Endothelium-derived Relaxing Factor Activity Research Articles

Similarity between the vasorelaxing activity of dinitrosyl iron cysteine complexes and endothelium-derived relaxing factor

Dinitrosyl iron complexes with cysteine (DNIC) induced a concentration-dependent relaxation of pre-contracted (norepinephrine, 10 −7 M) de-endothelialized ring segments of rat aorta. The vasodilator response was more similar to acetycholine (ACh)-induced relaxation in intact aortic rings than to nitric oxide (NO)-induced relaxation. The time course of tone recovery after maximal concentrations (10 −5 M) of DNIC was similar to the time course of tone recovery after endothelium-dependent relaxation induced by ACh, whereas the restoration of tone after NO was much faster. Vessel tone was restored by oxyhemoglobin (10 −5 M) in all cases. The results suggest that DNIC with cysteine may function as endothelium-derived relaxing factor in the vessels.

Endothelium-derived nitric oxide and cyclooxygenase products modulate corpus cavernosum smooth muscle tone.

Relaxation of penile corpus cavernosum smooth muscle is controlled by nerve and endothelium derived substances. In this study, endothelium-dependent relaxation of corporal smooth muscle was characterized and the role of arachidonic acid products of cyclooxygenase in endothelium-dependent relaxation was examined. Endothelium removal from rabbit corpora was performed by infusion with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate and was confirmed by transmission electron microscopy. Strips of human and rabbit corporal tissues were studied in the organ chambers for isometric tension measurement. The accumulation of cyclic guanosine monophosphate (cGMP) and the release of eicosanoids from corporal tissue was measured by radioimmunoassay and correlated to smooth muscle relaxation. Our study showed that relaxation of corpus cavernosum tissue to acetylcholine, bradykinin and substance P was endothelium-dependent; potentiated by indomethacin; and inhibited by NG-monomethyl-L-arginine, methylene blue or LY83583. Relaxation to papaverine and sodium nitroprusside was endothelium-independent, and unaffected by NG-monomethyl-L-arginine. Relaxation to vasoactive intestinal polypeptide was partially endothelium-dependent; potentiated by indomethacin; attenuated by NG-monomethyl-L-arginine or methylene blue. The tissue level of cGMP was enhanced by acetylcholine and nitric oxide. Methylene blue inhibited both basal and drug-stimulated levels of cGMP. The release of eicosanoids was enhanced by acetylcholine and blocked by indomethacin. In conclusion, nitric oxide or a closely related substance accounts for the activity of endothelium-derived relaxing factor in the corporal tissue. Inhibition of the release of eicosanoids potentiates the relaxing effect of nitric oxide. Nitric oxide increases tissue cGMP which appears to modulate corporal smooth muscle relaxation.

S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds.

Endothelium-derived relaxing factor (EDRF) activity has been attributed to the highly labile nitric oxide radical (NO). In view of the fact that the plasma and cellular milieux contain reactive species that can rapidly inactivate NO, it has been postulated that NO is stabilized by a carrier molecule that preserves its biological activity. Reduced thiol species are candidates for this role, reacting readily in the presence of NO to yield biologically active S-nitrosothiols that are more stable than NO itself. Because sulfhydryl groups in proteins represent an abundant source of reduced thiol in biologic systems, we examined the reaction of several sulfhydryl-containing proteins of diverse nature and function upon exposure to authentic NO and EDRF. We demonstrate that S-nitroso proteins form readily under physiologic conditions and possess EDRF-like effects of vasodilation and platelet inhibition. These observations suggest that S-nitrosothiol groups in proteins may serve as intermediates in the cellular metabolism of NO and raise the possibility of an additional type of cellular regulatory mechanism.

Endothelium-derived hyperpolarizing factor (EDHF).

In vascular tissues, many types of stimulants which release endothelium-derived relaxing factor (EDRF) hyperpolarized the smooth muscle membrane in an endothelium-dependent manner. The hyperpolarizations were resistant to inhibitors of the actions of EDRF, thereby suggesting that they were elicited by an unidentified substance called endothelium-derived hyperpolarizing factor (EDHF). The EDHF induced hyperpolarization was produced by an increase in K(+)-permeability of the membrane, and was one of the factors to induce endothelium-dependent vasodilations. Release of EDHF required an increase in endothelial Ca2+, due either to release of Ca2+ from intracellular storage or to its influx from the external media.

Endothelium-dependent tolerance to ethanol-induced contraction of rat aorta: effect of inhibition of EDRF action and nitric oxide synthesis.

