Inhibition Of Endothelium-dependent Relaxation Research Articles

Barbiturates inhibit endothelium-dependent and independent relaxations mediated by cyclic GMP.

The inhibitory effects of volatile and local, but not intravenous, anesthetics on endothelium-dependent relaxations of blood vessels have been demonstrated in vitro by several investigators. The aim of this study was to determine the effects of barbiturates on endothelium-dependent arterial relaxation and elucidate the mechanism(s) responsible. Canine mesenteric arteries and rat aortae were isolated, and tension changes in helical strips were recorded. Endothelium-dependent relaxations elicited by acetylcholine and bradykinin in canine mesenteric arteries, and those by acetylcholine in rat aortae, were significantly attenuated by thiopental (3 x 10(-4) M) pretreatment. Sodium nitroprusside (SNP)-induced, endothelium-independent relaxations were significantly attenuated by thiopental (10(-4)-3 x 10(-4) M). The effects of pentobarbital were less marked than those of thiopental. Acetylcholine (10(-5) M)-stimulated levels of 3',5'-cyclic guanosine monophosphate (cGMP) in rat aortae were reduced significantly by thiopental and pentobarbital (both 10(-3) M), and SNP (3 x 10(-7) M)-stimulated levels were reduced by thiopental (3 x 10(-4)-10(-3) M) and pentobarbital (10(-3) M). We conclude that barbiturates inhibit cGMP-mediated endothelium-dependent and independent arterial relaxations. Inhibition of endothelium-dependent relaxation by barbiturates may be mediated by their effects on vascular smooth muscle itself and not on endothelium.

Predominant role for nitric oxide in the relaxation induced by acetylcholine in human uterine artery.

The effect of acetylcholine on the isolated human uterine artery rings was investigated. Acetylcholine (10(-10) M to 6 x 10(-5) M) induced concentration- and endothelium-dependent relaxation (pD2 = 7.4 +/- 0.02, maximal response was 77.5 +/- 6.3% of relaxation induced by papaverine at 3 x 10(-4) M) of the pre-contracted arterial segments. Indomethacin (10(-5) M), diethylcarbamazine (10(-4) M) and tetra-ethylammonium (3 x 10(-4) M) had no effects on acetylcholine-evoked relaxation. Methylene blue (10(-5) M) and NG-monomethyl-L-arginine (L-NMMA) (3 x 10(-6) to 3 x 10(-5) M) antagonized relaxation induced by acetylcholine. The inhibition of endothelium-dependent relaxation by L-NMMA (10(-5) M) was reversed by L-arginine (10(-5) M) but not by D-arginine (10(-4) M). It is concluded that in uterine artery acetylcholine induces endothelium-dependent relaxation of isolated uterine artery is probably mediated via endothelial nitric oxide formation.

Inhibition of Endothelium-Dependent Relaxation by Free Hemoglobin in the Human Internal Thoracic Artery

Hemolysis and postoperative hypertension are well recognized complications of cardiopulmonary bypass (CPB). The rise in the concentration of total and free hemo globin is directly related to the duration of CPB. It is well established that free hemoglobin binds and inactivates endothelium-derived relaxing factor, and the authors have studied the effects of low concentrations of free hemoglobin on the endothelium-dependent and endothelium-independent relaxation of the human internal thoracic artery (ITA). In the presence of free hemoglobin the endothelium-dependent relaxation induced by acetylcholine was reduced by 80% with 0.5 μM free hemoglobin (P< 0.01) and by 52% with 0.1 μM free hemoglobin (P<0.05). Sodium nitroprusside, endothelium indepen dent, produced relaxation of almost 100%. These results indicate that relatively low concentrations of free hemoglobin significantly inhibit endothelium-dependent but not endothelium-independent relaxation. This may influence blood flow in the ITA and may be a contributory factor in postoperative hypertension.

Protein kinase C inhibitors prevent impairment of endothelium-dependent relaxation by oxidatively modified LDL.

