[The inhibitory effect and the mechanism of ethanol absorption by zinc complex in mouse gastrointestinal tract].

The inhibitory effect of ethanol absorption by zinc cysteine (Cys-Zn) and zinc acetylcysteine (ACys-Zn) were studied in mice. This mice orally received Cys-Zn, ACys-Zn, Cys or Zn acetate, and after 1 hr, 14C-ethanol was given orally or intraperitoneally (i.p.). At various times, blood was drawn from the tail vein and 14C-radioactivities determined. Cys-Zn and ACys-Zn inhibited the appearance of 14C-radioactivities in blood following oral 14C-ethanol loading, but Cys and Zn acetate caused no changes as compared to the control. Cys-Zn pretreatment did not induce any change in the blood 14C-radioactivity when ethanol was given i.p. The levels of 14C-radioactivity and zinc in the gastrointestinal tract after oral administration (adm) of Cys-Zn and 14C-ethanol were significantly higher than those of the control for 7 hr. At 72 hr after adm, the urinary and fectal excretions of 14C-radioactivity were low when Cys-Zn was given at the dose of 5.0% or 0.5%. The excretion of 14C-radioactivity through expiration in both cys-Zn and control mice was about 28% of the dose at 5 hr after adm. Distributions of 14C-radioactivities in other organs of Cys-Zn treated mice were lower then those of the control. In the vitro study, Cys-Zn stimulated the metabolism of ethanol to acetaldehyde and acetic acid in the 9,000 g supernatant of the small intestine. The results suggest that zinc complex shows a long-term adhesive and permeable action on the gastrointestinal tract in mice, and this inhibited ethanol absorption.

Role of nitric oxide in the convulsions following the coadministration of enoxacin with fenbufen in mice.

Effects of nitric oxide (NO) synthase inhibitors on the enoxacin-induced convulsions were examined in mice pretreated with fenbufen. 7-nitroindazole markedly suppressed the incidence of convulsions, whereas L-arginine did not modify the convulsions at all. The suppression of the convulsions by 7-nitroindazole was not reversed by the pretreatment of L-arginine. Brain NO synthase activity was significantly raised at 30 min after enoxacin when combined with fenbufen. The increased NO synthase activity was found to be suppressed by the pretreatment of 7-nitroindazole. These findings suggest that endogenous NO may be involved as a proconvulsant substance in the development of enoxacin-induced convulsions in mice pretreated with fenbufen.

[The inhibitory effect and the mechanism of ethanol absorption by L-carnosine zinc complex in mouse gastrointestinal tract].

The effect of L-carnosine-zinc complex(Car-Zn) on ethanol absorption was investigated after oral administration(adm) to mice. One hour after oral adm of Car-Zn, and 14C-ethanol was given orally or intraperitoneally(i.p.). After passage of time, the blood was drawn from the tail vein, and 14C-radioactivities determined. The Car-Zn showed a dose-dependent inhibition of the appearance of 14C-radioactivities in blood following oral 14C-ethanol loading, while Zn acetate did not induce any alteration as compared with control. Car-Zn pretreatment did not induce any change in the blood 14C-radioactivity when ethanol was given i.p. The 14C-radioactivity and zinc in gastrointestinal tract after oral adm of Car-Zn and 14C-ethanol showed significantly higher levels than those of control for 7 hr. Distribution of 14C-radioactivities in other organs of Car-Zn treated mice were lower than those of control 3 hr after adm, while it was similar or higher than those of control 7 hr after adm. The excretions of 14C-radioactivity through expiration in Car-Zn group was a lower than that of control. Also, the urinary and fecal excretions of 14C-radioactivity were low values at 5.0% and 0.5% of the administered dose 72 hr after adm, respectively. Also, the 14C-radioactivities remaining in the organs did not detect or were very low values. In vitro study, Car-Zn stimulated the metabolism of ethanol to acetaldehyde and acetic acid in 9,000 g supernatant of small intestine. The major route of excretions of 14C-radioactivity may be excreted into the expired air. The results suggest that Car-Zn shows a long-term adhesive and permeable action on gastrointestinal tract in the mouse; as a result, this may inhibited ethanol absorption.

Effects of liposome size on penetration of dl-tocopherol acetate into skin.

