Prolonged effect of a novel S-nitrosated glyco-amino acid in endothelium-denuded rat femoral arteries: Potential as a slow release nitric oxide donor drug

Article abstract of DOI:10.1038/sj.bjp.0701557

1. The vasodilator properties of a novel S-nitrosated glyco-amino acid (RIG200) were investigated in isolated rat femoral arteries and compared with those of the parent S-nitrosothiol compound, S-nitroso-N-acetylpenicillamine (SNAP). 2. Spectrophotometric analysis revealed that 2.5 mM solutions of RIG200 decomposed more slowly (half-life (t(1/2)) = 216.2 ± 26.7 min) than SNAP (t(1/2) = 37.2 ± 13.8 min) in Krebs buffer at 24°C. Furthermore, the rate of decomposition of SNAP, but not of RIG200, was significantly reduced by the Cu(I) chelator, neocuproine. We concluded that the relative stability of RIG200 is due, at least in part, to its resistance to trace Cu(I)-catalyzed decomposition. Nitric oxide (NO) generation from SNAP and RIG200 was confirmed by use of an NO electrode. 3. Experiments to investigate the vasodilator effects of RIG200 were carried out on isolated femoral arteries taken from adult male Wistar rats (400-550 g). Lengths of artery (7-8 mm long) were cannulated, dissected free and perfused at constant flow rate (0.6 ml min^-1) with Krebs buffer. Vessels were precontracted with phenylephrine (10.2 ± 0.3 μM) and developed pressures of 91.8 ± 4 mmHg, detected upstream by a differential pressure transducer. 4. Concentration-dependent vasodilator responses to bolus injections of SNAP or RIG200 (10 μl; 10^-8-10^-3 M) made into the perfusate of endothelium-intact vessels were transient, recovering the preinjection pressure in < 20 min. 5. Responses to equivalent bolus injections of SNAP in endothelium-denuded vessels were also transient but those in response to concentrations of RIG200 > 10^-5 M were sustained. Responses to 10^-3 M RIG200 were sustained for periods > 4 h. Sustained vasodilatation was reversed by the NO scavenger, ferrohaemoglobin (10 μM) but was unaffected by the NO synthase inhibitor, N(ω)-nitro-L-arginine methyl ester (200 μM), indicating involvement of NO from a source other than NO synthase. 6. We suggest that a possible explanation for the prolonged effect of RIG200 is retention of the compound by the vascular wall, facilitated by endothelial denudation. Slow decomposition of RIG200 in situ would release sufficient NO to maintain a 'vasodilator tone' which persists for more than 4 h. Selective retention by damaged vessels could have important therapeutic implications with regard to targeted delivery of NO, restoring protection to areas deprived of endogenous NO, whilst avoiding unwanted hypotension.

Full text of DOI:10.1038/sj.bjp.0701557

1997 Stockton Press All rights reserved 0007 ± 1188/97 $12.00
British Journal of Pharmacology (1997) 122, 1617 ± 1624
Prolonged e€ect of a novel S-nitrosated glyco-amino acid in
endothelium-denuded rat femoral arteries: potential as a slow
release nitric oxide donor drug
1I.L. Megson, *I.R. Greig, {G.A. Gray, D.J. Webb & *A.R. Butler
Clinical Pharmacology Unit & Research Centre, University of Edinburgh, Western General Hospital, Edinburgh, EH4 2XU;
*School of Chemistry, University of St. Andrews, St. Andrews, Fife, KY16 9ST and {Department of Pharmacology, University of
Edinburgh, 1 George Square, Edinburgh, EH8 9JZ, Scotland
1
The vasodilator properties of a novel S-nitrosated glyco-amino acid (RIG200) were investigated in
isolated rat femoral arteries and compared with those of the parent S-nitrosothiol compound, S-nitroso-
N-acetylpenicillamine (SNAP).
2
Spectrophotometric analysis revealed that 2.5 mM solutions of RIG200 decomposed more slowly
(half-life (t1/2)=216.2+26.7 min) than SNAP (t1/2=37.2+13.8 min) in Krebs bu€er at 248C.
Furthermore, the rate of decomposition of SNAP, but not of RIG200, was signi®cantly reduced by
the Cu(I) chelator, neocuproine. We concluded that the relative stability of RIG200 is due, at least in
part, to its resistance to trace Cu(I)-catalyzed decomposition. Nitric oxide (NO) generation from SNAP
and RIG200 was con®rmed by use of an NO electrode.
3
Experiments to investigate the vasodilator e€ects of RIG200 were carried out on isolated femoral
arteries taken from adult male Wistar rats (400 ± 550 g). Lengths of artery (7 ± 8 mm long) were
cannulated, dissected free and perfused at constant ¯ow rate (0.6 ml min71) with Krebs bu€er. Vessels
were precontracted with phenylephrine (10.2+0.3 mM) and developed pressures of 91.8 + 4 mmHg,
detected upstream by a di€erential pressure transducer.
