The inducing NO-vasodilation by chemical reduction of coordinated nitrite ion in cis-[Ru(NO2)L(bpy)2]+ complex

Article abstract of DOI:10.1039/b803441a

The synthesis of [Ru(NO₂)L(bpy)₂]⁺ (bpy = 2,2′-bipyridine and L = pyridine (py) and pyrazine (pz)) can be accomplished by addition of [Ru(NO)L(bpy)₂](PF₆) ₃ to aqueous solutions of physiological pH. The electrochemical processes of [Ru(NO₂)L(bpy)₂]⁺ in aqueous solution were studied by cyclic voltammetry and differential pulse voltammetry. The anodic scan shows a peak around 1.00 V vs. Ag/AgCl attributed to the oxidation process centered on the metal ion. However, in the cathodic scan a second peak around -0.60 V vs. Ag/AgCl was observed and attributed to the reduction process centered on the nitrite ligand. The controlled reduction potential electrolysis at -0.80 V vs. Ag/AgCl shows NO release characteristics as judged by NO measurement with a NO-sensor. This assumption was confirmed by ESI/MS⁺ and spectroelectrochemical experiment where cis-[Ru(bpy) ₂L(H₂O)]²⁺ was obtained as a product of the reduction of cis-[Ru^II(NO₂)L(bpy)₂] ⁺. The vasorelaxation observed in denuded aortic rings pre-contracted with 0.1 μmol L^-1 phenylephrine responded with relaxation in the presence of cis-[Ru^II(NO₂)L(bpy)₂]⁺. The potential of rat aorta cells to metabolize cis-[Ru^II(NO ₂)L(bpy)₂]⁺ was also followed by confocal analysis. The obtained results suggest that NO release happens by reduction of cis-[Ru^II(NO₂)L(bpy)₂]⁺ inside the cell. The maximum vasorelaxation was achieved with 1 × 10^-5 mol L^-1 of cis-[Ru^II(NO₂)L(bpy)₂] ⁺ complex.

Full text of DOI:10.1039/b803441a

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PAPER
www.rsc.org/dalton | Dalton Transactions
The inducing NO-vasodilation by chemical reduction of coordinated nitrite ion
in cis-[Ru(NO2)L(bpy)2]+ complex
Zeˆnis Novais da Rocha,a Mario Se´rgio Pereira Marchesi,b Josiane Cristina Molin,b Claure N. Lunardi,b
Katrina M. Miranda,c Lusiane Maria Bendhack,b Peter C. Fordd and Roberto Santana da Silva*b,e
Received 28th February 2008, Accepted 12th June 2008
First published as an Advance Article on the web 7th July 2008
DOI: 10.1039/b803441a
The synthesis of [Ru(NO2)L(bpy)2]+ (bpy = 2,2ꢀ-bipyridine and L = pyridine (py) and pyrazine (pz))
can be accomplished by addition of [Ru(NO)L(bpy)2](PF6)3 to aqueous solutions of physiological
pH. The electrochemical processes of [Ru(NO2)L(bpy)2]+ in aqueous solution were studied by cyclic
voltammetry and differential pulse voltammetry. The anodic scan shows a peak around 1.00 V vs.
Ag/AgCl attributed to the oxidation process centered on the metal ion. However, in the cathodic scan a
second peak around −0.60 V vs. Ag/AgCl was observed and attributed to the reduction process
centered on the nitrite ligand. The controlled reduction potential electrolysis at −0.80 V vs. Ag/AgCl
shows NO release characteristics as judged by NO measurement with a NO-sensor. This assumption
was confirmed by ESI/MS+ and spectroelectrochemical experiment where cis-[Ru(bpy)2L(H2O)]2+ was
obtained as a product of the reduction of cis-[RuII(NO2)L(bpy)2]+. The vasorelaxation observed in
denuded aortic rings pre-contracted with 0.1 lmol L−1 phenylephrine responded with relaxation in the
presence of cis-[RuII(NO2)L(bpy)2]+. The potential of rat aorta cells to metabolize
cis-[RuII(NO2)L(bpy)2]+ was also followed by confocal analysis. The obtained results suggest that NO
release happens by reduction of cis-[RuII(NO2)L(bpy)2]+ inside the cell. The maximum vasorelaxation
was achieved with 1 × 10−5 mol L−1 of cis-[RuII(NO2)L(bpy)2]+ complex.