The mechanism(s) of inhibition of endothelium-dependent relaxation (EDR) by oxidized low-density lipoprotein (Ox-LDL) was examined in isolated porcine coronary arteries and rabbit aortas. Incubation with Ox-LDL but not native LDL caused the inhibition of thrombin- or acetylcholine-induced EDR, whereas A23187-induced EDR was preserved after incubation with Ox-LDL. Lysophosphatidylcholine (lysoPC), which was abundant in Ox-LDL and was found to be transferred from Ox-LDL to endothelial cells, also caused the inhibition of EDR in response to thrombin or acetylcholine but not to A23187. Ox-LDL depleted of lysoPC, which was prepared by phospholipase B, failed to inhibit the vasorelaxation. Coincubation with staurosporine or calphostin C, potent inhibitors of protein kinase C, attenuated the EDR inhibition by Ox-LDL or lysoPC. Phorbol 12-myristate 13-acetate, a specific protein kinase C activator, caused the EDR inhibition, and its effect was attenuated by staurosporine or calphostin C. Furthermore, lysoPC was capable of activating protein kinase C purified from cultured porcine endothelial cells. In conclusion, protein kinase C activation plays a role in the inhibition of surface receptor-mediated EDR by Ox-LDL, and lysoPC transferred from Ox-LDL to endothelial cells may be involved in the activation of protein kinase C.

Inhibition by a stable factor derived from neutrophils of endothelium-dependent relaxation in rat aorta

Polymorphonuclear leukocytes (PMNs) may play an important role in many pathophysiological states. The effect of a factor derived from PMNs on endothelium-dependent relaxation was studied using rat aortic rings in organ chambers. PMNs were obtained from cardiac surgical patients and healthy volunteers. After incubation in Krebs solution for 3 h, supernatants of PMN suspensions were isolated and used to pretreat the aortic rings for 30 min. The results showed that the supernatants derived from 1 x 10(4) to 5 x 10(6) cells/ml PMNs produced a concentration-dependent inhibition of endothelium-dependent relaxation to acetylcholine but not endothelium-independent relaxation to sodium nitroprusside. The effect could not be prevented by oxygen free radical scavenger superoxide dismutase (150 U/ml), catalase (1,200 U/ml), or mannitol (20 mM) used alone or in combination. Heating the supernatants at 95 degrees C for 30 min did not reduce the inhibitory effect. L-Arginine at 3 x 10(-5) to 3 x 10(-3) M did not significantly reverse the inhibitory effect of the PMN-derived factor. In conclusion, this study reveals that a heat-stable factor derived from human PMNs potently inhibits acetylcholine-induced endothelium-dependent relaxation but not sodium nitroprusside-induced endothelium-independent relaxation in rat aorta. This inhibitory effect is not caused by oxygen free radicals, a limitation of nitric oxide precursor or other unstable factors.

Tumor Necrosis Factor Inhibits Stimulated but Not Basal Release of Nitric Oxide

Tumor necrosis factor alpha (TNF alpha) increases nitric oxide (NO) synthase in vascular endothelium, but it inhibits endothelium-dependent relaxation (EDR) of vascular smooth muscle. We tested whether TNF alpha inhibits the response to, or release of, NO in bovine pulmonary artery (BPA) using the technique of perfusion-superfusion bioassay and ozone chemiluminescence. Effluent from the perfused BPA with endothelium (donor)-relaxed endothelium-rubbed bovine coronary artery (BCA) (detector). Moreover, effluent from the donor stimulated with acetylcholine (ACh) or bradykinin (BK) (0.001 to 100 nmol) relaxed the detector. Direct application of these agonists to the detector failed to produce relaxation. Basal and agonist-stimulated effluent from the donor treated with L-NG-monomethylarginine (LNMMA) (100 microM) suppressed effluent-mediated relaxation of the detector. ACh and BK released LNMMA-inhibitable nitrite and nitrate from the BPA. Thus, the effluent contained NO. Exposure of the donor to TNF alpha (1.25 micrograms/ml) for 60 min did not affect basal release of NO, but it attenuated bioassayable and chemiluminescence-detectable NO release by ACh and BK. The inhibition of NO release was directly related to the magnitude of inhibition of EDR by ACh and BK. Thus, TNF alpha selectively inhibits receptor-mediated release of NO without affecting basal release of NO. This effect differs from that of L-arginine-based inhibitors of NO and represents a unique physiologic mechanism of regulation of NO in the endothelium.

High density lipoprotein reverses inhibitory effect of oxidized low density lipoprotein on endothelium-dependent arterial relaxation.

