Nak Doo Kim

Procyanidins in crataegus extract evoke endothelium-dependent vasorelaxation in rat aorta.

The extract of Crataegus, a mixture of flavonoids and procyanidins extracted from hawthorn, Crataegus oxyacantha, L. and C. monogyna Jacq., relaxed vascular tone or increased production of cyclic GMP in the rat aorta, but flavonoid components of Crataegus extract, hyperoside, rutin and vitexin, did not affect the vascular tone. The aim of the present study was to characterize the endothelium-dependent relaxation elicited by procyanidins fractionated from Crataegus extract in isolated rat aorta. Procyanidins caused endothelium-dependent relaxation which was associated with the production of cyclic GMP. Both responses to these procyanidins were inhibited by methylene blue or N(G)-nitro-L-arginine, but not by indomethacin. Relaxation in response to procyanidins was not affected by atropine, diphenhydramine, [D-Pro2,D-Trp7,9]substance P, propranolol, nifedipine, verapamil and glibenclamide, but were markedly reduced by tetraethylammonium. These findings showed that procyanidins in Crataegus extract may be responsible for the endothelium-dependent nitric oxide-mediated relaxation in isolated rat aorta, possibly via activation of tetraethylammonium-sensitive K+ channels.

GINSENOSIDES OF THE PROTOPANAXATRIOL GROUP CAUSE ENDOTHELIUM-DEPENDENT RELAXATION IN THE RAT AORTA

The vasoactive effects of several ginsenosides, purified from Panax ginseng, were tested in the rat aorta. Ginsenosides from the protopanaxatriol group and its purified ginsenosides Rg1 and Re cause endothelium dependent relaxation which is associated with the formation of cyclic GMP. Both responses to these protopanaxatriol-containing ginsenosides are inhibited by methylene blue, an inhibitor of soluble guanylate cyclase and of nitric oxide synthase. In contrast, ginsenosides from the protopanaxadiol group and its purified ginsenosides Rb1 and Re do not affect vascular tone or production of cyclic GMP in the rat aorta. These findings demonstrate that ginsenosides from the protopanaxatriol group but not from the protopanaxadiol group enhance the release of nitric oxide from endothelial cells and may contribute to the beneficial effect of ginseng on the cardiovascular system.

GINSENOSIDE RG3 MEDIATES ENDOTHELIUM-DEPENDENT RELAXATION IN RESPONSE TO GINSENOSIDES IN RAT AORTA: ROLE OF K+ CHANNELS

The aim of the present study was to characterize the endothelium-dependent relaxation elicited by ginsenosides, a mixture of saponin extracted from Panax ginseng, in isolated rat aorta. Relaxations elicited by ginsenosides were mimicked by ginsenoside Rg1 and ginsenoside Rg1, two major ginsenosides of the protopanaxatriol group. Ginsenoside Rg3 was about 100-fold more potent than ginsenoside Rg1. The endothelium-dependent relaxation in response to ginsenoside Rg3 was associated with the formation of cycle GMP. These effects were abolished by N(G)-nitro-L-arginine and methylene blue. Relaxations in response to ginsenoside Rg3 were unaffected by atropine, diphenhydramine, [D-Pro2, D-Trp7,9]substance P, propranolol, nifedipine, verapamil and glibenclamide but were markedly reduced by tetraethylammonium. Tetraethylammonium modestly reduced the relaxation induced by sodium nitroprusside. These findings indicate that ginsenoside Rg3 is a major mediator of the endothelium-dependent nitric oxide-mediated relaxation in response to ginsenosides in isolated rat aorta, possibly via activation of tetraethylammonium-sensitive K+ channels.

Ginsenoside Rg3 inhibits phenylephrine‐induced vascular contraction through induction of nitric oxide synthase

Ginsenoside Rg3 (Rg3) isolated from Panax ginseng relaxes vessels and exerts a cytoprotective effect. In view of the fact that nitric oxide (NO) is involved in vascular hyporeactivity and immunostimulation, the effects of total ginsenosides (GS) and Rg3 on the vascular responses and the expression of inducible nitric oxide synthase (iNOS) were investigated. Vasocontraction of endothelium‐denuded aortic ring was induced by phenylephrine with or without GS or Rg3. The expression of iNOS was assessed by Western blot and RT–PCR analyses. NF‐κB activation was monitored by gel shift, immunoblot and immunocytochemical analyses. Incubation of the endothelium‐denuded aortic ring with GS or Rg3 inhibited phenylephrine‐induced vasocontraction, which was abrogated by NOS inhibition. GS or Rg3 increased NO production in aortic rings, but Rb1, Rc, Re and Rg1 had no effect. Aortic rings obtained from rats treated with GS or Rg3 responded to phenylnephrine to a lesser extent, while producing NO to a larger extent, than those from control animals. GS or Rg3 induced iNOS in vascular smooth muscle. Rg3 induced iNOS with increase in NO production in Raw264.7 cells. Rg3 increased NF‐κB DNA binding, whose band was supershifted with anti‐p65 and anti‐p50 antibodies, and elicited p65 nuclear translocation, which was accompanied by phosphorylation and degradation of I‐κBα. PKC regulated iNOS induction by Rg3. In conclusion, Rg3 relaxes vessels as a consequence of NO production, to which iNOS induction contributes, and iNOS induction by Rg3 accompanied NF‐κB activation, which involves phosphorylation and degradation of I‐κBα and nuclear translocation of p65.

