Determinants of renal afferent arteriolar actions of bradykinin: evidence that multiple pathways mediate responses attributed to EDHF
Xuemei Wang,
Greg Trottier, and
Rodger Loutzenhiser
Smooth Muscle Research Group, Department of Pharmacology and
Therapeutics, University of Calgary, Calgary, Alberta, Canada T2N 4N1
Submitted 28 March 2003
; accepted in final form 30 April 2003
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ABSTRACT
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The determinants of bradykinin (BK)-induced afferent arteriolar
vasodilation were investigated in the in vitro perfused hydronephrotic rat
kidney. BK elicited a concentration-dependent vasodilation of afferent
arterioles that had been preconstricted with ANG II (0.1 nmol/l), but this
dilation was transient in character. Pretreatment with the nitric oxide
synthase inhibitor N
-nitro-L-arginine
methyl ester (100 µmol/l) and the cyclooxygenase inhibitor ibuprofen (10
µmol/l) did not prevent this dilation when tone was established by ANG II
but fully blocked the response when tone was established by elevated
extracellular KCl, which suggests roles for both NO and endothelium-derived
hyperpolarizing factor (EDHF). We had previously shown that the EDHF-like
response of the afferent arteriole evoked by ACh was fully abolished by a
combination of charybdotoxin (ChTX;10 nmol/l) and apamin (AP; 1 µmol/l).
However, in the current study, treatment with ChTX plus AP only reduced the
EDHF-like component of the BK response from 98 ± 5 to 53 ± 6%
dilation. Tetraethylammonium (TEA; 1 mmol/l), which had no effect on the
EDHF-induced vasodilation associated with ACh, reduced the EDHF-like response
to BK to 88 ± 3% dilation. However, the combination of TEA plus ChTX
plus AP abolished the response (0.3 ± 1% dilation). Similarly,
17-octadecynoic acid (17-ODYA) did not prevent the dilation when it was
administered alone (77 ± 9% dilation) but fully abolished the EDHF-like
response when added in combination with ChTX plus AP (-0.5 ± 4%
dilation). These findings suggest that BK acts via multiple EDHFs: one that is
similar to that evoked by ACh in that it is blocked by ChTX plus AP, and a
second that is blocked by either TEA or 17-ODYA. Our finding that a component
of the BK response is sensitive to TEA and 17-ODYA is consistent with previous
suggestions that the EDHF released by BK is an epoxyeicosatrienoic acid.
arteriole; endothelium-derived hyperpolarizing factor; acetylcholine; 17-octadecynoic; epoxyeicosatrienoic acids; tetraethylammonium; charybdotoxin; apamin; potassium channels
THE ENDOTHELIUM PLAYS AN IMPORTANT role in modulation of
vascular reactivity and transmission of signals along blood vessels. A number
of vasodilator agents including ACh and bradykinin (BK) elicit vasodilation in
an indirect manner by releasing endothelium-derived relaxing factors (EDRFs)
such as nitric oxide (NO), prostacyclin (PGI2), and less-defined
factors that act through hyperpolarization [endothelium-derived
hyperpolarizing factors (EDHFs)]. Although the term EDHF implies a factor that
is released from the endothelium, in terminal arterioles such as the afferent
arteriole, it is quite possible that the responses ascribed to EDHF are
mediated by endothelial hyperpolarization and electrical coupling of the
endothelial layer to the underlying smooth muscle myocytes (e.g.,
3,
18,
30). For the purposes of the
present report, we continue to use the conventional term EDHF or EDHF-like
while acknowledging that the responses may not actually involve a released
factor. The properties of the EDHF that contribute to the component of the
dilation that is insensitive to inhibition of both nitric oxide synthase (NOS)
and cyclooxygenase (COX) vary among different vascular beds
(23). Moreover, the relative
contribution of EDHF vs. NOS and COX products varies between vascular beds and
between different endothelium-dependent vasodilators
(23,
34). In regard to the renal
circulation, knowledge of the relative contributions of NO, PGI2,
and EDHF to the renal microvascular actions of different endothelium-dependent
vasodilators and the nature of the renal EDHF(s) involved is limited.
We recently demonstrated
(36) that the EDHF associated
with the renal microvascular response to ACh is similar to that seen in
several other vascular beds in that it is abolished by a combination of
charybdotoxin (ChTX) and apamin (AP). This EDHF-like component accounted for
95% of the initial phasic vasodilatory response to ACh but did not contribute
to the sustained phase of the vasodilation nor did it contribute to the
efferent arteriolar actions of ACh. Although the pharmacological properties of
the renal EDHF associated with ACh were similar to those described for other
vascular beds, these properties differed from the properties of the EDHF
associated with BK-induced renal vasodilation. Specifically, we found 1 mmol/l
tetraethylammonium (TEA) to have no effect on the EDHF associated with ACh,
whereas several other laboratories found TEA to attenuate the EDHF component
of the BK response (10,
26,
29). Moreover, Imig et al.
