European Journal of Pharmacology 546 (2006) 120 – 126
www.elsevier.com/locate/ejphar
EDHF-mediated rapid restoration of hypotensive response to acetylcholine
after chronic, but not acute, nitric oxide synthase inhibition in rats
Kaushik M. Desai a,⁎, Venkat Gopalakrishnan a , Linda M. Hiebert b ,
J. Robert McNeill a , Thomas W. Wilson c
a
Department of Pharmacology, University of Saskatchewan, Saskatoon, Canada
Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Canada
Department of Medicine, Royal University Hospital, University of Saskatchewan, Saskatoon, Canada
b
c
Received 24 March 2006; received in revised form 22 June 2006; accepted 27 June 2006
Available online 5 July 2006
Abstract
Several in vitro studies have shown that endothelium-dependent vasodilatation is maintained by endothelium-derived hyperpolarizing factor
(EDHF) or prostacyclin in vessels isolated from endothelial nitric oxide synthase knockout mice. Since this has not been addressed by in vivo
studies, we sought to define the magnitude and the onset time of this compensation by recording blood pressure responses to endotheliumdependent vasodilators in rats treated acutely or chronically with the NOS inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME). Groups of male
Sprague–Dawley rats were given plain water (control) or L-NAME (0.7 mg/ml) in drinking water for 1 day, 5 days, 3 wks or 6 wks. Dosedependent hypotensive responses to acetylcholine, bradykinin and sodium nitroprusside were determined in anesthetized rats before and after
acute intravenous infusion of either L-NAME or a combination of apamin plus charybdotoxin that would selectively inhibit EDHF. Acute LNAME treatment increased the mean arterial pressure and inhibited acetylcholine- and bradykinin-induced fall in blood pressure in control but not
in chronic L-NAME treated rats. The endothelium-dependent hypotensive responses to acetylcholine and bradykinin were restored in rats treated
with L-NAME after a time period of 24 h along with increased sensitivity to sodium nitroprusside and reduced plasma nitrate + nitrite levels. While
apamin + charybdotoxin pretreatment inhibited the responses to acetylcholine and bradykinin in both acute and chronic L-NAME treated groups, it
was more pronounced in the latter group. In conclusion, chronic inhibition of nitric oxide synthase results in the development of a compensatory
hypotensive response to acetylcholine within 24 h and this is mediated by EDHF.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Nitric oxide; EDHF; Endothelium; Vasodilation; Nitric oxide synthase; In vivo
1. Introduction
Acetylcholine induces an endothelium-dependent vasodilatation and a transient hypotensive response in vivo (Furchgott and
Zawadzki, 1980; Rees et al., 1990). Endothelial nitric oxide
synthase (eNOS) mediates this in vivo response to acetylcholine
through nitric oxide (NO) release causing stimulation of soluble
guanylate cyclase and formation of cyclic guanosine monophosphate (cGMP) (Murad, 1994; Rees et al., 1990; Wang et al.,
⁎ Corresponding author. A120 Health Sciences Building, Department of
Pharmacology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK,
Canada S7N 5E5. Tel.: +1 306 966 2723; fax: +1 306 966 1440.
E-mail address: k.desai@usask.ca (K.M. Desai).
0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2006.06.072
1993). In vitro experiments have shown that in large conduit
vessels such as the aorta, acetylcholine-induced vasodilatation is
predominantly mediated by NO (Freitas et al., 2003; Nagao et al.,
1992; Shimokawa et al., 1996). However, in small resistance type
vessels such as the mesenteric, hindlimb, coronary and brain pial
vessels, besides NO, other mediators such as the endotheliumderived hyperpolarizing factor (EDHF) or prostacyclin, contribute to the endothelium-dependent vasodilator response to agonists
(Brandes et al., 2000; Gödecke et al., 1998; Lamping et al., 2000;
Meng et al., 1996). EDHF has been proposed to mediate its
vasodilator action through the initial activation of small conductance and intermediate conductance calcium-activated potassium channels (KCa) that are present on the endothelium and are
sensitive to inhibition by a combination of optimal concentrations
K.M. Desai et al. / European Journal of Pharmacology 546 (2006) 120–126
of apamin and charybdotoxin (Busse et al., 2002; Garland and
Plane, 1996).