We have recently reported that oxidized low density lipoprotein (ox-LDL) inhibits endothelium-dependent arterial relaxation through its increased lysophosphatidylcholine (LPC). In this study we examined whether high density lipoprotein (HDL) has any effect on the inhibition of endothelium-dependent relaxation by ox-LDL in isolated strips of rabbit thoracic aorta. Both low density lipoprotein (LDL) and HDL were isolated from normal human plasma, and LDL was oxidized by exposure to copper. Preincubation of arterial strips with ox-LDL (0.1-0.5 mg protein/ml) inhibited endothelium-dependent relaxation to acetylcholine (ACh) in a concentration-dependent manner. HDL (1 mg protein/ml) by itself had no effect on the relaxation to ACh. In the presence of HDL, the inhibition by ox-LDL was markedly reduced. Although synthetic L-alpha-palmitoyl LPC (5 micrograms/ml) completely abolished a relaxation to ACh, the preincubation of arterial strips with HDL completely prevented the LPC-induced inhibition. Moreover, a relaxation to ACh was almost completely recovered when the strips were washed with buffer containing HDL even after LPC-induced inhibition had occurred. HDL markedly reduced the incorporation of [1-14C]palmitate-labeled LPC ([14C]LPC) into cultured bovine aortic endothelial cells and promoted the release of cell-incorporated [14C]LPC into the medium, resulting in a reduction of the remaining [14C]LPC in the cells. Agarose electrophoresis after incubation of a mixture of ox-LDL labeled with [14C]LPC and unlabeled HDL demonstrated a transfer of [14C]LPC from ox-LDL to HDL. These results indicate that HDL reverses the ox-LDL-induced impairment of endothelium-dependent relaxation by removing LPC from ox-LDL and preventing LPC from acting on the endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Oral NG-nitro-L-arginine in conscious dogs: 24 hour hypertensive response in relation to plasma levels

Haemodynamic changes after oral administration of 30 mg/kg N(G)-nitro-L-arginine (L-NNA) were studied in conscious chronically instrumented mongrel dogs throughout a 24 h observation period in order to evaluate the long-term efficacy of L-NNA-induced inhibition of endothelium-dependent relaxation and its relation to plasma L-NNA level. Diastolic blood pressure remained elevated for the entire 24 h observation period, but systolic blood pressure was raised only up to the 6 h value. The hypertensive response was accompanied by bradycardia. The increase in blood pressure and the plasma L-NNA level both reached their maxima at 3 h. The plasma L-NNA level at the end of the observation period was diminished by only 21.7% with respect to the maximum increase, whilst the maximum increase in mean arterial blood pressure was attenuated by 72.2% at 24 h. These data show a dissociation between plasma L-NNA level and the respective blood pressure.

Chronic hypoxia selectively augments rat pulmonary artery Ca2+ and K+ channel-mediated relaxation.

The initiating event in hypoxic pulmonary hypertension is felt to be sustained hypoxic vasoconstriction, ultimately leading to vascular remodeling and fixed pulmonary hypertension. During the initial vasospastic phase endogenous vasodilatory pathways may serve to ameliorate the development of pulmonary hypertension. However, various studies in the systemic and pulmonary circulations have shown that chronic hemodynamic stress alters both endothelial and smooth muscle cell function. The effect of chronic hypoxia in rats was therefore tested on three major vasodilatory pathways: 1) endothelium-dependent relaxation (using endothelium-derived relaxing factor agonists and antagonists); 2) smooth muscle cell cyclic nucleotide-mediated relaxation [using guanosine and adenosine 3',5'-cyclic monophosphate (cGMP and cAMP) agonists]; and 3) ion channel-dependent relaxation (using K+ channel agonists and Ca2+ channel antagonists). It was found that short-term exposure (72 h) to hypoxia caused augmentation of K+ and Ca2+ channel-dependent relaxation with no effect on endothelium-dependent or cyclic nucleotide-mediated relaxation. More prolonged exposure (4-5 wk) was additionally associated with inhibition of endothelium-dependent relaxation and smooth muscle cell cGMP-mediated relaxation. There was no effect on either basal modulation of tone by the endothelium, cAMP-mediated relaxation, or systemic vessel relaxation. It is concluded that an early response to hemodynamic stress in the pulmonary circulation is alteration in smooth muscle cell ion channel function and/or Ca2+ homeostasis.

Nitric oxide synthesis inhibitors attenuate calcitonin gene-related peptide endothelium-dependent vasorelaxation in rat aorta

The effect of inhibitors of nitric oxide synthesis were examined on the endothelium-dependent relaxation induced by acetylcholine and human α-calcitonin gene-related peptide (CGRP) on rat isolated aortic rings preconstricted with noradrenaline. The endothelium-dependent vasorelaxation induced by acetylcholine and CGRP was inhibited by N G-monomethyl-L-arginine (L-NMMA) and N G-nitro-L-arginine (L-NOARG) in a concentration-related fashion. The inhibition of endothelium-dependent relaxation by L-NMMA and L-NOARG was partially reversed by L-arginine but not by D-arginine for both acetylcholine and CGRP. The data presented suggests that CGRP, like acetylcholine; relaxes the rat aorta via the release of nitric oxide.

