Downregulation of vascular soluble guanylate cyclase induced by high salt intake in spontaneously hypertensive rats.

1. Cyclic guanosine monophosphate (cyclic GMP)-mediated mechanism plays an important role in vasodilatation and blood pressure regulation. We investigated the effects of high salt intake on the nitric oxide (NO) - cyclic GMP signal transduction pathway regulating relaxation in aortas of spontaneously hypertensive rats (SHR). 2. Four-week-old SHR and normotensive Wistar-Kyoto rats (WKY) received a normal salt diet (0.3% NaCl) or a high salt diet (8% NaCl) for 4 weeks. 3. In aortic rings from SHR, endothelium-dependent relaxations in response to acetylcholine (ACh), adenosine diphosphate (ADP) and calcium ionophore A23187 were significantly impaired by the high salt intake. The endothelium-independent relaxations in response to sodium nitroprusside (SNP) and nitroglycerin were also impaired, but that to 8-bromo-cyclic GMP remained unchanged. On the other hand, high salt diet had no significant effects on the relaxations of aortic rings from WKY. 4. In aortas from SHR, the release of NO stimulated by ACh was significantly enhanced, whereas the production of cyclic GMP induced by either ACh or SNP was decreased by the high salt intake. 5. Western blot analysis showed that the protein level of endothelial NO synthase (eNOS) was slightly increased, whereas that of soluble guanylate cyclase (sGC) was dramatically reduced by the high salt intake. 6. These results indicate that in SHR, excessive dietary salt can result in downregulation of sGC followed by decreased cyclic GMP production, which leads to impairment of vascular relaxation in responses to NO. It is notable that chronic high salt intake impairs the sGC/cyclic GMP pathway but not the eNOS/NO pathway.

Oxidants in cigarette smoke extract modify low-density lipoprotein in the plasma and facilitate atherogenesis in the aorta of Watanabe heritable hyperlipidemic rabbits.

Epidemiological studies have shown that cigarette smoking is a major cause of atherosclerosis. Oxidants as well as nicotine in cigarette smoke have been implicated in atherogenesis. To clarify the mechanism involved, we examined the chronic effects of nicotine and nicotine-free cigarette smoke extracts (CSE) on oxidative modification of low-density lipoprotein (LDL) in the plasma of Watanabe heritable hyperlipidemic rabbits and atherogenesis in the aorta. CSE was prepared by bubbling the gas phase of smoke (1 ml/three cigarettes) into phosphate buffer saline, and 3 ml of this CSE was injected daily into the ear vein of the rabbit for five months. The rabbits treated with CSE showed an increase in lipid peroxide levels, estimated as thiobarbituric acid reactive substances (TBARS), with a corresponding decrease in vitamin E levels in the plasma. They also showed enhanced oxidative modification of LDL, assessed by anion-exchange HPLC, incorporation into macrophages and measurement of TBARS. These events could be efficiently prevented by administering vitamin E (150 mg/kg/day, p.o.). Nicotine alone (0.5 mg/kg/day, s.c.) led to a temporary increase in the plasma triglyceride level. At the end of the experiment, CSE but not nicotine had caused progression of atherosclerotic lesions together with accumulation of cholesteryl ester in the thoracic aorta, while vitamin E had significantly prevented such atheromatous formation. These results indicate that oxidants in CSE can promote the development of atherosclerosis through oxidative modification of plasma LDL, particularly in hypercholesterolemia, and offer evidence for increased vitamin E utilization in smokers.

2-Arachidonoylglycerol, a candidate of endothelium-derived hyperpolarizing factor.

We investigated whether 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, is involved in acetylcholine- and calcium ionophore A23187-induced relaxations in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME) and indomethacin, which is considered to be mediated by endothelium-derived hyperpolarizing factor (EDHF). In rabbit mesenteric arterial rings pre-constricted with noradrenaline, 2-arachidonoylglycerol caused concentration-dependent relaxation. The 2-arachidonoylglycerol-induced relaxations were not affected by endothelium removal. N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-caroxamide (SR141716A) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morholinyl-1H-pyrazole-3-carboxamide (AM281), cannabinoid CB(1) receptor antagonists, significantly attenuated 2-arachidonoylglycerol-induced relaxation and the acetylcholine-induced relaxation only slightly, but not the calcium ionophore A23187-induced relaxation. On the other hand, charybdotoxin plus apamin, K(+) channel blockers, significantly attenuated acetylcholine and calcium ionohore A23187-induced relaxations but not 2-arachidonoylglycerol-induced relaxations. These results suggest that 2-arachidonoylglycerol can cause relaxations via cannabinoid CB(1) receptors, but is not involved in EDHF-mediated relaxations.

