Vol. 280, Issue 6, H2462-H2469, June 2001
SPECIAL TOPIC
EDHF mediates flow-induced dilation in skeletal muscle
arterioles of female eNOS-KO mice
An
Huang1,
Dong
Sun1,
Mairead A.
Carroll2,
Houli
Jiang2,
Carolyn J.
Smith3,
Joseph A.
Connetta3,
John R.
Falck4,
Edward G.
Shesely5,
Akos
Koller1, and
Gabor
Kaley1
Departments of 1 Physiology, 2 Pharmacology, and
3 Pathology, New York Medical College, Valhalla, New York
10595; 4 Department of Biochemistry, University of Texas
Southwestern Medical Center, Dallas, Texas 75235; and 5 Division
of Hypertension and Vascular Research, Henry Ford Hospital,
Detroit, Michigan 48202
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ABSTRACT |
Vasodilation to increases in flow was
studied in isolated gracilis muscle arterioles of female endothelial
nitric oxide synthase (eNOS)-knockout (KO) and female wild-type (WT)
mice. Dilation to flow (0-10 µl/min) was similar in the two
groups, yet calculated wall shear stress was significantly greater in
arterioles of eNOS-KO than in arterioles of WT mice. Indomethacin,
which inhibited flow-induced dilation in vessels of WT mice by ~40%,
did not affect the responses of eNOS-KO mice, whereas miconazole and
6-(2-proparglyoxyphenyl)hexanoic acid (PPOH) abolished the responses.
Basal release of epoxyeicosatrienonic acids from arterioles was
inhibited by PPOH. Iberiotoxin eliminated flow-induced dilation in
arterioles of eNOS-KO mice but had no effect on arterioles of WT mice.
In WT mice, neither
N
-nitro-L-arginine methyl ester
nor miconazole alone affected flow-induced dilation. Combination of
both inhibitors inhibited the responses by ~50%.
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one
(ODQ) alone inhibited flow-induced dilation by ~49%. ODQ + indomethacin eliminated the responses. Thus, in arterioles of female WT
mice, nitric oxide and prostaglandins mediate flow-induced dilation.
When eNOS is inhibited, endothelium-derived hyperpolarizing factor
substitutes for nitric oxide. In female eNOS-KO mice, metabolites of
cytochrome P-450, via activation of large-conductance
Ca2+-activated K+ channels of smooth muscle,
mediate entirely the arteriolar dilation to flow.
nitric oxide; prostaglandins; hyperpolarizing factor; cytochrome
P-450 metabolites; potassium channels
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INTRODUCTION |
NITRIC OXIDE (NO),
prostaglandins, and endothelium-derived hyperpolarizing factor (EDHF)
represent three major endothelial factors involved in the local
regulation of vascular tone (4, 14). Attributing specific
effects to each of these factors is hampered by the fact that all three
mediators may be simultaneously synthesized in and/or released from
endothelial cells in response to a single stimulus, e.g., shear stress
(21) or vasoactive agents (16). The
mechanisms by which release of NO or prostaglandins is activated by
alterations in wall shear stress (WSS) during changes in blood flow
have been extensively studied (4, 21). The release of EDHF
in response to vasoactive agents, as well as to hemodynamic stimuli
such as pulsatile stretch, has also been demonstrated in a variety of
vascular beds (1, 16, 28). However, little experimental
evidence has been provided to suggest that the release of EDHF can be
stimulated by shear stress, a physiologically relevant stimulus in vivo.
The term EDHF may prove to represent a group of different factors,
since not all EDHF-mediated vascular responses display the same
behavior or the same sensitivity to pharmacological agents (6, 9,
11, 22, 33). In coronary, cerebral, renal, and skeletal muscle
circulations, EDHF has been characterized as a cytochrome
P-450 (CYP) epoxygenase metabolite(s) of arachidonic acid
[epoxyeicosatrienoic acids (EETs)], thought to hyperpolarize vascular
smooth muscle by opening K+ channels (1, 10, 12,
16).