Ginsenosides evoke endothelium-dependent vascular relaxation in rat aorta

1. Ginsenoside (10-100 mg/kg, i.v.) lowered blood pressure in a dose-dependent manner in rats. 2. Ginsenoside (10(-5)-3 x 10(-4) g/ml) relaxed the aorta after contractions were induced by 10(-6) M phenylephrine in the aorta with endothelium but not in that without endothelium. 3. The relaxation induced by ginsenoside was attenuated by 3 x 10(-7) M methylene blue (MB) and 10(-4) M NG-monomethyl-L-arginine (L-NMMA) but not inhibited by 10(-5) M indomethacin. 4. Ginsenoside (10(-4) g/ml for 2 min) increased the accumulation of cGMP in rings with endothelium. L-NMMA and MB inhibited the accumulation of cGMP induced by ginsenoside. 5. These data suggest that vascular relaxations induced by ginsenoside are mediated by release of endothelium-drived nitric oxide which enhances the accumulation of cGMP.

Inhibition of cytochrome P450 2E1 expression by 2-(allylthio)Pyrazine, a Potential chemoprotective agent: hepatoprotective Effects

Cytochrome P450 2E1 (P450 2E1) is active in both the detoxification and activation of small organic molecules. The effects of 2-(allylthio)pyrazine (2-AP) on P450 2E1-catalytic activity and the expression of rat hepatic P450 2E1 were examined. 2-AP competitively inhibited 4-nitrophenol hydroxylase activity in vitro (Ki, 12 microM). 2-AP treatment of rats (200 mg/kg/day, p.o., 1-3 days old) resulted in 20-30% decreases in the rates of P450 2E1-specific metabolic activities. Immunoblot analysis also revealed that hepatic microsomes isolated from 2-AP-treated rats showed substantial decreases in P450 2E1 level. 2-AP-suppressed isoniazid (INH)-inducible hepatic P450 2E1 levels, as shown by both metabolic activities and immunoblot analyses. Thus, 2-AP was effective in suppressing both constitutive and inducible P450 2E1 expression. Northern blot analysis showed that 2-AP transiently suppressed the hepatic P450 2E1 mRNA level, suggesting that suppression in P450 2E1 expression by 2-AP may be mediated in part by transcriptional inactivation. Hepatoprotective effects of 2-AP against toxicants were monitored in mice. 2-AP pretreatment prior to the administration of lethal doses of acetaminophen (AAP) or INH substantially reduced toxicant-induced mortality. Whereas serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were markedly elevated after AAP administration (i.e. 9-20-fold), 2-AP pretreatment of animals before AAP administration resulted in >95% decreases in elevated serum aminotransferase activities. 2-AP was also effective against CCl4-induced hepatotoxicity. Whereas CCl4 treatment caused 35-70-fold increases in aminotransferase activities, treatment of mice with 2-AP (>10 mg/kg) resulted in the blocking of CCl4-induced liver toxicity. The hepatoprotective effect of 2-AP was in part due to 2-AP-induced elevation of hepatic GSH levels. Whereas AAP or CCl4 treatment resulted in 70-80% reduction in hepatic GSH levels, pretreatment of mice with 2-AP caused a 40-210% elevation in hepatic GSH levels, as compared with either AAP or CCl4 alone. 2-AP pretreatment also reduced AAP- or CCl4-induced increases in lipid peroxidation in a dose-dependent manner. The results of these metabolic activities and of immunoblot and RNA blot analyses demonstrate that 2-AP is efficacious in suppressing constitutive and inducible P450 2E1 expression and effective in protecting against toxicant-induced liver toxicity.

Inhibition of cytochrome P4502E1 expression by organosulfur compounds allylsulfide, allylmercaptan and allylmethylsulfide in rats