A compensatory increase in EDHF and or prostacyclin-mediated vasodilatation to acetylcholine has been demonstrated in blood
vessels of eNOS knockout mice (Busse et al., 2002; Gödecke et al.,
1998; Iwakiri et al., 2002; Koller et al., 1994; Lamping et al., 2000;
Meng et al., 1996; Sun et al., 1999) and high salt treated rats
(Katusic, 2002; Sofola et al., 2002). In hypertensive patients,
impaired NO release is compensated by an endothelium-derived
hyperpolarizing vasodilator mediator (Taddei et al., 1999). In
animal models of hypertension, atherosclerosis, hyperlipidemia
and diabetes as well as in clinical settings with patients with these
disease conditions, a large number of studies have shown evidence of endothelial dysfunction, measured as a reduced level of
NO-mediated endothelium-dependent vasodilatation, while others
have shown unimpaired endothelium-dependent vasodilation
(Boulanger, 1999; Brunner et al., 2005; Chan et al., 2000). This
controversy remains unresolved. The relative roles of NOindependent endothelial mediators such as EDHF and/or prostacyclin contributing to endothelium-dependent hypotensive response under such in vivo situations have not been adequately
explored. The time-course of development of compensatory hypotensive response, especially after in vivo inhibition of NOS, has
not been reported. Here, we attempted to outline the time when the
compensatory mechanisms begin and/or when endothelial dysfunction, measured as reduced hypotensive responses to endothelium-dependent agonists, develops after acute and chronic
inhibition of NOS over different time periods in Sprague–Dawley
rats. In this study, we have used Nω-nitro-L-arginine methyl ester
(L-NAME) to inhibit NO formation in adult rats, a situation that
more closely, though not ideally, reflects the gradual impairment
of NO production that can occur in disease states, as opposed to eNOS knockout animals.
2. Methods and materials
The experimental protocols used here were approved and
carried out under the guidelines of the Animal Care Committee
of the University of Saskatchewan and conform with the Guide
for the Care and Use of Laboratory Animals published by the
US National Institutes of Health (NIH Publication No. 85-23,
revised 1996).
2.1. In vivo experiments
Male Sprague–Dawley rats weighing 300–350 g were used.
Different groups of rats (n = 4–6 each group) were given plain
drinking water (control) or Nω-nitro-L-arginine methyl ester (LNAME, 0.7 mg/ml, corresponding to a daily intake of approximately 65 mg/kg) (Ribeiro et al., 1992) in drinking water for
1 day, 3 days, 5 days, 3 weeks or 6 weeks. They were anaesthetized with intraperitoneal (i.p.) thiopental sodium, 100 mg/kg
(dissolved in saline at 25 mg/ml) (Laight et al., 2000). The rat was
placed on a heated pad to maintain the temperature at 37 °C
measured by a rectal probe. The trachea was cannulated and the
rat was allowed to breathe spontaneously. The right carotid artery
and the left jugular vein were cannulated with polythene cannulas
121
(Portex Ltd., Hythe, England). The carotid cannula (id 0.4, od
0.8 mm) was filled with heparinised saline (50 U/ml) and
connected to a pressure transducer to record mean arterial pressure
using the Powerlab data acquisition system (AD Instruments Pvt.
Ltd., Sydney, Australia). Hypotensive responses to endotheliumdependent agonists, acetylcholine and bradykinin, in vivo were
recorded as a transient fall in mean arterial pressure (Laight et al.,
2000; Rees et al., 1990). The jugular vein cannula (id 0.5, od
0.63 mm) was used to administer drugs as intravenous (i.v.) bolus
injections. After a 30 min stabilization period, responses were
obtained to various hypotensive agents. Enough time was allowed
between responses for the mean arterial pressure to recover to the
resting level.
2.2. Responses to drugs
Drug doses were calculated as μg/kg body weight. Drugs were
injected i.v. in a volume of 0.4 ml/kg body weight and flushed
with 0.1 ml saline. Dose-related responses were obtained to
acetylcholine (0.02 to 2 μg/kg), bradykinin (0.1 to 30 μg/kg), and
to NO-releasing endothelium-independent vasodilators nitroglycerine (0.1 to 50 μg/kg) and sodium nitroprusside (0.2 to 20 μg/kg).
A dose that represented the mid-part of the dose–response curve
was selected for some agents, for single dose responses in some
cases. Thus, responses to acetylcholine (0.02 to 2 μg/kg, whole
dose–response curve), bradykinin (3 μg/kg), nitroglycerine (5 μg/
kg) and sodium nitroprusside (2 μg/kg) were obtained before
(control) and after L-NAME (100 mg/kg i.v.) (Rees et al., 1990) or
apamin (25 μg/kg i.v.) + charybdotoxin (25 μg/kg i.v.) administration in different groups of rats. The minimum effective doses
for apamin plus charybdotoxin were determined in preliminary
experiments based on previously used doses (Shinde et al., 2005).
Apamin plus charybdotoxin were used at only one time-point of
chronic L-NAME treatment in one group of rats since their cost
was very high for in vivo type studies.