Our previous studies demonstrated that, whereas endothelial NO and
prostaglandins mediate flow-dependent dilation in gracilis muscle
arterioles in male wild-type (WT) mice, an upregulation of
prostaglandin synthesis is solely responsible for the maintenance of
this response in male endothelial NO synthase (eNOS)-knockout (KO) mice
(32). Estrogen was shown to potentiate shear
stress-dependent dilation of arterioles, suggesting that the
NO-stimulating effect of the hormone is one of the key mechanisms that
underlies the gender difference in the regulation of arteriolar tone
(17). In addition, estrogen increases coronary blood flow
and improves vascular dysfunction by opening Ca2+-dependent
K+ (KCa) channels (26) and by
potentiating EDHF-mediated, agonist-induced vasorelaxation
(23). The question, therefore, arose as to whether there
is a gender difference in the compensatory mechanisms that accounts for
the preservation of flow-induced dilation in eNOS-KO mice. We
hypothesized that, because of a targeted disruption of the gene
encoding eNOS, endothelial mediators other than NO maintain a
close-to-normal dilation to flow/shear stress in skeletal muscle arterioles of female mice. Thus we aimed to elucidate the nature of the
endothelial mediators responsible for flow-induced dilation in gracilis
muscle arterioles of female eNOS-KO and WT mice and then to identify
the gender difference, if any, in the endothelial regulatory mechanisms.
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METHODS |
Animals.
Heterozygous eNOS (+/
) mice, originally developed by Shesely et al.
(30), were interbred to generate eNOS-WT (+/+) and homozygous mutant (/) mice. Mice were genotyped by Southern analysis of DNA as described previously (30). All
protocols were approved by the Institutional Animal Care and Use
Committee of New York Medical College and conform to the National
Institutes of Health and American Physiological Society guidelines for
the use and care of laboratory animals. eNOS-KO and WT mice were bred in the Department of Comparative Medicine at New York Medical College.
Experimental setup.
Experiments were conducted on isolated gracilis muscle arterioles of
female eNOS-KO and WT mice. The mice were killed and the vessels were
dissected and isolated as described previously (16, 32).
Experimental procedures.
Changes in diameter of arterioles in response to increases in flow were
studied at 80 mmHg of perfusion pressure. Perfusate flow was increased
from 0 to 10 µl/min in 2 µl/min steps.
In the first series of experiments, the role of prostaglandins in
flow-induced dilation was assessed by using indomethacin (Indo,
10
5 M), an inhibitor of cyclooxygenase, after control
flow-diameter curves were obtained.
In the second series of experiments, the role and interaction of
metabolites of CYP and eNOS in flow-induced dilations were assessed by
using miconazole (MCZ, 2 × 106 M) and
6-(2-proparglyoxyphenyl)hexanoic acid (PPOH, 105 M),
inhibitors of CYP epoxygenase (14, 34), and
N-nitro-L-arginine methyl ester
(L-NAME, 104 M), an inhibitor of NOS. After
control experiments, MCZ or L-NAME was administered first
for 30 min before the experiments were repeated. Flow-induced responses
were then studied once more in the additional presence of
L-NAME or MCZ. In a separate group of experiments, the
effect of metabolites of CYP on flow-induced dilation in arterioles of
eNOS-KO mice was confirmed further by using PPOH, thought to be a more
specific inhibitor of CYP epoxygenase (34).
In the third series of experiments, the contribution of EDHF to
flow-induced responses was evaluated by performing the experiments before and after extraluminal administration of iberiotoxin (IBTX, 2 × 108 M), a blocker of large-conductance
KCa channels.
In the forth series of experiments, the participation of the
L-arginine-NO-cGMP pathway in the flow-induced responses of
arterioles from WT mice was tested by using L-NAME or
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 3 × 105 M), an inhibitor of guanylate
cyclase. The inhibitors were administered alone or simultaneously with
MCZ or Indo.
Passive diameter.
At the conclusion of each experiment, the suffusion solution was
changed to a Ca2+-free solution containing 1 mM EGTA.
Vessels were incubated for 10 min to reach maximal diameter at 80 mmHg
of perfusion pressure.
Quantitation of EETs.