In vitro ethanol induces a dose-dependent contraction of the aorta. Tolerance to this effect of ethanol is expressed by a rightward shift of the dose-response curve in aorta from animals intoxicated with ethanol for 2 days. The expression of tolerance in the aorta is dependent upon the presence of functional endothelial cells which suggests that tolerance is mediated, in part, by endothelium-derived relaxing factor (EDRF). To test this hypothesis, three inhibitors of EDRF action and an inhibitor of nitric oxide synthesis were studied for their ability to alter tolerance to ethanol-induced contraction of the aorta. In vitro pretreatment of aortic rings with gossypol (10(-6)-10(-5) M), pyrogallol (10(-5) M), hemoglobin (10(-6) M), and NG-nitro-L-arginine (NOARG, 10(-4) M) inhibited endothelium-dependent relaxation induced by carbachol. The inhibition of carbachol-induced relaxation produced by pyrogallol was reversed by superoxide dismutase (SOD, 45 units/ml). In vitro pretreatment of rings obtained from ethanol-treated rats with gossypol, pyrogallol, hemoglobin, or NOARG inhibited the expression of ethanol tolerance, shifting the ethanol dose-response curve to control values. SOD reversed the effect of pyrogallol pretreatment. None of the antagonists significantly altered the ethanol dose-response curve of aortic rings obtained from control animals. These data support the hypothesis that tolerance to ethanol-induced contraction of the aorta is mediated by the release of EDRF from endothelial cells.

Endothelium-derived relaxing factor and the pulmonary circulation

Endothelium-derived relaxing factor (EDRF) is probably identical to nitric oxide (NO) and is released by the vascular endothelium both in the basal unstimulated state and in response to a wide range of physical and chemical stimuli. Since it was first described 10 years ago, evidence is accumulating that it is an important modulator of vascular smooth muscle tone. EDRF acts on the pulmonary vascular bed as on the systemic circulation. EDRF release to pharmacologic stimuli is impaired in pulmonary arteries from patients with chronic hypoxemia. This impairment is associated with severity of respiratory failure and of structural change of vessel walls. Disturbance of EDRF activity may be important in the pathophysiology of pulmonary vascular disease. This brief review describes the current status of experimental studies concerning the possible role of EDRF on the pulmonary circulation in normal conditions and in the pathogenesis of pulmonary hypertension.

Mechanism of action of angiotensin converting enzyme inhibitors on endothelial function in hypertension.

Hypertension is associated with an endothelial dysfunction characterized by an increased endothelium-dependent contraction and a decreased endothelium-dependent relaxation. Angiotensin converting enzyme (ACE) inhibition with cilazapril or captopril can remarkably improve the endothelial function in spontaneously hypertensive rats (SHRs). The goal of the present study was to investigate whether ACE inhibitors were acting by decreasing endothelium-dependent contraction or by increasing endothelium-dependent relaxation. Endothelial function was estimated by calculating the ratio of maximal contraction to serotonin on isolated aortic rings with endothelium to maximal contraction on paired rings without endothelium, termed the serotonin ratio. The serotonin ratio was greater than 1 in SHRs, indicating the release of a vasoconstrictor substance by the endothelium. This substance was identified as prostaglandin (PG) H2, because the serotonin ratio was significantly decreased by thromboxane (TX) A2/PGH2 receptor antagonists but not by TXA2 synthetase inhibitors. Two weeks of treatment of SHRs with cilazapril led to a marked decrease in the serotonin ratio, although acute administration of cilazaprilat was without any effect. However, after 2 weeks of treatment, the serotonin ratio still could be lowered further by TXA2/PGH2 receptor antagonists, indicating that cilazapril did not act by inhibition of PGH2 synthesis. In contrast, the effect of a 4-week treatment with cilazapril could be completely reversed by inhibiting the action of endothelium-derived relaxing factor with methylene blue. The same result was found after treatment with captopril. We speculate that ACE inhibitors improve endothelial function in SHRs not by inhibiting the synthesis of PGH2 but by increasing the release or the action of endothelium-derived relaxing factor.

Nitric oxide synthesis in endothelial cells: evidence for a pathway inducible by TNF-alpha.