Liposomes were prepared from hydrogenated lecithin (H-PC) by sonication (S) or injection (I) of H-PC dissolved in ethanol containing dl-tocopherol acetate (VEA). The effects of liposomes on the dermal absorption of VEA were studied. The particle diameter of S-liposomes was smaller than that of I-liposomes. The penetration of liposomal H-PC into the skin was much higher for S-liposomes than for I-liposomes 30 min after application to the arms of healthy human volunteers and also to hairless rat back skin. The penetration of 14C-VEA into hairless rat back skin was higher from the liposomes than from free VEA, and the 14C-VEA penetration was higher from S-liposomes than from I-liposomes. 3H-Dipalmitoylphosphatidylcholine and 14C-VEA, which had been entrapped in liposomes, were not detected in plasma. H-PC inhibited the peroxidation of skin lipids. H-PC enhanced the penetration of VEA into the skin, but the degree of enhancement depended on the size of the liposomes, indicating that this liposomal characteristic was an important factor in dermal absorption and/or penetration.

Effects of ethanol on phospholipases in the mouse brain, heart and liver.

The activities of microsomal phospholipase A(2) (PLA(2)) and C (PLC) from mouse brain, heart and liver were determined using the substrate 1-palmitoyl-2-N-(4-nitrobenzo-2-oxa-1,3-diazole amino caproyl-phosphatidylcholine (NBD-PC), and the effects of chronic ethanol treatment (ethanol) as well as in vitro addition of various n-alcohols including ethanol on these activities were evaluated. Microsomal membrane fluidity was estimated by diphenylhexatriene anisotropy (gamma). The microsomes from the brain and heart of ethanol-treated mice showed significantly higher PLA(2) activity than those from controls. The brains of ethanol group showed significantly higher PLC activity, while the heart showed significantly lower PLC activity than those of controls. The microsomes from the brain and heart of ethanol-treated mice showed significantly reduced gamma values compared to those of controls. The addition of ethanol in vitro to microsomes was found to increase PLC activity in these tissues, while it decreased PLA(2) activity in a dose-dependent manner. The other n-alcohols showed similar effects on PLA(2) and PLC activity in the live microsomes, while decreases were observed the gamma values in a dose-dependent manner. These results suggest that the change in the membrane fluidity associated with addition of alcohols is a prerequisite for the changes in PLA(2) and PLC activities. In addition, our findings suggest that these changes may play a major role in the cellular injury associated with chronic ethanol treatment in the mouse.

[Effect of ethanol on expression of nitric oxide synthases in the cerebral culture cells from chick embryo].

The effect of ethanol on nitric oxide synthase (NOS) activity was examined histochemically and biochemically in cultured cerebral cells of chick embryos. The cells were isolated from 13- to 14-day-old chick embryos to which 10% ethanol (ethanol group) or saline (control) had been injected on the 3rd day of embryogenesis. Expression of NADPH diaphorase in cultured cells was stained using nitro blue tetrazolium (NBT). The activity of NOS was observed by the following 2 assays: NADPH diaphorase activity was determined using the substrate NBT with the cofactor NADPH, and NOS activity was determined by measurement as radiochemical activity of the conversion of [3H]L-arginine to [3H]L-citrulline. The number of isolated cells and viability from one cerebrum of chick embryo were 3 approximately 4 x 10(6) and more than 97%, respectively. In the neuronal cells, moderately positive expression of NADPH diaphorase was first detected on about the 3rd day of culture in both the control and ethanol group. The NADPH diaphorase and NOS activities in the isolated cells were higher in the ethanol group than in the control group. The NADPH diaphorase and NOS activities were significantly higher in the ethanol group than in the control group on the 4th and 2nd day of culture, respectively. These findings suggest that NO-released by elevated NADPH diaphorase activity and NOS activity is responsible for the induction of neuronal cell disorders attributed to chronic ethanol damage.

Effect of chronic ethanol on phospholipase A2- and C-activity in chick embryo brain, heart and liver.

The activities of phospholipase A2 (PLA2) and C (PLC) in subcellulal fractions from chick embryo brain, heart and liver were determined using the substrate 1-palmitoy 1-2-N-(4-nitrobenzo-2-oxa-1, 3-diazole amino caproyl-phsphatidylcholine (NBD-PC), and the effect of chronic ethanol treatment on these activities was evaluated. PLA2 and PLC activities of each fraction were assayed by measuring release of N- (4-nitrobenzo-2-oxa-1, 3-diazole) amino caproic acid (NBD-caproic acid) and of 1-palmitoy 1-2-NBD amino caproyl glycerol (NBD-DG) from exogenous NBD-PC. The microsomal membrane fluidity was estimated from diphenylhexatriene anisotropy (gamma). Cytosolic, mitochondorial, and microsomal subcellular fractions were prepared by differential centrifugation of homogenates of the brain, heart, and liver. Microsomal subcellular fractions from the brain, heart and liver of ethanol treated chick embryo showed significantly higher PLA2 and PLC specific activities than did corresponding fractions from non-treated chick embryo. Mitochondrial subcellular fractions from the brain and heart of ethanol-treated chick embryo also showed significantly higher PLA2 and PLC specific activities than the corresponding control fractions. Microsomal fractions from the brain and heart of ethanol-treated chick embryo decreased significantly the gamma than those of control. These results suggest that the change in the membrane fluidity is an apparent prerequisite for the changes of PLA2 and PLC activities.

