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

European Journal of Pharmacology, 211 (1992) 313-317

© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00

EJP 52288

Similarity between the vasorelaxing activity of dinitrosyl iron cysteine complexes and endothelium-derived relaxing factor Y u r i P. V e d e r n i k o v t P e t e r I. M o r d v i n t c e v , I r i n a V. M a l e n k o v a a n d A n a t o l y F. V a n i n 1 Cardiology Research Center, USSR Academy of Medical Sciences, Moscow, U.S.S.R. and Institute of Chemical Physics, U.S.S.R. Academy of Sciences, Moscow, U.S.S.R.

Received 27 June 1991, revised MS received 19 November 1991, accepted 26 November 1991

Dinitrosyl iron complexes with cysteine (DNIC) induced a concentration-dependent relaxation of pre-contract (norepinephrine, 10 -7 M) de-endothelialized ring segments of rat aorta. The vasodilator response was more similar

acetylcholine (ACh)-induced relaxation in intact aortic rings than to nitric oxide (NO)-induced relaxation. The time course tone recovery after maximal concentrations (10 -5 M) of DNIC was similar to the time course of tone recovery af endothelium-dependent relaxation induced by ACh, whereas the restoration of tone after NO was much faster. Vessel tone restored by oxyhemoglobin (10 -5 M) in all cases, The results suggest that DNIC with cysteine may function as endothelium-~ rived relaxing factor in the vessels.

Dinitrosyl iron complexes (with cysteine); EDRF (endothelium-derived relaxing factor); Nitric oxide (NO); Vasorelaxation

1. Introduction Palmer et al. (1987) and Ignarro et al. (1987) showed that endothelium-derived relaxing factor ( E D R F ) might be identical to nitric oxide (NO) or to a labile nitrosocompound. Other investigators have come to the conclusion that E D R F is not identical to N O but contains N O as an active principle (Shikano et al., 1988; Rubanyi et al., 1988; Myers et al., 1990; Vedernikov et al., 1990). It was shown that N O as a component of E D R F was one order of magnitude more active than free N O (Myers et al., 1990). Thus E D R F seems to be a NOcontaining compound in which N O is stabilized, thereby enabling N O to be an extremely active vasorelaxant. Recently, nitrosothiols were suggested to be the most probable candidate for E D R F (Rubanyi et al., 1988; Ignarro, 1989; Myers et al., 1990). However, this is unlikely, firstly, because nitrosothiols are not formed in aqueous medium at neutral pH, and secondly, because they decompose rapidly under these conditions. These compounds are stable only in acid medium and it was suggested that nitrosothiols are formed and localized

Correspondence to: A.F. Vanin, Institute of Chemical Physics, U.S.S.R. Academy of Sciences, Kosygin Street 4, 117334 Moscow, U.S.S.R.

in vacuoles with an acidic internal environment insi the cell (Ignarro, 1989). Another hypothesis was t[ E D R F might be a more complex compound includi both NO, thiol-containing substances, and non-her iron. Thus E D R F might be a nitrosyl iron complex wi thiol-containing ligands (Vanin, 1991). Such comple~ could be formed in animal ceils and tissues in a neut~ aqueous environment under the influence of exo~ nous producers of N O (Vanin et al., 1967; Woolt and Commoner, 1970; Vanin et al., 1971; Nagata et 1973; Vanin, 1987). These complexes have recen been shown to be formed in macrophages after b synthesis from endogenous sources had been induc (Lancaster and Hibbs, 1990; Pellat et al., 1990). The complexes were dinitrosyl iron complexes (DNIC) w~ paired protein thiol groups or low molecular wei~ compounds such as cysteine or glutathione with t formula Fe(NO)e(RS) 2. They are characterized electron spin resonance (ESR) signals with g ± = 2.0! gH = 2.012, gay 2.03, and are usually named 2.03 co: plexes. In the present study comparative data on the vaso~ laxing activity of D N I C with cysteine, E D R F stirc lated by acetylcholine (ACh), and N O are present~ The main aims of this study were to verify whetl~ inclusion of N O into D N I C stabilizes it, and whetl: D N I C has vasorelaxing activity. =

