Cholesterol feeding impairs endothelium-dependent relaxation of rabbit aorta

Experiments were designed to assess the effect of cholesterol feeding on the endothelium-mediated relaxation of the rabbit aorta to acetylcholine. Age-matched male New Zealand white rabbits were fed either a 2% cholesterol diet or standard rabbit chow. The animals were anaesthetized with sodium pentobarbitone and sacrificed after 4 and 8 weeks on these diets. Rings were prepared from the proximal thoracic aorta and examined in tissue baths. These rings were contracted first with norepinephrine (-6 log mol/L) and acetylcholine was added to demonstrate the endothelium-mediated relaxation. The endothelium-dependent relaxation was significantly less in aortas from rabbits fed the 2% cholesterol diet than in aortas from animals fed the conventional diet. This impairment of relaxation was apparent after both 4 and 8 weeks of cholesterol feeding. In both groups of animals no relaxation was seen in rings from which the endothelium was removed. These results show that cholesterol feeding leads to an impairment of endothelium-mediated relaxation of the rabbit aorta to acetylcholine.     

Possible mechanism of captopril induced endothelium-dependent relaxation in isolated rabbit aorta

The mechanism of captopril, an angiotensin converting enzyme (ACE) inhibitor with sulfhydryl group (SH) in its structure, to produce an endothelium-dependent vasorelaxation was studied. In rabbit aorta with intact endothelium and precontracted with phenylephrine, captopril and superoxide dismutase (SOD) produced dose-dependent relaxation. Lisinopril, an ACE inhibitor without a -SH group in its structure, did not produce endothelium-dependent relaxation. It was observed that captopril, like SOD, produced the relaxation by protecting the EDRF from getting inactivated by superoxide anions as pyrogallol and methylene blue inhibited both the captopril and SOD-mediated relaxation. The free radical scavenging action of captopril is further substantiated by the observation that captopril, but not lisinopril, inhibited FeCl₃/ascorbic acid-induced lipid peroxidation in whole tissue homogenates of rabbit aorta to a level comparable to that of SOD. These results suggest that endothelium-dependent vasodilation produced by captopril may be due to its ability to scavenge superoxide anion and this property may be ascribed to the -SH group present in its structure.     

Vasodilatory and anti-inflammatory effects of the aqueous extract of rhubarb via a NO-cGMP pathway

While conducting an in vitro screen of various medicinal plant extracts, an aqueous extract of rhubarb (Rheum undulatum L, AR) was found to exhibit a distinct vasorelaxant activity. AR induced a concentration-dependent relaxation of the phenylephrine-precontracted aorta. This effect disappeared with the removal of functional endothelium. Pretreatment of the aortic tissues with N(G)-nitro-L-arginine methyl ester (L-NAME), methylene blue, or 1H-[1,2,4]-oxadiazole-[4,3-alpha]-quinoxalin-1-one (ODQ) inhibited the relaxation induced by AR. Incubation of human umbilical vein endothelial cells (HUVECs) with AR increased the production of cGMP in a dose-dependent manner, but this effect was blocked by pretreatment with L-NAME and ODQ, respectively. AR treatment attenuated TNF-alpha-induced NF-kappaB p65 translocation in HUVECs in a dose-dependent manner. In addition, AR suppressed the expression levels of adhesion molecules including ICAM-1 and VCAM-1 induced by TNF-alpha in HUVECs. TNF-alpha-induced MCP-1 expression was also attenuated by the addition of AR. This attenuation was blocked by pretreatment with either L-NAME or ODQ. AR treatment inhibited cellular adhesion of U937 cells onto HUVECs induced by TNF-alpha. Taken together, the present study suggests that AR dilates vascular smooth muscle and suppresses the vascular inflammatory process via endothelium-dependent NO/cGMP signaling.     

Correlations between the endothelium-dependent relaxation of the aorta and arterial pressure in rats with myocardial infarction

The effect of experimental myocardial infarction on endothelium-dependent relaxation was studied on isolated rat aorta and compared with the dynamics of arterial pressure (AP). It was shown that the endothelium-dependent relaxation of aorta was increased 1.8 times 3 h following the myocardial infarction. Simultaneously the drop in AP which had begun immediately following the experimental infarction became maximal. In 24 h both the indices were restored practically to the initial level. There was a significant negative correlation between the extent of endothelium-dependent relaxation and AP. It was suggested that the increase in endothelium-dependent relaxation could influence vascular tone, the drop in AP, and, finally, the development of cardiogenic shock in myocardial infarction in man.     