Nitric oxide (NO) release accounts for the biological activity of endothelium-derived relaxing factor. Given that tumor necrosis factor-alpha (TNF-alpha) has been implicated as an important mediator in septic shock, we explored whether TNF-alpha enhances L-arginine-dependent synthesis of NO and L-citrulline in endothelial cells. The release of NO was detected in a coincubation bioassay where measurement of guanosine 3',5'-cyclic monophosphate (cGMP) production in reporter monolayers, namely glomerular mesangial cells or fetal lung fibroblasts, reflected activation of soluble guanylate cyclase. Reporter monolayer cGMP content was greater in the presence of TNF-alpha-treated bovine aortic and renal artery endothelial cells than in the presence of vehicle-treated endothelial cells. TNF-alpha-stimulated endothelium-dependent increases in reporter monolayer cGMP content were first evident at 8 h and maximal at 16-24 h. In addition, TNF-alpha-stimulated endothelium-dependent increases in reporter monolayer cGMP content were abrogated by hemoglobin and methylene blue, blunted by N omega-nitro-L-arginine and augmented by superoxide dismutase and the calcium agonist bradykinin. These observations suggested that TNF-alpha enhanced release of NO. Furthermore, the formation of L-[14C]citrulline from L-[14C]arginine, as determined by quantitative cation-exchange chromatography and thin-layer chromatography, was enhanced by TNF-alpha in a time- and concentration-dependent manner. Thus it is evident that endothelial cells release NO for a prolonged period in response to TNF-alpha and transiently when stimulated with calcium agonists. The prolonged release of NO from TNF-alpha-stimulated endothelial cells may be implicated in the pathogenesis of septic shock.

Diminished endothelium-derived relaxing factor activity in an experimental model of chronic heart failure.

Abnormalities in vasomotor tone, including enhanced vasoconstriction at rest and diminished vasodilation in response to various stimuli, develop as a consequence of chronic heart failure. This study was undertaken to evaluate whether a specific local mechanism, namely endothelium-derived relaxing factor (EDRF) activity, might be impaired in an experimental model of chronic heart failure. Segments of thoracic aorta (TA) and pulmonary artery (PA) were isolated from a group of rats that had hemodynamic evidence of heart failure 10 weeks after ligation of the left coronary artery (n = 25) and from a group of sham-operated control rats (n = 18). Both endothelium-dependent and endothelium-independent vascular responses were assessed by exposing arterial segments to increasing concentrations of agonists. All studies were performed in the presence of 10 microM indomethacin to avoid the influence of vasoactive prostanoids. The dose-response curve for EDRF-mediated relaxation to acetylcholine was shifted rightward in rats with heart failure, and the concentrations of acetylcholine required to achieve 50% maximal relaxation (EC50) were increased compared with those of control rats in both TA and PA segments. Additionally, the dose-response curve for relaxation to ADP was shifted rightward with significantly increased EC50 in PA segments from rats with heart failure. In contrast, EDRF-mediated relaxation to the calcium ionophore A23187 was similar in the groups. Endothelium-independent relaxation to nitroglycerin was slightly increased in TA but not PA segments in the heart-failure group. Basal EDRF activity, as assessed by the increase in force after exposure to hemoglobin, was diminished in PA segments from rats with heart failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced endothelium-derived relaxing factor activity in pregnant, spontaneously hypertensive rats

Pregnancy is normally associated with vasodilation that, in hypertensive animals such as the spontaneously hypertensive rat, causes a profound decrease in blood pressure. To test the possibility that enhanced basal endothelium-derived relaxing factor activity has a role in the vasodilation of pregnancy, we measured the changes in mean arterial pressure and heart rate induced by NG-monomethyi-L-arginine, a specific inhibitor of endothelium-derived relaxing factor synthesis, in conscious nonpregnant and pregnant (postmating day 20 to 21) normotensive Wistar-Kyoto and spontaneously hypertensive rats. NG-monomethyi-L-arginine caused similar dose-dependent increases in mean arterial pressure in nonpregnant and pregnant Wistar-Kyoto rats, but the accompanying decrease in heart rate was significantly greater in nonpregnant rats than in pregnant ones. In the spontaneously hypertensive rats, NG-monomethyi-L-arginine caused significantly greater dose-dependent increases in mean arterial pressure in pregnant compared with nonpregnant rats; there were no differences in the decreases in heart rate. These pressor responses were partially reversed by excess L-arginine but not D-arginine. Indomethacin had no effect on the pressor response to NG-monomethyi-L-arginine or the depressor response to L-arginine after NG-monomethyi-L-arginine. Therefore basal endothelium-derived relaxing factor plays a role in vascular tone and blood pressure regulation in vivo, and pregnancy may be associated with enhanced basal endothelium-derived relaxing factor activity in the hypertensive spontaneously hypertensive rats.

