Distinct Endothelium-Derived Hyperpolarizing Factors
Emerge In Vitro and In Vivo and Are Mediated in Part via
Connexin 40 –Dependent Myoendothelial Coupling
Markus Boettcher, Cor de Wit
See Editorial Commentary, pp XX–XX
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Abstract—The endothelium-derived hyperpolarizing factor (EDHF) contributes critically to the regulation of vascular tone.
Its dependency on direct signaling through myoendothelial gap junctions composed of connexins (Cx) is controversially
discussed. We studied the impact of Cx40 in EDHF-type dilations in vivo and in vitro (wire and pressure myography)
in small arteries (A. gracilis) using different Cx40-deficient mouse models. Acetylcholine induced prominent
EDHF-type dilations (inhibition of NO synthase and cyclooxygenase) of ⬇90% (maximum effect) in wild-type and
Cx40-deficient vessels (Cx40⫺/⫺) in vitro under isobaric conditions. In contrast, under isometric conditions, EDHF-type
relaxations were nearly abrogated in Cx40⫺/⫺ (9⫾3%) but only slightly reduced in wild-type vessels (45⫾4%;
P⬍0.05). Vessels expressing Cx45 instead of Cx40 exhibited similarly reduced relaxations (13⫾1%), demonstrating
that Cx45 cannot replace Cx40 functionally. The necessity of Cx40 in EDHF-type dilations under isometric conditions
was verified by the attenuation in vessels being specifically deficient for Cx40 in endothelial cells (Cx40fl:TIE2-Cre:
17⫾3%; Cx40-floxed controls: 67⫾6%; P⬍0.05). Nevertheless, EDHF-type dilations were Cx40 independent when
studied isobarically. The EDHF-type dilation in vivo resembled the isobaric situation, being virtually Cx40 independent
and similar powerful. Distinct EDHF mechanisms can be distinguished by their Cx40 dependency. A powerful EDHF
is present in vivo and in vitro under isobaric conditions but is lacking in wire myography (isometric conditions). Herein,
a less potent EDHF depends on Cx40 and may represent signaling through myoendothelial gap junctions. We suggest
that distinct EDHFs (even in the same artery) explain partially the controversy on the role of myoendothelial gap
junctions in EDHF signaling. (Hypertension. 2011;57:00-00.) ● Online Data Supplement
Key Words: myoendothelial coupling 䡲 gap junctions 䡲 connexins 䡲 microcirculation
䡲 endothelium-derived hyperpolarizing factor
T
he endothelium is the key player in arteries to control the
contractile state of the adjacent smooth muscle and
thereby vascular diameter. This function is achieved by the
release of NO, prostaglandins, and the endothelium-derived
hyperpolarizing factor (EDHF). Of these autocaids, NO
predominates in large conducting arteries, whereas the importance of EDHF increases as the size of the arteries
decreases.1–5 NO and prostaglandins are well characterized
with respect to chemistry and signaling cascades, but the
nature of EDHF is controversially discussed.6 – 8 By definition, EDHF-type dilations are associated with smooth muscle
hyperpolarization, which is followed by its relaxation, most
likely through decreasing the open probability of voltagedependent L-type Ca2⫹-channels. Because EDHF-type dilations require an endothelial hyperpolarization through activation of Ca2⫹-activated K⫹-channels (KCa)9 –11 as an initial
step the smooth muscle hyperpolarization may be caused by
a direct charge transfer from the endothelium into the smooth
muscle, because they are coupled heterocellularly by myoendothelial gap junctions.6,12–14
Gap junction forming proteins constitute a family named
connexins (Cx), of which Cx40, Cx37, Cx43, and Cx45 are
expressed in vascular cells and build tightly sealed intercellular channels between adjacent cells. Morphologically,
myoendothelial gap junctions were visualized,15,16 and specifically Cx40 and Cx37 have been located in myoendothelial
gap junctions in rat cerebral vessels.17,18 Functional experiments verified dye transfer and spreading of hyperpolarization from the endothelium into the smooth muscle, which
suggests effective heterocellular coupling.19 –22 Although in
many experiments nonspecific blockade of gap junctions
effectively abrogated EDHF-type dilations, this does not
provide compelling evidence, because such blockers also
affect ion channels and interrupt homocellular coupling
Received October 29, 2010; first decision November 14, 2010; revision accepted January 24, 2011.
From the Institut für Physiologie, Universität zu Lübeck, Lübeck, Germany.
Correspondence to Cor de Wit, Physiologisches Institut, Universität zu Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. E-mail
[email protected]
© 2011 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.110.165894
1
2
Hypertension
A
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CA (A. femoralis)
80
Relaxation [%]
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60
Control
LN/Indo
+ KCl
40
20
0
*
#
#
SA (A. gracilis)
Control
LN/Indo
+ KCl
*
#
*
*
*
#
#
*
*
*
#
#
*
#
#
*
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-8
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ACh [log mol/L]
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within the endothelium and the smooth muscle layer, the
latter being specifically important in larger vessels.22 Recently, Mather et al23 have demonstrated using antibodies
targeted against Cx40 that specifically the function of Cx40 is
required to sustain an EDHF-type dilation in small mesenteric
arteries of rats studied in vitro. In marked contrast, EDHFtype dilations elicited by acetylcholine (ACh) are preserved
in the cremaster microcirculation in Cx40-deficient mice in
vivo. 24,25 In keeping with this, ACh injected intra-arterially in
awake animals decreased blood pressure similarly in the
absence of NO in wild-type (wt) and Cx40-deficient mice,
suggesting an intact EDHF-type dilation in the overall circulation.26 In addition, electrophysiological assessment of the
membrane potential of endothelial and smooth muscle cells in
arterioles in vivo argued against well-coupled vascular cells
in vivo.27
Because in vitro and in vivo studies delivered such controversial data for the significance of myoendothelial coupling in EDHF-type dilations, we aimed to investigate the
impact of Cx40 in EDHF-type dilations in small arteries
depending on the experimental setting including in vivo and
in vitro conditions by using diverse Cx40-deficient mouse
models. We report herein that EDHF-type dilations depend
on Cx40 only under isometric conditions in vitro, suggesting,
in addition to myoendothelial coupling, a potent EDHF-type
dilatory mechanism under isobaric conditions in vitro, which
is importantly also present in vivo.
