Yasumitsu Yamanaka

The protective effect of glycyrrhizin against injury of the liver caused by ischemia-reperfusion

Effects of glycyrrhizin (GR) on an injury of the liver caused by ischemia-reperfusion in rats were determined. In the liver ischemia-reperfusion model, levels of serum AST, ALT and LDH, lipid peroxides in the liver tissue, and blood superoxide dismutase activity were significantly increased. On the contrary, total glutathione content in the liver tissue was decreased. When rats were given GR 100 mg/kg for 10 days, GR suppressed the elevation of the lipid peroxide level, serum AST, ALT, LDH level, and the decrease in glutathione content during the period of reperfusion. The suppressive effect of GR was similar with that of α-tocopherol (VE). GR showed neither 1,1-diphenyl-2-picrylhydrazyl (DPPH) nor 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OOH radical-trapping ability, but exhibited DMPO-OH radical-trapping action, while, VE exhibited both DPPH and DMPO-OOH radical-trapping ability. These results indicate that the hydroxyl radical trapping action of GR is the likely mechanism suppressing liver injury caused by ischemia-reperfusion.

Release of dopamine by perfusion with 1-methyl-4-phenylpyridinium ion (MPP(+)) into the striatum is associated with hydroxyl free radical generation.

In Parkinsons disease (PD), the dopamine (DA) neuronal cell death in the nigrostriatal system has been proposed to be mediated by reactive oxygen radicals such as hydroxyl radicals (.OH). This.OH production may cause lipid peroxidation of cell membranes leading to neuronal cell death. This paper report that the DA-selective neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP(+)), (1 nmol/microl per min for 1 h) infusion into the striatum of rats induces elevation of extracellular DA and.OH formation. These elevations seem to induce lipid peroxidation of striatum membranes, as detected by increases in non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) levels. To test the involvement of DA release in the.OH generation and lipid peroxidation, the rats were pretreated with reserpine (5 mg/kg, i.v., 24 h before MPP(+) or without MPP(+)) to deplete presynaptic DA. Reserpine treatment alone did not change the levels of DA or 2,3-DHBA, while the combined treatment with both MPP(+) and reserpine clearly decreased 2,3-DHBA, as well as DA levels, compared to those in the group treated with MPP(+) alone. After injection into reserpinized rats, DA at various doses (2, 5 and 10 microM) small increased 2,3-DHBA levels dose-dependently, as compared to the MPP(+) alone-treated group. These results clearly indicate that MPP(+) perfusion into the striatum increases extracellular DA levels and this increase may concomitantly induce the formation of reactive free oxygen radicals, such as.OH free radicals. These events may contribute, at least in part, to the nigrostriatal neurons cell death after MPP(+).

Intracranial microdialysis of salicyclic acid to detect hydroxyl radical generation by monoamine oxidase inhibitor in the rat

The effect of pargyline, a monoamine oxidase inhibitor, on the generation of hydroxyl free radicals (OH) was investigated using striatal microdialysis. Salicylic acid in Ringers solution (0.5 nmol microliter-1 min-1) was infused through a microdialysis probe to detect the generation of hydroxyl radicals (OH) as reflected by the formation of dihydroxybenzoic acid (DHBA) in the striatum. When pargyline (100 nmol microliter-1 min-1) was infused in rat brain, the level of 3,4-dihydroxyphenylacetic acid (DOPAC) gradually decreased in a time-dependent manner. In addition, a marked elevation of DHBA was observed. The present results indicate that accumulation of dopamine (DA) in the extracellular fluid elicited by pargyline can be auto-oxidized, which in turn leads (possibly by an indirect mechanism) to the formation of cytotoxic OH free radicals.

Stimulation of α1‐adrenoceptors and Protein Kinase C‐mediated Activation of Ecto‐5′‐Nucleotidase in Rat Hearts in vivo

1 To determine whether protein kinase C (PKC)‐mediated activation of ecto‐5′‐nucleotidase would increase interstitial adenosine concentrations in the rat heart in vivo, we made use of the microdialysis technique and a flexibly mounted probe, which was implanted in the left ventricular myocardium and perfused with Tyrode solution. 2 The baseline level of dialysate adenosine was 0.51 ± 0.09 μM (n= 16). Perfusion of adenosine 5′‐monophosphate (AMP, 100 μM) through the probe increased the dialysate adenosine concentration markedly to 9.25 ± 0.46μM (n= 15). αβ‐Methyleneadenosine 5′‐diphosphate (AOPCP, 100 μM), an inhibitor of ecto‐5′‐nucleotidase, abolished the AMP‐induced increase in dialysate adenosine, but did not affect the baseline level of adenosine. These observations suggest that the dialysate adenosine obtained during the perfusion with AMP, but not the baseline levels of adenosine, originated from the dephosphorylation of AMP by ecto‐5′‐nucleotidase. Thus, the level of adenosine measured during AMP perfusion gives an index of the activity of ecto‐5′‐nucleotidase in the tissue. 3 Noradrenaline (10 μm) increased the adenosine concentration measured in the presence of 100 μm AMP (i.e. the activity of ecto‐5′‐nucleotidase) by 38.7 ± 9.6 % (n= 5, P < 0.05), an increase which was inhibited by an antagonist of the α1adrenoceptor (prazosin, 50 μM) or of PKC (chelerythrine, 10 μm). Further application of either the α1‐adrenoceptor agonist methoxamine (100 μM) or the diacylglycerol analogue 1,2‐dioctanoyl‐sw‐glycerol (DOG, 100 μm) also increased the adenosine concentration by 35.1 ± 10.0% (n= 6, P < 0.05) or 40.6 ± 8.3% (n= 5, P < 0.05), respectively. 4 The presence of okadaic acid (50 μm), an inhibitor of protein phosphatase, enhanced the noradrenaline‐induced increase in adenosine concentration by 112.4 ± 35.9% (n= 4, P < 0.05), to a level significantly (P < 0.05) greater than the increase caused by noradrenaline alone (38.7 ± 9.6%). 5 These data provide the first evidence that α1‐adrenoceptor stimulation and the subsequent activation of PKC can increase adenosine concentrations in interstitial spaces of ventricular muscle in vivo, through activation of endogenous ecto‐5′‐nucleotidase.

