Endothelium-dependent Relaxation Of Rabbit Aorta Research Articles

Inhibition by diethylcarbamazine of acetylcholine-induced endothelium-dependent relaxation of rabbit aorta: are leukotrienes involved?

Acetylcholine caused relaxations of preconstricted rabbit aortic strips if the endothelium was intact. These relaxations were reversed in a concentration-dependent manner by diethylcarbamazine, an inhibitor of slow-reacting substance and leukotriene formation. However, when exogenous leukotrienes (LTA4, LTB4, LTC4 and LTD4) were added to the precontracted arterial strips none of them caused relaxation, showing that they are unlikely to be involved as mediators in the acetylcholine-induced relaxation of rabbit aorta.

Homocysteinylation of Metallothionein Impairs Intracellular Redox Homeostasis

Heart disease and stroke are major causes of death and morbidity in North America, and they exact high personal, community, and health care costs. Most heart attacks and strokes are caused by thrombosis superimposed on disrupted atherosclerotic lesions, a process known as atherothrombosis.1,2 A number of risk factors are known to accelerate atherothrombosis, including hypercholesterolemia, smoking, diabetes, hypertension, and obesity. Numerous clinical and epidemiological studies have established hyperhomocysteinemia as an independent risk factor for cardiovascular disease and stroke.3–8 Patients with inborn errors of methionine metabolism caused by deficiencies in cystathionine β-synthase or 5,10-methylenetetrahydrofolate reductase present with severe hyperhomocysteinemia and have a 50% chance of developing a major vascular event by the age of 30 years if untreated.6 This vascular risk is substantially decreased by homocysteine-lowering therapy (dietary supplementation with folic acid, B-vitamins, and/or betaine), even if the treatment does not completely normalize total plasma homocysteine levels.8 Unlike severe hyperhomocysteinemia, mild hyperhomocysteinemia attributable to deficiencies in dietary folic acid and/or B-vitamins is common in the general population. Despite the association between hyperhomocysteinemia and increased cardiovascular risk, several recent clinical trials have failed to show a preventative benefit of homocysteine-lowering therapy in cardiovascular patients with mild hyperhomocysteinemia.9,10 Although not completely understood, the results of these studies could imply that homocysteine-lowering therapy is ineffective in patients with established cardiovascular disease or that vitamin therapy has other, potentially adverse effects that neutralize its homocysteine-lowering benefit.11 See page 49 A direct causal relationship between hyperhomocysteinemia and atherosclerosis has been reported in diet- and/or genetic-induced mouse models of hyperhomocysteinemia.12–15 Previous studies have also reported that human cells relevant to atherothrombotic disease, including vascular endothelial cells and smooth muscle cells, are particularly sensitive to the cytotoxic effects of homocysteine that may result from the limited capacity of these cells to …

Probucol Protects against Hypochlorite-induced Endothelial Dysfunction: IDENTIFICATION OF A NOVEL PATHWAY OF PROBUCOL OXIDATION TO A BIOLOGICALLY ACTIVE INTERMEDIATE

Atherosclerosis is associated with endothelial dysfunction and a heightened state of inflammation characterized, in part, by an increase in vascular myeloperoxidase and proteins modified by its principal oxidant, hypochlorous acid (HOCl). Here we examined whether probucol could protect against endothelial dysfunction induced by the two-electron oxidant HOCl. Hypochlorous acid eliminated endothelium-dependent relaxation of rabbit aorta, whereas endothelial function and tissue cGMP was preserved and elevated, respectively, in animals pretreated with probucol. Exogenously added probucol also protected against HOCl-induced endothelial dysfunction. In vitro, HOCl oxidized probucol in a two-phase process with rate constants k(1) = 2.7 +/- 0.3 x 10(2) and k(2) = 0.7 +/- 0.2 x 10(2) m(-1) s(-1) that resulted in a dose- and time-dependent accumulation of probucol-derived disulfoxide, 4,4'-dithiobis(2,6-di-tert-butyl-phenol) (DTBP), DTBP-derived thiosulfonate, disulfone, and sulfonic acid, together with 3,3',5,5'-tetra-tert-butyl-4,4'-diphenoquinone (DPQ) as determined by high performance liquid chromatography and mass spectrometry. Like HOCl, selected one-electron oxidants converted probucol into DTBP and DPQ. Also, dietary and in vitro added DTBP protected aortic rings from HOCl-induced endothelial dysfunction and in vitro oxidation by HOCl gave rise to the thiosulfonate, disulfone, and sulfonic acid intermediates and DPQ. However, the product profiles of the in vitro oxidation systems were different from those in aortas of rabbits receiving dietary probucol or DTBP +/- HOCl treatment. Together, the results show that both probucol and DTBP react with HOCl and protect against HOCl-induced endothelial dysfunction, although direct scavenging of HOCl is unlikely to be responsible for the vascular protection by the two compounds.