by chemical reduction. Mechanisms have been proposed for the
1
Introduction
in vivo conversion of free NO2− to NO, for example nonenzymatic
disproportionation by stomach acid15 and enzymatic reduction
of nitrite to NO by xanthine oxidoreductase, hemoglobin or
myoglobin16–19 have all been proposed as biologically significant.
Nitric oxide (NO) is a stable free molecule produced in various
physiological tissues and has become recognized as a major effec-
tor molecule in a diverse array of physiological and pathological
processes.1 In addition, certain disease states are associated with
nitric oxide deficiencies, and this has raised interest in exogenous
donors that can deliver NO to specific targets under external
stimulation.2–8 Among them are metal nitrosyl coordination com-
pounds LxM(NO) successfully employed as NO delivery agents
by accessing the reduction potential of the nitrosyl complex or by
photochemical stimulation.2–12
Perhaps the most studied NO donors among metal nitrosyls
are those based on ruthenium.5–12 However, a limiting factor is
the tendency for RuII–NO+ species to undergo electrophilic attack
by hydroxide or water to form the nitro analog RuII–NO2 (eqn
(1)), so such species may function as NO-donors only at low
pH (<4.0).5 Although the nitrite ruthenium species might still
serve as photochemical NO delivery agents,13,14 it is less clear that
the coordinated nitrite ligand could be activated to generate NO
LxRuII–NO+ + OH− (or H2O) ꢀ LxRuII–NO2
−
+ H+ (or 2 H+)
(1)
In the present study, we consider the possibility that nitrite
coordinated to ruthenium(II) could induce the vasodilation effect
by reduction of RuII–NO2 to provide a source of exogenous
−
NO. Here we present some electrochemical and pharmacological
results involving the use of cis-[RuII(NO2)L(bpy)2]+ complexes
(L = pyridine (1) and pyrazine (2)) as nitric oxide delivery agents.
2
Experimental section
2.1 Apparatus
The ultraviolet-visible (UV-Vis) spectra were recorded on a Hitachi
U-3501 and Genesys-2 apparatus from Spectronic. Infrared (IR)
spectra were recorded on a Protege´ 460 series FTIR spectrometer,
using solid samples pressed in KBr pellets. The pH measurements
were made using a 430 pH meter from Corning.
aInstituto de Qu´ımica da Universidade Federal da Bahia – Campus Ondina,
Salvador, Bahia, Brazil
bFaculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Universidade de
Sa˜o Paulo, Av. do Cafe´ s/n, CEP 14040-903, Ribeira˜o Preto, SP, Brazil.
E-mail: silva@usp.br
The electrochemical study was performed with a potentiostat–
cChemistry Department, University of Arizona, Tucson, AZ 85719, USA
R
galvanostat AUTOLABꢀ PGSTAT 30 model consisting of a
dDepartment of Chemistry and Biochemistry, University of California, Santa
Barbara, Santa Barbara, CA 93106-9510, USA
conventional three electrode cell with a Pt wire auxiliary electrode,
an Ag/AgCl reference electrode, and a glassy carbon working
electrode. The supporting electrolyte for electrochemistry in
aqueous solution was 0.1 mol L−1 KCl.