314 2. Materials and methods

2.1. The preparation of DNIC with cysteine F e S O a - 7 H e O , 1 mg per ml in physiological salt solution (PSS), was mixed with cysteine, either 1.5 or 15 mg per ml in PSS, and NO gas under pressure (300 mm Hg), after the system had been evacuated to 10-1 mm Hg. After 5 min of intensive mixing of FeSO 4 and cysteine in an atmosphere of NO gas, the latter was pumped out. The DNIC solution obtained was frozen in liquid nitrogen for storage and transportation. DNIC solutions were thawed immediately before use. In these solutions the molar ratios of Fe 2+ to cysteine were 1 : 2 or 1:20, and the latter complex gave an ESR signal with g~v = 2.03. Comparison, by the double integration method, of the intensity of this signal with that of the ESR signal of a known concentration of the nitroxyl radical, N-oxyl-2,2',6,6'-tetramethyl-4-piperidinol, in aqueous solution revealed that all the Fe 2+ in the solution was included in DNIC. The quantitive estimation of NO by the 'acid' method with ESR spectroscopy (Vedernikov et al., 1990) showed that the NO content (in a molar ratio) was twice that of Fe 2+, i.e. all NO was bound in DNIC 1 : 20. DNIC solutons with Fe 2÷ : cysteine in 1 : 2 ratio gave a weak singlet ESR signal at g~v = 2.03. On a molar basis, the content of NO in these solutions was equal to the Fe 2÷ content. At a 1 : 2 ratio of Fe z+ : cysteine, dimeric and polimeric species of DNIC with weak ESR signal are formed (McDonald et a1.,1965; Burbaev and Vanin, 1970).

the ring segments was increased by addition of a su maximal concentration of norepinephrine (10 -7 1~ and, after stabilization, the agents were cumulative added to the organ bath in incremental steps of 0 logarithmic unit. The experiments were performed parallel in the absence and presence of superoxic dismutase (SOD) (30 U / m l ) , which was added to tl organ bath before application of the agents studied.

2.3. Materials The agents used were norepinephrine (Serv~ acetylcholine (Sigma), atropine (Serva), superoxide di mutase from bovine erythrocytes (Serva), and bovil hemoglobin (Serva) reduced to obtain oxyhemoglob according to Martin et al. (1985). NO was obtained 1 reaction of FeSO 4 with NaNO 2 in acid aqueo' medium (Burbaev and Vanin, 1970). The NO solutk was prepared as described elsewhere (Vedernikov al., 1989) and the NO content was measured by tl acid method (Vedernikov et al., 1990).

2.4. Statistical evaluation The data are presented as experimental curves or means + S.E.M. and were analyzed with Studen~ paired sample t-tests. The concentrations given in tl text are the final concentrations in the organ bath. TI number of experiments is the number of isolated ve sels from different animals.

3. Results

2.2. Organ bath experiments Thoracic aortas of Wistar rats (220-250 g) were removed and cleaned of all fat and connective tissue in ice-cold PSS of the following composition (in mM): NaC1 118, KCI 4.7, CaC1 z 2.52, MgSO 4 1.64, N a H C O 3 24.88, K H 2 P O 4 1.18, sodium pyruvate 2.0, Na2-EDTA 0.026, glucose 11.0. Ring segments of 4 mm in width were cut. The endothelium was removed by gentle rolling of the segments over a wooden stick covered with cotton. The endothelium was preserved only in the segments in which ACh-induced endothelium-dependent relaxation was studied. Segments of aorta were placed in organ baths of 10 ml volume filled with PSS. The temperature of the organ bath solution was thermostatically maintained at 37°C and the pH at 7.35-7.4 by continuous bubling with a gas mixture of 95% 0 2 + 5% CO 2. Changes in isometric tension were recorded with UC-2 transducers (Gould, USA) on a dynograph R-711 (Beckman, USA). Passive tension was maintained at the level of 2 g during the equilibration period of 60-90 min, during which time the organ bath solution was exchanged every 15 rain. The tone of

