In vivo effects of the controlled NO donor/scavenger ruthenium cyclam complexes on blood pressure

In vivo effects of the controlled NO donor/scavenger

ruthenium cyclam complexes on blood pressure$

Fabiana G. Marcondesa, Alessander A. Ferrob, Adriana Souza-Torsonia,Marie Sumitania, Michael J. Clarkec, Douglas W. Francod,

Elia Tfounib,*, Marta H. Kriegera,*,1

aDepartamento de Fisiologia e Biofısica, Instituto de Biologia UNICAMP. Campus Universitario Zeferino Vaz,

13083-970, Barao Geraldo, Campinas-SP, BrazilbDepartamento de Quımica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto,

Universidade de Sao Paulo. Av. dos Bandeirantes, 3900. 14040-901- Ribeirao Preto, SP, BrazilcMerkert Chemistry Center, Boston College, Chestnut Hill, MA 02467, USA

dInstituto de Quımica de Sao Carlos Universidade de Sao Paulo. Av.Dr. Carlos Botelho,

1465 CEP 13560-970, Sao Carlos, SP, Brazil

Received 19 June 2001; accepted 6 December 2001

Abstract

Ruthenium(II/III) complexes able to bind and release NO. were tested in vivo, in conscious Wistar

rats instrumented for continuous blood pressure (BP) measurement and administration of in bolus

injections (5 to 100 nmol/Kg i.v.) of trans-[RuIICl(NO+)(cyclam)](PF6)2 (cyclam-NO) or sodium

nitroprusside (SNP). For normotensive rats, cyclam-NO produced a sustained 10% BP reduction of

basal MAP during 7 ± 0.4 to 11 ± 0.3 min. In acute hypertensive rats, cyclam-NO produced BP

reduction 3-fold larger than in normotensive rats and similar to that of SNP (maximal effect: 41 ± 1.3

vs. 45 ± 2.2 mmHg, respectively). However, the duration of the effect of cyclam-NO was 13 to 21-fold

longer than that of SNP. The hypotensive effect of cyclam-NO was fully blocked in presence of

continuous infusion of a NO. scavenger, carboxy-PTIO (6 mmol/Kg/min), or of the inhibitor of cGMP

0024-3205/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved.

PII: S0024 -3205 (02 )01528 -X

$ Previous results of this work were presented at the First International Conference. Biology, Chemistry and

Therapeutic Applications of Nitric Oxide. San Francisco, California, USA, June 2000. Nitric Oxide: Biology and

Chemistry, 2000, 4, 181-182.

* Corresponding authors. Current Address: Departamento de Ciencias Exatas e Tecnologicas- UESC, Rodovia

Ilhues-Itabuna, Km 16, 45650-000, Ilhues/BA. Tel.: +55-16-602-3748, +55-16-602-3877; fax: +55-16-633-8151.

E-mail addresses: [email protected] (E. Tfouni), [email protected] (M.H. Krieger).1 Tel.: +55-19-3788-6189; fax: +55-192893124.

Life Sciences 70 (2002) 2735-2752

activation, methylene blue (83 nmol/Kg/min), or of the cyclam-NO precursor, trans-[RuCl(tfms)(cy-

clam)](tfms) (cyclam-tfms) (500 mmol/Kg/min). The long lasting BP reduction of cyclam-NO can be

interpreted in terms of a slower rate of NO. release (k-NO = 2.2 � 10�3 s�1 at 35 �C) followingchemical reduction (E00 = 0.10 V vs NHE). In summary, cyclam-NO showed an hypotensive effect

around 20 times longer than SNP in either normotensive or hypertensive rats, which was completely

inhibited by methylene blue or carboxy-PTIO. Continuous infusion of cyclam-tfms completely

blocked the hypotensive effect of cyclam-NO by scavenging the NO. released by the reduced cyclam-

NO. D 2002 Elsevier Science Inc. All rights reserved.

Keywords: NO donor; NO scavenger; Nitric oxide; Cyclam; Ruthenium nitrosyl; Hypotensive effect; Nitroprusside

Introduction

Nitric oxide (nitrogen monoxide, NO), a lipid-soluble and unstable gas [1], is one of the

most fascinating substances in biological chemistry. The NO molecule plays a significant role

in a variety of physiological and pathological processes in different cells and tissues, such as

neurotransmission, blood pressure (BP) control, inhibition of platelet aggregation and im-

munological responses [2,3]. Several of the biological actions of NO are attributed to its

stimulation of soluble guanylate cyclase that acts on GTP to produce cGMP, leading to

vascular relaxation [4]. Therefore, the NO-dependent relaxation of vascular smooth muscle

cell (VSMC) may be regulated locally. This may represent the simplest, essential mechanism

for adaptation of the cardiovascular system in maintaining normal blood pressure [5]. The

importance of the balance between the release, uptake, and degradation of NO for the main-

tenance of normal blood pressure is evident i) in that administration of the NO synthase

inhibitors, such as L-NAME (NG-nitro-L-arginine methyl ester), markedly increase blood

pressure in animals [5,6]; and ii) when occurs decrease of NO bioavailability in the vascular

function resulting from oxidative inactivation by excessive production of the superoxide

anion [7,8].

