Multiple functions of nitric oxide in pathophysiology and ... - CiteSeerX

Multiple functions of nitric oxide in pathophysiology and microbiology: analysis by a new nitric oxide scavenger Hiroshi

Maeda,

Departments

Takaaki

of Microbiology,

Akaike,

Masaki

Urology,

*

and

Yoshida

Medicine,

NO

and

Based on derivatives, ical states

was

oxidized

by

PTIO,

yielding

one

mole

plication

of

recent

work

role of NO. NO analyzed study of this

has or

Kumamoto,

to be done

scavengers

Here

we

by

the

on the in

review NO

unique

Japan

view

clinical of

various

apvitally

aspects

scavengers,

NO

this

of

focusing

scavenger,

on

Ff10.

2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl.

diseases are now and carboxy-PTIO

CHEMISTRY:

Words: vascular

nitric oxide permeability

scavenger PTIO viral pathogenesis

endotoxin

-

shock

REACTION

OF PTIO

2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (Ff10) is a blue solid compound that

explicated. showed

significant inhibitory activity against a series of biological actions of NO: (1) endothelium-dependent vascular relaxation in an ex vivo system, (2) pathogenicity of NO produced excessively in endotoxin shock in rats and in influenza virus pneumonitis in mice, and (3) enhanced vascular permeability in solid tumors mediated by NO. PTIO directly extinguishes NO generated by NO synthase (NOS) without affecting NOS activity, which is a clear contrast to NOS inhibitors. Therefore, characterization of this unique mode of action of PTIO appears to be helpful not only in understanding of the pathophysiological role of NO but also in the treatment of various diseases caused by excessive production of NO.J. Leukoc. Biol. 56: 588-592; 1994.

tumor

much inhibitors

of Medicine,

each

the potent NO-scavenging activity of PTIO the diverse functions of NO under physiologas well as various pathological conditions such

as endotoxin shock and viral It was found that PTIO

Key

event,

functions

Sugat

School

of any

important our

Moritaka

University

In any

Abstract: A wide range of biological functions of nitric oxide (NO) was analyzed using a newly discovered nitric oxide scavenger, 2-phenyl-4,4,5,5-tetramethylimidazolineoxyl-1-oxyl-3-oxide (PTIO) or its water-soluble derivative carboxy-PTIO. The chemistry is very simple in that of NO2

and

Kumamoto

AND NO has

been

used

N0 posed with

in a polluted atmosphere as a solid stable to air in a canister. We found that it can NO in an aqueous system [3]. This reaction of Ff10 with NO seems to

stoichiometric

manner,

yielding limidazoline-1-oxyl (Ff1) and ducted in an oxygen-depleted Ff10 is only slightly soluble derivatives,

such

phenyl

as

carboxy-FFIO,

has

carboxy-PTIO group

carboxy-FT 10 3%, respectively. ble oil and can lation, allowing [4]. Details of tion [3]. Ff10 spin resonance in Figure 2.

to detect

indicator exreact rapidly proceed

in

a

2-phenyl-4,4,5,5-tetramethyNO2 at 1:1 when it was consolution (Fig. 1). Because in water, more water-soluble were also synthesized [3]; group at the R position of the

a COOH as shown in Figure 1. Solubilities of Ff10 and in water are less than 0.03% and more than Ff10 can be soluble in biocompatible edibe injected intraperitoneally in an oily formuslow release and prolonged bioavailability the chemistry are given in our earlier publicaand Ff1 give completely different electron (ESR) signals at room temperature, as shown

INTRODUCTION Biological

functions

diverse,

and

portance

of

or NO Ff10, radical, and

crucial the

use

scavengers an imidazolineoxyl can oxidize

completely

well as in vivo The advantage NOS

of

inhibitors

oxide

(NO)

are

so

INHIBITORY DEPENDENT

profound,

N-oxide

NO

[3-7]. of this that

to NO2 biological

NO

derivative

directly

over

the

in Figure 3, we examined whether Ff10 can nulthe acetylcholine-induced vasorelaxation of rabbit aorta had been precontracted with phenylephrine. Stimulaby acetylchoiine is known to mediate release of NO from endothelial cells during this vasorelaxation. Both Ff10 and As

manner in

vitro

widely

extinguishes

NO

as

used

carboxy-VfIO

and

vasorelaxation

showed arginine prevented

hence restrains its biological actions; on the contrary, NOS inhibitors do not remove NO directly from the system but inhibit generation of NO and L-citrulline from L-arginine. NOS inhibitors, which have been extensively used, are the substrate (L-arginine) analogues text, a number of problems may sible inhibition of NOS in vivo,

for NOS [1, 2]. In this conbe anticipated upon irreverwhich might cause excessive

Journal

of Leukocyte

Biology

Volume

56,

November

inhibited in

no

a

similarly

dose-dependent

acetylcholine-induced manner.

