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A macrocyclic nitrosyl ruthenium complex is a NO donor that induces rat aorta relaxation
NITRIC OXIDE
Biology and Chemistry
Nitric Oxide 10 (2004) 83–91 www.elsevier.com/locate/yniox
A macrocyclic nitrosyl ruthenium complex is a NO donor that induces rat aorta relaxation Daniella Bonaventura,a Fabiana de S. Oliveira,a Vanessa Togniolo,a Antonio C. Tedesco,b Roberto S. da Silva,a and Lusiane M. Bendhacka,* a
Laborat orio de Farmacologia, Depto. de Fısica e Quımica, Faculdade de Ci^encias Farmac^euticas de Ribeir~ao Preto, USP Av. do Cafe s/no, 14040-903 Ribeir~ao Preto, SP, Brazil b Depto. de Quımica, FFCLRP-USP, 14040-903 Ribeir~ao Preto, SP, Brazil Received 21 October 2003; received in revised form 17 March 2004
Abstract The vasorelaxation induced by a nitrosyl macrocyclic ruthenium complex, proposed as a new nitric oxide (NO) carrier, was studied in rat isolated aorta. The compound trans-[RuCl([15]aneN4 )NO]2þ was characterized by elemental analysis, UV–visible spectrum, and infrared spectrum. Based on the electrochemical process, the reduction of the compound was followed by NO release, which was also observed using norepinephrine as a reducing agent and NO released was analyzed by a sensor. Vasorelaxation induced by this NO donor was studied and compared to those obtained with sodium nitroprusside (SNP). The relaxation induced by the compound was concentration-dependent in denuded rat aortas and occurred only in pre-contracted arteries with norepinephrine. The macrocyclic compound induced relaxation with a similar efficacy as SNP, although the potency of SNP was slightly greater. The time to reach maximum relaxation (595 s) was longer than that of SNP (195 s). Relaxation was completely abolished by oxyhemoglobin, a known NO scavenger. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Macrocyclic nitrosyl ruthenium complex; Nitric oxide donor; Sodium nitroprusside; Vasorelaxation; Rat aorta
Nitric oxide (NO), a lipid-soluble and unstable gas, is one of the most fascinating substances in biological chemistry. NO has been identified as an important signaling molecule playing a crucial role in the cardiovascular and nervous systems [1–4]. The action of NO has also been found in a variety of physiological and pathological processes in different cells and tissues, such as neurotransmission, blood pressure control, inhibition of platelet aggregation, and immunological responses [5,6]. Most of the effects of NO are mediated via the stimulation of soluble guanylyl cyclase (sGC), which catalyses conversion of guanosine-50 -trisphosphate (GTP) to cyclic guanosine-30 ; 50 -monophosphate (cGMP) [7]. Despite the great importance of NO in the biological system, its pharmacological studies have been limited due to its high reactivity and short half-life [8,9]. The * Corresponding author. Fax: +55-16-6332960. E-mail address: [email protected] (L.M. Bendhack).
1089-8603/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2004.03.004
free radical nature of NO has hindered the understanding of many problems associated with its diffusional delays or associated with the effect that NO presents on its action sites. The development of molecules that can release NO can overcome this kind of problem and also be potentially developed as therapeutic agents. One of the most common compounds used as a NO delivery agent in pharmacological and clinical studies is the sodium nitroprusside salt (SNP)— [Fe(CN5 )NO]2 [10,11]. The therapeutic properties of SNP are related to NO release, which occurs via the redox conversion of SNP to its one or two electron-reduced forms [12–14]. SNP requires cellular metabolism to produce NO and extracellular release of NO is not the mechanism for the relaxant effect of SNP. On the other hand, SNP releases cyanide in addition to NO which exerts multiple effects including the activation of sGC and cell toxicity that is an important limitation of the pharmacological use of
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Fig. 1. (A) Molecular structure of trans-[RuCl([15]aneN4 )NO]2þ ; (B) Chem 3Dd calculated trans-[RuCl([15]aneN4 )NO]2þ structure (hydrogen atoms are omitted).
SNP [15]. Furthermore, it is very important to develop compounds that display relatively low thermal reactivity, but could become active yielding NO under a physical or chemical stimulation. Research in the NO field has expanded considerably in the last few years, particularly with respect to the application of new NO drugs to medicine. Metalonitrosyl complexes, for example, are a class of compounds that can be used as agents potentially capable of NO releasing in vivo [16–18]. The strategy used by specialists is to synthesize compounds that act as a NO carrier for activation of the release of NO in a controlled manner, of the order of minutes. These compounds are particularly relevant as vasodilators, since the slow release of NO could avoid the reflex side effects on the cardiovascular system usually present when drugs such as SNP are used. The vasodilator effects of compounds such as organic nitrites (e.g., nitroglycerine, NTG) as well inorganic SNP are mediated by the generation of NO [19]. Although SNP and NTG are widely used in the treatment of various cardiovascular disorders, there are many limitations for their use, and both classes of compounds have undesirable side effects [20]. Continuous exposure to nitroglycerine leads to the development of nitrate tolerance [21] characterized by an impairment of the NO-cGMP signaling cascade due to oxidative stress [15]. High doses of SNP or its prolonged use are associated with the accumulation of cyanide and the metabolite thiocyanate [22], which could impair hepatic function. Several new metal complexes have been studied as a new class of NO donors [23] including nitrosyl ruthenium complexes some of which exhibit low toxicity and are quite soluble in aqueous solutions. Recently, Lang et al. [24] have found that [RuCl(cyclam)NO]2þ , where cyclam
is a tetraazamacrocyclic ligand of 14 members, is stable in physiological conditions and can act as vasodilator agent due to the NO release. The physical chemistry properties of a macrocyclic complex type [RuL(MACROCYCLE)L0 ]nþ seem to be dependent on the size of the MACROCYCLE [25], which influence very greatly the properties of resulting metal complexes. Thus, we decide to study some kinetic NO releasing properties and present pharmacological assays for a new macrocyclic nitrosyl ruthenium complex, which was characterized as trans-[RuCl([15]aneN4 )NO]2þ , where [15]aneN4 is a tetraazamacrocyclic ligand of 15 members (Fig. 1). The synthesis trans-[RuCl([15]aneN4 )NO]2þ complex was recently described [26]. Furthermore, a comparison of the thermodynamic and photochemical properties of trans[RuCl([15]aneN4 )NO]2þ and trans-[RuCl(cyclam)NO]2þ complexes was proved to be different [26]. Considering the chemical characteristics of the trans[RuCl([15]aneN4 )NO]2þ complex, the aim of the present work was to study the effect of the NO donor of nitrosyl ruthenium complex as a pro-drug that could release NO only when it is induced and to investigate the possible mechanisms of vascular relaxation. The vasorelaxation induced by this inorganic NO donor was studied and its effects were compared to those obtained with SNP.