Influence of hypoxia and anoxia on endothelium-dependent dilatation response of rat's aorta exposed to low-intensity gamma irradiation

The study of isolated segments of aorta has shown that prolongated exposure of rats to gamma-radiation with 50 cGy dose (with a dose rate of 2.8 x 10(7) Gy/s) causes the decrease in endothelium-dependent dilatation responses to M-cholinoreceptors stimulation. During oxygenation the post-radiation changes are displayed within one month, and under functional loads, specifically, during perfusion by hypoxic and anoxic solutions--in more remote terms of the post-radiation period.     

Correlation of lysophosphatidylcholine-induced vs spontaneous relaxation to cyclic GMP levels in rabbit thoracic aorta

We demonstrated previously that 10(-5)M micellar solution of lysophosphatidylcholine (LPC) produced relaxation of rabbit thoracic aorta in a dose-dependent manner. It was also noticed that histamine (HA)-precontracted strips relaxed spontaneously in the absence of LPC. We now measured the cyclic GMP changes during these two types of relaxation. Results indicate that while LPC-induced relaxation was mediated through cyclic GMP, spontaneous relaxation was not. The vasodilating effect of LPC was not due to its interference with cellular viability by solubilizing the membrane, because repeated additions of LPC produced identical degrees of relaxation and after three consecutive additions of LPC, the strips continued to relax to the same degree with acetylcholine (Ach) demonstrating the functional integrity of the endothelium. LPC/Cholesterol dispersions (1:0.5 mole percent) relaxed the strips to the same extent as the micellar solution of LPC, while 1:1 mole percent did not. The results stress the role of cyclic GMP in LPC-induced relaxation. They further suggest that LPC/Cholesterol ratio may determine the availability of LPC and regulate LPC-mediated processes.     

Role of phospholipase C and diacylglyceride lipase pathway in arachidonic acid release and acetylcholine-induced vascular relaxation in rabbit aorta

ACh stimulates arachidonic acid (AA) release from membrane phospholipids of vascular endothelial cells (ECs). In rabbit aorta, AA is metabolized through the 15-lipoxygenase pathway to form vasodilatory eicosanoids 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA) and 11,12,15-trihydroxyeicosatrienoic acid (THETA). AA is released from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by phospholipase A2 (PLA2), or from phosphatidylinositol (PI) by phospholipase C (PLC) pathway. The diacylglycerol (DAG) lipase can convert DAG into 2-arachidonoylglycerol from which free AA can be released by monoacylglycerol (MAG) lipase or fatty acid amidohydrolase (FAAH). We used specific inhibitors to determine the involvement of the PLC pathway in ACh-induced AA release. In rabbit aortic rings precontracted by phenylephrine, ACh induced relaxation in the presence of indomethacin and N(omega)-nitro-L-arginine (L-NNA). These relaxations were blocked by the PLC inhibitor U-73122, DAG lipase inhibitor RHC-80267, and MAG lipase/FAAH inhibitor URB-532. Cultured rabbit aortic ECs were labeled with [14C]AA and stimulated with methacholine (10(-5) M). Free [14C]AA was released by methacholine. Methacholine decreased the [14C]AA content of PI, DAG, and MAG fractions but not PC or PE fractions. Methacholine-induced release of [14C]AA was blocked by U-73122, RHC-80267, and URB-532 but not by U-73343, an inactive analog of U-73122. The data suggested that ACh activates PLC, DAG lipase, and MAG lipase pathway to release AA from membrane lipids. This pathway is important in regulating vasodilatory eicosanoid synthesis and vascular relaxation in rabbit aorta.     

A model for demonstration of reversal of impairment of endothelium-dependent relaxation in the cholesterol-fed rabbit.

