Regional measurements of NO formed in vivo during brain ischemia.

Nitric oxide formed in vivo in the rat brain regions of hippocampus, striatum, neocortex and cerebellum was spin trapped and measured ex vivo by cryogenic electron paramagnetic resonance spectroscopy. In non-ischemic control animals the rate of nitric oxide (NO) formation in the individual brain regions ranged from 15 to 42 pmol.g-1.min-1. During exposure to global ischemia for 7 min the generation of NO increased in all parts of the brain. In the hippocampus the rate of NO formation during ischemia increased by 6-fold from a control rate of 19 pmol.g-1.min-1. This increase was attenuated 47% by pretreatment with the NO synthase antagonist 7-nitroindazole, whereas pretreatment with the non-NMDA receptor anatogonist NBQX and the Ca2+ channel blocker NS638 did not influence the NO formation. The data show that short-duration ischemia elicits a significant, NO-synthase-dependent formation of NO in all brain regions.

In vivo nitrate tolerance is not associated with reduced bioconversion of nitroglycerin to nitric oxide.

BACKGROUND: In vitro data suggest that reduced bioconversion of nitroglycerin (NTG) to nitric oxide (NO) contributes to the development of vascular and hemodynamic tolerance to NTG. We examined the in vivo validity of this hypothesis by measuring NTG-derived NO formation by in vivo spin-trapping of NO in vascular tissues from nitrate-tolerant and -nontolerant rats. METHODS AND RESULTS: Five groups (n = 6 to 8 each) of conscious chronically catheterized rats received NTG (0.2 or 1 mg/h IV) for 72 hours (nitrate-tolerant groups). Four other groups received either NTG vehicle (placebo, for 72 hours) or were left untreated (control). Nitrate tolerance was substantiated by a reduced (55% to 85%) hypotensive response to NTG in vivo and a reduced relaxation to NTG in isolated aortic rings. NTG-derived NO formation in aorta, vena cava, heart, and liver was measured as NOFe(DETC)2 and NO-heme complexes formed in vivo during 35 minutes combined with ex vivo cryogenic electron spin resonance spectroscopy. NO formation was significantly (P < .05) increased in all tissues in nitrate-tolerant rats in an NTG dose-dependent manner. Furthermore, the amount of NO formed from a bolus dose of NTG (6.5 mg/kg over 20 minutes) was similar in nitrate-tolerant and -nontolerant rats. CONCLUSIONS: The results suggest that vascular and hemodynamic NTG tolerance occurs despite high and similar rates of NO formation by NTG in tolerant and nontolerant target tissues. This finding is compatible with the assumption that reduced biological activity of NO, rather than reduced bioconversion of NTG to NO, contributes to in vivo development of nitrate tolerance.

S-nitrosation of serum albumin by dinitrosyl-iron complex.

The objective of this study was to identify a potential mechanism for S-nitrosation of proteins. Therefore, we assessed S-nitrosation of bovine serum albumin by dinitrosyl-iron-di-L-cysteine complex [(NO)2Fe(L-cysteine)2], a compound similar to naturally occurring iron-nitrosyls. Within 5-10 min, (NO)2Fe(L-cysteine)2 generated paramagnetic albumin-bound dinitrosyl-iron complex and S-nitrosoalbumin in a ratio of 4:1. Although S-nitroso-L-cysteine was concomitantly formed in low amounts, its concentration was not sufficient to account for formation of S-nitrosoalbumin via a trans-S-nitrosation reaction. Low oxygen tension did not affect S-nitrosation by the dinitrosyl-iron complex thus excluding the involvement of oxygenated NOx-species in the nitrosation reaction. Blockade of albumin histidine residues by pyrocarbonate, which prevented formation of dinitrosyl-iron-albumin complex, did not inhibit S-nitrosation of albumin. Thus, S-nitrosation of albumin by (NO)2Fe(L-cysteine)2 can proceed by direct attack of a nitrosyl moiety on the protein thiolate, without previous binding of the iron. We conclude that protein-bound dinitrosyl-iron complexes detected in high concentrations in certain tissues provide a reservoir of S-nitrosating species, e.g. low molecular dinitrosyl iron complexes.

Specificity of different organic nitrates to elicit NO formation in rabbit vascular tissues and organs in vivo.