Original recordings, and mean dose-response curv for NO, DNIC and ACh in the presence and in tl absence of SOD are presented in figs. 1 and 2, respe tively. As shown in the figures, the vasorelaxing effe of the agents studied was more pronounced in tl presence of SOD. This is in agreement with data the inhibitory effect of superoxide radicals on NO-pr ducing vasodilator actions. From the data presented in fig. 2, the ratios of tl relaxation (in percent) induced by concentrations DNIC and ACh to the relaxation (in percent) producl by the same concentrations of exogenous NO in tl presence and in the absence of SOD were calculatq (fig. 3). These data show that DNIC was more effecti as a vasorelaxant than free NO. The difference vasorelaxing effects was most clearly seen with k concentrations of the agents studied when SOD w not present. Increasing the concentration of agents 10 -6 M abolished the differences between the vasot laxing effects of DNIC, ACh and free NO. In t] presence of SOD, the difference between the vasor laxing activity of the agents was less pronounced, ev,

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concentration (10 -5 M) of the agents (fig. 1). TI restoration of tone after D N I C was very slow and d not reach the preceding level after 6 0 - 9 0 min. Tt might be due to the persistance of low molecul weight D N I C in the solution, or to the possible form tion of D N I C with paired thiol groups of proteins the vessel walls, resulting in the transfer of Fe(N(3 from low molecular weight D N I C to protein thi groups (Kleshchev et al., 1985). G r a d u a l disintegrati~ of lOW molecular weight and protein D N I C could d liver free N O into the bath solution. T h e presence N O in the solution was confirmed by the acid meth( (Vedernikov et al., 1990). Fifteen minutes after DN] had been a d d e d to the solution, the N O content w decreased 10-fold. This rate of D N I C disintegration sufficient to provided prolonged relaxation of the ve sel. Hemoglobin, 10 -5 M a d d e d to the organ bath, z stored vessel tone completely, independently of t] extent of tone recovery at the time of hemoglob administration (fig. 4).

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Fig. 1. Effect of nitric oxide, NO (A) dinitrosyl iron complex with cysteine, DNIC 1 : 20 (B) and acetylcholine, ACh (C) in the absence (-SOD) or in presence (+ SOD) of superoxide d ismutase, SOD, 30 U/ml, on the tension of isolated ring segments of rat aorta induced by norepinephrine (NE), 10-7 M. The dots show the application of the agents (in -log M), atropine (Atr), 10-5 M and hemoglobin (Hb), 10 -5 M. Dotted line shows passive tension before addition of NE. Vertical bars, 1 g; horizontal bars, 5 min. Here and in the following figures the concentration of DNIC is expressed as the concentration of NO in the complexes.

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in the low concentration range. T h e activity ratio of A C h to that of equimolar concentrations of D N 1 C was 3- to 5-fold higher in the low concentration range (fig. 3). It is important to note that the concentration of free NO, or o t h e r substances released from endothelial cells by A C h , could not be estimated in these experiments. C o m p a r i s o n of the activity ratio o f D N I C and A C h in the absence of S O D to that of free N O in the presence of S O D did not show any difference between these agents (fig. 3). This means that N O contained in D N I C is stabilized to the same extent as free N O is in

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the presence o f SOD. As can be seen from figs. 2 and 3, the vasorelaxing activity of D N I C preparations with different cysteine content (1 : 2 or 1 : 20) did not differ, T h e vasorelaxing activity of D N I C and E D R F was similar in comparison with N O activity u n d e r our experimental conditions. This similarity was also seen in the dynamics of vessel tone recovery after a maximal