Suitable indices for evaluating the intensity of tumor metastasis in a mouse experimental metastatic model.

We investigated the time course of developmental changes of tumor metastasis in mice intravenously inoculated with B16-F10 melanoma cells. The mice showed a normal blood profile at 7 and 14 days after the injection, while at 21 days they developed severe anemia and thrombocytopenia. The number of metastatic lung nodules in the mice at 7 and 14 days after the cell injection was 68 +/- 12 and 326 +/- 39 (mean +/- S. E. of 5 mice), respectively, while it could not be accurately counted at 21 days owing to a fusion of the nodules. The wet weight and the melanin content of the lung markedly increased at 21 days. These results suggest that an abnormal blood profile, lung weight and the melanin content of the lung are suitable indices for the late stage of the experimental metastatic model, while the number of lung nodules is suitable for the early stage.

Inhibitory effects of water extracts from fruiting bodies of cultured Cordyceps sinensis on raised serum lipid peroxide levels and aortic cholesterol deposition in atherosclerotic mice.

We investigated the effects of the water extracts of the fruiting bodies of cultured Cordyceps sinensis (WECS) on lipid metabolism in mice fed an atherogenic diet. WECS was orally administered at doses of 50, 100 and 200 mg/kg/day for 12 weeks. WECS showed no toxic effects on the growth rate, liver or kidney weights of the mice. Mice fed the atherogenic diet showed marked increases in serum lipid and lipid peroxide levels and also aortic cholesterol levels, particularly cholesteryl ester level, a major lipid constituent in atherosclerotic lesions. WECS significantly suppressed the increased serum lipid peroxide level but not other lipid levels in a dose-dependent manner. WECS also suppressed the increased aortic cholesteryl ester level in a dose-dependent manner. These results suggest that WECS prevents cholesterol deposition in the aorta by inhibition of LDL oxidation mediated by free radicals rather than by reduction in serum lipid level. WECS may exert beneficial effects on the formation of the atherosclerotic lesion induced by oxidative stress with few side effects.

Antioxidant activity of the extracts from fruiting bodies of cultured Cordyceps sinensis.

Cordyceps sinensis is one of the most valued herbs in traditional Chinese medicine. We investigated the antioxidant activities of the cultured fruiting bodies of Cordyceps sinesis. The water and ethanol extracts of Cordyceps sinensis were found to possess a potent antioxidant activity. The scavenging effects of the extracts on superoxide were very weak, but the extracts moderately inhibited malondialdehyde formation via hydroxyl radical induced by SIN-1, a peroxynitrite generator. Of the extracts examined, the hot water extract (70 degrees C for 5 min) showed the greatest oxygen free radical scavenging activity. Also, when low-density lipoprotein (LDL) was incubated with macrophages in the presence of CuCl2 (1 microM), the hot water extract showed a strong inhibitory effect against lipid peroxidation in the medium and consequent accumulation of cholesteryl ester in macrophages. Their activities were comparable to that of authentic Cu/Zn SOD. These results suggest that the extracts of cultured Cordyceps sinensis possess potent antioxidant and anti-lipid peroxidation activities and inhibit accumulation of cholesteryl ester in macrophages via suppression of LDL oxidation.

Altered endothelium-dependent responsiveness in the aortas and renal arteries of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of non-insulin-dependent diabetes mellitus.