Endothelial cells modulate renin secretion from isolated mouse juxtaglomerular cells.

Utilizing cocultures of mouse renal juxtaglomerular cells with bovine microvascular endothelial cells, we have examined whether endothelial cells exert direct influence on renin secretion from renal juxtaglomerular cells. In the presence of endothelial cells both spontaneous and forskolin (10 microM) or isoproterenol (10 microM) stimulated renin release were markedly attenuated. The stimulatory effect of the calmodulin antagonist calmidazolium (10 microM) on renin secretion was not altered by endothelial cells, whereas the stimulatory effect of ethylisopropylamiloride (50 microM) an inhibitor of sodium-proton exchange was enhanced in the presence of endothelial cells. Indomethacin (10 microM) and NG-monomethyl-l-arginine (NMMA) (1 mM) used to inhibit cyclooxygenase activity and production of endothelium-derived relaxing factor (EDRF) decreased spontaneous renin release in the presence of endothelial cells only, but had no effect on forskolin stimulated renin secretion. Endothelin (1 microM) inhibited cAMP stimulated renin release both in the absence and in the presence of endothelial cells. ATP (10 microM) which acts on both endothelial and juxtaglomerular cells via purinergic P2 receptors inhibited cAMP stimulated renin release only in the absence but not in the presence of endothelial cells. This modulatory effect of endothelial cells was no altered by indomethacin nor by NMMA. Taken together, our findings provide first evidence for a local control function of the endothelium on cAMP stimulated renin secretion from renal juxtaglomerular cells, which could in part be mediated by endothelin.

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 ralaxine 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.

Comparison of responses to angiotensin II of dog mesenteric arteries and veins

Responses to angiotensin II (All), arachidonic acid (AA) and prostaglandin (PG) I 2 were compared in helical strips of dog mesenteric arteries and veins. Arterial strips contracted in response to AII, whereas venous strips responded with a transient, slight contraction followed by a moderate relaxation. The peptide-induced responses were abolished by treatment with saralasin. but were not influenced by ONO3708, an inhibitor of vasoconstrictor PG actions, or by endothelium denudation. Treatment with indomethacin potentiated the contractile response of the arteries and reversed the relaxant response of veins to a contraction. The concentration of exogenous PGI 2 methylester needed to produce a tension development similar to that induced by PGI 2 released by AII was greater in the mesenteric arteries than in the veins. The amount of 6-keto PGF 1 α in the bathing media measured by radioimmunoassay was increased in the arteries and veins stimulated by AII. Exogenously applied PGI 2 elicited relaxations of similar magnitude in the arteries and veins. AA-induced relaxations were greater in the veins; indomethacin suppressed the arterial and venous relaxations. The magnitude of the endothelium-dependent relaxation induced by A23187 was similar in the arteries and veins. It appears that the heterogenous responses to AII of dog mesenteric arteries and veins are due mainly to the difference in the AII receptors responsible for smooth muscle contraction and also to the difference in the ability to produce and liberate PGI 2. The synthesis and the action of EDRF (endothelium-derived relaxing factor) does not seem to differ in the arteries and veins.

A simple and sensitive bioassay method for detection of EDRF with RFL-6 rat lung fibroblasts.

Detailed characteristics of a new bioassay method for detection and quantification of endothelium-derived relaxing factor (EDRF) are described. Guanosine 3',5'-cyclic monophosphate (cGMP) responses of RFL-6 rat fetal lung fibroblast cells were utilized to estimate the activity of nitric oxide (NO) and EDRF. The conditioned medium from bovine aortic endothelial (BAE) cells cultured in tissue culture plates was quickly transferred to RFL-6 incubations to determine EDRF. In the presence of superoxide dismutase, RFL-6 cells cultured in six-well tissue culture plates exhibited very high sensitivities to both NO and EDRF; e.g., they responded to NO at a concentration as low as 2 nM and the basal release of EDRF from 1-2 X 10(6) BAE cells. Based on the lower detection limit of the radioimmunoassay for cGMP, calculations reveal that 100-200 fmol of NO and the basal EDRF release from 1-2 X 10(5) BAE cells can be detected with RFL-6 cells by choosing smaller culture wells. Thus this method is more sensitive than any other currently available. Furthermore, it may be widely used, since the instrumentation required is presently available in many laboratories. This bioassay technique for EDRF and NO is sensitive, simple, and quite useful for the evaluation of experimental conditions and compounds that regulate EDRF release from various endothelial cells and tissues.

