Biosynthesis Of Endothelium-derived Relaxing Factor Research Articles

Increased vasodilator response to acetylcholine of renal blood vessels from diabetic rats.

The vasodilator effect of acetylcholine (ACh) and nitroprusside and the vasoconstrictor effect of noradrenaline was assessed in the perfused kidney of streptozocin diabetic rats. Compared with control animals injected with acidified saline, the renal vasoconstrictor effect of noradrenaline was increased in diabetic rats both in terms of the dose required to produce 50% of the maximal effect (EC50) and in the maximal response achieved. The renal vasodilator effect of ACh (but not nitroprusside) was similarly enhanced in diabetic animals. The effect of ACh (but not nitroprusside) in the perfused kidney of both control and diabetic rats was reduced or abolished by mepacrine (10 microM), metyrapone (10 microM) or methylene blue (100 microM) suggesting that ACh exhibits vasodilator activity in the rat kidney by virtue of releasing endothelium derived relaxing factor (EDRF). These results are in contrast to previous published reports demonstrating reduced biosynthesis of EDRF in the aorta of diabetic rats. The mechanism which underlies the increased renal vascular response to ACh is not known. However, increased endothelial cell turnover or cholinoceptor number, elevated activity of enzyme(s) which synthesis EDRF or hyperresponsiveness of vascular smooth muscle to released EDRF should all be considered.

Pharmacologic differentiation between endothelium-dependent relaxations sensitive and resistant to nitro-L-arginine in coronary arteries.

We investigated whether formation of endothelium-derived relaxing factor (EDRF) and endothelium-derived hyperpolarizing factor (EDHF) in porcine and bovine endothelial cells (PAECs) was stimulated by different kinin receptors and studied pharmacologic differences and similarities between the two types of bradykinin-induced relaxation of bovine or porcine coronary arteries. Cultured PAECs were used for [3H]bradykinin binding assay and for measurement of the endothelial free [Ca2+]i by the fura-2/AM method. In organ bath studies with strips of bovine and porcine coronary arteries (endothelium intact), changes in length were recorded and cyclic GMP was measured by radioimmunoassay (RIA). Two bradykinin binding sites were detected, suggesting the presence of two subtypes of B2 kinin receptors. Bradykinin increased [Ca2+]i, and this action was antagonized by the B2 kinin receptor antagonist Hoe 140 and the K channel inhibitor tetrabutylammonium (TBA). Hoe 140 competitively antagonized the relaxing effects of bradykinin, whereas a B1 antagonist was inactive. L-omega N-nitro-arginine (L-NNA) diminished one part of bradykinin-induced relaxation and abolished the increases in cyclic GMP; TBA inhibited another part of the relaxing effect and attenuated (but not significantly) increases in cyclic GMP, and Hoe 140 completely inhibited relaxation and increases in cyclic GMP. The results indicate that the bradykinin response is mediated by biosynthesis of EDRF, which is sensitive to L-NNA, and of EDHF, which is sensitive to TBA.

Sulfhydryl-depleting agents, but not deferoxamine, modulate EDRF action in cultured pulmonary arterial cells.

The potential role of intracellular sulfhydryls and iron on the biological activity of endothelium-derived relaxing factor (EDRF) released basally from bovine pulmonary arterial endothelial (BPAE) cells was investigated in a cultured cell bioassay system, by measuring N omega-nitro-L-arginine-sensitive guanosine 3',5'-cyclic monophosphate (cGMP) accumulation in rabbit pulmonary arterial smooth muscle (SM) cells. The role of sulfhydryls in the biosynthesis of EDRF was studied by selectively exposing the endothelial cells to thiol-depleting agents. Both N-ethylmaleimide (NEM) and maleic acid diethyl ester (DEM) inhibited EDRF-induced cGMP accumulation in a dose-dependent manner. To study the requirement of SM thiols in the metabolism of EDRF to a stimulator of cGMP formation, SM were selectively exposed to NEM and DEM before bioassay with control, untreated BPAE. DEM and NEM inhibited cGMP formation in response to EDRF by 30 and 68%, respectively. The requirement of SM sulfhydryls was further investigated in the stimulation of SM cGMP accumulation elicited by nitrosothiols [S-nitroso-L-cysteine, S-nitroso-mercaptoproprionic acid, and sodium nitroprusside (SNP)]. NEM pretreatment of SM cells abolished cGMP responses to all vasodilators; DEM did not affect the nitrosothiol responses but reduced by 30% the cGMP accumulation to SNP. The role of iron in the endothelial synthesis of EDRF was assessed by chelating endothelial low-molecular-weight iron compounds. Exposure of BPAE to deferoxamine mesylate had no effect on cGMP accumulation in SM, suggesting that deferoxamine-available iron is not necessary for the endothelial stimulation of SM cGMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Blockade of endothelium-derived relaxing factor synthesis with N G-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 N G-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 contribues substantially to the control of large capacitance veins in vivo and that L-NAME increases venous reactivity to both norepinephrine and glyceryltrinitrate.

