An Endothelium-Derived Hyperpolarizing FactorLike Factor Moderates Myogenic

Constriction of Mesenteric Resistance Arteries in the Absence of Endothelial Nitric Oxide

SynthaseDerived Nitric Oxide
Ramona S. Scotland, Sharmila Chauhan, Patrick J.T. Vallance and Amrita Ahluwalia
Hypertension. 2001;38:833-839
doi: 10.1161/hy1001.092651
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright 2001 American Heart Association, Inc. All rights reserved.
Print ISSN: 0194-911X. Online ISSN: 1524-4563

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An Endothelium-Derived Hyperpolarizing FactorLike

Factor Moderates Myogenic Constriction of Mesenteric
Resistance Arteries in the Absence of Endothelial Nitric
Oxide SynthaseDerived Nitric Oxide
Ramona S. Scotland, Sharmila Chauhan, Patrick J.T. Vallance, Amrita Ahluwalia
AbstractMyogenic tone is an important determinant of vascular tone and blood flow in small resistance arteries of certain
vascular beds. The role of the endothelium in myogenic responses is unclear. We hypothesized that endothelium-derived
NO release modulates myogenic constriction in small resistance arteries and that mesenteric small arteries from mice
with targeted disruption of the gene for endothelial NO synthase (eNOS) (knockout mice) demonstrate greater myogenic
tone than do wild-type mice. Third-order mesenteric arteries (200 m) were isolated and mounted in a pressure
myograph. Internal diameter was recorded over a pressure range of 10 to 80 mm Hg. Removal of the endothelium
significantly (P0.05) enhanced the magnitude of myogenic constriction in wild-type mice. Similarly, pretreatment of
arteries with N G-nitro-L-arginine methyl ester (L-NAME; 300 mol/L) produced a comparable significant (P0.05)
increase in myogenic tone, whereas indomethacin (5 mol/L) had no effect. eNOS knockout arteries also exhibited
myogenic constriction. Neither L-NAME nor indomethacin had any effect on myogenic tone in the arteries of eNOS
knockout mice. However, blockade of potential endothelium-derived hyperpolarizing factorlike mechanisms via
inhibition of K flux using either apamin (100 nmol/L) with charybdotoxin (100 nmol/L), Ba2 (30 mol/L) with
ouabain (1 mmol/L), or 18-glycyrrhetinic acid (100 mol/L) significantly (P0.01) enhanced myogenic constriction.
This study demonstrates that basal endothelium-derived NO modulates myogenic tone in mesenteric small arteries of
wild-type mice. However, eNOS knockout arteries display normal myogenic responsiveness despite the absence of basal
NO activity. The data suggest that this compensatory effect is due to the activity of an endothelium-derived
hyperpolarizing factor to normalize vascular tone. (Hypertension. 2001;38:833-839.)
Key Words: nitric oxide endothelium mice nitric oxide synthase autoregulation

major determinant of vascular tone is termed autoregulation: a local regulatory mechanism independent of
neuronal and humoral systemic reflexes. Autoregulation describes the functional capacity of blood vessels to respond to
local metabolic changes and intraluminal forces generated at
the vessel wall (pressure [transmural force] and flow [shear
stress]) (for reviews, see Davis and Hill1 and Bevan2). The
response to transmural pressure is termed myogenic tone and
describes the ability of certain blood vessels to respond to
elevations in transmural pressure by constricting.3 This response predominates in resistance arteries (500 m)4 and is
considered to be due to a direct effect on smooth muscle,
resulting in depolarization5 and a consequent increase in
smooth muscle intracellular Ca2. However, the exact mechanisms are unclear.
Numerous studies have excluded a role for the endothelium
in mediation of the myogenic response (see Meininger and
Davis6). Recently, however, there has been renewed interest

