score was not significantly changed in the 5,7-DHT (4 micrograms/side)-treated rats. Taken together, these findings suggest that the alcohol drinking behavior is more influenced by the ACC DA activity than 5-HT activity. Changes in alcohol drinking behavior might be related to the compensatory mechanism for the rat to restore the original rewarding properties.

Role of alcohol dehydrogenase in rat ethanol elimination kinetics.

Ethanol elimination in rats following bolus intravenous administration (0.5, 1, 2, 3 g/kg body weight) was investigated with and without pyrazole pretreatment. Elimination time was significantly longer in the pyrazole-pretreated group than in the control. Simultaneous multilines fitting of two-compartment models to the ethanol concentration-time courses proved that the two-compartment model with Michaelis-Menten elimination kinetics was optimum to the curves in both groups. the V(max) value (0.41 +/- 0.05 mg/ml/hr) in the pyrazole-pretreated group was significantly lower than that (0.70 +/- 0.03 mg/ml/hr) in the control. The K(m) value (1.44 +/- 0.12 mg/ml) in the pyrazole-pretreated group was significantly higher than the control (0.07 +/- 0.01 mg/ml), similar to the in vitro value in hepatic alcohol dehydrogenase (ADH). This higher K(m) value in the pyrazole-treated group than the in vitro value in the microsomal ethanol oxidizing system (MEOS) suggests that pyrazole-insensitive pathways may involve pathways other than MEOS. The first-order elimination rate from the two-compartment model with parallel first-order and Michaelis-Menten kinetics was a very low value of 10(-5)min(-1) ,which shows that a pathway with a higher K(m) than blood ethanol concentration does not contribute to in vivo ethanol elimination. The relative contribution of the pyrazole-sensitive pathway calculated from the ratio of total clearance in two groups was 83% at a low ethanol concentration of 0.5 mg/ml and 60% at a high concentration of 5.0 mg/ml. The K(m) value (0.02 mg/ml) from fitting the double Michaelis-Menten model is similar to that in vitro in ADH3. These findings suggest that the ADH3 pathway plays a major role in ethanol elimination.

The patient's right to information in Japan--legal rules and doctor's opinions.

A questionnaire survey concerning informed consent was administered among Japanese physicians in Yamaguchi prefecture. The survey results showed that even though these Japanese physicians are willing to give their patients sufficient information to obtain informed consent, the discretion of the physician to provide information is still prevalent. The survey also revealed that Japanese physicians believe that information regarding the treatment to be administrated should be fully disclosed both in case when the treatment is still experimental and when it is established among specialists. Finally, the survey showed that despite the liberal attitude of the Japanese physicians toward informed consent, they are reluctant to make medical records accessible to the patients. It was found that when Japanese physicians were faced with special cases such as prior to administering high-risk diagnostic procedures, prior to disclosing the presence of cancer in their patients, or when faced with patients unwilling to receive treatment, the involvement of the patient's family members in the decision-making process was preferred so as not to aggravate the patient's emotional anxiety. With respect to cancer patients, the survey suggested that many Japanese physicians believe that cancer operations performed without informed consent from the patient should be legal. Finally, the survey concluded that, although the concept of individualized informed consent has been generally accepted among physicians, the involvement of family members in the decision-making process and a conservative attitude toward disclosure of information are still prevalent in Japan.

Parallel first-order and Michaelis-Menten elimination kinetics of ethanol. Respective role of alcohol dehydrogenase (ADH), non-ADH and first-order pathways.

Elimination kinetics of ethanol without (control group) and with pyrazole [alcohol dehydrogenase (ADH) inhibitor] pretreatment was studied with changing the i.v. dose amount to evaluate the respective role of ADH and non-ADH pathways in a rabbit. The moment analysis of the blood ethanol concentration-time curves showed that the normalized area under the blood ethanol concentration-time curve and the first moment increase with increasing dose amount in the control and pyrazole-pretreated groups. These increases suggested the capacity-limited elimination of ethanol through pyrazole-insensitive non-ADH pathways as well as through ADH pathway as pyrazole would fully block the oxidation of ethanol through ADH pathway. The simultaneous multiline fitting using time curves after five different doses also was attempted to determine the pharmacokinetic model by the application of minimum Akaike's information criterion estimation. Akaike's information criterion, consequently, showed the minimum for a two-compartment model with parallel first-order and Michaelis-Menten elimination kinetics. The computer analysis using this model yielded almost the same values of the volume of distribution and of the first-order elimination rate constant between both groups. The distribution of ethanol and the first-order elimination process were not influenced by pyrazole treatment. Km (0.57 mg/ml) of the pyrazole-pretreated group was higher than Km (0.03 mg/ml) of the control group. These results suggest that ADH pathway is readily saturated and non-ADH pathways are unsaturated over the wide range of concentration. The first-order process as well as non-ADH pathways are concluded to occupy the considerable part in the ethanol elimination at higher blood concentration.

A case of sudden death by decidual cell embolism.

A 35-year-old multipara died suddenly of a pulmonary embolism about 12 h after delivery. The morphological features and the entry site of the emboli into the circulation suggested that they were decidual cells. Intact decidual cells accounted for only a minority of the emboli: the great majority were cells that had lost their nuclei and/or had been fragmented. The presence of embolized areas, accompanied by fibroblasts and newly formed capillaries, suggested that the embolization process had started before the beginning of labor. However, no symptoms suggesting embolism had been recorded on the clinical chart.

Acute effects of carbon monoxide and cyanide on hepatic mitochondrial function.

The effects of carbon monoxide and cyanide on the hepatic redox state and energy charge were investigated. Rats were used for the experiment under pentobarbital anesthesia. Immediately after laparotomy, a rat was placed in an animal chamber made of a transparent plastic box and exposed to a test gas for 3 min. Every test gas was produced in a gas chamber connected to the animal chamber with a flexible tube. HCN was produced from NaCN and H2SO4. In the CO inhalation experiment, various amounts of CO were introduced into the gas chamber. Immediately after an exposure, about 2 g liver was frozen in situ with a precooled clamp. Oozed blood from the wound surface was sampled. Concentrations of ATP, ADP, AMP, acetoacetate, and beta-hydroxybutyrate in hepatic mitochondria were determined, and the redox state and the energy charge were calculated. For cyanide as well as CO, significant negative correlations were found between the concentration in the blood and the redox state. The same held true for the energy charge. The redox state showed a slight increase at low concentrations of both gases; however, thereafter it began to decrease sharply with increases in concentrations. When concentrations of the toxicant in the blood reached certain levels, a kind of turning point, beyond which the redox state does not decrease any more, was observed. It was about 40% for HbCO and about 2.0 micrograms/ml for cyanide, and the points seemed to be related to the concentrations, beyond which cells are irreversibly damaged. On the other hand, the energy charge did not change at low concentrations. With an increase in toxicant concentrations, the energy charge decreased drastically. The rate of decrease in the energy charge became higher when blood concentrations exceeded certain levels. It was about 40% for HbCO and 2.0 micrograms/ml for cyanide. The presence of low levels of blood cyanide did not affect the relationship between the energy charge and the HbCO concentration.