Ff1,

however,

effect. L-Arginine analogues, N#{176}-monomethyl-L(L-NMMA) and ]V5’-nitro-L-arginine (L-NNA) the relaxation as expected.

Abbreviations: NO, stitutive NOS; iNOS, limidazoline-l-oxyl-3-oxide;

nitric oxide; NOS, nitric oxide synthase; cNOS, coninducible NOS; P110, 2-phenyl-4,4,5,5-tetramethyESR, electron spin resonance; L-NMMA, .N’monomethyl-L-arginine; L-NAME, N#{176}-nitro-L-arginine methyl ester; LNNA, N-nitro-L-arginine; LPS, lipopolysaccharide; IFN-, interferon-’y; IL-I, interleukin-l; TNF-a, tumor necrosis factor a.

inhibition of vascular, hepatocytic, o? neuronal function [constitutive/endothelial/neuronal NOS (cNOS) or the inducible isoform of NOS (iNOS)]. Second, long-term use might interfere with the L-arginine/urea-ornithine cycle, with unforeseen metabolic consequences.

588

shown

lify that tion

is a stable

in a stoichiometric activity of NO

scavenger

Ff10

that

EFFECT OF FF10 ON ENDOTHELIUMVASORELAXATION: EX VIVO

EXPERIMENT

in various biological systems that the imof nitric oxide synthase (NOS) inhibitors is increasing [1, 2]. We have found that

abolish

is

nitric

Reprint Kumamoto

1994

requests:

Hiroshi

University

School

Maeda,

of Medicine,

Department

Kumamoto

of

Microbiology,

860, Japan.

P110 derivatIves

100

0 +

N02

+

6#{149} R

=

H: 2-phenyl-4,4,5,5-tetramethyl-

R

=

COOH:

imidazoline-1

(PTIO)

-oxyl-3-oxide

60

carboxy-PTIO

I Fig. 1. Structures NO.

of PTIO

and

carboxy-PTIO

and

the

reaction

scheme

with

I::

During Ff10 to generation

the acetylcholine-induced relaxation, addition of the organ bath yielded a significantly increased of Ff1 due to reaction with NO, which was released from endothelial cells by stimulation with acetylcholine. L-NMMA not only prevented consumption of Ff10 but also inhibited the generation of Ff1 in this vascular system,

indicating

the

THERAPEUTIC ENDOTOXIN

direct

reaction

of Ff10

with

NO

EFFECT OF FF10 AGAINST SHOCK IN RATS BY SCAVENGING

[31.

0

NO

30

Hypotension with decreased peripheral vascular resistance in endotoxin shock is now known to be mediated through excessive production of NO. We examined whether Ff10 can rescue such an animal model in pathological conditions. Endotoxin [lipopolysaccharide (LPS), Escherichia coli] was administered as an intravenous (i.v.) bolus to male Wistar rats at a dose of 10 mg/kg/0.2 ml of saline, which is almost fivefold higher than the LD50 in rats. After 90 mi carboxyFf10 at various doses (0.056-1.7 mg/kg/mm) was infused at

mm.

6 ml/h for 60 blood pressure and the survival 90 mm arterial

after blood

Time profiles of changes were monitored as demonstrated rate of rats at 12 h is shown

the administration pressure was

10, the 60 mm

a1=

bioassay (n

conditions

- 3-4).

mT

A*)

effect

and

PT!.

(A) Spectrum

given

aorta

elsewhere

shock

(Table

became 1)

PATHOGENIC SIGNIFICANCE VIRAL DISEASES

a

= 0.44

mT

In

of PTIO;

(B)

induced [3].

(jiM)

and

NOS

inhibitors

by acetylcholine.

