Inhibitor Of Endothelium-derived Relaxing Factor Synthesis Research Articles

The Interaction between Endothelium-Derived Relaxing Factor (EDRF) and Eicosanoids in the Regulation of the Mesenteric Microcirculation

Locally produced eicosanoids and endothelium-derived factors are believed to be the mediators of vascular tone of various vascular beds including the mesentery. Using a small vessel isometric myograph which allows direct measurement of microvascular reactivity, the interaction of eicosanoids and endothelium-derived relaxing factor (EDRF) in regulating vascular tone of mesenteric microcirculation of the rat was characterized. The microvascular response to various vasoactive agents before and after inhibition of prostacyclin production with indomethacin (INDO, 5 μM) and inhibition of EDRF synthesis with Nω-nitro-dcl026;-arginine methyl ester (dcl026;-NAME, 50 μM) was compared. Analysis of dose-response curves for prostaglandin F2α (PGF2α), U46619, a stable thromboxane analog, and norepinephrine (NE) after pretreatment with INDO demonstrated that inhibition of endogenous eicosanoids significantly attenuated the vasoconstrictor response to PGF2α and U46619 but not to NE. Inhibition of EDRF synthesis with dcl026;-NAME potentiated the vasoconstrictor response to PGF2α, U46619, and NE. These results suggest that EDRF acts as the primary mediator of vasodilator tone in the mesenteric microcirculation rather than vasodilator cyclooxygenase products such as prostacyclin. It also appears that the vasoconstrictor action of PGF2α and U46619 may be mediated by a release of an endogenous indomethacin-sensitive factor.

Inhibition of nitric oxide synthesis augments centrally induced sympathetic coronary vasoconstriction in cats

The principal effect of sympathetic activation on the coronary circulation is an alpha-adrenergic coronary vasoconstriction in the presence of beta-receptor blockade. Secondary effects include vasodilation due to beta-adrenoceptor stimulation and alpha 2-mediated release of endothelium-derived relaxing factor (EDRF) from the coronary vascular endothelium. We hypothesized that blockade of nitric oxide synthesis (nitro-L-arginine methyl ester, L-NAME) would augment coronary vasoconstriction to sympathetic stimulation as a result of a decrease in alpha 2-mediated EDRF release. In chloralose-anesthetized cats, hypothalamic stimulation produced increases in coronary vascular resistance [maximum 26 +/- 9% (SE)] and arterial pressure (41 +/- 7%) and a decrease in coronary blood flow velocity (15 +/- 6%). L-NAME (3 mg/kg iv) increased baseline arterial pressure from 69 +/- to 92 +/- 7 mmHg (P < 0.05). After L-NAME, a greater increase in coronary vascular resistance (55 +/- 20%, P < 0.05), a decrease in coronary blood flow velocity (24 +/- 7%, P < 0.05), and a similar pressor response (34 +/- 7%) were observed in response to hypothalamic stimulation. L-Arginine reversed the effect of L-NAME on coronary vasoconstriction to hypothalamic stimulation. Similar increases in arterial pressure (from 73 +/- 3 to 91 +/- 5 mmHg, P < 0.05) with vasopressin (0.01-0.05 U/min) failed to enhance coronary vasoconstriction to activation in anterior hypothalamus. We conclude that inhibition of EDRF synthesis augments centrally induced sympathetic coronary vasoconstriction in the cat.

Contribution of endothelium-derived relaxing factor to exercise-induced vasodilation in humans.