Changes in the levels of several endogenous opioid peptides in dog cerebrospinal fluid following morphine administration.

The cisterna magna of dogs anesthetized with sodium Surital was fitted with a cannula, and cerebrospinal fluid (CSF) was withdrawn before (control) and one hour after the s.c. injection of 10 mg/kg of morphine sulfate (morphine). The CSF from control and morphine-treated dogs was purified initially by gel filtration. Each fraction was submitted to opiate bioassay procedures, followed by high performance liquid chromatography (HPLC) purification on a mu-Bondapak C18 column. Two of the CSF fractions from HPLC purification showed greater opiate-like activity after morphine treatment than that in controls. One fraction contained morphine, the other an unknown peptide. This latter fraction produced a dose-dependent effect in the mouse tail-flick test. This fraction did not show radioimmunoreactivity to methionine (met)- or leucine (leu)-enkephalins, but showed a small amount of reactivity to beta-endorphin and dynorphin (1-13). Further purification of this fraction by HPLC yielded a fraction with five peaks, which upon amino acid analysis were found to contain small peptides. Met- and Leu-enkephalins, beta-endorphin and dynorphin (1-13)-like immunoreactivity in the fraction in which the respective standard was eluted by HPLC was significantly increased after a single administration of morphine. Based on these results, it is suggested that morphine at an antinociceptive dose causes the release of endogenous opioid peptides and may also stimulate the biosynthesis of their precursor molecules, pre-pro-opiomelanocortin, pre-pro-enkephalin A and pre-pro-enkephalin B.

Effect of ethanol on mouse brain microsomal phospholipid turnover.

In an investigation of the effects of chronic and acute ethanol administration on phospholipid synthesis, mice subjected to chronic or acute ethanol administration were intraventricularly injected with 1,332 kBq of 2-3H-glycerol (3H-glycerol) and 4,070 kBq of 32P-phosphoric acid 15 min after the ethanol injection. One hour later, mice were sacrificed, and brain microsomes were prepared for analysis of incorporation of radioactivity into phospholipids. Chronic ethanol treatment significantly decreased incorporation of 3H-glycerol and 32P-phosphoric acid into phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol plus phosphatidylserine. However, acute ethanol treatment had no marked effect on incorporation of 3H-glycerol or 32P-phosphoric acid into any of these phospholipids. On the other hand, chronic ethanol administration had no significant effect on incorporation of the radioisotopes into triphosphoinositide (TPI) or diphosphoinositide (DPI) in microsomal fractions, or the 3H- and 32P-TPI/DPI ratios. However, acute ethanol administration decreased the incorporation of 3H-glycerol into DPI and TPI, but did not change the TPI/DPI ratio; it also significantly increased the 32P-TPI/DPI ratio and decreased 32P-phosphoric acid incorporation into DPI but did not significantly affect 32P-phosphoric acid incorporation into TPI. Chronic ethanol administration is thought to have altered the turnover of phospholipids in the microsomal membrane, thereby affecting both the levels and turnover of neurotransmitters. In addition, the change of labeled TPI/DPI ratio observed after acute ethanol treatment may reflect nicotinic receptor activity in the mouse brain.

Effects of mushroom toxins on glycogenolysis; comparison of toxicity of phalloidin, alpha-amanitin and DL-propargylglycine in isolated rat hepatocytes.