Experimental procedures Measurements The pH measurements were made using a DM 20 pH meter (Digimed). The UV–vis spectra were recorded on
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a Hitachi U-3501. IR spectra were recorded on a Protege 460 series FT-IR spectrometer, using solid samples pressed in KBr pellets. The NO measurements were performed using the ISO-NO NO-meter and the DUO-18 acquisition board. The sensitivity of this apparatus ranges from 1 nmol/L to 20 lmol/L, with a 2 mm sensor, which directly detects NO concentration by an amperometric technique. The setup was calibrated following a published procedure [27–29]. Synthesis of the complexes The recrystallized complex salts trans-[RuCl2 ([15] aneN4 )]Cl and trans-[RuCl([15]aneN4 )NO]Cl were prepared in accordance with the procedure published by Oliveira et al. [30]. The trans-[RuCl([15]aneN4 )NO]PF6 complex was characterized by IR mNO 1884 cm1 , UV–Vis (1 lmol/L HCl): 268 nm (e ¼ 4:42 103 mol1 L cm1 ) and 352 nm (e ¼ 2:40 102 mol1 L cm1 ). Elemental analysis calculated for C11 H26 N5 Cl3 ORu: C, 29.24; H, 5.80; N, 15.50. Found: C, 29.10; H, 5.87; N, 15.35. The trans-[RuCl([15]aneN4 )(H2 O)]þ complex was synthesized in situ by reduction of trans-[RuCl2 ([15]aneN4 )]þ with zinc amalgam in 0.1 mol/L trifluoromethanesulfonic acid (HTFMS). The free chloride obtained by reduction of dichloro(macrocyle)ruthenium(III) was successively removed from the solution by passing in an ionic exchange column. The aquachloro(1,4,8,12-tetraazacyclopentadecane)ruthenium(II) synthesized was characterized by cyclic voltammetry method and compared to the published results [25]. The formal reduction potential was found as +0.24 V vs Ag/AgCl in 0.1 mol/L HTFMS in agreement with the previously described trans[RuCl([15]aneN4 )(H2 O)]þ [25]. Vessel preparation Male Wistar rats (400–450 g) were killed by decapitation in accordance with the Ethical Animal Committee, Ribeir~ ao Preto Campus, University of S~ ao Paulo, Brazil. The thoracic aorta was quickly removed, dissected free, and cut into 4 mm long rings. Since the response to trans-[RuCl([15]aneN4 )NO]2þ does not require intact endothelium, in the present study we investigated the relaxation induced by this compound in endothelium-denuded arteries. The endothelium was mechanically removed by gently rolling the lumen of the vessel on a thin wire. The aortic rings were placed between two stainless-steel stirrups and connected to an isometric force transducer (Letica Scientific Instruments) to measure tension in the vessels. The rings were placed in a 10 ml organ chamber containing Krebs solution with the following composition (mmol/L): NaCl 130, KCl 4.7, KH2 PO4 1.2, MgSO4 1.2, NaHCO3 14.9, glucose 5.5, CaCl2 1.6. The solution was maintained at pH 7.4 gassed with 95% O2 and 5% CO2 at 37 °C.
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The rings were initially stretched to a basal tension of 1.5 g (previously determined by length–tension relationship experiments), before allowing them to equilibrate for 60 min in the bath fluid, which was changed every 15– 20 min. Endothelial integrity was qualitatively assessed by the degree of relaxation caused by acetylcholine (ACh, 1 lmol/L) in the presence of contractile tone induced by phenylephrine (0.1 lmol/L) [31]. Since our studies required endothelium-denuded aortas, the rings were discarded if there was any degree of relaxation, to avoid the possible influence of endothelial factors. Experimental protocols Relaxant effect of trans-[RuCl([15]aneN4 )NO]2þ after pre-contraction with norepinephrine To examine whether trans-[RuCl([15]aneN4 )NO]2þ induces smooth muscle cell relaxation, aortic rings were pre-contracted with 0.1 lmol/L norepinephrine and when the contraction had reached a plateau, trans[RuCl([15]aneN4 )NO]2þ (0.1 nmol/L to 100 lmol/L) was cumulatively added. Similar protocol was accomplished for SNP. Time-course for the relaxation induced by trans[RuCl([15]aneN4 )NO]2þ trans-[RuCl([15]aneN4 )NO]2þ (100 lmol/L) was added to the organ chamber when a stable contraction in response to 0.1 lmol/L norepinephrine was achieved. Time-course for relaxation induced by this compound was evaluated and similar protocol was accomplished for SNP. Relaxant effect of trans-[RuCl([15]aneN4 )NO]2þ after pre-contraction with KCl In another set of experiments, cumulative concentration–response curves for trans-[RuCl([15]aneN4 )NO]2þ (0.1 nmol/L to 100 lmol/L) were obtained after precontraction with 60 mmol/L KCl in the absence of a reductor agent. Contribution of potassium channels to the relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ Tetraethylammonium (TEA 100 lmol/L, 1 mmol/L, and 5 mmol/L), a non-selective potassium channel blocker, was used to study the contribution of potassium channels to trans-[RuCl([15]aneN4 )NO]2þ -induced relaxation. TEA was added 30 min before the addition of norepinephrine (0.1 lmol/L). Subsequently, cumulative concentration–response curves for trans-[RuCl([15] aneN4 )NO]2þ were constructed in the presence of TEA. Effect of oxyhemoglobin on the relaxation of trans[RuCl([15]aneN4 )NO]2þ The oxidation of hemoglobin was performed as previously described [32]. Oxyhemoglobin (HbO2 10 lmol/L),
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a nitric oxide scavenger, was added 30 min before the addition of 0.1 lmol/L norepinephrine. Subsequently, cumulative concentration–response curves to trans[RuCl([15]aneN4 )NO]2þ were constructed. Statistical analysis Data are expressed as means SEM. In each set of experiments, n indicates the number of rats studied. Two pharmacological parameters, maximal effect (Emax ) and pD2 , obtained from concentration–response curves for trans-[RuCl([15]aneN4 )NO]2þ and SNP were used to analyze the data. Emax was considered as the maximal amplitude response reached in the concentration-effect curves for relaxant agents. We consider the maximal relaxing effect for 15 ane when the concentration used reached the baseline. The concentrations of agents producing a half-maximal relaxation amplitude (EC50 ) were determined after logit transformation of the normalized concentration–response curves and were reported as the negative logarithm (pD2 ) of the mean of individual values for each tissue using the GraphPad Prism version 3.0 (GraphPad Software Corporation San Diego, CA). Statistical significance was tested by one way ANOVA (post-test: Newman–Keuls) and StudentÕs t test, and values of p < 0:05 were considered to be significant. Drugs Acetylcholine, norepinephrine, tetraethylammonium, phenylephrine, and sodium nitroprusside were purchased from Sigma Chemical (St. Louis, MO, USA). Hemoglobin was purchased from Calbiochem–Novabiochem (La Jolla, CA, USA). Reagents RuCl3 3H2 O, 1,4,8,12-tetraazacyclopentadecane ([15] aneN4 ) was purchased as a highly pure reagent from Aldrich Chemicals and used as supplied, sodium nitrite was purchased from Mallinckrodt. Double distilled H2 O was used for all experiments. All preparations and measurements were carried out under an argon atmosphere and protected from light.