in the endothelium as a modulator rather than as a mediator of

myogenic constriction.6 Studies of mesenteric vessels from
spontaneously hypertensive rats suggest that endothelium
removal enhances myogenic constriction,7 possibly as a
consequence of the loss of vasodilator endothelium-derived
NO. In addition, NO synthase (NOS) inhibitors potentiate
pressure-induced constriction of large, but not small, hamster
cremaster arterioles in vivo.8 Similarly, in the presence of
estrogen, NOS inhibition modulates myogenic constriction of
rat arteries (see Wellman et al9). The aim of the present study
was to investigate the role of NO in pressure-mediated
responses in small vessels to test the hypothesis that endothelial NOS (eNOS) activity is involved in the modulation of
myogenic responses in isolated resistance arteries. Because
highly specific inhibitors for each NOS isoform10 are not yet
available, we conducted experiments in vessels from mice
with targeted disruption of the eNOS gene to assess the
importance of this particular NOS isoform.

Received November 7, 2000; first decision December 4, 2000; revision accepted March 23, 2001.
From the Centre for Clinical Pharmacology, University College London, The Rayne Institute, London, United Kingdom.
Correspondence to Dr Amrita Ahluwalia, Centre for Clinical Pharmacology, University College London, The Rayne Institute, 5 University St, London,
United Kingdom WC1E 6JJ. E-mail a.ahluwalia@ucl.ac.uk
2001 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org

833
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Methods
Animals and Tissue Collection
Male (25 to 30 g) wild-type (F1 between male SV129 and female
C57BL/6J) and mutant mice lacking eNOS11 were bred in-house. All
experiments were conducted according to the Animals (Scientific
Procedures) Act 1986, United Kingdom. Mice were killed by neck
dislocation, and the mesentery was removed and placed in cold PSS
composed of (in mmol/L) NaCl 119, KCl 4.7, CaCl202H2O 2.5,
MgSO407H2O 1.2, NaHCO3 25, KH2PO4 1.2, and glucose 5.5.
Third-order arteries were cleansed of surrounding fat and mounted in
either a pressure or a tension myograph.

Studies of Pressure-Mediated Responses In Vitro

Vessels were mounted in a 10-mL perfusion myograph chamber onto
2 opposing PSS-filled cannulas. The chamber was continuously
superfused with PSS at 10 mL/min (37C, pH 7.4), gassed with 21%
O2/5% CO2 in N2, and placed on the stage of an inverted microscope
(Nikon, TMS). The vessel was visualized using a video camera
(VM-902; Hitachi Denshi Ltd), and the internal diameter was
determined using a video dimension analyzer (Living Systems Inc)12
and recorded on a pen chart recorder (BS-272; Gould Electronics
Ltd). After equilibration, vessels were pressurized to 80 mm Hg;
those that did not develop spontaneous myogenic tone were rejected.
Endothelial integrity was tested using acetylcholine (10 mol/L);
vessels not demonstrating 50% reversal of myogenic tone in
response to acetylcholine were rejected. In vessels from eNOS
knockout mice, acetylcholine does not always produce sufficient
reversal of tone13; in these instances, the endothelium-dependent
vasodilator bradykinin (1 mol/L) was tested. Reversal of myogenic
tone of 50% was taken as indicative of intact endothelium.
Pressure-diameter curves were constructed under no-flow conditions in the absence and then in the presence of drug treatment,
Ca2-free PSS containing 2 mmol/L EGTA, or endothelium denudation. At the conclusion of each experiment, vessels were bathed in
Ca2-free PSS containing 2 mmol/L EGTA at 80 mm Hg to determine passive diameter. Endothelium removal was achieved by the
injection of air.14 U46619 (11,9-epoxymethano-PGH2, 10
nmol/L) was used to elevate tone, and acetylcholine (10 mol/L)
was used to test endothelium integrity. Vessels were considered
endothelium denuded when acetylcholine reversal of tone was
10%. Smooth muscle sensitivity was tested using S-nitroso-Nacetyl penicillamine (SNAP; 10 mol/L).
The NOS and cyclooxygenase inhibitors N G-nitro-L-arginine
methyl ester (L-NAME; 300 mol/L) and indomethacin (5 mol/L)
were used to determine the roles of NO and prostanoids, respectively. Involvement of endothelium-derived hyperpolarizing factor
(EDHF) was determined using apamin (100 nmol/L) and charybdotoxin
(100 nmol/L),15 the gap junction inhibitor 18-glycyrrhetinic acid (100
mol/L),16 or Ba2 (30 mol/L) and ouabain (1 mmol/L).17 Apamin and
charybdotoxin in PSS were perfused (10 L/min) through the artery and
left in vessel contact for 30 minutes. All other agents were superfused
for 30 minutes before construction of the second pressure curve.