While the importance of investigating drugs, which act as NO donors is well recognized

[9,10], its unstable nature and short half-life, 0.1 to 6 seconds [11], present challenges from a

pharmacological perspective. Furthermore, the mechanism and extent of dissociation from

NO donors is not fully understood, and such compounds are sometimes unstable with respect

to temperature and light [12]. As a consequence, there is considerable interest in kinetically

and thermodynamically stable compounds that can, with minimal toxic effects, release or

scavenge NO in vivo.

A compound that acts as a NO carrier and could be activated to release nitric oxide in a

controlled manner, in the order of minutes, would be of great relevance. The vasodilating

effects of nitrovasodilators, such as organic nitrites (e.g. nitroglycerine, NTG) and

inorganic sodium nitroprusside (SNP) are mediated by generation of NO [13]. Although

SNP and NTG are widely used in the treatment of various cardiovascular disorders and

F.G. Marcondes et al. / Life Sciences 70 (2002) 2735–27522736

reducing blood pressure during anesthesia, there are limitations to their use, and both

classes of compounds have undesirable side effects [13,14]. Continuous exposure to ni-

troglycerine leads to the development of nitrate resistance and nitrate tolerance. Tolerance

to NTG is a clinically important problem that requires periodic discontinuation to restore

effectiveness [14]. High doses, prolonged use, or use of SNP in patients with compromised

hepatic function, are associated with the accumulation of cyanide and its metabolite thio-

cyanate [13].

Several new metal complexes have been studied recently as NO donors or scavengers,

including nitrosyl ruthenium complexes, nitrite complexes, and related species [15-39],

some of which exhibit low toxicity and are quite robust in aqueous solutions even in the

presence of O2. Coordinated NO, in the nitrosonium (NO+) form, may be released, in vitro,

after activation through irradiation by light at a specific wavelength [25,38-41] or a selective

chemical reduction [42] of the nitrosonium site to the nitrosyl (NO.) form. Recently [33], the

complex trans-[RuCl(NO)(cyclam)](PF6)2 (cyclam-NO) (cyclam = 1,4,8,11-tetraazocyclote-

tradecane) has been isolated and its reactivity studied. The slow loss of NO (k-NO = 6.1 �10�4s�1, at 25 �C, in vitro) from the cyclam-NO complex [33] following a biologically

accessible one-electron reduction (E0 = �0.1 V vs NHE [33]), led us to investigate the

possible hypotensive effect of this complex in biological systems. The cyclam-NO or

SNP complexes were administered in the presence and absence of the NO scavenger

carboxy-PTIO [43], or methylene blue [44], an inhibitor of the synthesis of c-GMP,

and trans-[RuCl(tfms)(cyclam)](tfms) (cyclam-tfms). Since the cyclam-NO complex can be

synthesized by reacting cyclam-tfms with NO [33], the latter complex was tested as a

potential NO scavenger. The biological results are interpreted in light of the chemistry of

these complexes.

Materials and methods

In conducting this research the authors adhered to NIH Animal Care guidelines for use of

animals. All surgical procedures were performed under anesthesia produced by ketamine

(40 mg/Kg i.p., Ketalar, Parke Davis) in conjunction with xylazine (6mg/Kg i.m., Rompum,

Bayer). Adult male Wistar rats (250-300 g) were supplied by Centro de Bioterismo da

Universidade Estadual de Campinas CEMIB-UNICAMP. Cyclam-NO and cyclam-tfms com-

plexes were synthesized as described [33,40].

Arterial pressure and heart rate

Polyethylene cannulae (PE10 connected to PE50) filled with heparinized saline were

implanted into the abdominal aorta (through the femoral artery) for recording blood

pressure and into the iliac vein for administration of drugs or vehicle (pH 7.4 PBS, 0.1 M

phosphate buffer, 0.15 M saline). After catheterization, the rats received a single dose

(40000 U) of penicillin G procaine and were kept in individual home cages under a 12 h

light/dark cycle, receiving standard rat chow and tap water ad libitum. To complete animal

F.G. Marcondes et al. / Life Sciences 70 (2002) 2735–2752 2737

recovery, the drug administration section was performed 24 hours after the catheterization.