Release of endothelium-derived relaxing factor (EDRF) from the vascular endothelium may contribute to skeletal muscle arteriolar vasodilation during exercise. The present study was undertaken to test this hypothesis. Ten normal subjects underwent brachial arterial catheter insertion and instrumentation of their forearm to measure plethysmographic forearm blood flow. Forearm blood flow was then measured at rest, during two levels of wrist flexion exercise (0.2 and 0.4 W), and during 0.4-W exercise with concurrent infusion of norepinephrine (100 ng.min-1 x 100 ml forearm volume-1). Measurements were made with and without infusion of N-monomethyl-L-arginine (L-NMMA) (0.1-0.2 mg.min-1 x 100 ml forearm volume-1), a specific inhibitor of EDRF synthesis. Infusion of L-NMMA reduced resting forearm blood flow (control: 2.5 +/- 0.4 vs. L-NMMA: 1.5 +/- 0.1 ml.min-1 x 100 ml-1) and markedly reduced forearm blood flow response to acetylcholine (5 micrograms.min-1 x 100 ml forearm volume-1) (control: 20.2 +/- 2.9 vs L-NMMA: 4.0 +/- 1.0 ml.min-1 x 100 ml-1; both P < 0.01). However, L-NMMA had no significant effect on flow responses to exercise (0.2 W: 8.3 +/- 1.1 vs. 8.3 +/- 1.2; 0.4 W: 13.8 +/- 1.7 vs. 13.5 +/- 1.7; 0.4 W + norepinephrine: 10.3 +/- 2.4 vs. 9.4 +/- 2.6 ml.min-1 x 100 ml-1; all P = NS). These findings suggest that release of EDRF from the vascular endothelium contributes to skeletal muscle arteriolar vasodilation at rest but does not contribute to the arteriolar vasodilation produced by exercise.

Endothelin and endothelium‐derived relaxing factor control of basal renovascular tone in hydronephrotic rat kidneys.

1. In order to investigate the control of renal vascular tone by endothelin (ET) and endothelium-derived relaxing factor (EDRF) under basal conditions, we infused intravenously anti-ET-1/3 antibodies (a-ET-1/3) and NG-nitro-L-arginine methyl ester (L-NAME) in split hydronephrotic rat kidneys. 2. A 25 min I.V. infusion of a-ET-1/3 (4.0 x 10(-13) mol kg-1 min-1) induced a time-dependent vasodilatation of arcuate (16.5%) and interlobular arteries (18.6%) as well as an increase of glomerular blood flow (GBF) by 32%. 3. Inhibition of EDRF synthesis by L-NAME produced a marked vasoconstriction of arcuate arteries (17.1%) and efferent (20.1%) arterioles and a decrease of GBF by 43%. 4. Co-infusion of a-ET-1/3 and L-NAME induced efferent vasoconstriction by 19.5%, whereas preglomerular vessel diameters remained unchanged. 5. The specificity of a-ET-1/3 effects was confirmed by simultaneous I.V. application of a-ET-1/3 and ET-1 (160 ng I.V.) which produced no significant vascular effects. Injection of ET-1 alone constricted arcuate arteries and decreased glomerular blood flow by 25%. 6. Experiments in normal rat kidneys with a-ET-1/3 I.V. revealed an increase of renal blood flow by 21%. 7. Our results demonstrate a physiological control of basal vascular tone in larger preglomerular arterioles by ET and EDRF. Efferent arteriolar tone is predominantly controlled by EDRF.

Effects of oxygen and exogenous L-arginine on EDRF activity in fetal pulmonary circulation.

To determine whether L-arginine, the precursor of endothelium-derived relaxing factor (EDRF), increases vasodilator activity in the fetal pulmonary circulation, we studied its effects on basal pulmonary vascular tone and on pulmonary vasodilation stimulated by oxygen and acetylcholine (ACh) in chronically prepared late-gestation fetal lambs. L-Arginine infusion (30-300 mg over 10 min) into the left pulmonary artery (LPA) increased blood flow (18-57%) without changing pulmonary artery pressure. To determine whether O2-induced vasodilation involves EDRF and is augmented by L-arginine treatment, we infused L-arginine or NG-nitro-L-arginine (L-NNA), an inhibitor of EDRF synthesis, while increasing fetal PO2 6 Torr by delivering 100% O2 to the ewe for 120 min. In controls, LPA blood flow progressively increased from 106 +/- 13 ml/min (baseline) to 257 +/- 34 ml/min (peak) at 40 min of increased PO2 (P < 0.05, baseline vs. peak) but steadily returned toward baseline during the next hour. Treatment with L-NNA markedly attenuated O2-induced pulmonary vasodilation (P < 0.05 vs. control). L-Arginine infusion did not augment or sustain the O2-induced vasodilator response. We also examined whether L-arginine could sustain pulmonary vasodilation to ACh, another EDRF-dependent stimulus, and found that the EDRF substrate neither potentiated nor sustained the ACh response. We conclude that: in the fetal lung 1) exogenous L-arginine is a fetal pulmonary vasodilator, 2) increased PO2 augments EDRF activity in the fetal lung, and 3) supplemental L-arginine does not sustain either O2- or ACh-induced vasodilation.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal versus femoral hemodynamic response to endothelium-derived relaxing factor synthesis inhibition.