This investigation was undertaken to determine whether it was possible to restore endothelium-dependent relaxation (EDR) in the cholesterol-fed rabbit model of atherosclerosis following discontinuation of the cholesterol. New Zealand white rabbits, approximately 8 weeks of age, were randomized into (i) control group (9 animals fed a standard rabbit diet) and (ii) experimental group (27 animals: fed the same diet supplemented with 2.5% cholesterol). The experimental animals were restored to the standard diet after 3 weeks. EDR to acetylcholine (-9.0 to -5.0 log mol/L) was examined in the experimental animals at 3, 7, and 15 weeks after commencement of the study (n = 9 at each stage) and the nine control animals examined after 7 weeks. At the end of 7 weeks, EDR to acetylcholine (-6.0 log mol/L) was significantly (p less than 0.05) impaired in the experimental group (34.3 +/- 3.8%) compared with that in the control group (79.8 +/- 3.0%). The loss of EDR was not apparent in the experimental group at 3 weeks (relaxation: 81.7 +/- 4.7%). At the end of 15 weeks, the EDR was significantly restored in the experimental group (relaxation: 63.6 +/- 5.1%). These findings demonstrate that it is possible to reverse the loss of EDR that occurs with cholesterol feeding in the rabbit by limiting the period of exposure to a high cholesterol diet.     

有氧运动训练影响大鼠胸主动脉环内皮依赖性舒张功能机制的研究

目的:探讨有氧运动对大鼠胸主动脉内皮依赖性舒张功能影响的机制。方法:12只SD大鼠随机分为有氧运动和对照两组(n=6),经过8周每天1 h的游泳训练后(每周5天),测定比较2组间大鼠胸主动脉舒张功能的改变。结果:有氧运动组一氧化氮(NO)和前列环素PGI2途径胸主动脉舒张功能的Rmax值较对照组明显提高(P<0.05)。结论:有氧运动对大鼠胸主动脉舒张功能的有益影响,主要是由NO和PGI2途径介导的。     

Persistent impairment of endothelium-dependent relaxation to acetylcholine and progression of atherosclerosis following 6 weeks of cholesterol feeding in the rabbit

The synthesis and (or) release of endothelium-dependent relaxant factor released by acetylcholine is impaired in New Zealand white rabbits fed an atherogenic diet. Experiments were designed to investigate whether the synthesis and (or) release of the endothelium-dependent relaxant factor from rabbit aortas are restored after reversal from an atherogenic diet to a non-atherogenic diet. Atherosclerosis was induced by feeding a diet containing lipids and 2% cholesterol for 6 weeks. Rabbits were sacrificed after 6 weeks on the atherogenic diet and 36 weeks after return to a standard laboratory diet. Synthesis and (or) release of the factor from the thoracic aorta was assayed using a bioassay system. The relaxant responses produced in the assay tissue were impaired both in the acute stage and after 36 weeks on non-atherogenic food. This impaired relaxation is probably due to a persistent functional abnormality in the aortic endothelium resulting in the failure to synthesize and (or) release endothelium-dependent relaxation factor 36 weeks after induction of atherosclerosis.     

Chronic ouabain treatment increases the contribution of nitric oxide to endothelium-dependent relaxation

The aim of this study was to analyze the contribution of nitric oxide, prostacyclin and endothelium-dependent hyperpolarizing factor to endothelium-dependent vasodilation induced by acetylcholine in rat aorta from control and ouabain-induced hypertensive rats. Preincubation with the nitric oxide synthase inhibitor N-omega-nitro-l-arginine methyl esther (L-NAME) inhibited the vasodilator response to acetylcholine in segments from both groups but to a greater extent in segments from ouabain-treated rats. Basal and acetylcholine-induced nitric oxide release were higher in segments from ouabain-treated rats. Preincubation with the prostacyclin synthesis inhibitor tranylcypromine or with the cyclooxygenase inhibitor indomethacin inhibited the vasodilator response to acetylcholine in aortic segments from both groups. The Ca²⁺-dependent potassium channel blocker charybdotoxin inhibited the vasodilator response to acetylcholine only in segments from control rats. These results indicate that hypertension induced by chronic ouabain treatment is accompanied by increased endothelial nitric oxide participation and impaired endothelium-dependent hyperpolarizing factor contribution in acetylcholine-induced relaxation. These effects might explain the lack of effect of ouabain treatment on acetylcholine responses in rat aorta.     