The effects of phalloidin and alpha-amanitin as toxins of Amanita species and DL-propargylglycine identified from A. abrupta on the glycogenolysis in isolated rat hepatocytes were investigated. Phalloidin decreased glycogen content and activated phosphorylase a activity remarkably. alpha-Amanitin also decreased glycogen content significantly but activated phosphorylase a activity slightly. DL-Propargylglycine slightly affected glycogenolysis. Phalloidin, which most affected glycogenolysis among the three compounds mentioned above, elevated cytosolic Ca2+ concentration ([Ca2+]i) and 45Ca uptake into cells. Phalloidin depressed slightly 3H-inositol incorporation into phosphatidylinositol (PI) and remarkably phosphatidylinositol 4,5-bisphosphate (PIP2) but increased phosphoinositides breakdown. These results suggest that phalloidin alters phosphoinositides turnover and intracellular Ca2+ homeostasis, subsequently activates phosphorylase a, resulting in glycogenolysis in isolated rat hepatocytes.

Relationship between incorporation of choline into phosphatidylcholine and morphine binding sites.

Calcium (25 mM)-stimulated incorporation of choline into phosphatidylcholine (PC) and 3H-morphine (3H-M) binding was studied using cerebral crude synaptosomal fractions from acute morphine-treated (naive), morphine-dependent and morphine-withdrawn mice. Mice became physically dependent on morphine after ingesting morphine-mixed food. Morphine dependence inhibited the incorporation of 14C-choline into PC. The injection of naloxone (1 mg/kg) to morphine-dependent mice also inhibited the incorporation of 14C-choline into PC. The injection of morphine (10 mg/kg) to morphine-withdrawn mice stimulated the incorporation of 14C-choline into PC, but had no effect, either on acute morphine-treated mice or on morphine-dependent mice. Naloxone (1 mg/kg), morphine (10 mg/kg), morphine dependence and opiate withdrawal (20 hrs after the last exposure to morphine in food) all caused about a 25% decrease in 3H-M (10(-7) M) binding to mouse brain synaptosomes. The injection of 10 mg/kg of morphine 45 min before death caused an additional 20% decrease in 3H-morphine binding in the dependent and withdrawn mice, but not in the naive mice. Stereospecific binding (SSB) of 3H-M was evaluated with respect to: 1) the effect of naloxone treatment in vivo on 3H-M binding in vitro, 2) the effect of morphine treatment in vivo on 3H-M binding in vitro; and 3) the differences between 3H-M bound (in vitro) and both naloxone and morphine treatment (in vivo). SSB activity was high in morphine-withdrawn mice. These differences may be caused by changes in the opiate receptors related to tolerance and withdrawal, and may entail between PC and membrane receptors.

Effect of ethanol on phospholipid turnover and calcium mobilization in chick embryos.

Effects of ethanol on [3H]inositol and [14C]choline incorporation into phosphatidylinositol (PI) and phosphatidylcholine (PC), free intrasynaptosomal Ca2+ ([Ca2+]i) and synaptosomal 45Ca2+ uptake, were investigated in the brain and heart of 17-day-old chick embryos to which a 10% ethanol solution had been injected on the 3rd day of embryogenesis. In brain synaptosomes, ethanol increased the incorporation of [3H]inositol and [14C]choline into PI and PC, increased [Ca2+]i, and decreased 45Ca2+ uptake. On the other hand, in heart synaptosomal membrane, ethanol decreased the incorporation of [3H]inositol and [14C]choline into PI and PC, decreased [Ca2+]i, and increased 45Ca2+ uptake. Ethanol stimulated in vitro [3H]inositol and [14C]choline incorporation into PI and PC in the brain and heart in both the control and ethanol-treated groups. However, addition of ethanol did not affect the release of 45Ca2+ from the synaptosomal membrane of either organ in either group. Addition of ethanol inhibited 45Ca2+ uptake in a dose-dependent manner in the brain but not in the heart. In both organs, there was a relationship between phospholipid turnover and [Ca2+]i after ethanol.

Effect of ethanol on calcium-uptake and phospholipid turnover by stimulation of adrenoceptors and muscarinic receptors in mouse brain and heart synaptosomes.

