Naunyn-Schmiedeberg’s Arch Pharmacol (2003) 367 : 532–537
DOI 10.1007/s00210-003-0711-x
O R I G I N A L A RT I C L E
Pouya Movahed · Edward D. Högestätt · Jesper Petersson
Effect of hypoxia on vasodilator responses
to S-nitroso-N-acetylpenicillamine and levcromakalim
in guinea pig basilar artery
Received: 24 September 2002 / Accepted: 7 February 2003 / Published online: 25 March 2003
© Springer-Verlag 2003
Abstract Ischaemic stroke is characterised by reduction
of blood flow, tissue hypoxia, energy depletion and neu-
ronal death. Drugs causing vasodilatation of cerebral ar-
teries may potentially enhance blood supply to the isch-
aemic area and improve clinical outcome. However, va-
sodilators could also reduce cerebral blood flow in the
ischaemic region by acting on blood vessels in non-isch-
aemic tissue, a phenomenon known as blood flow steal.
To explore whether these drugs could act selectively on
cerebral blood vessels in a hypoxic environment, we ex-
amined the effect of hypoxia on vasodilator responses to
the nitric oxide (NO) donor S-nitroso-N-acetylpenicil-
lamine (SNAP) and the ATP-dependent potassium chan-
nel (KATP) opener levcromakalim in guinea-pig basilar ar-
teries contracted by endothelin-1. Hypoxia considerably
enhanced the vasodilator responses to SNAP, while those
to levcromakalim were unaffected. In the presence of the
NO synthase inhibitor NG-nitro-L-arginine, hypoxia no
longer enhanced the vasodilator response to SNAP and
suppressed responses to levcromakalim. The results show
that the NO donor SNAP, but not the KATP opener levcro-
makalim, is a more effective vasodilator of cerebral arter-
ies contracted by endothelin-1 during hypoxia than under
control conditions. Hypoxia-induced inhibition of basal
NO synthesis could explain this enhancement of the va-
sodilator response to SNAP. Thus, NO donors may have a
selective effect on blood vessels in ischaemic brain areas
and therefore warrant further evaluation as therapeutic
agents in cerebral ischaemia.
Keywords K channel · Endothelium · Cerebral
vasculature · Hypoxia · NO donor · KATP opener
P. Movahed · E. D. Högestätt
Department of Clinical Pharmacology, Lund University Hospital,
221 85 Lund, Sweden
J. Petersson (✉)
Department of Neurology, Malmö University Hospital,
205 02 Malmö, Sweden
Fax: +46-40-337870,
e-mail: jesper.petersson@skane.se
Introduction
Ischaemic stroke is characterised by regional loss of blood
flow, tissue hypoxia, energy depletion and subsequent
neuronal death. In the search for therapeutic strategies,
much effort has been invested in the area of neuroprotec-
tion. However, ischaemic and post-ischaemic hypoperfu-
sion represent key pathophysiological events in acute
stroke, directly affecting neuronal survival (Ames et al.
1968; Levy et al. 1979). Pharmacological modulation of
cerebrovascular tone could potentially enhance blood sup-
ply to the ischaemic area and thus improve clinical out-
come. A general concern with vasodilators is, however,
their tendency to lower systemic blood pressure, an effect
that may negatively influence cerebral perfusion particu-
larly in ischaemic regions with disturbed cerebrovascular
autoregulation (Fujita et al. 1997; Ahmed et al. 2000). As
shown in the coronary circulation (Schulz et al. 1985), a
vasodilator may also compromise the blood supply to the
ischaemic area by way of acting on vessels in non-isch-
aemic regions, a phenomenon referred to as blood flow
steal. Thus, an optimal vasodilator in stroke should have a
selective action not only on the cerebrovascular bed, but
also on arteries in the ischaemic region to avoid haemo-
dynamically significant shunting of blood flow towards
non-ischaemic tissue.
Cerebral ischaemia causes structural damage to the en-
dothelium, inhibits endothelium-dependent vasodilatation
and increases the formation of the potent vasoconstrictor
endothelin-1 (Faraci and Heistad 1998; Stauton et al. 1999).
We have previously reported that hypoxia abolishes endo-
thelium-dependent vasorelaxation mediated by nitric ox-
ide (NO) in guinea-pig basilar artery (Petersson et al.
1998). Hypoxia, ischaemia and endothelial damage are
known to modulate the effects of vasoactive agents
(Auch-Schwelk and Vanhoutte 1991; Bari et al. 1996;
Husken et al. 1997; Petersson et al. 1997a). When target-
ing pharmacological agents for use in cerebral ischaemia,
it is therefore important to assess their effects under hy-
poxic conditions.
 533