Systemic inhibition of endothelium-derived relaxing factor (EDRF) synthesis leads to acute hypertension and increased peripheral vascular resistance. The changes in vascular resistance are not evenly distributed to all vascular beds. In this study, we compared the renal and femoral hemodynamic responses to EDRF synthesis inhibition. Renal blood flow (RBF) and femoral blood flow (FBF) were assessed in the presence and absence of DuP 753, an angiotensin II receptor antagonist. Inhibition of EDRF synthesis by a bolus dose of Lw-nitroarginine methyl ester (L-NAME) increased blood pressure (BP) by 21 +/- 1 mm Hg (p < 0.001) and decreased RBF by 32 +/- 5% (from 5.9 +/- 0.5 to 3.9 +/- 0.3 ml/min/g kidney weight; p < 0.005) while FBF remained unchanged (9.5 +/- 0.4 versus 9.4 +/- 0.4 ml/min). Renal vascular resistance (RVR) increased by 83 +/- 16% (p < 0.001), compared with only a 24 +/- 6% increase in femoral vascular resistance (FVR; p < 0.005). To eliminate the influence of systemic hypertension, we returned organ perfusion pressure to pre-L-NAME levels by partial aortic constriction. The kidney maintained RBF by decreasing RVR by 8 +/- 2% (p < 0.02), while FBF decreased by 15 +/- 5% (p < 0.01). When rats were pretreated with DuP 753, L-NAME still increased BP by 22 +/- 2 mm Hg, but RVR increased by only 26 +/- 5% (from 13.2 +/- 1.6 to 16.8 +/- 2.7; p < 0.01) and RBF did not change. DuP 753 had no effect on the femoral vascular response to L-NAME.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of BRL 38227, sodium nitroprusside and verapamil on collateral perfusion following acute arterial occlusion in the rabbit isolated ear.

1. We have used an isolated, buffer-perfused, rabbit ear model of acute arterial occlusion to investigate the effects of the nitrovasodilator sodium nitroprusside, the potassium channel activator BRL 38227 (the active (-)-enantiomer of cromakalim) and the calcium antagonist, verapamil, on collateral perfusion in the absence of pharmacological tone. 2. Verapamil was the most potent vasodilator (EC50 = 72.6 +/- 32.0 nM) of 5-hydroxytryptamine/histamine-induced tone in the rabbit isolated perfused ear. Sodium nitroprusside and BRL 38227 were less potent with respective EC50 values of 488 +/- 75 nM and 296 +/- 40 nM. Following inhibition of endothelium-derived relaxing factor (EDRF) synthesis, the potency of BRL 38227 was significantly (P less than 0.001) increased with an EC50 of 55.6 +/- 5.0 nM. 3. BRL 38227 at 500 nM and 3 microM induced substantial increases in collateral perfusion following arterial ligation in the absence of pharmacological tone compared to control. Furthermore 3 microM BRL 38227 completely reversed the attenuation of collateral perfusion which followed inhibition of EDRF synthesis with 100 microM NG-nitro-L-arginine methyl ester (L-NAME). 4. Sodium nitroprusside (500 nM and 3 microM) induced modest improvements in collateral perfusion in the early stages after arterial occlusion. 5. Verapamil did not influence collateral perfusion at either of the concentrations used (50 nM and 3 microM), even though it was a potent vasodilator. 6. The results of this study indicate that BRL 38227, and to a much lesser extent sodium nitroprusside, selectively improve collateral perfusion following arterial occlusion, even in the presence of effects of EDRF on acute collateralization, while verapamil has no effect. Furthermore, BRL 38227 also improves collateral perfusion following inhibition of EDRF synthesis. It remains to be established whether BRL 38227 has beneficial actions in acute arterial occlusion in vivo.