(16) observed BK to release
epoxyeicosatrienoic acids (EETs) from the renal vasculature, and the
hyperpolarization elicited by EETs in isolated myocytes and the preglomerular
vasodilatory actions of EETs are blocked by TEA
(38). When interpreted in
concert with our observations, these findings suggest that BK may release an
EDHF that is distinct from that associated with ACh. In support of this
premise, Fulton et al. (11)
observed that the cytochrome P-450 inhibitor
5,8,11,14-eicosatetraynoic acid (ETYA) attenuated the response of the isolated
perfused kidney to BK but did not alter the response to ACh.
Thus in the present study, we used the in vitro perfused hydronephrotic rat
kidney model to investigate the determinants of the renal afferent arteriolar
response to BK, and we compared these results to previous studies that
employed this model to characterize the EDHF associated with ACh. Our findings
suggest the presence of at least two differing pathways that mediate the COX-
and NOS-independent EDHF-like responses in the renal microcirculation. One
component is abolished by ChTX plus AP. The second pathway is resistant to
this treatment but is blocked by either TEA (1 mmol/l) or the cytochrome
P-450 inhibitor 17-octadecynoic acid (17-ODYA).
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METHODS
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Unilateral hydronephrosis was induced to facilitate direct observations of
renal afferent arteriolar actions of BK (see Ref.
21 for detailed description of
model). The use of animals complied with the Canadian Council on Animal Care
regulations. The left ureters of 6- to 7-wk-old male Sprague-Dawley and
Long-Evans rats were ligated under halothane-induced anesthesia to induce
hydronephrosis. After 6-8 wk, the renal artery was cannulated in situ and the
hydronephrotic kidney was excised and transferred to a heated chamber on the
stage of an inverted microscope with continuous perfusion. The perfusate, a
DMEM (GIBCO Life Technologies, Gaithersburg, MD) that contained (in mmol/l) 30
bicarbonate, 5 glucose, and 5 HEPES, was equilibrated with 95% air-5%
CO2. Temperature and pH were maintained at 37°C and 7.40,
respectively. Medium was circulated through a heat exchanger to a pressurized
reservoir connected to the renal arterial cannula. Perfusion pressure, which
was monitored within the renal artery, was maintained at 80 mmHg. The kidneys
were allowed to equilibrate from surgical manipulations for at least 1 h
before experimental protocols were initiated. Cortical afferent arterioles
that originated from interlobular arteries <50 µm in diameter were
selected for study. Diameter was measured over a 20- to 30-µm segment near
the midpoint of the vessel and was determined by online image processing at a
sampling rate of
3 Hz. Mean diameter values were then averaged over the
plateau or peak of the response. Typically, each determination was derived
from the mean of 50-100 individual measurements of the average diameter over
the vessel segment.
All agents were added directly to the perfusate. The venous effluent
emptied into the perfusion chamber, thus agents reached both the luminal and
adluminal surfaces of the arterioles. Experiments that employed AP and ChTX
required the use of a recirculating perfusion system as previously described
(36). In all other
experiments, single-pass perfusion was employed. The data are expressed as
means ± SE. Differences between treatment groups were assessed by
Bonferroni's t-test and one-way ANOVA. P values <0.05
were considered significant. The two rat strains, Sprague-Dawley and
Long-Evans, were used based on availability. No significant differences were
observed between these two strains, and the data were combined.
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RESULTS
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BK-induced vasodilation. The concentration-dependent actions of BK
on the afferent arteriole are depicted in
Fig. 1. ANG II (0.1 nmol/l),
which was used to establish basal tone, reduced diameter values from 16.2
± 0.9 to 4.7 ± 0.5 µm(P < 0.001; n = 6).