Studies of Vascular Reactivity to NO Donors

Arteries were mounted in an automated isometric tension myograph
(JP Trading) and bathed in PSS at 37C.18 After equilibration19 in
U-46619 (0.03 to 1 mol/L)precontracted vessels, relaxation
concentration-response curves to SNAP (1 nmol/L to 30 mol/L)
were constructed in the absence or presence of charybdotoxin and
apamin or Ba2 and ouabain.

Data Analysis
All values are expressed as the arithmetic meanSEM. Statistical
analysis was performed by paired or unpaired Students t test or
ANOVA followed by Bonferronis correction for multiple comparisons. Differences were considered significant at P0.05.
An expanded Methods section can be found in an online data
supplement available at http://www.hypertensionaha.org.

Comparison of Structural and Reactivity Parameters in

Mesenteric Resistance Arteries Isolated From eNOS Wild-Type
and Knockout Mice
Reactivity Parameter

Wild-Type Mice

Knockout Mice

Basal diameter, m

100.13.4 (n36)

104.64.6 (n24)

Passive diameter, m

186.55.7 (n36)

190.06.6 (n24)

Wall thickness, m

14.10.6 (n36)

15.80.6 (n24)

10 nmol/L U46619 (%) endo

50.46.0 (n6)

42.19.0 (n9)

10 nmol/L U46619 (%) endo

40.33.8 (n6)

SNAP

87.610.3 (n6)

95.31.2 (n10)

Responses to SNAP are calculated as percent of passive diameter:

DSNAP/D0100%. Responses to U46619 are calculated as percent decrease in
diameter: 100%(DU46619/D0100), where D0 is the diameter at 80 mm Hg in
Ca2-free PSS in the presence (endo) or absence (endo) of the endothelium.
All comparisons are PNS.

Materials
U46619 was purchased from BIOMOL, and all other drugs were
from Sigma Chemical Co.

Results
Myogenic constriction occurs in the arteries of both wild-type
and eNOS knockout mice. Basal (at 10 mm Hg) or passive (at
80 mm Hg) diameter and wall thickness in wild-type (n36)
and eNOS knockout (n24) arteries did not differ significantly (Table). Figure 1A shows that stepwise increases in
intraluminal pressure, up to 60 mm Hg, causes sequential
increase in internal diameter in an artery from a wild-type
mouse. At 60 mm Hg, the diameter plateaus, and subsequent
increases in pressure result in decreases in diameter. The
passive pressure-diameter curve in wild-type (n7) and
knockout (n8) tissues (Figures 1B and 1C) demonstrates
that myogenic tone is dependent on Ca2 influx in small
arteries of both strains. The pressure-wall tension response in
wild-type tissues plateaus at 60 mm Hg. However, in the
absence of extracellular Ca2, wall tension increases linearly
(Figure 1B, inset). The active myogenic constrictor response
appeared greater in wild-type than in eNOS knockout arteries.

Endothelium-Derived NO Moderates Myogenic

Responses in Wild-Type Arteries
Endothelium removal significantly (n6, P0.05) increased
myogenic tone (Figure 2A) and abolished dilator responses to
acetylcholine without affecting the response to U46619 or
SNAP (Table). L-NAME treatment (n6) also significantly
(P0.05) enhanced myogenic tone (Figure 2B), whereas
indomethacin (n6) (Figure 2C) had no effect.