4
Concentration-dependent vasodilator responses to bolus injections of SNAP or RIG200 (10 ml;
1078 ± 1073 M) made into the perfusate of endothelium-intact vessels were transient, recovering the pre-
injection pressure in 520 min.
5
Responses to equivalent bolus injections of SNAP in endothelium-denuded vessels were also transient
M
but those in response to concentrations of RIG200 41075 M were sustained. Responses to 1073
RIG200 were sustained for periods 44 h. Sustained vasodilatation was reversed by the NO scavenger,
ferrohaemoglobin (10 mM) but was una€ected by the NO synthase inhibitor, No-nitro-L-arginine methyl
ester (200 mM), indicating involvement of NO from a source other than NO synthase.
6
We suggest that a possible explanation for the prolonged e€ect of RIG200 is retention of the
compound by the vascular wall, facilitated by endothelial denudation. Slow decomposition of RIG200 in
situ would release sucient NO to maintain a `vasodilator tone' which persists for more than 4 h.
Selective retention by damaged vessels could have important therapeutic implications with regard to
targeted delivery of NO, restoring protection to areas deprived of endogenous NO, whilst avoiding
unwanted hypotension.
Keywords: Nitric oxide; S-nitrosothiols; vasodilatation; RIG200
Introduction
Nitric oxide (NO) synthesized in the endothelium of blood
vessels (Palmer et al., 1987; 1988; Palmer & Moncada, 1989) is
recognised to be an important factor in control of local blood
¯ow and of blood pressure in animals (Aisaka et al., 1989; Rees
et al., 1989; Gardiner et al., 1990; Chu et al., 1991) and man
(Vallance et al., 1989; Haynes et al., 1993). In addition, NO is
known to inhibit platelet aggregation and adhesion (Radomski
et al., 1987a,b; Vallance et al., 1989; Haynes et al., 1993),
smooth muscle mitogenesis (Garg & Hassid, 1989) and
monocyte adhesion (Lefer, 1997). In view of these properties,
endothelial dysfunction resulting in reduced NO synthesis is
thought to play an important role in atherogenesis (Chappell et
al., 1987; Harrison et al., 1987; Forstermann et al., 1988;
Guerra et al., 1989). Physical damage to, or removal of, the
endothelium during percutaneous transluminal angioplasty
(PCTA) is a major contributory factor in the high incidence of
restenosis following the procedure (Langford et al., 1994).
Organic nitrates (such as glyceryl trinitrate) have been used
for many years in the treatment of angina (Fung & Bauer,
1994). Their actions are mediated by NO generated in situ by a
mechanism which may involve reduced tissue thiols (Needle-
man et al., 1973; Ignarro et al., 1981). However, the
therapeutic use of organic nitrates is limited by the
development of nitrate tolerance, possibly due to the depletion
of reduced tissue thiols essential to their metabolism (Fung &
Bauer, 1994). Sodium nitroprusside (SNP) is another NO
donor drug which can be used as a post-operative anti-
hypertensive agent. The mechanism of NO release from SNP is
poorly understood but it is known not to decompose
spontaneously in solution at physiological pH unless exposed
to light (Butler & Glidewell, 1987). It has been suggested that
in vivo decomposition is tissue-dependent, and may involve
speci®c membrane-bound proteins (Kowaluk et al., 1992).
SNP administration requires careful titration and prolonged
use can result in cyanide poisoning (Butler & Glidewell, 1987).
Neither organic nitrates nor SNP can be targeted at areas of
endothelial damage and their use is often associated with
systemic hypotension. The possibility of localizing supply of
NO to areas of the vasculature devoid of endothelium-derived
NO is a more desirable therapeutic option.
S-Nitrosothiols (general formula R-S=NO) decompose
spontaneously in solution to their disulphide form, generating
1 Author for correspondence.

1618
I.L. Megson et al
Prolonged effect of a novel S-nitrosothiol
NO in the process (Williams, 1985), and present a potential
alternative to organic nitrates, particularly as they do not appear
to engender vascular tolerance (Harowitz et al., 1983; Bauer &
Fung, 1991). However, the therapeutic potential of many
existing S-nitrosothiols, like S-nitroso-N-acetylpenicillamine
(SNAP; Figure 1a) and S-nitrosoglutathione (GSNO) is limited
by the rapid and unpredictable nature of their decomposition
due to the catalytic e€ect of trace Cu(I) ions (Dicks et al., 1996;
Gordge et al., 1996). Accelerated decomposition in the presence
of Cu(II) (De Man et al., 1996) is now thought to be mediated by
prior reduction to Cu(I) by thiols. In vivo, decomposition may
also be accelerated by direct transfer of NO+ to reduced tissue
thiols (transnitrosation; Askew et al., 1995) and by enzyme-
dependent mechanisms (Askew et al., 1995; Gordge et al., 1996).
Therapeutic e€ects of existing S-nitrosothiols are, therefore,
potentially variable and short-lived.