Fig. 2. Electronic spectrum of trans-[RuCl([15]aneN4 )NO]2þ in aqueous solution (solid line) and after reduction at )0.50 V vs Ag/AgCl (dash line). Concentration of the trans-[RuCl([15]aneN4 )NO]2þ complex ¼ 0.21 mmol/L. Inset: Differential pulse voltammogram of trans[RuCl([15]aneN4 )NO]2þ in 0.1 mol/L trifluoromethanesulfonic acid.
electrochemical characteristics of the trans-[RuCl([15] aneN4 )NO]2þ complex in aqueous solution were studied using a differential pulse voltammogram, which exhibited a large peak at 0.276 V vs Ag/AgCl, with a probable contribution of the reduction of the nitrosyl ligand which was inferred by comparison to similar systems [33]. The electro-reduction of trans-[RuCl([15]aneN4 )NO]2þ was performed in 0.1 mol/L trifluoromethanesulfonic acid or in phosphate buffer solution, pH 7.40, at )0.50 V vs Ag/ AgCl. The product of this reaction was characterized in situ by UV–Vis spectrum and attributed to the formation of trans -[RuCl([15]aneN4 )H2 O]þ (Fig. 2), by comparison to the synthesized aquachloro(1,5,8,12-tetraazacyclopentadecane)ruthenium(II) complex [25]. Based on the electrochemical process, the reduction of trans-[RuCl([15]aneN4 )NO]2þ was also studied using norepinephrine as a reducing agent, a known vasoconstrictor agent. The NO release from the nitrosyl ruthenium complex during reduction was analyzed by a sensor, which directly detects NO concentrations by an amperometric technique. A similar experiment was carried out with [Fe(CN)5 NO]2 (SNP) and the results for both complexes were compared (Fig. 3). Relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ and SNP in rat aortic rings pre-contracted with norepinephrine
Results The nitrosyl ruthenium complex was characterized by elemental analysis, UV–Visible spectrum (UV–Vis), and infrared spectrum (FT-IR). FT-IR is frequently used to characterize the oxidation state of the metal and NO ligands in ruthenium complexes [18]. The NO frequency of trans- [RuCl([15]aneN4 )NO]2þ reported in this paper is 1884 cm1 which indicates that formally a linear NOþ is coordinated to the ruthenium(II) center. The
As shown in Fig. 4, the relaxation induced by this compound was concentration-dependent in denuded rat aortas pre-contracted with 0.1 lmol/L norepinephrine. SNP also induced concentration-dependent relaxation, but was more potent than trans-[RuCl([15]aneN4 )NO]2þ (pD2 : 7.97 0.07, n ¼ 6 and 5.03 0.2, n ¼ 6, respectively). Both compounds, however, induced the same Emax (105.86 3.34%, n ¼ 6 for SNP and 98.35 1.22%, n ¼ 6 for trans-[RuCl([15]aneN4 )NO]2þ ).
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Fig. 3. Time-dependent nitric oxide generation from trans[RuCl([15]aneN4 )NO]2þ (line A) and sodium nitroprusside (line B) in buffer solution at pH 7.40. Concentration of the trans[RuCl([15]aneN4 )NO]2þ complex ¼ SNP ¼ 0.21 mmol/L.
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Fig. 5. Time-course for sodium nitroprusside (SNP) and trans[RuCl([15]aneN4 )NO]2þ -induced relaxation. Denuded thoracic aortic rings were pre-contracted with 0.1 lmol/L norepinephrine and 0.1 lmol/ L SNP (m, n ¼ 7) or 100 lmol/L trans-[RuCl([15]aneN4 )NO]2þ (s, n ¼ 8) were added. Data are means SEM of n experiments performed on preparations obtained from different animals.
Fig. 4. Effects of trans-[RuCl([15]aneN4 )NO]2þ and sodium nitroprusside (SNP) on rat thoracic aorta pre-contracted with norepinephrine. The arteries were pre-contracted with 0.1 lmol/L norepinephrine and trans-[RuCl([15]aneN4 )NO]2þ (, n ¼ 6) or SNP (j, n ¼ 6) was added cumulative (0.1 nmol/L to 100 lmol/L and 0.1 nmol/L to 0.3 lmol/L, respectively). Data are means SEM of n experiments performed on preparations obtained from different animals.
Fig. 6. Effects of trans-[RuCl([15]aneN4 )NO]2þ on rat thoracic aorta pre-contracted with norepinephrine and KCl. The arteries were precontracted with norepinephrine (n, n ¼ 6) or 60 mmol/L KCl (.; n ¼ 6) and trans-[RuCl([15]aneN4 )NO]2þ was cumulatively added (0.1 nmol/L to 100 lmol/L). Data are means SEM of n experiments performed on preparations obtained from different animals.
Time-course for the relaxation induced by trans[RuCl([15]aneN4 )NO]2þ and sodium nitroprusside
Effect of tetraethylammonium on relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ
The time to reach maximum relaxation was longer for trans-[RuCl([15]aneN4 )NO]2þ (595 s, n ¼ 8) than for SNP (195 s, n ¼ 7), as shown in Fig. 5.
To investigate whether the relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ involves potassium channels, we studied the effect of the following concentrations of TEA. As shown in Fig. 7, TEA (100 lmol/L) shifted the concentration–response curves for trans[RuCl([15]aneN4 )NO]2þ to the right and reduced the maximum effect from 98.28 0.70 to 61.44 3.72% and pD2 values from 5.12 0.16 to 4.46 0.12 (n ¼ 6). Similarly, at 1 mmol/L, TEA reduced the maximum effect to 39.40 2.01% and pD2 to 4.51 0.13 (n ¼ 6) and 5 mmol/L TEA reduced the maximum effect to 33.27 5.69% and pD2 to 4.74 0.27 (n ¼ 6) in the
Relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ in rat aortic rings pre-contracted with KCl In preparations pre-contracted with 60 mmol/L KCl, relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ was inhibited when compared to relaxation observed in aortic rings pre-contracted with norepinephrine (pD2 ¼ 7.97 0.07, n ¼ 6), as shown in Fig. 6.
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the relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ was completely abolished by oxyhemoglobin.
Discussion
Fig. 7. Effects of tetraethylammonium (TEA) on the relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ in denuded rat aortic rings. The figure illustrates the effects of TEA (control: n; n ¼ 6; TEA 100 lmol/L: m; n ¼ 5; TEA 1 mmol/L: d; n ¼ 5 and TEA 5 mmol/L: r; n ¼ 8) on trans-[RuCl([15]aneN4 )NO]2þ -induced relaxation. Tissues were incubated with TEA for 30 min prior to the application of 0.1 lmol/L norepinephrine. Data are means SEM of n experiments performed on preparations obtained from different animals.
concentration–response curves for trans-[RuCl([15]aneN4 )NO]2þ , when compared to control curves. However, at 1 or 5 mmol/L, TEA did not alter the pD2 values when compared to those obtained with 100 lmol/L TEA. Effect of oxyhemoglobin on trans-[RuCl([15]aneN4 ) NO]2þ induced relaxation To investigate whether the relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ could be due to NO release, we studied the effect of this complex in the presence of 10 lmol/L oxyhemoglobin. As shown in Fig. 8,
Fig. 8. Effects of oxyhemoglobin (HbO2 ) on the relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ in denuded rat aortic rings. The figure illustrates the effects of HbO2 10 lmol/L HbO2 (control: n; n ¼ 6; 1 lmol/L: m; n ¼ 8) on trans-[RuCl([15]aneN4 )NO]2þ -induced relaxation. Tissues were incubated with HbO2 for 30 min prior to the application of 0.1 lmol/L norepinephrine. Data are means SEM of n experiments performed on preparations obtained from different animals.