K Channel Blockers Have Little Effect on

Myogenic Constriction of Wild-Type Arteries
Charybdotoxin and apamin treatment enhanced myogenic
constriction only at 70 mm Hg (n5, P0.05) in wild-type
arteries. This drug combination did not alter U-46619
induced constriction (49.32.9% in the absence and
51.25.3% in the presence) but attenuated acetylcholineinduced dilatation (63.714.8% in the absence and
41.815.8% in the presence, P0.05) and relaxation to
SNAP (pEC50 7.40.2 in the absence and 6.90.3 in the

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eNOS Knockout and Myogenic Tone

835

Discussion

Figure 1. Effect of intraluminal pressure on internal diameter in

mesenteric small arteries from eNOS wild-type and knockout
mice. A, Representative trace of a control pressure-diameter
curve in wild-type arteries. Pressure-diameter and pressure-wall
tension curves (inset) in normal PSS () and Ca2-free PSS () in
arteries of wild-type (B) and eNOS knockout (C) mice. *P0.05,
**P0.01, and ***P0.001.

presence, n5, P0.001). In contrast, ouabain and Ba2

(n4) or 18-glycyrrhetinic acid (n4) did not alter myogenic responses (Figures 2E and 2F) or relaxation to SNAP
(n5).

Inhibitors of EDHF Responses Increase Myogenic

Constriction of eNOS Knockout Arteries
Myogenic tone of eNOS knockout arteries was not altered by
NOS (n5, Figure 3A) or cyclooxygenase (n5, Figure 3B)
inhibition. However, myogenic tone was significantly increased in the presence of charybdotoxin and apamin (n4,
P0.01, Figure 3C), Ba2 and ouabain (n5, P0.01, Figure
3D), or 18-glycyrrhetinic acid (n4, P0.05, Figure 3E).
18-Glycyrrhetinic acid also abolished acetylcholine dilatation (47.812.0% in the absence and 0% in the presence)
without altering U-46619 induced constriction (41.011%
and 35.56.6%, respectively) in eNOS knockout arteries.

The myogenic response is a major physiological determinant

of smooth muscle tone and hence vascular resistance.4 The
role of the endothelium as mediator or modulator of this
response is controversial. The results of the present study
demonstrate that endothelium-derived NO, produced from
eNOS, is an important modulator of myogenic tone in murine
small mesenteric arteries. In addition, in the absence of
eNOS, an EDHF-like factor compensates for the loss of NO.
These results imply that in situations of chronic eNOS
suppression, EDHF is upregulated and may represent a
mechanism whereby the vasculature maintains a moderating
vasodilator influence over constrictor tone of resistance
arteries.
Third-order mesenteric small arteries from eNOS wildtype mice exhibited a typical pressure-dependent decrease in
diameter within a physiologically relevant pressure range.
Normal in vivo intraluminal pressure in arteries of the size
used in the present study (ie, 100 m) is thought to be 60%
to 80% of mean arterial pressure (see Mulvany and Aalkjaer18
for a review). Therefore, with a mean arterial pressure of 95
to 100 mm Hg, intraluminal pressure of these arteries should
range from 57 to 80 mm Hg (ie, within the range tested). The
myogenic constrictor response first described by Bayliss in
1902 is considered to involve influx of extracellular Ca2,5
and consistent with this hypothesis, myogenic tone was
abolished in the absence of extracellular Ca2. The pressurepassive diameter curve (Figure 1B) demonstrates that the
vessels that were studied develop myogenic tone at pressures
of 40 mm Hg, which is similar to responses in vessels of
most other species, including humans.20 The presence of
myogenic constriction is associated with a plateau in wall
tension consistent with the suggestion that wall tension may
be the stimulus and regulated variable for myogenic
constriction.1
Small arteries of eNOS knockout mice also exhibited
pressure-dependent constriction that is dependent on the
presence of extracellular Ca2. However, the absolute magnitude of this response was less than that of wild-type arteries.
This did not appear to be due to any gross abnormalities in
vessel wall structure consequent to eNOS deletion, because
no differences were evident between the 2 strains in basal and
passive diameters or wall thickness. It is unlikely that the
difference seen was an artifact due to the pressure range used,
because the mean arterial pressure of the eNOS knockout is
110 mm Hg,11 and therefore the physiological pressure
range for small arteries in these animals falls well within the
intraluminal pressures used. An alternative explanation for
the apparent suppression in myogenic tonus may be that in
the absence of eNOS, other dilator factors are upregulated,
such that an overcompensation for the loss of basal NO
activity is produced. Indeed, this appears to be the case in the
present study.
The importance of the endothelium was assessed by
construction of pressure-diameter curves before and after
endothelium removal, achieved by bolus injection of air
through the vessel. The vessel was considered denuded if
dilatation to the endothelium-dependent dilator acetylcholine
was lost. This procedure did not damage the underlying