Cytochrome P4502E1 (CYP2E1) is active in both detoxication and activation of small organic molecules. The effects of organosulfur compounds including allylsulfide (AS), allylmercaptan (AM) and allylmethylsulfide (AMS) on the expression of CYP2E1 were examined in rats. 4-Nitrophenol, aniline hydroxylase and N-nitrosodimethylamine demethylase activities, the rates of which represent the level of CYP2E1, decreased in hepatic microsomes isolated from rats treated with AS in a time-dependent manner by 45% to 90%, as compared to control. Pyrazine-induced hepatic microsomes exhibited approximately 5-fold increases in CYP2E1-catalysed metabolic activities, whereas the hepatic microsomes obtained after treatment of animals with both AS and pyrazine showed rates comparable to or less than those in control microsomes. AM or AMS suppressed constitutive and pyrazine-inducible levels of CYP2E1 similarly to AS. Immunoblot analyses of hepatic microsomes, using an anti-CYP2E1 antibody, showed that AS, AM and AMS significantly suppressed constitutive levels of CYP2E1 apoprotein after 24, 48 and 72 hr. Time-dependent induction of CYP2E1 by pyrazine was also completely blocked by treatment of animals with AS throughout the experimental period, as evidenced by immunoblot analysis. The levels of CYP2E1 apoprotein in the hepatic microsomes isolated from animals treated with both AM and pyrazine, or with both AMS and pyrazine were comparable to those in control hepatic microsomes at days 1-3 post-treatment. Treatment of rats with each of these organosulfur compounds caused no significant changes in the levels of CYP2E1 mRNA, as assessed by slot and northern blot analyses, suggesting that post-transcriptional regulation may be associated with the suppression of CYP2E1 apoprotein levels. The results of metabolic activities, immunoblot analyses and RNA blot analyses demonstrated that these organosulfur compounds are effective in suppressing constitutive and inducible expression of CYP2E1.

The ginsenoside Rg3 evokes endothelium-independent relaxation in rat aortic rings: role of K+ channels.

The purpose of the present study was to characterize the mechanism underlying the direct relaxing activity of ginsenosides on vascular smooth muscle. The total ginsenoside mixture, ginsenosides from either the protopanaxadiol group or the protopanaxatriol group, and the ginsenoside Rg3 from the protopanaxatriol group caused a concentration-dependent relaxation of rat aortic rings without endothelium contracted with 25 x 10(-3) M KCl but affected only minimally those contracted with 60 x 10(-3) M KCl. Ginsenoside Rg3 was the most potent relaxing agonist. Relaxations elicited by ginsenoside Rg3 were markedly reduced by tetraethylammonium, a blocker of non-selective K+ channels, but not by glibenclamide, a blocker of ATP-sensitive K+ channels. Ginsenoside Rg3 significantly inhibited Ca2+-induced concentration-contraction curves and the 45Ca2+ influx in aortic rings incubated with 25 x 10(-3) M KCl whereas these responses were not affected in rings incubated with 60 x 10(-3) M KCl. Ginsenoside Rg3 caused a time- and concentration-dependent efflux of 86Rb from aortic rings that was inhibited by tetraethylammonium but not by glibenclamide. These findings indicate that ginsenoside Rg3 is a potent inhibitor of vascular smooth muscle tone and that this effect seems to be due to an inhibition of Ca2+ influx and stimulation of K+ efflux, possibly via activation of tetraethylammonium-sensitive K+ channels.

Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase.

We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10μg/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.

Pharmacokinetics and pharmacodynamics of bumetanide after intravenous and oral administration to rats: Absorption from various GI segments

Bumetanide, 2, 8, and 20 mg/kg, was administered both intravenously and orally to determine the pharmacokinetics and pharmacodynamics of bumetanide in rats (n=10–12). The absorption of bumetanide from various segments of GI tract and the reasons for the appearance of multiple peaks in plasma concentrations of bumetanide after oral administration were also investigated. After iv dose, the pharmacokinetic parameters of bumetanide, such ast1/2 (21.4, 53.8 vs. 127 min),CL (35.8, 19.1 vs. 13.4 ml/min per kg),CLNR (35.2, 17.8 vs. 12.6 ml/min per kg) andVSS (392, 250 vs. 274 ml/kg) were dose-dependent at the dose range studied. It may be due to the saturable metabolism of bumetanide in rats. After iv dose, 8-hr urine output per 100g body weight increased significantly with increasing doses and it could be due to significantly increased amounts of bumetanide exreted in 8-hr urine with increasing doses. The total amount of sodium and chloride exreted in 8-hr urine per 100g body weight also increased significantly after iv dose of 8 mg/kg, however, the corresponding values for potassium were dose-independent. After oral administration, the percentages of the dose excreted in 24-hr urine as unchanged bumetanide were dose-independent. Bumetanide was absorbed from all regions of GI tract studied and approximately 43.7, 50.0, and 38.4% of the orally administered dose were absorbed between 1 and 24 hr after oral doses of 2, 8, and 20 mg/kg, respectively. Therefore, the appearance of multiple peaks after oral administration could be mainly due to the gastric emptying patterns. The percentages of bumetanide absorbed from GI tract as unchanged bumetanide for up to 24 hr after oral doses of 2, 8, and 20 mg/kg (96.2, 95.4 vs. 98.2%) were not significantly different, suggesting that the problem of precipitation of bumetanide in acidic gastric juices or dissolution may not contribute significantly to the absorption of bumetanide after oral administration. Urine output per 100g body wt increased at oral doses of 8 and 20 mg/kg.