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.     

Blockade of endothelium-derived relaxing factor synthesis with NG-nitro-L-arginine methyl ester leads to enhanced venous reactivity in vivo.

This study was performed to examine whether endothelium-derived relaxing factor (EDRF) influences venous tone and reactivity in vivo. The inferior vena cava and abdominal aorta were studied simultaneously under continuous haemodynamic monitoring in anaesthetised rabbits. In addition, a 20-MHz intravascular ultrasound catheter was placed in the vena cava for on-line two-dimensional imaging of vessel cross-sectional area and calculation of wall stress (T(ension) = P(mean) * r(adius)/2). This approach enabled simultaneous visualisation of both venous (CA(ven)) and aortic (CA(art)) cross-sectional area with continuous recording of vessel dimensions. Measurements were made before and after administration of NG-nitro-l-arginine methyl ester (L-NAME; 10 mg.kg i.v.), a specific inhibitor of EDRF biosynthesis. After L-NAME there was a significant increase in central venous pressure and a decrease in CA(ven). On the arterial side, L-NAME caused a significant increase in mean pressure and CA(art), resulting in a significantly augmented arterial wall stress. The venodilatation elicited by increasing doses of glyceryltrinitrate was markedly enhanced after L-NAME. Norepinephrine caused a parallel shift of the dose-response curve for CA(ven) in the presence of a lower baseline value. These results suggest that EDRF contributes substantially to the control of large capacitance veins in vivo and that L-NAME increases venous reactivity to both norepinephrine and glyceryltrinitrate.     

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.     

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.     

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.     

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.     

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.     

Cholera toxin augments the release of endothelium-derived relaxing factor evoked by bradykinin and the calcium ionophore A23187.

1. Experiments were designed to examine the effect of cholera toxin and forskolin on the release of relaxing factor(s) from superfused cultured endothelial cells under basal conditions and upon stimulation with bradykinin, adenosine diphosphate or the calcium ionophore A23187. 2. Exposure of cultured porcine aortic endothelial cells to cholera toxin (30 micrograms/ml, for 3 hr) and forskolin (10(-6) M, for 45 min) significantly increased the intracellular content in cyclic AMP. Cholera toxin but not forskolin stimulated the accumulation of cyclic GMP. 3. Exposure to cholera toxin did not modify the basal release of endothelium-derived relaxing factor nor that induced by adenosine diphosphate, but significantly increased that evoked by bradykinin and the calcium ionophore A23187. Forskolin did not significantly affect the basal or the stimulated release of endothelium-derived relaxing factor. 4. These results suggest that cholera toxin potentiates the release of endothelium-derived relaxing factor (presumably nitric oxide) from endothelial cells by a mechanism other than augmented production of cyclic AMP.     

Potassium-free solution prevents the action but not the release of endothelium-derived relaxing factor

Exposure to a K+-free solution reversibly inhibited the acetylcholine-induced relaxation in perfused canine femoral artery segments with endothelium and in superfused bioassay femoral artery rings without endothelium. Infusion of 6.7 mM K+ into the K+-free perfusate downstream of the perfused artery restored the acetylcholine-induced relaxation in the bioassay ring, whereas no relaxation was observed in the perfused segment still exposed to the K+-free solution. These data demonstrate that the K+-free solution depresses the endothelium-dependent relaxation to acetylcholine in femoral arteries by preventing the action, but not the production, of endothelium-derived relaxing factor.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Pranidipine enhances relaxation produced by endothelium-derived relaxing factor in carotid artery

The effects of pranidipine, a novel dihydropyridine-type Ca(2+)-channel antagonist, on acetylcholine-induced endothelium-dependent relaxation were investigated in isolated carotid artery of the guinea-pig. In arteries contracted with high-K(+) solution ([K(+)](0)=28.8 mM) containing noradrenaline, the relaxation was inhibited by N(omega)-nitro-L-arginine, indicating an involvement of endothelium-derived relaxing factor. Pranidipine (10(-9)-10(-7) M) augmented the relaxation in a concentration-dependent manner. Sodium nitroprusside produced a relaxation in arteries contracted with high-K(+) solution containing noradrenaline, in an endothelium-independent manner, and the relaxation was enhanced by pranidipine. 1H-[1,2,4] oxadiazolo [4, 3-a] quinoxalin-l-one (ODQ), an inhibitor of nitric oxide-sensitive guanylate cyclase, attenuated the relaxation produced by acetylcholine or sodium nitroprusside. In the presence of ODQ, pranidipine did not enhance the acetylcholine-induced relaxation. The relaxation produced by endothelium-derived hyperpolarizing factor was inhibited by pranidipine, with no alteration of the hyperpolarization. Thus, pranidipine augments the nitric oxide-induced relaxation, possibly by enhancing the mechanisms related to cyclic GMP.     

Simultaneous release of endothelium-derived relaxing and contracting factors induced by noradrenaline in normotensive rats

1. Noradrenaline produced a biphasic response in indomethacin (10(-5) M)-treated aorta isolated from normotensive rats. 2. The relaxation phase is enhanced by previous treatment of the animals with estrogen. 3. Blockade of the relaxant effect elicited by supramaximal concentration of noradrenaline was observed when 10(-5) M of methylene blue was present in the bathing fluid for 15 min. 4. Noradrenaline caused a simultaneous release of endothelium-derived relaxing and contracting factors in aorta from normotensive rats.