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.     

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。     

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.     

Thimerosal blocks stimulated but not basal release of endothelium-derived relaxing factor (EDRF) in dog isolated coronary artery.

1. The effect of an acetly-coA lysolecithin acyltransferase inhibitor, thimerosal, on the release of endothelium-derived relaxing factor (EDRF) was examined in the greyhound isolated coronary artery. 2. Thimerosal (1-10 microM) relaxed fully, ring segments of coronary artery which were contracted with the thromboxane A2-mimetic, U46619 (30 nM). The response was endothelium-dependent, slow in both onset and time to reach maximum. The maximum relaxation to the highest concentration of thimerosal (10 microM) was maintained for 10-20 min before the tissue slowly regained active force (1-2 h) to the same or higher level as that prior to the addition of thimerosal. At this time the endothelium-dependent relaxation responses to acetylcholine (ACh), substance P (SP), bradykinin (BK) and the calcium ionophores, ionomycin and A23187 were abolished. The endothelium-dependent contractions to the nitric oxide synthase inhibitors, NG-nitro-L-arginine (L-NNA; 10-100 microM) and NG-monomethyl-L-arginine (L-NMMA: 10-100 microM), however, were unaffected. 3. Thimerosal (10 microM) did not affect the relaxation curve to sodium nitroprusside (SNP) nor the contraction curve to the thromboxane A2-mimetic, U46619. 4. Both the relaxation response to thimerosal and the selective block of the relaxation responses to stimulated EDRF release were unaffected by either indomethacin (10 microM) or superoxide dismutase (150 u ml-1). 5. L-NNA (100 microM) significantly blocked the relaxation curves to thimerosal and A23187 but not that to SNP.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of basal and agonist-stimulated release of endothelium-derived relaxing factor from different arteries

1. The release of endothelium-derived relaxing factor (EDRF) from rabbit aorta and pig coronary artery vessels in response to acetylcholine (ACh), substance P (SP) and the calcium ionophore A23187 has been studied by means of a bioassay cascade system. 2. A technique is described which allows the quantification of EDRF release rates from vessels of different sizes, perfused at different flow rates and with different donor-recipient transient times. 3. Rabbit aorta and pig coronary arteries, perfused at flow rates which equalize endothelial shear stress, released EDRF at a similar basal rate. 4. In response to ACh, rabbit aortic endothelium released EDRF at a significantly greater maximum rate than pig coronary artery endothelium. 5. In response to SP, both endothelium types released EDRF; SP was a significantly more potent agonist in pig coronary artery than in rabbit aorta, but maximum SP-induced EDRF release from rabbit aorta was twice that of pig coronary artery. 6. These data indicate that different endothelium types can release EDRF at widely different rates, according to the agonist used, and that the previously obtained lack of relaxant response to ACh in pig coronary artery was due to a lack of EDRF release rather than concomitant smooth muscle constriction.     

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.     

Release and vascular activity of endothelium-derived relaxing factor in atherosclerotic rabbit aorta.

A diet containing 0.3% cholesterol was given to male New Zealand rabbits for 16 weeks; this produced atherosclerotic lesions (fatty streaks) on 80% of the intimal surface of the thoracic aorta and on 45% of the intimal surface of the abdominal aorta. The endothelium-dependent relaxations induced by acetylcholine, substance P and ionophore A23187 were inhibited in the atherosclerotic aortas. Besides the endothelium-independent relaxations induced by nitroglycerine, the relaxations induced by atrial natriuretic peptide (ANF) were also significantly reduced in the more atherosclerotic thoracic aorta. In bioassay experiments it was found that acetylcholine and substance P caused a smaller release of endothelium-derived relaxing factor (EDRF) from atherosclerotic thoracic aortas than from control thoracic aortas: the EDRF released by the vasodilators evoked less relaxation in atherosclerotic detector abdominal aortas than in control detector abdominal aortas. Nitric oxide evoked significantly less transient relaxation in the atherosclerotic thoracic and abdominal aortas than in the respective control tissues. The data indicate that as experimental atherosclerosis in the rabbit progresses, both vascular activity and EDRF release become affected; this leads to a complete loss of endothelium-dependent relaxation in the more atherosclerotic blood vessels.     

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.     

Inhibition by L-glutamine of the release of endothelium-derived relaxing factor from cultured endothelial cells.

L-Glutamine (0.02-2 mM) but not D-glutamine (0.2 mM and 2 mM) inhibited the release of endothelium-derived relaxing factor (EDRF) from bovine aortic endothelial cells cultured in the presence or absence of L-arginine. Inhibition was maximal at a concentration of 200 microns, and was reversed by L-arginine (50 microns) but not D-arginine (100 microns). L-Glutamic acid (2 mM) or ammonium chloride (1 mM), putative products of the metabolism of L-glutamine in endothelial cells, had no effect on EDRF release. L-Glutamine (0.2 mM and 2 mM) but not D-glutamine (2 mM), L-glutamic acid (2 mM) or ammonium chloride (1 mM) also inhibited the generation of L-arginine in endothelial cells. Thus, L-glutamine inhibits EDRF release by preventing the generation of L-arginine.     