Blood pressure was continuously recorded in conscious freely moving unrestrained rats

using a pressure transducer (Gould) and an amplifier attached to the intra-arterial cannula,

and the measurements were acquired in a computer for posterior analysis (DI220 AT-

CODAS data acquisition system, DataQ Instruments Co.). The systolic, diastolic, and

mean arterial pressures (MAP), and the heart rate (HR, peak-to-peak counting) in the basal

condition and during drugs administration were calculated using the Advanced CODAS

software, from which the maximum DMAPs were obtained. The total duration of the

effect refers to the elapsed time from the initial of the BP decrease up to total basal

pressure recovery.

Drug administration

Since SNP is light sensitive, appropriate dilutions of the drug in cold phosphate buffered

saline were prepared immediately before use, protected from light, to prevent its photolysis.

This procedure was standardized to the other complexes, cyclam-NO and cyclam-tfms,

although they are photochemically inert [40], and stable [33,40]. Before the intravenous

administration of cyclam-NO, a control record of blood pressure during approximately

20 minutes was performed until stabilization of pressure and heart rate. Blood pressure

variations after in bolus injection of SNP (MW = 298, Sigma Chemical), cyclam-NO (MW =

802) or cyclam-tfms (MW = 634) were determined in 31 normotensive rats. Equimolar doses

of cyclam-NO, cyclam-tfms and SNP were used. The doses of SNP were chosen following

similar experiments described in the literature [13]. The ranges of doses (5, 10, 25, 30 or

100 nmol/Kg i.v.) of each drug were injected in a randomized sequence protocol. Each rat

received 6-10 injections of PBS or PBS containing SNP, or cyclam-NO, or cyclam-tfms, each

injected in a volume of 0.1 ml. Intervals between administrations of different doses were made

after complete recovery of basal values of blood pressure (3 to 5 min for SNP and � 30 min

for cyclam-NO and cyclam-tfms).

Acute hypertension model

The hypotensive effect (DMAP) of cyclam-NO (10 nmol/Kg i.v. in bolus) was tested in

the presence of continuous infusion with phenylephrine (or the PBS vehicle) made by a

pump (Cole Parmer model HP74900-25). The infusion of 0.11 mg/Kg/min phenylephrine

(L-phenylephrine hydrochloride, Sigma Chemical)) was performed over 1 hour to obtain an

approximately 30% increase in basal blood pressure.

NO scavenger infusion

In another group of 7 rats, the effect of in bolus injection of 5 nmol/Kg of cyclam-NO was

tested during infusion of 0.6 or 6 mmol/Kg/min of carboxy-PTIO ([2-(4-carboxyphenyl)-

4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide], MW 277, Calbiochem). Other 6 rats were

tested in the presence of 83 nmol/Kg/min of methylene blue (C16H18ClN3S, MW 320, Sigma


Chemical) and, finally, in another group of 5 rats, a 5 nmol/Kg in bolus injection of cyclam-

NO was tested during infusion of 50, 250 or 500 nmol/Kg/min of cyclam-tfms.

Evaluation of the toxicity of cyclam-NO

The cytotoxicity of cyclam-NO was evaluated according to standardized methods. The

assays were carried out on a lung fibroblast cell line derived from Chinese hamsters (V-79)

established in culture. Periodic plating was performed as soon as the cells reached the

standardized confluence density. DMEM essential medium was used for culture, sup-

plemented with 10% bovine fetal serum (BFS), 100 IU/ml penicillin and 100 mg/ml

streptomycin sulfate. The material was incubated at 37 �C in a humid atmosphere containing

5% CO2 in a Forma Scientific incubator. Plating was performed with 3�104 viable cells/ml/

well on a 96-well plate. After incubation at 37 �C for 48 h in DMEM supplemented with

10% serum, the culture was exposed for 24 h to different cyclam-NO concentrations in the

0.1-3 mM concentration range. The nucleic acid content (NAC) was used for the direct

evaluation of the number of viable cells. The numbers of cells in the control and the

complexes samples were estimated from the total NAC according to the method of Cingi

et al. [45] The metabolic activity of these cells was evaluated by a colorimetric method

based on the reduction of the tetrazolium salt MTT, according to the method of Mosmann

et al. [46].

Statistical analysis

All values are expressed as mean ± S.E.M. The MAP values obtained before and after drug

administration were compared by using the STAT SOFT Inc. software Statistic version 5,

using the one way ANOVA followed by the Duncan test. Differences were considered

statistically significant when P < 0.05.