Apamin-sensitive K+ currents mediate arachidonic acid-induced relaxations of rabbit aorta.

Arachidonic acid induces an endothelium-dependent relaxation of the rabbit aorta that is blocked by lipoxygenase inhibitors. The cellular vasodilatory mechanisms activated by arachidonic acid metabolites remain undefined. In rabbit thoracic aortic rings pretreated with indomethacin (10 micromol/L) and contracted with phenylephrine, arachidonic acid (0.1 to 100 micromol/L) induced concentration-dependent relaxations. Maximal relaxations averaged 45+/-3% and were inhibited by increasing extracellular K+ (30 mmol/L, 15+/-5%; P<0.001) or incubation with apamin (100 nmol/L, 26+/-7%; P<0.05) but not incubation with charybdotoxin (100 nmol/L, 41+/-5%). In aortic strips with an intact endothelium that were treated with phenylephrine, arachidonic acid (10 micromol/L) increased the membrane potential from -28.7+/-1.3 to -37.8+/-3.0 mV (P<0.01). Preincubation with apamin did not alter basal membrane potential but inhibited arachidonic acid-induced hyperpolarization (-31.5+/-1.5 mV). Incubation of rabbit aortic segments with apamin or charybdotoxin did not alter [14C]arachidonic acid metabolism. Whole-cell outward K+ currents from isolated rabbit aortic smooth muscle cells averaged 43.0+/-4.8 pA/pF at 60 mV and were significantly decreased to 35.7+/-4.2 pA/pF by apamin (P<0.001). Subsequent addition of charybdotoxin further decreased maximal currents to 14.4+/-2.3 pA/pF. Addition of 11,12,15-trihydroxyeicosatrienoic acid increased the outward whole-cell K+ current. In inside-out patches of aortic smooth muscle, apamin inhibited the calcium activation (100 to 300 nmol/L; P<0.001) of a small-conductance K+ channel (approximately 24 pS). These results suggest that arachidonic acid induces endothelium-dependent hyperpolarization and relaxation of rabbit aorta through activation of smooth muscle, apamin-sensitive K+ currents.

Mechanisms of endothelial dysfunction after ionized radiation: selective impairment of the nitric oxide component of endothelium-dependent vasodilation.

(1) Gamma radiation impairs vascular function, leading to the depression of endothelium-dependent vasodilatation. Loss of the nitric oxide (NO) pathway has been implicated, but little is known about radiation effects on other endothelial mediators. (2) This study investigated the mechanisms of endothelial dysfunction in rabbits subjected to whole-body irradiation from a cobalt(60) source. (3) The endothelium-dependent relaxation of rabbit aorta evoked by acetylcholine (ACh) or A23187 was impaired in a dose-dependent manner by irradiation at 2 Gy or above. Inhibition was evident 9 days post-irradiation and persisted over the 30 day experimental period. (4) Endothelium-independent responses to glyceryl trinitrate (GTN), sodium nitroprusside (SNP) and 3-morpholino-sydnonimine (SIN-1) were suppressed over a similar dose range at 7-9 days post-irradiation, but recovered fully by 30 days post-irradiation. (5) In healthy vessels, ACh-induced relaxation was inhibited by L-N(omega)-nitroarginine (L-NA; 3 x 10(-4) M) and charybdotoxin (10(-8) M) plus apamin (10(-6) M) but resistant to indomethacin, indicating the involvement of NO and endothelium-derived hyperpolarizing factor (EDHF). Supporting this, ACh caused smooth muscle hyperpolarization that was reduced by L-NA and charybdotoxin plus apamin. (6) In irradiated vessels, responses to ACh were insensitive to L-NA but abolished by charybdotoxin plus apamin, indicating selective loss of NO-mediated relaxation. (7) In animals treated shortly after irradiation with the antioxidant, alpha-tocopherol acetate, the NO-dependent relaxation was restored without effect on the EDHF-dependent component. (8) The results imply that radiation selectively impairs the NO pathway as a consequence of oxidative stress, while EDHF is able to maintain endothelium-dependent relaxation at a reduced level.