Endogenous S-nitrosothiols have been identi®ed in both
human plasma (Stamler et al., 1992a,b) and tissue extracts
(Gaston et al., 1993) but their role is, as yet, unclear. GSNO
has shown a degree of tissue speci®city, inhibiting platelet
aggregation more readily than causing vasodilatation (Ra-
domski et al., 1992; De Belder et al., 1994) and acting as a
more potent vasodilator in arteries than veins (MacAllister et
al., 1995). GSNO has also been shown to have potentially
bene®cial e€ects with regard to inhibition of platelet
aggregation associated with PCTA of coronary arteries
(Langford et al., 1994).
a
S—NO
H3C
H3C
CO2H
CH3COHN
S-nitroso-N-acetylpenicillam ine
b
S—NO
H3C
OAc
H
N
H3C
O
A
CH3COHN
AcO
CH OAc
2
O
O
c
N-(S-nitroso-N-acetylpenicillam ine)-2-am ino-2-deoxy-
1,3,4,6,-tetra-O-acetyl-β-D-glucopyranose
Figure 1 Structural formulae and full generic names for SNAP (a)
and RIG200 (b). Note that RIG200 contains a SNAP moiety linked
by a peptide bond to glucosamine tetra-acetate.
Here, we investigate the vasodilator properties of a novel S-
nitrosated glyco-amino acid (RIG200; Figure 1b) in rat
isolated femoral arteries. RIG200 is SNAP coupled to
glucosamine tetra-acetate by an amide bond. The molecule is
di€erent from those described recently (Ramirez et al., 1996) in
that the glucose moiety is acetylated at the 1, 3, 4 and 6
positions. It was envisaged that non-polar glucosamine tetra-
acetate would cause steric hindrance of Cu(I)-catalyzed
decomposition and also increase lipid solubility of RIG200.
These features would result in slow release of NO from
RIG200, independent of Cu(I) concentration, and could
facilitate uptake of RIG200 into vascular tissue, altering the
mode of NO delivery.
Biological activity of SNAP and RIG200
Preparation Experiments were carried out on isolated
segments of femoral artery from adult male Wistar rats
(400 ± 550 g; n = 36). The perfusion system used was similar
to that described previously for rat tail artery perfusion
(Flitney et al., 1992). Brie¯y, animals were killed by cervical
dislocation and both femoral arteries were exposed and
cannulated immediately distal to the epigastric arterial branch.
Cannulated arterial segments (7 ± 8 mm long) were dissected
free and transferred to perspex organ bath chambers (1 ml
volume) at 378C where they were perfused (0.6 ml min71
;
Gilson minipuls 3, Anachem, Luton, U.K.) and superfused
(1 ml min71, Watson Marlow 302S; Watson Marlow, Fal-
mouth, U.K.) with fresh oxygenated Krebs bu€er solution.
Twin vessels were precontracted with phenylephrine (PE) and
perfusion pressure was monitored by a di€erential pressure
transducer (T; Sensym SCX 15ANC, Farnell Electronic
Components, Leeds, U.K.) located upstream.
Methods
Decomposition of SNAP and RIG200 in vitro
The apparatus permits exclusive drug delivery to the
luminal surface of the vessel by bolus injection (10 ml) through
a resealable rubber septum into the perfusate immediately
upstream of the vessel (transit time to artery *3 s, through
lumen *300 ms). Injections of vehicle (Krebs bu€er) had no
e€ect on perfusion pressure. Vasodilator responses in control
vessels could be compared to those perfused with supra-
maximal concentrations of either the recognised NO scaven-
ger, ferrohaemoglobin (Martin et al., 1985; Hb; 10 mM), or the
NO synthase (NOS) inhibitor, No-nitro-L-arginine methyl
ester (Rees et al., 1990; L-NAME; 200 mM). Where possible,
two vessels from each animal were used in parallel; one
receiving SNAP and the other RIG200.
SNAP and RIG200 (2.5 mM) diluted in oxygenated (95% O2,
5% CO2) Krebs bu€er (composition in mM: NaCl 118,
KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25
and glucose 5.5) were incubated in the dark at 248C. Care was
taken to use the same Krebs solution to dilute SNAP and
RIG200 in order to ensure identical copper ion content. The
decrease in absorbance at a wavelength (l) of 341 nm was
measured by use of Phillips PU 8720 ultraviolet/visible scan-
ning spectrophotometer (path length=1 cm).
Experiments at 248C were repeated in the presence of either
an intermediate concentration of CuSO4 (1 mM; Askew et al.,
1995) or the speci®c Cu(I) chelator, neocuproine (NCu; 1 mM;
Dicks et al., 1996) in order to establish the e€ect of Cu(I) on
the rate of decomposition of RIG200 and SNAP.
Experimental protocols All experiments were carried out in a
darkened laboratory in order to protect photolabile drugs and
to prevent photorelaxation of vessels (Megson et al., 1995). All
drugs were dissolved and diluted in PE-containing Krebs
solution and kept on ice before use.