Results

Cytotoxicity assay

Fig. 1 shows the V79 cell viability after treatment with cyclam-NO evaluated by two

different assays, MTT and NAC. These tests show that cyclam-NO is particularly non-toxic,

especially when compared with SNP (IC50 = 0.06 mM, Fig. 1, panel B). The IC50 value for

cyclam-NO was not achieved experimentally up to 3.0 mM (Fig. 1, panel A).

Hypotensive effect in conscious normotensive rats

Fig. 2, panel A, shows the effect of intravenous in bolus injections of different

concentrations of cyclam-NO, SNP or cyclam-tfms on mean arterial pressure (MAP) of

conscious normotensive rats. The vasodilator effect upon administration of cyclam-NO


(5.0, 25.0, 30.0, 50.0 and 100 nmol/Kg) on MAP varied from �7 ± 0.4 to �11 ± 0.3 mmHg,

which correspond to an average of ~ 8% decrease of the basal MAP. The highest MAP

reduction, which was produced at 30.0 nmol/Kg, was �11 ± 0.3 mmHg (P < 0.05). When

the doses administered were higher than 30.0 nmol/Kg, the magnitude of the MAP

response did not increase further (Fig. 2, panel A). The hypotensive effect of cyclam-

NO was significantly lower than that produced by equimolar doses of SNP (P < 0.01). The

BP reductions by SNP varied from �12.6 ± 0.8 to �36 ± 1.6 mmHg, and correlated with

dose (P < 0.05). In bolus administration, cyclam-tfms did not result in any significant MAP

variation (Fig. 2, panel A). Fig. 2, panel B, shows the total time duration of the effect on

MAP, produced by the above compounds. The duration of the effect produced by different

doses of SNP varied from 0.5 ± 0.04 to 0.8 ± 0.1 min, almost without dose correlation,

Fig. 1. MTT (dashed lines) and NAC (solid lines) cell viability tests in cultures of fibroblast-like V79 cells. The

percentage of living cells in relations to the control after incubation for 48 h at 37 �C, are plotted versus the

complex concentration (SNP (panel A) or cyclam-NO (panel B)). Data reported as Mean ± S.E.M. of 6 assays.


whereas those produced by cyclam-NO varied from 6.4 ± 1.4 to 10.6 ± 2.4 min (Fig. 2, panel

B). This duration of cyclam-NO effect was, on average, 13-fold longer than the effect of

SNP, reaching a maximum of 21-fold at 25.0 nmol/Kg (P < 0.05). The latency (time to reach

maximal BP decrease) of the effect for SNP varied from 0.12 ± 0.02 to 0.18 ± 0.03 min,

whereas for cyclam-NO varied from 0.62 ± 0.10 to 1.3 ± 0.18 min.

Hypotensive effect in conscious hypertensive rats

Fig. 3, panel A, shows the BP reduction produced by in bolus injection of different

concentrations of cyclam-NO or SNP in acute hypertensive rats under continuous

infusion of phenylephrine (0.11 mg/Kg/min) and in normotensive rats under continuous

PBS infusion. There was no significant effect of PBS on the BP of normotensive rats.

The phenylephrine infusion resulted in a BP increase from 107 ± 2.7 mmHg to 160 ±

Fig. 2. Effect of intravenous in bolus injections of cyclam-NO, SNP or cyclam-tfms on MAP of conscious

normotensive rats. Panel A: effect of different concentrations of the complexes on MAP. Panel B: duration of the

effect of different complexes concentrations. Data reported as Mean ± S.E.M. of 16 rats.


3.9 mmHg (P < 0.05), which lasted for one hour. The effect of SNP upon the BP re-

duction of hypertensive rats was slightly higher than in normotensive rats (P < 0.05 for

5 nmol/Kg dose). However, the effect of cyclam-NO is similar to that of SNP, but

shows a clearer dose-response effect than in normotensive rats. The BP reduction of

SNP varies from �28 ± 1.5 to �45 ± 2.2 mmHg, while that of cyclam-NO from �32 ±

0.5 to �41 ± 1.3 mmHg (significant versus PBS infusion, P < 0.05). Fig. 3, panel B,

shows that in hypertensive rats the duration of the SNP effect varies from 2.8 ± 0.3 to

4.9 ± 0.4 min, while that of cyclam-NO increases from 13 ± 5 to 21 ± 8 min, reaching

a maximum of 21 ± 8 min at 25.0 nmol/Kg and then decreases to 4.5 ± 0.9 min at 100

nmol/Kg. However, these variations are not statistically significant and may only reflect

a tendency.

Fig. 3. Effect of intravenous in bolus injections of cyclam-NO, SNP or cyclam-tfms on MAP of conscious

hypertensive rats (phenylephrine, 0.11 mg/Kg/min i.v.). Panel A: effect of different concentrations of the

complexes on MAP; dashed lines refer to the control performed with normotensive rats under PBS infusion; solid

lines refer to hypertensive rats. Panel B: duration of the effect at different complexes concentrations. Data reported

as Mean ± S.E.M. of 7 rats.