C-6-Sulfidopeptide leukotrienes are unlikely to be involved in the endothelium dependent relaxation of rabbit aorta by acetylcholine

Acetylcholine (ACh) induced dilation of precontracted strips of rabbit aorta by a mechanism dependent on an intact endothelium, probably by releasing an unknown endothelial relaxing factor (ERF). The relaxation was completely inhibited by the lipoxygenase inhibitor nor-dihydroguaiaretic acid (10⁻⁵ M) but not by the cyclo-oxygenase inhibitor indomethacin (10⁻⁵ M). The aortic strips were found to release small amounts of a material with a leukotriene-like activity. Its action on the guinea pig ileum was antagonized by FPL 55712 (10⁻⁶ M). However, FPL 55712 (10⁻⁶ - 10⁻⁴ M) did not alter the response of rabbit aortic strips to ACh. Also when decreasing intracellular concentrations of glutathion (GSH) by incubating the strips with diethylmaleat or 2-cyclohexen-1-one (both 10⁻³ M) the vasodilator response could still be elicited. Leukotriene (LT) C₄ and LTD₄ (10⁻⁹ - 10⁻¹⁰ M) were found to be ineffective on oartic strips under basal or induced tension. The same held true for LTE₄ ( 10⁻⁹ - 10⁻⁷ M). At 10⁻⁶ M, however, LTE₄ induced slight relaxations of the vascular tissues. For reasons discussed this is likely to be a pharmacological action independent of the effects of endogenous ERF (e.g. inhibition of the formation of the LTE₄ precursor LTD₄ by high extracellular GSH concentrations did not reverse the ACh-induced vasodilation). It is concluded from these data, that C-6-sulfidopeptide leukotrienes, although probably produced by vascular tissue, are unlikely to be involved in the ACh-induced relaxation of rabbit aorta.     

Dysfunction of endothelium-dependent relaxation to insulin via PKC-mediated GRK2/Akt activation in aortas of ob/ob mice

In diabetic states, hyperinsulinemia may negatively regulate Akt/endothelial nitric oxide synthase (eNOS) activation. Our main aim was to investigate whether and how insulin might negatively regulate Akt/eNOS activities via G protein-coupled receptor kinase 2 (GRK2) in aortas from ob/ob mice. Endothelium-dependent relaxation was measured in aortic rings from ob/ob mice (a type 2 diabetes model). GRK2, β-arrestin2, and Akt/eNOS signaling-pathway protein levels and activities were mainly assayed by Western blotting. Plasma insulin was significantly elevated in ob/ob mice. Insulin-induced relaxation was significantly decreased in the ob/ob aortas [vs. age-matched control (lean) ones]. The response in ob/ob aortas was enhanced by PKC inhibitor or GRK2 inhibitor. Akt (at Thr(308)) phosphorylation and eNOS (at Ser(1177)) phosphorylation, and also the β-arrestin2 protein level, were markedly decreased in the membrane fraction of insulin-stimulated ob/ob aortas (vs. insulin-stimulated lean ones). These membrane-fraction expressions were enhanced by GRK2 inhibitor and by PKC inhibitor in the ob/ob group but not in the lean group. PKC activity was much greater in ob/ob than in lean aortas. GRK2 protein and activity levels were increased in ob/ob and were greatly reduced by GRK2 inhibitor or PKC inhibitor pretreatment. These results suggest that in the aorta in diabetic mice with hyperinsulinemia an upregulation of GRK2 and a decrease in β-arrestin2 inhibit insulin-induced stimulation of the Akt/eNOS pathway and that GRK2 overactivation may result from an increase in PKC activity.     