Release of endothelium-derived relaxing factor is modulated both by frequency and amplitude of pulsatile flow

We have dissociated the effects of frequency and amplitude of pulsatile flow on flow-induced release of endothelium-derived relaxing factor (EDRF) using cascade bioassay. Rat aortic segments were buffer perfused with a peristaltic pump at a constant mean flow rate of 9 ml/min. EDRF activity in effluent was measured by relaxation of endothelium-denuded rabbit aortic rings preconstricted by phenylephrine. Pulse frequency was varied over the range 0.1-12 Hz at a constant amplitude of 2 mmHg; pulse amplitude was varied over the range 2-16 mmHg at a constant frequency of 0.1 Hz. Relaxation of the detector vessel depended on frequency of flow through the donor; peak response occurred between 4.2 and 6 Hz and was approximately three times greater than that induced at lower or higher frequencies. In contrast, increases in pulse pressure amplitude (maximum 16 mmHg) monotonically augmented constriction of partially preconstricted detector tissue by up to 10%. Incubation of the donor vessel with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthesis, or removal of its endothelium by rubbing, abolished both the frequency- and the amplitude-dependent effects observed in the detector tissue, indicating that these were mediated by changes in EDRF release. Increasing the amplitude of the pressure pulse also reduced mean perfusion pressure (by up to 50%), implying distension of donor vessel since mean flow rate was constant. This fall in pressure was not affected by incubation with L-NAME or removal of endothelium, indicating that it was not dependent on EDRF activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the acetylcholine-induced relaxation of canine isolated basilar artery by potassium-conductance blockers

Canine basilar artery rings precontracted with 5-hydroxytryptamine (0.1-0.5 microM) relaxed in the presence of acetylcholine (25-100 microM), sodium nitroprusside (0.1 microM), or stimulation of the electrogenic sodium pump by restoration of extracellular K+ (4.5 mM) after K(+)-deprivation. Acetylcholine-induced relaxation is believed to be caused by the release of endothelium-derived relaxing factor (EDRF) and is prevented by mechanical removal of the endothelium, while relaxations induced by sodium nitroprusside or restarting of the sodium pump are endothelium-independent. Acetylcholine-induced relaxation was selectively blocked by pretreatment of the tissue with the nonselective K+ conductance inhibitors, 4-aminopyridine (4-AP, 3 mM), Ba2+ (1 mM), and tetraethylammonium (20 mM), 4-AP also blocked ACh-mediated relaxation in muscles contracted with elevated external K+. Relaxation of 5-hydroxytryptamine-induced contraction by sodium nitroprusside, or by addition of K+ to K(+)-deprived muscle, was not affected by 4-AP. Relaxation of basilar artery with acidified sodium nitrite solution (containing nitric oxide) was reduced by 4-AP. These results suggest that 4-AP and possibly Ba2+ inhibit acetylcholine-induced endothelium-dependent relaxation by inhibition of the action of EDRF on the smooth muscle rather than through inhibition of release of EDRF. The increase in K+ conductance involved in acetylcholine-induced relaxation is not due to ATP-inhibited K+ channels, as it is not blocked by glyburide (10(-6) M). Endothelium-derived relaxant factor(s) may relax smooth muscle by mode(s) of action different from that of sodium nitroprusside or by hyperpolarization due to the electrogenic sodium pumping.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of endothelial dysfunction in the pathogenesis of reperfusion injury after myocardial ischemia

Endothelial dysfunction occurs after myocardial ischemia and reperfusion characterized by a marked reduction in endothelium-dependent relaxation (EDR) due to reduced release or action of endothelium-derived relaxing factor (EDRF). This reduced EDR occurs in coronary rings isolated from cats 2.5 min after reperfusion and in isolated perfused cat hearts 2.5 min after reperfusion. No decrease in EDR occurs before reperfusion in either preparation, suggesting that this impairment in EDR occurs during reperfusion. The decrease in EDR occurs soon after the generation of superoxide radicals by the reperfused coronary endothelium. Accumulation of neutrophils and myocardial cell injury does not occur until 3-4.5 h after reperfusion. Thus, endothelial generation of superoxide radicals acts as a trigger mechanism for endothelial dysfunction which is then amplified by neutrophil adherence and diapedesis into the ischemic region enhancing post-reperfusion ischemic injury. Agents that preserve endothelial function or inhibit neutrophil activation (e.g., superoxide dismutase, prostacyclin analogs, TGF-beta, antibodies to adhesive proteins) can protect against endothelial dysfunction and myocardial injury, if administered before reperfusion.

