Modulation of pulmonary artery contraction by endothelium-derived relaxing factor

In isolated rat pulmonary artery rings, both endothelial denudation and treatment with 10(-5) M hemoglobin inhibited relaxation to acetylcholine and increased contractile sensitivities, i.e. decreased the EC50s, to KCl, angiotensin II and norepinephrine. Denudation caused similar inhibition of acetylcholine relaxation and potentiation of KCl and norepinephrine contractions in isolated bovine pulmonary arteries. These results indicate that endothelium-derived relaxing factor plays a significant role in modulating the contractile sensitivity of isolated pulmonary arteries to at least some agonists.     

Differences in basal endothelium-derived relaxing factor activity in different artery types

Mechanical responses and calcium influx were measured in order to compare basal endothelium-derived relaxing factor (EDRF) activity in isolated preparations of rabbit aorta, rat aorta, and dog coronary artery. EDRF activity was characterized by endothelium-dependent mechanical relaxation and reduction of 45Ca influx which could be blocked by EDRF inhibitors. In resting preparations, the mechanical effects of basal EDRF were negligible in all preparations, and a small effect on calcium influx was demonstrated only in rat preparations. In agonist-constricted preparations, basal EDRF activity had only a small mechanical effect in rabbit preparations but markedly depressed constriction in rat and dog preparations; likewise, it had no demonstrable effect on calcium influx in rabbit preparations but had a marked effect in rat and dog preparations. In both resting and agonist-stimulated rabbit preparations, endothelium caused a cyclooxygenase product-dependent increase in calcium influx. Thus, basal EDRF activity has little or no effect in resting preparations and little or no effect in agonist-stimulated rabbit aorta preparations, but a marked effect in agonist-stimulated rat aorta and dog coronary preparations.     

Phorbol ester-induced release of endothelium-derived relaxing factor

The effects of a phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB), on contraction of isolated vascular smooth muscle of rat aorta were examined. DPB (100 nM-1 microM) induced a concentration-dependent contraction in resting aorta. DPB (3-100 nM) also induced a concentration-dependent relaxation of the norepinephrine-induced contraction. The contractile effect of DPB was potentiated whereas the relaxant effect was inhibited by endothelium removal or by methylene blue, suggesting that lower concentrations of DPB release endothelium-derived relaxing factor to inhibit contraction in rat aorta.     

Modulation of alpha-adrenergic-induced contractions by endothelium-derived relaxing factor in rat aorta.

1. In rat thoracic aorta, endothelium removal produced a significant increase of the maximal contraction (Emax) and of the pD2 value (-log ED50) induced by norepinephrine, phenylephrine and clonidine, and did not affect the maximal contractile response to 70 mM KCl. 2. Clonidine did not induce a contraction in aorta with intact endothelium, but after endothelium removal, the contractile response was 94.8% of the Emax produced by norepinephrine in aorta with endothelium. 3. Pre-incubation with methylene blue (10(-5) M) and hemoglobin (0.02%), which inhibit EDRF effects, produced the same effects as the mechanical removal of endothelium on the contractile responses to alpha-adrenergic agonists. 4. These results suggest that EDRF formation and release is an important factor in the modulation of alpha-adrenergic-induced vasoconstriction.     

Perfused rabbit heart: endothelium-derived relaxing factor in coronary arteries

The study was performed to demonstrate the presence of the endothelium-derived relaxing factor (EDRF), previously found to be active in isolated artery preparations in vitro, in the coronary arteries of rabbits remaining in contact with the beating perfused heart. This was accomplished by following the response of the preconstricted obtuse marginal coronary artery to topically administered acetylcholine (ACH). Both intact arteries and arteries in which the endothelium had been removed were studied. Preconstriction of the obtuse marginal coronary artery was accomplished by topical application (spray) of histamine (10 microM), in 2 ml Krebs-Henseleit (KH) solution. This resulted in a decrease in internal diameter of the coronary artery, a decline in coronary flow, and an increase in total coronary vascular resistance. The principal difference between intact coronary arteries and those in which the endothelium had been removed was in the response to ACH. In arteries with intact endothelium preconstricted with histamine, topical administration (spray) of ACH (0.6 mM in 2 ml KH solution) caused, as compared with histamine, an increase in coronary flow, a significant decrease in total coronary vascular resistance, and a rise in internal vascular diameter. Arteries in which the vascular endothelium had been removed, as compared to those pretreated with histamine, responded to topical administration of ACH (0.6 mM in 2 ml KH solution) with a decrease in coronary flow, coronary vascular resistance, and internal vascular diameter. No change in the dose-response curve to histamine between the two preparations was noted. The results demonstrate that coronary vascular endothelium exerts a protective effect in coronary arteries remaining in contact with the perfused beating heart.     