SKF-525A Does Not Inhibit Release of Endothelium-Derived Relaxing Factor from Rat Thoracic Aorta and Dog Mesenteric and Femoral Artery

SKF-525A (proadifen) inhibits endothelium-dependent relaxations induced by acetylcholine, arachidonic acid and the calcium ionophore A23187. This suggests that SKF-525A is an inhibitor of endothelium-derived relaxing factor (EDRF) and that EDRF may be a product of arachidonic acid metabolism formed via a cytochrome P-450-dependent pathway or that EDRF release is dependent on cytochrome P-450. We tested this postulate using both isolated rings of rat thoracic aorta and dog mesenteric and femoral artery and the perfusion-superfusion bioassay. Rings of rat thoracic aorta and dog mesenteric and femoral artery with intact endothelium were precontracted with an EC50 concentration of norepinephrine (0.1 nmol/l) or U46619 (0.05 mumol/l) and the relaxation to acetylcholine (ACh), bradykinin, adenosine triphosphate (ATP) or nitroglycerin (GTN) were obtained before, 30 min after addition of, and 30 min after washout of SKF-525A (50 mumol/l). SKF-525A inhibited ACh-induced endothelium-dependent relaxation of rat aortic rings and endothelium-dependent relaxation of the dog mesenteric and femoral artery produced by ACh and ATP, but did not affect relaxation to bradykinin or GTN. The inhibitory effect on SKF-525A on ACh and ATP-induced relaxation was partially reversed upon its washout from the muscle chamber. Pretreatment of the blood vessels with ibuprofen (1 mumol/l) did not attenuate SKF-525A-mediated inhibition of the relaxations to any agonist. Selective exposure of dog femoral artery (donor) to SKF-525A (50 mumol/l) for 60 min did not affect the relaxation responses of endothelium-rubbed coronary artery (bioassay tissue) to basal EDRF nor to the effluent from donor tissues stimulated with ACh (10-1,000 pmol), ATP (1-100 nmol) or bradykinin (3-100 pmol). The results show that SKF-525A exhibited a reversible inhibition of endothelium-dependent relaxation by a smooth muscle mechanism unrelated to the generation of EDRF from endothelium.

Inhibition of endothelium-dependent relaxation by oxidized low-density lipoproteins

Inhibition of endothelium-dependent relaxation by oxidized low-density lipoproteins

Inhibition of endothelium dependent relaxation by N-methylarginine, N-nitroarginine, and methylene blue

Inhibition of endothelium dependent relaxation by N-methylarginine, N-nitroarginine, and methylene blue

Interaction between endothelin-1 and endothelium-derived relaxing factor in human arteries and veins.

Endothelin-1 is a 21-amino acid endothelial vasoconstrictor peptide that may be the physiological antagonist of endothelium-derived relaxing factor (EDRF). Endothelin-1 (10(-11)-3 x 10(-7) M) evoked potent contractions of isolated internal mammary arteries, internal mammary veins, and saphenous veins, which were enhanced in internal mammary veins as compared with internal mammary arteries (concentration shift, 6.3-fold; p less than 0.05) but not in the saphenous veins. Endothelial removal augmented the response to the peptide (at 3 x 10(-7) M) in internal mammary arteries (p less than 0.05) but not in veins. In the artery, EDRF released by acetylcholine or bradykinin reversed endothelin-1-induced contractions; in saphenous veins, both agonists were much less effective compared with the artery and veins contracted with norepinephrine (p less than 0.005-0.01). This inhibition of endothelium-dependent relaxations in veins occurred at half-maximal contractions but was most prominent at maximal contractions to the peptide. Nitric oxide similarly inhibited contractions to endothelin-1 and norepinephrine in internal mammary arteries, whereas in veins that were contracted with endothelin-1 but not with norepinephrine, the relaxations were blunted (p less than 0.005). The nitric oxide donor SIN-1 and sodium nitroprusside induced complete relaxations of internal mammary arteries but were less effective in veins contracted with endothelin-1 (p less than 0.005). Thus, in normal human arteries, EDRF inhibits endothelin-1-induced contractions, whereas the peptide specifically attenuates the effects of EDRF and nitrovasodilators in veins. This may be important in pathological conditions associated with increased levels of endothelin-1 and in veins used as coronary bypass grafts.