Inhibitory effect of unstimulated neutrophils on platelet aggregation by release of a factor similar to endothelium-derived relaxing factor (EDRF)

Previous studies have indicated a possible role for polymorphonuclear leukocytes (PMNLs) in the maintenance of hemostasis and vascular tone. We now demonstrate that unstimulated isolated PMNLs maintained at 37 degrees inhibited human platelet aggregation in a concentration- and time-dependent fashion. In addition, PMNLs increased platelet cyclic GMP concentrations. The platelet aggregation inhibitory effect of PMNLs was potentiated by superoxide dismutase and attenuated by hemoglobin and methylene blue. This inhibitory effect of PMNLs was not observed in 48-hr-old killed cells and was not modulated by aspirin treatment or by adenosine deaminase. These observations suggest that human PMNLs maintained at 37 degrees produce a substance with biological characteristics similar to those of the endothelium-derived relaxing factor.     

The basal and stimulated release of the endothelium-derived relaxing factor from isolated pig coronary arteries does not interfere with the vascular release of superoxide

Oxygen-derived free radicals, in particular superoxide anions, are known to inactivate the endogenous vasodilator endothelium-derived relaxing factor (EDRF) which is probably identical with the gaseous radical nitric oxide. It is possible that EDRF is not the target of superoxide anions but may also be an endogenous scavenger of this radical.Superoxide anions generated by the vessel wall were measured by a modified lucigenin-enhanced chemiluminescence technique in isolated pig coronary artery rings with intact endothelium.The addition of bovine superoxide dismutase, a scavenger of superoxide anions, decreased the chemiluminescence signal by 40 ± 26% (mean ± SD; P < 0.05; n = 21) indicating reduced generation/release of superoxide anions. In contrast, pretreatment of coronary artery rings with diethyldithiocarbamate, an inhibitor of the intrinsic copper-zinc superoxide dismutase, increased the chemiluminescence response by 136 ± 128°10 (P < 0.05; n = 21). This increase in the chemiluminescence response induced by diethyldithiocarbamate-pretreatment was almost abolished in the presence of added bovine superoxide dismutase. Specific inhibition of the EDRF release with nitro-l-arginine (100 M) did not affect the chemiluminescence response. On the other hand, stimulation of the EDRF release by substance P (10 nM) or addition of the endothelium-mediated relaxant bradykinin (0.1 M) did not affect the chemiluminescence response. Stimulation of the EDRF release with serotonin (0.1 M) significantly reduced the photon emission by 15 ± 16% (n = 27). However, this effect of serotonin on the chemiluminescence response could not be prevented by specific inhibition of the EDRF release with nitro-l-arginine (100 M) but could be prevented by buffering the acidic serotonin solution with NaOH to pH 7.4.Our results suggest that basal and agonist-stimulated release of EDRF in isolated pig coronary artery rings does not interfere with the basal generation/release of superoxide anions derived from the vascular wall. Correspondence to: A. Mugge at the above address     

Inhibition of the release of endothelium-derived relaxing factor in vitro and in vivo by dipeptides containing NG-nitro-L-arginine.

1. We have shown that dipeptides containing NG-nitro-L-arginine (NO2Arg) inhibit the biosynthesis of endothelium-derived relaxing factor (EDRF) in vitro and in vivo. 2. In anaesthetized rats, intravenous administration at 1-30 mg kg-1 of the methyl ester of NO2Arg, NO2-Arg-L-phenylalanine (NO2Arg-Phe), L-alanyl-NO2Arg (Ala-NO2Arg) or NO2Arg-L-arginine (NO2Arg-Arg) produced dose-related increases in mean arterial blood pressure (MABP) which were unaffected by D-arginine (D-Arg; 20 mg kg-1 min-1 for 15 min), but prevented by co-infusions of L-arginine (L-Arg; 20 mg kg-1 min-1 for 15 min) or by their parent dipeptides. 3. NO2Arg methyl ester, NO2Arg-Phe methyl ester or Ala-NO2Arg methyl ester (10 mg kg-1, i.v.) also inhibited the reduction in MABP caused by the endothelium-dependent vasodilator, acetylcholine (30 micrograms kg-1 min-1 for 3 min), but not those induced by glycerly trinitrate (20 micrograms kg-1 min-1 for 3 min) or iloprost (6 micrograms kg-1 min-1 for 3 min) which act directly on the vascular smooth muscle. 4. Moreover, NO2Arg methyl ester, NO2Arg-Phe methyl ester or NO2Arg-Arg methyl ester (100 microM) inhibited the acetylcholine-induced relaxation of rabbit aortic strips, and NO2Arg-Phe methyl ester (30 microM) blocked the stimulated (bradykinin, 30 pmol) release of EDRF from bovine aortic endothelial cells grown on microcarrier beads.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition by alpha-adrenoceptor agonists of renin release in vitro.