Although some nitrosyl ruthenium complexes have been found to be NO delivery agents, numerous species are unstable in physiological conditions or present side reactions leading to different products in addition to the production of NO [18,34,35]. To avoid the side reaction, macrocyclic complexes have been suggested as nitric oxide delivery agent [25]. In this way, the trans[RuCl(cyclam)NO]2þ has been isolated and some physiological properties studied [24]. These macrocyclic complexes show slow NO release after reduction of coordinated nitrosyl group. Considering that the size of the macrocycle also influences the characteristic of ruthenium macrocyclic complexes, we decided to study some pharmacological properties of trans-[RuCl ([15]aneN4 )NO]2þ . We, herein, report trans-[RuCl([15] aneN4 )NO]2þ to be a stable compound in oxygenated physiological saline solution for at least a week, as observed by its unchangeable ultraviolet–visible spectrum. The electronic spectrum of an aqueous solution of trans-[RuCl([15]aneN4 )NO]2þ displays a band at 268 and 352 nm, which changes under controlled potential reduction (Fig. 2). The shift observed in the absorption spectra after the reduction of trans-[RuCl([15]aneN4 ) NO]2þ in aqueous solution was attributed to the release of nitric oxide bonded to the ruthenium(II). The resulting electronic spectrum after full reduction was due to the formation of trans-[RuCl ([15]aneN4 )(H2 O)]þ , which electronic spectrum was compared to the synthesized aqua-complex, previously described [25]. Similar results were also observed when zinc amalgam or norepinephrine was used as reducing agents, permitting us to propose the mechanism as described in Scheme 1. The investigation of the induced NO-releasing pathway was also supported by in situ NO detection when an aqueous phosphate buffer solution of trans-[RuCl ([15]aneN4 )NO]2þ was submitted to reduction with norepinephrine (Fig. 3). The signal recorded by the NO sensor rose quickly when the reduction was initiated. As the experiment was carried out in an argon atmosphere, there was no consumption of NO, which conducts a stable current after the total release of NO from the nitrosyl ruthenium complex. A similar experiment was performed with SNP. The nitric oxide time-dependent graph of SNP shows a small current in comparison to the trans-[RuCl([15]aneN4 )NO]2þ complex (Fig. 3) for the same concentration used in both species. This dependence on NO concentration may be related to the low stability of SNP in physiological solution, due to the nucleophilic attack of hydroxide ion in the nitrosyl ligand originating a nitro group, as previously described
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Scheme 1.
[36]. The side reaction involving consumption of NO after release from SNP is also detected, leading to a current that decreases as a function of time. This procedure is not detected in the process observed with NO release from trans-[RuCl([15]aneN4 )NO]2þ (Fig. 3). The new NO donor, trans-[RuCl([15]aneN4 )NO]2þ , induces relaxation in a concentration-dependent manner in aortic rings pre-contracted with norepinephrine, which was compared to SNP. Relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ was less potent than that induced by SNP, however both donors presented similar efficacy in relaxing the rat aorta. SNP is a known potent, rapid, and efficiently nitrovasodilator compound [15,37,38] which is therapeutically used in hypertensive emergencies, heart failure, and for controlled hypotension during surgery. SNP is thought to produce its vasorelaxant action, at least in part, by the release of NO into the vascular smooth muscle cell. Norepinephrine induces vascular contraction by activating a1 adrenoceptors on the vascular smooth muscle, resulting in the activation of at least two different signal transduction cascades: the phospholipase C-induced phosphoinositol breakdown with a subsequent release of calcium from intracellular stores and the activation of calcium channels in the outer plasma membrane leading to a calcium influx [39]. The time necessary to obtain the complete concentration–effect curve for relaxation with trans-[RuCl ([15]aneN4 )NO]2þ was longer than that for SNP. The time-course for the maximum relaxation indicates that the relaxation effect of SNP developed faster than for trans-[RuCl([15]aneN4 )NO]2þ . L opez-L opez et al. [38] observed, in pulmonary arteries of male piglets, that the time-course of relaxant responses to a single concentration of SNP was 250 s. The differences between our results and those observed by Lopez-Lopez et al. [38] may be related to the vascular bed or animal species. These authors suggest that extracellular release of NO is not the mechanism for relaxant effect of SNP. Thus, these findings may explain why the time-course for relaxation is slower for trans-[RuCl([15]aneN4 )NO]2þ than for SNP. In accordance with these authors Kowaluk et al. [40] demonstrated in studies with bovine coronary arterial smooth muscle cells, that SNP requires cellular metabolism to produce NO, and that a membrane catalyzes NO generation associated NOgenerating activity. Furthermore, Angulo et al. [37]
demonstrated that glycosylated human hemoglobin (GHHb) did not modify the vasodilatations of rat aorta induced by SNP; however, GHHb inhibited the vasorelaxant effects of exogenous NO. Another important finding in this study is that this new NO donor compound requires chemical reduction to induce relaxation. This mechanism was demonstrated when the concentration–response curves for trans[RuCl([15]aneN4 )NO]2þ were studied in the absence of the reducing agent. The preparations were pre-contracted with KCl, a mechanism not mediated by receptors, but by cell membrane depolarization, which results in the opening of voltage-dependent calcium channels [41,42]. The increased concentrations of intracellular calcium ultimately induce contraction as a result of activation of calcium-sensitive protein kinases, such as the calmodulin-dependent myosin light chain kinase that phosphorylates the light chain of myosin which is associated with the development of tension [43]. Since the relaxation induced by trans-[RuCl([15] aneN4 )NO]2þ was completely abolished by the addition of a high concentration of KCl to the extracellular medium, two hypothese could be proposed: (1) that potassium channels are more important for trans[RuCl([15]aneN4 )NO]2þ relaxation, or (2) that the effect observed for trans-[RuCl([15]aneN4 )NO]2þ could be related to the absence of reducing agent. NO induces vascular smooth muscle relaxation through the activation of soluble guanylyl cyclase, which transforms GTP to cGMP. Thus, the cGMP produced acts as a second messenger activating PKG, which in turn phosphorylates various proteins, producing vasorelaxation [44]. The increase in cGMP levels and the consequent activation of PKG reduces intracellular calcium concentrations by different mechanisms such as potassium channel activation and inhibition of Ca2þ L-type channels [45] or direct activation of potassium channels by NO [46]. To verify whether the absence of relaxation for trans[RuCl([15]aneN4 )NO]2þ is due to the absence of reductor agent or blockage of potassium channels, concentration–response curves for trans-[RuCl([15] aneN4 )NO]2þ were obtained in the presence of TEA, a non-selective potassium channel blocker. Our data suggest that the participation of potassium channels sensitive to TEA is important for the relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ but it did not abolish
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the relaxation. Furthermore, these results indicate that trans-[RuCl([15]aneN4 )NO]2þ needs to be chemically reduced to release NO and induce relaxation and that the potassium channels sensitive to TEA also participate of this relaxation. We also investigated whether the relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ is really due to NO release, based on the scavenger properties of oxyhemoglobin on the relaxant response induced by trans[RuCl([15]aneN4 )NO]2þ . Classic study demonstrated that micromolar or higher (non physiological) concentrations of free oxyhemoglobin bind and inactivate NO [47]. In our study, the exposure of the aortic rings to oxyhemoglobin abolished the relaxation elicited by trans-[RuCl([15]aneN4 )NO]2þ . L opez-L opez et al. [38] observed, in piglet pulmonary arteries, that oxyhemoglobin, which is unable to penetrate into the cells and only scavenges extracellular NO, markedly inhibited exogenous NO relaxation and had no effect on SNPinduced relaxation. Interestingly, these results confirm that NO release from trans-[RuCl([15]aneN4 )NO]2þ occurs outside the smooth muscle cells. Taken together, the present findings demonstrated that the new complex trans-[RuCl([15]aneN4 )NO]2þ induced vascular relaxation in the presence of reducing agent and that the relaxation induction time-course for trans-[RuCl([15]aneN4 )NO]2þ is longer than that for SNP. We also conclude that potassium channels sensitive to TEA contribute to this relaxation and that this ruthenium complex releases NO outside the vascular smooth muscle cells.
Acknowledgments This work was supported by grants from FAPESP, CNPq, and Pronex.