2.3. Measurement of plasma nitrate plus nitrite
Levels of plasma nitrate plus nitrite were obtained by the
Griess method using the Nitrate/Nitrite Colorimetric Assay Kit
(Cayman Chemical, Ann Arbor, MI). 0.5 ml jugular vein blood
samples were collected into 10% sodium citrate. The tubes were
centrifuged at 4 °C and 3000 rpm for 5 min, and the recovered
plasma was stored at − 70 °C. Subsequently, the samples were
thawed and transferred to Nanosep® 10K Omega centrifuge
filter tubes (Pall Corp., Ann Arbor, Michigan) which employ
10 kDa molecular weight cut-off filters. Tubes were centrifuged
at 4 °C and 15,000 g for 20 min to remove the hemoglobin. The
filtrate was then transferred in 40 μl aliquots to a 96 well plate,
with each sample performed in duplicate. To each well, 10 μl of
Enzyme Cofactor mixture and 10 μl of nitrate reductase mixture
were added, and the plate was covered and incubated at room
temperature for 3 h. After the incubation was complete, 50 μl
each of 2% sulphanilamide in 5% phosphoric acid and 0.2%
naphthylethylenediamine dihydrochloride was added to each
well. After allowing 10 min for optimal color development, we
read the absorbance at 540 nm using the Anthos II Plate Reader.
122
K.M. Desai et al. / European Journal of Pharmacology 546 (2006) 120–126
Table 1
Basal values of mean arterial pressure (mean ± S.E.M.) in different groups of rats
Treatment group
Mean arterial pressure (mmHg)
Control
Acute L-NAME
Chronic L-NAME — 6 weeks
Chronic L-NAME — 3 weeks
Chronic L-NAME — 5 days
Chronic L-NAME — 1 day
Apa + ChTx
L-NAME + Apa + ChTx
107 ± 5 (n = 10)
149 ± 3 (n = 6)a
135 ± 5 (n = 5)b
133 ± 5 (n = 6)b
133 ± 2 (n = 7)b
138 ± 3 (n = 4)b
111 ± 8 (n = 4)
144 ± 2 (n = 4)a,c
Chronic L-NAME was given in drinking water (0.7 mg/ml) for the time
indicated. Acute L-NAME (100 mg/kg), apamin (25 μg/kg) and charybdotoxin
(25 μg/kg) were administered intravenously.
ω
L-NAME — N -nitro-L-arginine methyl ester, Apa — apamin, ChTx —
charybdotoxin, n = number of rats, aP < 0.001, bP < 0.01 vs. control group,
c
P < 0.01 vs. Apa + ChTx group.
2.4. Materials
Acetylcholine, Nω-nitro-L-arginine methyl ester (L-NAME),
apamin, indomethacin, sodium nitroprusside dihydrate and
bradykinin acetate were purchased from Sigma-Aldrich Canada
Ltd. (Oakville, Ontario, Canada). Charybdotoxin was purchased from Ana Spec (San Jose, CA). Nitroglycerine was
purchased from SABEX (Boucherville, Québec, Canada).
Thiopental sodium was from Abbott Laboratories Ltd. (SaintLaurent, Québec, Canada).
in a group of rats that received acute L-NAME infusion (i.v.)
subsequent to receiving chronic L-NAME treatment (Fig. 1A and
B). The mean arterial pressures were significantly higher after
acute L-NAME, compared to their respective control groups, in
both chronic L-NAME treatment group (155± 3 vs. 135± 5 mmHg,
P < 0.05, n = 5 each) and acute L-NAME treatment group (153± 3
vs. 103 ± 9 mmHg, P < 0.001, n = 5 each). In the chronic L-NAME
treated rats, a single dose of sodium nitroprusside, 2 μg/kg,
induced a greater hypotensive response (40± 3% of the baseline,
n = 5, P < 0.01) compared to the responses seen in the control
group (29 ± 2% of the baseline, n = 5). Plasma nitrate + nitrite
levels were significantly lower (P < 0.01) in chronic L-NAME
treated group (1.54± 0.31 μM, n = 6) compared to the control
group (4.55 ± 0.73 μM, n = 6).
In preliminary experiments we have ascertained that
pretreatment with indomethacin (10 mg/kg i.p.) did not affect
either the basal mean arterial pressure values or the responses to
acetylcholine in either the control group or in the chronic LNAME treated rats (data not shown).
3.2. Chronic L-NAME for 3 weeks
Acute L-NAME administration led to a significant rightward
shift in acetylcholine-evoked fall in mean arterial pressure
2.5. Data analysis
Hypotensive responses were calculated as % fall in mean
arterial pressure with respect to the baseline mean arterial
pressure before each response. We, and others, have shown that
the dose-dependent percent fall in mean arterial pressure after
acetylcholine, bradykinin, sodium nitroprusside and nitroglycerine is unrelated to the baseline mean arterial pressure (Laight
et al., 2000; Weldon et al., 1995). This is also supported by the
fact that the mean arterial pressure values were comparable in
the rats given acute L-NAME alone and in the rats that were
given L-NAME chronically (Table 1). The former group showed
inhibition of hypotensive responses to acetylcholine, the later
did not, as discussed in results. The values are expressed as
mean ± SEM with the number of experiments (n) shown in
brackets. The data was analyzed for statistical significance
using one-way analysis of variance (ANOVA) with Dunnett's
post-test, or Student's unpaired two-tailed t-test, as appropriate.