A variety of alpha-adrenergic agonists encompassing a broad range of concentrations were used to investigate the existence and nature of a putative alpha-adrenergic mechanism inhibitory to renin release, which may operate at the level of the juxtaglomerular apparatus. For this purpose rat renal cortical tissue incubated in vitro was used. Concentrations of noradrenaline, adrenaline and methoxamine of 10(-6), 10(-5), 10(-4) and 10(-3) M caused significant dose-related inhibition of renin release. The inhibition of release by these doses was reversed completely by 10(-4) M phentolamine. In contrast, phenylephrine, oxymetazoline and clonidine did not inhibit renin release. The results support the concept of an alpha-adrenergic mechanism inhibitory to renin release and show that high concentrations of alpha-adrenergic agonist are required for its operation in vitro. The manner in which this inhibitory mechanism affects renin release under physiological circumstances remains to be demonstrated.     

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.     

Vascular smooth muscle sensitivity to endothelium-derived relaxing factor is different in different arteries.

1. The relaxation responses of pre-constricted pig coronary artery (PCA) and rabbit aorta (RA) without endothelium, to endothelium-derived relaxing factor (EDRF) released from either a PCA or RA with intact endothelium have been studied by use of a bioassay cascade system. Effects of EDRF have been compared with sodium nitroprusside (NaNP) and 8-bromo-cyclic GMP. 2. The time course of changes in cyclic GMP levels in response to EDRF in PCA and RA have also been studied. 3. EDRF (released from a PCA or RA) caused significantly greater relaxation in the PCA than the RA, whether 5-hydroxytryptamine or high extracellular potassium was used as the constrictor agonist. 4. These differences in sensitivity to EDRF were paralleled by NaNP but not 8-bromo-cyclic GMP. 5. Cyclic GMP levels peaked earlier in the RA (30s) than in the PCA (180s) but the peak levels were significantly greater in the PCA (2.45 fold) than the RA (1.48 fold). 6. These data show that the previously described differences in EDRF activity between different artery types can be explained in part by differences in the responsiveness of the smooth muscle to EDRF.     

Inhibitory role of endothelium-derived relaxing factor in rat and human pulmonary arteries.

1. The inhibitory role of endothelium-derived relaxing factor was studied in both rat and human pulmonary arteries in vitro by inhibiting its synthesis with the L-arginine analogue NG-monomethyl-L-arginine (L-NMMA). 2. In rat pulmonary arteries, L-NMMA pretreatment (10-300 microM) dose-dependently inhibited acetylcholine-induced relaxation (which is endothelium-dependent). NG-monomethyl-D-arginine (D-NMMA, 100 microM) was without effect. L-Arginine, but not D-arginine, dose-dependently reversed this inhibition. L-NMMA had no effect on relaxation induced by sodium nitroprusside. 3. In human small pulmonary arteries L-NMMA (100 microM) pretreatment similarly inhibited the acetylcholine-induced relaxation but had no effect on the sodium nitroprusside-induced relaxation. 4. In both rat and human pulmonary arteries, L-NMMA, but not D-NMMA, always caused contraction of preconstricted tissues whereas it had no effect on baseline tone. In the rat this contraction was completely prevented by prior treatment with L-arginine. 5. L-NMMA (100 microM) pretreatment mimicked the effect of endothelium removal on phenylephrine-induced vasoconstriction, both resulting in an increase in tension development at each concentration of phenylephrine. This enhancement was greatest at low concentrations of phenylephrine but was still present even at the highest concentrations. Pretreatment with L-NMMA (100 microM) also significantly increased the responses to single doses of phenylephrine. 6. These results suggest that endothelium-derived relaxing factor from endothelial cells both mediates the relaxation response to acetylcholine and also acts as a physiological brake against vasoconstriction in pulmonary vessels.     

Differential selectivity of endothelium-derived relaxing factor and nitric oxide in smooth muscle.

The selectivity of endothelium-derived relaxing factor (EDRF) and nitric oxide (NO) on smooth muscle relaxation was examined and compared. EDRF released from was examined and compared. EDRF released from bovine pulmonary arterial endothelium (BPAE) in culture and NO were superfused over vascular, tracheal, gastrointestinal and uterine smooth muscle. EDRF relaxed vascular smooth muscle but not tracheal, gastrointestinal or uterine smooth muscle. NO relaxed vascular and gastrointestinal smooth muscle but not tracheal or uterine smooth muscle. There was a differential selectivity between the relaxant effect of EDRF and NO on smooth muscle.     

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.