Cyclosporin A inhibits endothelium-dependent, prostanoid-induced relaxation in human subcutaneous resistance vessels

The effect of cyclosporin on endothelium-dependent relaxation of human subcutaneous resistance vessels was investigated. Resistance vessels were obtained from normal subjects undergoing routine surgery. Acetylcholine-induced relaxation was shown to consist of at least two components, one mediated through vasodilatory prostanoids, the other through endothelium-derived relaxing factor (EDRF). Incubation with cyclosporin inhibited endothelium-dependent relaxation, as assessed by the response to acetylcholine. The inhibitory effect of cyclosporin on the acetylcholine response was most marked in the presence of methylene blue, which is an inhibitor of EDRF. This suggests that the inhibition of endothelium-dependent relaxation by cyclosporin is mediated by an effect on vasodilatory prostanoids.

L-Arginine causes whereas L-argininosuccinic acid inhibits endothelium-dependent vascular smooth muscle relaxation

This study examined the actions of L-arginine, a putative precursor of endothelium-derived nitric oxide, and arginine analogs on endothelium-dependent relaxation of isolated rings of bovine pulmonary artery. L-Arginine did not consistently relax arterial rings unless rings were first rendered refractory to endothelium-dependent relaxation by pretreatment with 1 μM A23187 for 45 min. L-Arginine-elicited relaxation was endothelium-dependent, antagonized by oxyhemoglobin or methylene blue, and unaffected by indomethacin. L-Argininosuccinic acid caused endothelium-dependent contractions and irreversible inhibition of endothelium-dependent but not nitroglycerin-elicited relaxation, which was not overcome by addition of L-arginine. Inhibition of endothelium-dependent relaxation by L-N G-monomethyl arginine, however, was reversible and overcome by L-arginine. Therefore, endothelium-dependent relaxants may cause arginine depletion in endothelial cells and endogenous argininosuccinic acid may modulate the biosynthesis of endothelium-derived nitric oxide from arginine.

A specific inhibitor of nitric oxide formation from l‐arginine attenuates endothelium‐dependent relaxation

1. The role of L-arginine in the basal and stimulated generation of nitric oxide (NO) for endothelium-dependent relaxation was studied by use of NG-monomethyl L-arginine (L-NMMA), a specific inhibitor of this pathway. 2. L-Arginine (10-100 microM), but not D-arginine (100 microM), induced small but significant endothelium-dependent relaxations of rings of rabbit aorta. In contrast, L-NMMA (1-300 microM) produced small, endothelium-dependent contractions, while its enantiomer NG-monomethyl-D-arginine (D-NMMA; 100 microM) had no effect. 3. L-NMMA (1-300 microM) inhibited endothelium-dependent relaxations induced by acetylcholine (ACh), the calcium ionophore A23187, substance P or L-arginine without affecting the endothelium-independent relaxations induced by glyceryl trinitrate or sodium nitroprusside. 4. The inhibition of endothelium-dependent relaxation by L-NMMA (30 microM) was reversed by L-arginine (3-300 microM) but not by D-arginine (300 microM) or a number of close analogues (100 microM). 5. The release of NO induced by ACh from perfused segments of rabbit aorta was also inhibited by L-NMMA (3-300 microM), but not by D-NMMA (100 microM) and this effect of L-NMMA was reversed by L-arginine (3-300 microM). 6. These results support the proposal that L-arginine is the physiological precursor for the basal and stimulated generation of NO for endothelium-dependent relaxation.

Selective Inhibition of Endothelium-Dependent Relaxation by Gold-Containing Compounds

Gold-containing compounds inhibited endothelium-dependent relaxation mediated by acetylcholine and the calcium ionophore A23187 in isolated rabbit thoracic aortic rings. Auranofin was the most potent gold-containing compound studied, producing approximately 40% inhibition of vascular relaxation at a concentration of 1 microM. Concentration-response curves to vascular relaxation produced by sodium nitroprusside were significantly shifted 5-fold to the left in the presence of auranofin. However, concentration-response curves to vascular relaxation produced by nitric oxide or isoproterenol were unaffected by auranofin. A series of other gold-containing compounds also inhibited endothelium-dependent relaxation with varying degrees of potency, but none approaching that observed with auranofin. These findings reveal that certain gold-containing compounds are potent and selective inhibitors of endothelium-dependent relaxation.