Methods
Experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals
and approved by the local government. Relaxations of conducting
and small arteries (A. gracilis) in wt, Cx40⫺/⫺, Cx40KI45, Cx40fl, and
Cx40fl:TIE2-Cre mice were examined. Cx40⫺/⫺ mice lack Cx40
globally, whereas it is replaced by Cx45 in Cx40KI45. In Cx40fl, it
is flanked by loxP sites, which were used to generate endothelial
cell-specific deficiency using a Cre recombinase driven by the TIE2
promoter (Cx40fl:TIE2-Cre).28,29 Mice were anesthetized and killed
and arteries isolated for in vitro studies using wire and pressure
myography. After pressurization (small arteries: 60 mm Hg; conducting arteries: 80 mm Hg), vessels were preconstricted using
norepinephrine or potassium (KCl, 50 mmol/L) and, subsequently,
relaxations were induced by ACh or sodium nitroprusside (SNP;
traces: Figure S1, available in the online Data Supplement at
http://hyper.ahajournals.org). NO synthase and cyclooxygenase were
blocked by L-nitro-arginine (LN; 300 ␮mol/L) and indomethacin
(3 ␮mol/L). Relaxations were normalized to the initial increase in
-7
-6
Figure 1. EDHF-type dilations are pronounced in small arteries (SAs, B) as
compared with conducting arteries (CAs,
A). ACh induced concentration-dependent
relaxations in preconstricted isometrically
mounted CAs (A, n⫽10 to 14) and SAs
(B, n⫽13 to 16) from wt mice were
reduced in the presence of LN/Indo (300
and 3 ␮mol/L). The remaining response
was abrogated after preconstriction using a
high potassium solution (⫹KCl; 50 mmol/L).
*P⬍0.05 LN/Indo vs control, #P⬍0.05 KCl
vs LN/Indo, paired t test.
ACh [log mol/L]
force (or decrease in diameter) on vasoconstriction. In additional
experiments, the A. gracilis was exposed, preconstricted, and examined in vivo in anesthetized mice. Data are given as mean⫾SEM.
Comparisons within groups were performed using a paired t test and
between groups using ANOVA followed by Bonferroni correction.
Additional details are given in the online Data Supplement, together
with Figures S1 through S5).
Results
EDHF-Type Dilations in Large Conducting and
Small Arteries During Isometric Conditions
Large conducting arteries (n⫽14) mounted in a wire myograph and studied isometrically exhibited a diameter of
241⫾30 ␮m at 80 mm Hg. Preconstriction with norepinephrine (NE; 1 ␮mol/L) increased force from 1.33⫾0.08 to 2.60⫾
0.42 mN/mm. From this level, ACh induced a concentrationdependent relaxation (at 1 ␮mol/L to 1.60⫾0.12 mN/mm).
After blockade of NO synthase and cyclooxygenase (LN/
Indo, 300 and 3 ␮mol/L, respectively), force increase in
response to NE was reduced (from 1.20⫾0.08 to 1.68⫾0.10
mN/mm, P⫽0.05) and ACh-induced relaxations were
strongly reduced (at 1 ␮mol/L to 1.53⫾0.08 mN/mm; Figure
1A). However, endothelium-independent relaxations (SNP)
remained unaffected (Figure S2). Preconstriction with K⫹
solution (50 mmol/L, presence of LN/Indo) instead of NE
completely prevented relaxations in response to ACh but also
reduced responses to SNP.
Small arteries (A. gracilis) exhibited a diameter of 137⫾
28 ␮m at 60 mm Hg (n⫽16) corresponding to 0.49⫾0.03
mN/mm. NE (1 ␮mol/L) increased force to 1.17⫾0.10
mN/mm, and from this level ACh relaxed the vessels concentration dependently (at 1 ␮mol/L to 0.69⫾0.07 mN/mm).
After LN/Indo, constrictions on NE were similar (from
0.33⫾0.04 to 1.18⫾0.11 mN/mm) but ACh-induced relaxations were reduced (at 1 ␮mol/L to 0.82⫾0.08 mN/mm;
Figure 1B). However, ACh relaxations resistant to LN/Indo
were significantly larger in small compared with conducting
arteries at 0.03 to 0.30 ␮mol/L ACh. These LN/Indo-resistant
ACh responses were abrogated after preconstriction with
depolarizing K⫹ solution (Figure 1B). Relaxations to SNP
were not attenuated by LN/Indo and were reduced in the
presence of K⫹ (Figure S2).
Cx40 Expression in Gracilis Artery
Immunohistochemistry demonstrated Cx40 expression at the
inner vessel wall. Fluorescence was detected only at cell
Boettcher and de Wit
A
Relaxation [%]
B
C o n tr o l
80
60
wt
Cx40 -/Cx40KI45
*
*
*
*
20
#
0
-8
#
#
-7
#
-6
-8
#
*
*
#
*
-7
#
*
Figure 2. Cx40 is required in EDHF-mediated
dilations under isometric conditions. ACh
induced a concentration-dependent relaxation
in preconstricted small arteries isolated from
Cx40⫺/⫺ (n⫽12), as well as in vessels
expressing Cx45 instead of Cx40 (Cx40KI45;
n⫽9) in the wire myograph (A, control), which
was nearly abrogated after LN/Indo (B). At
both conditions, relaxations were reduced in
both genotypes compared with wt mice
(replotted from Figure 1B). *P⬍0.05 vs wt.
-6
ACh [log mol/L]
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borders, and cellular morphology indicated that Cx40 is
expressed in endothelial cells and located in the membrane.
Staining was absent in vessels from Cx40-deficient mice
(Cx40⫺/⫺) with intact endothelium (Figure S3).
Cx40 Is Crucial for EDHF-Type Dilations Under
Isometric Conditions
The role of Cx40 in EDHF-type dilations was studied in small
arteries (diameter of 128⫾18 ␮m) from Cx40⫺/⫺ mice
(n⫽12). After preconstriction (NE: from 0.40⫾0.05 to
0.62⫾0.06 mN/mm), ACh relaxed these vessels concentration dependently; however, relaxation was significantly reduced compared with wt mice in the presence of NO and
prostaglandins. LN/Indo nearly abrogated the relaxation on
ACh in Cx40⫺/⫺ (Figure 2). These strongly blunted relaxations are also reflected in significantly reduced maximum
effect (EMax) values (Table). In contrast, endotheliumindependent relaxations (SNP) were not attenuated in
Cx40⫺/⫺ and unaltered by LN/Indo (Figure S4). These
experiments suggest that Cx40 is required to support an
EDHF-type dilation at these experimental conditions.