Species-dependent differences in the nature of endothelium-derived vascular relaxing factor

Effluents from perfused acetylcholine-relaxed endothelium segments of rabbit aorta (or canin femoral artery) contained endothelium-derived relaxing factors (EDRF) which dilated the endothelium-free segments of rabbit femoral artery (or side branches of canine femoral artery). The half-life of EDRF was 24 ± 3 s for the rabbit and 49 ± 5 s for the canine system. Nordihydroguaiaretic acid was less effective against the formation of canine EDRF than of rabbit EDRF. These findings suggest species differences in the nature of EDRF.     

内皮舒张因子抑制剂对低氧收缩离体猪冠脉的影响(英文)

左旋硝基精氨酸,NLA (0.2 mmol·L~(-1))可阻断离体猪冠脉依内皮性缺氧收缩反应,用左旋精氨酸,L-Arg(2mmol·L~(-1))预处理可显著降低NLA的抑制作用,四乙胺,TEA(10mmol·L~(-1))和格列本脲,Gli(1 μmol·L~(-1))对缺氧收缩反应无明显影响,而Cro-makalim,Cro(1 μmol·L~(-1))则可抑制缺氧冠脉收缩。     

Release of endothelium-derived relaxing factor is inhibited by 8-bromo-cyclic guanosine monophosphate

Endothelial cells are known to contain both soluble and particulate guanylate cyclase, but the functional role of cyclic guanosine monophosphate (cGMP) in endothelial cells remains unknown. We have investigated the effects of 8-bromo-cGMP on endothelium-dependent relaxations to acetylcholine, substance P, ATP, and the calcium ionophore A23187, and on endothelium-independent relaxations to sodium nitroprusside and glyceryl trinitrate (GTN). The ability of each of these agents to relax phenylephrine-preconstricted rings of rabbit aorta was tested in the absence and presence of 8-bromo-cGMP. In the presence of 8-bromo-cGMP, a greater concentration of phenylephrine had to be used to produce a similar level of tone and then endothelium-dependent relaxations to acetylcholine and substance P were inhibited, whereas endothelium-dependent relaxations to ATP and A23187 were unaffected. Endothelium-independent relaxations to sodium nitroprusside and GTN were only inhibited at the highest concentrations of nitroprusside and GTN. These results suggest that: (a) increasing GMP levels in endothelial cells inhibit agonist-induced release of endothelium-derived relaxing factor (EDRF); (b) a negative feedback mechanism may exist whereby EDRF stimulates soluble guanylate cyclase in endothelial cells to inhibit its own release; and (c) ATP does not induce EDRF release via phosphoinositol hydrolysis.     

Platelet inhibition by endothelium-derived relaxing factor from the rabbit perfused aorta.