eDepartamento de Qu´ımica, Faculdade de Filosofia, Cieˆncias e Letras de
Ribeira˜o Preto, Universidade de Sa˜o Paulo, Av. dos Bandeirantes 3900,
CEP 14040-901, Ribeira˜o Preto, SP, Brazil
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The HPLC system consisted of a Shimadzu (Kyoto, Japan) liq-
uid chromatography equipped with a LC-10AD pump, Rheodyne
injector model 7125, SPD-10AV UV-Vis detector, and C-R6A
integrator. A CLC-ODS column (250 × 4.6 mm i.d.; Shimadzu,
Japan) with 5 lm particle size linked in an octadecyl chain was
used. Isocratic elution with (%) 20 : 80 ethanol : water containing
0.1 mol L−1 of phosphate buffer solution, pH 7.4, was used at a
constant flow-rate of 1.0 ml min−1, at room temperature. Samples
for analysis were dissolved in the mobile phase and 25 lL volumes
were injected throughout the experiments and used to check the
purity of the complexes.
The NO release was detected and measured with an ISO-NOP
NO-meter from World Precision Instruments that directly detects
NO concentration by an amperometric technique. The sensitivity
of this apparatus ranges from 1 nmol L−1 to 20 lmol L−1, with a
2 mm sensor.
2.5 Pharmacological experimental protocols
2.5.1 Vessel preparation. Male Wistar rats (400–450 g) were
killed by decapitation in accordance with the Ethical Animal
Committee, Ribeira˜o Preto Campus, University of Sa˜o Paulo,
Brazil. The thoracic aorta was quickly removed, dissected free, and
cut into 4 mm long rings. Since the response to nitro ruthenium
complexes 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 and the aortic rings were placed between two stainless-
steel stirrups and connected to an isometric force transducer
(Letica Scientific Instruments) in order to measure tension in the
vessels. The rings were placed in a 10 mL organ chamber contain-
ing Krebs solution with the following composition (mol L−1
103): NaCl 130, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, NaHCO3
14.9, glucose 5.5, and CaCl2 1.6. The solution was maintained
×
◦
at pH 7.4 gassed with 95% O2 and 5% CO2 at 37 C. 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, which was
changed every 15–20 min. Endothelial integrity was qualitatively
assessed by the degree of relaxation caused by acetylcholine (ACh,
1 × 10−6 mol L−1) in the presence of contractile tone induced by
phenylephrine (1 × 10−7 mol L−1).7 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.
2.2 Chemicals and reagents
RuCl3·3H2O, 2,2ꢀ-bipyridine (bpy), pyridine (py) and pyrazine (pz)
were purchased as high purity reagents from Aldrich Chemicals
and used as supplied. Fluo-3 AM was obtained from Sigma Chem.
Co. (St Louis, MO, USA).
2.3 NO-meter calibration
The calibration curve of the selective electrode was constructed by
several dilutions of a known volume of a saturated nitric oxide
solution in 10.0 mL of a phosphate buffer solution (pH 7.4)
previously degassed. For each volume added, the current value in
nA was recorded. The NO concentration was calculated according
to reported molar fraction solubility of NO (2.1 × 10−3 mol L−1 at
25 ◦C).7,14
Nitric oxide gas was produced by the reaction of copper metal
with HNO3 solution (50%) and was passed through a 10 mol L−1
KOH solution to remove any trace of NO2 species. Nitric oxide
2.5.2 Experimental protocols.
2.5.2.1 Relaxant effect of cis-[RuII(NO2)L(bpy)2]+ after
pre-contraction with phenylephrine. To examine whether cis-
[RuII(NO2)L(bpy)2]+ induces smooth muscle cell relaxation,
aortic rings were pre-contracted with 0.1 lmol L−1 phenyle-
phrine (Phe). When the contraction had reached a plateau cis-
[RuII(NO2)L(bpy)2]+ (0.1–50 lmol L−1) was cumulatively added.