The effect of ethanol treatment on mouse brain and heart synaptosomal 45Ca uptake and the incorporation of [3H]inositol and [14C]choline into phosphatidylinositol (PI) and phosphatidylcholine (PC) were investigated. Ethanol in drinking water (15%) was given to mice for 3 weeks. The consumption of ethanol increased gradually during treatment but food intake was almost the same as control. The body weight of ethanol-treated mice was slightly less than that of control. The synaptosomal lipid peroxidation level of ethanol-treated mice was almost the same as control in the brain and heart. On the other hand, the synaptosomal glutathione level of ethanol-treated mice was higher than control in both brain and heart. The 45Ca uptake of brain and heart from ethanol-treated mice was 87% and 216% of control mice, respectively. Not only ethanol but also norepinephrine (NE), carbachol (Carb), or isoproterenol (IsoPro) added in vitro increased 45Ca uptake in all cases. The incorporation of [3H]inositol into PI in the brain and heart synaptosomes of ethanol-treated mice was 150% and 113% of control, respectively. The incorporation of [14C]choline into PC in the brain and heart of ethanol-treated mice was 104% and 125% of control, respectively. In vitro addition of ethanol, NE, Carb or IsoPro to brain synaptosomes increased the incorporation of [3H]inositol and [14C]choline into PI and PC, respectively, in both control and ethanol-treated mice. In the case of heart synaptosomes, NE and Carb increased the incorporation of [3H]inositol and [14C]choline into phospholipids in control mice but not ethanol-treated mice. However, IsoPro increased the incorporation by both control and ethanol-treated heart synaptosomes. These results suggest that alpha-adrenoceptors and the cholinergic system of the heart play important roles in modulating the toxic effects of ethanol.

Effect of ethanol on brain biogenic amines and phospholipids in the chick embryo.

The role of biogenic amines and phospholipids in the acute and chronic effects of ethanol on chick embryo development were studied. Injection of ethanol into chicken eggs on the third day of embryonic development produced no deficits in body or brain weight in the 17-day-old embryo. Spontaneous fetal motility of the 17-day old embryo did not differ between vehicle- and ethanol-treated groups, but acute administration of ethanol decreased motility. Ethanol decreased dopamine and homovanillic acid, while the level of serotonin and 5-hydroxyindoleacetic acid did not change in the 17-day-old chick embryo brain. Furthermore, lysophosphatidylcholine, phosphatidylcholine and phosphatidyl-ethanolamine turnover was decreased by ethanol. Ethanol decreased embryo viability in a dose-dependent manner as early as day 7. At this time, radiolabeled ethanol could be detected in the embryo. Thereafter, the levels of radiolabeled ethanol in whole embryos or embryo brains revealed continual exposure to ethanol throughout development. Our data suggest that monoaminergic neurons are involved in the development of ethanol tolerance through phospholipid synthesis in the chick embryo brain.

In vitro toxicity test of poisonous mushroom extracts with isolated rat hepatocytes.

Effects of poisonous mushroom extracts on isolated rat hepatocytes were studied. Though no significant decrease in the cell viability was observed during the incubation of hepatocytes with the extracts at a concentration of 5% (v/v) of Amanita abrupta, A. gymnopus, and A. virosa caused marked decreases in the intracellular glutathione content in sharp contrast to the extracts of A. volvata and A. flavipes. Comparative toxicity tests were carried out for the effects of the extract of A. abrupta, dl-propargylglycine, and alpha-amanitin. The extract of A. abrupta at a concentration of 1% (v/v) caused a marked decrease in the glycogen content, a noticeable elevation in the phosphorylase alpha activity, and a slight acceleration of lipid peroxidation in the hepatocytes. Although dl-propargylglycine decreased the intracellular glutathione content progressively with the incubation time, a significant effect of the chemical on lipid peroxidation and the glycogen content was observed only after prolonged incubation at a concentration of 5 mM. On the other hand, alpha-amanitin exerted a little effect on the hepatocytes at 1 microM. These results have indicated that the intoxication by the extract of A. abrupta on the hepatocytes might not due to independently each component, dl-propargylglycine and alpha-amanitin, but combined effect of these components or unidentified substances.

Influence of morphine, beta-endorphin and naloxone on the synthesis of phosphoinositides in the rat midbrain.

The effects of morphine, beta-endorphin and naloxone on the initial incorporation of 32Pi and [3H]glycerol into TPI, DPI and PI were measured in discrete subcellular fractions of the rat midbrain. Morphine and beta-endorphin significantly increased microsomal 32PI incorporation into TPI and PI but not DPI. Although neither morphine nor beta-endorphin significantly affected the levels of [3H]TPI or [3H]DPI, both agents significantly increased [3H]PI levels. All of the significant effects induced by morphine were blocked by naloxone treatment and were decreased after chronic morphine administration. However, naloxone treatment alone also mimicked all the effects of morphine except the increased incorporation of [3H]glycerol into PI. It was also found that chronic morphine treatment significantly increased the incorporation of 32Pi into synaptic TPI and DPI. This effect, however, did not show regional specificity being found in both cortical and subcortical synaptic membranes. Overall, the results suggest that the mechanisms of opioid action are closely associated with changes in the turnover of the brain phosphoinositides.