A P value less than 0.05 was deemed significant.
3. Results
3.1. Chronic L-NAME for 6 weeks
The hypotensive responses to acetylcholine and bradykinin
were inhibited with a significant rightward shift in their dose–
response curves in rats that received acute L-NAME treatment. In
contrast, the dose–response curves to acetylcholine and bradykinin remained unaffected in chronic L-NAME treatment group or
Fig. 1. Dose–response curves for acetylcholine (ACh, A) and bradykinin (BK, B)
evoked hypotension in Sprague–Dawley rats after 6 wks of chronic nitric oxide
synthase (NOS) inhibition. Two groups of rats (n = 6 each) were given either plain
drinking water (Control) or N ω-nitro-L-arginine methyl ester (L-NAME, 0.7 mg/ml
water) for 6 wks (Chronic L-NAME). After determining the responses to ACh and
BK, both groups were given acute L-NAME (100 mg/kg, i.v.) and the responses
were repeated. ⁎P < 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001 vs. paired response in the same
rat before acute L-NAME.
K.M. Desai et al. / European Journal of Pharmacology 546 (2006) 120–126
123
inhibited the response to a single dose of bradykinin, 3 μg/kg,
selected from the mid-part of its dose–response curve, in the
control group of rats. In the chronic L-NAME group the response
to bradykinin was comparable to the control group and was not
inhibited by acute L-NAME treatment (Fig. 2B).
The responses to single doses of nitroglycerine (5 μg/kg) and
sodium nitroprusside (2 μg/kg), selected from the mid-part of
their dose–response curves, were significantly greater in chronic
L-NAME treated group compared to the control group; acute LNAME treatment did not modify these responses (Fig. 2C). In the
control group, after acute L-NAME treatment the hypotensive
responses to nitroglycerine and sodium nitroprusside were greater. These observations are consistent with the data reported earlier
(Moncada et al., 1991).
3.3. Chronic L-NAME for 5 days
When L-NAME was given for 5 days, the fall in mean
arterial pressure evoked by acetylcholine was comparable or
even greater than that observed in the control group (Fig. 3A).
Administration of acute L-NAME to control rats increased the
mean arterial pressure significantly whereas in the chronic L-
Fig. 2. Dose–response curves for acetylcholine (ACh)-induced hypotension (A)
and hypotensive responses to single doses (selected from the mid-part of their
dose–response curves) of bradykinin (BK, B), nitroglycerine (NTG, C) and
sodium nitroprusside (SNP, C) in Sprague–Dawley rats after 3 wks of chronic
NOS inhibition. Two groups of rats (n = 6 each) were given either plain drinking
water (Control) or N ω-nitro-L-arginine methyl ester (L-NAME, 0.7 mg/ml
water) for 3 wks (Chronic L-NAME). After taking responses to all the agonists,
both groups were given acute (ac.) L-NAME (100 mg/kg, i.v.) and the responses
were repeated. ⁎P < 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001 vs. paired response in the
same rat before acute L-NAME (A and B) or chronic L-NAME vs. paired plain
water control group (C).
(Fig. 2A). However, 3 weeks of chronic L-NAME treatment
failed to alter the dose–response curve to acetylcholine from
its respective control group. Acute L-NAME treatment given to
chronic L-NAME treated rats for 3 weeks did not inhibit the
fall in mean arterial pressure to acetylcholine (Fig. 2A). Acute
L-NAME administration led to a significant increase in the
mean arterial pressure only in the control group (146 ± 2 vs.
100 ± 3 mmHg, P < 0.001, n = 4) but not in the chronic L-NAME
group (143 ± 7 vs. 133 ± 5 mmHg, n = 6). Acute L-NAME also
Fig. 3. Dose–response curves for acetylcholine (ACh)-induced hypotension (A)
and mean arterial pressure (MAP, B) in Sprague–Dawley rats. Two groups of rats
(n = 5 each) were given plain drinking water (Control) for 1 day or 5 days (not
shown) and two groups (n = 5–6 each) were given Nω-nitro-L-arginine methyl
ester (L-NAME, 0.7 mg/ml water) for 1 day or 5 days. After taking responses to
ACh, both groups were given acute L-NAME (100 mg/kg, i.v.) and the responses
were repeated. Acute L-NAME did not inhibit the responses to ACh in the 1 day
or the 5 day chronic L-NAME groups (not shown). ⁎P < 0.05, ⁎⁎P < 0.01,
⁎⁎⁎P < 0.001 vs. paired response in the same rat before acute L-NAME (A) or vs.
control group (B).