2.5.2.2 Time-course for the relaxation induced by cis-
[RuII(NO2)L(bpy)2]+ (L = py and pz) complex. The nitro
ruthenium complexes 50 lmol L−1 (L = pz) and 30 lmol L−1 (L =
py) were added to the organ chamber when a stable contraction in
response to 0.1 lmol L−1 phenylephrine was achieved. Time-course
for relaxation induced by cis-[RuII(NO2)L(bpy)2]+ was evaluated.
2.5.2.3 Effect of oxyhemoglobin on the relaxation induced
by cis-[RuII(NO2)L(bpy)2]+ (L = pyridine, 4-picoline and pi-
razine) complex. The oxidation of hemoglobin was performed
as previously described.21 Oxyhemoglobin (HbO2 10 lmol L−1),
an extracellular NO scavenger, was added 30 min before the
addition of the contractile agonist chosen (0.1 lmol L−1 phenyle-
phrine). Subsequently, cumulative concentration–effect curves
(0.1–50 lmol L−1) were obtained.
−
stock solution was prepared by degassing 10 mL of distilled water
in which the NO gas was bubbled for 30 min.
2.4 Synthesis of the complexes
The complexes cis-[Ru(NO)(NO2)(bpy)2](PF6)2 and cis-
[Ru(NO)L(bpy)2](PF6)2 (L
=
py and pz) were prepared
according to methods previously described.20 The complexes were
characterized by UV-Vis and infrared spectroscopy, and then
compared to the published results.20 [RuII(bpy)2(py)NO+](PF6)3:
UV-Vis k (nm) (log e) 334 (3.70), 300 (4.17), and FTIR mNO
1947 cm−1; cis-[RuII(bpy)2(pz)NO+](PF6)3: UV-Vis k (nm) (log e)
432 (3.34), 338 (3.74), 300 (4.26), and FTIR mNO 1950 cm−1.
The cis-[RuII(NO2)L(bpy)2]+ cations, with L = py and pz,
were synthesized in situ by dissolving the nitrosyl species in a
0.01 mol L−1 phosphate buffer solution at pH 7.4. Low-resolution
mass spectra of the cis-[RuII(NO2)L(bpy)2]+ cations with L = py
(m/z = 539.07) and pz (m/z = 540.07) were obtained using a VG
Fisons Platform II single quadrupole mass spectrometer with an
electrospray ionization source run with a Fisons Masslinks data
system.
2.6 Cell culture
Male Wistar rats (400–450 g) were killed by decapitation and
the aortas were quickly dissected and longitudinally opened. The
endothelium and the adventitia were removed. Vascular smooth
muscle cells were isolated by enzymatic digestion from aorta.
Briefly, the aortas were dissected and longitudinally opened. The
endothelium and the adventitia were removed and the tissue
was minced into small pieces and incubated in Ca2+-free Hanks
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solution containing 0.03 mg mL−1 collagenase Type II-S. The
tissue was gently shaken in this solution for 25 min at 37 ◦C
and bubbled with carbogen mixture (95% O2, 5% CO2). After
incubation, 10 mg mL−1 bovine serum albumin (type I) was added
to the vessel fragments in Ca2+-free Hanks solution, and cells were
released by mechanical dispersion with a Pasteur pipette. The
resultant cell suspension was centrifuged at 1000 rpm for 3 min and
resuspended in Dulbecco’s modified Eagle’s medium (DMEM)
containing 2 mmol L−1 glutamine, 20 mmol L−1 HEPES (pH 7.4),
10 000 U mL−1 penicillin, and 10 000 lg mL−1 streptomycin. The
cells were plated on glass coverslips and kept in a humidified 37 ◦C
incubator gassed with 5% CO2. The cells were used 3 h after plating
and were maintained in a serum-free medium.
2.6.1 Ca2+ imaging with fluorescent probe and confocal mi-
croscopy.