Prevention of LDL α-tocopherol consumption, cholesterol oxidation, and vascular endothelium dysfunction by polyphenolic compounds from red wine

Red wine polyphenolic compounds (RWPCs) have been demonstrated to possess antioxidant properties, and several studies have suggested that they might constitute a relevant dietary factor in the protection from coronary heart disease. The aim of the present study was to determine further the mechanism by which RWPCs can prevent the formation of vasoactive compounds in oxidized LDL. RWPCs were obtained from the Cabernet-Sauvignon grape variety. Human LDL was oxidized in the presence of CuSO 4 (ox-LDL). Vascular reactivity studies were conducted on rabbit aortic rings. RWPCs significantly reduced the formation of 7β-hydroxycholesterol and 7-ketocholesterol and in a lower extent the emergence of lysophosphatidylcholine in ox-LDL. The ability of RWPCs to prevent cholesterol oxide formation was directly dependent on the LDL α-tocopherol content. Once the LDL α-tocopherol has been consumed, RWPCs were no longer effective, indicating that RWPCs act by sparing endogenous α-tocopherol. As a consequence of the preservation of the endogenous α-tocopherol content of LDL, RWPCs could prevent the inhibition of the acetylcholine-mediated endothelium-dependent relaxation of rabbit aorta which was linked to a direct effect on NO release. Independently of a treatment with ox-LDL, RWPC exerted a concentration-dependent and persistent inhibitory effect on the norepinephrine-induced contraction of rabbit aorta. In conclusion, RWPCs can preserve a normal vascular reactivity by acting at different stages of the cascade that leads to lipid oxidation, endothelium dysfunction and vasospasm.

Homocysteine-induced inhibition of endothelium-dependent relaxation in rabbit aorta: role for superoxide anions.

Hyperhomocysteinemia is associated with endothelial dysfunction, although its mechanism is unknown. Isometric tension recordings and lucigenin chemiluminescence were used to assess the effects of homocysteine exposure on endothelium-dependent and -independent relaxation in isolated rabbit aortic rings and superoxide anion (O(2)(-)) production by cultured porcine aortic endothelial cells, respectively. Homocysteine (0.1 to 10 mmol/L) produced a significant (P<0.001) concentration- and time-dependent inhibition of endothelium-dependent relaxation in response to both acetylcholine and the calcium ionophore A23187. Only the intracellular O(2)(-) scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron, 10 mmol/L) significantly (P<0.001) inhibited the effect of homocysteine on acetylcholine- and A23187-induced relaxation. Incubation of porcine aortic endothelial cells with homocysteine (0.03 to 1 mmol/L for up to 72 hours) caused a significant (P<0.001) time-dependent increase in the O(2)(-) released by these cells on the addition of Triton X-100 (1% [vol/vol]), with levels returning to values comparable to those of control cells at the 72-hour time point. These changes in O(2)(-) levels were associated with a time-dependent increase in endothelial cell superoxide dismutase activity, becoming significant (P<0.001) after 72 hours. Furthermore, the homocysteine-induced increase in endothelial cell O(2)(-) levels was completely inhibited (P<0.001) by the concomitant incubation with either Tiron (10 mmol/L), vitamin C (10 micromol/L), or vitamin E (10 micromol/L). These data suggest that the inhibitory effect of homocysteine on endothelium-dependent relaxation is due to an increase in the endothelial cell intracellular levels of O(2)(-) and provide a possible mechanism for the endothelial dysfunction associated with hyperhomocysteinemia.