In the present study, we examined the effects of hy- tion was tested according to Smirnow-Kolmogorow. Two-tailed
poxia on vasodilator responses to the ATP-dependent potas- Student’s t-test or analysis of variance (ANOVA) followed by
Bonferroni-Dunn’s post hoc test was used for statistical compari-
sium channel (KATP) opener levcromakalim and the NO son; a p value less than 0.05 was considered as statistically signif-
donor S-nitroso-N-acetylpenicillamine (SNAP). Drugs act- icant. Due to study design, only comparisons between control and
ing through these distinct molecular mechanisms seem to hypoxic conditions are presented.
have beneficial effects in cerebral ischaemia as shown in
Drugs. Acetylcholine chloride, endothelin-1, glibenclamide and
initial experimental (Heurteaux et al. 1993; Takaba et al. NG-nitro-L-arginine were purchased from Sigma, St. Louis, MO,
1997; Reshef et al. 1998) and clinical studies (Joshi et al. USA; apamin from Alomone labs, Jerusalem, Israel; charybdo-
1997; Butterworth et al. 1998). toxin from Latoxan, Rosans, France; indomethacin (Confortid)
from Dumex, Copenhagen, Denmark; levcromakalim from Smith-
kline Beecham, UK; ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxa-
line-1-one) from Tocris Cookson, St. Louis, MO, USA. S-nitroso-
Materials and methods N-acetylpenicillamine was obtained from Schwarz Pharma, Mon-
heim, Germany.
Vascular preparations. Guinea pigs of male gender (300 g) where
killed by CO2 asphyxia followed by exsanguination. After removal
of the brainstem, the basilar artery was dissected and flushed with
physiological salt solution (PSS; composition: NaCl 119 mM, KCl Results
4.6 mM, CaCl2 1.5 mM, MgCl2 1.2 mM, NaHCO3 15 mM, NaH2PO4
1.2 mM and D-glucose 6 mM) to remove blood products. SNAP
Tension experiments. Tension experiments were performed in or- The NO donor SNAP induced concentration-dependent
gan baths containing PSS. The temperature was maintained at
37°C. To provide oxygenation and a pH of 7.4, the PSS in the or-
relaxations in guinea-pig basilar arteries (Fig. 1a). The
gan baths was continuously bubbled with carbogen. Ring prepara-
tions (1–2 mm long) of the arteries were gently thread on two
stainless steel wires (Högestätt et al. 1983). One of the wires was
connected to a force-displacement transducer (model FT03 C,
Grass Instruments, USA) for isometric tension recording. The out-
put from the transducer was displayed on a polygraph (model 7D,
Grass Instruments, USA).
The vessel segments were allowed to equilibrate for 60 min un-
der a passive load of 2–4 mN (corresponding to a wall tension of
1–2 mN/mm). A 60 mM K+ solution (PSS with an equimolar
amount of NaCl substituted with KCl) was then introduced to as-
sess the contractile capacity of the preparation. Inhibitors were
added 30 min prior to relaxation. With the exception of the combi-
nation of charybdotoxin and apamin, which occasionally induced a
contraction, none of the inhibitors affected the basal tension level.
Endothelin-1 was used to contract the arteries, since this contrac-
tile agent induces stable contractions under both control and hy-
poxic conditions in this preparation (Petersson et al. 1998). The
level of contraction was titrated in each experiment aiming at
90–110% of the initial contraction obtained with 60 mM K+. Final
concentrations of endothelin-1 were 4 nM (–log M 8.5±0.06) in
controls and 10 nM (–log M 8.0±0.02) under hypoxic conditions.
Absolute levels of pre-contraction induced by endothelin-1 were
4.68±0.31 mN and 4.24±0.26 mN under control and hypoxic con-
ditions, respectively (n=49). Acetylcholine (0.01–100 µM), levcro-
makalim (0.01–10 µM) or SNAP (0.01–10 µM) were added cumu-
latively to elicit relaxations. Indomethacin (10 µM) was present
throughout.