1. In the present study we assessed the formation of nitric oxide (NO) from classical and thiol-containing organic nitrates in vascular tissues and organs of anaesthetized rabbits, and established a relationship between the relaxant response elicited by nitroglycerin (NTG) and NO formation in the rabbit isolated aorta. Furthermore, the effect of isolated cytochrome P450 on NO formation from organic nitrates was investigated. 2. Rabbits received diethyldithiocarbamate (DETC; 200 mg kg-1 initial bolus i.p. and 200 mg kg-1 during 20 min, i.v.) and either saline, or one of the following organic nitrates: nitroglycerin (NTG, 0.5 mg kg-1), isosorbide dinitrate (ISDN), N-(3-nitratopivaloyl)-L-cysteine ethylester (SPM 3672), S-carboxyethyl-N-(3-nitratopivaloyl)-L-cysteine ethylester (SPM 5185), at 10 mg kg-1 each. After 20 min the animals were killed, blood vessels and organs were removed, and subsequently analyzed for spin-trapped NO by cryogenic electron spin resonance (e.s.r.) spectroscopy. 3. In the saline-treated control group, NO remained below the detection limit in all vessels and organs. In contrast, all of the nitrates tested elicited measurable NO formation, which was higher in organs (liver, kidney, heart, lung, spleen) (up to 4.8 nmol g-1 20 min-1) than in blood vessels (vena cava, mesenteric bed, femoral artery, aorta) (up to 0.7 nmol g-1 20 min-1). Classical organic nitrates (NTG, ISDN) formed NO preferentially in the mesenteric bed and the vena cava, while the SPM compounds elicited comparable NO formation in veins and arteries. 4. Using a similar spin trapping technique, NO formation was assessed in vitro in phenylephrine-precontracted rabbit aortic rings. The maximal relaxation elicited by a first exposure (10 min) to NTG (0.3 to 10 microM) was positively correlated (r = 0.8) with the net increase (NTG minus basal) of NO spin-trapped during a second exposure to the same concentration of NTG in the presence of DETC. 5. Cytochrome P450 purified from rabbit liver enhanced NO formation in a NADPH-dependent fashion from NTG, but not from the other nitrates, as assessed by activation of purified soluble guanylyl cyclase. 6. We conclude that the vessel selective action of different organic nitrates in vivo reflects differences in vascular NO formation. Thus, efficient preload reduction by classical organic nitrates can be accounted for by higher NO formation in venous capacitance as compared to arterial conductance and resistance vessels. In contrast, NO is released from cysteine-containing nitrates (SPMs) to a similar extent in arteries and veins, presumably independently of an organic nitrate-specific biotransformation. Limited tissue bioavailability of NTG and ISDN might account for low NO formation in the aorta, while true differences in biotransformation seem to account for differences in NO formation in the other vascular tissues.

In vivo spin trapping of glyceryl trinitrate-derived nitric oxide in rabbit blood vessels and organs.

BACKGROUND: The objectives of this study were (1) to assess glyceryl trinitrate (GTN)-derived nitric oxide (NO) formation in vascular tissues and organs of anesthetized rabbits in vivo, (2) to establish a correlation between tissue NO levels and a biological response, and (3) to verify biotransformation of GTN to NO by cytochrome P-450. METHODS AND RESULTS: NO was trapped in tissues in vivo as a stable paramagnetic mononitrosyl-iron-diethyldithiocarbamate complex [NOFe(DETC)2]. After removal of the tissues, NO was determined by cryogenic electron spin resonance spectroscopy. NO formation in vitro was assessed by spin trapping and by activation of soluble guanylyl cyclase. The GTN-elicited decrease in coronary perfusion pressure was monitored in isolated, constant-flow perfused rabbit hearts. NO was not detected in control tissues. In GTN-treated rabbits, NO formation was higher in organs than in vascular tissues and higher in venous than in arterial vessels. In isolated hearts, ventricular NO levels and decreases in coronary perfusion pressure achieved by GTN were closely correlated. Purified cytochrome P-450 catalyzed NO formation from GTN in a P-450-NADPH reductase- and NADPH-dependent fashion. CONCLUSIONS: Since GTN-derived NO formation in myocardial tissue correlates to the GTN-elicited vasodilator response, we conclude that GTN-derived NO detected in vivo correlates with the systemic effects of GTN. Therefore, the higher rate of NO formation detected in veins compared with arteries explains the preferential venodilator activity of GTN. High NO formation in cytochrome P-450-rich organs in vivo and efficient NO formation from GTN by cytochrome P-450 in vitro highlights the importance of this pathway for NO formation from GTN in the intact organism.