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Figure 2. Pressure-diameter curves in

eNOS wild-type mesenteric arteries in
the absence () and presence () of endothelium (A), 300 mol/L L-NAME (B),
5 mol/L indomethacin (C), 100 nmol/L
apamin with 100 nmol/L charybdotoxin
(D), 30 mol/L Ba2 with 1 mmol/L
ouabain (E), or 18-glycyrrhetinic acid
(100 mol/L) (F). The mean passive
diameter at 80 mm Hg (ie, in Ca2-free
PSS) for vessels was 20318 m (A),
20717 m (B), 18014 m (C), 1705
m (D), 16612.3 m (E), and 1658.8
m (F). *P0.05 and **P0.01.

smooth muscle because vasoconstriction to U46619 and

dilation to the endothelium-independent dilator SNAP were
unaffected. Removal of the endothelium from eNOS wildtype arteries greatly increased the magnitude of myogenic
constriction. These findings are in agreement with a recent
study in mesenteric small arteries of spontaneously hypertensive rats in which myogenic tone was greater in the absence
of the endothelium.21 In contrast, studies in a variety of other
vessel types show no effect of endothelium removal on the
pressure-diameter relationship.6 The difference between these
studies and the present study may relate to the method of
endothelium removal that was used; many of the studies used
chemical rather than physical removal procedures. Alternatively, the differences may reflect true differences in endothelial regulation of myogenic tone between species or
vascular beds.
NO is an important mediator of vascular autoregulation. In
isolated resistance arteries, changes in flow (shear stress)
result in eNOS-derived NO release from the endothelium to
produce dilatation and thereby normalize shear stress.14,22 In
the present study, we demonstrated that NO is also involved

in transmural pressure-mediated responses. Inhibition of NO

synthesis in eNOS wild-type arteries significantly decreased
intraluminal diameter only in the pressure range at which
myogenic tone was observed (ie, 40 mm Hg). The lack of
effect of L-NAME at intraluminal pressures of 50 mm Hg
indicates that the extent of basal NO release has minimal
impact on vascular tone of these vessels at low pressures. In
keeping with previous results,9 the increase in magnitude of
the myogenic response after NOS inhibition was similar to
the effect of endothelium removal. Inhibition of cyclooxygenase, using indomethacin, had no effect on myogenic tone.
Together, these findings show that the endothelium functionally moderates myogenic tone and that the endotheliumderived factor responsible for this effect is NO.
Because NOS inhibitors show little selectivity for the
different NOS isoforms, we used mice with targeted disruption of the eNOS gene to determine whether modulation of
myogenic constriction was provided by eNOS activity. We
focused on eNOS because it is the main isoform found in the
endothelium. Unlike mesenteric eNOS wild-type arteries,
NOS inhibition had no effect on the pressure-diameter rela-

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eNOS Knockout and Myogenic Tone