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Biology and Chemistry
Nitric Oxide 10 (2004) 83–91 www.elsevier.com/locate/yniox
A macrocyclic nitrosyl ruthenium complex is a NO donor that induces rat aorta relaxation Daniella Bonaventura,a Fabiana de S. Oliveira,a Vanessa Togniolo,a Antonio C. Tedesco,b Roberto S. da Silva,a and Lusiane M. Bendhacka,* a
Laborat orio de Farmacologia, Depto. de Fısica e Quımica, Faculdade de Ci^encias Farmac^euticas de Ribeir~ao Preto, USP Av. do Cafe s/no, 14040-903 Ribeir~ao Preto, SP, Brazil b Depto. de Quımica, FFCLRP-USP, 14040-903 Ribeir~ao Preto, SP, Brazil Received 21 October 2003; received in revised form 17 March 2004
Abstract The vasorelaxation induced by a nitrosyl macrocyclic ruthenium complex, proposed as a new nitric oxide (NO) carrier, was studied in rat isolated aorta. The compound trans-[RuCl([15]aneN4 )NO]2þ was characterized by elemental analysis, UV–visible spectrum, and infrared spectrum. Based on the electrochemical process, the reduction of the compound was followed by NO release, which was also observed using norepinephrine as a reducing agent and NO released was analyzed by a sensor. Vasorelaxation induced by this NO donor was studied and compared to those obtained with sodium nitroprusside (SNP). The relaxation induced by the compound was concentration-dependent in denuded rat aortas and occurred only in pre-contracted arteries with norepinephrine. The macrocyclic compound induced relaxation with a similar efficacy as SNP, although the potency of SNP was slightly greater. The time to reach maximum relaxation (595 s) was longer than that of SNP (195 s). Relaxation was completely abolished by oxyhemoglobin, a known NO scavenger. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Macrocyclic nitrosyl ruthenium complex; Nitric oxide donor; Sodium nitroprusside; Vasorelaxation; Rat aorta
Nitric oxide (NO), a lipid-soluble and unstable gas, is one of the most fascinating substances in biological chemistry. NO has been identified as an important signaling molecule playing a crucial role in the cardiovascular and nervous systems [1–4]. The action of NO has also been found in a variety of physiological and pathological processes in different cells and tissues, such as neurotransmission, blood pressure control, inhibition of platelet aggregation, and immunological responses [5,6]. Most of the effects of NO are mediated via the stimulation of soluble guanylyl cyclase (sGC), which catalyses conversion of guanosine-50 -trisphosphate (GTP) to cyclic guanosine-30 ; 50 -monophosphate (cGMP) [7]. Despite the great importance of NO in the biological system, its pharmacological studies have been limited due to its high reactivity and short half-life [8,9]. The * Corresponding author. Fax: +55-16-6332960. E-mail address: [email protected] (L.M. Bendhack).
1089-8603/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2004.03.004
free radical nature of NO has hindered the understanding of many problems associated with its diffusional delays or associated with the effect that NO presents on its action sites. The development of molecules that can release NO can overcome this kind of problem and also be potentially developed as therapeutic agents. One of the most common compounds used as a NO delivery agent in pharmacological and clinical studies is the sodium nitroprusside salt (SNP)— [Fe(CN5 )NO]2 [10,11]. The therapeutic properties of SNP are related to NO release, which occurs via the redox conversion of SNP to its one or two electron-reduced forms [12–14]. SNP requires cellular metabolism to produce NO and extracellular release of NO is not the mechanism for the relaxant effect of SNP. On the other hand, SNP releases cyanide in addition to NO which exerts multiple effects including the activation of sGC and cell toxicity that is an important limitation of the pharmacological use of
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Fig. 1. (A) Molecular structure of trans-[RuCl([15]aneN4 )NO]2þ ; (B) Chem 3Dd calculated trans-[RuCl([15]aneN4 )NO]2þ structure (hydrogen atoms are omitted).
SNP [15]. Furthermore, it is very important to develop compounds that display relatively low thermal reactivity, but could become active yielding NO under a physical or chemical stimulation. Research in the NO field has expanded considerably in the last few years, particularly with respect to the application of new NO drugs to medicine. Metalonitrosyl complexes, for example, are a class of compounds that can be used as agents potentially capable of NO releasing in vivo [16–18]. The strategy used by specialists is to synthesize compounds that act as a NO carrier for activation of the release of NO in a controlled manner, of the order of minutes. These compounds are particularly relevant as vasodilators, since the slow release of NO could avoid the reflex side effects on the cardiovascular system usually present when drugs such as SNP are used. The vasodilator effects of compounds such as organic nitrites (e.g., nitroglycerine, NTG) as well inorganic SNP are mediated by the generation of NO [19]. Although SNP and NTG are widely used in the treatment of various cardiovascular disorders, there are many limitations for their use, and both classes of compounds have undesirable side effects [20]. Continuous exposure to nitroglycerine leads to the development of nitrate tolerance [21] characterized by an impairment of the NO-cGMP signaling cascade due to oxidative stress [15]. High doses of SNP or its prolonged use are associated with the accumulation of cyanide and the metabolite thiocyanate [22], which could impair hepatic function. Several new metal complexes have been studied as a new class of NO donors [23] including nitrosyl ruthenium complexes some of which exhibit low toxicity and are quite soluble in aqueous solutions. Recently, Lang et al. [24] have found that [RuCl(cyclam)NO]2þ , where cyclam
is a tetraazamacrocyclic ligand of 14 members, is stable in physiological conditions and can act as vasodilator agent due to the NO release. The physical chemistry properties of a macrocyclic complex type [RuL(MACROCYCLE)L0 ]nþ seem to be dependent on the size of the MACROCYCLE [25], which influence very greatly the properties of resulting metal complexes. Thus, we decide to study some kinetic NO releasing properties and present pharmacological assays for a new macrocyclic nitrosyl ruthenium complex, which was characterized as trans-[RuCl([15]aneN4 )NO]2þ , where [15]aneN4 is a tetraazamacrocyclic ligand of 15 members (Fig. 1). The synthesis trans-[RuCl([15]aneN4 )NO]2þ complex was recently described [26]. Furthermore, a comparison of the thermodynamic and photochemical properties of trans[RuCl([15]aneN4 )NO]2þ and trans-[RuCl(cyclam)NO]2þ complexes was proved to be different [26]. Considering the chemical characteristics of the trans[RuCl([15]aneN4 )NO]2þ complex, the aim of the present work was to study the effect of the NO donor of nitrosyl ruthenium complex as a pro-drug that could release NO only when it is induced and to investigate the possible mechanisms of vascular relaxation. The vasorelaxation induced by this inorganic NO donor was studied and its effects were compared to those obtained with SNP.