Endothelial control of long bone vascular resistance.

This in vitro study investigates whether intraosseous endothelial cells can regulate long bone blood flow by secretion of vasodilator prostaglandin and EDRF (endothelium-derived relaxing factor). Canine tibia were perfused through the nutrient artery at a constant flow rate, and the increases in perfusion pressure caused by standard doses of norepinephrine were recorded first under control conditions and then during acetylcholine infusion. Acetylcholine attenuated the norepinephrine pressure responses (-62 +/- 3%). This attenuating effect of acetylcholine was partially abolished by inhibition of prostaglandin synthesis (-20 +/- 6%) and completely abolished by inhibition of EDRF synthesis (+73 +/- 43%) or combined inhibition of prostaglandin and EDRF synthesis (+134 +/- 30%). These results are statistically significant (p less than 0.0001) and suggest that both EDRF and vasodilator prostaglandin are synthesized by intraosseous endothelial cells, and can modify long bone vascular resistance. Thus, as in other organs, intraosseous endothelial cells may provide bone with an autoregulatory control mechanism and enable it to respond to a diverse group of vasodilator stimuli.

Collateral perfusion: the role of endothelium-derived relaxing factor and effects of vasodilators.

An in vitro rabbit ear model has been used to investigate the role of endothelium-derived relaxing factor (EDRF) in collateral perfusion after acute arterial occlusion and also the effects of vasodilators. Collateral perfusion of an arterial segment isolated between occlusions was assessed by x-ray microangiography and was found to develop in a time-dependent manner. Inhibition of EDRF synthesis with NG-nitro-L-arginine methyl ester (L-NAME) greatly impaired collateral perfusion, indicating that the development of collateral perfusion was dependent on EDRF activity. This inhibitory effect was reversed by an excess of L-arginine. Further studies using vasodilators with different modes of action indicated that BRL 38227 (the active enantiomer of cromakalim, a potassium channel activator) substantially enhanced collateral perfusion, sodium nitroprusside had early beneficial effects, and verapamil had no effect.

Impairment of Endothelium-Dependent Pulmonary-Artery Relaxation in Chronic Obstructive Lung Disease

Endothelial cells release endothelium-derived relaxing factor (EDRF) in a variety of vascular beds, including the pulmonary circulation. However, the role of EDRF-mediated pulmonary-artery relaxation in chronic hypoxic lung disease is unknown. We studied endothelium-dependent relaxation mediated by EDRF in vitro in pulmonary arteries that had been obtained from 22 patients undergoing heart-lung transplantation for end-stage chronic obstructive lung disease. Control pulmonary arteries were obtained from 15 patients undergoing lobectomy for lung carcinoma who did not have evidence of other chronic lung disease. The responses of all vascular rings (external diameter, 1.2 to 3.4 mm) to the endothelium-dependent vasodilators acetylcholine and adenosine diphosphate were studied immediately after lung excision. Pulmonary arterial rings from the patients with chronic lung disease developed a greater tension (2.19 +/- 0.16 g) in response to phenylephrine (10(-6) M) than the rings from control patients (1.28 +/- 0.18 g, P less than 0.05). Inhibition of EDRF synthesis by treatment with NG-monomethyl-L-arginine (10(-4) M) eliminated this difference, increasing the tension in the rings from the controls (P less than 0.01) but not in those from the patients with chronic lung disease. Rings from control patients relaxed in response to cumulative doses (10(-10) to 10(-5) M) of acetylcholine (maximal relaxation, 81.3 +/- 3.9 percent) and adenosine diphosphate (maximal relaxation, 85.3 +/- 2.6 percent). By contrast, rings from patients with chronic obstructive lung disease achieved only 41.3 +/- 4.8 percent of maximal relaxation in response to acetylcholine (n = 32) and 49.4 +/- 5.5 percent in response to adenosine diphosphate (n = 24) (P less than 0.001, as compared with control rings). Rings from both the controls and the patients with chronic lung disease relaxed similarly in response to the endothelium-independent vasodilator sodium nitroprusside (10(-4) M). There was an inverse correlation between the degree of intimal thickening and the level of maximal relaxation of the rings from the patients with chronic lung disease (r = -0.60, P less than 0.001). Maximal relaxation was also related directly to the partial pressure of arterial oxygen before transplantation (r = 0.68, P less than 0.01) and inversely to the partial pressure of arterial carbon dioxide before transplantation (r = -0.55, P less than 0.01), but not to the forced expiratory volume in one second (r = 0.19, P not significant). Endothelium-dependent pulmonary-artery relaxation in vitro is impaired in arteries from patients with end-stage chronic obstructive lung disease. Such impairment may contribute to the development of pulmonary hypertension in chronic hypoxic lung disease.