Data

are

shown

on

vas-

Details as means

of SE

±

many

viral

diseases,

apparent

against

endotoxin-

[5].

mT

complicated

OF NITRIC

interactions

OXIDE

between

IN

vi-

rus and host are reported and immunological effects of the host on virus propagation are known to be involved in the pathogenesis of the disease. There is evidence that soluble factors such as proinflammatory cytokines, proteases, kinins, and free radicals, which are produced through the host’s immune responses, indirectly participate in the pathogenesis

lmT of PTIO

thoracic

derivatives

= 0.98

MnO

spectra

are

PT!0

a

MnO

2. ESR

of rabbit

of the

of carboxy-PTIO

induced

Fig.

effect

a dose of 0.056 mg carboxy-FfI0/kg/min or above. Infusion of carboxy-FfI0 at a dose of 0.17 mg/kg/mm restored the mean blood pressure during the infusion period, and 1.7 mg/kg/mm completely prevented the blood pressure drop immediately after the infusion was started. Bolus i.v. injection of carboxy-PTIO was found to be ineffective because of rapid renal clearance of this compound (data not shown). Administration of carboxy-FTIO (i.v. infusion of 1.7 mg/kg/mm for 60 mm) to normal rats did not have any significant effect on blood pressure and kidney functions. This observation indicates a clear contrast to L-NMMA which led to elevation of blood pressure. The therapeutic

mean Hg at

0.82

300

Concentration Fig. 3. Inhibitory cular relaxation

of mean arterial in Figure 4, in Table 1. Even

of carboxy-Ff maintained above

100

spectrum

[8-12].

of PT!.

Maeda

et al.

NO

scavenger

and

multiple

functions

of NO

589

previously shown that the kallikrein-kinin cascade is activated in solid tumors and bradykinmn thus formed facilitates vascular permeability and hence sustains rapid tumor growth [16, 17]. We examined this effect by injecting Ff10 intraperitoneally

(i.p.)

found

that

the

tumor

(S-180)

was

inhibitory

effect

enhanced

vascular

in

most

tumor

ity on Lewis

NO lung

noma

in

in

suppressed was

with

permeability

mice

permeability

more

observed

types,

(Y.

tumor-bearing

vascular

than

50%

[4].

tumors

H.

Maeda,

A similar this

is observed

permeabilS-180 > B-16 mela-

dependence

Noguchi,

solid

Although

L-NAME.

in solid

and

in

of vascular in experimental tumors: colon 38 carcinoma >

the

varies greatly carcinoma, mice

S-180

enhanced

et al.,

unpublished

observation).

PRE

0.5

1.0

1.5

Time Fig.

2.0

after

2.5

3.0

3.5

4.0

4.5

5.0

Our hanced

5.5

(C8)

of LPS (hr)

injection

pig,

4. Effect

of carboxy-PT!O on the LPS-induced hypotension (A) Control rats; (0) endotoxin-shock rats without treatment; shock rats treated with carboxy-PT!0 at doses of 1.7, 0.17, mg/kg/mm, respectively. Data are shown as means ± SE (n5).

in rats

pharmacokinetic

[5].

More

duced in tumor tissue vascular permeability ates cNOS activation

ON THE

the survival rate of mice was treatment with both NOS inhibitors AIwnitroLarginine methyl ester, (L-NAME) and L-NMMA, and Ff10 (unpublished observation). As reported earlier [2], NO reacts with O2 very rapidly to form peroxynitrite, which is a highly reactive and oxidizing molecule. Therefore, it seems that the orchestrated network of NO

with

mechanism

oxygen in the

by

radicals process

(O2) of tissue

is the injury

cardinal in

some

PERMEABILITY

Vascular

permeability

of tumor

MEDIATED and

normal

Journal

of Leukocyte

Biology

Volume

as

diffusion,

recep-

completely

differ-

are

may be also together with in endothelium

EFFECT

CYTOTOXIC

operative bradykinmn, [1].

in augmented which medi-

OF NO

diseases.

BY NO tissues

56,

is usually

November

I.

Effects

of Carboxy-PTIO Endotoxin Shock

on

of

Survival

rated

P value

Vehicle Carboxy-PTIO Carboxy-PT!O

(1.7 mg/kg/mm) (0.17 mg/kg/mm)

0/8 8/8 8/8