Hypotensive effect in presence of a scavenger or GMPc blockage in normotensive

conscious rats

Fig. 4 shows the BP reduction of in bolus administration of cyclam-NO or SNP under

infusion of PBS, 83 nmol/Kg/min methylene blue, 6 � 102 or 6 � 103 nmol/Kg/min carboxy-

PTIO. Compared to the effect in PBS, the hypotensive effect of SNP was decreased 16 ± 5%,

18 ± 4% and 23 ± 4 % under continuous infusion of methylene blue, and 6 � 102 and 6 �103 nmol/Kg/min carboxy-PTIO, respectively. Notably, a larger decrease in effect is observed

for cyclam-NO. Compared to the effect in PBS, the hypotensive effect of cyclam-NO

decreased 47 ± 7 % and 80 ± 11 % for 6 � 102 and 6 � 103 nmol/Kg/min carboxy-PTIO,

respectively. Methylene blue completely blocks the BP reduction of cyclam-NO.

Hypotensive effect of cyclam-NO in the presence of cyclam-tfms in normotensive

conscious rats

Fig. 5 shows the BP reduction of in bolus administration of 5 nmol/Kg cyclam-NO under

continuous infusion of PBS, 50, 250 or 500 nmol/Kg/min of cyclam-tfms in normotensive

rats. The cyclam-tfms infusion per se (1 ml/h) does not change the BP of normotensive rats.

A marked decrease of the hypotensive effect of cyclam-NO occurs with increasing

Fig. 4. Influence of methylene blue endovenous infusion (83 nmol/Kg/min i.v.) and of carboxy-PTIO infusion (6

� 102 or 6 � 103 nmol/Kg/min i.v.) on the hypotensive effect produced by 5 nmol/Kg i.v. in bolus injection of

cyclam-NO or SNP in normotensive conscious rats. Data reported as Mean ± S.E.M. of 7 rats for methylene blue

and 6 rats for carboxy-PTIO (P < 0.05 compared to PBS group).


concentration of cyclam-tfms. Compared to PBS infusion, the hypotensive effect was

reduced to 41 ± 12 % and 66 ± 14 % with 50 and 250 nmol/Kg/min cyclam-tfms, respec-

tively. Remarkably, the hypotensive effect of cyclam-NO was practically completely blocked

(97 ± 12 %) by a concentration of cyclam-tfms (500 nmol/Kg/min) a 100 times higher than

that of cyclam-NO.

Discussion

The results described in this paper refer to the BP reduction effect of cyclam-NO on

normotensive and hypertensive rats and can be interpreted at least partly on the chemical

properties of the complexes involved [33,40], as shown below.

Hypotensive effects in normotensive and hypertensive rats

Cyclam-NO is water soluble, quite stable, and in solution ionizes to yield trans-

[RuIICl(NO+)(cyclam)]2+, which can be reduced to trans-[RuIICl(cyclam)(NO.)]+ (Eq. 1):

trans�½RuIIClðNO2

ÞðcyclamÞ�2þ þ e� ��!k1 trans�½RuIIClðcyclamÞðNO*Þ�þ ð1Þ

Fig. 5. Influence of PBS, 50, 250 or 500 nmol/Kg/min cyclam-tfms endovenous infusion on the hypotensive effect

produced by 5 nmol/Kg i.v. in bolus injection of cyclam-NO in normotensive conscious rats. Data reported as

Mean ± S.E.M. of 8 rats.(P < 0.05 for comparison versus PBS group).


The formation of trans-[RuIICl(cyclam)(NO.)]+ is followed by the rapid loss of chloride by

first-order kinetics [33], with k = 1.5 s�1, forming trans-[RuII(H2O)(cyclam)(NO.)]+ (Eq. 2):

trans�½RuIIClðcyclamÞðNO*Þ�þ þ H2O

!k2 trans�½RuIIðH2 OÞðcyclamÞðNO*Þ�2þ þ Cl� ð2Þ

This chloride loss is suppressed by 0.1 M chloride, and both, trans-[RuII(H2O)(cyclam)

(NO.)]+ and trans-[RuIICl(cyclam)(NO.)]+ release NO. (Eqs. 3 and 4) with specific rate

constants of 6.2 � 10�4 s�1 at 25 �C, and 2.2 � 10�3 s�1 at 35 �C, which are independent of

chloride concentration up to 0.25 M [33,40]:

trans�½RuIIðH2OÞðcyclamÞðNO*Þ�2þ þ H2O

!k3 trans�½RuIIðH2OÞ2ðcyclamÞ�2þ þ NO* ð3Þ

trans�½RuIIClðcyclamÞðNO*Þ�þ þ H2O

! trans�½RuIIClðH2OÞðcyclamÞ�þ þ NO* ð4Þ

This slow rate of release allowed the observation of the EPR spectra of the coordinated NO.

in trans-[RuII(H2O)(cyclam)(NO.)]+ [34].