Induction of hypoxia. Hypoxia was achieved by bubbling the PSS

in the organ bath with a mixture of N2 (95%) and CO2 (5%). A box
was placed over the organ baths and continuously flushed with
N2/CO2 to minimise oxygen exchange with the surrounding air.
Drugs were added through elastic membranes situated on top of the
box above each organ bath. Relaxation experiments were performed
after 30 min of hypoxia when the pO2 is approximately 5.9 mm Hg
under these experimental conditions (Petersson et al. 1998).

Calculations and statistics. The negative logarithm of the drug

concentration eliciting half-maximal relaxation (pEC50) was calcu-
lated by non-linear regression (GraphPad Prism 3.0). Emax refers to
the maximal relaxation achieved (100% denotes a complete rever-
sal of the contraction). The area under the curve (AUC) was calcu-
lated according to the trapezoid principle (GraphPad Prism 3.0).
Results are given as arithmetic mean values ± standard error of the
mean (SEM). Individual relaxation experiments were analysed and
the parameters were put in a table. The normality of the distribu-
Fig. 1 S-nitroso-N-acetylpenicillamine (SNAP)-induced relaxations
under control (black circles) and hypoxic (white circles) conditions
in the a absence (n=7) or b presence (n=6) of NG-nitro-L-arginine
(L-NOARG; 0.3 mM) in arteries contracted by endothelin-1. Indo-
methacin (10 µM) was present in all experiments. Data are pre-
sented as means ± SEM
534
Table 1 Relaxations induced by S-nitroso-N-acetylpenicillamine (SNAP) and levcromakalim in cerebral arteries under control or hy-
poxic conditions. AUC area under the curve. n.d. not determined
Agonist Treatment Control Hypoxia

n AUC pEC50 Emax % n AUC pEC50 Emax %

SNAP Vehicle 7 61±20 n.d. 59±13 7 165±17* 6.9±0.4 92±2*
L-NOARG (0.3 mM) 6 191±20 6.9±0.2 97±3 6 128±29 6.2±0.4 84±9
Levcromakalim Vehicle 10 82±24 6.3±0.2 53±4 8 136±26 6.7±0.3 70±11
L-NOARG (0.3 mM) 7 174±21 6.8±0.2 95±3 7 54±15* n.d. 29±7*
Indomethacin (10 µM) was present in all experiments Statistical comparisons were made between control and hypoxic
Data given as means ± SEM conditions (*p<0.05)

Fig. 3 Relaxations induced by levcromakalim in the presence

(white circles; area under the curve (AUC)=25±29) or absence
(black circles; AUC=160±25) of 10 µM glibenclamide (n=5;
p<0.05). Indomethacin (10 µM) and L-NOARG (0.3 mM) were
present in all experiments. Data are presented as means ± SEM

Levcromakalim

The KATP activator levcromakalim induced concentration-

Fig. 2 Levcromakalim-induced relaxations under control (black
circles) and hypoxic (white circles) conditions in the a absence
(n=8–10) or b presence (n=7) of L-NOARG (0.3 mM) in arteries
contracted by endothelin-1. Indomethacin (10 µM) was present in
all experiments. Data are presented as means ± SEM
guanylate cyclase inhibitor ODQ (10 µM) abolished these
responses (n=6). Exposure to hypoxia enhanced the SNAP-
induced vasorelaxation (Fig. 1a; Table 1). This effect of
hypoxia was not observed when the experiments were
performed in the presence of the NO synthase inhibitor
NG-nitro-L-arginine (L-NOARG; 0.3 mM; Fig. 1b; Table 1).
dependent relaxations (Fig. 2a; Table 1). The KATP block-
ing agent glibenclamide (10 µM) attenuated these re-
sponses (Fig. 3). Exposure to hypoxia did not significantly
affect the levcromakalim-induced vasorelaxation (Fig. 2a;
Table 1). However, hypoxia inhibited the vasodilator re-
sponse to levcromakalim when the experiments were per-
formed in the presence of L-NOARG (Fig. 2b; Table 1).
Endothelium-derived NO
The vasodilator response to acetylcholine in the presence
of the potassium channel inhibitors charybdotoxin and
apamin is mediated by endothelium-derived NO in the
guinea-pig basilar artery (Petersson et al. 1997b). Expo-
sure to hypoxia abolished the acetylcholine-induced va-
sorelaxation in the presence of these potassium channel
inhibitors (Fig. 4a; controls: AUC=157±27, pEC50=6.6±0.3
and Emax=87±6%; hypoxia: AUC=1±1 and Emax=3±2%; n=6;
p<0.05).