1. The platelet inhibiting activity of endothelium-derived relaxing factor (EDRF) released by the perfused thoracic aorta of the rabbit was investigated. 2. The aortic effluent superfused a ring of the abdominal aorta without endothelium in order to bioassay EDRF. Aliquots of effluent were collected on rabbit washed platelets and aggregation induced by U-46619 was measured after 1 min. Prostacyclin (PGI2) was monitored by radioimmunoassay of 6-oxo-prostaglandin F1 alpha. 3. Acetylcholine (ACh) caused a dose-dependent secretion of EDRF, PGI2 and anti-aggregating activity. Plasma and methylene blue suppressed the platelet inhibition by the effluent. 4. The PGI2 content of the effluent was not sufficient to account for all the anti-aggregating activity. However, the platelet inhibition disappeared when PGI2 formation was blocked with indomethacin. 5. Compression of the thoracic aorta increased the EDRF content in the effluent. A transient secretion of anti-aggregating activity was then observed in aortic effluent in the absence of PGI2. This activity coincided with the presumed EDRF peak in the effluent. 6. Superoxide dismutase enhanced the ACh-induced EDRF content and revealed secretion of an anti-aggregating substance when PGI2 formation was blocked. Pretreatment of the platelets with subthreshold concentrations of PGI2, or the cyclic GMP phosphodiesterase inhibitor RX-RE 56, also revealed the release of a labile platelet inhibitor in response to ACh. 7. The results indicate that EDRF released by fresh aortic endothelium may suppress platelet aggregation, particularly when PGI2 is present.     

Pharmacological reactivity of human epicardial coronary arteries: characterization of relaxation responses to endothelium-derived relaxing factor.

1. Human epicardial coronary artery rings, freshly obtained from cardiac transplant patients, were examined for their responses to endothelium-derived relaxing factor (EDRF)-releasing agents. 2. Functional antagonism profoundly influenced relaxation responses in this tissue. Increasing force with concentrations of U46619 above 3 nM (40% of maximum contraction response) resulted in a reduction of the maximum response to four vasorelaxants which relax vascular smooth muscle via different mechanisms: the EDRF-releasing agents, substance P and bradykinin; the endothelium-independent nitro-vasodilator, sodium nitroprusside (SNP); and the beta-adrenoceptor agonist, isoprenaline. 3. Substance P, histamine, bradykinin and the Ca2+ ionophores ionomycin and A23187 all caused concentration- and endothelium-dependent relaxation in vessels pre-contracted with the thromboxane A2-mimetic, U46619 (3 nM) to an active force optimal for relaxation responses. Nifedipine (0.1 microM), added to prevent spontaneous contractions, had no effect or relaxation responses to substance P, bradykinin and histamine. 4. Substance P was the most potent of the EDRF-releasing agents examined and all agents except for bradykinin caused near-maximal relaxation. Bradykinin caused only 46.2% +/- 7.3% relaxation. Responses were abolished when the endothelium was removed and, except for histamine, were not significantly affected by indomethacin (3-10 microM, P > 0.05). Histamine (0.1-10 microM) caused a concentration-dependent contraction of arterial rings without endothelium. 5. The L-arginine analogues NG-nitro-L-arginine (L-NOARG, 0.1 mM) and NG-monomethyl-L-arginine (L-NMMA, 0.1 mM) both caused no further contraction in arteries precontracted with U46619 (3 nM) and were in general, poor inhibitors of responses to EDRF agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pranidipine, a 1,4-dihydropyridine calcium channel blocker that enhances nitric oxide-induced vascular relaxation

Pranidipine, a long acting 1,4-dihydropyridine calcium channel blocker, prolongs nitric oxide (NO)-mediated relaxation of rat aorta; it prolongs acetylcholine-induced relaxation in presence of endothelium as well as nitroglycerin-induced relaxation in absence of endothelium. In rat aorta the effect of pranidipine on NO-mediated relaxation is cyclic guanosine monophosphate (cGMP)-independent, but in guinea pig carotid artery the same effect of pranidipine is cGMP-dependent. It has been reported that in co-cultured human endothelial and smooth muscle cells pranidipine, at a higher concentration (10(-6) M), enhances vasorelaxant effect of NO by blocking NO decomposition. The enhancement of NO action by pranidipine differs from the direct NO-releasing action of other 1,4-dihydropyridines. It is expected that enhancement of NO-induced vasodilatation will lead to a venodilator action in vivo and less peripheral edema. The target organ protective effects of pranidipine are also reviewed in this article.     