Parathyroid hormone-induced changes in cyclic nucleotide levels during relaxation of the rabbit [correction of rat] aorta

Parathyroid hormone is a potent vasodilator in vivo and relaxes vascular tissue in vitro. Since parathyroid hormone action in kidney and bone is thought to be mediated by stimulation of cellular cyclic AMP production, the present study was designed to monitor changes in cyclic AMP and cyclic GMP in vascular tissue during relaxation by parathyroid hormone. Rabbit aortic strips were quick-frozen at various times after exposure to parathyroid hormone and the percent relaxation and cyclic nucleotide levels were determined. Cyclic AMP concentrations were elevated about 3-fold within 30 seconds after treatment with hormone. This corresponded to a 10% relaxation of the norepinephrine-contracted tissue. After five minutes, cyclic AMP was still elevated 2-fold above basal and the relaxation response was maximal (36%). The cyclic AMP and relaxation responses to parathyroid hormone were markedly potentiated by forskolin or methylisobutylxanthine. Parathyroid hormone produced a small but significant increase in cyclic GMP concentrations only at early time points whereas sodium nitroprusside substantially increased cyclic GMP and relaxed the strips at all times studied. The increase in cyclic AMP levels after exposure to parathyroid hormone occurred prior to or coincident with the onset of relaxation of the aortic strips. These findings are supportive of the hypothesis that the vascular actions of parathyroid hormone involve cyclic AMP.     

Subcellular distribution of hexokinase isoenzymes in pancreatic islet cells exposed to digitonin after incubation at a low or high concentration of D-glucose

It was recently proposed that stimulation of pancreatic islet by D-glucose results in the translocation of glucokinase from the perinuclear area to the cell periphery, where the enzyme might conceivably interact with either the glucose transporter GLUT-2 or some other proteins and, by doing so, become better able to express its full catalytic activity. To explore the possible interaction between glucokinase and the cell boundary, dispersed rat pancreatic islet cells were preincubated for 60 min at a low (2.8 mM) or high (16.7 mM) concentration of D-glucose, then exposed for 1 min to digitonin (0.5 mg/ml) and eventually centrifuged through a layer of oil for separation of the cell pellet from the supernatant fraction containing the material released by digitonin. Under these conditions, the bulk of lactate dehydrogenase and glutamate dehydrogenase activities were recovered in the supernatant fraction and cell pellet, respectively. The measurement of hexokinase isoenzyme activities in th e two subcellular fractions, as conducted at low or high hexose concentrations and in either the absence or presence of exogenous hexose phosphates (3.0 mM glucose 6-phosphate and 1.0 mM fructose 1-phosphate) indicated a preferential location of the low-Km hexokinase in the cell pellet and of the high-Km glucokinase in the cytosolic fraction. Such a distribution pattern failed to be significantly affected by the concentration of D-glucose used during the initial incubation of the dispersed islet cells. These findings argue against the view that the glucose-induced translocation of glucokinase would result in any sizeable binding of the enzyme to a plasma membrane-associated protein. (Mol Cell Biochem 175: 131–136, 1997)     

Na+-K+ pump activation inhibits endothelium-dependent relaxation by activating the forward mode of Na+/Ca2+ exchanger in mouse aorta

The effect of Na+-K+ pump activation on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) was examined in mouse aorta and mouse aortic endothelial cells (MAECs). The Na+-K+ pump was activated by increasing extracellular K+ concentration ([K+]o) from 6 to 12 mM. In aortic rings, the Na+ ionophore monensin evoked EDR, and this EDR was inhibited by the Na+/Ca2+ exchanger (NCX; reverse mode) inhibitor KB-R7943. Monensin-induced Na+ loading or extracellular Na+ depletion (Na+ replaced by Li+) increased [Ca2+]i in MAECs, and this increase was inhibited by KB-R7943. Na+-K+ pump activation inhibited EDR and [Ca2+]i increase (K+-induced inhibition of EDR and [Ca2+]i increase). The Na+-K+ pump inhibitor ouabain inhibited K+-induced inhibition of EDR. Monensin (>0.1 microM) and the NCX (forward and reverse mode) inhibitors 2'4'-dichlorobenzamil (>10 microM) or Ni2+ (>100 microM) inhibited K+-induced inhibition of EDR and [Ca2+]i increase. KB-R7943 did not inhibit K+-induced inhibition at up to 10 microM but did at 30 microM. In current-clamped MAECs, an increase in [K+]o from 6 to 12 mM depolarized the membrane potential, which was inhibited by ouabain, Ni2+, or KB-R7943. In aortic rings, the concentration of cGMP was significantly increased by acetylcholine and decreased on increasing [K+]o from 6 to 12 mM. This decrease in cGMP was significantly inhibited by pretreating with ouabain (100 microM), Ni2+ (300 microM), or KB-R7943 (30 microM). These results suggest that activation of the forward mode of NCX after Na+-K+ pump activation inhibits Ca2+ mobilization in endothelial cells, thereby modulating vasomotor tone.     