Fig. 4 Acetylcholine-induced relaxations a in the presence of
0.1 µM charybdotoxin and 0.1 µM apamin (n=6) and b in the pres-
ence of 0.3 mM L-NOARG (n=5) under control (black circles) or
hypoxic (white circles) conditions in arteries contracted with en-
dothelin-1. Indomethacin (10 µM) was present in all experiments.
Data are presented as means ± SEM
Endothelium-derived hyperpolarising factor
The vasodilator response to acetylcholine in the presence
of L-NOARG and indomethacin is mediated by endothe-
lium-derived hyperpolarising factor (EDHF) in the guinea-
pig basilar artery (Petersson et al. 1997b). Exposure to hy-
poxia did not significantly affect the acetylcholine-in-
duced vasorelaxation in the presence of L-NOARG and
indomethacin (Fig. 4b; controls: AUC=185±24, pEC50=
6.9±0.3 and Emax=97±2%; hypoxia: AUC=133±22, pEC50=
6.3±0.3 and Emax=83±4%; n=5).
Discussion
In the present study, the NO donor SNAP and the KATP
opener levcromakalim induced concentration-dependent
535
relaxations under both standard and hypoxic conditions.
However, the vasodilator responses to these drugs were
differently affected by hypoxia. SNAP-induced relaxations
were more pronounced under hypoxic conditions, while
vasodilatation induced by levcromakalim was not signifi-
cantly affected by removal of oxygen. Consistent with our
earlier findings, hypoxia abolished the acetylcholine-in-
duced vasorelaxation mediated by endothelium-derived
NO (Petersson et al. 1998). This probably reflects that
oxygen is required for synthesis of NO (Moncada and
Higgs 1993). Loss of basal NO formation under hypoxic
conditions provides a likely explanation for the change in
potency of SNAP. In the presence of oxygen, basal release
of endothelium-derived NO and subsequent activation of
guanylate cyclase probably lessens the capacity for fur-
ther activation of this relaxant pathway. To test this hy-
pothesis, the experiments were repeated in the presence of
the NO synthase inhibitor L-NOARG. Indeed, L-NOARG
abolished the hypoxia-induced enhancement of the va-
sodilator response to SNAP. These experiments clearly in-
dicate that activation of the NO-guanylate cyclase path-
way is a very effective vasodilator principle under condi-
tions of low pO2.
The role of NO in acute ischaemic stroke is complex.
As a vasodilator, anti-thrombotic and antioxidative agent,
NO may confer protective effects in the central nervous
system, but NO may also be involved in the generation of
neurotoxic oxygen radicals (Lipton et al. 1998; Chiueh
1999). In mice, deletion of the gene encoding endothelial
NO synthase increases the infarcted area in stroke (Huang
et al. 1994). Lack of endothelial NO may facilitate an un-
timely vasoconstriction in stroke. On the other hand, dele-
tion of the gene encoding neuronal NO synthase reduces
the infarcted area in stroke, consistent with a neurotoxic
effect of NO (Huang et al. 1994). Nevertheless, it has
been reported that early administration of NO-donors can
reduce the damaged area after focal ischaemic injury in-
duced by occlusion of the middle cerebral artery in spon-
taneously hypertensive rats (Zhang et al. 1994). Initial
studies on patients indicate that the NO donor sodium ni-
troprusside may improve regional cerebral blood flow in
acute ischaemic stroke and reverse vasospasm after sub-
arachnoid haemorrhage (Butterworth et al. 1998; Thomas
and Rosenwasser 1999).
Activators of KATP are potent vasodilators (Standen et
al. 1989), showing selectivity towards cerebral arteries
(Ryman et al. 1993; Petersson et al. 2000). These va-
sodilators also protect against neuronal injury in different
models of cerebral ischaemia, presumably by way of acti-
vation of neuronal KATP (Heurteaux et al. 1993; Takaba et
al. 1997; Reshef et al. 1998). Furthermore, the KATP opener
levcromakalim has been shown to alleviate vasospasm in
subarachnoid haemorrhage in dog, consistent with a direct
vasodilator effect (Sugai et al. 1999). In the present study,
hypoxia did not significantly affect the vasodilator re-
sponse to levcromakalim in the guinea-pig basilar artery.
A clear depression of the levcromakalim-induced vasore-
laxation was, however, seen when the experiments were