Once precontracted, endothelial function was assessed by
use of the endothelium-dependent vasodilator, carbachol
(CCh). Bolus injections of supra-maximal concentrations of
CCh (10 ml; 10 mM) into the perfusate of endothelium-intact
vessels caused transient vasodilatations similar to those
described in rat tail arteries (Flitney et al., 1992). In experiments
where the endothelium was removed, air was passed through
the lumen until such time as the vessel was unresponsive to CCh
Generation of NO from SNAP and RIG200 in vitro
SNAP and RIG200 1 mM in Krebs bu€er were stirred in the
dark at 248C. NO generation was measured using an NO
probe (ISO-NO World Precision Instruments, Stevenage,
Herts, U.K.), calibrated with NaNO2 (1 ± 25 mM) acidi®ed with
1 mM ascorbic acid (Gaston et al., 1993). The e€ect of CuSO4
(1 mM) or NCu (100 mM) on NO generation was also investi-
gated. A higher concentration of NCu was necessary in the
electrode experiments to scavenge Cu(I) derived from added
(1 mM) rather than trace Cu(II).

I.L. Megson et al
Prolonged effect of a novel S-nitrosothiol
1619
(5 ± 10 min). Removal of the endothelium invariably caused an
increase in pressure due to loss of endothelium-derived NO
synthesis. Pressure was restored to its original level by
appropriate reduction in PE concentration (*0.56original
concentration). Selected vessels were taken for histological
staining to con®rm endothelial removal (5 mm paran sections,
®xed in formalin (10%; 24 h) and stained with haematoxylin
and eosin). Final assessment of the integrity of the endothelium
was achieved at the end of experiments where vessels were
perfused with ferrohaemoglobin (Hb; 10 mM). Perfusion
pressure in endothelium-intact vessels rose considerably higher
than pretreatment pressure, indicating the presence of
endothelium-derived NO, whilst endothelium-denuded vessels
failed to develop pressures above pretreatment levels.
violet/visible spectral analysis revealed an absorption peak at
71
l=341 nm where e = 640 M cm71. RIG200 is soluble in
Krebs bu€er at concentrations 53 mM after ultrasonication
(1 min).
Analysis of results
Signals from the pressure transducers were processed by a
MacLab/4e analogue-digital converter and displayed through
`Chart' software (AD Instruments, Sussex, U.K.) on a Ma-
cintosh Performa 630 microcomputer. Vasodilator response
amplitude was expressed as a % of PE-induced pressure ex-
isting before the ®rst in a series of drug applications (%
pressure change; negative values represent relaxation, positive
Vasodilator responses to bolus injections of SNAP and
RIG200 Bolus injections of increasing concentrations of
SNAP or RIG200 (10 ml; 1078 ± 1073 M) were made sequen-
tially into the perfusate of precontracted, endothelium-intact
or -denuded vessels. Responses were deemed to have recovered
once pressure was maintained for more than 2.5 min, at which
time the next concentration was injected. Time intervals
between injections of SNAP and RIG200 were matched for
each individual experiment. Responses to 1073 M concentra-
tions were then allowed to recover for periods of between
15 min and 4 h, after which vessels were perfused with Hb
(10 mM).
a
1.0
SNAP
SNAP+NCu
SNAP+CuSO4
0.8
0.6
0.4
0.2
0.0
The e€ect of Hb on vasodilator responses to SNAP and
RIG200 In order to assess the role of NO in SNAP- and
RIG200-induced vasodilatation, sequential bolus injection
experiments were also carried out in endothelium-denuded
vessels perfused with Hb (10 mM).
The e€ect of L-NAME on sustained vasodilator responses to
RIG200 The possible role of the inducible isoform of NOS
(iNOS) in sustaining RIG200-induced vasodilatation in
endothelium-denuded vessels was assessed by examining the
e€ect of perfusing vessels with the NOS inhibitor, L-NAME
(200 mM; 3 h), on responses to a single bolus injection (1073M)
of RIG200 in endothelium-denuded vessels. Perfusion com-
menced 1 h after the bolus injection of RIG200.
0
10
20
30
40
50
60
Tim e (m in)
b
Drugs and reagents S-Nitrosothiols were synthesized as
described below in the School of Chemistry (University of
St. Andrews, St. Andrews, Scotland). All other chemicals were
obtained from Sigma Ltd. (Poole, Dorset, U.K.) Met-Hb was
reduced to the ferro- form with sodium dithionite as described
previously (Martin et al., 1985).
1.0
0.8
0.6
0.4
0.2
0.0
SNAP was prepared by an established method (Field et al.,
1978). Ultraviolet/visible spectral analysis con®rmed an
absorption peak at l=341 nm, characteristic for S-nitro-
sothiols. The disulphide products formed during decomposi-
tion of S-nitrosothiols do not absorb at this wavelength. The
extinction coecient (e) at this wavelength was 1168 M71 cm71
SNAP is soluble in Krebs bu€er at concentrations 510 mM.
.