Table.
#
#
ACh [log mol/L]
3
LN /Indo
wt
Cx40-/Cx40KI45
*
40
Distinct EDHF-Type Dilations In Vitro and In Vivo
Next, we evaluated whether Cx45 can compensate for the
lack of Cx40 by isolating arteries from animals in which
Cx40 is replaced by Cx45 (Cx40KI45), which leads to Cx45
expression in lieu of Cx40.30 ACh induced concentrationdependent relaxations in preconstricted (NE: from 0.44⫾0.04
to 1.00⫾0.08 mN/mm) small arteries from Cx40KI45 mice
(n⫽9; diameter: 121⫾12 ␮m). However, the responses were
attenuated compared with wt mice and were not different
from Cx40⫺/⫺ vessels in the presence of NO and prostaglandins. Similar to Cx40⫺/⫺, LN/Indo nearly abolished the
ACh-induced relaxation (Figure 2, for EMax see Table).
Relaxations induced by SNP were slightly attenuated in
comparison with wt mice but not different from Cx40⫺/⫺
vessels (Figure S4). This suggests that expression of Cx45
instead of Cx40 cannot rescue the ACh-induced EDHF-type
dilation assessed under isometric conditions.
EDHF-Type Dilations Are Cx40 Independent in
These Small Arteries During Isobaric Conditions
and In Vivo
EDHF-type dilations in this exact same artery (A. gracilis)
were also studied in vitro under isobaric conditions and in
EMax and EC50 of ACh-Induced Dilations
Isometric
Genotype
Treatment
EMax, %
78⫾3
Isobaric
EC50, nmol/L
EMax, %
EC50, nmol/L
30⫾6
89⫾6
55⫾16
Wt
Control
Wt
LN/Indo
45⫾4*
21⫾9
95⫾5‡
47⫾12
Cx40⫺/⫺
Control
40⫾5†
27⫾16
85⫾5‡
23⫾7
Cx40⫺/⫺
LN/Indo
29⫾6
82⫾6‡
33⫾12
Cx40KI45
Control
Cx40KI45
LN/Indo
13⫾1*†
Cx40fl
Control
73⫾5
54⫾19
89⫾5‡
16⫾5‡
Cx40fl
LN/Indo
67⫾6
48⫾20
101⫾4‡
26⫾5
Cx40fl:TIE2-Cre
Control
63⫾4
43⫾14
91⫾6‡
29⫾9
Cx40fl:TIE2-Cre
LN/Indo
17⫾3*†
15⫾13
93⫾6‡
44⫾15
9⫾3*†
25⫾4†
20⫾16
7⫾5
Data show EMax and EC50 of ACh-induced dilations observed in vessels mounted in a wire
(isometric) or a pressure myograph (isobaric).
*P⬍0.05 vs control condition.
†P⬍0.05 vs control mice (wt or Cxfl).
‡P⬍0.05 vs isometric condition.
4
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In vivo
}
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vivo in anesthetized mice. Wt arteries (n⫽6; diameter:
195⫾21 ␮m) were cannulated and preconstricted (NE,
1 ␮mol/L) at an intraluminal pressure of 60 mm Hg, which
led to a diameter of 160⫾12 ␮m. From this level, arteries
dilated concentration dependently to ACh (189⫾9 ␮m at
1 ␮mol/L), achieving almost complete dilation at 3 ␮mol/L.
In contrast to isometric conditions, these responses were not
attenuated by LN/Indo (from 162⫾11 to 193⫾7 ␮m at
1 ␮mol/L; Figure 3A), emphasizing the contribution of
EDHF at these conditions. Cx40⫺/⫺ arteries (n⫽6; diameter:
181⫾26 ␮ m) dilated from the preconstricted state
(144⫾12 ␮m) likewise to ACh (to 177⫾11 ␮m at 1 ␮mol/L)
and attained at these isobaric conditions also a near fully
relaxed state at 3 ␮mol/L. Most interestingly, dilations
remained unaffected after LN/Indo in Cx40⫺/⫺ arteries (Figure 3A; 1 ␮mol/L: from 146⫾9 to 176⫾12 ␮m), indicating
the presence of a powerful EDHF that acts independent of
Cx40 at these isobaric conditions. Preconstriction with depolarizing K⫹ solution in the presence of LN/Indo prevented
dilations in both genotypes (Figure 3A).
Gracilis arteries studied in vivo exhibited maximal diameters
of 135⫾19 ␮m in wt mice (n⫽3) and 117⫾16 ␮m in Cx40⫺/⫺
mice (n⫽9). To mimic the in vitro setting, NE (1 ␮mol/L) was
applied. In this setting, ACh dilated wt (1 ␮mol/L: from 58⫾9
to 104⫾20 ␮m) and Cx40⫺/⫺ vessels (1 ␮mol/L: from 43⫾4 to
101⫾7 ␮m) before and after LN/Indo (Figure 3B). Differences
were not found at any condition revealing a similar EDHF-type
dilation in both genotypes. Thus, dilations in vivo were similar
to the situation in vitro during isobaric conditions. The EDHFtype dilation was also unaltered in Cx40⫺/⫺ mice in vivo in the
absence of blood flow and in the presence of pressure achieved
by a downstream ligation (Figure 3B) or after removal of the
ligature and restoration of blood flow (data not shown). These
experiments highlight that EDHF-type dilations are independent
of Cx40 and blood flow in vivo in these small arteries.
Endothelial Cx40 Is Required Under
Isometric Conditions
The specific contribution of endothelial Cx40 was studied
using animals in which Cx40 was selectively deleted therein
using the Cre/lox system (Cx40fl:TIE2-Cre). The successful
deletion of Cx40 in endothelial cells was verified by immu-
Figure 3. EDHF-mediated dilations are independent of Cx40 during isobaric conditions in
vitro and in vivo. ACh initiates a concentrationdependent dilation in preconstricted isolated
small arteries studied using pressure myography in wt and Cx40⫺/⫺ mice (A, each n⫽6).