2.6.1.1 Dye loading and treatments. To assess [Ca2+]c intensity,
on the day of the experiment vascular smooth muscle cells were
loaded with Fluo-3 AM (10 lmol L−1, Sigma Probes) for 30 min
at room temperature. Excess dye was removed by washing out
the dye with bath solution and allowing 30 min for intracellular
desterification of Fluo-3 AM.
Fig. 1 Differential pulse voltammogram of cis-[Ru(NO2)(bpy)2(py)]+ in
Krebs solution at pH 7.4.
of RuII to RuIII (inset, Fig. 2) by comparison to the results of similar
compounds.20 In the cathodic scan (1.00 to −0.80 V vs. Ag/AgCl) a
new signal appears with less pronounced intensity around −0.60 V
vs. Ag/AgCl (Fig. 1).
2.6.1.2 Microscopy and image analysis. The cells were imaged
in Hanks buffer (pH 7.4). [Ca2+]c was assessed by a confocal
scanning laser microscope (Leica TCS SP5). Fluo-3 AM fluores-
cence was excited with the 488 nm line of an argon ion laser and
the emitted fluorescence was measured at 510 nm. Time course
software was used to capture images of the cells at intervals of 2 s
(xyt) in Live Data Mode acquisition. By applying the computer
software of the LSCM, the intensities of intracellular maximum
and minimum fluorescence were measured. From this data, the
initial fluorescence value at t = 0 s was used for F0 and the final
value of fluorescence for F. In this way the %IF was obtained for
each protocol:
%IF = (F/F0) × 100
where F0 = control fluorescence intensity of Fluo-3 AM and
F = fluorescence intensity of Fluo-3 AM after NO donor cis-
[Ru(NO2)L(bpy)2]+ stimulation on the vascular smooth muscle
cell for 240 s.
The NO donor cis-[Ru(NO2)L(bpy)2]+ (100 lmol L−1) was
added to the cell chamber and the IF was measured every 2 s.
Fig. 2 UV-Vis spectrum of cis-[Ru(NO2)(bpy)2(py)]+ in phosphate buffer
solution pH 7.4 (red) and after controlled potential electrolysis at
−0.8 V vs. Ag/AgCl (black). Inset: Differential pulse voltammogram
of cis-[Ru(NO2)(bpy)2(py)]+; full line: cis-[Ru(NO2)(bpy)2(py)]+; dotted
line: cis-[RuII(OH)(bpy)2(py)]+; dashed line: during controlled potential
electrolysis of cis-[RuII(NO2)(bpy)2(py)]+ at −0.8 V vs. Ag/AgCl.
3
Results and discussion
The direct electroreduction of nitrite ion at most electrode surfaces
leads to the formation of NO and N2O in 0.05 mol L−1 H2SO4 and
NH3 in pH 7.0 buffer solution.22–25 In contrast, we report here
that the cis-[RuII(NO2)L(bpy)2]+ complexes are reduced in a single
electrochemical process to generate nitric oxide at physiological
pH; thus this process could be used as a source of NO in biological
medium.
The electrochemistry of cis-[RuII(NO2)L(bpy)2]+ was investi-
gated by cyclic voltammetry and differential pulse voltammetry.
The cyclic voltammograms of the cis-[RuII(NO2)L(bpy)2]+ com-
plexes exhibit unusual behavior in repetitive scans, which is better
defined by a differential pulse voltammogram (Fig. 1). The anodic
scan shows a peak for cis-[RuII(NO2)L(bpy)2]+ (L = py, E =
1.00 V; L = pz, E = 1.10 V vs. Ag/AgCl) attributed to the oxidation
The potential controlled electrolysis shows significant change on
the anodic scans. The second and consecutive scans increase the
peak height at ∼0.40 V vs. Ag/AgCl (Fig. 2, inset) with subsequent
2+/+
decrease in peak intensity for the {Ru–NO2}
process. For
example the electronic spectrum of the species generated from
reduction of cis-[RuII(NO2)(py)(bpy)2]+ shows new bands at 470,
328(sh), 287 and 240 nm (Fig. 2).