Experimental procedures Measurements The pH measurements were made using a DM 20 pH meter (Digimed). The UV–vis spectra were recorded on
D. Bonaventura et al. / Nitric Oxide 10 (2004) 83–91
a Hitachi U-3501. IR spectra were recorded on a Protege 460 series FT-IR spectrometer, using solid samples pressed in KBr pellets. The NO measurements were performed using the ISO-NO NO-meter and the DUO-18 acquisition board. The sensitivity of this apparatus ranges from 1 nmol/L to 20 lmol/L, with a 2 mm sensor, which directly detects NO concentration by an amperometric technique. The setup was calibrated following a published procedure [27–29]. Synthesis of the complexes The recrystallized complex salts trans-[RuCl2 ([15] aneN4 )]Cl and trans-[RuCl([15]aneN4 )NO]Cl were prepared in accordance with the procedure published by Oliveira et al. [30]. The trans-[RuCl([15]aneN4 )NO]PF6 complex was characterized by IR mNO 1884 cm1 , UV–Vis (1 lmol/L HCl): 268 nm (e ¼ 4:42 103 mol1 L cm1 ) and 352 nm (e ¼ 2:40 102 mol1 L cm1 ). Elemental analysis calculated for C11 H26 N5 Cl3 ORu: C, 29.24; H, 5.80; N, 15.50. Found: C, 29.10; H, 5.87; N, 15.35. The trans-[RuCl([15]aneN4 )(H2 O)]þ complex was synthesized in situ by reduction of trans-[RuCl2 ([15]aneN4 )]þ with zinc amalgam in 0.1 mol/L trifluoromethanesulfonic acid (HTFMS). The free chloride obtained by reduction of dichloro(macrocyle)ruthenium(III) was successively removed from the solution by passing in an ionic exchange column. The aquachloro(1,4,8,12-tetraazacyclopentadecane)ruthenium(II) synthesized was characterized by cyclic voltammetry method and compared to the published results [25]. The formal reduction potential was found as +0.24 V vs Ag/AgCl in 0.1 mol/L HTFMS in agreement with the previously described trans[RuCl([15]aneN4 )(H2 O)]þ [25]. Vessel preparation Male Wistar rats (400–450 g) were killed by decapitation in accordance with the Ethical Animal Committee, Ribeir~ ao Preto Campus, University of S~ ao Paulo, Brazil. The thoracic aorta was quickly removed, dissected free, and cut into 4 mm long rings. Since the response to trans-[RuCl([15]aneN4 )NO]2þ does not require intact endothelium, in the present study we investigated the relaxation induced by this compound in endothelium-denuded arteries. The endothelium was mechanically removed by gently rolling the lumen of the vessel on a thin wire. The aortic rings were placed between two stainless-steel stirrups and connected to an isometric force transducer (Letica Scientific Instruments) to measure tension in the vessels. The rings were placed in a 10 ml organ chamber containing Krebs solution with the following composition (mmol/L): NaCl 130, KCl 4.7, KH2 PO4 1.2, MgSO4 1.2, NaHCO3 14.9, glucose 5.5, CaCl2 1.6. The solution was maintained at pH 7.4 gassed with 95% O2 and 5% CO2 at 37 °C.
85
The rings were initially stretched to a basal tension of 1.5 g (previously determined by length–tension relationship experiments), before allowing them to equilibrate for 60 min in the bath fluid, which was changed every 15– 20 min. Endothelial integrity was qualitatively assessed by the degree of relaxation caused by acetylcholine (ACh, 1 lmol/L) in the presence of contractile tone induced by phenylephrine (0.1 lmol/L) [31]. Since our studies required endothelium-denuded aortas, the rings were discarded if there was any degree of relaxation, to avoid the possible influence of endothelial factors. Experimental protocols Relaxant effect of trans-[RuCl([15]aneN4 )NO]2þ after pre-contraction with norepinephrine To examine whether trans-[RuCl([15]aneN4 )NO]2þ induces smooth muscle cell relaxation, aortic rings were pre-contracted with 0.1 lmol/L norepinephrine and when the contraction had reached a plateau, trans[RuCl([15]aneN4 )NO]2þ (0.1 nmol/L to 100 lmol/L) was cumulatively added. Similar protocol was accomplished for SNP. Time-course for the relaxation induced by trans[RuCl([15]aneN4 )NO]2þ trans-[RuCl([15]aneN4 )NO]2þ (100 lmol/L) was added to the organ chamber when a stable contraction in response to 0.1 lmol/L norepinephrine was achieved. Time-course for relaxation induced by this compound was evaluated and similar protocol was accomplished for SNP. Relaxant effect of trans-[RuCl([15]aneN4 )NO]2þ after pre-contraction with KCl In another set of experiments, cumulative concentration–response curves for trans-[RuCl([15]aneN4 )NO]2þ (0.1 nmol/L to 100 lmol/L) were obtained after precontraction with 60 mmol/L KCl in the absence of a reductor agent. Contribution of potassium channels to the relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ Tetraethylammonium (TEA 100 lmol/L, 1 mmol/L, and 5 mmol/L), a non-selective potassium channel blocker, was used to study the contribution of potassium channels to trans-[RuCl([15]aneN4 )NO]2þ -induced relaxation. TEA was added 30 min before the addition of norepinephrine (0.1 lmol/L). Subsequently, cumulative concentration–response curves for trans-[RuCl([15] aneN4 )NO]2þ were constructed in the presence of TEA. Effect of oxyhemoglobin on the relaxation of trans[RuCl([15]aneN4 )NO]2þ The oxidation of hemoglobin was performed as previously described [32]. Oxyhemoglobin (HbO2 10 lmol/L),
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a nitric oxide scavenger, was added 30 min before the addition of 0.1 lmol/L norepinephrine. Subsequently, cumulative concentration–response curves to trans[RuCl([15]aneN4 )NO]2þ were constructed. Statistical analysis Data are expressed as means SEM. In each set of experiments, n indicates the number of rats studied. Two pharmacological parameters, maximal effect (Emax ) and pD2 , obtained from concentration–response curves for trans-[RuCl([15]aneN4 )NO]2þ and SNP were used to analyze the data. Emax was considered as the maximal amplitude response reached in the concentration-effect curves for relaxant agents. We consider the maximal relaxing effect for 15 ane when the concentration used reached the baseline. The concentrations of agents producing a half-maximal relaxation amplitude (EC50 ) were determined after logit transformation of the normalized concentration–response curves and were reported as the negative logarithm (pD2 ) of the mean of individual values for each tissue using the GraphPad Prism version 3.0 (GraphPad Software Corporation San Diego, CA). Statistical significance was tested by one way ANOVA (post-test: Newman–Keuls) and StudentÕs t test, and values of p < 0:05 were considered to be significant. Drugs Acetylcholine, norepinephrine, tetraethylammonium, phenylephrine, and sodium nitroprusside were purchased from Sigma Chemical (St. Louis, MO, USA). Hemoglobin was purchased from Calbiochem–Novabiochem (La Jolla, CA, USA). Reagents RuCl3 3H2 O, 1,4,8,12-tetraazacyclopentadecane ([15] aneN4 ) was purchased as a highly pure reagent from Aldrich Chemicals and used as supplied, sodium nitrite was purchased from Mallinckrodt. Double distilled H2 O was used for all experiments. All preparations and measurements were carried out under an argon atmosphere and protected from light.