A phorbol ester inhibits the release of endothelium-derived relaxing factor

The effect of the phorbol ester phorbol-12,13-dibutyrate (PDB), an activator of protein kinase C, on endothelium-dependent relaxation was studied in noradrenaline-constricted isolated aortic ring preparations of the rabbit. Endothelium-dependent relaxation induced by acetylcholine or substance P was inhibited by PDB (greater than or equal to 10(-7) M). Endothelium-dependent relaxation induced by the calcium ionophore A23187 (7.5 X 10(-8) and 10(-7) M) was unaffected by PDB (to 10(-6) M). The mechanical responses to acetylcholine or sodium nitroprusside in endothelium-denuded rings were not altered by PDB (to 10(-6) M). The results suggest a role for protein kinase C in receptor-mediated EDRF release mechanisms.     

Simultaneous measurement of endothelium-derived relaxing factor by bioassay and guanylate cyclase stimulation.

1. Endothelium-derived relaxing factor (EDRF) released by cultured endothelial cells (EC) from bovine aortae was measured by bioassay using pre-contracted strips of rabbit aorta and by radioimmunoassay of guanosine 3':5'-cyclic monophosphate (cyclic GMP) produced by stimulation of bovine lung soluble guanylate cyclase. 2. Bradykinin (Bk, 3 and 30 pmol) injected through a column of EC caused release of EDRF as detected by bioassay and increased cyclic GMP concentrations. Superoxide dismutase (SOD, 15 u ml-1) increased the amount of EDRF detected by the activation of soluble guanylate cyclase. 3. In the absence of endothelial cells, nitric oxide (NO, 1-2 microM), arachidonic acid (AA, 3-30 microM) or sodium nitroprusside (SNP, 1-100 microM) stimulated guanylate cyclase. Superoxide dismutase strongly increased the stimulation of guanylate cyclase induced by NO, but had little effect on the stimulation induced by SNP and no effect on the stimulation induced by AA. 4. Oxyhaemoglobin (10-300 microM) abolished the stimulation of guanylate cyclase by EDRF, NO or SNP but was much less effective as an inhibitor of AA-induced stimulation of guanylate cyclase. 5. These results demonstrate that measurement of guanylate cyclase stimulation by radioimmunoassay is a viable method for detecting EDRF release, especially useful when the drugs used interfere with bioassay tissues.     

Stimulation of soluble guanylate cyclase by endothelium-derived relaxing factor from cultured endothelial cells

Bovine endothelial cells, grown on microcarrier beads and superfused with a saline solution, were stimulated with thimerosal or bradykinin to release endothelium-derived relaxing factor (EDRF). EDRF activity in the effluent was assayed in endothelium-denuded rabbit aorta. The stimulation of purified soluble guanylate cyclase in test tubes by the EDRF-containing effluent amounted to 90-fold of basal activity and its time course correlated with that of the dilator response of the aorta. After preincubation of endothelial cells with gossypol the EDRF-induced dilator response as well as the stimulation of guanylate cyclase was suppressed.     

Selective inhibition by barbiturates of the synthesis of endothelium-derived hyperpolarizing factor in the rabbit carotid artery.

1. Several lines of evidence suggest that both volatile and intravenous anaesthetics may interfere with the synthesis and release of endothelium-derived vasoactive factors. We have investigated the effects of three different barbiturates on the release of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in phenylephrine (1 microM)-preconstricted, endothelium-intact ring segments of the rabbit carotid artery. The segments were pretreated with the cyclo-oxygenase inhibitor, diclofenac (1 microM), to prevent the formation of vasoactive prostanoids, such as prostacyclin (PGI2). 2. Acetylcholine (ACh) elicited a concentration-dependent relaxation (EC50 0.15 microM) in control segments which was not significantly different from the relaxant responses of segments pretreated with methohexitone (0.03-0.3 mM), phenobarbitone (0.1-0.3 mM) or thiopentone (0.1-0.3 mM). 3. Inhibition of NO synthesis with NG-nitro-L-arginine (0.1 mM) significantly reduced the maximum relaxant response to ACh from 96 to 40%. This NO/PGI2-independent relaxation appeared to be mediated by the release of EDHF, since it was strongly diminished in the presence of the K+Ca inhibitors, tetrabutylammonium (1-3 mM) and charybdotoxin (10 nM), following preconstriction with potassium calcium (40 mM) or removal of the endothelium. Thiopentone or methohexitone markedly attenuated the EDHF-mediated relaxant response to ACh, while phenobarbitone had no effect. The endothelium-independent relaxation elicited by sodium nitroprusside (0.01-10 microM), on the other hand, was only marginally affected by these anaesthetics. 4. The cytochrome P450 inhibitor, clotrimazole (3-100 microM), mimicked the inhibitory effect of thiopentone and methohexitone on the NO/PGI2-independent relaxant response to ACh.(ABSTRACT TRUNCATED AT 250 WORDS)     