conducted in the presence of the NO synthase inhibitor
536
L-NOARG. Hypoxia also attenuated the vasodilator re-
sponse to levcromakalim in rat aorta (Husken et al. 1997),
and global cerebral ischemia impaired the vasodilatation
of cerebral arterioles in situ to the KATP opener aprikalim
is in piglets (Bari et al. 1996).
The reason why hypoxia failed to enhance the va-
sodilator response to levcromakalim is unclear, but hy-
poxia-induced changes of the intracellular levels of nu-
cleotides may be involved. KATP is reciprocally regulated
by the intracellular concentrations of ATP and ADP (Ash-
croft and Gribble 1998), and hypoxia-induced changes of
these nucleotides favour channel opening (Taggart and
Wray 1998). As shown in the rat middle cerebral artery,
hypoxic vasodilatation, i.e. relaxation induced by lower-
ing of pO2, is eliminated by glibenclamide, implying acti-
vation of KATP at least during short periods of hypoxia
(Fredricks et al. 1994). An intrinsic activation of KATP
during hypoxia, which causes a hyperpolarisation of the
vascular smooth muscle cell (Taggart and Wray 1998),
may lessen the vasodilator effect of levcromakalim, thus
outbalancing any positive effect of inhibition of NO syn-
thesis on the levcromakalim-induced vasorelaxation. This
could explain the apparent hypoxia-induced inhibition of
the vasodilator response to levcromakalim in the presence
of L-NOARG. In contrast, vasodilator responses mediated
by activation of guanylate cyclase may be more resistant
to hypoxia-induced changes in membrane potential. Con-
sistent with our earlier findings, hypoxia did not signifi-
cantly affect the EDHF-mediated vasorelaxation (Peters-
son et al. 1998), which does not involve KATP (Taylor and
Weston 1988; Zygmunt et al. 1994). Thus, although hy-
perpolarisation seems an effective vasodilator mechanism
in the cerebral vasculature, other mechanisms than activa-
tion of KATP may be preferable in the setting of cerebral
ischaemia.
During the first hours in ischaemic stroke, there is lit-
tle blood flow in the core of the infarction, while the isch-
aemic penumbra zone is characterised by blood flow in-
sufficient for maintaining neuronal function (Astrup et al.
1981). The penumbra is thus an obvious target for drugs
improving tissue perfusion. The present study shows that
the NO donor SNAP is more effective at low than at high
pO2, suggesting that SNAP may have a selective action on
the vasculature in ischaemic brain regions. Levcromakalim
does not seem to show this selectivity towards vessels in a
hypoxic environment. Interestingly, in a study performed
on patients undergoing cerebral angiography, intra-arterial
administration of the NO donor sodium nitroprusside, at a
dose inducing vasodilatation in the coronary and brachial
circulations, did not increase cerebral blood flow (Joshi et
al. 1997). These findings suggest that cerebral arteries with
intact endothelium are relatively insensitive to the vaso-
dilatory action of NO donors during normoxia. This ob-
servation is in line with the results of the present study
and indicates that a steal phenomenon within the cerebral
circulation is unlikely to occur with an NO donor.
In conclusion, the NO donor SNAP was more effective
as a vasodilator during hypoxia than under control condi-
tions in guinea-pig basilar arteries contracted by endothe-
lin-1, whereas the KATP opener levcromakalim produced
similar vasorelaxation. Hypoxia-induced inhibition of en-
dogenous NO synthesis and subsequent down regulation
of the basal guanylate cyclase activity could explain this
enhancement of the vasodilator response to SNAP. Thus,
NO donors may have a selective effect on blood vessels in
ischaemic brain areas and therefore warrant further evalu-
ation as therapeutic agents in cerebral ischaemia (Bath et
al. 2000).
Acknowledgements This work was supported by the Swedish
Research Council (grant no. 11582.), the Heart and Lung Founda-
tion and the Medical Faculty of Lund (ALF). J.P. was supported by
the Medical Faculty of Lund (ALF). The study was approved by
the local ethics committee for experimental animal studies.
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