RIG200 was prepared by mixing equimolar amounts of
glucosamine tetra-acetate synthesized by the method pre-
viously described (Bergmann & Zervas, 1931) and N-acetyl-
D,L-penicillamine in dichloromethane. Addition of an equi-
molar amount of the coupling agent, 1-cyclohexyl-3-(2-
morpholino-ethyl) carbodiimide metho-p-toluene suphonate,
resulted in the formation of a white precipitate. The mixture
was stirred for 24 h, ®ltered, and the ®ltrate washed (1 M HCl,
saturated NaHCO3, water), dried (MgSO4), evaporated and
recrystallized from methanol (46% yield). The product was
then nitrosated in 50% methanol/water by NaNO2 acidi®ed
with HCl. The green solution formed was extracted into
dichloromethane and evaporated in an oil. Trituration with
ether gave a dark green solid (88% yield). The identity of the
product was con®rmed by use of mass spectrometry (EPSRC
Mass Spectroscopy Service, University College, Wales, U.K.)
and nuclear magnetic resonance (n.m.r.) spectroscopy. Ultra-
RIG200
RIG200+NCu
RIG200+CuSO4
0
10
20
30
40
50
60
Tim e (m in)
Figure 2 Mean absorbance (A), expressed as a fraction of A at time
0 (A0) measured at l=341 nm for 2.5 mM solutions of SNAP (a) and
RIG200 (b) in Krebs solution alone, Krebs solution+1 mM
neocuproine (NCu) and Krebs solution+1 mM copper sulphate
(CuSO4) at 248C plotted against time (all n=6). Half-lives (t1/2) for
decomposition derived from semi-logarithmic plots of the above data
are given in the text. Vertical lines show s.e.mean.

1620
I.L. Megson et al
Prolonged effect of a novel S-nitrosothiol
represent constriction). Data are given as % pressure change
both at the peak of responses and following response recovery
as de®ned earlier. Mean values are given +s.e.mean.
P values stated in the text were obtained by two-factor,
repeated dose ANOVAs except where stated otherwise.
Unpaired, two-tail student's t-tests and Mann-Whitney U
(MWU) non-parametric tests were also used where appro-
priate. P50.05 was accepted as statistically signi®cant.
(91.8+4.0 mmHg; n=52). Endothelium intact vessels dilated
in response to 10 mM bolus injections of CCh (25.5+3.3%
relaxation; n=22). Pressure in PE-contracted vessels increased
by 52.3+9.3% (n=14) when perfused with 10 mM Hb,
con®rming the presence of a functional endothelium.
Endothelium removal by perfusion of air abolished
responses to CCh and caused an increase in PE-induced
pressure of 75.4+11.3% (n=30). Pressure in these vessels
failed to rise signi®cantly above baseline pressure when
perfused with Hb (+4.5+4.8%; n=14).
Results
Examination of histological sections of vessels con®rmed
the presence of endothelium in intact vessels and successful
removal in those perfused with air.
Decomposition of SNAP and RIG200 in vitro
Absorbance (A) was plotted as a fraction of A at time 0 (Ao).
Half-lives (t1/2) of SNAP and RIG200 were derived from linear
plots of log A/Ao against time. Figure 2 shows the rate of
decomposition of SNAP (a) and RIG200 (b) in oxygenated
Krebs bu€er at 248C, as assessed by monitoring absorbance at
341 nm. The rate of SNAP decomposition (t1/2=37.2+
13.8 min) was signi®cantly faster than that of RIG200
(t1/2=216.2+26.7 min, P=0.006, Mann Whitney U non-
parametric test). Decomposition of SNAP was more variable
than that of RIG200 in Krebs alone and was signi®cantly re-
duced in the presence of NCu (1 mM; t1/2=57+6.1 min,
P=0.04, Mann Whitney U test). NCu did not a€ect the rate of
RIG200 decomposition (t1/2=227+24.45 min, P=0.68, Mann
Whitney U test). In the presence of CuSO4 (1 mM), there was
accelerated decomposition of both SNAP (t1/2=10.0+3.2 min,
P = 0.01, Mann Whitney U test) and RIG200 (t1/2=55.1+
8.9 min, P=0.006, Mann Whitney U test).
Vasodilator responses to bolus injections of SNAP and
RIG200 Bolus micro-injections of SNAP into the perfusate
of endothelium-intact vessels caused concentration-dependent,
transient vasodilatations (Figures 4a and 5a). Responses to
moderate concentrations of SNAP typically recovered to
pressures higher than pre-injection pressure. The e€ect
subsided, but pressure often remained slightly elevated
(+11.2+8.1% for 1075 M SNAP; P=0.042). Responses to
the highest concentration of SNAP (1073 M) caused relaxa-
tions of 775.2+4.7% and generally recovered fully in
a
SNAP (+endothelium )
–3
–4
–8
–7
–5
–6
Generation of NO from the decomposition of SNAP and
RIG200 (1 mM) was con®rmed with an isolated NO electrode
(Figure 3). In Krebs bu€er alone at 248C, SNAP generated
more NO than RIG200, re¯ected in NO concentrations at
equilibrium of 5.9+0.9 and 3.8+0.7 mM, respectively
(P=0.028, Student's unpaired t test). The amount of NO
measured in solution increased to 11.7+2.7 and 6.2+1.1 mM,
respectively, on addition of CuSO4 (1 mM). The increase was
reversed by adding excess NCu (100 mM), yielding NO
concentrations of 3.5+0.6 and 3.2+0.8 mM, respectively (all
n=5).