The ESI/MS+ spectrum of the cis-[RuII(NO2)(py)(bpy)2]+ in
aqueous solution shows parent M+ peaks at m/z 539.08 con-
sistent with the above formula. Under reduction, this peak
gradually decreases with the concomitant appearance of a new
peak at m/z 510.00, a molecular weight consistent with that of
4284 | Dalton Trans., 2008, 4282–4287
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cis-[RuII(OH)(py)(bpy)2]+. Therefore, this new species was con-
cluded to be the reduction product of 1.
The nitric oxide release derived from reduction of the nitro
ruthenium complex was also examined by using a NO-sensor
electrode and chemiluminescence techniques. Chemiluminescence
experiments show that NO is the product of cis-[RuII(NO2)-
(py)(bpy)2]+ reductive decomposition in aqueous solution. Solu-
tions of 1 (50 mmol L−1) were incubated in H2O and aliquots were
injected into the reaction chamber of a commercial chemilumines-
cence nitric oxide analyzer (NOA) containing a KI/HOAc solu-
tion at pH 5.4. Under these conditions, cis-[RuII(NO2)(py)(bpy)2]+
showed a current increase, which was attributed to NO re-
lease (Fig. 3). The full reduction potential electrolysis of cis-
[RuII(NO2)(py)(bpy)2]+ performed in Krebs solution of pH 7.4
in the presence of the NO-sensor electrode showed variation of
the measured currents that is consistent with a NO release process
(Fig. 4).
Scheme 1
The effect of the NO originating from the reduction of cis-
[Ru(NO2)(bpy)2(py)]+ was observed from the pharmacological
viewpoint by means of vasorelaxation. It is known that free
NO is a bioregulator for the control of vascular tone, and
vasodilation has been demonstrated in the NO release from several
nitrosyl ruthenium complexes.7,14 The dose-dependence of added
1 was performed in order to achieve the ideal concentration to
cause maximum vasorelaxation, presumably via an intracellular
reduction mechanism. The experiments were carried out both
in the absence and presence of oxyhemoglobin, a known NO-
scavenger.7,14 In these experiments the aortic rings were pre-
contracted with 0.1 lmol L−1 phenylephrine, and when the
contraction had reached the plateau, cis-[Ru(NO2)(bpy)2(py)]+
(0.1–30 lmol L−1) was cumulatively added (Fig. 5). The maximum
vasorelaxation (105.16
1.69%, number of experiments n =
5) was achieved with 1 × 10−5 mol L−1 of 1. The presence of
oxyhemoglobin had only moderate effects on the vasodilation
results with maximum relaxation of 83.88
6.83%, P < 0.05.
This is a good indication that the NO production from 1 is
largely intracellular, since oxyhemoglobin would be a very efficient
scavenger of the extracellular NO, if it were formed from 1 in
solution. Similar results were obtained for 2, with maximum
relaxation in rat aortas of 101.55
0.56%, n = 8, with 1 ×
10−5 mol L−1 2; with oxyhemoglobin the maximum relaxation was
90.56 3.48%, n = 10.
Fig. 3 NO release from cis-[Ru(NO2)L(bpy)2]+ complexes. ● for L = py;
ꢀ for L = pz.
Fig. 5 Relaxation induced by the NO donor cis-[Ru(NO2)(bpy)2(py)]+
([M]). Concentration–response curves for the NO donor were constructed
in denuded aortic rings pre-contracted with 100 nmol L−1 phenylephrine in
the absence (ꢁ, n = 5) or after addition of 10 lmol L−1 oxyhemoglobin (ꢂ,
n = 7). Data are means SEM of n experiments performed on preparations
obtained from different animals. * P < 0.05.