Magnesium inhibits basal release of endothelium-derived relaxing factor in canine coronary arteries

Atrial natriuretic factor (ANF) had been found in brain tissues. Its role and the mechanisms by which it is produced and functions in the brain were not clear. We have initiated in vitro studies to find whether it is released from brain tissue and to elucidate the mechanism of its release. ANF was found to be released from rat hypothalamus by a depolarizing concentration of potassium and by a calcium-dependent mechanism. The ANF released was found to be predominantly a low molecular weight form. A small amount of high molecular weight form was also released. These results suggest that ANF produced in brain tissues is released, by a depolarization-induced and calcium-mediated mechanism, presumably from neuronal cells.     

N omega-nitro-L-arginine: a potent inhibitor of endothelium-derived relaxing factor formation

Endothelium-derived relaxing factor (EDRF) released from cultured endothelial cells was assayed by examining changes in cyclic GMP levels of rat lung fibroblasts. N omega-nitro-L-arginine and NG-monomethyl-L-arginine inhibited basal and A23187-, ATP- and melittin-induced EDRF release, and the inhibition was prevented with L-arginine. The IC50 values of N omega-nitro-L-arginine and NG-monomethyl-L-arginine for EDRF release evoked with 1 microM A23187 were 230 nM and 16 microM, respectively. N omega-nitro-L-arginine and NG-monomethyl-L-arginine did not affect cyclic GMP accumulation in the fibroblasts with atrial natriuretic factor or sodium nitroprusside. Thus, N omega-nitro-L-arginine is 70 times more potent than NG-monomethyl-L-arginine as a specific inhibitor of EDRF formation/release.     

Contamination of adenosine deaminase by superoxide dismutase. Stabilization of endothelium-derived relaxing factor.

We have measured cyclic GMP accumulation in co-cultures of bovine aortic endothelial cells and rat smooth muscle cells as an index of endothelium-derived relaxing factor (EDRF) production. Adenosine deaminase (EC 3.5.4.4, Sigma type VI) produced a 5- to 10-fold increase in the basal and bradykinin-stimulated cyclic GMP content of co-cultures but had no effect on smooth muscle cells alone. Cyclic GMP accumulation in response to adenosine deaminase was not blocked by adenosine deaminase inhibitors or affected by adenosine, the products of adenosine deamination (inosine and ammonia), or adenosine receptor antagonists. Since superoxide anion is known to destroy EDRF and nitric oxide (NO) (which is similar or identical to EDRF in composition), we tested for superoxide dismutase (SOD, EC 1.15.1.1) in single lots of eight commercial sources of adenosine deaminase by measuring inhibition of the superoxide-mediated reduction of cytochrome c. SOD activity was found in all sources of adenosine deaminase, but varied widely. One lot of Sigma type VI enzyme contained 0.08 units SOD/unit adenosine deaminase. The EC50 values of purified SOD (0.23 units/mL) and Sigma type VI adenosine deaminase (2.1 units/mL) needed to increase the cyclic GMP content of co-cultures differed by a similar factor, 0.11. Thus, the SOD activity in adenosine deaminase is sufficient to account for its effect on cyclic GMP accumulation. One lot of Boehringer Mannheim adenosine deaminase contained much less SOD contamination (0.006 units SOD/unit adenosine deaminase) and produced much less accumulation of cyclic GMP in co-cultures. Cyclic GMP accumulations in response to adenosine deaminase and SOD were both abolished by the NO synthetase inhibitor NG-monomethyl-L-arginine (0.1 mM), consistent with the idea that these enzymes act by stabilizing EDRF. Adenosine deaminase and the SOD activity contaminating it were found to have similar molecular masses of 33-34 kD as assessed by gel permeation chromatography. When run under reducing conditions to dissociate homodimeric SOD into monomers, a 16.6 kD peptide which co-migrates with purified cupro-zinc SOD was visible in silver-stained sodium dodecyl sulfate-polyacrylamide gels of the Sigma type VI but not the Boehringer Mannheim adenosine deaminase. We conclude that commercial sources of adenosine deaminase are variably contaminated by SOD. Since EDRF is synthesized by many tissues, the use of adenosine deaminase contaminated with SOD may produce numerous effects not attributable to the deamination of adenosine.     