Role of cGMP mechanisms in response of rat pulmonary arteries to hypoxia

We have demonstrated previously that in response to hypoxia, isolated rat pulmonary arteries show an initial endothelium-dependent relaxation followed by an endothelium-independent transient contraction. In the presence of increased extracellular Ca2+, both of these responses were enhanced in endothelium-intact arteries. Nitro-L-arginine, a blocker of the biosynthesis of endothelium-derived relaxing factor (EDRF), abolished the initial endothelium-dependent relaxation and Ca(2+)-induced enhancement of hypoxic contraction in endothelium-intact arteries but did not alter responses in endothelium-denuded vessels. Inhibition of prostaglandin formation with indomethacin had no effect on the hypoxia-elicited responses. Preincubation with LY 83583, an inhibitor of guanylate cyclase activation, abolished the initial hypoxia-elicited relaxation and subsequent contraction. M & B 22948, a guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase inhibitor, decreased tone under O2 but not under N2, causing an apparent enhancement of the contraction to hypoxia. Thus the modulation of hypoxic responses by the endothelium is dependent on changes in EDRF production, and a decrease in smooth muscle cGMP not involving an EDRF mechanism appears to mediate the endothelium-independent hypoxic contraction observed in the isolated rat pulmonary artery.

Functional hyperemia of skeletal muscle: role of endothelium.

The role of the endothelium and endothelium-derived vasoactive factors in the development of skeletal muscle functional hyperemia was studied. Experiments were performed in anesthetized dogs. Functional hyperemia of isometrically contracted gastrocnemius muscle was produced by direct electric stimulation. Five series of experiments were conducted to investigate the effect of (a) de-endothelialization of gastrocnemius vessels, (b) inhibition of prostanoids biosynthesis by indomethacin, (c) inhibition by gossipol of lipoxygenase, which mediates biosynthesis of endothelium-derived relaxing factor (EDRF), (d) inhibition of cGMP formation by methylene blue, and (e) stimulation of EDRF biosynthesis of L-arginine. The gastrocnemius muscle work was accompanied by an increase in blood flow by 6.8 +/- 0.6 ml/min/N (8 Hz) and by 2.5 +/- 0.2 ml/min/N) (40 Hz). Chemical deendothelialization of gastrocnemius vessels decreased functional hyperemia, and blood flow increased by only 1.2 +/- 0.3 ml/min/N (8 Hz) and by 0.5 +/- 0.1 ml/min/N (40 Hz). Cyclooxygenase inhibition by indomethacin had no significant effect on functional hyperemia, whereas methylene blue and gossipol inhibited functional hyperemia by 4.2- and 6.6-fold, respectively, and L-arginine significantly elevated blood flow by 12.6 +/- 1.4 ml/min/N (8 Hz) and by 3.5 +/- 0.2 ml/min/N (40 Hz) during skeletal muscle work. Thus, it can be concluded that the endothelium plays an important role in functional hyperemia of skeletal muscle, probably through the release of EDRF.

Role of intracellular thiols in release of EDRF from cultured endothelial cells.