Direct Evidence that Protein Kinase C Mediates the Acute Inhibitory Effect of Oxidised Low Density Lipoprotein on Acetylcholine-induced Endothelium-dependent Relaxation of Rabbit Aorta

This study further examines the involvement of protein kinase C and superoxide anions in the inhibition of agonist-induced endothelium-dependent relaxation by oxidised low density lipoprotein (LDL). Pretreatment of rabbit aorta rings with either oxidised low density lipoprotein or phorbol dibutyrate (PDB) inhibited acetylcholine-induced endothelium-dependent relaxation, but not endothelium-independent relaxations to glyceryl trinitrate. Prior exposure of the rings to either of the specific protein kinase C inhibitors Ro 31-8220 or Ro 31-7549 prevented or reduced respectively the inhibition of acetylcholine-induced relaxation by oxidised LDL or PDB. Neither inhibitor alone affected acetylcholine-induced relaxation. We directly demonstrate for the first time that oxidised, but not native, LDL produces a prolonged activation of protein kinase C in endothelial cells, which was prevented by pretreatment with Ro 31-8220. Oxidised or native LDL, or Ro 31-8220 increased superoxide anion production by rabbit aorta...

Pathogenesis of acute myocardial infarction. Novel regulatory systems of bioactive substances in the vessel wall.

Rupture of the lipid-rich atheromatous plaque, intraplaque hemorrhage, and intraluminal thrombus are three pathological hallmarks most commonly recognized in the infarct-related coronary artery at the site of acute myocardial infarction. Rupture of the atheromatous plaque is closely related to but does not fully explain the genesis of occlusive intracoronary thrombus formation and thus the development of acute myocardial infarction. Besides a variety of hematologic disorders, one should emphasize the role of the platelet-derived mediators that promote an environment where thrombosis and vasoconstriction occur, including TXA2, serotonin, ADP, platelet-derived growth factor, tissue factor, and the diminished availability of those natural endogenous substances that inhibit platelet aggregation, such as EDRF, tissue plasminogen activator, and PGI2. PGI2 released from vascular endothelial cells is extremely unstable. Our group provided the first evidence that HDL stabilizes PGI2 through the newly discovered function of Apo A-I, which is associated with the surface of HDL particles and identified as PGI2 stabilizing factor. Decrease in HDL-associated Apo A-I in patients with unstable angina and during the acute phase of myocardial infarction indicates that HDL plays an important role in preventing coronary atherosclerosis and intracoronary thrombus formation by stabilizing PGI2 in addition to the generally accepted biochemical property of HDL to prevent the accumulation of cholesterol by mobilizing free cholesterol from tissues or macrophages. There is also a PGI2 synthesis-stimulating factor in serum that has not yet been identified chemically. EDRF or nitric oxide provides another important regulating system in the vessel wall. Lipoproteins are inhibitors of endothelium-dependent relaxation of rabbit aorta.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelium-dependent relaxation in rabbit aorta after cold storage

The extent of preservation of endothelial, smooth muscle and neurogenic function following cold storage was studied in rabbit thoracic aorta. Relaxation responses to acetylcholine and sodium nitroprusside were compared between fresh aortic rings and rings that had been stored in a refrigerator for 2–8 days at 4°C. In fresh aortic rings, the addition of acetylcholine to precontracted vessels resulted in dose-dependent relaxation. The magnitude of relaxation was gradually decreased after 4–8 days of cold storage. Relaxation in response to sodium nitroprusside did not change. Following cold storage contractions of aortic rings in response to noradrenaline and phenylephrine were not reduced. Contractile responses induced by transmural nerve stimulation were gradually attenuated with the length of cold storage. Electron microscopy after 4 days showed partial damage of endothelial cells (slightly vacuolized mitochondria). After 8 days, endothelial cells were destroyed; only membranous material was present. The structure of smooth muscle cells was only partially changed even after 8 days. Sympathetic nerve endings on the 4th day were partially, but on the 8th day completely destroyed. These results suggest that after cold storage of rabbit aorta, the gradual reduction of endothelium-dependent relaxation is probably caused by a decrease in production of the endothelium-derived relaxing factor due to the destruction of endothelial cells.