Different patterns of release of endothelium-derived relaxing factor and prostacyclin.

1. Release of endothelium derived relaxing factor (EDRF) and prostacyclin (PGI2) from endothelial cells (EC) cultured from bovine aortae was measured by bioassay and radioimmunoassay, respectively, during infusions (10 min) of bradykinin (BK), adenosine diphosphate (ADP), arachidonic acid (AA), alkaline buffers and the free-bases (FB) of L-arginine or D-arginine. Release of EDRF from the luminally perfused rabbit aorta was also measured during infusions (10 min) of acetylcholine (ACh), substance P and ADP. 2. Bradykinin (10 or 30 nM) infused through the column of EC induced release of both EDRF and PGI2, neither of which was maintained for the duration of the infusion. 3. ADP (1.6 or 4 microM) infused through the column of EC induced release of a EDRF which was maintained for the duration of the infusion and a release of PGI2 which lasted for a much shorter period. 4. Arachidonic acid (30 or 90 microM) infused through the column of EC caused a sustained release of EDRF and PGI2, both of which outlasted the infusion of AA. 5. L-Arginine FB, D-arginine FB or alkaline buffer infused through the column of EC released EDRF, but only small amounts of PGI2. The release of EDRF outlasted the period of infusion and was due to an increase in the pH of the Krebs solution perfusing the EC. 6. Infusions of ACh (0.25-1 microM) or ADP (4-16 microM) caused a sustained release of EDRF from the luminally-perfused rabbit aorta, whereas infusion of substance P (3.3-10 microM) caused only a transient release of EDRF. 7. These results show that distinct patterns of EDRF release exist to different agonists in both cultured and in situ EC, and that EDRF and PGI2 do not necessarily follow the same time course of release. Furthermore, sustained release of EDRF does not require the constant infusion of the precursor, L-arginine, whereas sustained release of PGI2 only occurs when AA, the precursor of PGI2, is present in the extracellular medium.     

Effect of hydroxocobalamin on vasodilatations to nitrergic transmitter, nitric oxide and endothelium-derived relaxing factor in guinea-pig basilar artery

In endothelium-denuded guinea-pig isolated basilar artery preparations, hydroxocobalamin (30, 100 and 300 μM) concentration-dependently inhibited the vasodilator responses to exogenous nitric oxide (NO), whereas the vasodilator responses to nitrergic nerve stimulation were slightly reduced by high (100 and 300 μM) but not by the low (30 μM) concentration of hydroxocobalamin. Vasodilatation in response to sodium nitroprusside (10–100 nM) was totally abolished by 300 μM hydroxocobalamin. In endothelium-intact preparations, vasodilator responses to acetylcholine (0.3–3 μM) were significantly reduced or abolished by hydroxocobalamin (30–300 μM). The mean reduction by hydroxocobalamin of relaxations to acetylcholine was significantly greater than that of the equivalent response evoked by nitrergic nerve stimulation. The findings suggest that the nitrergic transmitter in the guinea-pig basilar artery may be quantitatively less susceptible than the endothelium-derived relaxing factor to the NO scavenger hydroxocobalamin.