Release Of Endothelium-dependent Relaxing Factor Research Articles

New methods to evaluate endothelial function: Non-invasive method of evaluating endothelial function in humans.

Evidence has accumulated that impairment of vascular endothelial function is the initial step in the development of atherosclerosis. One important finding is impairment of the release of endothelium-dependent relaxing factor, which is now thought to be nitric oxide or its related substances, from endothelial cells. Flow-mediated dilatation has been known to be endothelium-dependent, and this can be detected during reactive hyperemia by high-resolution ultrasound in superficial arteries. Several coronary risk factors have been reported to be significantly related with decreased flow-mediated dilatation. We studied the association between the accumulation of coronary risk factors (hyperlipidemia, diabetes mellitus, hypertension, and current smoking habitus) and vascular endothelial function. The lower incidence of atherosclerosis in women before the menopause than that in men is an established epidemiological observation. Short-term estrogen therapy improves endothelial function in postmenopausal women. However, there are few reports on its long-term effects on endothelial function. Furthermore, we determined whether a reduced dosage of estrogen may maintain its beneficial effects. A similar improvement was also observed while women were on hormone replacement therapy even at the reduced dosage. Our results indicate that even at half the dose of estrogen, hormone replacement therapy may improve endothelial function in postmenopausal women.

Mechanism of pathophysiological effects of diesel exhaust particles on endothelial cells

The suspension of diesel exhaust particles (DEP) inhibited endothelium-dependent relaxation (EDR). The mechanism of the impairment of EDR by DEP was investigated with cultured porcine endothelial cells (PEC) and NO synthase (NOS) cell free system. Incubation of PEC with DEP (50–150 μg/ml) for 10–30 min did not induce cell damage. Bradykinin-induced endothelium-dependent relaxing factor (EDRF) release from PEC was bioassayed by cyclic GMP formation in RFL-6 cells. A 10-min preincubation of PEC with DEP (0.1–100 μg/ml) inhibited EDRF release. NOS activity from rat cerebellum cytosol was measured either by the conversion of 3H- l-arginine to 3H- l-citrulline or the NO 2 − formation. A 10-min preincubation of NOS with DEP (0.1–100 μg/ml) did not affect the formation of 3H- l-citrulline. In contrast, it inhibited NO 2 − formation. These results suggest that DEP neither induced cell damage nor inhibited EDRF release from PEC, but DEP scavenged NO to block its physiological action.

Extracellular ATP and bradykinin increase cGMP in vascular endothelial cells via activation of PKC.

Vasodilation by agents such as bradykinin and ATP is dependent on nitric oxide, the endothelium-dependent relaxing factor (EDRF). The release of EDRF results in elevation of cGMP in endothelial and smooth muscle cells (9). The signaling pathway that leads to increases in cGMP is not completely understood. The role of protein kinase C (PKC) in the elevation of cGMP induced by ATP and bradykinin was studied in cultured porcine aortic endothelial cells, by measuring PKC phosphorylation of a substrate and by measuring cGMP levels by radioimmunoassay. Extracellular ATP and bradykinin simultaneously elevated cGMP levels and PKC activity. The PKC inhibitors staurosporine, calphostin C, and Cremophor EL (T. Tamaoki and H. Nakano. Bio/Technology 8: 732-735, 1990; F. K. Zhao, L. F. Chuang, M. Israel, and R. Y. Chuang. Biochem. Biophys. Res. Commun. 159: 1359-1367, 1989) prevented the elevation of cGMP elicited by ATP and reduced that produced by bradykinin. Cremophor did not affect the elevation of cGMP by nitroprusside, an agent that directly increases guanylate cyclase activity (9). The PKC activator phorbol 12-myristate 13-acetate, but not a phorbol ester analog inactive on PKC, also elevated cGMP levels. These results suggest that EDRF agonists elevate cGMP in endothelial cells via PKC stimulation.

Effects of calcium dobesilate on the synthesis of endothelium-dependent relaxing factors in rabbit isolated aorta.

1. Some cardiovascular disturbances which occur in diabetics are a consequence of alterations in vascular contractility as well as in endothelium-dependent relaxation. 2. Calcium dobesilate (DOBE) is a drug used in diabetic retinopathy and its mechanism of action is not yet understood. 3. The aim of this study was to investigate the effects of DOBE on synthesis and release of endothelium-dependent relaxing factor (EDRF) and endothelium-dependent hyperpolarizing factor (EDHF) in rabbit isolated aorta. 4. Endothelium-dependent relaxation induced by acetylcholine (ACh) (10(-8)-(10(-5) M) increased in the presence of DOBE 10(-5) M only when vascular endothelium was kept intact. 5. NG-nitro-L-arginine methyl ester (L-NAME; 10(-8)-10(-4) M progressively decreased the enhancing effect of DOBE on endothelium-dependent relaxation whereas it was progressively increased by L-Arg. 6. DOBE 10(-5) M increased in a non-significant manner endothelium-dependent relaxation induced by ACh when the arteries were incubated with both L-NAME 10(-4) M and indomethacin 10(-5) M. 7. DOBE (10(-6) M and 10(-5) M) was able to scavenge superoxide anion radicals generated by the hypoxanthine/xanthine oxidase reaction. 8. These results provide evidence that DOBE is able to affect the vascular disorders associated with diabetes mellitus since it enhances the synthesis of endothelium-dependent relaxing factors.