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~00 9i 8~ 7, 6~ 5~ 8i '7J 6J 5 -log IONIC1:21 I'1 -10g tONICl: Z0) H Fig. 2. Dose-response curves of relaxing activity of nitric oxide, 1~ (A), acetylcholine, ACh (B) and dinitrosyl iron complexes with c teine, DNIC 1 : 2 (C) and DNIC 1 : 20 (D) in the absence ( • ) and the presence ( I ) of superoxide dismutase, SOD, 30 U/ml, isolated ring segments of rat aorta. Abscissa: -log of concentrati in moles. Ordinate: percent relaxation of tension induced by no pinephrine (NE), 10-7 M. n = 7 in each experimental group. "I level of stabilized tension after NE and after addition of SOD in 1 experimental groups (0 tension) was (in grams): NO, 2.9 + 0.2; NC SOD, 3.5 + 0.3; DNIC 1 : 2, 3.6___0.3; DNIC 1 : 2 + SOD, 3.7 + ( DNIC 1:20, 3.05+0.2; DNIC I:20+SOD, 3.1-+0.3; ACh, 3.3-+( ACh + SOD, 3.1 + 0.1, * P < 0.05, ** P < 0.02, *** P < 0. ** ** P < 0.002, ***** P < 0.001.

316 The recovery of vessel tone after ACh-indueed

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dothelium-dependent relaxation was rather slow and, as in case of DNIC, was accelerated by hemoglobin. This slow recovery of vessel tone might be due to a maintained production of E D R F by ACh. Atropine (10 .5 M), a blocker of muscarinic receptors, which in endothelial cells are responsible for E D R F release, led to only a partial recovery of tone. Addition of hemoglobin (10 -5 M) restored the tone completely (fig. 1C). In separate experiments, a 15-min pretreatmerit of aortic rings with atropine (10 -5 M) abolished the ACh-induced endothelium-dependent relaxation (data not shown). In contrast to the recovery of tone after DNIC and A Ch, the recovery of vessel tone after NO (10 -5 M) was rather fast and reached a new stable level in 5-7 min, which was 15-20% lower than the tone before NO was added. Hemoglobin (10 -5 M) restored vessel tone

completely (not shown). However, determination

of NO in the solution by the acid method (Vedernikov

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et al., 1990) did not reveal any marked decrease in N content during 30 min of incubation. Apparently, tl method of NO determination used was not adequa and could not discriminate beween NO and nitrite solution. The latter might result from NO oxidatic followed by NO formation in an acidic environment the presence of a reducing agent. Such a reduci: agent could be the thiosulfate added to the ac medium for NO determination (Vedernikov et a 1990). We checked this possibility, preincubating N (10 -5 M) in PSS for 10 min. This resulted in a 23-fold decrease in vasorelaxing activity compared wi that of the initial NO solution. Apparently, some N had been transformed into nitrite anions, which ev, in a concentration of 10 -5 M possess very weak vasm laxing activity (data not shown).

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- I o O 1'1 Fig. 3. Ratios of the percentage relaxation of rat aorta ring segments produced by acetylcholine (11), dinitrosyl iron complexes with cysteine, DNIC 1:2 (©) and DNIC 1:20 (e) to the percentage relaxation induced by the same concentrations of NO in the absence ( - SOD) (A) and in the presence ( + SOD) (B) superoxide dismutase, SOD, 30 U / m l . Dotted lines show the ratios calculated for the effects of the agents in the absence of SOD to the effects of NO in the presence of SOD (A). Abscissa: - log of agent concentrations in moles. Ordinate: ratio of the relaxing effect of the respective concentrations in relative units. Contraction was evoked by norepinehrine

(10-7M).

The data presented show that DNIC, independen of its cysteine content, is a more effective vasorelaxa than free NO in isolated rat aorta segments placed organ baths. The active principle of DNIC, as well EDRF, is NO. NO included in DNIC is more star than free NO. Moreover, complex formation makes possible to deliver more NO from these complexes its target, the soluble guanylate cyclase in vessel s m o o muscle ceils. N O m a y be released from DNIC as a result of tl oxidation of thiol groups of ligands. The releas, of

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Fe(NO) 2 is not as stable as D N I C and quickly produces free NO. It seems important to emphasize that inclusion of N O in D N I C e n s u r e s a stabilization comparable to the

effect of addition of 30 U / m l SOD. In this connection we suggest that even at the low SOD activity in the vessel tissues, the formation of NO-bearing complexes, such DNIC, is necessary to transport N O from endothelium to the target cells. It is probable that these complexes may function like E D R F . The similarity between D N I C and E D R F w a s e s p e cially noticeable with regard to the dynamics of vessel tone restoration after the addition of maximal concentrations of D N I C and ACh (fig. 1). The observation that the ACh-induced relaxation w a s n o t completely abolished by atropine suggests that, before atropine was added, an agent(s) had accumulated in the vessel tissue and caused a long-lasting relaxation. Since hemoglobin restored vessel tone, it seems logical to suggest that this agent is a producer of NO, probably D N I C formed in the vessel tissue.