Figure 1A depicts a
representative tracing that illustrates the transient character of the
BK-induced vasodilation in this setting. At concentrations of 0.1, 1, 10, and
100 nmol/l, BK increased diameter values to peaks of 7.0 ± 0.7, 10.1
± 0.7, 14.0 ± 1.0, and 15.9 ± 0.9 µm, respectively
(Fig. 1B). The data
were converted to percent reversal of the ANG II-induced vasoconstriction
(percent vasodilation). The corresponding values were 20 ± 3, 48
± 5, 81 ± 5, and 98 ± 5% vasodilation
(Fig. 1C). Although
the afferent arteriole consistently exhibited transient BK-induced
vasodilation, sustained responses were frequently observed in the interlobular
artery (ILA). Figure 2 depicts
a representative example of a sustained response. Of eight ILAs studied, four
exhibited transient dilations (mean initial diameter, 67 ± 11 µm)
and four exhibited sustained responses (mean diameter, 88 ± 6
µm).

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Fig. 1. A: original tracings illustrate the transient vasodilation evoked
by increasing concentrations of bradykinin ([BK]) in the afferent arteriole.
B and C: concentration-response relationship is expressed as
both diameter and percent vasodilation. Vessels were preconstricted with 0.1
nmol/l ANG II.
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Fig. 2. Representative tracing depicts sustained BK-induced vasodilation in an
interlobular artery (ILA).
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In a separate series of kidneys (n = 7), we assessed the effects
of pretreatment with 10 µmol/l ibuprofen and 100 µmol/l
N
-nitro-L-arginine methyl ester
(L-NAME) on the response to BK. As shown in
Fig. 3B and subsequent
figures (i.e., see Figs. 5,
6,
7), inhibition of COX and NOS
had little effect on basal afferent arteriolar diameter. Similar findings were
reported in past publications using this model
(35,
36). Moreover, we also
observed this treatment to have no effect on basal perfusate flow of the
isolated perfused normal rat kidney in the absence of an added vasoconstrictor
(13). As further depicted in
Fig. 3, inhibition of COX and
NOS had very modest effects on the profile of the response to BK (100 nmol/l),
which suggests a prominent role of additional factors such as EDHF. In the
control kidneys, BK (100 nmol/l) elicited a peak dilation of 97 ± 2%
(from 6.2 ± 1.1 to 16.4 ± 0.4 µm; basal, 16.7 ± 0.6
µm; n = 7). In the kidneys pretreated with L-NAME and
ibuprofen, BK elicited a peak dilation of 83 ± 4% (n = 7;
P = 0.009; from 5.4 ± 0.3 to 15.5 ± 0.7 µm; basal,
16.9 ± 1.7 µm). The vasodilatory responses decayed with a similar
time course in these two settings. For example at 4 and 6 min, diameter
measurements of controls (10.8 ± 2.7 and 7.1 ± 1.2 µm) were
not statistically different from those of kidneys pretreated with
L-NAME (5.3 ± 0.6 and 4.8 ± 0.5 µm; P =
0.06 and P = 0.11, respectively).

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Fig. 3. BK elicits a phasic vasodilation of the afferent arteriole regardless of
the presence or absence of
N -nitro-L-arginine methyl ester
(L-NAME). A: original tracings of vessels preconstricted
with 0.1 nmol/l ANG II illustrate control response obtained in the presence of
10 µmol/l ibuprofin (left) and the response after the addition of
100 µmol/l L-NAME (right). B: mean data depict
time course of the response to BK (added at t = 0) in
controls (ibuprofen treated) and in vessels pretreated with 100 µmol/l
L-NAME (and ibuprofen).
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Fig. 5. In contrast with our observations with ACh, the endothelium-derived
hyperpolarizing factor (EDHF)-like response to BK was not prevented by the
combination of 10 nmol/l charybdotoxin (ChTX) plus 1 µmol/l apamin (AP).
A: original tracing. B: mean data. C: mean data are
compared with the effects of AP plus ChTX on EDHF-like response to ACh (data
taken from Ref. 36).
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Fig. 6. A: tetraethylammonium chloride (TEA; 1 mmol/l) had no effect on
the EDHF-like response to BK when administered alone (left). Mean
data are shown (right; n = 5). B: however, when TEA
treatment was combined with 10 nmol/l ChTX plus 1 µmol/l AP, the response
was totally eliminated (n = 7). C: effects of TEA alone, AP
plus ChTX, and the combination of TEA plus AP plus ChTX are shown. All
experiments were performed in the presence of L-NAME plus
ibuprofen. ANG II indicates application of 0.1 nmol/l ANG II.
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Fig. 7. A: pretreatment with 17-octadecynoic acid (ODYA; 50 µmol/l)
alone did not prevent the EDHF-like response to BK (left). Mean data
are shown (right; n = 4). B: however, when combined
with 10 nmol/l ChTX plus 1 µmol/l AP, ODYA eliminated this response
(n = 6). C: summary of the effects of ODYA alone and ODYA
plus AP plus ChTX. All experiments were performed in the presence of
L-NAME plus ibuprofen.