To quantitate the basal release of EETs by arterioles (control group),
as well as the specific inhibitory effect of PPOH (3 × 105 M) on this release (PPOH group), arterioles isolated
from three WT mice from each group (~13 µg protein/ml) were
incubated in the presence of NADPH (1 M), Indo (3 × 105 M), and dibromododecynylmethylsulfimide (3 × 105 M), an inhibitor of 
-hydroxylase, at 37°C for
1 h. The vessel and media eicosanoids were extracted after
addition of 4.5 ng of a mixture of D8-EETs (8,9-, 11,12-, and 14,15-EET) as internal standards.
Purification of EETs.
The EETs were purified using reverse-phase HPLC and derivatized and
quantitated by negative chemical ionization gas chromatography (GC)-mass spectroscopy (MS) as described previously (7).
Briefly, the samples were extracted twice with two volumes of acidified ethyl acetate (pH 4.0) and evaporated to dryness. The samples were
purified by reverse-phase HPLC on a C18 µ-Bondapak column (4.6 × 24 mm) using a linear gradient from
acetonitrile-water-acetic acid (62.5:37.5:0.05%) to acetonitrile
(100%) over 20 min at a flow rate of 1 ml/min. Fractions containing
EETs were collected on the basis of the elution profile of standards
monitored by ultraviolet absorbance (205 nm). The fractions were
evaporated to dryness and derivatized for GC-MS analysis.
Derivatization and MS analyses.
Pentafluorobenzyl esters were prepared by the addition of
-bromo-2,3,4,5,6-pentafluorotoluene (pentafluorobenzylbromide, 5 µl; Aldrich) and N,N-diisopropylethylamine (5 µl;
Aldrich) to a sample dissolved in acetonitrile (100 µl), and the
derivatization was continued at room temperature for 30 min. Samples
were dissolved in isooctane, and 1-µl aliquots were injected into a
GC (model HP-5890, Hewlett-Packard) column (15.0 m, 0.25 mm ID, 0.25 µm film thickness; DB-1, Supelco) using a temperature program ranging from 150 to 300°C at a rate of 10°C/min. Methane was used as a reagent gas at a flow resulting in a source pressure of 1.3 Torr, and
the MS (model 5989A, Hewlett-Packard) was operated in electron capture
chemical ionization mode. The endogenous EETs were identified (ion
mass-to-charge ratio = 319) by comparison of GC retention times
with authentic D8-EETs (mass-to-charge ratio = 327) standards and quantitated by calculating the ratio of abundance.
Chemicals.
All chemicals were obtained from Sigma (St. Louis, MO). PPOH was
dissolved in ethanol at 102 M and further diluted with
physiological saline solution.
Calculations and statistics.
Changes in diameter in response to increases in flow/shear stress in
each vessel were normalized by its passive diameter. WSS at each flow
rate and that required to cause 50% of maximal dilation
(WSS50) were calculated (32). Statistical
significance was calculated by repeated-measures ANOVA followed by the
Tukey-Kramer multiple-comparison test. Values are means ± SE.
When two or more vessels were studied from one animal, the responses
were averaged. The GC-MS data were analyzed by a group
t-test on logarithmic transformation of the data.
Significance level was taken at P < 0.05.
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RESULTS |
The characteristics of arterioles isolated from gracilis muscle of
female WT (N = 28) and eNOS-KO (N = 20)
mice are summarized in Table 1. Active
and passive diameters were significantly smaller in arterioles of
eNOS-KO mice than in arterioles of WT mice, whereas the basal
arteriolar tone, expressed as percentage of passive diameter, was not
significantly different in vessels of the two groups of mice.
Increasing flow from 0 to 10 µl/min elicited significant increases in
diameter of arterioles from WT and eNOS-KO mice. The maximal changes in
diameter were not significantly different (31.6 ± 1.1 and
29.9 ± 1.1 µm at 10 µl/min, respectively). The dilations of
arterioles from eNOS-KO and WT mice were nearly identical, as shown in
Fig. 1A, where the normalized
diameter as a function of perfusate flow is depicted. Increases in flow
elicited significantly greater increases in shear stress (12.3 ± 0.9 vs. 8.5 ± 0.5 at 10 µl/min flow) and resulted in greater
WSS50 (8.3 ± 0.8 vs. 5.9 ± 0.9 dyn/cm2) in arterioles of eNOS-KO mice than in arterioles
of WT mice (Fig. 1B). In the next series of experiments, the
endothelial mediators contributing to flow-induced dilation in
arterioles of WT and eNOS-KO mice (summarized in Figs.