Endothelium-dependent relaxation in pulmonary arteries after lung preservation and transplantation

Pulmonary hypertension is frequently seen after lung transplantation. To study how the release of the endothelium-dependent relaxing factor is affected by lung preservation and transplantation, porcine pulmonary arteries were investigated in organ baths. The arteries (1 mm in diameter) were taken from fresh nonperfused lungs (group I), lungs immediately after flush-perfusion with a low-potassium-dextran solution (group II), non-perfused lungs stored for 12 hours in low-potassium-dextran solution (group III), flush-perfused lungs stored for 12 hours in low-potassium-dextran solution (group IV), and group IV lungs after left lung transplantation and right pneumonectomy followed by 24 hours of reperfusion (group V). Stable contractions were induced with the thromboxane A 2 analogue U-46619. Acetylcholine was used to stimulate the release of endothelium-dependent relaxing factor. In vessel segments where the endothelium had been removed, acetylcholine elicited no response. In segments with intact endothelium, acetylcholine induced concentration-dependent relaxation; the maximum relaxation obtained was 91% ± 3% (I), 86% ± 3% (II), 85% ± 3% (III), 69% ± 5% (IV), and 69% ± 9% (V). Relaxation was significantly reduced in groups IV ( p < 0.01) and V ( p < 0.05) as compared with group I. Stable moderate pulmonary hypertension was present in all the transplanted lungs throughout the 24-hour observation period. It is concluded that the endothelium-mediated relaxation is significantly reduced after flush perfusion combined with 12 hours of storage in low-potassium-dextran solution. Lung transplantation, followed by 24 hours of reperfusion did not further impair the endothelium-dependent relaxation.

Effect of chronic smoking on endothelium-dependent vascular relaxation in humans.

1. Cigarette smoking is one of the major risk factors for the development of atherosclerosis. It is not clear, however, whether chronic cigarette smoking impairs the normal physiological function of the endothelium before the development of morphological vascular lesions. To test this, we investigated endothelium-dependent vascular relaxation in young habitual smoking subjects. 2. In 11 non-smokers and 10 habitual smokers we measured the changes in bilateral forearm blood flow, arterial blood pressure and forearm vascular resistance (ratio between mean arterial blood pressure and forearm blood flow) during three interventions: post-occlusive forearm hyperaemia, intrabrachial infusion of methacholine which causes vasodilatation by stimulating the release of endothelium-dependent relaxing factor, and intrabrachial infusion of sodium nitroprusside which causes vasodilatation independently from the endothelium by a direct effect on the vascular smooth muscle wall. 3. During infusion of the highest dose of methacholine, forearm vascular resistance decreased by 91.7 +/- 1.4% in the smokers and by 89.9 +/- 1.8% in the non-smokers. During infusion of sodium nitroprusside, forearm vascular resistance decreased by 80.0 +/- 3.8% in the smokers as compared with 80.7 +/- 6.1% in the non-smokers. There was no difference in basal forearm vascular resistance or in post-ischaemic reactive hyperaemia between smokers and non-smokers. Thus, vasodilatation induced by both methacholine and sodium nitroprusside was not significantly different between smokers and non-smokers. 4. We conclude that in young habitual cigarette smokers the endothelium-dependent vasodilation in the forearm seems to be preserved, suggesting that habitual smoking does not result in permanent endothelial dysfunction in the human forearm.

Computer system for the acquisition and analysis of vascular contractility application to a bioassay of endothelial cell function