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To further assess the contribution of a hyperpolarizing factor, we
determined the effects of these inhibitors on the actions of BK on arterioles
that had been preconstricted with elevated extracellular K+. As
illustrated in Fig. 4, BK
partially reversed the vasoconstrictor actions of 25 mmol/l KCl. Treatment
with KCl reduced diameter measurements from 18.7 ± 0.6 to 9.9 ±
0.7 µm, and BK caused a peak dilation to 14.7 ± 1.4 µm
(n = 5; P = 0.007) thus eliciting a 55 ± 11% dilation
(Fig. 4B, solid bar).
Pretreatment with ibuprofen alone (10 µmol/l) did not significantly alter
this response (50 ± 13% vasodilation,
Fig. 4B, open bar),
whereas treatment with L-NAME (100 µmol/l) abolished this action
(-0.1 ± 1.5% vasodilation). We interpret these findings as indicating
that the phasic vasodilatory response to BK seen in the presence of
KCl-induced vasoconstriction is mediated by NO production, and that the NOS-
and COX-independent actions of BK, which are seen when the afferent arteriole
is preconstricted by ANG II, were blocked by an elevation of extracellular
K+. The latter observation is consistent with a proposed EDHF-like
component of the afferent arteriolar actions of BK.

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Fig. 4. BK-induced vasodilation of afferent arterioles preconstricted with 25
mmol/l KCl. A: original tracing illustrates the transient nature of
this response. B: mean data (n = 5) illustrate lack of
effect of 10 µmol/l ibuprofen (Ibu) and complete abolition of response by
100 µmol/l L-NAME.
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Effects of K+-channel blocking agents on NOS- and
COX-independent actions of BK. We have shown that like BK, ACh elicits a
phasic vasodilator response that is independent of COX and NOS but is blocked
by elevated K+ (14,
36). In the case of ACh, this
EDHF-like response was abolished by a combination of 10 nmol/l ChTX plus 1
µmol/l AP. We therefore examined the effects of this treatment on the BK
response that is elicited in the presence of ibuprofen and L-NAME.
These findings are summarized in Fig.
5. In these experiments (n = 6), diameter measurements in
the presence of ibuprofen (10 µmol/l) and following treatment with
L-NAME (100 µmol/l) and then the combination of ChTX (10 nmol/l)
and AP (1 µmol/l) were 17.8 ± 0.8, 17.7 ± 0.8, and 17.3
± 0.8 µm, respectively. The subsequent administration of ANG II (0.1
nmol/l) reduced diameter values to 4.8 ± 0.8 µm. In this setting,
100 nmol/l BK caused a transient vasodilation to a peak value of 12.6 ±
1.2 µm (P = 0.003), which corresponds to a 46 ± 6%
vasodilation (P = 0.003 vs. 83 ± 4% vasodilation in the
presence of L-NAME and ibuprofen alone). Thus in contrast to our
previous findings with ACh (presented as solid bars in
Fig. 5C for
illustrative purposes; data taken from 36), ChTX plus AP attenuated but did
not abolish the EDHF-like response to BK.
We next examined the effects of TEA both alone and in combination with ChTX
plus AP. TEA is a nonselective K+-channel blocker at high
concentrations, but at a concentration of 1 mmol/l, TEA blocks
large-conductance Ca2+-activated K+ channels
(BKCa) while having minimal effects on other K+-channel
species (19). After treatment
with ibuprofen and then L-NAME (diameter, 15.2 ± 0.5 and
14.8 ± 0.5 µm, respectively; n = 5), the addition of 1
mmol/l TEA reduced the diameter to 13.6 ± 0.7 µm (P >
0.05). In this setting, ANG II reduced the diameter to 4.9 ± 0.6 µm,
and 100 nmol/l BK caused a transient dilation to 14.1 ± 0.6 µm (107
± 9% vasodilation vs. diameter in TEA alone; P = 0.0004). In a
separate series of seven kidneys, basal (ibuprofen) diameter values and
measurements after treatment with L-NAME and ChTX plus AP were 15.5
± 0.8, 15.1 ± 0.7, and 14.7 ± 0.8 µm, respectively
(Fig. 6B). The
addition of TEA (1 mmol/l) reduced diameter to 13.5 ± 0.7 µm. In
this setting, ANG II reduced diameter to 6.6 ± 0.6 µm, and the
administration of BK (100 nmol/l) had no effect (6.6 ± 0.6 µm, 0
± 1% vasodilation). Thus whereas TEA or ChTX plus AP only partially
attenuated the EDHF-like response to BK when added alone, these agents
abolished this response when added concurrently
(Fig. 6C).