124
K.M. Desai et al. / European Journal of Pharmacology 546 (2006) 120–126
NAME treated rats there was no further increase in the mean
arterial pressure (Fig. 3B).
3.4. L-NAME given acutely or for 1 day
The dose–response curves to acetylcholine-evoked hypotension were similar between L-NAME treated groups (L-NAME
given for 1 day or 5 days) and the control group (Fig. 3A). On the
other hand, acute L-NAME administration to the control group of
rats showed a significant level of inhibition with a rightward shift
in the dose–response curve to acetylcholine (Fig. 3A).
3.5. Apamin plus charybdotoxin inhibit the responses to
acetylcholine
The hypotensive response curves to acetylcholine were quite
similar between chronic L-NAME treatment (given for 3 days)
and the control group (Fig. 4). Preinfusion of apamin plus
charybdotoxin combination either in the control group or chronic
L-NAME treated group led to significant rightward shifts in the
dose–response curves to acetylcholine (Fig. 4). Interestingly, the
shift in the dose–response curve after apamin plus charybdotoxin
infusion was more pronounced in the chronic L-NAME treated
group compared to the shift attained in the control group. Administration of apamin plus charybdotoxin in acute L-NAME treated
rats was invariably lethal. Therefore, the responses to acetylcholine could not be determined after the inclusion of apamin plus
charybdotoxin combination in this group. There were no significant differences in the fall in mean arterial pressure evoked by a
fixed concentration of sodium nitroprusside before and after the
infusion of apamin plus charybdotoxin in the control (35 ± 2% vs.
28 ± 5%, n = 4 each) as well as chronic L-NAME treated groups
(40 ± 2% vs. 34 ± 9%, n = 3 each).
Fig. 4. Dose–response curves to acetylcholine (ACh)-induced hypotension after
chronic NOS inhibition for 3 days in Sprague–Dawley rats. Two groups of rats
(n = 4–6 each) were given plain drinking water (Control) for 3 days (pooled data
shown for control graph) while one group (n = 4) was given N ω-nitro-L-arginine
methyl ester chronically (Ch. L-NAME, 0.7 mg/ml water) for 3 days. After
taking responses to acetylcholine, one control group was given acute L-NAME
(Ac. L-NAME, 100 mg/kg, i.v., n = 6), while the other control group (n = 4) and
the chronic L-NAME group (n = 3) were given apamin (Apa, 25 μg/kg i.v.) plus
charybdotoxin (ChTx, 25 μg/kg i.v.) and the responses were repeated. ⁎P<0.05,
⁎⁎P < 0.01, ⁎⁎⁎P<0.001 vs paired response in the same rat before acute l-NAME
or apamin plus charybdotoxin.
4. Discussion
We report the rapid development of a compensatory endothelium-dependent agonist-induced hypotension within one day
after chronic NOS inhibition. This compensatory hypotension is
mediated via activation of endothelial calcium-activated potassium channels, probably through the release of EDHF. Endothelial
dysfunction, in terms of reduced agonist-induced endotheliumdependent hypotension was not observed even after 6 weeks of
chronic NOS inhibition. To our knowledge this time-frame of
compensatory changes in endothelial function in vivo is being
reported for the first time in the literature.
The intravenous injection of a small dose of acetylcholine
produces a transient fall in blood pressure owing to generalized
vasodilatation. A considerably larger dose is required to elicit
bradycardia or block of AV nodal conduction from a direct action
of acetylcholine on the heart (Brown and Taylor, 2006). We used
doses of acetylcholine up to 2 μg/kg body wt. (Laight et al., 1998),
less than previously reported use (Rees et al., 1989). Moreover,
for the entire dose range of acetylcholine that we have used, the
heart rate did not change at the peak of the hypotensive response
as compared to the baseline value (unpublished data) (Rees et al.,
1989). Thus, bradycardia or tachycardia does not seem to be
modulating the response. Also, the hypotensive response is
endothelium-dependent since acetylcholine produces contraction
of vascular smooth muscle in the absence of the endothelium
(Furchgott and Zawadzki, 1980).