Effects of lysolipids and oxidatively modified low density lipoprotein on endothelium-dependent relaxation of rabbit aorta.

Exposure of isolated arteries to oxidatively modified low density lipoprotein (LDL) has been reported to suppress endothelium-dependent relaxation (EDR). To determine whether lipid degradation products in oxidized LDL contribute to impaired relaxation, we have tested the responsiveness of isolated rabbit aortas to endothelium-dependent relaxants (acetylcholine, ATP, and calcium ionophore A23187) and nitroglycerin before and after 2-hour incubations with selected lipids and LDL preparations. Concentrations (10 microM) of lecithin, phosphatidylserine, lysophosphatidylserine, sphingomyelin, phosphatidic acid, palmitate, arachidonate, and auto-oxidized arachidonate had no effect on EDR. Concentrations (10 microM) of lysolecithin, lyso-platelet activating factor, and sphingosine significantly suppressed endothelium-dependent relaxation. Native LDL (100 micrograms/ml incubation buffer) containing only small amounts of lysophosphatidylcholine exerted no effect on EDR. In contrast, LDL preparations oxidatively modified by exposure to cultured endothelial cells or copper inhibited EDR. When modified LDL was depleted of its lysolecithin by treatment with a selective phospholipase B (lysolecithinase), the inhibitory effects were attenuated. In contrast, native LDL accumulating lysolecithin under the influence of a phospholipase A2 (lecithinase) exerted inhibitory effects mimicking those of oxidized LDL. Lipids and lipoproteins had no effect on the responsiveness to nitroglycerin, an endothelium-independent vasodilator. We conclude that lysolecithin in oxidatively modified LDL contributes importantly to its vasomotor effects.

Endothelial cells metabolize NG-monomethyl-L-arginine to L-citrulline and subsequently to L-arginine

NG-monomethyl-L-arginine (MeArg) inhibits the release of endothelium-derived relaxing factor (EDRF) from endothelial cells (EC) and the formation of nitric oxide (NO) from L-arginine (Arg) in EC and activated macrophages. We have compared the inhibitory potency of MeArg to that of Nω-nitro-L-arginine (NO2Arg), a more potent inhibitor of EDRF synthesis in vitro. NO2Arg (100 μM) was significantly more potent than MeArg in inhibiting the endothelium-dependent relaxation of rabbit aorta induced by acetylcholine. MeArg and NO2Arg (10 and 30 μM) also inhibited the release of EDRF from bovine aortic cultured EC. In the anaesthetized rat in vivo, the pressor effect of NO2Arg (3 and 10 mg kg−1) was significantly larger and longer lasting than that of MeArg. These differences in potency could be due to the extensive metabolism of MeArg but not NO2Arg to L-citrulline (Cit) and subsequently to Arg by EC. The enzyme responsible for the conversion of MeArg to Cit had the characteristics of a novel deiminase, NG,NG-dimethylarginine dimethylaminohydrolase, recently isolated from rat kidney.

Lipoproteins are inhibitors of endothelium-dependent relaxation of rabbit aorta

The present study was performed to investigate plasma inhibitors of endothelium-dependent relaxation other than hemoglobin and low-density lipoprotein (LDL). We purified an inhibitor that contained a protein of 28,000 Da from human plasma by ammonium sulfate precipitation and serial chromatography. NH2-terminal sequence analysis revealed the protein to be homologous with human apolipoprotein A-I (Apo A-I), a major apolipoprotein of high-density lipoprotein (HDL). Very low-density lipoprotein (VLDL), LDL, and HDL obtained from rabbit plasma reversed endothelium-dependent relaxation of rabbit aorta induced by acetylcholine (ACh) and A23187 but did not inhibit relaxations induced by nitroglycerin or nitric oxide. These inhibitory activities were lost by delipidation of lipoproteins, and there were no differences in the inhibitory activity among these three lipoproteins on the basis of phospholipid concentration. Moreover, phospholipids such as phosphatidylcholine, phosphatidylinositol, and sphingomyelin reversed relaxations by ACh and A23187. Thus all lipoproteins inhibit endothelium-dependent relaxation, and this nonspecific inhibition seems to be due to the inhibition of production or release of endothelium-derived relaxing factor by phospholipids in the lipoprotein complex.