The availability of intracellular reduced thiols, such as L-cysteine or glutathione (GSH), may be critically important for the biosynthesis of endothelium-derived relaxing factor (EDRF). We have, therefore, investigated the effects of various sulfhydryl (SH) reagents, such as 1-chloro-2,4-dinitrobenzene (CDNB), diamide, 2,2'-dithiodipyridine (DTDP), or N-ethyl-maleimide (NEM), on the release of EDRF from cultured endothelial cells. None of the SH reagents tested affected the flow-induced EDRF release, but DTDP and NEM inhibited the release of EDRF stimulated by ADP, ionomycin, or poly-L-lysine. In contrast, NG-nitro-L-arginine methyl ester, an inhibitor of EDRF biosynthesis, blocked both the flow- and agonist-induced release of EDRF. Although NEM substantially potentiated the flow-induced release of prostacyclin (PGI2), probably due to a blockade of the reacylation of arachidonic acid, it inhibited the stimulated release of PGI2, whereas diamide did not significantly affect either release. Like CDNB or diamide, NEM, but not DTDP, caused a significant decrease in endothelial GSH. In contrast, both NEM and DTDP, but not CDNB or diamide, inhibited the ADP-induced mobilization of intracellular calcium, suggesting that they act on specific target proteins involved in endothelial cell calcium homeostasis rather than intracellular free SH groups. Moreover, the selective inhibition by these two SH reagents of the stimulated release of EDRF implies that a fundamental regulatory difference exists between agonist- and flow-induced EDRF biosynthesis.

Nitroarginine-sensitive and -insensitive components of the endothelium-dependent relaxation in the guinea-pig carotid artery.

In the guinea-pig carotid arteries, nitroarginine elevated the resting tension (greater than 3 x 10(-6) M) and enhanced the noradrenaline (NA)- and high-potassium (high-K, 29.6 mM) induced contractions (greater than 10(-7) M), in a concentration-dependent manner, with no significant change in the resting membrane potential and depolarizations elicited by NA or high-K. ACh (10(-6) M) relaxed the muscles precontracted with NA or high-K by 96 or 46% of the contraction, respectively. In the presence of nitroarginine (10(-5) M) for 1-3 h, the ACh-induced relaxation was reduced to 40 or 0% of the NA- or high-K-contractions, respectively. In tissues contracted with NA and exposed to nitroarginine, the ACh-induced relaxation changed from a sustained to a transient form. ACh relaxed the muscles to a similar extent, at any given level of tension, as elevated by different concentrations of NA to 1-3 times the level produced by 10(-6) M NA, either in the presence or absence of nitroarginine. ACh (greater than 10(-8) M) produced a transient hyperpolarization of the membrane, in an endothelium-dependent manner, and the responses were blocked by atropine (10(-6) M) or high-K solution, but not by NA or nitroarginine. We propose that 1) endothelium-derived hyperpolarizing factor (EDHF) is produced by pathways independent of the biosynthesis of endothelium-derived relaxing factor (EDRF), 2) the sustained release of EDRF maintains the muscle tone at a low level, and 3) the endothelium-dependent relaxation is produced by both EDRF and EDHF, and they elicit sustained and transient relaxations, respectively.

Is the bradykinin-induced Ca 2+ influx and the formation of endothelium-derived relaxing factor mediated by a G protein?

In cultured porcine aortic endothelial cells bradykinin produced a long-lasting Ca 2+ influx. In contrast to the G protein-independent Ca 2+ entry evoked by ionomycin or digitonin, bradykinin-induced Ca 2+ influx was antagonized by Ni 2+ with an IC 50 value of about 50 μM. Since identical IC 50 values for Ni 2+ were found when Ca 2+ entry was induced by sodium fluoride or GTPγS, we suggest that stimulation of G protein(s) results in the activation of the same Ca 2+ channels as stimulation by bradykinin. This conclusion is supported by our findings that inhibition of GTPase by mepacrine amplified bradykinin-stimulated Ca 2+ influx, but did not interfere with the effect of the Ca 2+ ionophore A23187. Similar to its effect on Ca 2+ influx, mepacrine also potentiated endothelium-derived relaxing factor (EDRF) formation by bradykinin and sodium fluoride, but did not affect A23187-induced EDRF biosynthesis. We therefore suggest that in endothelial cells the bradykinin-induced Ca 2+ influx and the resulting formation of EDRF are regulated by a G protein.

