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.     

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.     

A comparison of endothelium-derived relaxing factor activity in the coronary and renal arteries of the pig.

A cascade bioassay system has been used to quantify the basal and receptor-mediated endothelium-derived relaxing factor (EDRF) activity of pig coronary artery and pig renal artery. When exposed to EDRF released from a common donor vessel, pig coronary artery smooth muscle showed a greater sensitivity to EDRF than pig renal artery, and these differences were paralleled by differential responses to sodium nitroprusside. When mounted as donors in the bioassay, and EDRF detected using a common recipient, pig coronary artery and renal artery endothelium showed similar basal EDRF release rates. EDRF release in response to substance P was greater from pig coronary artery donors, but EDRF release in response to bradykinin was greater from pig renal artery donors. The data indicate that differences in EDRF response and EDRF release occur in different vessels, and that certain EDRF-releasing agents may exert regional vasodilator effects.     

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.     

Human red blood cells inhibit endothelium-derived relaxing factor (EDRF) activity

We have used a bioassay-cascade system to investigate the inhibitory effects of human red blood cells on EDRF activity. The vascular smooth muscle relaxant effect of EDRF released from an endothelium-intact pig coronary artery by the calcium ionophore A23187 was inhibited by washed red cells. This inhibition of EDRF activity by red cells was similar to that expected from their haemoglobin content. This study provides further evidence that in vivo EDRF acts as a local autocoid.     

5-Hydroxytryptamine does not release endothelium-derived relaxing factor in rat isolated coronary arteries

Removal of the endothelium from isolated rat proximal and distal coronary artery segments shifted the 5-HT concentration-response curve to the left without affecting, the maximal contractile response. 5-HT had no relaxing effect in 10(-5) M prostaglandin F2 alpha-precontracted vessels with an intact endothelium in the presence of 10(-5) M ketanserin. The spontaneous myogenic tone increased in both proximal and distal coronary artery segments after the endothelium had been removed. Indomethacin (10(-5) M) reduced the response of the proximal coronary artery segments to 5-HT by 35% but indomethacin had no effect on the 5-HT concentration-response curve of the distal coronary artery segments. Indomethacin relaxed precontracted (40 mM potassium) proximal coronary artery segments independently of the presence of the endothelium, suggesting a non-specific relaxing effect of indomethacin in these arteries. It is concluded that rat coronary artery endothelium is unresponsive to 5-HT because it lacks 5-HT1 receptors. The increased 5-HT sensitivity and spontaneous myogenic tone of endothelium-denuded rat coronary arteries is probably due to the elimination of the relaxing stimulus mediated by spontaneously released endothelium-derived relaxing factor.     

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.     

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.     

Interactions between endothelium-derived relaxing factors in the rat hepatic artery: focus on regulation of EDHF

1. In rat isolated hepatic arteries contracted with phenylephrine, acetylcholine and the calcium ionophore A23187 each elicit endothelium-dependent relaxations, which involve both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). However, the contribution of prostanoids to these responses, and the potential interaction between EDHF and other endothelium-derived relaxing factors have not been examined.
2. In the presence of the NO synthase inhibitor N^G-nitro-L-arginine (L-NOARG, 0.3 mM) and a mixture of charybdotoxin (0.3 μM) and apamin (0.3 μM), inhibitors of the target potassium (K) channel(s) for EDHF, acetylcholine and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 each induced a concentration-dependent and almost complete relaxation, which was abolished in the additional presence of indomethacin (10 μM). Thus, in addition to EDHF and NO, a relaxing factor(s) generated by cyclo-oxygenase (COX) contributes to endothelium-dependent relaxation in the rat hepatic artery.
3. The resting membrane potentials of endothelium-intact and endothelium-denuded vascular segments were −57 mV and −52 mV, respectively (P>0.05). In intact arteries, the resting membrane potential was not affected by L-NOARG plus indomethacin, but reduced to −47 mV in the presence of charybdotoxin plus apamin. Acetylcholine and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (10 μM each) elicited a hyperpolarization of 13 mV and 15 mV, respectively. The hyperpolarization induced by these agents was not affected by L-NOARG plus indomethacin (12 mV and 14 mV, respectively), but reduced in the presence of charybdotoxin plus apamin (7 mV and 10 mV, respectively), and abolished in the combined presence of charybdotoxin, apamin and indomethacin.
4. The NO donor 3-morpholino-sydnonimine (SIN-1) induced a concentration-dependent relaxation, which was unaffected by charybdotoxin plus apamin, but abolished by the selective soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 10 μM). SIN-1 (10 μM) did not alter the resting membrane potential in endothelium-denuded vascular segments.
5. The COX-dependent relaxation induced by acetylcholine was abolished following exposure to 30 mM KCl, but unaffected by glibenclamide (10 μM). The prostacyclin analogue iloprost induced a concentration-dependent relaxation, which was also abolished in 30 mM KCl and unaffected by the combined treatment with glibenclamide, charybdotoxin and apamin. Iloprost (10 μM) induced a glibenclamide-resistant hyperpolarization (8 mV with and 9 mV without glibenclamide) in endothelium-denuded vascular segments.
6. Exposure to SIN-1 or iloprost did not affect the EDHF-mediated relaxation induced by acetylcholine (i.e. in the presence of L-NOARG and indomethacin). Replacement of L-NOARG with the NO scavenger oxyhaemoglobin (10 μM) or the soluble guanylate cyclase inhibitor ODQ (10 μM) or methylene blue (10 μM), which all significantly inhibited responses to endothelium-derived NO, did not affect the acetylcholine-induced relaxation in the presence of indomethacin, indicating that endogenous NO also does not suppress EDHF-mediated responses.
7. These results show that, in addition to EDHF and NO, an endothelium-derived hyperpolarizing factor(s) generated by COX contributes significantly to endothelium-dependent relaxation in the rat heptic artery. Neither this factor nor NO seems to regulate EDHF-mediated responses. Thus, EDHF does not serve simply as a `back-up'' system for NO and prostacyclin in this artery. However, whether EDHF modulates the NO and COX pathways remains to be determined.