Role of the endothelium in modulation of the acetylcholine vasoconstrictor response in porcine coronary microvessels

The aim was to investigate the role of the endothelium in modulating the acetylcholine response in porcine coronary microvessels and compare the results with simultaneously studied large coronary arteries. Coronary microvessels [104 (SEM 3.3) microns; range 38-150] were removed from fresh porcine hearts and studied in vitro during no flow constant pressure conditions. Endothelium derived relaxing factor (EDRF) activity and the role of the endothelium in modulating the acetylcholine response in microvessels was assessed by measuring changes in intraluminal diameter using a video tracking device. Large coronary arteries were simultaneously studied using conventional isometric ring techniques. Fresh porcine hearts were obtained from a local slaughterhouse. Acetylcholine was a potent vasoconstrictor (EC50 = 0.17 microM) of passively distended microvessels. The effects of EDRF were studied by either inactivation with haemoglobin or inhibition of EDRF synthesis with N-omega-nitro-L-arginine. Preconstricted microvessels exposed to either N-omega-nitro-L-arginine or haemoglobin constricted further, consistent with basal release of EDRF. Neither drug affected passively distended microvessels. The acetylcholine vasoconstrictor response was potentiated after exposure of microvessels to either drug. Atropine, but not indomethacin, blocked the acetylcholine response in microvessels. As with microvessels, acetylcholine was a vasoconstrictor (EC50 = 0.3 microM) of large coronary arteries. In contrast to microvessels, indomethacin antagonised acetylcholine vasoconstriction in vessels with intact endothelium. Bioassay experiments using indomethacin-treated large epicardial donor artery segments showed basal release of EDRF but no EDRF release in response to acetylcholine. The results show the microvessels and large coronary arteries are similar in their vasoconstrictor response to acetylcholine, that both release EDRF basally, and that vasoconstriction to acetylcholine is importantly modulated by the endothelium. In large arteries, acetylcholine does not stimulate EDRF release and, in contrast to microvessels, a cyclo-oxygenase product influences the vasoconstrictor action of acetylcholine.

Effects of endothelium-derived relaxing factor and nitric oxide on rat mesangial cells

We have investigated whether endothelium-derived relaxing factor (EDRF) and nitric oxide (NO), a substance proposed to be one of the EDRFs, could elicit biochemical and biological responses in rat glomerular mesangial cells (MC). In wells with MC alone, guanosine 3',5'-cyclic monophosphate (cGMP) levels were 2.6 +/- 0.6 fmol/microgram protein, and bradykinin did not affect these levels, whereas in coincubation experiments with bovine aortic EC and rat MC, cGMP levels in MC increased to 44.6 +/- 21 fmol/micrograms protein after bradykinin stimulation (P less than 0.05). This effect was potentiated by superoxide dismutase and inhibited by hemoglobin and L-NG-monomethyl arginine, a specific inhibitor of EDRF synthesis. Increases in cGMP were also observed when MC were incubated directly with NO and were potentiated by superoxide dismutase and inhibited by hemoglobin. We also tested whether NO could inhibit angiotensin II (ANG II)-induced reductions in cross-sectional area (CSA) of MC. When MC were exposed to ANG II only, 65% of the cells underwent a significant reduction in CSA, as measured by digital image analysis. However, when MC were incubated with ANG II and NO, only 10% of cells responded (P less than 0.04). These studies demonstrate that EDRF and NO induce significant biochemical and functional responses in rat glomerular MC and suggest that communication between EC and MC may be important in regulation of glomerular function.