Hb
1 h
b
SNAP (–endothelium )
–6
–4
–5
–7
–3
–8
Biological activity of SNAP and RIG200
Hb
Vessel parameters Vessels were precontracted with PE
(10.2+0.3 mM) in order to give pressures of *90 mmHg
c
RIG200 (+endothelium )
–4
–3
–7
–5
–6
–8
2 µM
10 m in
Hb
RIG200
SNAP
d
RIG200 (–endothelium )
–7
–6
–8
–5
–4
–3
25 %
NCu
Hb
CuSO4
5 m in
SNAP/RIG200
Figure 3 Representative recording of NO in a stirred solution of
RIG200 or SNAP (1 mM) in Krebs bu€er at 248C, measured with an
isolated NO electrode. CuSO4 (1 mM) and NCu (100 mM) were added
to the solution as indicated. Baseline recordings at the beginning and
end of experiments were made in Krebs bu€er alone.
Figure
sequential bolus micro-injections (10 ml) of SNAP (a and b) or
RIG200 (c and d; log concentrations as indicated) into the
perfusate of endothelium-intact and endothelium-denuded vessels.
Perfusion with Hb (10 mM) in indicated by the solid bar.
4 Pressure recordings showing vasodilator responses to
M

I.L. Megson et al
Prolonged effect of a novel S-nitrosothiol
1621
520 min. However, some vessels (n=2/7) failed to recover to
pre-injection pressures even after washout periods of up to 1 h.
Perfusion with 10 mM Hb invariably caused pressure to rise
signi®cantly above pre-injection pressures (+45.1+ 15.1%;
P=0.001).
Endothelium removal did not signi®cantly increase vessel
sensitivity to SNAP (P=0.75). However, the contractile e€ect
often seen in endothelium-intact vessels following SNAP-
induced vasodilatations was not seen in denuded vessels
(Figures 4b and 5b). Responses to the highest concentration
of SNAP (1073 M) in some vessels (n=2/7) failed to recover to
pre-injection pressures even after washout periods of up to 1 h.
Perfusion with 10 mM Hb following this concentration of
SNAP caused rapid recovery to pre-injection pressures
(+7.9+3.8%) in those vessels which had failed to show full
spontaneous recovery (Figure 4b).
and 5c). Perfusion of these vessels with Hb invariably caused
a rapid rise in pressure to +74.1+5.4% (P=0.001; two-
tailed, unpaired Student's t test) above pre-injection levels.
Responses in endothelium-denuded vessels also showed full
recovery following 1078 ± 1077 M microinjections but there-
after failed to recover to pre-injection pressure (Figures 4d
and 5d). The sustained vasodilator e€ect of moderate and
high concentrations of RIG200 was concentration-dependent,
with perfusion pressure recovering to only 48.0+5.2% of pre-
injection pressure following a 1073 M bolus injection. The
response persisted for 44 h and was fully reversed on
perfusion with Hb, reaching pressures +1.0+3.2% of pre-
injection levels. The rate of pressure recovery on perfusion
with Hb was often noticeably slower in vessels treated with
RIG200 than SNAP.
Vessels were less sensitive to RIG200 than SNAP in either
endothelium-intact (P=0.022) or endothelium-denuded vessels
(P=0.014), corresponding to greater stability of RIG200 in
Responses to RIG200 in endothelium-intact vessels were
transient and similar in nature to those for SNAP (Figures 4c
a
c
+25
+25
0
–25
–50
0
–25
–50
–75
–75
Peak am plitude
Peak am plitude
Recovery
Recovery
–100
–100
–9
–8
–7
–6
–5
–4
–3
–2
–9
–8
–7
–6
–5
–4
–3
–2
Bolus conc. (log M)
Bolus conc. (log M)
b
d
+25
0
+25
0
–25
–50
–75
–100
–25
–50
–75
–100
Peak am plitude
Recovery
Peak am plitude
Recovery
–9
–8
–7
–6
–5
–4
–3
–2
–9
–8
–7
–6
–5
–4
–3
–2
Bolus conc. (log M)
Bolus conc. (log M)
Figure 5 Log-dose response curves showing the vasodilator e€ect (% pressure change) of bolus micro-injections (10 ml) of (a and b) SNAP or
(c and d) RIG200 on endothelium-intact (a and c) and -denuded (b and d) rat femoral arteries. Peak amplitude and recovery of responses are
illustrated (all n=8). Points shown are means and vertical lines indicate s.e.mean.

1622
I.L. Megson et al
Prolonged effect of a novel S-nitrosothiol
vitro. Relaxations in response to 1073 M RIG200 were similar
in amplitude to those to SNAP (75.0+3.0% relaxation).
Endothelium-removal did not signi®cantly alter sensitivity to
RIG200 (P=0.66).