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. 5. In endothelial cells grown in L-Arg-deficient medium, L-Arg-containing dipeptides such as L-Arg-LPhe, L-Ala-L-Arg or L-Arg-L-Arg increased both the basal and stimulated release of EDRF. Moreover, the L-Arg containing dipeptides, but not their NO2Arg analogues, were rapidly cleaved by these cells. 6. Thus, dipeptides containing NO2Arg can directly interfere with the biosynthesis of EDRF in vitro and in vivo. Moreover, the potentiation of EDRF release from endothelial cells deprived of L-Arg by dipeptides containing L-Arg suggests that such peptides may serve as an additional or alternative substrate for the biosynthesis of EDRF.

The Biosynthesis of Endothelium-Derived Relaxing Factor by Endothelial Cells as a Means of Removing Excess Nitrogen

Summary: The amino acid l-arginine (l-Arg) is the substrate for the biosynthesis of nitric oxide (NO) by a variety of cells, including endothelial cells, cytotoxic macrophages, and brain synaptosomes. Although the enzymatic conversion of l-Arg to NO and its cellular regulation is at present the focus of intensive investigations, little is known about the concentration, synthesis, and metabolism of l-Arg by cells capable of producing NO. We have therefore studied the relationship between the metabolism of l-Arg and the biosynthesis of endothelium-derived relaxing factor (EDRF), i.e., NO, in bovine aortic cultured endothelial cells (ECs). The intracellular concentration of l-Arg in cultured ECs was 0.1-0.2 mM, compared with 2-4 mM in freshly isolated ECs from bovine or rabbit aorta. When placed into physiologic buffer solutions, cultured ECs generated l-Arg, and this process was linked to an increase in the release of EDRF. Studies with l-[14C]Arg-labeled ECs showed that the generation of l-Arg was inversely related to a dilution of the l-Arg pool with nonlabeled l-Arg derived from an intracellular source. ECs deficient in l-Arg metabolized l-citrulline (l-Cit) and l-argininosuccinate to l-Arg. Dipeptides containing l-Arg or l-Cit were rapidly cleaved to yield l-Arg orl-Cit, respectively. When superfused with Krebs' solution, ECs maintained their l-Arg level despite the continuous flow-induced release of EDRF. When incubated in Krebs' solution in the presence of the calcium ionophore A 23187, there was only a transient rise in l-Cit, the by-product of EDRF biosynthesis, suggesting a recyclingof l-Cit to l-Arg. The generation of l-Arg was also inversely related to a rapid decrease in intracellular l-glutamine (l-Gln), and both the generation of l-Arg and the release of EDRF were inhibited by l-Gln, a major constituent of the culture medium employed. Cultured ECs did not produce l-ornithine (l-Orn) or urea and d id not metabolize l-Orn to l-Cit. However, they contained and released substantial amounts of ammonia, which was attributable in part to the metabolism of l-Gln. This, instead of a complete urea cycle, cultured ECs possess an l-Arg-l-Cit cycle, which is sensitive to l-Gln and linked to both the biosynthesis of EDRF and the removal of excess nitrogen. It is also possible to imply that the metabolism of l-Arg and hence its availability is an important regulatory factor for the biosynthesis of EDRF.

Multigram Synthesis of NG-Methyl-(L)-Arginine and Its Analytical Characterization

Abstract A multigram preparation and analytical characterization of NG-methyl-(L)-arginine (L-NMMA) 1, the most important inhibitor of the biosynthesis of Endothelium-Derived Relaxing Factor (EDRF), is reported.