These findings suggest that two components may contribute to the EDHF-like
response to BK: one that (as previously shown for ACh) is blocked by ChTX plus
AP and a second that is sensitive to TEA. BK has been shown to stimulate the
release of EETs from the renal vasculature
(16), and 11,12-EET has been
reported to elicit afferent arteriolar vasodilation by a mechanism that is
blocked by 1 mmol/l TEA (17,
38). We therefore determined
whether the cytochrome P-450 inhibitor 17-ODYA would also block the
component of the BK response that was insensitive to ChTX plus AP. The results
of these experiments are summarized in Fig.
7. Alone or in combination with TEA, 17-ODYA (50 µmol/l) did
not prevent the EDHF-like response to BK. After treatment with ibuprofen,
L-NAME, and 50 µmol/l 17-ODYA, diameter measurements were 17.0
± 0.9, 16.8 ± 0.9, and 16.0 ± 1.2 µm, respectively.
ANG II reduced diameter to 6.0 ± 0.5 µm, and 100 nmol/l BK elicited
a peak dilation to 13.4 ± 1.0 µm, which corresponds to a 77 ±
9% vasodilation (n = 4; Fig.
7A). Similarly, after treatment with ibuprofen and
L-NAME (diameters, 15.18 ± 1.1 and 14.8 ± 1.0 µm,
respectively), the addition of 1 mmol/l TEA reduced diameter to 13.8 ±
1.4 µm (P = 0.10) and addition of 17-ODYA reduced diameter to 11.5
± 1.5 µm (P = 0.02). In this setting, ANG II reduced
diameter to 4.0 ± 0.5 µm, and 100 nmol/l BK caused a transient
dilation to 11.6 ± 1.1 µm (n = 5; P = 0.002; 106
± 13% vasodilation of ANG II response vs. 17-ODYA alone, data not
shown). Thus the inhibitory effects of TEA and 17-ODYA on BK responses were
not additive. In a separate series of six kidneys, basal (ibuprofen) diameter
measurements and values after treatment with L-NAME and ChTX plus
AP were 15.6 ± 0.5, 15.1 ± 0.5, and 14.2 ± 0.6 µm
(Fig. 7B). The
addition of 17-ODYA (50 µmol/l) reduced diameter to 12.5 ± 0.8 µm
(P = 0.01). In this setting, ANG II reduced diameter to 5.2 ±
0.2 µm, and the administration of BK (100 nmol/l) had no effect (5.2
± 0.3 µm, 0 ± 6% vasodilation). Accordingly, as seen with
TEA, 17-ODYA abolished that component of the EDHF-like response to BK that was
resistant to ChTX plus AP. These results are summarized in
Fig. 7C.
Contribution of NO to vasodilation elicited by BK. As shown in
Fig. 4, during KCl-induced
vasoconstriction, BK elicited a dilation that was NO dependent (e.g., blocked
by L-NAME). Nevertheless, L-NAME had little effect on
the magnitude or profile of the BK-induced vasodilation when ANG II was used
to establish basal tone (see Fig.
3). To further assess the contribution of NO in this setting, we
examined the response elicited following pretreatment with ibuprofen, TEA, and
ChTX plus AP, which would block the COX- and EDHF-dependent components, and
compared these results to those in which L-NAME was also present
(see Fig. 6A). These
findings are summarized in Fig.
8, A and B. Diameter measurements were 16.9
± 0.5 µm in the presence of ibuprofen and 16.8 ± 0.5 and 15.7
± 0.1 µm following ChTX plus AP and TEA, respectively. The addition
of ANG II reduced diameter to 7.4 ± 1.2 µm, and the subsequent
administration of BK resulted in a maximal dilation to 12.3 ± 1.0
µm, which corresponds to a 62 ± 7% vasodilation (n = 5).
These findings suggest that, as seen during KCl-induced vasoconstriction, NO
production contributes to the transient BK-induced afferent arteriolar
vasodilation that is observed during ANG II-induced vasoconstriction.

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Fig. 8. A: original tracing (left) shows that pretreatment with
ibuprofen, TEA, and plus 10 nmol/l ChTX plus 1 µmol/l AP eliminated the
response to BK in the presence of L-NAME but not in the absence of
the nitric oxide synthase (NOS) inhibitor. Thus it appears that NO production
contributes to the phasic vasodilation produced by BK. Mean data (n =
5) are also depicted (right). B: responses (n = 7)
in presence and absence of L-NAME are compared.