The reduction potential of trans-[RuIICl(NO+)(cyclam)]2+, of -0.10 V vs NHE [33] is

accessible to glutathione [47], flavins, and other single electron reductants that may ultimately

be reduced by NADH (�0.32 V vs NHE) [48]. Considering that in blood, chloride loss is

suppressed, then, the hypotensive effects observed in conscious rats are consistent with the

release of NO. from trans-[RuIICl(cyclam)(NO.)]+, following the reduction of trans-

[RuIICl(NO+)(cyclam)]2+.

The hypotensive effect of cyclam-NO is smaller in normotensive rats (Fig. 2), when

compared to the effect produced by SNP. For hypertensive rats, the effects of cyclam-NO and

SNP are similar (Fig. 3). However, for both, normotensive and hypertensive rats, the duration

of the effects of cyclam-NO are longer than those of SNP. This difference is consistent with the

differences in the rates of release of NO. between SNP and cyclam-NO. The specific rate

constant for the release of NO. from SNP through reduction activation is not accessible in the

literature. However, it is well known that this rate is fast, and it is consistent with effecting a BP

decrease similar to that of trans-[RuII(NO+)(NH3)4(P(OEt)3)]3+ [49,50], which releases NO.

three to four orders of magnitude faster (k = 0.97 s�1 at 25 �C and 5.0 s�1 at 37 �C [49,50])

than cyclam-NO (2.2 � 10�3 s�1 at 35 �C [40]). Both, SNP and trans-[RuII(NO+)(NH3)4(P(OEt)3)]

3+are reported to show a similarly short-lived BP reduction [49,50]. Fig. 2 shows the

effect of SNP on BP compared to that of cyclam-NO. The slower rate of release of NO. from

trans-[RuIICl(cyclam)(NO.)]+, compared with these other two complexes, is consistent with a

longer duration of the hypotensive effect of cyclam-NO. It should be noted that the longer

time-course of the hypotensive effect of the cyclam-NO complex might overcome some

possible regulatory blood pressure mechanisms, such as the baroreflex [6].


While chromium and molybdenum nitrosyl complexes have been reported to be efficient

potent vasodilators in vivo and in vitro, with actions compared to that of SNP [15], no

comparisons with cyclam-NO could be made due to the different animal models assayed in

each case. For the chromium and molybdenum complexes anesthetized rats were used,

whereas for cyclam-NO, the rats were conscious. Earlier reports indicated that some Ru

nitrosyl complexes, such as [Ru(NO)Cl5]2� and [Ru(NO)(NH3)5]

3+ relax vascular smooth

muscle, but none were as effective as SNP in relaxing aortic strips and in decreasing

arterial blood pressure in anesthetized cats [51]. The authors also report toxic ip LD50s for

these complexes.

Hypotensive effects in the presence of methylene blue and carboxy-PTIO

Continuous infusion for 30 minutes of methylene blue (83 nmol/Kg/min) alone does not

alter the MAP values of normotensive rats [50], whereas that of carboxy-PTIO (6 � 103

nmol/Kg/min) raises the MAP 15 % [50]. In the presence of methylene blue, a soluble

guanylyl cyclase inhibitor [44,52], the vasodilator effect of SNP is only partly blocked, as

expected [44], whereas that of the cyclam-NO complex is fully blocked, consistent with

NO. release and involvement of the NO/cGMP pathway for in vivo vasorelaxation.

However, methylene blue may act not only by inhibition of the activation of guanylate

cyclase [52], but also through other pathways, such as extra cellular generation of

superoxide anion [53]. Nevertheless, a similar, but less pronounced effect is observed when

carboxy-PTIO, which traps NO. [43], is present during the administration of cyclam-NO.

For SNP, the effect of carboxy-PTIO is only slightly more pronounced than with methylene

blue. For both complexes the effect is larger for greater concentrations of carboxy-PTIO,

although it should be noted that for cyclam-NO the effect of carboxy-PTIO is not as large as

that of methylene blue. This difference could possibly be larger if we take into account that

carboxy-PTIO alone increases the MAP 15 %. Direct comparison of effects is difficult,

since different mechanisms are operative for each drug. Nevertheless, these results are

consistent with carboxy-PTIO acting as a NO. scavenger (the rate constant of reaction of

carboxy-PTIO with NO. is �104 M�1 s�1) [43], and imply NO. release and involvement of

the NO/cGMP pathway for in vivo vasorelaxation. These results strongly suggest that

cyclam-NO delivers NO. in vivo following reduction, and that its hypotensive effect is

primarily dependent on cGMP, in contrast to the other NO-dependent metabolic action of

SNP [54].