Delivery of maximal and supra-maximal concentrations of
both SNAP and RIG200 was limited by their saturation points
in Krebs bu€er (saturated at *361073 M), preventing
calculation of EC50 values.
responses (P=0.019) and abolished the sustained vasodilator
e€ect of RIG200 (Figure 6b).
The e€ect of L-NAME on sustained vasodilator responses to
RIG200 Single bolus micro-injections of RIG200 (1073 M) in
endothelium-intact vessels caused transient vasodilatations
which recovered to 95.3+4.2% in 530 min (Figure 7a;
n=8). Equivalent micro-injections of RIG200 in endothe-
lium-denuded vessels caused relaxations which only recovered
to 48.2+7.9% of pre-injection pressure 4 h after injection
(Figure 7b; n=6). Sustained vasodilatation was inhibited by
Hb, as described previously, and the e€ect was reversed on
washout (Figure 7b; n=6). However, sustained vasodilatation
in endothelium-denuded vessels was not signi®cantly a€ected
by L-NAME (Figure 7c), with perfusion pressure recovering to
45.3 + 3.2% of pre-injection pressure 4 h after bolus washout
(n=6; P=0.47 compared to untreated vessels; unpaired
Student's t test).
The e€ect of Hb on vasodilator responses to SNAP and RIG200
in denuded vessels The peak amplitude of responses to bolus
injections of SNAP in endothelium-denuded vessels was
signi®cantly attenuated, but not abolished, in the presence of
Hb (P=0.001; Figure 6a). Similarly, perfusion of endothelium-
denuded vessels with Hb caused inhibition of RIG200-induced
a
+25
Discussion
Our results show that the novel S-nitrosated glyco-amino acid,
RIG200, decomposes spontaneously in solution to release NO
in vitro and is capable of causing NO-mediated vasodilatation
in rat isolated femoral arteries. Decomposition was found to
be slower than that of the parent compound, SNAP, but was
not susceptible to trace Cu(I) catalysis in vitro. Bolus injections
of both SNAP and RIG200 produced transient responses in
endothelium-intact, rat isolated femoral arteries. SNAP-in-
duced vasodilatations in endothelium-denuded vessels were
transient whilst those induced by RIG200 were sustained for
up to 4 h.
0
–25
–50
Spectrophotometric analysis showed RIG200 to be 5 fold
more stable than SNAP in Krebs bu€er solution (Figure 2).
The rate of SNAP decomposition in vitro is notoriously
variable, a feature which is now understood to be due to
di€erences in the amounts of trace Cu(I) present in solutions
(Dicks et al., 1996). It has been suggested that a small
proportion of Cu(II) is reduced to Cu(I) by thiols present as
impurities in solutions of S-nitrosothiols and that it is Cu(I)
which catalyzes S-nitrosothiol decomposition (Dicks et al.,
1996). Our results show that SNAP decomposition was
signi®cantly slower in the presence of the Cu(I)-chelator,
NCu, con®rming the involvement of Cu(I)-catalysis. In
contrast, the rate of RIG200 decomposition was una€ected
Peak am plitude
–75
Recovery
–100
–9
–8
–7
–6
–5
–4
–3
–2
Bolus conc. (log M)
b
+25
0
a
b
–25
–50
–75
–100
Hb
c
Peak am plitude
Recovery
25 %
30 m in
–9
–8
–7
–6
–5
–4
–3
–2
L-NAME
Bolus conc. (log M)
Figure 6 Log-dose response curves showing the vasodilator e€ect
(% pressure change) of bolus micro-injections (10 ml) of (a) SNAP or
(b) RIG200 in rat endothelium-denuded femoral arteries whilst being
perfused with 10 mM Hb. Peak amplitude and recovery of responses is
illustrated (all n=8). Points show means and vertical lines indicate
s.e.mean.
Figure 7 Pressure recordings showing vasodilator responses to single
bolus micro-injections of 1073 M RIG200 in (a) an endothelium-
intact vessel in the absence of L-NAME (b) an endothelium-denuded
vessel in the absence of L-NAME but perfused with Hb (10 mM) for
the period indicated and (c) an endothelium-denuded vessel perfused
with L-NAME for the period indicated (3 h).

I.L. Megson et al
Prolonged effect of a novel S-nitrosothiol
1623
by NCu, indicating that it was resistant to trace Cu(I)-
catalyzed decomposition. However, addition of a relatively
high concentration of Cu(II) (1 mM) accelerated decomposition
of both compounds.
RIG200-treated vessels (Figure 4d) may indicate that NO
generated by RIG200 is less accessible to Hb than that derived
either from the endothelium or from SNAP. The inhibitory
e€ect of Hb on sustained vasodilatation was reversible on
washout (Figure 7b), indicating continuing NO generation
after Hb treatment.
NO generation from both SNAP and RIG200 in vitro was
con®rmed by use of an isolated NO electrode. RIG200
generated less NO than SNAP in Krebs bu€er alone,
con®rming its comparative stability under these conditions
(Figure 3). Both SNAP and RIG200 decomposition was
accelerated in the presence of Cu(II), an e€ect that was
reversed by addition of the Cu(I) chelator, NCu to the bu€er.