Characterization of furoxans as a new class of tolerance-resistant nitrovasodilators.

The vasodilator effects of C92-4609 (4-hydroxymethyl-furoxan-3-carboxamide, CAS 1609), C92-4678(4-phenyl-furoxan-3-carboxylic acid (pyridyl-3-yl-methyl)-amide), C92-4679 (3-phenyl-furoxan-4-carboxylic acid (pyridyl-3-yl-methyl)-amide) and C93-4759 (3-hydroxymethyl-furoxan-4-carboxamide) were studied in the isolated rabbit femoral artery and jugular vein. All furoxans were potent vasodilators in the femoral artery (EC50 0.1-50 microM), while they were less potent in the jugular vein by at least one order of magnitude. Apart from C92-4679, the vasodilatory potency of the furoxans correlated well with their nitric oxide (NO)-releasing capacity which was estimated both by stimulation of purified soluble guanylyl cyclase activity and electron spin resonance spectroscopy with a trapping agent for NO. The hypothesis that furoxans stimulate soluble guanylyl cyclase in the smooth muscle by spontaneously releasing NO was supported by the marked attenuation of their vasodilator effect in the presence of oxyhaemoglobin (10 microM) or following treatment with methylene blue (30 microM). In contrast to earlier findings, NO release from these furoxans was not thiol-dependent, as demonstrated for C92-4609, the relaxant effect of which in the femoral artery was not altered in the presence of N-acetyl-L-cysteine (1 mM). Moreover the KCa+ channel inhibitor, tetrabutylammonium (3 mM), but not the KATP+ channel inhibitor, glibenclamide (3 microM), significantly attenuated the dilator response to C92-4679 in the femoral artery. Pretreatment of these segments with the cytochrome P450 inhibitor, SKF525a (30 microM), also reduced the C92-4679-induced relaxation in this vascular bed.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of 15N omega-hydroxy-L-arginine and ESR and 15N-NMR studies for the elucidation of the molecular mechanism of enzymic nitric oxide formation from L-arginine.

N omega-Hydroxy-L-arginine (2) was prepared by a multi-stage synthesis; the key step was the addition of hydroxylamine to the protected cyanamide 8. The presence of N-hydroxyguanidines was confirmed, above all, by 15N-NMR investigations. 15N omega-Hydroxy-L-arginine (2) was converted quantitatively to 15NO by NO synthases from macrophages. 15NO was identified by ESR-spectroscopy. These experiments confirm that 15N omega-hydroxy-L-arginine (2) is an intermediate in the biosynthesis of NO from arginine (1) and that the N-hydroxylated N-atom is present in the NO formed.

Nitric oxide promotes seizure activity in kainate-treated rats.

L-Arginine-derived nitrogen monoxide (NO) formation was determined in different regions of the rat brain during kainate-induced seizures. NO was trapped in vivo as a paramagnetic mononitrosyl-iron diethyldithiocarbamate complex, the concentration of which was determined ex vivo by cryogenic electron spin resonance spectroscopy. Basal NO formation (0.3-0.8 nmol g-1 tissue 30 min-1) was detected in the brain of control rats. In kainate-injected rats NO formation was increased six-fold within 30-60 min in the amygdala/temporal cortex region, and up to 12-fold, though more slowly, in the remaining cortex. The kainate-elicited convulsions and NO formation were attenuated in animals pretreated with either 7-nitroindazole, a specific inhibitor of neuronal NO synthase, or diazepam. These findings identify NO as a proconvulsant mediator in kainate-evoked seizures.

Biotransformation of sodium nitroprusside into dinitrosyl iron complexes in tissue of ascites tumors of mice.