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DISCUSSION
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The results of the present study indicate that BK evokes a transient
vasodilatory response in the renal afferent arteriole and that this action
involves both NO-dependent and NO-independent mechanisms. The NO-independent
component of the vasodilation that remained following NOS blockade had
features similar to those ascribed to an EDHF in that it was blocked by either
elevated external K+ or a combination of agents known to block
Ca2+-activated K+ (KCa) channels.
A major finding of this study is that the pharmacological characteristics of
this EDHF-like component of the afferent arteriolar response to BK differ
fundamentally from the previously observed characteristics of the EDHF-like
response of the same vessel to ACh
(36). In concert, these
observations suggest that multiple factors or multiple mechanisms contribute
to the vasodilator responses ascribed to EDHF in the renal
microcirculation.
We suggest that the EDHF-like response to BK consists of two components,
one of which was blocked by a combination of ChTX plus AP. We had previously
shown that this combination fully abolished the EDHF-like response of the
afferent arteriole to ACh
(36). Indeed, treatment with
ChTX plus AP has been shown to block the vasodilator responses attributed to
EDHF in a variety of blood vessels under diverse conditions (for review, see
Refs. 23,
34). At the concentrations
employed, these toxins are known to block small- and intermediate-conductance
KCa channels (32,
33). It has been suggested by
a number of investigators that these K+-channel blockers may
attenuate EDHF-like responses by blocking K+ channels that are
present on the endothelium rather than on the vascular myocyte (e.g.,
4,
5,
7). An elegant demonstration of
this was provided by Doughty et al.
(5), who reported that ChTX and
AP inhibit EDHF responses when selectively applied to the endothelial cells
within the vessel lumen but not when selectively applied to the outer surface
of a perfused mesenteric artery. These authors interpret these observations to
suggest that these agents act on the endothelium rather than the smooth muscle
myocytes. Accordingly, in small vessels, in which such EDHF-like responses are
more prevalent, endothelial agonists may activate K+ channels in
the endothelium, and the resultant hyperpolarization may be transmitted to the
underlying smooth muscle layer via electrical communication through
myoendothelial gap junctions
(30). An obligate role of
myoendothelial gap junctions in responses attributed to EDHF is suggested by
several studies (e.g., 3,
18,
30).
The present study did not specifically address this issue. However, we
previously demonstrated that the combination of ChTX plus AP not only blocks
the transient EDHF-like component of the afferent arteriolar dilation evoked
by ACh but also inhibits the sustained phase of the ACh response that is
dependent on NO formation
(36). This latter observation
is consistent with the premise that endothelial K+-channel
activation and the resultant hyperpolarization contribute to
Ca2+ entry (see Ref.
24 for review). Thus by
interfering with this mechanism, the blockade of endothelial K+
channels by ChTX and AP would be anticipated to inhibit the sustained
component of NO formation as we have observed. Accordingly, our previous
finding that ChTX plus AP blocks the sustained NO-dependent component of the
ACh response would be consistent with the postulate put forward by others
(4,
5,
7) that the K+
channels affected by these toxins are in the endothelium. ACh and BK are both
known to release intracellular Ca2+ stores in the
endothelium by stimulating phospholipase C, increasing levels of inositol
1,4,5-trisphosphate (IP3), and activating IP3 receptors
present in the endoplasmic reticulum. Thus these two agents could activate the
same population of KCa channels in the endothelium
(24). Such a scheme (see
Fig. 9) could explain our
finding that although the characteristics of the EDHF-like response to BK and
ACh were quite different (e.g., see Fig.
3), a common component of the response to each of these agents was
blocked by ChTX and AP. Although the present study does not provide a direct
test of this postulate, our findings would be consistent with this scheme.

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Fig. 9. A proposed model that is consistent with our findings. BK and ACh release
Ca2+ from the endoplasmic reticulum via inositol
1,4,5-trisphosphate formation. Transient elevation in intracellular
Ca2+ activates low- and intermediate-conductance
Ca2+-activated K+ channels in the
endothelium, and the resultant hyperpolarization is transmitted to the
underlying myocytes via myoendothelial coupling. This pathway, which is shared
by BK and ACh, is blocked by the combined treatment with ChTX plus AP. In
addition, BK-receptor activation results in the liberation of arachidonic acid
(AA), which is converted to epoxyeicosatrienoic acids (EETs) by cytochrome
P-450 (Cyt P450). Released EETs activate large-conductance
Ca2+-activated K+ channels in the vascular
myocytes. Thus unlike ACh, the EDHF-like response to BK is not fully blocked
by ChTX plus AP. However, this second component of the BK response can be
eliminated by either 17-ODYA or 1 mmol/l TEA.