2-5
and Figs. 6 and 7, respectively) were
investigated.
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Fig. 1.
Normalized diameter of gracilis muscle arterioles
(A) and calculated shear stress (B) as a function
of perfusate flow in female wild-type (WT; 54 vessels from 28 animals)
and endothelial nitric oxide synthase (eNOS)-knockout (KO; 32 vessels
from 17 animals) mice. *Significant difference between the two curves
(by ANOVA).
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Fig. 2.
Normalized diameter of gracilis muscle arterioles of
female WT (12 vessels from 6 animals) mice as a function of perfusate
flow in control conditions and in the presence of indomethacin (Indo,
105 M). PD, passive diameter. *Significant difference
from control.
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Fig. 3.
Normalized diameter of gracilis muscle arterioles of
female WT mice as a function of perfusate flow in the control
condition, in the presence of
N-nitro-L-arginine methyl ester
(L-NAME, 104 M) and L-NAME + miconazole (MCZ, 2 × 106 M; A; 9 vessels
from 5 animals), or in the presence of MCZ and MCZ + L-NAME (B; 8 vessels from 5 animals).
*Significant difference from control and L-NAME or from
control and MCZ.
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Fig. 4.
Normalized diameter of gracilis muscle arterioles of
female WT (16 vessels from 8 animals) mice as a function of perfusate
flow in the control condition, in the presence L-NAME + MCZ, and in the presence of L-NAME + MCZ + Indo. *Significant difference from control and from
L-NAME + MCZ + Indo.
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Fig. 5.
Normalized diameter of gracilis muscle arterioles of
female WT mice as a function of perfusate flow in the control condition
and in the presence of
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one
(ODQ, 105 M), ODQ + MCZ, and ODQ + Indo
(A; 13 vessels from 7 animals) and in the presence of
iberiotoxin (IBTX, 2 × 108 M; B; 9 vessels from 5 animals). *Significant difference from control and from
ODQ + Indo.
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Fig. 6.
Normalized diameter of gracilis muscle arterioles of
female eNOS-KO mice as a function of perfusate flow in the control
condition or in the presence of Indo (12 vessels from 6 animals) or MCZ
(9 vessels from 5 animals; A) or
6-(2-proparglyoxyphenyl)hexanoic acid (PPOH, 105 M;
B; 9 vessels from 3 animals). *Significant difference from
control and from Indo.
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Fig. 7.
Normalized diameter of gracilis muscle arterioles of
female eNOS-KO mice (9 vessels from 5 animals) as a function of
perfusate flow in the control condition and in the presence of IBTX.
*Significant difference from control.
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The role of prostaglandins in the mediation of flow-induced dilation in
vessels of WT mice is shown in Fig. 2. Indo, which did not affect basal
tone in arterioles of either strain of mouse, significantly inhibited
flow-induced dilation by ~40% (P < 0.05) in
arterioles of WT mice. In a separate group of experiments (Fig. 3), the
roles of NO and CYP metabolites in the mediation of flow-induced dilation in WT arterioles were assessed by using
L-NAME and MCZ, respectively. Neither L-NAME
alone nor MCZ alone affected flow-induced dilations and the basal tone
of vessels. Combination of both inhibitors, however, significantly
inhibited the responses (by ~50%; Fig. 3). The remaining portion of
the dilator responses was essentially eliminated by additional
administration of Indo (Fig. 4). ODQ, an inhibitor of guanylate
cyclase, was then used to confirm the role of the NO-cGMP pathway in
the mediation of flow-induced dilations in control conditions. Figure
5A shows that, unlike L-NAME, ODQ alone, while
having no effect on basal tone, significantly inhibited flow-induced
dilation by (~49%), which was not affected further by MCZ. However,
the residual portion of the responses in the presence of ODQ was
abolished by additional administration of Indo. IBTX, a blocker of
large-conductance KCa channels, a target of CYP
metabolites/EDHF in smooth muscle (16), did not affect the
response in control conditions (Fig. 5B).