Hypotensive effects in the presence of cyclam-tfms

The cyclam-tfms complex is water soluble and undergoes fast aquation in water resulting

in trans-[RuIIICl(H2O)(cyclam)]2+ (Eq. 5).

trans�½RuIIIClðtfmsÞðcyclamÞ�ðtfmsÞ

��!H2O

Hþ trans�½RuIIIClðH2OÞðcyclamÞ�2þ þ 2 tfms ð5Þ


This last complex, trans-[RuIIICl(H2O)(cyclam)]2+, has a pKa of 3.3 [55], and in water and at

physiological pH is in the hydroxo form, trans-[RuIIICl(OH)(cyclam)]+ (Eq. 6).

trans�½RuIIIClðH2OÞðcyclamÞ�2þ] trans�½RuIIIClðOHÞðcyclamÞ�þ þ Hþ ð6Þ

Then, trans-[RuIIICl(OH)(cyclam)]+ reacts with NO. to form trans-[RuIICl(NO+)(cyclam)]2+

(Eq. 7):

trans� ½RuIIIClðOHÞðcyclamÞ�þ þ NO*

! trans�½RuIIClðNOþÞðcyclamÞ�2þ þ OH� ð7Þ

In fact, the synthesis of the cyclam-NO complex from cyclam-tfms follows Eqs. 4-6 [33,40],

and, thus, cyclam-tfms is the precursor of cyclam-NO. Thus, the blockage of the MAP

decrease effected by the NO. released from the reduction of cyclam-NO, in the presence of

cyclam-tfms (500 times excess) is consistent with cyclam-tfms readily resulting in a NO.

scavenger, trans-[RuIIICl(OH)(cyclam)]+ (Eqs. 5-7). It can be noticed that, for a fixed

cyclam-NO concentration, increasing concentrations of the tfms complex scavenge the

released NO., thereby attenuating the BP lowering effect, and fully blocking the effect with a

500 times excess of cyclam-tfms. In addition, this is also strong evidence that cyclam-NO

decreases the MAP through release of NO.. Other Ru complexes had been proposed and

reported to behave as NO. scavengers, especially polyaminocarboxylates [16-18,27], of

which, [Ru(Hedta)Cl] and [Ru(Hedta)(H2O)] appeared to be efficient NO. scavengers,

resulting in the respective nitrosyl complex [Ru(NO)(Hedta)]+. However, it should be

mentioned that the specific rate constant for the release of NO., after reduction in the edta

complex is 7.3 � 10�3 s�1 at 25 �C [56], one order of magnitude faster than that for the

cyclam-NO complex (6.1 � 10�4 s�1) [33]. Furthermore, it should be noted that the

concentration of cyclam-tfms (500 nmol/Kg/min) to fully block the MAP reduction effect of

cyclam-NO is approximately one order of magnitude smaller than the most efficient

concentration of carboxy-PTIO (6 � 103 nmol/Kg/min).

The cyclam complexes system

After reduction of the cyclam-NO complex and the subsequent reactions (Eqs. 1-4),

trans-[RuIICl(H2O)(cyclam)]+, as the predominant species in blood, and trans-[RuII(H2O)2(-

cyclam)]2+ are formed. These complexes are easily oxidized (E vs NHE = �0.050 V for the

chloro complex [33], and +0.155 V at pH 1 [57], +0.132 V at pH 4.32 [33,40], and ��0.2 V

at pH 7 for the diaqua [55]) by dissolved oxygen (E = +0.815 V at pH 7) or some other

available and accessible oxidizing species present in biological systems, to trans-[RuIIICl

(H2O)(cyclam)]2+ and trans-[RuIII(H2O)2(cyclam)]3+ (pKa = 1.1 [58]), (Eq. 8),

trans�½RuIIðH2OÞ2ðcyclamÞ�2þ ! trans�½RuIIIðH2OÞ2ðcyclamÞ�3þ þ e� ð8Þ


which at pH � 7 are in the form of trans-[RuIIICl(OH)(cyclam)]+ and trans-[RuIII(OH)

(H2O)(cyclam)]2+ (Eqs. 6 and 9)

trans�½RuIIIðH2OÞ2ðcyclamÞ�3þ] trans�½RuIIIðOHÞðH2OÞðcyclamÞ�2þ þ Hþ ð9Þ

trans-[RuIIICl(OH)(cyclam)]2+ reacts with NO. to form trans-[RuIICl(NO+)(cyclam)]2+