Fig. 2. Electronic spectrum of trans-[RuCl([15]aneN4 )NO]2þ in aqueous solution (solid line) and after reduction at )0.50 V vs Ag/AgCl (dash line). Concentration of the trans-[RuCl([15]aneN4 )NO]2þ complex ¼ 0.21 mmol/L. Inset: Differential pulse voltammogram of trans[RuCl([15]aneN4 )NO]2þ in 0.1 mol/L trifluoromethanesulfonic acid.
electrochemical characteristics of the trans-[RuCl([15] aneN4 )NO]2þ complex in aqueous solution were studied using a differential pulse voltammogram, which exhibited a large peak at 0.276 V vs Ag/AgCl, with a probable contribution of the reduction of the nitrosyl ligand which was inferred by comparison to similar systems [33]. The electro-reduction of trans-[RuCl([15]aneN4 )NO]2þ was performed in 0.1 mol/L trifluoromethanesulfonic acid or in phosphate buffer solution, pH 7.40, at )0.50 V vs Ag/ AgCl. The product of this reaction was characterized in situ by UV–Vis spectrum and attributed to the formation of trans -[RuCl([15]aneN4 )H2 O]þ (Fig. 2), by comparison to the synthesized aquachloro(1,5,8,12-tetraazacyclopentadecane)ruthenium(II) complex [25]. Based on the electrochemical process, the reduction of trans-[RuCl([15]aneN4 )NO]2þ was also studied using norepinephrine as a reducing agent, a known vasoconstrictor agent. The NO release from the nitrosyl ruthenium complex during reduction was analyzed by a sensor, which directly detects NO concentrations by an amperometric technique. A similar experiment was carried out with [Fe(CN)5 NO]2 (SNP) and the results for both complexes were compared (Fig. 3). Relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ and SNP in rat aortic rings pre-contracted with norepinephrine
Results The nitrosyl ruthenium complex was characterized by elemental analysis, UV–Visible spectrum (UV–Vis), and infrared spectrum (FT-IR). FT-IR is frequently used to characterize the oxidation state of the metal and NO ligands in ruthenium complexes [18]. The NO frequency of trans- [RuCl([15]aneN4 )NO]2þ reported in this paper is 1884 cm1 which indicates that formally a linear NOþ is coordinated to the ruthenium(II) center. The
As shown in Fig. 4, the relaxation induced by this compound was concentration-dependent in denuded rat aortas pre-contracted with 0.1 lmol/L norepinephrine. SNP also induced concentration-dependent relaxation, but was more potent than trans-[RuCl([15]aneN4 )NO]2þ (pD2 : 7.97 0.07, n ¼ 6 and 5.03 0.2, n ¼ 6, respectively). Both compounds, however, induced the same Emax (105.86 3.34%, n ¼ 6 for SNP and 98.35 1.22%, n ¼ 6 for trans-[RuCl([15]aneN4 )NO]2þ ).
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Fig. 3. Time-dependent nitric oxide generation from trans[RuCl([15]aneN4 )NO]2þ (line A) and sodium nitroprusside (line B) in buffer solution at pH 7.40. Concentration of the trans[RuCl([15]aneN4 )NO]2þ complex ¼ SNP ¼ 0.21 mmol/L.
87
Fig. 5. Time-course for sodium nitroprusside (SNP) and trans[RuCl([15]aneN4 )NO]2þ -induced relaxation. Denuded thoracic aortic rings were pre-contracted with 0.1 lmol/L norepinephrine and 0.1 lmol/ L SNP (m, n ¼ 7) or 100 lmol/L trans-[RuCl([15]aneN4 )NO]2þ (s, n ¼ 8) were added. Data are means SEM of n experiments performed on preparations obtained from different animals.
Fig. 4. Effects of trans-[RuCl([15]aneN4 )NO]2þ and sodium nitroprusside (SNP) on rat thoracic aorta pre-contracted with norepinephrine. The arteries were pre-contracted with 0.1 lmol/L norepinephrine and trans-[RuCl([15]aneN4 )NO]2þ (, n ¼ 6) or SNP (j, n ¼ 6) was added cumulative (0.1 nmol/L to 100 lmol/L and 0.1 nmol/L to 0.3 lmol/L, respectively). Data are means SEM of n experiments performed on preparations obtained from different animals.
Fig. 6. Effects of trans-[RuCl([15]aneN4 )NO]2þ on rat thoracic aorta pre-contracted with norepinephrine and KCl. The arteries were precontracted with norepinephrine (n, n ¼ 6) or 60 mmol/L KCl (.; n ¼ 6) and trans-[RuCl([15]aneN4 )NO]2þ was cumulatively added (0.1 nmol/L to 100 lmol/L). Data are means SEM of n experiments performed on preparations obtained from different animals.
Time-course for the relaxation induced by trans[RuCl([15]aneN4 )NO]2þ and sodium nitroprusside
Effect of tetraethylammonium on relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ
The time to reach maximum relaxation was longer for trans-[RuCl([15]aneN4 )NO]2þ (595 s, n ¼ 8) than for SNP (195 s, n ¼ 7), as shown in Fig. 5.
To investigate whether the relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ involves potassium channels, we studied the effect of the following concentrations of TEA. As shown in Fig. 7, TEA (100 lmol/L) shifted the concentration–response curves for trans[RuCl([15]aneN4 )NO]2þ to the right and reduced the maximum effect from 98.28 0.70 to 61.44 3.72% and pD2 values from 5.12 0.16 to 4.46 0.12 (n ¼ 6). Similarly, at 1 mmol/L, TEA reduced the maximum effect to 39.40 2.01% and pD2 to 4.51 0.13 (n ¼ 6) and 5 mmol/L TEA reduced the maximum effect to 33.27 5.69% and pD2 to 4.74 0.27 (n ¼ 6) in the
Relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ in rat aortic rings pre-contracted with KCl In preparations pre-contracted with 60 mmol/L KCl, relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ was inhibited when compared to relaxation observed in aortic rings pre-contracted with norepinephrine (pD2 ¼ 7.97 0.07, n ¼ 6), as shown in Fig. 6.
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the relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ was completely abolished by oxyhemoglobin.
Discussion
Fig. 7. Effects of tetraethylammonium (TEA) on the relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ in denuded rat aortic rings. The figure illustrates the effects of TEA (control: n; n ¼ 6; TEA 100 lmol/L: m; n ¼ 5; TEA 1 mmol/L: d; n ¼ 5 and TEA 5 mmol/L: r; n ¼ 8) on trans-[RuCl([15]aneN4 )NO]2þ -induced relaxation. Tissues were incubated with TEA for 30 min prior to the application of 0.1 lmol/L norepinephrine. Data are means SEM of n experiments performed on preparations obtained from different animals.
concentration–response curves for trans-[RuCl([15]aneN4 )NO]2þ , when compared to control curves. However, at 1 or 5 mmol/L, TEA did not alter the pD2 values when compared to those obtained with 100 lmol/L TEA. Effect of oxyhemoglobin on trans-[RuCl([15]aneN4 ) NO]2þ induced relaxation To investigate whether the relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ could be due to NO release, we studied the effect of this complex in the presence of 10 lmol/L oxyhemoglobin. As shown in Fig. 8,
Fig. 8. Effects of oxyhemoglobin (HbO2 ) on the relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ in denuded rat aortic rings. The figure illustrates the effects of HbO2 10 lmol/L HbO2 (control: n; n ¼ 6; 1 lmol/L: m; n ¼ 8) on trans-[RuCl([15]aneN4 )NO]2þ -induced relaxation. Tissues were incubated with HbO2 for 30 min prior to the application of 0.1 lmol/L norepinephrine. Data are means SEM of n experiments performed on preparations obtained from different animals.