Maturational changes in endothelium-derived relaxing factor activity of ovine pulmonary arteries in vitro

To examine mechanisms underlying fetal pulmonary vascular vasodilator responses and maturational changes in endothelial function, we studied effects of endothelium-dependent (acetylcholine, ACh: adenosine diphosphate, ADP; and A23187) and -independent (sodium nitroprusside, SNP) vasodilators on tone of small (third generation) pulmonary artery rings isolated from late-gestation fetal, newborn, and adult sheep. Changes in isometric force of phenylephrine-contracted rings were measured after the cumulative addition of vasodilators in baths aerated with 21% O2 and 5% CO2. Pulmonary artery rings from fetal lambs demonstrated minimal relaxation to ACh, ADP, and A23187, achieving only 17 +/- 3, 14 +/- 5, and 23 +/- 8% relaxation, respectively. In contrast, fetal rings relaxed completely (100%) to SNP. Rings from newborn and adult animals had significantly greater maximal relaxation in response to ACh. ADP, and A23187 than fetal rings (at least P less than 0.05 for each comparison with fetal rings), but responses to SNP were not different. Hemoglobin (10(-5) M), an inhibitor of endothelium-derived relaxing factor, caused less augmentation of phenylephrine contraction in fetal than adult pulmonary artery rings (11 +/- 4% vs. 49 +/- 8%; P less than 0.01). We conclude that in comparison with pulmonary artery rings from postnatal animals, fetal pulmonary artery rings have diminished endothelium-derived relaxation factor activity. We speculate that maturational changes in endothelial cell function contribute to ontogenetic differences in pulmonary vasoreactivity.

Shear stress-induced release of nitric oxide from endothelial cells grown on beads.

An in vitro bioassay system was developed to study endothelium-mediated, shear stress-induced, or flow-dependent generation of endothelium-derived relaxing factor (EDRF). Monolayers of aortic endothelial cells were grown on a rigid and large surface area of microcarrier beads and were packed in a small column perfused with Krebs bicarbonate solution. The perfusate was allowed to superfuse three endothelium-denuded target pulmonary arterial strips arranged in a cascade. Fluid shear stress caused a flow-dependent release of EDRF from the endothelial cells. The action of EDRF was abolished by oxyhemoglobin and methylene blue, and the generation of EDRF in response to shear stress was markedly inhibited or abolished by NG-nitro-L-arginine, by NG-amino-L-arginine, by calcium-free extracellular medium, and by depleting endothelial cells of endogenous L-arginine. Addition of L-arginine to arginine-deficient but not arginine-containing endothelial cells rapidly restored the capacity of shear stress and bradykinin to generate EDRF. These observations indicate that fluid shear stress causes the generation of EDRF with properties of nitric oxide from aortic endothelial cells and that the bioassay system described may be useful for studying the mechanism of mechanochemical coupling that leads to nitric oxide generation.

Partial Purification and Characterization of a Ca2+/Calmodulin-Dependent Endothelium-Derived Relaxing Factor-Forming Enzyme from Porcine Cerebellum

Summary: l-Arginine-derived nitric oxide (NO) or a labile NO-containing compound accounts for the biological activity of endothelium-derived relaxing factor (EDRF). We recently demonstrated the enzymatic conversion of l-arginine into EDRF in endothelial cells by means of a bioassay, using purified soluble guanylate cyclase as a detection system, and suggested that the EDRF-forming enzyme may be regulated by the intracellular concentration of free Ca2+. In the present article, we describe the partial purification and characterization of this enzyme from porcine cerebellum. EDRF formation was assayed by the determination of the activation of soluble guanylate cyclase; in addition, the conversion of l-arginine into l-citrulline was measured. From crude porcine cerebellum supernatant, we purified the EDRF-forming enzyme about sevenfold with 30% recovery. Guanylate cyclase was activated about 100-fold by l-arginine in the presence of the partially purified protein, which required nicotinamide adenine dinucleotide phosphate (NADPH), Ca2+, and calmodulin for enzyme activity. With regard to the cofactor concentrations producing half-maximal effects, a good correlation was found between EDRF formation and the Ca2+/calmodulin-dependent conversion of l-arginine into l-citrulline.