We have found that treatment of various types of murine ascites tumors with sodium nitroprusside (SNP) both in vitro (1.5 mM) and in vivo (25 mg/kg; i.p.) results in formation of EPR-detectable dinitrosyl nonheme iron complexes (DNIC) with RS- groups of proteins (g perpendicular = 2,037; g parallel = 2,012). The DNIC were mainly localized in the ascitic extracellular fluid. The appearance of DNIC was unaffected by an inhibitor of nitric oxide synthase, N omega-nitro-L-arginine (1 mM), but abolished with a SH-blockator, p-chloromercuribenzoate (0.1 mM). No DNIC formation was observed when ascitic fluid (after tumor cells separation) was incubated with SNP alone, but with SNP and L-cysteine (10 mM). Thus, ascites tumor cells contribute to the transformation of SNP into DNIC and thiols are essential in this process.

Epr evidence of nitric oxide production by the regenerating rat liver.

Nitric oxide (NO) production in the regenerating liver was estimated from the intensity of the electron paramagnetic resonance (e.p.r.) signal of the mononitrosyl complexes of iron and diethylthiocarbamate (DETC). Preformed complexes of intracellular non-heme Fe2+ and added DETC served as a trap for endogenously produced NO. The time-dependent changes of NO production were connected with the periodicity of liver regeneration. The first increase in NO production occurred ca. 1 h after partial hepatectomy (PHE). The second and more pronounced peak of NO production was observed about 6 h after PHE, when the hepatocytes entered the first cell cycle; it originated mainly from these cells. The following minimum of NO synthesis coincided with the maximal rate of DNA synthesis. The third gradual rise of NO production was seen at the end of the investigated period that covered the G2 + M phases, the transit from the first to the second cell cycle of the hepatocytes and the entrance of the nonparenchymal cells into proliferation.

Formation and release of dinitrosyl iron complexes by endothelial cells.

The release of dinitrosyl non-heme iron complexes from cytotoxic macrophages accounts for NO-mediated iron loss. We have now investigated whether or not a similar mechanism operates in endothelial cells. Following stimulation with bradykinin or calcium ionophore A23187 NO and intracellular dinitrosyl iron complexes were detected by ESR spectroscopic analysis of frozen cells. In addition, endothelial cells released dinitrosyl iron complexes which bound to extracellular albumin. In transferrin and iron-free medium stimulation of endothelial cells by bradykinin or thimerosal resulted in a loss of non-heme iron. These effects were prevented by inhibition of NO synthase. Thus NO generated by the constitutive NO synthase appears to be incorporated into dinitrosyl iron complexes, which potentially account for endothelium-dependent relaxation.

The relationship between L-arginine-dependent nitric oxide synthesis, nitrite release and dinitrosyl-iron complex formation by activated macrophages.

We identified the source of the nitrogen included into nitric oxide (NO) and studied the relationship between formation of NO, intracellular dinitrosyl ferrous iron complex (DNIC) and release of nitrite by murine bone-marrow-derived macrophages stimulated with E. coli lipopolysaccharide (LPS). NO was trapped in the cell membrane by iron-diethyldithiocarbamate complex (FeDETC) and was detected as a paramagnetic NOFe(DETC)2 complex by electron paramagnetic resonance (EPR) spectroscopy. Macrophages stimulated for 7 h up to 48 h with LPS and then incubated for 2 h with DETC exhibited an anisotropic EPR signal of axial symmetry with g-factor values g perpendicular = 2.035, g parallel = 2.02 and a triplet hyperfine structure (hfs) at g perpendicular characteristic for NOFe(DETC)2. In cells incubated with [15NG]L-arginine instead of [14NG]L-arginine the EPR signal of [15N]OFe(DETC)2 was detected with a doublet hfs at g perpendicular, indicating that NO was generated exclusively from the terminal guanidino-nitrogen of extracellular L-arginine. The ratio of NO formation and of nitrite release changed with time of exposure to LPS, nitrite exceeding NO at early stages of macrophage activation, and NO exceeding nitrite at later stages. DNIC with thiolate ligands (0.5 nmol/10(7) cells) was observed in stimulated macrophages not loaded with DETC. Furthermore, DNIC released from macrophages was trapped in the extracellular medium by bovine serum albumin (BSA) (1 nmol/10(7) cells per 2 h) by formation of a paramagnetic DNIC with BSA. DNIC release not only provides a route for iron loss from activated macrophages, but may also play a role in the cytotoxic and microbiostatic activity of macrophages.