In conclusion, the data obtained are consistent with the hypothesis that E D R F might be D N I C with thiolcontaining ligands. However the validity of this hypothesis can only be established by direct analysis of the composition of E D R F .

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Martin,W., G.M. Jothianandan and R. Furchgott, 1985, Selecti, blockade of endothelium-derived relaxing factor and glycel trinitrate-induced relaxation by hemoglobin and by methylel blue in the rabbit aorta, J. Pharmacol. Exp. Ther. 232, 708. McDonald, C.C., W.D. Phillips and H.F. Mower, 1965, An electr~ spin resonance study of some complexes of iron, nitric oxide ar anionic ligands, J. Am. Chem. Soc. 87, 3319. Myers, P.R., R.L. Minor, R. Guerra, J.N. Bates and D.G. Harriso 1990, Vasorelaxant properties of the endothelium-derived rela ing factor more closely resemble S-nitrosocysteine than nit~ oxide, Nature 345, 161. Nagata, C., Y. loki, M. Kodama and Y. Tagashira, 1973, Frq radical-induced in rat liver by a chemical carcinogen, N-meth~ N-nitro-N-nitrosoguanidine, Ann. N.Y. Acad. Sci. 222, 1031. Palmer, R.M.J., A.G. Ferrige and S. Moncada, 1987, Nitric oxil release accounts for the biological activity of endothelium-derM

relaxing factor, Nature 327, 524.

Pellat, C., Y. Henry and J.-C. Drapier, 1990, IFN-y-activatc macrophages: detection by electron paramagnetic resonance

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dence that endothelium-derived relaxing factor may be identk with an S-nitrosothiol and not with free nitric oxide, Circulati~ (Suppl.) 80, Abstr. II-281. Shikano, K., J. Long, E.H. Oblstein and B.A. Berkowitz, 19~ Comparative pharmacology of endothelium-derived relaxing f~ tor and nitric oxide, J. Pharmacol. Exp. Ther. 347, 873. Vanin, A.F., 1987, Sources of non-heine iron capable of formil nitrosyl complexes in animal tissues, Biofizika U.S.S.R. 31, 128 Vanin, A.F., 1991, Endothelium-derived relaxing factor is a nitro~ iron complex with thiol ligands (Hypothesis), FEBS Letts. 289, Vanin, A.F., L.A. Blumenfeld and A.G. Chetverikov, 1967, El: study of non-heme iron complexes in cells and tissues, Biofizi U.S.S.R. 12, 829. Vanin, A.F., L.N. Kubrina, I.L. Lisovskaya, I.V. Malenkova and A., Chetverikov, 1971, Endogenic nitrosyl non-heme and heme ir, complexes in cells and tissues, Biofizika U.S.S.R. 16, 650. Vedernikov, Ju.P., T. GrS.ser and A.F. Vanin, 1989, Similar endoth lium-independent arterial relaxation by carbon monoxide a: nitric oxide, Biomed. Biochim. Acta 48, 595. Vedernikov, Ju.P., P.I. Mordvintcev, I.V. Malenkova and A.F. Van: 1990, Endothelium-derived relaxing factor is not identical nitric oxide, in: Nitric Oxide from L-Arginine. A Bioregulatc System, eds. S. Moncada and E.A. Higgs (Exerpta Medica, A] sterdam, New York, Oxford) p. 373. Woolum, J.C. and B. Commoner, 1970, Isolation and identificati, of a paramagnetic complex from the livers of carcinogenic-treat rats, Biochim. Biophys. Acta 201, 131.