Although some nitrosyl ruthenium complexes have been found to be NO delivery agents, numerous species are unstable in physiological conditions or present side reactions leading to different products in addition to the production of NO [18,34,35]. To avoid the side reaction, macrocyclic complexes have been suggested as nitric oxide delivery agent [25]. In this way, the trans[RuCl(cyclam)NO]2þ has been isolated and some physiological properties studied [24]. These macrocyclic complexes show slow NO release after reduction of coordinated nitrosyl group. Considering that the size of the macrocycle also influences the characteristic of ruthenium macrocyclic complexes, we decided to study some pharmacological properties of trans-[RuCl ([15]aneN4 )NO]2þ . We, herein, report trans-[RuCl([15] aneN4 )NO]2þ to be a stable compound in oxygenated physiological saline solution for at least a week, as observed by its unchangeable ultraviolet–visible spectrum. The electronic spectrum of an aqueous solution of trans-[RuCl([15]aneN4 )NO]2þ displays a band at 268 and 352 nm, which changes under controlled potential reduction (Fig. 2). The shift observed in the absorption spectra after the reduction of trans-[RuCl([15]aneN4 ) NO]2þ in aqueous solution was attributed to the release of nitric oxide bonded to the ruthenium(II). The resulting electronic spectrum after full reduction was due to the formation of trans-[RuCl ([15]aneN4 )(H2 O)]þ , which electronic spectrum was compared to the synthesized aqua-complex, previously described [25]. Similar results were also observed when zinc amalgam or norepinephrine was used as reducing agents, permitting us to propose the mechanism as described in Scheme 1. The investigation of the induced NO-releasing pathway was also supported by in situ NO detection when an aqueous phosphate buffer solution of trans-[RuCl ([15]aneN4 )NO]2þ was submitted to reduction with norepinephrine (Fig. 3). The signal recorded by the NO sensor rose quickly when the reduction was initiated. As the experiment was carried out in an argon atmosphere, there was no consumption of NO, which conducts a stable current after the total release of NO from the nitrosyl ruthenium complex. A similar experiment was performed with SNP. The nitric oxide time-dependent graph of SNP shows a small current in comparison to the trans-[RuCl([15]aneN4 )NO]2þ complex (Fig. 3) for the same concentration used in both species. This dependence on NO concentration may be related to the low stability of SNP in physiological solution, due to the nucleophilic attack of hydroxide ion in the nitrosyl ligand originating a nitro group, as previously described
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89
Scheme 1.
[36]. The side reaction involving consumption of NO after release from SNP is also detected, leading to a current that decreases as a function of time. This procedure is not detected in the process observed with NO release from trans-[RuCl([15]aneN4 )NO]2þ (Fig. 3). The new NO donor, trans-[RuCl([15]aneN4 )NO]2þ , induces relaxation in a concentration-dependent manner in aortic rings pre-contracted with norepinephrine, which was compared to SNP. Relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ was less potent than that induced by SNP, however both donors presented similar efficacy in relaxing the rat aorta. SNP is a known potent, rapid, and efficiently nitrovasodilator compound [15,37,38] which is therapeutically used in hypertensive emergencies, heart failure, and for controlled hypotension during surgery. SNP is thought to produce its vasorelaxant action, at least in part, by the release of NO into the vascular smooth muscle cell. Norepinephrine induces vascular contraction by activating a1 adrenoceptors on the vascular smooth muscle, resulting in the activation of at least two different signal transduction cascades: the phospholipase C-induced phosphoinositol breakdown with a subsequent release of calcium from intracellular stores and the activation of calcium channels in the outer plasma membrane leading to a calcium influx [39]. The time necessary to obtain the complete concentration–effect curve for relaxation with trans-[RuCl ([15]aneN4 )NO]2þ was longer than that for SNP. The time-course for the maximum relaxation indicates that the relaxation effect of SNP developed faster than for trans-[RuCl([15]aneN4 )NO]2þ . L opez-L opez et al. [38] observed, in pulmonary arteries of male piglets, that the time-course of relaxant responses to a single concentration of SNP was 250 s. The differences between our results and those observed by Lopez-Lopez et al. [38] may be related to the vascular bed or animal species. These authors suggest that extracellular release of NO is not the mechanism for relaxant effect of SNP. Thus, these findings may explain why the time-course for relaxation is slower for trans-[RuCl([15]aneN4 )NO]2þ than for SNP. In accordance with these authors Kowaluk et al. [40] demonstrated in studies with bovine coronary arterial smooth muscle cells, that SNP requires cellular metabolism to produce NO, and that a membrane catalyzes NO generation associated NOgenerating activity. Furthermore, Angulo et al. [37]
demonstrated that glycosylated human hemoglobin (GHHb) did not modify the vasodilatations of rat aorta induced by SNP; however, GHHb inhibited the vasorelaxant effects of exogenous NO. Another important finding in this study is that this new NO donor compound requires chemical reduction to induce relaxation. This mechanism was demonstrated when the concentration–response curves for trans[RuCl([15]aneN4 )NO]2þ were studied in the absence of the reducing agent. The preparations were pre-contracted with KCl, a mechanism not mediated by receptors, but by cell membrane depolarization, which results in the opening of voltage-dependent calcium channels [41,42]. The increased concentrations of intracellular calcium ultimately induce contraction as a result of activation of calcium-sensitive protein kinases, such as the calmodulin-dependent myosin light chain kinase that phosphorylates the light chain of myosin which is associated with the development of tension [43]. Since the relaxation induced by trans-[RuCl([15] aneN4 )NO]2þ was completely abolished by the addition of a high concentration of KCl to the extracellular medium, two hypothese could be proposed: (1) that potassium channels are more important for trans[RuCl([15]aneN4 )NO]2þ relaxation, or (2) that the effect observed for trans-[RuCl([15]aneN4 )NO]2þ could be related to the absence of reducing agent. NO induces vascular smooth muscle relaxation through the activation of soluble guanylyl cyclase, which transforms GTP to cGMP. Thus, the cGMP produced acts as a second messenger activating PKG, which in turn phosphorylates various proteins, producing vasorelaxation [44]. The increase in cGMP levels and the consequent activation of PKG reduces intracellular calcium concentrations by different mechanisms such as potassium channel activation and inhibition of Ca2þ L-type channels [45] or direct activation of potassium channels by NO [46]. To verify whether the absence of relaxation for trans[RuCl([15]aneN4 )NO]2þ is due to the absence of reductor agent or blockage of potassium channels, concentration–response curves for trans-[RuCl([15] aneN4 )NO]2þ were obtained in the presence of TEA, a non-selective potassium channel blocker. Our data suggest that the participation of potassium channels sensitive to TEA is important for the relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ but it did not abolish
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the relaxation. Furthermore, these results indicate that trans-[RuCl([15]aneN4 )NO]2þ needs to be chemically reduced to release NO and induce relaxation and that the potassium channels sensitive to TEA also participate of this relaxation. We also investigated whether the relaxation induced by trans-[RuCl([15]aneN4 )NO]2þ is really due to NO release, based on the scavenger properties of oxyhemoglobin on the relaxant response induced by trans[RuCl([15]aneN4 )NO]2þ . Classic study demonstrated that micromolar or higher (non physiological) concentrations of free oxyhemoglobin bind and inactivate NO [47]. In our study, the exposure of the aortic rings to oxyhemoglobin abolished the relaxation elicited by trans-[RuCl([15]aneN4 )NO]2þ . L opez-L opez et al. [38] observed, in piglet pulmonary arteries, that oxyhemoglobin, which is unable to penetrate into the cells and only scavenges extracellular NO, markedly inhibited exogenous NO relaxation and had no effect on SNPinduced relaxation. Interestingly, these results confirm that NO release from trans-[RuCl([15]aneN4 )NO]2þ occurs outside the smooth muscle cells. Taken together, the present findings demonstrated that the new complex trans-[RuCl([15]aneN4 )NO]2þ induced vascular relaxation in the presence of reducing agent and that the relaxation induction time-course for trans-[RuCl([15]aneN4 )NO]2þ is longer than that for SNP. We also conclude that potassium channels sensitive to TEA contribute to this relaxation and that this ruthenium complex releases NO outside the vascular smooth muscle cells.
Acknowledgments This work was supported by grants from FAPESP, CNPq, and Pronex.
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