This result con®rms that Cu(I) derived from added Cu(II) is
the catalyst for decomposition of both SNAP and RIG200. In
addition, NCu caused a considerable reduction of NO
generation from SNAP, but not RIG200, in Krebs bu€er,
further evidence supporting the proposal that trace Cu(I),
which may be derived from de-ionized water, glassware and
salts, is sucient to accelerate decomposition of SNAP but not
RIG200. It is apparent, therefore, that RIG200 is inherently
more stable than SNAP in Krebs bu€er solution, due in part to
its resistance to trace Cu(I) catalysis.
Decomposition of both SNAP and RIG200 would be
expected to be greater in vivo since Cu(II) levels in human
serum are 12 ± 24 mM of which 1 ± 2 mM is unbound (Lenter,
1984). The relatively slow, trace Cu(I)-resistant decomposition
of RIG200 in vitro is advantageous in limiting the undesirable
variability in NO generation and the resultant handling
diculties experienced with SNAP, and may in¯uence its
biological activity.
Bolus injections of SNAP or RIG200 into the perfusate of
precontracted, endothelium-intact rat isolated femoral arteries
induced transient vasodilatations (Figures 4 and 5). Vessels
were signi®cantly less sensitive to RIG200 than to SNAP,
perhaps re¯ecting the relative stability of RIG200 in solution.
NO is known to inhibit synthesis of endogenous NO by
endothelial NOS and this process may be responsible for the
modest vasconstriction seen following responses to bolus
injections of intermediate concentrations (1075 ± 1074 M) of
either SNAP or RIG200 in endothelium-intact vessels (Figures
4a,c and 5a,c). Absence of equivalent vasoconstriction in
endothelium-denuded vessels (Figures 4b,d and 5b,d) is further
evidence for NO-induced inhibition of NOS by NO derived
from S-nitrosothiols.
Endothelium-removal did not signi®cantly a€ect vasodila-
tations induced by bolus injections of SNAP or RIG200
(Figures 4a,c and 5a,c) and responses to both compounds were
signi®cantly inhibited by Hb (Figure 6a,b). However,
responses to intermediate and high concentrations of
RIG200 in endothelium-denuded vessels recovered at a slower
rate (Figure 4d) and caused a sustained depression of tone
(Figures 4d and 5d). Pre-injection pressure was restored by
perfusing with Hb, con®rming that sustained vasodilatation is
entirely NO-mediated. The slow rate of recovery observed in
Vasodilatation to a single bolus injection of 1073 M RIG200
in endothelium-intact vessels typically recovered to pre-
injection pressure within 30 min (Figure 7a). Equivalent
responses in endothelium-denuded vessels caused sustained
vasodilatation which showed only partial recovery after 4 h
washout (Figure 7b). Sustained responses were una€ected by
L-NAME treatment (Figure 7c), excluding induction of iNOS
as a possible mechanism for the e€ect.
A possible explanation for our results is that SNAP-
induced, transient vasodilatations are caused primarily by
spontaneous release of NO in the perfusate, irrespective of the
presence of endothelium. Similarly, transient responses to
RIG200 in endothelium-intact vessels are due to spontaneous
release of NO in the perfusate. However, responses to RIG200
in endothelium-denuded vessels appeared to comprise two
elements; a transient component due to spontaneous NO
release in the perfusate and a sustained component due to
release of NO from RIG200, retained within the tissue long
after the bolus has passed through the lumen. It is apparent
that the sustained component of vasodilator responses to
RIG200 is speci®c to endothelium-denuded vessels. We suggest
that endothelial cells present a barrier to RIG200 retention but
that in endothelium-denuded vessels, RIG200 may be retained
in the tissue underlying the endothelium (the internal elastic
lamina and/or the media). Possible retention of RIG200 by the
sub-endothelium may be particularly important in view of
emerging evidence that the barrier function of endothelium is
altered by oxidized low density lipoprotein (Rangaswamy et
al., 1997), which is known to accumulate in atherosclerotic
lesions (Haberland et al., 1988).
The stability of RIG200 in vitro is a distinct advantage over
existing S-nitrosothiols with regard to its handling, and may be
a factor in increasing the duration of vasodilator responses in
vivo. In addition, the ability of RIG200 to cause selectively
sustained vasodilatation in vessels where the endothelium has
been removed could have important implications in cardio-
vascular diseases, where endothelial damage is a contributory
factor, and may be useful in preventing restenosis of vessels
following surgical procedures like PCTA and CABG. The
unique properties of RIG200 require con®rmation in human
vessels and ultimately in vivo but RIG200 potentially presents
new therapeutic opportunities for S-nitrosothiols as slow-
release NO donor drugs.
I.L.M. is a British Heart Foundation Junior Research Fellow (FS/
95061). We also acknowledge support from the Rollo Trust.
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(Received July 10, 1997
Revised August 20, 1997
Accepted September 11, 1997)