We examined endothelium-dependent relaxation in the aortas and renal arteries of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of non-insulin-dependent diabetes mellitus, in comparison with non-diabetic Long-Evans Tokushima Otsuka rats as controls. Acetylcholine-induced relaxation in both arteries was attenuated, and the attenuation was restored to the control level by indomethacin. The relaxation was inhibited completely in the aortas, but only partially in renal arteries by N(G)-nitro-L-arginine methyl ester, and the degree of the latter inhibition was greater in OLETF rats than in the controls. The relaxation was inhibited by aminoguanidine in both arteries of OLETF rats but not in the controls. Serum NO(2) plus NO(3) levels significantly increased in OLETF rats. These results suggest that impairment of relaxation in OLETF rat arteries is due to increased release of contractile factors but not decreased release of nitric oxide.

Evidence of modified LDL in the plasma of hypercholesterolemic WHHL rabbits injected with aqueous extracts of cigarette smoke.

Cigarette smoking is a major risk factor for atherosclerosis, and oxidative modification of low density lipoprotein (LDL) is a key event in the development of atherosclerosis. The aim of the present study was to determine whether modified LDL would be formed in the plasma of Watanabe heritable hyperlipidemic (WHHL) rabbits injected with nicotine-free cigarette smoke extracts (CSE). In order to assess this, cigarette smoke-modified LDL (CS-LDL) was prepared by incubation of rabbit native LDL (N-LDL) with CSE for 24 h. The oxidative modification in CS-LDL was well established by the reduced ratio between two LDL subfractions (LDL2/LDL3) separated by anion-exchange HPLC, together with the fast migration in the anodic direction in agarose gel electrophoresis and the increased lipid peroxide levels. Very similar modification was noted with mildly oxidatively modified LDL prepared by incubation of N-LDL with 5 µM CuCl(2) for 1 h. When WHHL rabbits (n=4) intravenously received a single injection of CSE, the ratio of LDL2/LDL3 was markedly reduced compared with the control rabbits (n=4) while total cholesterol levels in the plasma gradually decreased until 24 h after the injection. These results suggest that oxidatively modified LDL, probably like CS-LDL, is produced in the plasma of WHHL rabbits injected with CSE.

Functional evidence for anti-oxidant action of fluvastatin on low-density lipoprotein using isolated macrophages and aorta.

1. Fluvastatin has been reported to have not only a hypocholesterolaemic effect, but also a protective effect on low-density lipoprotein (LDL) from oxidation. We functionally evaluated the anti-oxidant effect of fluvastatin on oxidation of LDL by copper ions in vitro using mouse macrophages and rabbit aorta preparations. 2. After native LDL (N-LDL) from rabbit plasma had been pre-incubated in the presence or absence of fluvastatin (10 micromol/L) for 4 h, the N-LDL was mildly oxidized by incubation with 5 micromol/L CuCl2 for 5 h and two oxidized LDL, fluvastatin-pretreated (Flu-OxLDL) and -non-treated (OxLDL), were prepared. The level of thiobarbituric acid-reactive substances (TBARS) in Flu-OxLDL and OxLDL markedly increased compared with N-LDL. The degree of increment was significantly less in Flu-OxLDL than OxLDL. 3. When macrophages were incubated with Flu-OxLDL or OxLDL, the amount of cholesteryl ester that accumulated in the macrophages markedly increased compared with N-LDL. The degree of increment was significantly less in Flu-OxLDL than OxLDL. 4. Acetylcholine-induced endothelium-dependent relaxations in rabbit aortic rings were impaired in the presence of either Flu-OxLDL or OxLDL. The degree of impairment was significantly less in Flu-OxLDL. 5. The increased TBARS level, facilitated cholesteryl ester accumulation in macrophages and impaired endothelium-dependent relaxation elicited by OxLDL were not affected by simultaneous treatment with fluvastatin (10 micromol/L). 6. These findings indicate that fluvastatin can protect plasma LDL from oxidative modification and, thereby, prevent cholesterol accumulation in macrophages and endothelial dysfunction in blood vessels. This additional anti-oxidative effect of fluvastatin may be beneficial for preventing the progression of atherosclerosis.

Peroxynitrite-generating species: good candidate oxidants in aqueous extracts of cigarette smoke.