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We previously found that treatment with ChTX plus AP fully blocked the
EDHF-like response to ACh
(36); however, in the present
study, we found that this treatment alone was not sufficient to prevent the
EDHF-like response to BK (see Fig.
5). The component of the EDHF-like response to BK that remained
after treatment with ChTX plus AP was completely eliminated by treatment with
either 1 mmol/l TEA or 50 µmol/l 17-ODYA. In the absence of ChTX and AP,
TEA or 17-ODYA administered either alone or in combination did not block the
EDHF-like response to BK. We interpret these observations to indicate that a
second, ChTX- and AP-insensitive but TEA- and 17-ODYA-sensitive component
contributes to the EDHF-like afferent arteriolar response to BK. The
properties of this second component are consistent with the suggestion put
forward by other investigators that the EDHF-like response to BK involves the
elaboration of an EET (9,
12,
16,
17,
28,
38) that in turn activates the
TEA-sensitive BKCa channels
(1,
17,
38) present in the vascular
myocytes (e.g., see Fig.
9).
Growing evidence implicates cytochrome P-450 products,
particularly the EETs, as candidate EDHFs (reviewed in Refs.
23,
28,
34). This is particularly true
of EDHF responses evoked by BK. Fulton et al.
(11) demonstrated that the
cytochrome P-450 inhibitor ETYA attenuated the response of the
isolated perfused kidney to BK. Imig and co-workers
(16) demonstrated not only
that cytochrome P-450 inhibitors prevented the EDHF-like response of
the afferent arteriole to BK, but also that BK treatment augmented EET
production by isolated renal vascular tissues. Moreover, Imig et al.
(17) found 11,12-EET to dilate
the rat ILA and juxtamedullary afferent arteriole via a COX-independent
mechanism, whereas 8,9-EET had no effect on these vessels. Zou et al.
(38) reported that 11,12-EET
activated a K+ channel in isolated renal vascular myocytes and
elicited vasodilation in the intact vessels, and both of these actions were
abolished by 1.0 mmol/l TEA, a finding consistent with our observation that
17-ODYA and 1.0 mmol/l TEA abolished the same component of the EDHF-like
response to BK. Gebremedhin et al.
(12) elegantly demonstrated,
using a bioassay system, that BK released a substance from a perfused bovine
coronary artery that stimulated BKCa channels in a downstream
myocytes and that the release of this factor was blocked by endothelium
removal or treatment with 17-ODYA. More recently, Archer et al.
(1) identified 11,12-EET as a
likely EDHF candidate in human internal mammary arteries and demonstrated that
this agent activates BKCa channels in this preparation. Fisslthaler
et al. (9) demonstrated that
chronic treatment of native coronary artery endothelial cells with
-naphthoflavone enhanced cytochrome P-450 (2C) expression
levels, augmented 11,12-EET production, and enhanced both the vasodilator
response and the hyperpolarization induced by BK in coronary artery segments.
These findings and many others (see Refs.
23,
27,
28,
34) support the postulate that
BK stimulates endothelial production of an epoxygenase product that in turn
can evoke vasodilation by activating TEA-sensitive BKCa channels
present in the underlying vascular myocytes.
It is important to emphasize that although our findings are consistent with
this postulate in regard to the actions of BK, we did not see a similar
profile of the EDHF-like response of the afferent arteriole to ACh. Thus ChTX
and AP treatment alone were sufficient to prevent the EDHF-like response to
this agent (see Ref. 36).
These observations suggest that there are multiple components to the EDHF-like
response and that the relative contributions of these two components or
pathways may differ with different endothelial-dependent vasodilator agents.
Very few studies have compared the determinants of the EDHF component of the
vasodilator response to differing agents. However, Fulton et al.
(11) previously reported that
ETYA prevented the response of the isolated perfused kidney to BK but did not
prevent the response of this preparation to ACh and suggested differing
pathways (27). Frieden et al.
(10) also observed marked
differences in the pharmacological profile of the EDHF associated with BK vs.
substance P, in that the response to the latter did not involve the cytochrome
P-450 pathway. Moreover, the determinants of the EDHF-like response
to an agent may vary with blood vessel type. In regard to the latter, it is
important to note that a number of studies report EDHF-like responses to BK
that are not prevented by cytochrome P-450 inhibition alone (e.g.,
Refs. 2,
6,
22,
25). However, a further
complication is that a concomitant activation of the two EDHF-like pathways
(see Effects of K+-channel blocking agents on NOS- and
COX-independent actions of BK) may obscure interpretations of experiments
in which only a single pathway is inhibited. The present study is an example
of this phenomenon, in that we could only demonstrate a major effect of
blocking the cytochrome P-450 pathway or 1.0 mmol/l TEA when the
vessels were concomitantly treated with ChTX plus AP.