Cardiac microdialysis of salicylic acid .OH generation on nonenzymatic oxidation by norepinephrine in rat heart.

The effect of pargyline, a monoamine oxidase inhibitor, on the generation of hydroxyl free radicals (.OH) was investigated using cardiac microdialysis. Salicylic acid in Ringers solution (0.5 nmol x microL(-1) x min(-1)) was infused directly through a microdialysis probe to detect the generation of .OH as reflected by the formation of dihydroxybenzoic acid (DHBA) in the myocardium of anesthetized rats. When pargyline (100 nmol x microL(-1) x min(-1)) was infused in rat heart, the level of norepinephrine (NE) gradually increased in a time-dependent manner and an increase of DHBA was also observed. When NE was administered to the pargyline pretreated animals, a marked elevation in the levels of 2,3- and 2,5-DHBA formation was obtained, as compared to the group treated with NE only, showing a positive linear correlation between NE and .OH formation trapped as 2,3-DHBA (R2 = 0.981) or 2,5-DHBA (R2 = 0.984) in the dialysate. NE clearly produced an increase in .OH formation. These results indicate that accumulation of NE in the extracellular fluid elicited by pargyline can be auto-oxidized, which in turn, leads (possibly by an indirect mechanism) to the formation of cytotoxic .OH free radicals.

Protective effect of histidine on para-nonylphenol-enhanced hydroxyl free radical generation induced by 1-methyl-4-phenylpyridinium ion (MPP+) in rat striatum.

The present study examined the antioxidant effect of histidine, a singlet oxygen ((1)O(2)) scavenger, on para-nonylphenol (an environmental estrogen-like chemical)-enhanced hydroxyl radical (.OH) generation induced by 1-methyl-4-phenylpyridinium ion (MPP+) in extracellular fluid of rat striatum. Rats were anesthetized, and sodium salicylate in Ringers solution (0.5 nmol/microl/min) was infused through a microdialysis probe to detect the generation of.OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. Introduction of para-nonylphenol (10 microM) significantly enhanced MPP+ -induced.OH generation. Histidine (25 mM) decreased the para-nonylphenol-enhanced.OH formation. Although the level of MPP+ -induced.OH formation trapped as DHBA after para-nonylphenol treatment increased, para-nonylphenol failed to increase either the level of dopamine and DHBA formation in the reserpinized animals. These results indicate that para-nonylphenol and MPP+ -enhanced.OH generation was based on 1O(2) production, and histidine may have a preventive effect on para-nonylphenol and MPP+ -induced.OH generation in rat striatum.

Effects of anti-free radical intervention on phosphatidylcholine hydroperoxide in plasma after ischemia-reperfusion in the liver of rats

The present study set out to investigate whether plasma phosphatidylcholine hydroperoxide (PCOOH) levels could accurately reflect lipid peroxidation linking to liver damage due to ischemia--reperfusion. PCOOH is a primary peroxidative product of phosphatidylcholine (PC), which is the most important functional lipid in the hepatocellular membrane, and may mediate oxidative stress. We quantified PCOOH and PC in the plasma and liver of rats subjected to hepatic ischemia-reperfusion by chemiluminescence detecting HPLC (CL-HPLC) method. Plasma PCOOH levels showed no significant rise in either the ischemia only group or in the sham-operation group, compared to controls (0.7 nmol/mL plasma). At 60 min subsequent to reperfusion, the PCOOH levels in plasma and liver, as well as the levels of several serum markers of liver injury [lactic dehydrogenase (LDH), glutamic-oxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT)] increased in proportion to the duration of ischemia (up to 60 min). During periods of reperfusion following 30 min of ischemia, plasma PCOOH increased biphasically (2 nmol/mL; 12-24 hr duration of reperfusion), and generally ran parallel to that in the liver after more than 60 min of reperfusion. Dose-dependent protective effects against warm ischemia (30 min)-reperfusion (12 hr) injury were clearly demonstrated in the groups treated with allopurinol, diclofenac Na, ascorbic acid (V.C), alpha-tocopherol and coenzyme Q10, but not in those treated with r-h-superoxide dismutase or betamethasone. The rises in plasma PCOOH and serum GOT, GPT and LDH of the ischemia-reperfused rats were ameliorated most in the group pretreated with diclofenac Na, and next most in the group pretreated with V.C. These results indicate that the plasma PCOOH levels are a useful index both for liver cell damage induced by oxygen free radicals generated during ischemia-reperfusion, and to investigate the efficacy of drugs against oxidative stress.