(Eq. 7), and it is reasonable to expect that trans-[RuIII(OH)(H2O)(cyclam)]2+, being similar

to trans-[RuIIICl(H2O)(cyclam)]2+ can react with NO. in a similar manner (Eq. 10),

resulting in trans-[RuII(OH)(NO+)(cyclam)]2+ (pKa of trans-[RuII(NO+)(H2O)(cyclam)]2+ is

3.0 [33,40].

trans� ½RuIIIðOHÞðH OÞðcyclamÞ�2þ þ NO*

! trans� ½RuIIðOHÞðNOþÞðcyclamÞ�2þ þ H2O ð10Þ

In addition to reducing species, oxidizing species are also present in biological systems,

and, thus, conceivably the formed trans-[RuIICl(H2O)(cyclam)]+ and trans-[RuII(H2O)2(cyclam)]2+, resulting from the cyclam-NO administration, can be oxidized to yield the

NO. scavengers trans-[RuIIICl(OH)(cyclam)]+ and trans-[RuIII(OH)(H2O)(cyclam)]2+.

Therefore, since the NO. is released slowly from the reduced cyclam-NO, the resulting

trans-[RuIIICl(OH)(cyclam)]+ and trans-[RuIII(OH)(H2O)(cyclam)]2+ complexes scavenge

the NO., forming trans-[RuIICl(NO+)(cyclam)]2+ and trans-[RuII(OH)(NO+)(cyclam)]2+

(Eqs. 7 and 10) and, thus, attenuating the BP decrease. The NO. scavenging action is

dependent on the trans-[RuIIICl(OH)(cyclam)]+, trans-[RuIII(OH)(H2O)(cyclam)]2+ and NO.

concentrations, and, thus, is more important at higher concentrations. Two factors at least are

involved in these concentrations. They should increase with increasing concentrations of

cyclam-NO and with time due to the slow release of NO. and formation of trans-

[RuIII(OH)(H2O)(cyclam)]2+. There is no full blockage of the hypotensive effect because

the scavenger concentration does not reach the 500 times of cyclam-NO concentration,

needed to fully block the effect.

However, this is not straightforward, as it may seem. Other chemical considerations may

also be involved, and which may contribute to a lack of a dose-response in normotensive rats,

as other chemical equilibria occur that may interact with biological regulatory mechanisms.

For example, while dissociation is suppressed at blood chloride levels, trans-[RuII(OH)

(NO+)(cyclam)]2+ may form inside cells. As this has a low reduction potential (�0.46 V vs

NHE at pH 7 [33,40]) it would be not biologically relevant. Consequently the various

possible ruthenium nitrosyl species may interact differently with available biological

reductants in different compartments. The net result would be dependent on the rate

constants, the nature and concentration of the species involved, and the possible different

biological regulatory mechanisms acting in normotensive and hypertensive rats.

The behavior of the above described cyclam complexes may have important consequences,

since at least conceivably, the pair of complexes cyclam-NO and cyclam-tfms may result in a

NO. buffering system, or lead to the design of one such system.


Conclusion

Cyclam-NO administration results in BP reduction, especially in hypertensive rats, with a

hypotensive effect 20 times more prolonged than SNP either in normotensive or hypertensive

rats. The hypotensive effect of cyclam-NO is totally inhibited by the unspecific GMPc

inhibitor methylene blue and also by c-PTIO. The continuous infusion of cyclam-tfms is able

to fully block the hypotensive effect of cyclam-NO by scavenging the NO. released from this

last complex after reduction. Finally, the above-mentioned chemical properties of cyclam-

NO, strongly suggest that the long term BP reduction, with cyclam-NO, comes from the slow

release of NO. from this complex after its reduction in vivo. The results are also consistent

with the NO. scavenging by trans-[RuIII(OH)Cl(cyclam)]+, formed from cyclam-tfms. In

addition, the possible attenuation of the BP reduction, from the NO. scavenging by trans-

[RuIII(OH)(H2O)(cyclam)]2+, possibly formed in the subsequent in vivo reactions of cyclam-

NO, would be beneficial in limiting higher concentrations of NO.. The above results may

play an important role in biological applications. The long-term action of cyclam-NO, which

makes it a sort of controlled NO. donor, can be important, under some pathophysiological

conditions, where high concentrations of NO. are harmful, whereas lower and lasting

concentrations are beneficial.

Acknowledgments

The authors thank CAPES, FAPESP, CNPq, FAEP-UNICAMP, NIH GM26390, for grants

and fellowships.

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In vivo effects of the controlled NO donor/scavenger ruthenium cyclam complexes on blood pressure

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