Cigarette smoking is a well-known risk factor for atherosclerosis, but the mechanism of the adverse biological effect of smoking remains to be established. Cigarette smoke contains high concentrations of free radicals and oxidants. We show here that cigarette smoke extracts (CSE), prepared by bubbling the gas phase of smoke into phosphate-buffered saline, could convert tyrosine to 3-nitrotyrosine. The tyrosine nitration terminated 6 h after incubating tyrosine with CSE at 37 degrees C. These results indicate that the active oxidants in CSE are peroxynitrite-generating species like 3-morpholinosydnonimine (SIN-1), suggesting that they modify plasma lipoproteins and contribute to the pathogenesis of atherosclerosis.

Characterization of nitric oxide- and prostaglandin-independent relaxation in response to acetylcholine in rabbit renal artery.

1. We investigated the characterization of acetylcholine (ACh)-induced NG-nitro-L-arginine methyl ester (L-NAME)- and indomethacin (IND)-resistant relaxations, which can be mediated by endothelium-derived hyperpolarizing factor (EDHF), in rabbit renal arterial rings. 2. The relaxations were inhibited by SKF 525A, a cytochrome P450 inhibitor, but were not affected by other inhibitors, namely clotrimazole, 17-octadecynoic acid and alpha-naphthoflavone. Furthermore, 11,12-epoxyeicosatrienoic acid, a cytochrome P450 metabolite, did not relax arterial rings. 3. Arterial relaxations were significantly attenuated by charybdotoxin and iberiotoxin, but not by apamin, all K+ channel blockers. 4. In a sandwich bioassay experiment, ACh-induced L-NAME- and IND-resistant relaxations were not transferred to the detector site. 5. Relaxations were also significantly attenuated by 1-heptanol and 18 alpha-glycyrrhetinic acid, gap junctional coupling inhibitors. 6. These results indicate that, in the rabbit renal artery, L-NAME- and IND-resistant relaxations are mediated by factors other than cytochrome P450-derived arachidonic acid metabolites, which may be able to diffuse into the lumen but be partly transferred via myoendothelial gap junctions to adjacent vascular smooth muscle cells and relax muscles by opening high-conductance Ca(2+)-activated K+ channels.

Excessive salt or cholesterol intake alters the balance among endothelium-derived factors released from renal arteries in spontaneously hypertensive rats.

We investigated the vasorelaxation in renal arteries isolated from spontaneously hypertensive rats (SHRs) fed a basal, a high-salt, or a high-cholesterol diet for 8 weeks. In renal arterial rings from the control group, acetylcholine (ACh)-induced endothelium-dependent relaxations were markedly increased by indomethacin (IND) and ONO-3708, a prostaglandin H2/thromboxane A2-receptor antagonist, but not affected by OKY-046, a thromboxane A2 synthetase inhibitor. These increased relaxations were partially inhibited by either NG-nitro-L-arginine methyl ester (L-NAME) or charybdotoxin (CTX), and almost completely abolished by the combination of L-NAME plus CTX. The ACh-induced endothelium-dependent relaxations in the absence of IND were significantly attenuated by the high-salt intake but not affected by the high-cholesterol intake. The degrees of relaxations in the presence of IND were approximately equal among the three diet groups. On the other hand, the relaxations in the presence of IND plus L-NAME were significantly augmented by a high-cholesterol intake and abolished by a high-salt intake, and the relaxations in the presence of IND plus CTX were slightly reduced by a high-cholesterol intake and significantly augmented by a high-salt intake. The production of cyclic guanosine monophosphate (cGMP) in response to ACh was significantly decreased by a high-cholesterol intake and tended to be increased by a high-salt intake. These findings indicate that in the renal artery of SHRs, ACh causes production of a sufficient amount of nitric oxide (NO), together with a relaxing factor resembling endothelium-derived hyperpolarizing factors (EDHFs) and also endothelium-derived contracting factors (EDCFs), probably prostaglandin H2. Our results also suggest that excessive salt intake increases the release of EDCF and NO and decreases that of an EDHF-like factor, whereas excessive cholesterol intake increases release of an EDHF-like factor and decreases that of NO.

High-performance liquid chromatographic assay of hydroperoxide levels in oxidatively modified lipoproteins.