Vessels dilated maximally in the absence (control) as well as in the presence of LN/Indo but
not after K⫹ preconstriction. Differences
between genotypes were not detected. Similarly, Cx40 is not required for EDHF-mediated
dilations in the A. gracilis in vivo. ACh dilated
NA-preconstricted small arteries in anesthetized
wt (n⫽3) and Cx40⫺/⫺ mice (B, n⫽12) fully
before and after LN/Indo. Differences between
genotypes were not detected. Interruption of
blood flow by a temporary ligation of the vessel
downstream from the observation site did not
alter EDHF-type dilations in Cx40⫺/⫺ mice (B,
presence of LN/Indo; n⫽4).
nohistochemistry (Figure S5). In fact, in Cx40fl:TIE2-Cre
animals, staining was not detected, suggesting that Cx40 is
near exclusively expressed in the endothelium. In small
arteries obtained from control mice carrying a floxed Cx40
gene (Cx40fl; n⫽5) that were studied isometrically and
preconstricted using 3 ␮mol/L of NE (from 0.45⫾0.03 to
1.33⫾0.13 mN/mm), ACh induced a concentrationdependent relaxation (1 ␮mol/L to 0.69⫾0.07 mN/mm) that
remained unaffected by LN/Indo (1 ␮mol/L from 1.53⫾0.21
to 0.74⫾0.06 mN/mm, EMax: Table 1). Although AChinduced relaxations were intact in Cx40fl:TIE2-Cre vessels in
the presence of NO and prostaglandins, they were nearly
abrogated after LN/Indo (Figure 4, EMax: Table 1; 1 ␮mol/L
of ACh: Control: from 1.23⫾0.17 to 0.73⫾0.05 mN/mm;
LN/Indo: from 1.37⫾0.24 to 1.23⫾0.22 mN/mm). However,
SNP-induced relaxations were similar in both genotypes and
not attenuated by LN/Indo (before: 0.1 ␮mol/L, 41⫾7%
versus 42⫾11%; 10 ␮mol/L, 95⫾2% versus 94⫾5%; after:
0.1 ␮mol/L, 47⫾8% versus 47⫾9%; 10 ␮mol/L, 97⫾2%
versus 96⫾2%, Cx40fl and Cx40fl:TIE2-Cre, respectively).
Thus, endothelial cell-specific deletion of Cx40 results in a
loss of EDHF-type dilations during isometric conditions
similar to Cx40⫺/⫺ animals.
However, if Cx40fl:TIE2-Cre vessels were mounted isobarically and similarly preconstricted, ACh-induced dilations
were virtually independent of endothelial Cx40 and of a
larger magnitude compared with isometrically mounted vessels (Figure 4, EMax and EC50: Table 1). In nontreated vessels
and in the presence of LN/Indo, ACh dilated Cx40fl as well as
Cx40fl:TIE2-Cre small arteries to a fully relaxed state without
detectable differences (Figure 4). SNP-induced dilations were
also similar in both genotypes (data not shown). Therefore,
the dilations to ACh displayed a similar sensitivity toward
LN/Indo in both Cx40-deficient genotypes, global or endothelial-specific deletion, namely, an attenuation only during
isometric conditions.
Discussion
The present data demonstrate the importance of an EDHFtype dilation in response to endothelial stimulation using ACh
in small arteries in mice. Most importantly, we identified
distinct EDHF-type mechanisms. In vivo, as well as after
Boettcher and de Wit
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Distinct EDHF-Type Dilations In Vitro and In Vivo
-7
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ACh [log mol/L]
fl
Cx40 : TIE2-Cre (LN/Indo)
isometric
isobaric
*
60
40
20
Figure 4. EDHF-mediated dilations require
endothelial Cx40 in small arteries under isometric conditions but not during isobaric
conditions. ACh relaxed small arteries isolated from control mice with a floxed Cx40
gene (A and C, Cx40fl, n⫽6) or mice with
endothelial-cell specific Cx40 deletion (B
and D, Cx40fl:TIE2-Cre, n⫽7 to 8) under
isometric (filled) or isobaric conditions
(open) in untreated preparations (A and B)
or after LN/Indo (C and D). Dilations during
isobaric conditions were enhanced compared with isometric conditions in both genotypes and not attenuated by LN/Indo,
indicating powerful EDHF-type dilations in
isobarically mounted vessels, which were
fully preserved in animals lacking endothelial Cx40 (D). In contrast, the EDHF-type
dilation was nearly abrogated in Cx40fl:
TIE2-Cre (D) but present in control mice (C)
during isometric conditions, suggesting distinct EDHFs. At least n⫽6 for each genotype and condition, *P⬍0.05 for Emax.
0
-8
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isolation and isobaric examination of the gracilis artery, an
EDHF elicits a powerful dilation that is independent of Cx40.
In marked contrast, a secondary (and weaker) EDHF-type
dilation is uncovered under isometric conditions that requires
the presence of endothelial Cx40, because it is nearly absent
in mice lacking Cx40 globally or specifically in endothelial
cells. Thus, we conclude that myoendothelial gap junctions
containing Cx40 are crucially important in EDHF-type dilations, however, only in arteries studied isometrically because
of the lack of a more potent EDHF. This potent EDHF-type
dilation is Cx40 independent and prevails in the same vessels
examined in vivo.
In the conducting artery, ACh-induced dilations were
mainly mediated by NO and prostaglandins, similar to other
conducting vessels.2,3,31,32 However, also in mice with a
genetically disrupted NO pathway, a pronounced dilation
remains in smaller arteries and most prominently in arterioles,
suggesting a strong impact of EDHF-type responses.4 In line
with these findings, the dilation was less reduced by inhibition of NO and prostaglandins in the gracilis artery in the
present study. This remaining dilation could be abrogated by
preventing a K⫹-dependent hyperpolarization (presence of
high K⫹), exemplifying an EDHF-type dilation.
Recently, EDHF-type dilations have been attributed to
heterocellular current transfer through myoendothelial gap
junctions depending on Cx40 and/or Cx37.6,17,23,33 The pres-
ent study reveals striking differences in EDHF-type dilations
at different experimental conditions. In an isolated small
artery studied isobarically, as well as in the same vessel in
vivo, EDHF-type dilations were most prominent and virtually
independent of Cx40, because responses were identical in all
of the genotypes. In marked contrast, EDHF-type dilations
were nearly abrogated in Cx40-deficient vessels during isometric conditions, whereas only a minor attenuation was
observed after LN/Indo in wt mice verifying EDHF-type
dilations during this condition. Thus, this EDHF-type dilation
obviously requires Cx40. Interestingly, this “isometric”
EDHF-type dilation was also abrogated in animals exhibiting
an endothelial cell–specific Cx40 deficiency, suggesting that
Cx40 expressed in endothelial cells supports isometric
EDHF-type dilations. Because these latter mice are normotensive,34 the defect in EDHF-type dilation is not caused by
the hypertension that is seen in global Cx40 deficiency26 but
is truly related to the lack of Cx40 in endothelial cells. We
demonstrated previously that the role of Cx40 in conducted
dilations cannot be taken over by Cx45 in the microcirculation.30 This also holds true for the need of Cx40 in mediating
isometric EDHF dilations, because isometric EDHF cannot
be retrieved in mice carrying a replacement of Cx40 by Cx45,
which demonstrates that Cx45 cannot compensate functionally for the lack of Cx40 herein.