In arterioles of eNOS-KO mice, flow-induced dilation was independent of
prostaglandins, since Indo had no effect on the response. The response,
however, was eliminated by MCZ or PPOH (Fig. 6), suggesting an
involvement of the CYP pathway in the mediation of the responses to
flow. When arterioles from eNOS-KO mice were treated with IBTX,
flow-induced dilation was abolished (Fig. 7).
Figure 8 shows that the basal release of
EETs from gracilis muscle arterioles of mice was substantial, and PPOH
specifically inhibited this release by ~40%.
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Fig. 8.
Basal release of epoxyeicosatrienoic acids (EETs) from
gracilis muscle arterioles of female WT mice in the control condition
(n = 3) and in the presence of PPOH (3 × 105 M, n = 3). *Significant difference
from control.
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DISCUSSION |
The new findings of the present study suggest that in female
eNOS-KO mice the endothelium-dependent dilation of isolated skeletal muscle arterioles to flow/shear stress is close to normal and is likely
to be mediated by EETs, which could be viewed as EDHF. In contrast, in
arterioles of female WT mice, corelease of NO and prostaglandins is
responsible for the mediation of flow-induced dilation. Also, in
vessels of WT mice, when eNOS is inhibited by the acute administration
of L-NAME, EETs substitute for NO in the mediation of
flow-dependent dilation.
Our previous study demonstrated that EDHF is responsible for dilation
to ACh of skeletal muscle arterioles of male eNOS-KO mice, while NO
seems to account almost entirely for the dilation in vessels of
corresponding WT mice (16). A similar phenomenon has also
been observed regarding the participation of NO and EDHF in
agonist-induced dilations of porcine epicardial arteries and dog
coronary arterioles before and after acute inhibition of NO synthesis
(20, 25). Also, the hyperpolarization of coronary smooth
muscle elicited by ACh can be inhibited by exogenously applied
endothelial prostanoids (36). On the basis of these findings, it seems plausible that the significance of the contribution of EDHF to the regulation of vascular tone in physiological conditions is compromised by the presence of NO and prostaglandins, since EDHF-mediated responses appear only after inhibition of eNOS and/or cyclooxygenase (2, 3). In this context, the experimental model of mice deficient in the gene for eNOS becomes a valuable probe
in the investigation of the interactions among these enzyme systems.
Indeed, we previously found that, in male eNOS-KO mice, flow/shear
stress-induced dilation of skeletal muscle arterioles is mediated
exclusively by prostaglandins, instead of by both NO and prostaglandins
in control (WT) mice (32).
The effect of estrogen on enhancing the release of endothelial
mediators, especially NO, has been well documented (18). On the other hand, it was also reported that pregnancy induces a
significant increase in the activity of CYP (29) and
causes an upregulation of gap junction protein expression, which may well be responsible for the augmented ACh-induced dilation
(8). The importance of gap junctional communication in the
mediation of EDHF-induced vasodilation, especially in the microvessels, has attracted considerable attention (24). Moreover, it
was also reported that estrogen favors the contribution of EDHF over NO
in the mediation of agonist-induced vasodilation (13, 23).
Given that there is a negative interaction among the activity of
endothelial mediators and that female hormones favor EDHF-mediated responses, it was plausible to speculate that in female eNOS-KO mice
the synthesis/activity of EDHF is upregulated in response to shear
stress. To test this hypothesis, flow-induced dilation and the role of
endothelial factors mediating this response were investigated in
gracilis muscle arterioles of female eNOS-KO and WT mice, the type of
vessels that we previously studied in male littermates
(32).
The average age and body weight of the two strains of mice were
comparable; however, the active and passive diameters of arterioles of
eNOS-KO mice were significantly smaller than those of WT mice. As a
result, the basal tone of vessels in the two strains of mice was
comparable, indicating a similar responsiveness to intraluminal pressure.
Flow-induced dilation in arterioles of WT and eNOS-KO mice.
In response to increases in perfusate flow, arterioles of both strains
of mice exhibited substantial dilations that were similar in magnitude,
suggesting that, despite the absence of NO synthesis in endothelium of
arterioles from eNOS-KO mice, dilation to increases in flow is
essentially preserved. However, the effect of the lack of eNOS was
revealed by a significant leftward shift of the flow-shear stress curve
in eNOS-KO arterioles (Fig. 1B), indicating that these
vessels require a greater shear stress than those of WT mice to achieve
a dilation of similar magnitude. A greater WSS50 in vessels
of eNOS-KO mice is also indicative of a reduced endothelial sensitivity
to shear stress.