An anion-exchange HPLC method has been developed for the chemiluminescence (CL) assay of hydroperoxide (HPO) levels in native and oxidized low density lipoproteins (N- and Ox-LDLs, respectively) of Watanabe heritable hyperlipidemic (WHHL) rabbits. The method involves anion-exchange HPLC separation in N- and Ox-LDLs using a DEAE-glucomannan gel, and direct CL detection of HPOs in them without extraction of the lipids following postcolumn reaction with isoluminol, microperoxidase and Triton X-100. Addition of Triton X-100, which could solubilize lipids, were essential for the detection of HPOs in N- and Ox-LDLs. With an increase in the degree of oxidation, Ox-LDL was more retained on the DEAE-glucomannan gel with a concomitant increase in the CL intensity. The proposed method could analyze the HPO levels in N- and Ox-LDLs of WHHL rabbits without extraction of the lipids.

Both extracellular ATP and shear stress regulate the release of nitric oxide in rat caudal artery.

1. To elucidate the physiological role of nitric oxide (NO) in regulating vascular tone, the effects of NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, on the vasoconstrictor response to noradrenaline (NA) in rat caudal artery was examined. 2. NG-Nitro-L-arginine methyl ester significantly potentiated the NA-induced increase in perfusion pressure in the perfused caudal artery, but did not affect the NA-induced contraction in caudal artery ring preparations. In addition, an increase in perfusion pressure mechanically produced by a stepwise increase in flow rate was not affected by L-NAME. 3. Noradrenaline evoked a significant increase in the release of endogenous ATP and its metabolites from the perfused artery, whereas increased perfusion pressure as a result of increased flow rate did not evoke release of endogenous ATP. 4. In the presence of exogenously applied ATP, L-NAME significantly potentiated the increase in perfusion pressure produced by increased flow rate. 5. These results indicate that perfused vascular tone is regulated by endogenous NO and suggest that extracellular ATP may participate in the synthesis and release of NO by shear stress in endothelial cells in the rat caudal artery.

Activation of in vivo Kupffer cell function by oral administration of Cordyceps sinensis in rats.

We investigated the effect of water extracts of Cordyceps sinensis (WECS) on Kupffer cell function in rats. Rats were received a single i.v. injection of a colloidal carbon solution and then the clearance rate from the blood were measured. The rats had been daily administered with WECS, p.o. at a dose of 200 mg/kg for 25 days until the day before the injection of colloidal carbon. The half-life of the colloidal carbon in the blood of rats administered WECS 200 mg/kg was significantly shorter than that of the control rats. This suggests that accelerated function of Kupffer cells is partially involved in the anti-metastatic action of WECS.

Inhibitory effect of Cordyceps sinensis on spontaneous liver metastasis of Lewis lung carcinoma and B16 melanoma cells in syngeneic mice.

We investigated the effect of the water extract of Cordyceps sinensis (WECS) on liver metastasis of Lewis lung carcinoma (LLC) and B16 melanoma (B16) cells in mice. C57BL/6 mice were given a s.c. injection of LLC and B16 cells and sacrificed 20 and 26 days after tumor inoculation, respectively. WECS was daily administered p.o. to the mice in a dose of 100 mg/kg body weight (wt.) in the experiment of LLC and in a dose of 100 or 200 mg/kg body wt. in the experiment of B16 from one week before tumor inoculation to one day before the date of sacrifice. The tumor cells increased in the thigh in LLC-inoculated mice and in the footpad in B16-inoculated mice. The relative liver wt. of the tumor-inoculated mice significantly increased as compared to that of the normal mice due to the tumor metastasis, as verified by the hematoxylin-eosin staining pathological study in the LLC experiment. The relative liver wt. of the WECS-administered mice significantly decreased relative to that of the control mice in both the LLC and B16 experiments. WECS showed a strong cytotoxicity against LLC and B16 cells, while cordycepin (3'-deoxyadenosine), an active component of WECS, was not cytotoxic against these cells. These findings suggest that WECS has an anti-metastatic activity that is probably due to components other than cordycepin.

Evidence of modified lipoprotein in the plasma of Watanabe heritable hyperlipidemic rabbits by anion-exchange high-performance liquid chromatographic assay.