The effect of glibenclamide on the production of interstitial adenosine by inhibiting ecto-5′-nucleotidase in rat hearts

1 Adenosine exerts cardioprotective effects on the ischaemic myocardium. The production of adenosine in the ischaemic myocardium is attributed primarily to the enzymatic dephosphorylation of adenosine 5′‐monophosphate (AMP) by 5′‐nucleotidase. We determined the activity of 5′‐nucleotidase in rat hearts. The objective of the study was to determine the effects of ATP‐sensitive K+ (KATP) channel antagonists (glibenclamide and 5‐hydroxydecanoate) on the production of adenosine, by use of a flexibly mounted microdialysis technique. 2 Rats were anaesthetized and the microdialysis probe was implanted in the left ventricular myocardium, followed by perfusion with Tyrode solution. The baseline level of dialysate adenosine was 0.51±0.09 μM (n=16). Introduction of AMP (100 μM) through the probe increased the dialysate adenosine markedly to 9.79±0.43 μM (n=12, P<0.001 vs baseline), and this increase was inhibited by the ecto‐5′‐nucleotidase inhibitor, α,β‐methyleneadenosine 5′‐diphosphate (100 μM), to 0.76±0.12 μM (n=8). Thus, the dialysate adenosine noted during the perfusion of AMP originated from dephosphorylation of AMP by ecto‐5′‐nucleotidase, and the dialysate level of adenosine attained reflects the ecto‐5′‐nucleotidase activity in the tissue in situ. 3 Glibenclamide (0.1–100 μM) decreased the adenosine concentration measured during the perfusion of AMP (100 μM) in a concentration‐dependent manner (IC50=10.5 μM). In contrast, 5‐hydroxydecanoate (10–100 μM) did not affect the concentrations of dialysate adenosine, measured in the presence of AMP (100 μM). These results suggest that glibenclamide inhibits the activity of endogenous ecto‐5′‐nucleotidase and decreases the concentration of adenosine in the interstitial space of rat ventricular muscles in situ.

Nitric oxide enhances MPP+-induced hydroxyl radical generation via depolarization activated nitric oxide synthase in rat striatum

We examined the effect of N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, on extracellular potassium ion concentration ([K(+)](o))-enhanced hydroxyl radical (.OH) generation due to 1-methyl-4-phenylpyridinium ion (MPP(+)) was examined in the rat striatum. Rats were anesthetized, and sodium salicylate in Ringers solution (0.5 nmol/microl per min) was infused through a microdialysis probe to detect the generation of.OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. Induction of KCl (20, 70 and 140 mM) increased MPP(+)-induced.OH formation trapped as 2,3-dihydroxybenzoic acid (DHBA) in a concentration dependent manner. However, the application of L-NAME (5 mg/kg i.v.) abolished the [K(+)](o) depolarization-induced.OH formation with MPP(+). Dopamine (DA; 10 microM) also increased the levels of DHBA due to MPP(+). However, the effect of DA after application of L-NAME did not change the levels of DHBA. On the other hand, the application of allopurinol (20 mg/kg i.v., 30 min prior to study), a xanthine oxidase (XO) inhibitor was abolished the both [K(+)](o)- and DA-induced.OH generation. Moreover, when iron(II) was administered to MPP(+) then [K(+)](o) (70 mM)-pretreated animals, a marked increase in the level of DHBA. However, when corresponding experiments were performed with L-NAME-pretreated animals, the same results were obtained. Therefore, NOS activation may be no relation to Fenton-type reaction via [K(+)](o) depolarization-induced.OH generation. The present results suggest that [K(+)](o)-induced depolarization augmented MPP(+)-induced.OH formation by enhancing NO synthesis.

Cardiac microdialysis of salicylic acid to detect hydroxyl radical generation associated with sympathetic nerve stimulation.

We examined the relationship between norepinephrine (NE) and hydroxyl free radical (OH) generation on cardiac nerve stimulation. Salicylic acid in Ringers solution (0.5 nmol microliter-1 min-1) was infused directly through a microdialysis probe to detect the generation of hydroxyl radicals (OH) as reflected by the formation of dihydroxybenzoic acid (DHBA) in the myocardium of anesthetized rats. Sympathetic nerve stimulation increased the release of NE and the formation of DHBA. A positive linear correlation between the release of NE and the formation of 2,3-DHBA (R2 = 0.982) or 2,5-DHBA (R2 = 0.976) was observed. These data indicate that the sustained elevation of NE in the extracellular fluid can be auto-oxidized, which in turn leads (possibly by an indirect mechanism) to the formation of cytotoxic OH free radicals.