Effects of LY83583, nordihydroguaiaretic acid, and quinacrine on cyclic GMP elevation and inhibition of tension by muscarinic agonists in rabbit aorta and left atrium.

Elevation of cyclic GMP by muscarinic agonists has been suggested to be responsible for the negative inotropic effects of these agents in cardiac muscle, and for the endothelium-dependent relaxation caused by these agents in vascular smooth muscle. These relationships were studied by monitoring the effects of muscarinic agonists on tension and cyclic GMP levels in rabbit left atrial strips and aortic rings, in the presence and absence of the cyclic GMP lowering agent, LY83583. LY83583 completely blocked both the cyclic GMP increase and the relaxation caused by acetylcholine in rabbit aortic rings with intact endothelial cells. Acetylcholine-induced cyclic GMP elevation and relaxation in these preparations were also blocked by quinacrine and nordihydroguaiaretic acid (NDGA), but neither response was blocked by the 5-lipoxygenase inhibitor U-60257. In the experiments with rabbit left atrium, LY83583 blocked the acetylcholine-induced cyclic GMP elevation but did not block the negative inotropic effects of the drug. Quinacrine, NDGA, and a guanylate cyclase inhibitor, methylene blue, failed to block either the cyclic GMP increase or the decrease in contractile force caused by carbachol in atrial strips. These results support the suggestion that an increase in cyclic GMP may be responsible for the endothelium-dependent relaxation of rabbit aorta by muscarinic agonists, but not for the direct negative inotropic effects of these drugs in rabbit atrium. Muscarinic agents appear to increase cyclic GMP levels in rabbit atrium and aorta by different mechanisms. Although both are blocked by LY83583, they differ not only in their requirements for endothelial cells, but also in their susceptibility to other blocking agents.

Low-density lipoproteins inhibit endothelium-dependent relaxation in rabbit aorta.

The vascular endothelium, in response to pulsatile flow and vasoactive agents including acetylcholine, secretes the endothelium-derived relaxing factor (EDRF), a substance which regulates vascular tone. Recent interest in EDRF has focused on its possible dysfunction in atherosclerosis. In animal models of the disease, endothelium-dependent relaxation is markedly reduced. The continuous exposure of the endothelium in hyperlipidaemia to high concentrations of low-density lipoprotein (LDL), a known atherogenic risk factor, may explain this dysfunction. Here, we demonstrate that pathophysiological concentrations of LDL directly inhibit endothelium-dependent relaxation. Chemically modified LDL, in contrast, is inactive, implying that the inhibition is through a receptor-dependent mechanism.

Endothelium-dependent relaxation in experimental atherosclerosis in the rabbit.

The effect of feeding a diet supplemented with lipids and containing 2% cholesterol on the endothelium-dependent relaxation of rabbit aorta to acetylcholine was assessed. The effect of feeding a standard rabbit diet after an initial period of 2% cholesterol feeding was assessed also. Age-matched male, New Zealand white rabbits were fed either a 2% cholesterol diet or a standard rabbit diet. The animals were anesthetized with pentobarbitone sodium (25 mg/kg) and killed either at the beginning of the study (0 weeks) or at 4, 8, or 10 weeks. The animals in the reversal study were fed the 2% cholesterol diet for 6 weeks and killed after an additional 14 and 32 weeks on standard diet. The extent of atherosclerosis in the aorta was assessed by Sudan Red staining, estimation of tissue cholesterol, and light and electron microscopy. The relaxation response to acetylcholine was measured in rings of the thoracic aorta following precontraction with norepinephrine (-6.0 log mol/l). The relaxation was significantly impaired in aortas from rabbits fed the 2% cholesterol diet compared to aortas from animals fed the standard diet. The impairment of relaxation was apparent as early as 4 weeks after the start of the 2% cholesterol diet and remained impaired over the next 6 weeks. No improvement in endothelium-dependent relaxation was seen in rabbits on the reversal diet for 14 and 32 weeks. Thus, endothelium-dependent relaxation is attenuated in animals fed a 2% cholesterol diet, and the loss of relaxation persists for at least 32 weeks after the animals are returned to a standard diet.