The differing dependency of EDHF dilations on Cx40
suggests that, in fact, 2 distinct EDHFs can be discriminated,
6
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“isobaric” and isometric EDHF. This is further supported by
the greater efficacy of EDHF during isobaric conditions
eliciting larger dilations than isometric EDHF. The enhanced
efficacy can be observed in all Cx40-expressing mice (wt and
Cx40-floxed controls). Both EDHF-type dilations in fact
require hyperpolarization and K⫹-channel activation, because
they were abrogated in the presence of high K⫹. NO and
prostaglandins are less important in this small artery, and
isobaric EDHF can fully compensate for their lack, because
inhibition of NO synthase and cyclooxygenase did not reduce
ACh dilations at these conditions. In contrast, isometric
EDHF seems not to be of such overwhelming potency,
because NO and prostaglandin inhibition attenuated dilations
in wt mice under isometric conditions, suggesting that this
EDHF cannot compensate for the lack of NO. Conversely,
NO is also not able to replace the lack of isometric EDHF as
deduced from reduced dilations observed in untreated Cx40deficient arteries (Cx40⫺/⫺ and Cx40KI45) during isometric
conditions.
Isometric EDHF requires Cx40, suggesting a contribution
of myoendothelial coupling therein. Cx40 is mainly expressed in this small artery in endothelial cells, as shown by
its lack in Cx40fl:TIE2-Cre mice. Moreover, Cx40 seems not
to be expressed in smooth muscle, which suggests that the
myoendothelial junctions required for isometric EDHF are
composed of heterotypic gap junctions. Interestingly, Cx37
expression is strongly reduced in the aorta or even lacking in
arterioles in Cx40-deficient mice,35 and, thus, it is also
conceivable that Cx37 contributes to myoendothelial coupling. However, no matter which connexins actually form the
channel, the present experiments demonstrate that endothelial
Cx40 is required for the dilation mediated by isometric
EDHF. Thus, we suggest that, under certain circumstances,
direct signaling through myoendothelial gap junctions is
indeed a mechanism by which an EDHF-type response is
transferred between these cells (isometric EDHF). However,
this is not the main EDHF in mice in vitro during isobaric
conditions and in vivo. In these settings, a more powerful
isobaric EDHF takes center stage and is able to fully dilate the
arteries.
Why is the powerful isobaric EDHF lost during isometric
conditions? A key difference between isobaric and isometric
preparations is the change in wall tension during constriction
and dilation. In isometric conditions, wall tension increases
during constriction and decreases during dilation, whereas the
opposite is true during isobaric conditions. Such experimental
differences have been shown to modulate the sensitivity of rat
mesenteric arteries for vasoconstrictors.36 Modulatory effects
of experimental conditions have also been postulated for
EDHF-type dilations, such that distinct endothelial KCa channels hyperpolarize the endothelium. This also affected the
transfer of the hyperpolarization to the adjacent smooth
muscle, which relied either on myoendothelial coupling or the
Na⫹/K⫹ATPase defining distinct EDHF pathways.37 It is
currently unclear whether distinct KCa channels are keys for
isobaric or isometric EDHFs. However, in the skeletal muscle
microcirculation and in the carotid artery (isobaric conditions), KCa3.1 is crucial in EDHF-type responses.10,11 Thus,
one may speculate that KCa3.1 is important for isobaric
EDHF and is during isometric conditions unable to contribute
to isometric EDHF, as suggested.37 This leaves KCa2.3 as the
channel to provide the less powerful isometric EDHF, which
relies on Cx40-dependent myoendothelial gap junctions.
Most interestingly, in line with the present data, myoendothelial coupling was found to be associated with KCa2.3
activation.37
Perspectives
EDHF-type dilations may compose future therapeutic targets
in hypertension. We have revealed that separate EDHFs can
be distinguished by their dependency on Cx40 and experimental conditions. A primary, powerful EDHF prevails in
vivo independent of Cx40, whereas a secondary EDHF relies
on endothelial Cx40 and is only retrieved in the absence of
the primary EDHF. In our hands, this was observed studying
isolated vessels during isometric conditions, but other factors
related to wall tension or constriction may have similar
consequences. The need for Cx40 most likely reflects charge
transfer between vascular cells through myoendothelial gap
junctions. Distinct entities of EDHF provide a compelling
explanation for striking differences observed in vivo versus in
vitro with respect to EDHF-type dilations. Thus, experimenters need to define experimental conditions carefully to
separate distinct dilator pathways and to prevent missing a
powerful “primary EDHF.”
Acknowledgments
We thank Rita Meuer for excellent technical assistance and Toon van
Veen (University Medical Centre Utrecht, Utrecht, the Netherlands)
for generously providing mice carrying a floxed connexin40 gene.
Sources of Funding
This work was supported by Deutsche Forschungsgemeinschaft
(WI2071/2-1 to C.d.W.).
Disclosures
None.
References
1. Hwa JJ, Ghibaudi L, Williams P, Chatterjee M. Comparison of
acetylcholine-dependent relaxation in large and small arteries of rat mesenteric vascular bed. Am J Physiol. 1994;266:H952–H958.
2. Huang PL, Huang Z, Mashimo H, Bloch KD, Moskowitz MA, Bevan JA,
Fishman MC. Hypertension in mice lacking the gene for endothelial nitric
oxide synthase. Nature. 1995;377:239 –242.
3. Tomioka H, Hattori Y, Fukao M, Sato A, Liu M, Sakuma I, Kitabatake A,
Kanno M. Relaxation in different-sized rat blood vessels mediated by
endothelium-derived hyperpolarizing factor: importance of processes
mediating precontractions. J Vasc Res. 1999;36:311–320.
4. Koeppen M, Feil R, Siegl D, Feil S, Hofmann F, Pohl U, de Wit C.
cGMP-dependent protein kinase mediates NO- but not acetylcholineinduced dilations in resistance vessels in vivo. Hypertension. 2004;44:
952–955.