Endothelial mediators contributing to flow-induced dilation in
arterioles of female WT mice.
Flow-induced dilation in arterioles of WT female mice is mediated, in
part, by endothelial prostaglandins, as manifested by the significant
inhibition of the response by Indo administered in the control
condition (Fig. 2). Interestingly, L-NAME did not affect
the response, suggesting that in control conditions NO may not be
involved in the generation of flow-induced dilation. Additional
administration of the CYP blocker MCZ did, however, result in a ~50%
inhibition of the response (Fig. 3A). To evaluate the direct
role of CYP metabolites in the mediation of the responses, MCZ was then
given in the control condition. Similar to L-NAME, MCZ
alone did not affect the responses, unless it was administered simultaneously with L-NAME (Fig. 3B). We
interpret these findings to mean that there is a negative interaction
between NO and EDHF synthase, so that, in the presence of NO, EDHF
synthesis is suppressed. On the other hand, the inhibitory effect of
L-NAME on flow-dependent dilation could only be observed
when the activity of the CYP epoxygenase was blocked. This interaction
between the two enzyme systems, in response to flow/shear stress, seems
to be gender dependent, since it has not been found in arterioles from
male WT mice (32). The residual portion of the response in
the presence of L-NAME and MCZ was abolished by Indo,
confirming further the essential contribution of prostaglandins in the
response (Figs. 2 and 4), similar to that observed in male mice
(32).
The question remains as to whether and to what extent NO or CYP
metabolites are responsible for the mediation of flow-induced dilation
in arterioles of WT mice in control conditions. We assumed that if NO
is the primary mediator, then blocking cGMP with ODQ will affect the
NO-mediated portion of the response without MCZ having an additional
effect, since the activity of CYP would still be masked by NO. If, on
the other hand, CYP metabolites were primarily responsible via smooth
muscle K+ channel activation, the response would be
inhibited by IBTX, a specific blocker of the large-conductance
KCa channel. Results shown in Fig. 5 indicate that ODQ does
inhibit flow-induced dilation by ~49% in the control condition and
that the residual portion of the response is resistant to MCZ (Fig.
5A). The inability of KCa channel blockade with
IBTX to inhibit flow-induced dilation (Fig. 5B) argues
against the possibility that EDHF is the mediator of the response in
control conditions. Thus, in arterioles of female WT mice, it is NO,
together with prostaglandins, that mediates flow-induced responses. The
absence of NO after L-NAME treatment unmasks the activity
of CYP, eliciting EDHF formation, which then contributes to the
mediation of flow-induced dilation.
Recently, such an acute inhibition by NO of EDHF-induced coronary
arteriolar dilation to bradykinin in vivo has been reported (25). The underlying mechanism responsible for this
feedback inhibition of EDHF synthesis by NO released to flow or
agonists may be that NO interacts with the prosthetic heme group of CYP (4). Because in the present study the
prostaglandin-mediated component of the flow response was not affected
by NO, it is unlikely that a decreased endothelial Ca2+
concentration by NO, reducing phospholipase A2 activity,
would be responsible for the NO-mediated inhibition of EDHF synthesis (3, 4).
On the basis of previous studies (13, 17, 23), we propose
that the difference in hormonal status is responsible for the
differences observed between arterioles of male and female mice.
Because synthesis of NO and EDHF, in response to a variety of stimuli,
appears to be dependent on an increase in the intracellular concentration of Ca2+ and the formation of a
Ca2+-calmodulin complex (24) and because the
threshold endothelial concentration of Ca2+ required for
the activation of phospholipase is greater than that required for the
activation of eNOS (4, 27), it is conceivable that the
increases in endothelial Ca2+ concentration and calmodulin
synthesis by estrogen (18) activate not only the
L-arginine-NO pathway but also the phospholipase A2-CYP pathway when stimulated by shear stress, leading to
an EDHF-mediated dilation, a response that does, however, not occur in
vessels of male mice. Also, a positive correlation between the level of
estrogen and smooth muscle membrane hyperpolarization has been
demonstrated (15). Because there are no methods available to test CYP epoxygenase activity of microvessels in response to shear
stress, a stimulus that affects vascular responses by means of specific
signal transduction pathways different from those activated by other
stimuli (e.g., vasoactive agents), we could not explore the difference,
if any, in the activity of this enzyme in response to shear stress
between the arterioles of male and female mice. However, we found
substantial basal release of EETs from the arterioles, which was PPOH
sensitive (Fig. 8), providing the biochemical identification of these
arachidonic acid metabolites in the microvessels studied.