Endothelium-dependent relaxation of rabbit aorta by acetylcholine requires ethylenediaminetetraacetic acid.

Experiments were conducted to determine if ethylenediaminetetraacetic acid (EDTA) was essential for the acetylcholine (ACh)-induced relaxation of blood vessels. Isolated rabbit aortic rings were prepared for recording isometric tension. They were maintained in Krebs bicarbonate solution with various concentrations of EDTA. With EDTA concentrations of 0 or 0.003 mM, no ACh-induced relaxation was observed; only the contractile effect of ACh was seen. With 0.03 and 0.30 mM EDTA, ACh induced relaxation with EC50 values of 0.11 and 0.098 microM, respectively. Under the experimental conditions used, EDTA was essential for demonstration of ACh-induced relaxation.

The inhibitory effects of calmodulin antagonists on the endothelium-dependent relaxation in rabbit aorta.

Effects of trifluoperazine (TFP, 10(-5) M), trifluoperazine sulfoxide (TFP-SO, 10(-5) M), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7-, 10(-4) M), N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5, 10(-4) M) and verapamil (10(-5) M) on the endothelium-dependent relaxation of the contractions induced in the isolated rabbit aorta were studied. Carbachol (10(-6) - 10(-6) M) and A23187 (10(-8) M) relaxed the contraction due to norepinephrine (10(-6) M and histamine (10(-5) M) in the aorta with endothelium. TFP and W-7 completely inhibited the relaxation induced by carbachol and A23187. In contrast, TFP-SO and W-5, which have lower affinity to calmodulin, had little or no effect on either the carbachol- or A23187-induced relaxation. An organic calcium antagonist, verapamil, did not change the relaxation induced by carbachol or A23187. W-7 did not modify the relaxing effect of sodium nitroprusside (10(-8) - 10(-6) M) which is not mediated by endothelium. These results suggest that either formation or release of endothelium-derived relaxing factor involves the process mediated by calmodulin in the rabbit aorta.

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.

Effects of cytochrome P-450 inhibitors on endothelium-dependent relaxation in rabbit aorta.

Endothelium-dependent relaxation in rabbit aorta was inhibited by exposure to two cytochrome P-450 inhibitors, metyrapone and SKF-525A. Aortic rings were contracted to a stable plateau by addition of phenylephrine (10(-7) M). Relaxation was elicited by the cumulative addition of methacholine (3 X 10(-8) - 3 X 10(-6) M) or A23187 (10(-8) - 10(-6) M). Exposure to metyrapone (500 microM) or SKF-525A (10 micrograms/ml) was found to inhibit relaxation in response to concentrations of methacholine exceeding 10(-7) M. Maximal relaxation was inhibited 73% by metyrapone. Relaxation stimulated by concentrations of A23187 exceeding 10(-7) M was also found to be inhibited by both metyrapone and SKF-525A exposure. Maximum A23187-induced relaxation (55% of the phenylephrine contractile response) was inhibited 40% by metyrapone and 55% by SKF-525A. Arachidonic acid (10-100 microM) also elicited endothelium-dependent relaxation in rings pretreated with indomethacin (10 micrograms/ml) and contracted with phenylephrine. This relaxation response was abolished by exposure to metyrapone or SKF-525A. These results suggest that cytochrome P-450 may be involved in endothelium-dependent relaxation responses, perhaps by metabolizing arachidonic acid to active products.