The effectiveness of oral administration of L-NAME in the
drinking water (Ribeiro et al., 1992; Shinde et al., 2005) and the
inhibition of NOS are supported by several observations. In
chronic L-NAME treated rats there was a significant increase in
the mean arterial pressure, a reduction in plasma nitrate + nitrite
levels and no further increases in the mean arterial pressure after
acute L-NAME administration in chronic L-NAME treated rats,
especially after 5 days of L-NAME treatment. Moreover, after
chronic NOS inhibition, the responses to NO-releasing but endothelium-independent vasodilators such as sodium nitroprusside
and nitroglycerine were significantly enhanced indicating increased sensitivity of soluble guanylate cyclase after chronic NOS
inhibition (Moncada et al., 1991). Thus, in the presence of NOS
inhibition, the normal response to acetylcholine, as compared to
control rats, can be safely assumed to be mediated by non-NO
mediators such as EDHF and/or prostacyclin. In support of this
observation, acute L-NAME inhibited the responses to acetylcholine and bradykinin in the control rats but not in chronic LNAME treated rats (Figs. 1A,B and 2A,B). These kind of compensated normal responses have been reported in vitro in vessels
such as the coronary (Gödecke et al., 1998; Lamping et al., 2000),
brain pial (Meng et al., 1996), skeletal muscle (Koller et al., 1994),
the mesenteric (Sofola et al., 2002) and other vessels (Iwakiri
et al., 2002) in eNOS knockout mice.
The role of prostacyclin in the in vivo response to acetylcholine seems to be none or negligible since administration of
diclofenac (Brandes et al., 2000) or indomethacin (Rees et al.,
1990) to inhibit cyclooxygenase-mediated prostanoid production
did not affect the mean arterial pressure or the hypotensive responses to acetylcholine. The compensatory response was also
K.M. Desai et al. / European Journal of Pharmacology 546 (2006) 120–126
observed when bradykinin, another endothelium-dependent agonist, was used. This compensatory response was observed as early
as 1 day after NOS inhibition with L-NAME. However, acute
administration of L-NAME in control (given plain water) rats
significantly inhibited the responses to acetylcholine and bradykinin, indicating involvement of NO in the hypotensive response
to both agonists under normal circumstances. It should be pointed
out that acetylcholine- and bradykinin-induced hypotensive
responses taken even 3 h after acute L-NAME were significantly
inhibited. Normally, the responses to acetylcholine and bradykinin were determined 1 h after acute i.v. administration of a single
dose of L-NAME. The response after 1 day of L-NAME was close
to normal and was not significantly inhibited (Fig 3A). This
confirms that that the compensatory response by EDHF begins
within 1 day of NOS inhibition. It is noteworthy that the increase
in mean arterial pressure is not completely normalized even
6 weeks after chronic NOS inhibition (Table 1). When mean
arterial pressure is measured 3 or 6 weeks after NOS inhibition, it
is lower than the values for mean arterial pressure determined after
acute NOS inhibition (Table 1). This suggests differential regulation of the basal tone in resistance vessels, which determines the
mean arterial pressure, and the agonist-induced endothelium-dependent vasodilatation which determines acute changes in blood
flow to organs in response to neurohumoral regulation. Whether
this also means a more fundamental role of NO in basal tone and
steady-state blood pressure as compared to EDHF and a more
pronounced role of EDHF in acute blood flow changes remains to
be established. It is worth noting that most studies on EDHF relate
to its role in endothelium-dependent vasodilatation. This kind of
differential compensatory changes cannot be revealed by in vitro
experiments.
The compensated response to acetylcholine after chronic
NOS inhibition was almost completely inhibited by blockade of
endothelial KCa channels with the combination of apamin plus
charybdotoxin. The inhibition was greater in magnitude than
that in control rats by apamin plus charybdotoxin. This strongly
suggests a predominant involvement of KCa channels contributing to the compensated response during NOS inhibition. This
also agrees with several in vitro studies on eNOS knockout mice
which report a compensatory increase in the release of EDHF in
different vascular beds (Brandes et al., 2000; Huang et al., 2001;
Woodman et al., 2000) and in high salt treated Sprague–Dawley
rats (Katusic, 2002; Sofola et al., 2002). In the control rats the
response seems to be equally mediated by NO and KCa channels, probably activated by EDHF.
Interestingly, even after prolonged NOS inhibition by chronic
L-NAME treatment for 6 weeks, we did not see endothelial
dysfunction as indicated by an apparently normal endotheliumdependent hypotensive response. We did not look at other
indicators of endothelial dysfunction besides reduced endothelium-dependent responses in view of our specific objective. Thus,
endothelial dysfunction might develop only if the compensatory
vasodilation mediated by KCa channels or prostacyclin fails.
There are several animal models and human studies involving
different pathologies that show normal or impaired endothelium-dependent relaxation (Boulanger, 1999; Brunner et al.,
2005; Chan et al., 2000). These differences might reflect
125
different stages of the development of endothelial dysfunction.
Thus, it may be worthwhile to investigate the roles of EDHF as
well as prostacyclin signaling pathways besides NO signaling
in models of endothelial dysfunction. This may help better
planning of treatment approaches in cardiovascular disease
states.
In conclusion, the hypotensive response to endothelium-dependent agonists, such as acetylcholine and bradykinin, is rapidly
compensated within 1 day after chronic inhibition of nitric oxide
synthase. The compensatory relaxation is mediated by activation
of KCa channels. Endothelial dysfunction, measured as reduced
endothelium-dependent hypotensive response, does not develop
after inhibition of NOS activity for at least up to 6 weeks in
Sprague–Dawley rats.