Iron potentiates bacterial lipopolysaccharide-induced nitric oxide formation in animal organs.

Administration of an Fe(2+)-citrate complex to mongrel mice pretreated with lipopolysaccharide (LPS) from Salmonella typhosa increased LPS-induced NO formation in vivo in the liver, intestine, lung, heart, kidney and spleen by 10-20-fold. This process was monitored by the intensity of the EPR signal due to mononitrosyl iron complex (MNIC) formation with exogenous diethyldithiocarbamate (DETC) recorded in the tissues. The NO synthase inhibitor, NG-nitro-L-arginine, prevented this complex formation in the liver of mice treated with both LPS and Fe(2+)-citrate complex. Thus, administration of LPS and Fe(2+)-citrate complex to mice induced NO biosynthesis in this tissue via an L-arginine-dependent pathway, presumably by facilitating the entry of Ca2+ ions into NO-producing cells through Fe(2+)-induced cell membrane lesions.

Diethyldithiocarbamate inhibits induction of macrophage NO synthase.

We investigated whether sodium diethyldithiocarbamate (DETC), an inhibitor of the nuclear transcription factor kappa B (NFkappa B), modulates induction of NO synthase (NOS) in murine bone marrow-derived macrophages. A short exposure (between 1 and 16 h) of L929-cell medium-preconditioned macrophages to E. coli lipopolysaccharide (LPS) significantly increased the level of NOS mRNA, and elicited NO formation as detected by electron spin resonance spectroscopy and by the release of nitrite. DETC (0.1-1 mM) present during stimulation with LPS prevented the increase in NOS mRNA and the expression of NOS activity. These findings suggest that NFkappa B is involved in the signal transduction pathway linking stimulation of macrophages by LPS with transcription of the gene encoding inducible NOS.

Enzymic and nonenzymic release of NO accounts for the vasodilator activity of the metabolites of CAS 936, a novel long-acting sydnonimine derivative.

The molecular mechanism(s) underlying the vasodilator activity of CAS 936 (3-(cis-2,6-dimethylpiperidino)-N-(4-methoxybenzoyl)-sydn oni mine) and its metabolites 3-(cis-2,6-dimethylpiperidino)-sydnonimine (C87 3754) and N-(cis-2,6-dimethylpiperidino)-N-nitroso-2-aminoacetonitrile (C873786) was investigated. These compounds were tested for their relaxant activity in isolated rabbit arterial segments, activation of purified soluble guanylyl cyclase and release of nitric oxide (NO) in vitro and in vivo. C873754 and C873786 inhibited the noradrenaline-induced contraction and increased the cyclic GMP content of endothelium-denuded rabbit aortic and femoral segments, whereas CAS 936 was without effect. Similarly, both metabolites, but not CAS 936, activated purified soluble guanylyl cyclase (EC50 about 30 microM) and released NO in buffered aqueous solutions, as detected by electron spin resonance (esr) spectrometry. Both in vitro and in vivo an accumulation of NO was detected by esr spectrometry in vascular tissues exposed to the metabolites of CAS 936, whereas a significant release of NO from CAS 936 was only detected in the isolated rabbit liver, but not in vascular tissue. It is conceivable, therefore, that the metabolites of CAS 936 appearing in the systemic circulation after hepatic biotransformation induce vasodilatation by release of NO and activation of soluble guanylyl cyclase in vascular smooth muscle. Moreover, the activation of soluble guanylyl cyclase in vitro by the metabolites of CAS 936 was significantly enhanced by co-incubation with certain particulate fractions from bovine aortic endothelial and smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

NO accounts completely for the oxygenated nitrogen species generated by enzymic L-arginine oxygenation.