5. de Wit C, Wölfle SE. EDHF and gap junctions: important regulators of
vascular tone within the microcirculation. Curr Pharm Biotechnol. 2007;
8:11–25.
6. Griffith TM. Endothelium-dependent smooth muscle hyperpolarization:
do gap junctions provide a unifying hypothesis? Br J Pharmacol. 2004;
141:881–903.
7. Feletou M, Vanhoutte PM. EDHF: an update. Clin Sci (Lond). 2009;117:
139 –155.
8. de Wit C, Griffith TM. Connexins and gap junctions in the EDHF
phenomenon and conducted vasomotor responses. Pflugers Arch. 2010;
459:897–914.
Boettcher and de Wit
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
9. Crane GJ, Gallagher N, Dora KA, Garland CJ. Small- and intermediateconductance calcium-activated K⫹ channels provide different facets of
endothelium-dependent hyperpolarization in rat mesenteric artery.
J Physiol. 2003;553:183–189.
10. Wölfle SE, Schmidt VJ, Hoyer J, Köhler R, de Wit C. Prominent role of
KCa3.1 in endothelium-derived hyperpolarizing factor-type dilations and
conducted responses in the microcirculation in vivo. Cardiovasc Res.
2009;82:476 – 483.
11. Brähler S, Kaistha A, Schmidt VJ, Wölfle SE, Busch C, Kaistha BP,
Kacik M, Hasenau AL, Grgic I, Si H, Bond CT, Adelman JP, Wulff H,
de Wit C, Hoyer J, Köhler R. Genetic deficit of SK3 and IK1 channels
disrupts the endothelium-derived hyperpolarizing factor vasodilator
pathway and causes hypertension. Circulation. 2009;119:2323–2332.
12. Sandow SL, Tare M, Coleman HA, Hill CE, Parkington HC. Involvement
of myoendothelial gap junctions in the actions of endothelium-derived
hyperpolarizing factor. Circ Res. 2002;90:1108 –1113.
13. Sandow SL, Bramich NJ, Bandi HP, Rummery NM, Hill CE. Structure,
function, and endothelium-derived hyperpolarizing factor in the caudal
artery of the SHR and WKY rat. Arterioscler Thromb Vasc Biol. 2003;
23:822– 828.
14. Dora KA, Sandow SL, Gallagher NT, Takano H, Rummery NM, Hill CE,
Garland CJ. Myoendothelial gap junctions may provide the pathway for
EDHF in mouse mesenteric artery. J Vasc Res. 2003;40:480 – 490.
15. Sandow SL, Hill CE. Incidence of myoendothelial gap junctions in the
proximal and distal mesenteric arteries of the rat is suggestive of a role in
endothelium-derived hyperpolarizing factor-mediated responses. Circ
Res. 2000;86:341–346.
16. McSherry IN, Sandow SL, Campbell WB, Falck JR, Hill MA, Dora KA.
A role for heterocellular coupling and EETs in dilation of rat cremaster
arteries. Microcirculation. 2006;13:119 –130.
17. Haddock RE, Grayson TH, Brackenbury TD, Meaney KR, Neylon CB,
Sandow SL, Hill CE. Endothelial coordination of cerebral vasomotion via
myoendothelial gap junctions containing connexins 37 and 40. Am J
Physiol Heart Circ Physiol. 2006;291:H2047–H2056.
18. Sokoya EM, Burns AR, Setiawan CT, Coleman HA, Parkington HC, Tare
M. Evidence for the involvement of myoendothelial gap junctions in
EDHF-mediated relaxation in the rat middle cerebral artery. Am J Physiol
Heart Circ Physiol. 2006;291:H385–H393.
19. Emerson GG, Segal SS. Electrical coupling between endothelial cells and
smooth muscle cells in hamster feed arteries - role in vasomotor control.
Circ Res. 2000;87:474 – 479.
20. Coleman HA, Tare M, Parkington HC. K⫹ currents underlying the action
of endothelium-derived hyperpolarizing factor in guinea-pig, rat and
human blood vessels. J Physiol. 2001;531:359 –373.
21. Yamamoto Y, Klemm MF, Edwards FR, Suzuki H. Intercellular electrical
communication among smooth muscle and endothelial cells in guinea-pig
mesenteric arterioles. J Physiol. 2001;535:181–195.
22. Chaytor AT, Bakker LM, Edwards DH, Griffith TM. Connexin-mimetic
peptides dissociate electrotonic EDHF-type signalling via myoendothelial
and smooth muscle gap junctions in the rabbit iliac artery. Br J
Pharmacol. 2005;144:108 –114.
Distinct EDHF-Type Dilations In Vitro and In Vivo
7
23. Mather S, Dora KA, Sandow SL, Winter P, Garland CJ. Rapid endothelial
cell-selective loading of connexin 40 antibody blocks endotheliumderived hyperpolarizing factor dilation in rat small mesenteric arteries.
Circ Res. 2005;97:399 – 407.
24. de Wit C, Roos F, Bolz SS, Kirchhoff S, Krüger O, Willecke K, Pohl U.
Impaired conduction of vasodilation along arterioles in connexin40
deficient mice. Circ Res. 2000;86:649 – 655.
25. Milkau M, Köhler R, de Wit C. Crucial importance of the endothelial K⫹
channel SK3 and connexin40 in arteriolar dilations during skeletal muscle
contraction. FASEB J. 2010;24:3572–3579.
26. de Wit C, Roos F, Bolz SS, Pohl U. Lack of vascular connexin 40 is
associated with hypertension and irregular arteriolar vasomotion. Physiol
Genomics. 2003;13:169 –177.
27. Siegl D, Koeppen M, Wölfle SE, Pohl U, de Wit C. Myoendothelial
coupling is not prominent in arterioles within the mouse cremaster microcirculation in vivo. Circ Res. 2005;97:781–788.
28. Chadjichristos CE, Scheckenbach KE, van Veen TA, Richani Sarieddine
MZ, de Wit C, Yang Z, Roth I, Bacchetta M, Viswambharan H, Foglia B,
Dudez T, van Kempen MJ, Coenjaerts FE, Miquerol L, Deutsch U,
Jongsma HJ, Chanson M, Kwak BR. Endothelial-specific deletion of
Cx40 promotes atherosclerosis by increasing CD73-dependent leukocyte
adhesion. Circulation. 2010;121:123–131.