Attenuation of lipid peroxidation and hyperlipidemia by quercetin glucoside in the aorta of high cholesterol-fed rabbit

Antioxidative activity of dietary flavonoids is suggested to be, at least partly, responsible for a wide variety of their biological effects relating to anti-atherosclerosis. However, it is not known whether dietary flavonoids reach to the target site and act as antioxidants. In this study, we tried to evaluate the antioxidative effect of quercetin 3-O-beta-D-glucoside (Q3G), a typical flavonoid present in vegetables, in rabbit aorta. New Zealand White rabbits were fed a control diet (control group), 2.0% cholesterol diet (HC group) and 2.0% cholesterol plus 0.1% Q3G (HC + Q3G group) for one month. The amounts of total cholesterol, triacylglycerol and total fatty acids in both the plasma and aorta were significantly lower in the HC + Q3G group as compared with the HC group. Quercetin was detected in the aorta of the HC + Q3G group after enzymatic deconjugation, indicating that quercetin accumulated as conjugated metabolites in the aorta. The contents of TBA-reacting substances (TBARS) and cholesteryl ester hydroperoxides (CEOOH) in the aorta of the HC + Q3G group were significantly lower than those in the HC group. The aorta of HC + Q3G group was more resistant than that of HC group in copper ion-induced lipid peroxidation ex vivo. HC + Q3G group accumulated a higher amount of vitamin E per total cholesterol than HC group in the aorta. These results strongly suggest that quercetin glucosides accumulate in the aorta as their metabolites and attenuate lipid peroxidation occurring in the aorta, along with the attenuation of hyperlipidemia.     

Role of Rho kinases in PKG-mediated relaxation of pulmonary arteries of fetal lambs exposed to chronic high altitude hypoxia

An increase in Rho kinase (ROCK) activity is implicated in chronic hypoxia-induced pulmonary hypertension. In the present study, we determined the role of ROCKs in cGMP-dependent protein kinase (PKG)-mediated pulmonary vasodilation of fetal lambs exposed to chronic hypoxia. Fourth generation pulmonary arteries were isolated from near-term fetuses ( approximately 140 days of gestation) delivered from ewes exposed to chronic high altitude hypoxia for approximately 110 days and from control ewes. In vessels constricted to endothelin-1, 8-bromoguanosine-cGMP (8-Br-cGMP) caused a smaller relaxation in chronically hypoxic (CH) vessels compared with controls. Rp-8-Br-PET-cGMPS, a PKG inhibitor, attenuated relaxation to 8-Br-cGMP in control vessels to a greater extent than in CH vessels. Y-27632, a ROCK inhibitor, significantly potentiated 8-Br-cGMP-induced relaxation of CH vessels and had only a minor effect in control vessels. The expression of PKG was increased but was not accompanied with an increase in the activity of the enzyme in CH vessels. The expression of type II ROCK and activity of ROCKs were increased in CH vessels. The phosphorylation of threonine (Thr)696 and Thr850 of the regulatory subunit MYPT1 of myosin light chain phosphatase was inhibited by 8-Br-cGMP to a lesser extent in CH vessels than in controls. The difference was eliminated by Y-27632. These results suggest that chronic hypoxia in utero attenuates PKG-mediated relaxation in pulmonary arteries, partly due to inhibition of PKG activity and partly due to enhanced ROCK activity. Increased ROCK activity may inhibit PKG action through increased phosphorylation of MYPT1 at Thr696 and Thr850.     

Period of sensitivity of the rabbit fetus to the embryopathic and lethal action of D-glucose in intraovular injection]

We studied the effect of injection of D-glucose in rabbit foetus ovulary liquid at the 14th, 16th and 18th day of pregnancy. Injection of 14th and 16th day determined a high percentage of foetal death and limb necrosis. On and after the 18th day of pregnancy, the foetus is insensible to the treatment. The 18th day is, in the rabbit, the period of a new hormonal equilibre establishment.