     

Inhibition of collagen- and ADP-induced platelet aggregation by substance P in vivo: involvement of endothelium-derived relaxing factor.

We examined the effect of substance P, a potent stimulator of endothelium-derived relaxing factor (EDRF) release, on responses to collagen and adenosine 3',5'-diphosphate (ADP) in an in vivo model of platelet aggregation. Substance P inhibited platelet aggregation induced in vivo by both collagen and ADP. This anti-platelet effect was particularly pronounced against collagen-induced aggregation and was prevented by prior administration of haemoglobin (Hb), a known inhibitor of EDRF-mediated responses. Collagen-induced platelet aggregation in vitro was unaffected by a concentration of substance P equivalent to that achieved in plasma following in vivo administration. This study provides a clear demonstration of the anti-platelet activity of EDRF in vivo and an indication that its effectiveness may depend on the aggregating agent used.     

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.     

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.     

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.     

Effects of bradykinin in the rat isolated perfused heart: role of kinin receptors and endothelium-derived relaxing factor.

1. The effects of bradykinin (BK) in the microcirculation of the isolated perfused heart of the rat were examined. The kinin receptors mediating the effects of BK were characterized and the role of endothelium-derived relaxation factor (EDRF) and prostacyclin investigated. 2. The dose-related vasodilator responses elicited by bolus doses of BK (0.001-10.0 nmol) were competitively blocked by the selective kinin B2 receptor antagonist [D-Arg0,Hyp3, Thi5.8,D-Phe7]-bradykinin (pA2 = 6.8). Des-Arg9-bradykinin, a selective kinin B1 receptor agonist had no vasodilator activity at doses of up to 10 nmol. 3. L-NG-nitro arginine (100 microM; L-NOArg), an inhibitor of endothelium-dependent vasodilatation, reduced the duration but not the magnitude of the BK vasodilator response. This action of L-NOArg was not reversed by L-arginine (100 microM). 4. Superoxide dismutase (10 units ml-1), haemoglobin (10 microM) and methylene blue (MB; 1 microM), all known to modify EDRF-mediated responses, failed to alter the vasodilator action of BK. 5. Gossypol (1-15 microM), a presumed inhibitor of EDRF biosynthesis, caused a marked drop in perfusion pressure followed by vasoconstriction. These changes in coronary tone were accompanied by an irreversible depression of cardiac contractility and heart rate. Over the same concentration range gossypol abolished the vasodilator action of BK (1.0 nmol), however it also blocked the endothelium-independent vasodilator response to sodium nitroprusside (30 nmol) and the vasoconstrictor effect of endothelin-1 (10 pmol) which suggests non-specific toxic actions of gossypol.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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.     

Role of endothelium-derived relaxing factors in adrenomedullin-induced vasodilation in the rat kidney

The present study aimed to evaluate the contributions of endothelium-derived hyperpolarizing factor (EDHF), the nitric oxide (NO)-cGMP pathway, and prostaglandins to adrenomedullin-induced vasodilation in isolated rat kidney. Inhibition of the NO-cGMP pathway with N(omega)-nitro-L-arginine methyl ester (L-NAME) or 1H-[1,2,4]oxadiazolo-[4,3a]quinoxalin-1-one (ODQ) reduced the maximal vasodilator response to adrenomedullin by approximately 50%. Pretreatment of the vessels with the potassium channel inhibitor, tetraethylammonium or increased extracellular K(+), also decreased the maximal response to adrenomedullin by approximately 50%. The simultaneous administration of blockers of both endothelium-derived relaxing factors had a combined effect that almost suppressed adrenomedullin-induced vasodilation. The administration of indomethacin did not modify the renal response to adrenomedullin. Our results suggest that the vasodilator response to adrenomedullin in the isolated perfused kidney of rats is mediated by EDHF and NO to a similar extent. Our data also provide evidence that prostaglandins play no role in the vasodilator response to adrenomedullin in the renal vasculature.     

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.