A system for the digital acquisition and subsequent analysis of the tension developed by isolated blood vessels in response to an endothelial cell superfusate is reported. Tension of the isolated rat aortic rings was measured by strain gauge. Strain-gauge output was then amplified, and the analog signal was digitized on a 16-channel A/D board. Lab tech Notebook software was used to display and store the data. The sampling rate was 0.1 Hz, and the data was written concurrently to hard disk and printer. Both disk and printer output were accompanied by a time stamp for subsequent ease of retrieval. The endothelial cell bioassay system allowed measurement of changes in vascular tension after the release of endothelium-dependent relaxing factor, nitric oxide (EDRF-NO) from cultured cells. Cells were cultured on microcarrier beads, formed into columns, and perfused with physiological salt solution. Significant ( p < 0.05) relaxant responses occured after agonist stimulation with bradykinin (10 −8 M; E max −31.0% ± 8.2%), acetylcholine (10 −8 M; E max −33.2% ± 5.0%), and calcium ionophore A 23187 (10 −6 M; E max −55.7% ± 15.4%). These responses were dependent on EDRF-NO, as shown by both the lack of relaxation in the absence of endothelial cells, and that relaxation to A 23187 was overcome by hemoglobin (3 × 10 −6 M). Results were manipulated graphically to allow the superimposition of data and thereby provide a mean and standard error of the mean for the entire time course of each response. Thus, a system was produced where fidelity of data expression was not dependent on measurements made at single points, but on the sampling frequency of the acquisition system.

Pulmonary Vascular Reactivity to Endothelin-1 in Normal and Chronically Pulmonary Hypertensive Rats

The pulmonary vascular reactivity to endothelin-1 (ET-1) was assessed in rats previously exposed to 11% O2 (hypoxic) or room air (controls) for 3 weeks. In isolated control lung preparations studied during conditions of increased tone by U46619 (50 pmol/min) and treated with meclofenamate (3 microM), low doses of ET-1 (30 and 100 pM) reduced the pressor response to U46619 by 58 +/- 5% (p less than 0.01). Vasodilation induced by ET-1 was not abolished by the antagonist of endothelium-dependent relaxing factor (EDRF) NG-monomethyl-L-arginine (5 x 10(-4) M), which suppressed vasodilator response to ionophore A23187 (10(-8)-10(-7) M). Higher doses of ET-1 (300 and 1,000 pM) induced vasoconstriction during conditions of basal tone, and the pressor response to 300 pM ET-1 was enhanced by EDRF antagonists. Administration of ET-1 to lungs from hypoxic rats failed to cause pulmonary vasodilation and instead induced a greater pulmonary pressor response (300 pM) than in control rat lungs (7 +/- 1.5 vs. 1.6 +/- 0.5 mm Hg, p less than 0.01), which was not further potentiated by EDRF antagonists. Infusion of 300 pM ET to conscious catheterized animals induced a sustained increase in pulmonary resistance only in the hypoxic group (from 305 +/- 37 to 389 +/- 55 mm Hg/L/min, p less than 0.01) (n = 7). The results suggest that depending on the dose, ET-1 can cause pulmonary vasodilation (independent of EDRF release) or vasoconstriction (opposed by EDRF). During chronic hypoxic pulmonary hypertension, ET-1 behaves only as a pulmonary vasoconstrictor.

Tension-Induced Release of Endothelium-Derived Relaxing Factor; Possible Role in Establishment of Desensitization of Norepinephrine-Induced Contraction in Rat Aorta

In order to clarify the mechanisms involved in the endothelium-dependent development of desensitization of norepinephrine-induced contraction in rat aorta, we have tested the effect of repeated generation of tension without receptor stimulation. Even when tension alone, with a magnitude almost equal to that generated by norepinephrine, was applied to the endothelium-intact ring without norepinephrine, the ring became desensitized. In the absence of endothelium, the development of desensitization did not occur. Furthermore, L-NG-monomethyl arginine, which is an inhibitor of endothelium-dependent relaxing factor (EDRF) synthesis, prevented the occurrence of desensitization. It was even able to reestablish contractile force when added after the desensitization had developed, suggesting that an increased release of EDRF is necessary to produce the desensitization. Therefore, these results indicate that endothelium-dependent desensitization does not require adrenergic receptor stimulation, but rather that tension generation alone is sufficient to establish desensitization.

Does Endothelin-1 Mediate Endothelium-Dependent Contractions During Anoxia?

Endothelin-1 (ET-1) is a more potent constrictor of femoral venous than femoral arterial smooth muscle. By contrast, endothelium-dependent contractions to anoxia are more prominent in the artery. Unlike those to ET-1, the endothelium-dependent contractions evoked by anoxia are rapid in onset and readily reversible; they are antagonized by Ca2+ entry blockers. ET-1-induced responses are inhibited by endothelium-dependent relaxing factors in canine arteries, and to a lower extent in veins. ET-1 does not augment the production of cyclic GMP in cultured porcine aortic endothelial cells, or in canine arteries and veins, suggesting that the peptide does not evoke the release of endothelium-dependent relaxing factor (EDRF) in canine blood vessels. The endothelium-derived contracting factor (EDCF) released during anoxic contraction cannot be bioassayed, under conditions where ET-1 causes contraction of the bioassay tissue. No ET-1 appears to be released in the extracellular space during anoxic contractions. These findings do not support the hypothesis that ET-1 is the EDCF released by anoxia.