We searched for evidence for the presence of oxidatively modified low density lipoproteins (Ox-LDL) in the plasma of Watanabe heritable hyperlipidemic (WHHL) rabbits using an anion-exchange high-performance liquid chromatographic (HPLC) method newly developed for the assay of artificially modified lipoproteins. Various Ox-LDLs were prepared by incubation of native LDL (N-LDL) from rabbit plasma with 5 microM CuCl2 for 0, 1, 3 and 24 h, and separated by anion-exchange HPLC method using a DEAE-glucomannan gel column. With an increase in the incubation time, LDL was further oxidized and retained on the DEAE-glucomannan gel. This oxidized LDL migrated faster in the anodic direction in agarose gel electrophoresis, accompanied by an increase in lipid peroxide levels, estimated as thiobarbituric acid reactive substances (TBARS). There was a good agreement in the chromatographic and electrophoretic behaviors of the various Ox-LDLs. LDL from WHHL rabbits showed increases in TBARS levels, electrophoretic mobility on agarose gel electrophoresis and cholesteryl ester accumulation in mouse peritoneal macrophages compared to that from normolipidemic Japanese white (JW) rabbits. When LDLs from both animals were compared by the anion-exchange HPLC method using linear gradient or stepwise elution, LDL from WHHL rabbits had a longer retention time and was eluted by a higher concentration of sodium chloride. These results suggest that LDL in WHHL rabbit plasma may undergo mild oxidative modification.

Mechanisms of impairment of endothelium-dependent relaxation to acetylcholine in Watanabe heritable hyperlipidaemic rabbit aortas.

1. The mechanism of impairment of the endothelium-dependent relaxation in response to acetylcholine (ACh) in aortas from Watanabe heritable hyperlipidaemic (WHHL) rabbits was investigated using a modified sandwich (layered) technique. Intact aortas from WHHL rabbits or Japanese white (JW) rabbits as the control were used as donor strips of endothelium-derived relaxing factor (EDRF) and endothelium-denuded aortas from JW rabbits were used as detector strips. The EDRF released from a donor strip could be directly detected as the relaxation response in a detector strip. 2. The endothelium-dependent relaxations in all rabbit arteries were almost abolished by treatment with N(G)-nitro-L-arginine methyl ester (an inhibitor of nitric oxide synthase). 3. The ACh-induced endothelium-dependent relaxations in the donor strips were impaired in WHHL rabbits in comparison with relaxations in JW and heterozygous WHHL rabbits. Similarly, the relaxation in the detector strips induced by EDRF released from donor strips was reduced in WHHL rabbits. There was a good negative correlation between the aortic total cholesterol content in the donor strips and the degree of relaxation in the detector strips from WHHL rabbits. 4. The reduced relaxation in the detector strips when using donor strips with high cholesterol accumulation or atheromatous plaque was not affected by superoxide dismutase plus catalase (scavengers of superoxide anions), indomethacin (an inhibitor of cyclo-oxygenase), ONO-3708 (an antagonist of endoperoxide/thromboxane receptor) and 97-139 (an antagonist of endothelin ET(A) receptor). 5. These results suggest that the mechanism of impaired endothelium-dependent relaxations in atherosclerotic WHHL rabbit aortas may be due to the reduced amount of EDRF, probably nitric oxide, from the endothelium and not due to its inactivation by oxygen-derived free radicals or masking by increased production of endothelium-derived contracting factors.

Cigarette smoke-modified low density lipoprotein impairs endothelium-dependent relaxation in isolated rabbit arteries.

1. We investigated the effects of cigarette smoke-modified low-density lipoprotein (CS-LDL) on endothelium-dependent relaxations in various isolated rabbit arteries, and compared them with those of oxidized LDL (Ox-LDL). 2. In aorta rings, acetylcholine-induced endothelium-dependent relaxations were attenuated by preincubation with CS-LDL and Ox-LDL. Endothelium-independent relaxations induced by sodium nitroprusside were not modified. Similar changes were observed in rings of coronary and basilar arteries. 3. These findings indicate that CS-LDL, as well as Ox-LDL, may decrease the release or activity of endothelium-derived relaxing factor in vascular endothelial cells.