Acknowledgements
This work is supported by a group grant from the Heart and
Stroke Foundation of Saskatchewan and a CIHR grant to Dr
Gopalakrishnan.
References
Boulanger, C.M., 1999. Secondary endothelial dysfunction: hypertension and
heart failure. J. Mol. Cell. Cardiol. 31, 39–49.
Brandes, R.P., Schmitz-Winnenthal, F.H., Feletou, M., Gödecke, A., Huang, P.L.,
Vanhoutte, P.M., Fleming, I., Busse, R., 2000. An endothelium-derived hyperpolarizing factor distinct from NO and prostacyclin is a major endotheliumdependent vasodilator in resistance vessels of wild-type and endothelial NO
synthase knockout mice. Proc. Natl. Acad. Sci. U. S. A. 97, 9747–9752.
Brown, J.H., Taylor, P., 2006. Muscarinic receptor agonists and antagonists, In:
Brunton, L.L., Lazo, J.S., Parker, K.L. (Eds.), Goodman And Gilman's The
Pharmacological Basis of Therapeutics, 11th Edition. McGraw Hill, USA,
p. 184.
Brunner, H., Cockroft, J.R., Deanfield, J., Donald, A., Ferrannini, E., Halcox, J.,
Kiowski, W., Lüscher, T.F., Mancia, G., Natali, A., Oliver, J.J., Pessina, A.C.,
Rizzoni, D., Rossi, G.P., Salvetti, A., Spieker, L.E., Taddei, S., Webb, D.J.,
2005. Endothelial function and dysfunction. Part II: association with cardiovascular risk factors and diseases. A statement by the working group on
endothelins and endothelial factors of the European Society of hypertension.
J. Hypertens. 23, 233–246.
Busse, R., Edwards, G., Feletou, M., Fleming, I., Vanhoutte, P.M., Weston, A.H.,
2002. EDHF: bringing the concepts together. Trends Pharmacol. Sci. 23,
374–380.
Chan, N.N., Vallance, P., Colhoun, H.M., 2000. Nitric oxide and vascular responses in Type I diabetes. Diabetologia 43, 137–147.
Freitas, M.R., Schott, C., Corriu, C., Sassard, J., Stoclet, J.C., Andriantsitohaina,
R., 2003. Heterogeneity of endothelium-dependent vasorelaxation in conductance and resistance arteries from Lyon normotensive and hypertensive
rats. J. Hypertens. 21, 1505–1512.
Furchgott, R.F., Zawadzki, J.V., 1980. The obligatory role of endothelial cells in
the relaxation of arterial smooth muscle by acetylcholine. Nature 288,
373–376.
Garland, C.J., Plane, F., 1996. Relative importance of endothelium-derived
hyperpolarizing factor for the relaxation of vascular smooth muscle in different
arterial beds. In: Vanhoutte, P.M. (Ed.), Endothelium-Derived Hyperpolarizing
Factor 1. Harwood Academic Publishers, pp. 173–179.
Gödecke, A., Decking, U.K.M., Ding, Z., Hirchenhain, J., Bidmon, H.J.,
Gödecke, S., Schrader, J., 1998. Coronary hemodynamics in endothelial NO
synthase knockout mice. Circ. Res. 82, 186–194.
Huang, A., Sun, D., Carroll, M.A., Jiang, H., Smith, C.J., Connetta, J.A., Falck,
J.R., Shesely, E.G., Koller, A., Kaley, G., 2001. EDHF mediates flowinduced dilation in skeletal muscle arterioles of female eNOS-KO mice. Am.
J. Physiol. 280, H2462–H2469.
126
K.M. Desai et al. / European Journal of Pharmacology 546 (2006) 120–126
Iwakiri, Y., Cadelina, G., Sessa, W.C., Groszmann, R.J., 2002. Mice with
targeted deletion of eNOS develop hyperdynamic circulation associated with
portal hypertension. Am. J. Physiol. 283, G1074–G1081.
Katusic, Z.S., 2002. Back to the salt mines — endothelial dysfunction in hypertension and compensatory role of endothelium-derived hyperpolarizing
factor (EDHF). J. Physiol. 543 (Pt 1), 1.
Koller, A., Sun, D., Huang, A., Kaley, G., 1994. Corelease of nitric oxide and
prostaglandins mediates flow-dependent dilation of rat gracilis muscle arterioles. Am. J. Physiol. 266, H326–H332.
Laight, D.W., Kengatharan, K.M., Gopaul, N.K., Anggärd, E.E., Carrier, M.J.,
1998. Investigation of oxidant stress and vasodepression to glyceryl
trinitrate in the obese Zucker rat in vivo. Br. J. Pharmacol. 125, 895–901.