837

Figure 3. Pressure-diameter curves in eNOS knockout mesenteric arteries in the absence () and presence () of 300 mol/L L-NAME
(A), 5 mol/L indomethacin (B), 100 nmol/L apamin with 100 nmol/L charybdotoxin (C), 30 mol/L Ba2 with 1 mmol/L ouabain (D), and
18-glycyrrhetinic acid (100 mol/L) (E). The mean passive diameter at 80 mm Hg (ie, in Ca2-free PSS) for vessels was 21811 m
(A), 20014 m (B), 1707 m (C), 2057 m (D), and 15014.5 m (E). *P0.05 and **P0.01.

tionship of eNOS knockout arteries. However, contrary to

expectation, the magnitude of myogenic tone in these vessels
was not greater than that observed in wild-type arteries, and
if anything, it was less. These results imply developmental
adaptation and, possibly, overcompensation for the loss of
endothelium-derived NO. Indeed, other studies demonstrate
similar adaptation in other important physiological responses
in eNOS knockout animals. For example, the absence of
eNOS is compensated for by neuronal NOS in pial arteries23

and by cyclooxygenase in mesenteric arteries,24 both in the

response to acetylcholine25 and in flow-mediated responses in
gracilis muscle arterioles.26 However, in the present study,
neither nNOS nor cyclooxygenase compensated for the loss
of eNOS, because L-NAME and indomethacin had no effect.
An alternative endothelium-derived mediator that might
compensate for the loss of NO is EDHF. Similar to NO and
prostacyclin, EDHF is another prominent vasodilator factor
released from the endothelium in response to endothelium-

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dependent vasodilators such as acetylcholine and bradykinin.

EDHF causes vasodilatation by hyperpolarizing the adjacent
smooth muscle. In addition, studies suggest that the role of
EDHF in regulation of arterial vascular tone increases as the
diameter of the blood vessel decreases, such that EDHF has
little role in mediating endothelium-dependent vasodilatation
of conduit arteries but a major role in resistance arteries.27
Although it is clear that EDHF release and activity involve
alteration in K flux of both the endothelial and smooth
muscle cell, the exact mechanisms involved are uncertain and
highly controversial. Currently, the clearest method for identifying the involvement of EDHF can be achieved through the
combined inhibition of large and small conductance Ca2-activated K channels (using charybdotoxin and apamin,15
respectively). In the present study, replacement of the intraluminal solution with PSS containing charybdotoxin and
apamin profoundly enhanced myogenic constriction in eNOS
knockout arteries at pressures of 40 mm Hg. There is some
suggestion that large conductance Ca2-activated K channels
(BKCa) determine the extent of myogenic tone per se,26 and
therefore the increase in tone may not have been due solely to
the inhibition of EDHF. However, in the present study, the
channel blockers were administered intraluminally rather
than the abluminally, and therefore the effects seen are likely
to be due primarily to an effect on endothelial cells rather than
on smooth muscle cells. Indeed, previous studies in rat
mesenteric small arteries demonstrate that EDHF-mediated
relaxation to acetylcholine is inhibited by charybdotoxin and
apamin only when selectively applied to the lumen.28 Furthermore, these inhibitors do not affect myogenic constriction
or U-46619 induced tone when applied abluminally,28 supporting our conclusion that the effect seen in the present study
was due to inhibition of an EDHF response. The combination
of charybdotoxin and apamin also enhanced myogenic constriction of wild-type arteries, suggesting that at least part of
the response even in the presence of eNOS may be due to
hyperpolarization.29 However, in wild-type arteries, the effects of these inhibitors were far less than those seen in eNOS
knockout arteries. In wild-type arteries, charybdotoxin and
apamin partially blocked the responses to NO, as shown by
the suppression of SNAP dilatation in the presence of these
inhibitors, and this may explain the efficacy of these inhibitors against myogenic tone.
To provide additional support for a role of EDHF as a
moderator of myogenic constriction in eNOS knockout arteries, we studied other proposed inhibitors of EDHF. Inhibition
of inwardly rectifying K channels (Kir) with Ba2 and
Na,K-ATPase with ouabain blocks EDHF activity at the
smooth muscle cell in certain vascular preparations.15 In the
present study, this combination of inhibitors moderately
enhanced myogenic tone only in eNOS knockout arteries and
not in eNOS wild-type arteries. This suggests that Kir and
Na,K-ATPase may play a small role in mediation of the
EDHF component in eNOS knockout arteries. A similar
result was obtained using a selective blocker of gap junctions
(18-glycyrrhetinic acid). Recent theory suggests that endothelium-dependent hyperpolarization is consequent to passage, from the endothelial cell to the smooth muscle cell, of
a factor or electrotonic current via myoendothelial gap