29. Kisanuki YY, Hammer RE, Miyazaki J, Williams SC, Richardson JA,
Yanagisawa M. Tie2-Cre transgenic mice: a new model for endothelial
cell-lineage analysis in vivo. Dev Biol. 2001;230:230 –242.
30. Wölfle SE, Schmidt VJ, Hoepfl B, Gebert A, Alcolea S, Gros D, de Wit
C. Connexin45 cannot replace the function of connexin40 in conducting
endothelium-dependent dilations along arterioles. Circ Res. 2007;101:
1292–1299.
31. Sausbier M, Schubert R, Voigt V, Hirneiss C, Pfeifer A, Korth M,
Kleppisch T, Ruth P, Hofmann F. Mechanisms of NO/cGMP-dependent
vasorelaxation. Circ Res. 2000;87:825– 830.
32. Friebe A, Mergia E, Dangel O, Lange A, Koesling D. Fatal gastrointestinal obstruction and hypertension in mice lacking nitric oxidesensitive guanylyl cyclase. Proc Natl Acad Sci U S A. 2007;104:
7699 –7704.
33. Isakson BE, Duling BR. Heterocellular contact at the myoendothelial
junction influences gap junction organization. Circ Res. 2005;97:44 –51.
34. Wagner C, Jobs A, Schweda F, Kurtz L, Kurt B, Lopez MLS, Gomez RA,
van Veen TA, de Wit C, Kurtz A. Selective deletion of Connexin 40 in
renin-producing cells impairs renal baroreceptor function and is associated with arterial hypertension. Kidney Int. 2010;78:762–768.
35. de Wit C. Different pathways with distinct properties conduct dilations in
the microcirculation in vivo. Cardiovasc Res. 2010;85:604 – 613.
36. Buus NH, VanBavel E, Mulvany MJ. Differences in sensitivity of rat
mesenteric small arteries to agonists when studied as ring preparations or
as cannulated preparations. Br J Pharmacol. 1994;112:579 –587.
37. Dora KA, Gallagher NT, McNeish A, Garland CJ. Modulation of endothelial cell KCa3.1 channels during endothelium-derived hyperpolarizing
factor signaling in mesenteric resistance arteries. Circ Res. 2008;102:
1247–1255.
Distinct Endothelium-Derived Hyperpolarizing Factors Emerge In Vitro and In Vivo and
Are Mediated in Part via Connexin 40−Dependent Myoendothelial Coupling
Markus Boettcher and Cor de Wit
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Online Supplement
Distinct EDHFs emerge in vitro and in vivo and are mediated in part
via connexin40-dependent myoendothelial coupling
Markus Boettcher, Cor de Wit
Institut für Physiologie, Universität zu Lübeck, Lübeck, Germany
Address for correspondence:
Cor de Wit, MD PhD
Physiologisches Institut
Universität zu Lübeck
Ratzeburger Allee 160
23538 Lübeck, Germany
Telephone
+49-451-5004170
Telefax
+49-451-5004171
E-mail [email protected]
-2Methods
Animals:
All experiments were performed in accordance with the German animal protection law.
Relaxations of conducting (CA; A. femoralis) and small arteries (SA; A. gracilis) in wildtype
(wt, C57/BL6), Cx40-/-,1 Cx40KI45,2 Cx40fl and Cx40fl:TIE2-Cre were examined. The gene
encoding for Cx40 is globally lacking in Cx40-/- whereas it is replaced by Cx45 in Cx40KI45.
In Cx40fl, it is flanked by loxP-sites which was used to generate endothelial cell specific
Cx40-deficiency using a Cre-recombinase driven by the TIE2 promoter (Cx40 fl:TIE2-Cre).3,4
Mice were genotyped by PCR from tail tip biopsies using primers as described previously. 1-4
Preparations:
Mice were anesthetized by intraperitoneal injection of medetomidine (0.5mg/kg), midazolam
(5mg/kg) and fentanyl (0.05mg/kg). Anesthetized mice were killed and CA and SA were
isolated carefully for in vitro studies using wire and pressure myography. Ring segments were
mounted in a wire-myograph (510A Danish Myo Technology, Aarhus, Denmark; wire: 25µm
SA, 40µm CA) and normalized to a pressure of 60 mmHg (SA) or 80 mmHg (CA) after
equilibration to 37°C for 30min. Force was recorded at a sampling-rate of 2Hz (Fig.1A) using
software written in our laboratory. SA were mounted in a pressure myograph (in-house
development; adapted to an inverse microscope (Olympus IMT2, Hamburg, Germany)) and
pressurized to 60 mmHg. Outer diameter was measured automatically at a sampling-rate of
2Hz in images obtained with a CCD-camera (Kappa CF8/1, Kappa Optronics GmbH,
Gleichen, Germany) by software written in our laboratory (Fig.1). The organ bath contained
physiological salt solution (PSS) of the following composition (mmol/L): 119 NaCl, 4.7 KCl,
1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 25 NaHCO3, 0.03 EDTA and 5.5 glucose (gassed: 5%
CO2 balance O2; pH 7.4). Vessels which constricted only minimally (<0.15mN; <5µm) upon
norepinephrine (1 µmol/L) or did not dilate upon acetylcholine were discarded. Calcium-free
solution was applied in vessels studied under isobaric conditions to obtain the vessels
maximal diameter. In additional experiments, the A. gracilis (SA) was examined in vivo.
Therefore, the gracilis muscle was carefully freed of skin and moisturized with PSS
immediately. Mice were transferred onto the stage of a reflected light microscope equipped
with a CCD-camera adapted to an USB-frame-grabber (Dazzle DVC 90; Pinnacle Systems,
California) which allowed visualisation of the vessel in vivo. Image series of the SA were
taken before and 30, 60, and 90 s after application of vasoactive substances and diameters
were measured using AQuaL offline.5
Protocol and statistical analysis:
Vessels were preconstricted using norepinephrine (NE, 1µmol/L or 3µmol/L) or potassium
(KCl, 50mmol/L) and subsequently relaxations were induced by applying acetylcholine
(ACh) or sodium-nitroprusside (SNP) at increasing concentrations in a cumulative manner.
EDHF-type dilations were examined after incubation with L-Nitro-arginine (LN, 300µmol/L)
and indomethacin (indo, 3µmol/L) for >20min to block NO-synthase and cyclooxygenase.