Laight, D.W., Desai, K.M., Anggärd, E.E., Carrier, M.J., 2000. Endothelial
dysfunction accompanies a pro-oxidant, pro-diabetic challenge in the insulin
resistant, obese Zucker rat in vivo. Eur. J. Pharmacol. 402, 95–99.
Lamping, K., Nuno, D., Shesely, E.G., Maeda, N., Faraci, F., 2000. Vasodilator
mechanisms in the coronary circulation of endothelial nitric oxide synthasedeficient mice. Am. J. Physiol. 279, H1906–H1912.
Meng, W., Ma, J., Ayata, C., Hara, H., Huang, P.L., Fishman, M.C., Moskowitz,
M.A., 1996. ACh dilates pial arterioles in endothelial and neuronal NOS
knockout mice by NO-dependent mechanisms. Am. J. Physiol. 271,
H1145–H1150.
Moncada, S., Rees, D.D., Schulz, R., Palmer, R.M.J., 1991. Development and
mechanism of a specific supersensitivity to nitrovasodilators after inhibition
of vascular nitric oxide synthesis in vivo. Proc. Natl. Acad. Sci. U. S. A. 88,
2166–2170.
Murad, F., 1994. Regulation of cytosolic guanylyl cyclase by nitric oxide: the
NO-cyclic GMP signal transduction system. Adv. Pharmacol. 26, 19–33.
Nagao, T., Illiano, S., Vanhoutte, P.M., 1992. Heterogeneous distribution of
endothelium-dependent relaxations resistant to NG-nitro-L-arginine in rats.
Am. J. Physiol. 263, H1090–H1094.
Rees, D.D., Palmer, R.M., Moncada, S., 1989. Role of endothelium-derived nitric
oxide in the regulation of blood pressure. Proc. Natl. Acad. Sci. U. S. A. 86,
3375–3378.
Rees, D.D., Palmer, R.M., Schulz, R., Hodson, H.F., Moncada, S., 1990.
Characterization of three inhibitors of endothelial nitric oxide synthase in
vitro and in vivo. Br. J. Pharmacol. 101, 746–752.
Ribeiro, M.O., Antunes, E., de Nucci, G., Lovisolo, S.M., Zatz, R., 1992. Chronic
inhibition of nitric oxide synthesis. A new model of arterial hypertension.
Hypertension 20, 298–303.
Shimokawa, H., Yasutake, H., Fujii, K., Owada, M.K., Nakaike, R., Fukumoto,
Y., Takayanagi, T., Nagao, T., Egashira, K., Fujishima, M., Takeshita, A.,
1996. The importance of the hyperpolarizing mechanism increases as the
vessel size decreases in endothelium-dependent relaxations in rat mesenteric
circulation. J. Cardiovasc. Pharmacol. 28, 703–711.
Shinde, U.A., Desai, K.M., Yu, C., Gopalakrishnan, V., 2005. Nitric oxide
synthase inhibition exaggerates the hypotensive response to ghrelin: role of
calcium-activated potassium channels. J. Hypertens. 23, 779–784.
Sofola, O.A., Knill, A., Hainsworth, R., Drinkhill, M., 2002. Change in
endothelial function in mesenteric arteries of Sprague–Dawley rats fed a high
salt diet. J. Physiol. 543, 255–260.
Sun, D., Huang, A., Smith, C.J., Stackpole, C.J., Connetta, J.A., Shesely, E.G.,
Koller, A., Kaley, G., 1999. Enhanced release of prostaglandins contributes
to flow-induced arteriolar dilation in eNOS knockout mice. Circ. Res. 85,
288–293.
Taddei, S., Ghiadoni, L., Virdis, A., Buralli, S., Salvetti, A., 1999. Vasodilation
to bradykinin is mediated by an ouabain-sensitive pathway as a compensatory mechanism for impaired nitric oxide availability in essential hypertensive patients. Circulation 100, 1400–1405.
Wang, Y.-X., Poon, C.I., Pang, C.C.Y., 1993. Vascular pharmacodynamics of
NG-nitro-L-arginine methyl ester in vitro and in vivo. J. Pharmacol. Exp.
Ther. 267, 1091–1099.
Weldon, S.M., Winquist, R.J., Madwed, J.B., 1995. Differential effects of LNAME on blood pressure and heart rate responses to acetylcholine and
bradykinin in cynomolgus primates. J. Pharmacol. Exp. Ther. 272, 126–133.
Woodman, O.L., Wongsawatkul, O., Sobey, C.G., 2000. Contribution of nitric
oxide, cyclic GMP and K+ channels to acetylcholine-induced dilatation of rat
conduit and resistance arteries. Clin. Exp. Pharmacol. Physiol. 27, 34–40.