junctions.30 Similar to the other inhibitors of EDHF, 18glycyrrhetinic acid also enhanced myogenic constriction of
eNOS knockout arteries, with no effect in wild-type arteries,
but as with Ba2 and ouabain, the effect was moderate.
Together, the results suggest that hyperpolarization in part
mediates endothelium-induced modulation of myogenic constriction in both wild-type and eNOS knockout arteries but
that the factor or mechanism responsible for this effect is
different in the 2 species (ie, NO in wild-type and EDHF in
eNOS knockout arteries). This EDHF component involves
activation of large and small conductance Ca2-dependent K
channels with a minor role for Kir and Na,K-ATPase and
myoendothelial gap junctions. Ideally, measurement of membrane potential demonstrating hyperpolarization or rather
repolarization in response to increases in intraluminal pressure would provide additional support for a role for an EDHF.
However ,these experiments are technically difficult because
elevation of pressure results in diameter changes that would
dislodge an electrode impaled in the vessel wall, thus making
sustained measurement of membrane potential in a single
smooth muscle cell impossible. Regardless of this, the data
suggest that where eNOS activity is compromised or absent,
EDHF is upregulated as a compensatory mechanism to
preserve endothelium-derived vasodilator regulation of vascular tone. In support of such a back-up mechanism are
findings that demonstrate basal eNOS-derived NO inhibition
of EDHF release.31 In the present study, however, it is
unlikely that this is an acute back-up mechanism because in
arteries of wild-type mice, NOS inhibition produced a pronounced increase in myogenic tone: if NO-induced intrinsic
inhibition could be alleviated acutely, myogenic constriction
would be expected to remain relatively unchanged.
Finally, we attempted to investigate the effects of endothelium removal on myogenic responses of arteries from eNOS
knockout animals. However, using the same protocol as that
used for wild-type arteries, it was not possible to remove the
endothelium, despite reapplication of air up to 3 additional
times. Similar attempts to remove the endothelium with
detergent, such as Triton X or CHAPS, also failed, as
demonstrated by the fact that endothelium-dependent dilators
(acetylcholine and bradykinin) still caused relaxation. The
reason for the difficulty in removing the endothelium in
microvessels of eNOS knockout arteries is unclear and may
be worthy of investigation in its own right.
In conclusion, we have demonstrated that in the murine
mesenteric microcirculation, NO synthesized by eNOS in the
endothelium significantly modulates Bayliss-type autoregulation. Therefore, eNOS-derived NO release may provide an
important negative feedback mechanism in autoregulation of
resistance arteries, and this has implications for understanding how the loss of NO in disease states might affect tissue
and organ perfusion as well as blood pressure responses.
Moreover, in the chronic absence of eNOS-derived NO, an
EDHF-like factor or factors compensate for the lack of NO
such that a vasodilator influence over myogenic constriction
of resistance arteries is maintained. It remains to be determined whether these findings account for the observation that
EDHF-like responses are more prominent in humans with
hypertension in whom NO-mediated dilatation is impaired.32

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Scotland et al

Acknowledgments
This work was supported by Medical Research Council (UK) (R.
Scotland) and British Heart Foundation (S. Chauhan and Dr
Ahluwalia).

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