Fig. 6 illustrates the use of a suitably adapted NO-sensor
electrode to detect the formation of NO from the addition of
1 to the aorta tissues in the incubated flask at the same time
that vasodilation occurred. The current increase observed can be
attributed to NO diffusion from the aorta tissue and taken as
an indication of spontaneous NO production after addition of
cis-[Ru(NO2)(bpy)2(py)]+ (30 lmol L−1) to the flask.
Fig. 4 Chronoamperogram of cis-[Ru(NO2)(bpy)2(py)]+ in pH 7.4 Krebs
solution during reduction potential electrolysis at −0.80 V vs. Ag/AgCl.
Based on these NO-sensor electrode measurements we have
described the reduction process of cis-[RuII(NO2)L(bpy)2]+ as in
Scheme 1.
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Fig. 6 Time-course effects of cis-[Ru(NO2)(bpy)2(py)]+ on rat aorta
pre-contracted with phenylephrine. In the sustained contractions,
cis-[Ru(NO2)(bpy)2(py)]+ (30 lmol L−1, n = 5) was added. Data are
Fig. 8 Average fluorescence intensity (%) of vascular smooth muscle cells
preloaded with Fluo-3 AM in control cells (100%) and after addition of
cis-[Ru(NO2)(bpy)2(py)]+ (100 lmol L−1). * P < 0.001.
means
SEM of n experiments performed on preparations obtained
from different animals. Inset: NO measurements during vasodilatation
experiment in rat aorta rings.
vasodilator agent28 and similar to the values described in this work
for cis-[Ru(NO2)(bpy)2(py)]+.
The time-course of the maximum relaxation for 1 was reached
at 1140 s (n = 5) and for 2 it was reached at 1320 s (n = 7).
Conclusion
In addition, to gain a deeper insight into intracellular NO
formation, we examined the potential of rat aorta cells to
metabolize 1, as an example of cis-[RuII(NO2)L(bpy)2]+, using
confocal fluorescence microscopy. Measurements of changes in the
transient Ca2+ concentration according to the fluorescence emis-
sion intensity of Fluo-3 AM show that the contact between cis-
[Ru(NO2)(bpy)2(py)]+ and vascular smooth muscle cells induced
alterations. As shown in Fig. 7, the x–y scanned images show a
decrease in fluorescence emission intensity to 65.1 1.4%, n = 3 in
relation to the control (100%) (P < 0.001) after addition of 1. The
reduction of fluorescence intensity could be attributed to the basal
[Ca2+]c decreases where it was significantly different throughout the
experimental conditions used. Indeed, [Ca2+]c decreased during
In summary, our results suggest that cis-[Ru(NO2)L(bpy)2]+ is
a NO donor agent and induced a concentration-dependent,
endothelium-independent relaxation in rat aortic rings pre-
constricted by phenylephrine. The NO release occurs by a
reduction process and induces aorta relaxation by activation
of soluble guanylyl cyclase by a direct and indirect cGMP-
dependent mechanism. Maybe this compound could be used as
a pharmacological tool or in the future in clinical medicine.
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Fig. 7 Vascular smooth muscle cells were preloaded with Fluo-3 AM and
then stimulated with cis-[Ru(NO2)(bpy)2(py)]+(100 lmol L−1). Serial Ca2+
images of Fluo-3 fluorescence in a single VSMC were recorded at (A) 0,
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The vasorelaxation induced by NO involves mainly the ac-
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version of guanosine triphosphate (GTP) into cyclic guano-
sine monophosphate (cGMP).26 Acting as a second messenger,
cGMP activates a family of serine/threonine protein kinases
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relaxation by decrease of [Ca2+]c and Ca2+ desensitization of
the actin–myosin contractile system.27 The reduction of [Ca2+]c
induced by NO donors produced by a ruthenium complex was
previously described showing a decrease of 40.0 10.0% (n = 4,
P < 0.001) for cis-[RuCl(bpy)2(NO)](PF6).28 These values are
similar to that obtained for sodium nitroprusside, a known
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