When both of the EDHF-like or NO-independent mechanisms were blocked (by
the combined treatment with AP, ChTX, and 1 mmol/l TEA), BK elicited a phasic
vasodilation that could be blocked by further treatment with L-NAME
(see Fig. 8). Similarly, when
the afferent arteriole was preconstricted by elevation of extracellular
K+ (which would eliminate the effects of EDHF or
K+-channel activation), BK elicited a transient and NO-dependent
increase in diameter (see Fig.
4). These findings illustrate that both NO and EDHF contribute to
the phasic vasodilatory response of the afferent arteriole to BK. Moreover,
although K+-channel blockade (e.g., 1 mmol/l TEA, ChTX, AP) or
elevation of extracellular K+ eliminated the vasodilator actions of
EDHF, a significant component of the NO-dependent vasodilation persisted in
these settings. Thus while evidence suggests a prominent role of
BKCa channels in the vasodilatory actions of NO (e.g., Ref.
31), these data suggest that
additional mechanisms contribute to the response of the afferent arteriole to
NO and/or cGMP. It is widely acknowledged that multiple pathways contribute to
cGMP-dependent vasodilation and that the resultant smooth muscle relaxation
may involve both K+-channel-dependent and -independent mechanisms
(20).
Finally, we must comment on our observation that the cortical afferent
arterioles of our preparation exhibited a phasic or transient vasodilator
response to BK. The literature suggests a high degree of variability in regard
to the profile of the renal vascular responses to BK. Thus Edwards
(8) reported that the afferent
arteriole of the rabbit does not respond to BK, whereas Yu et al.
(37) found the same
preparation to exhibit vasodilation at very low BK concentrations and
vasoconstriction at concentrations above 0.1 nmol/l. Ihara et al.
(15) reported triphasic
responses of the porcine ILA to BK, characterized by an initial dilation that
was followed by a constriction and then a more slowly developing sustained
dilation. In each of these cases, the constriction was dependent on COX and
thromboxane, whereas in our studies, the transient nature of the response
persisted during COX inhibition. Imig et al.
(16) reported sustained
vasodilator responses of the blood-perfused juxtamedullary afferent arteriole.
It is possible that observed differences in the time course of BK responses
might reflect differences in the experimental preparations used, the impact of
acute trauma associated with the surgical preparations, the imposition of
hydronephrosis, species-related differences, or perfusate-type differences.
The possibility of regional differences in the profile of the response within
the renal microcirculation is also quite likely, as we observed sustained
responses in larger segments of the ILA in the present study (see
Fig. 2). Studies to address
regional differences in the temporal profile of the responses and any regional
differences in the determinants of the vasodilation produced by BK would be of
considerable interest.
In summary, the present study demonstrates that a component of the
EDHF-like response of cortical afferent arterioles in the in vitro perfused
hydronephrotic rat kidney preparation to BK is sensitive to inhibition by
either cytochrome P-450 inhibition by 17-ODYA or by blockade of
BKCa channels by 1.0 mmol/l TEA. These findings are consistent with
the proposed involvement of an EET in the EDHF-like response of this vessel to
BK (16,
17,
38). However, we also found an
additional component of the EDHF-like response to BK, and this component was
not prevented by 17-ODYA or TEA but was sensitive to inhibition by a
combination of ChTX plus AP. This ChTX- and/or AP-sensitive component of the
EDHF-like response was similar to that previously reported for ACh-induced
vasodilation of the afferent arteriole
(36). These findings cannot be
explained by a single EDHF. Thus based on these observations, we suggest that
multiple factors or hyperpolarizing pathways contribute to the EDHF-like
response of the renal afferent arteriole.
 |
ACKNOWLEDGMENTS
|
---|
This study was supported by a grant from the Heart and Stroke Foundation of
Alberta, the Northwest Territories and Nunavut. R. Loutzenhiser is a Senior
Medical Scholar of the Alberta Heritage Foundation for Medical Research.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: R. D. Loutzenhiser,
Dept. of Pharmacology and Therapeutics, Univ. of Calgary, Faculty of Medicine,
3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1 (E-mail:
rloutzen{at}ucalgary.ca).
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.
 |
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