Endothelial mediators contributing to flow-induced dilation in
arterioles of female eNOS-KO mice.
Flow-induced dilation in arterioles of female eNOS-KO mice is solely
mediated by metabolites of CYP, namely EETs, and is not affected by an
inhibitor of cyclooxygenase (Fig. 6). The response is completely
abolished by IBTX (Fig. 7), suggesting that the CYP-mediated dilation
to flow/shear stress is dependent on hyperpolarization of vascular
smooth muscle, via activation of large-conductance KCa channels.
The absence of NO may potentiate EDHF production in arterioles at two
different levels: acutely, as in WT mice given L-NAME or,
chronically, as in eNOS-KO mice (2, 4). Conversely, the
chronic inhibition of EDHF synthesis by NO may result from a decreased
expression of the gene for EDHF synthase. In lipopolysaccharide- or
interleukin-1
-treated vessels, it was reported that an increased production of inducible NO transcriptionally downregulates the putative
EDHF-forming enzyme (19). By the same token, the enhanced EDHF synthesis, due to the genetic loss of eNOS, may also
counteract the synthesis or activity of cyclooxygenase, leading to an
exclusively EDHF-mediated response. Indeed, similar results, i.e., a
solely MCZ/PPOH-sensitive response, instead of an
NO/prostaglandin-mediated response, to flow/shear stress, have been
obtained in gracilis muscle arterioles of female rats treated
chronically with L-NAME (35).
It was recently shown (33) that the resting membrane
potential of smooth muscle from arteries with regenerated endothelium was relatively depolarized, coincident with impaired NO-mediated responses (31), compared with control arteries from the
same heart, whereas the hyperpolarization to bradykinin was augmented. It was also demonstrated that the underlying vascular smooth muscle cells become depolarized after removal of the endothelial lining of
coronary arteries (5). On the basis of the aforementioned findings, it is possible that in eNOS-KO arterioles the solely EDHF-mediated flow-induced response may be due, at least in part, to an
augmented arteriolar response to EDHF secondary to a relative depolarization of smooth muscle. This hypothesis can also explain the
phenomenon that, despite a reduced sensitivity to shear stress, EDHF-mediated vasodilator responsiveness to flow is still maintained in
arterioles of female eNOS-KO mice. Thus the EDHF-mediated flow-induced response could be the result of an augmented arteriolar release of EDHF
or a greater response to EDHF due to a relative depolarization of
smooth muscle.
In conclusion, the present study demonstrates that, in female WT mice,
flow-induced dilation in skeletal muscle arterioles is mediated by
endothelial NO and prostaglandins. When NO is inhibited, EDHF becomes
the mediator of the response. In female eNOS-KO mice, the preserved
flow-induced dilation is mediated exclusively by EDHF. These findings
reveal a novel compensatory mechanism in arterioles of female mice
evoked by the absence of NO, by which EETs/EDHF contribute to the
maintenance of shear stress-sensitive regulation of skeletal muscle
arterioles and, consequently, peripheral resistance.
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ACKNOWLEDGEMENTS |
We appreciate the excellent secretarial assistance of Miriam Nunez
and Dana M. Spencer.
| |
FOOTNOTES |
This study was supported by American Heart Association Grant 9930244N,
American Heart Association New York State Affiliate Grant 9830015T, and
National Heart, Lung, and Blood Institute Grants HL-46813 and HL-43023.
Address for reprint requests and other correspondence: G. Kaley, Dept. of Physiology, New York Medical College, Valhalla, NY
10595 (E-mail: gabor-kaley{at}nymc.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 November 2000; accepted in final form 29 November 2000.
| |
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