Stimulation of Soluble Guanylate Cyclase by Endothelium-Derived Relaxing Factor Is Antagonized by Oxidized Low-Density Lipoprotein

Summary: Atherosclerosis is associated with a diminished dilatory response of blood vessels to endothelium-derived relaxing factor (EDRF). To clarify whether this impaired response may be due to a reduced biosynthesis of EDRF or a diminished responsiveness of the smooth muscle guanylate cyclase to EDRF, we investigated the effects of oxidized low-density lipoprotein (LDLox), a lipoprotein with atherogenic properties, on the activation of purified soluble guanylate cyclase by EDRF, as well as on the activity of the partially purified EDRF-forming enzyme. In the presence of LDLox, the effects of EDRF and other nitrocompounds on soluble guanylate cyclase were dose-dependently diminished, whereas no effect of LDLox on basal guanylate cyclase activity was observed. The inhibitory effect could be potentiated by preincubation of the enzyme with LDLox, indicating a direct interaction between the lipoprotein and guanylate cyclase. In contrast to its inhibitory effect on guanylate cyclase, LDLox did not affect the activity of the EDRF-forming enzyme. These data indicate that the impaired response of atherosclerotic blood vessels may be due to a diminished responsiveness of the smooth muscle guanylate cyclase rather than a reduced biosynthesis of EDRF.

Activation of G Protein Evokes Ca2+ Influx in Endothelial Cells Without Correlation to Inositol Phosphates

It is well etablished that the formation of endothelium-derived relaxing factor (EDRF) depends on an increase in intracellular free Ca 2+ concentration ([Ca 2+ ] i ) in vascular endothelial cells. The aim of our study was to investigate whether the origin of this increase in intracellular free calcium plays any role in this important physiological pathway. Therefore, we investigated the effects of bradykinin on [Ca 2] i in the presence and in the absence of external Ca 2+ and corraled these effects with bradykinin-induced EDRF biosynthesis also in the presence and the absence of external Ca 2+ .

Activation of G Protein Evokes Ca2+ Influx in Endothelial Cells Without Correlation to Inositol Phosphates

Summary: It is well established that the formation of endothelium-derived relaxing factor (EDRF) depends on an increase in intracellular free Ca2+ concentration ([Ca2+]i) in vascular endothelial cells. The aim of our study was to investigate whether the origin of this increase in intracellular free Ca2+ plays any role in this important physiological pathway. Therefore, we investigated the effects of bradykinin on [Ca2+]i in the presence and in the absence of external Ca2+ and correlated these effects with bradykinin-induced EDRF biosynthesis also in the presence and the absence of external Ca2+. Thereby, we found that the release of intracellularly stored Ca2+, on the one hand, and influx of external Ca2+, on the other hand, apparently differ in their effects on EDRF formation. Release of intracellularly stored Ca2+ by inosito-1,4,5-trisphosphate (IP3) results in a rapid increase in [Ca2+]i, and this seems to be necessary for a quick response for agonist-induced EDRF formation. However, Ca2+ influx, which is a sustained effect, seems to be important for a basal and long-lasting EDRF formation, which is involved in the regulation of vascular tone. Also, the mechanism of Ca2+ influx regulation in endothelial cells was investigated. Sodium fluoride, a known activator of G proteins, was used to investigate whether a G protein is involved in the mechanism of Ca2+ influx regulation. In our experiments, sodium fluoride increased [Ca2+]i and also induced an endothelium-dependent relaxation. Furthermore, the stimulation of IP3 formation induced by sodium fluoride was measured. The results indicate that neither a correlation between Ca2+ influx and formation of IP3 nor a depletion of intracellular Ca2+ stores appears to be involved in the mechanism of Ca2+ influx regulation. These findings may suggest the existence of a direct regulation of Ca2+ channels by a G protein.

The metabolism of L-arginine and its significance for the biosynthesis of endothelium-derived relaxing factor: cultured endothelial cells recycle L-citrulline to L-arginine.