We have assessed the stoichiometry of the nitric oxide (NO) synthase reaction by using a novel e.p.r. technique. NO generated by crude and partially purified NO synthase from endothelial cells and Escherichia coli-lipopolysaccharide-activated macrophages was trapped by a ferrous diethyldithiocarbamate complex dispersed in yeast. The paramagnetic ferrous mononitrosyl dithiocarbamate complex formed exhibited a characteristic e.p.r. signal at g perpendicular = 2.035 and g parallel = 2.02 with a triplet hyperfine structure (hfs) at g perpendicular. NO, 3-morpholinosydnonimine and S-nitroso-L-cysteine, but not nitrite or hydroxylamine, generated a similar e.p.r. signal. NO generated by NO synthase and by SIN-1 accumulated at a constant rate for 1 h, as measured by continuous e.p.r. registration at 37 degrees C. The formation of e.p.r.-detectable NO by NO synthases was inhibited by NG-nitro-L-arginine. Incubation with [15N]NG-L-arginine caused an e.p.r. signal with doublet hfs, indicating that the nitrosyl nitrogen derived exclusively from the guanidino nitrogen. The amount of NO generated by NO synthase as measured by e.p.r. technique was compared with formation of L-[3H]citrulline from L-[3H]arginine. NO and L-citrulline were detected at a 1:1 ratio with both NO synthase preparations. GSH and thiol depletion did not significantly affect NO synthase activity, excluding S-nitrosothiols as intermediates in the NO synthase reaction. We conclude that NO fully accounts for the immediate oxygenated nitrogen species derived from the enzymic oxygenation of L-arginine.

The source of non-heme iron that binds nitric oxide in cultivated macrophages.

In cultured macrophages (J 774 line) a decrease in iron-sulfur centers (ISC) was not observed after 5 min treatment with nitric oxide (NO) (10(-7) M NO/10(7) cells). The content of these centers was measured by electron spin resonance (ESR) spectroscopy at 16-60 K. However, the appearance of a characteristic ESR signal at g(av) = 2.03 indicated the formation of dinitrosyl iron complex (DNIC) in these cells. These findings suggest that loosely bound non-heme iron (free iron) but not iron from ISC is mainly involved in DNIC formation. ISC might release iron for DNIC formation after their destruction induced by the products of NO oxidation (NO2, N2O3, etc).

EPR evidence for nitric oxide production from guanidino nitrogens of L-arginine in animal tissues in vivo.

Administration of Fe(2+)-citrate complex (50 mg/kg of FeSO4 or FeCl2 plus 250 mg/kg of sodium citrate) subcutaneously in the thigh or Escherichia coli lipopolysaccharide (LPS, 1 mg/kg) intraperitoneally, (i.p.) to mice induced NO formation in the livers in vivo at the rate of 0.2-0.3 micrograms/g wet tissue per 0.5 h. The NO synthesized was specifically trapped with Fe(2+)-diethyldithiocarbamate complex (FeDETC2), formed from endogenous iron and diethyldithiocarbamate (DETC) administered i.p. 0.5 h before decapitation of the animals. NO bound with this trap resulted in the formation of a paramagnetic mononitrosyl iron complex with DETC (NO-FeDETC2), characterized by an EPR signal at g perpendicular = 2.035, g parallel = 2.02 with triplet hyperfine structure (HFS) at g perpendicular. This allowed quantification of the amount of NO formed in the livers. An inhibitor of enzymatic NO synthesis from L-arginine, NG-nitro-L-arginine (NNLA, 50 mg/kg) attenuated the NO synthesis in vivo. L-Arginine (500 mg/kg) reversed this effect. Injection of L-[guanidineimino-15N2]arginine combined with Fe(2+)-citrate or LPS led to the formation of the EPR signal of NO-FeDETC2 characterized by a doublet HFS at g perpendicular, demonstrating that the NO originates from the guanidino nitrogens of L-arginine in vivo.

Effect of diethyldithiocarbamate on the activity of nitric oxide-releasing vasodilators.

The effect of diethyldithiocarbamate (DETC, 10(-3) M) on the vasorelaxant activity of acetylcholine, nitric oxide (NO), nitrite, glyceryl trinitrate or dinitrosyl iron cysteine complexes was studied in isolated rat aortic rings contracted with norepinephrine. Pretreatment of these segments with DETC attenuated the vasorelaxation induced by vasodilators and prevented the subsequent restoration of vessel tone, whereas DETC added after the vasodilators induced a rapid restoration of tone. The inhibitory effect of DETC was due to the trapping of NO by a complex of DETC with Fe2+ formed in the tissue.

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 acetylcholine (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.