We have investigated the mechanism by which cultured endothelial cells generate L-arginine (L-Arg), the substrate for the biosynthesis of endothelium-derived relaxing factor. When Arg-depleted endothelial cells were incubated in Krebs' solution for 60 min, L-Arg levels were significantly (9.7-fold) elevated. The generation of L-Arg coincided with a substantial decrease (90%) in intracellular L-glutamine (L-Gln), whereas all other amino acids were virtually unaffected. Changes in calcium, pH, or oxygen tension had no effect on L-Arg generation, which was, however, prevented when the cells were incubated in culture medium containing L-Gln. L-Arg generated by endothelial cells labeled with L-[14C]Arg was derived from an unlabeled intracellular source, for the specific activity of the intracellular L-Arg pool decreased substantially (8.8-fold) over 60 min. Arg-depleted endothelial cells did not form urea or metabolize L-ornithine but converted L-citrulline (L-Cit) to L-Arg possibly via formation of L-argininosuccinic acid. Nondepleted cells stimulated with the calcium ionophore A23187 showed only a transient accumulation of L-Cit, indicating that L-Cit is recycled to L-Arg during the biosynthesis of endothelium-derived relaxing factor. The generation of L-Arg by Arg-depleted endothelial cells was partially (45%) blocked by protease inhibitors, and various Arg-containing dipeptides were rapidly cleaved to yield L-Arg. Thus, cultured endothelial cells recycle L-Cit to L-Arg and possibly liberate peptidyl L-Arg. The Arg-Cit cycle appears to be the equivalent in the endothelial cell to the formation of urea by the liver. The biosynthesis of endothelium-derived relaxing factor may, therefore, not only produce a powerful vasodilator but also relieve the endothelial cell of excess nitrogen.

The metabolism of L-arginine and its significance for the biosynthesis of endothelium-derived relaxing factor: L-glutamine inhibits the generation of L-arginine by cultured endothelial cells.

The mechanism by which L-glutamine (L-Gln) inhibits the release of endothelium-derived relaxing factor from bovine aortic cultured endothelial cells was investigated. The intracellular concentration of L-arginine (L-Arg) in Arg-depleted endothelial cells was inversely related to the level of L-Gln. Removal of L-Gln from the culture medium (usually containing L-Gln at 2 mM) abolished the inhibitory effect of the culture medium on L-Arg generation. L-Gln (0.2 and 2 mM) but not D-Gln inhibited the generation of L-Arg by both Arg-depleted and nondepleted endothelial cells. L-Gln did not interfere with the uptake of L-Arg or the metabolism of L-Arg-L-Phe to L-Arg but inhibited the formation of L-Arg from L-citrulline (L-Cit), L-Cit-L-Phe, and NG-monomethyl-L-arginine. L-Gln also inhibited the conversion of L-[14C]Cit to L-[14C]Arg by Arg-depleted endothelial cells. However, L-Gln did not inhibit the conversion of L-argininosuccinic acid to L-Arg by endothelial cell homogenates. Thus, L-Gln interferes with the conversion of L-Cit to L-Arg probably by acting on argininosuccinate synthetase rather than argininosuccinate lyase. L-Gln also inhibited the generation of L-Arg by the monocyte-macrophage cell line J774 but had no effect on the conversion of L-Cit to L-Arg by these cells. As the release of endothelium-derived relaxing factor from cultured and non-cultured endothelial cells is limited by the availability of L-Arg, endogenous L-Gln may play a regulatory role in the biosynthesis of endothelium-derived relaxing factor.

L-Glutamine inhibits the release of endothelium-derived relaxing factor from the rabbit aorta

The present study investigates the effect of the amino acid L-glutamine (L-Gln) on the release of endothelium-derived relaxing factor (EDRF) from the luminally perfused rabbit aorta and on endothelium-dependent relaxations of rabbit aortic strips. L-Gln (200 microM) had no effect on the acetylcholine (Ach)-induced release of EDRF from freshly prepared aortic tissues. The concentration of L-arginine (L-Arg) in endothelial cells isolated from these aortae was approximately 4 mM, as determined by HPLC analysis. After an initial equilibration period of 2 h and two consecutive infusions of Ach (55 microM for 15 min) at 2 and 3 h, L-Arg levels fell by 62 +/- 14% (n = 4). Under these conditions, L-Gln (200 microM) but not D-Gln (200 microM) inhibited the release of EDRF by 50 +/- 4% (n = 7). This effect of L-Gln was partially reversed by infusions of L-Arg (500 microM) but not D-Arg (500 microM). L-Gln (200 microM) but not D-Gln (200 microM) potentiated the inhibitory effect of N omega-nitro-L-arginine (30 microM), an inhibitor of EDRF biosynthesis, on Ach-induced relaxations of rabbit aortic strips, whereas L-Gln alone had no effect. Thus, L-Gln inhibits the release of EDRF from intact blood vessels presumably by interfering with the generation of L-Arg by the endothelium.

Biosynthesis of endothelium-derived relaxing factor: A cytosolic enzyme in porcine aortic endothelial cells Ca 2+-dependently converts L-arginine into an activator of soluble guanylyl cyclase

In the presence of porcine aortic endothelial cytosol, soluble guanylyl cyclase purified from bovine lung was activated by L-arginine up to 2.5-fold, with an EC 50 of about 6 μM. This activation was dependent on NADPH and Ca 2+. The EC 50 for Ca 2+ was about 60 nM. No effect of L-arginine on guanylyl cyclase was observed when the cytosolic proteins were heat-denaturated. The effect of L-arginine was inhibited by N G-monomethyl-L-arginine and hemoglobin. These results indicate that endothelial cells contain a cytosolic enzyme which is directly or indirectly regulated by Ca 2+ and converts L-arginine into a compound which in stimulating soluble guanylyl cyclase behaves similar to endothelium-derived relaxing factor.

Endothelium‐derived relaxing factor and the effects of acetylcholine and histamine on resistance blood vessels

1. The role of endothelium-derived relaxing factor (EDRF) in the action of vasodilator (acetylcholine, histamine, nitroprusside) and vasoconstrictor (noradrenaline, vasopressin) drugs on vascular resistance in the isolated perfused kidney and mesentery of the rat was studied. 2. Acetylcholine (EC50 = 0.18 +/- 0.05 nmol and 3.1 +/- 0.06 nmol, n = 8) and histamine (EC50 = 31.2 +/- 4.9 nmol and 46.2 +/- 3.9 nmol, n = 8) produced dose-related vasodilatation in noradrenaline-preconstricted (i.e. 'high tone') rat renal and mesenteric blood vessels. The response to both vasodilators (but not nitroprusside) was abolished by infusion of CHAPS (4.7 mg ml-1, 30 s). By use of an immunocytochemical staining procedure CHAPS was demonstrated to remove vascular endothelial cells lining intrarenal blood vessels. 3. Gossypol (3 microM), metyrapone (10 microM) and nordihydroguaiaretic acid, (NDGA, 30 microM), presumed inhibitors of EDRF biosynthesis, reduced or abolished the response to acetylcholine and histamine in perfused kidney and mesentery of the rat without affecting vasodilatation due to nitroprusside. Mepacrine (10 microM) similarly abolished the response to acetylcholine and histamine but in addition, reduced the response to nitroprusside in both preparations. 4. Methylene blue (100 microM), a presumed antagonist of the effect of EDRF, abolished vasodilatation due to acetylcholine and histamine and reduced the response to nitroprusside in perfused rat kidney and mesentery. Superoxide dismutase, SOD (15 u ml-1), was without effect. 5. While CHAPS treatment significantly augmented the vasoconstrictor response to both noradrenaline and vasopressin in perfused renal and mesenteric vessels this effect was not mimicked by metyrapone or gossypol suggesting that the enhanced effect of vasopressor agents in CHAPSperfused rat organs is due to the removal of a permeability barrier rather than impaired EDRF formation. 6. Responses to vasoconstrictor and vasodilator drugs in the perfused kidney and mesentery were obtained in the presence of indomethacin (8 microM) which produced in excess of 90% inhibition of prostacyclin (PGI2) release as measured by radioimmunoassay of 6-oxo-prostaglandin F1 alpha,. (6-oxo- PGF1 alpha) in the Krebs effluent. 7. We provide evidence that EDRF mediates the vasodilator response to acetylcholine and histamine in resistance blood vessels in perfused rat kidney and mesentery. The possibility that EDRF has a physiological role to play in regulating the calibre of resistance blood vessels is discussed.