Dilations were normalized to the initial increase in force (or decrease in diameter) upon
vasoconstriction. Maximal possible effect (EMax) and the concentration that produces a half
maximal effect (EC50) were calculated by nonlinear regression as described.6 All data are
given as mean±SEM. Comparisons within groups were performed using paired t-test and
between groups using ANOVA followed by a Bonferroni correction. Differences were
considered significant at a corrected error probability of P<0.05.
-3Immunostaining:
Cx40 expression was visualized in small arteries isolated from wt, Cx40 -/-, Cx40fl and
Cx40fl:TIE2-Cre mice by immunohistochemistry. Whole vessels were mounted onto
glasspipettes, tied with suture (11-0 Ethilon; Ethicon, germany), fixed using formaldehyde
(4.5%, 10min) or histochoice (20min; Amresco, Solon, USA), rinsed in PBS, and incubated in
blocking solution (1% BSA, 0.2% Triton-X; 2h). The primary antibody (1:400; antiCx40,
Chemicon, AB1726) was applied over night at 4°C in blocking solution. Vessels were rinsed
again by exchanging the bath solution and by perfusing the vessel before incubation with the
secondary antibody (1:800 for 1h; Alexa Fluor 594; invitrogen, USA). Small amounts of the
applied solutions were also luminally perfused through the cannulation pipette. Arteries were
transferred to microscope slides and examined using confocal microscopy (Leica TCS SP5,
Wetzlar, Germany). Every genotype was examined at least in triplicate. Immunostaining was
not detected in the absence of the primary antibody (not shown).
References
1. Kirchhoff S, Nelles E, Hagendorff A, Kruger O, Traub O, Willecke K. Reduced cardiac
conduction velocity and predisposition to arrhythmias in connexin40-deficient mice. Curr
Biol. 1998;8:299-302.
2. Alcolea S, Jarry-Guichard T, de Bakker J, Gonzalez D, Lamers W, Coppen S, Barrio L,
Jongsma H, Gros D, van Rijen H. Replacement of connexin40 by connexin45 in the
mouse: impact on cardiac electrical conduction. Circ Res. 2004;94:100-109.
3. Chadjichristos CE, Scheckenbach KE, van Veen TA, Richani Sarieddine MZ, de Wit C,
Yang Z, Roth I, Bacchetta M, Viswambharan H, Foglia B, Dudez T, van Kempen MJ,
Coenjaerts FE, Miquerol L, Deutsch U, Jongsma HJ, Chanson M, Kwak BR. Endothelialspecific deletion of Cx40 promotes atherosclerosis by increasing CD73-dependent
leukocyte adhesion. Circulation. 2010;121:123-131.
4. Kisanuki YY, Hammer RE, Miyazaki J, Williams SC, Richardson JA, Yanagisawa M.
Tie2-Cre transgenic mice: A new model for endothelial cell-lineage analysis in vivo. Dev
Biol. 2001;230:230-242.
5. Boettcher M, Gloe T, de Wit C. Semiautomatic quantification of angiogenesis. J Surg
Res. 2010;162:132-139.
6. de Wit C, von Bismarck P, Pohl U. Synergistic action of vasodilators that increase cGMP
and cAMP in the hamster cremaster microcirculation. Cardiovasc Res. 1994;28:15131518.
-4Supplemental Figures
Figure S1
A
B
ACh
diameter [µm]
force [mN]
2.0
1.5
1.0
NE
180
NE
170
ACh
160
150
0.5
140
0
200
400
600
time [s]
800 1000
0
400
800
1200
time [s]
Sample traces of preconstriction using norepinephrine (NE) and concentration-dependent
dilation in response to acetylcholine (ACh) in untreated small arteries from wt mice mounted
isometrically (A) or isobarically (B). Under isometric conditions constriction/relaxation is
detected as an increase/decrease in force whereas in isobaric conditions vessel diameter is
measured.
-5-
Figure S2
A
100
80
Relaxation [%]
B
CA (A. femoralis)
Control
LN/Indo
+ KCl
SA (A. gracilis)
*
Control
LN/Indo
+ KCl
*
*
#
60
40
#
20
#
#
#
#
*
#
#
#
#
#
#
#
0
-8
-7
-6
SNP [log mol/L]
-5
-8
-7
-6
-5
SNP [log mol/L]
Endothelium-independent dilations in response to the NO-donor sodium-nitroprusside (SNP)
in conducting arteries (CA, A. femoralis) and small arteries (SA, A. gracilis). SNP induced
concentration-dependent dilations in preconstricted isometrically mounted CA (A, n=10-14)
and SA (B, n=13-16) from wt mice that were not reduced in the presence of L-NA and
indomethacin (LN/Indo, 300 and 3 µmol/L). However, in the presence of high potassium
solution (+KCl, 50 mmol/L) dilations were attenuated in both vessels. *: P<0.05 LN/Indo vs.
control, #: P<0.05 KCl vs. LN/Indo, paired t-test.
-6-
Figure S3
Connexin40 (Cx40) is expressed in small arteries of wt mice (A) as judged by
immunohistochemistry in whole-mounted vessels. The specificity of the antibody is verified
by the lack of staining in Cx40-/- (C). Cell borders are brightly stained only at the inner edge
of the vessel (arrows; B,D: bright field images) suggesting that endothelial cells express Cx40
which is located in their membranes. Bar is 20 µm.
-7-
Figure S4
A
100
80
Relaxation [%]
B
Control
LN/Indo
wt
Cx40-/Cx40KI45
wt
Cx40-/Cx40KI45
*
60
40
*
*
*
*
*
*
20
0
-8
-7
-6
SNP [log mol/L]
-5
-8
-7
-6
-5
SNP [log mol/L]
Dilations in response to SNP in preconstricted SA isolated from Cx40 -/- (n=12) as well as in
vessels expressing Cx45 instead of Cx40 (Cx40KI45, n=9) in the wire myograph under
control conditions (A) and after LN/Indo (B). Data for wildtype vessels are replotted from
supplemental figure 2 for comparison. Endothelium-independent dilations were not attenuated
in Cx40-/- and only slightly reduced at the highest concentrations in Cx40KI45 mice before
(A) and after LN/Indo (B). *: P<0.05 vs. wt.
-8-
Figure S5
Cx40 is expressed in control mice carrying a floxed Cx40 gene (Cx40 fl, A-C) but lacking in animals
that express additionally a Cre-recombinase driven by the endothelial promotor TIE2 (Cx40 fl:TIE2Cre, D-E) verifying successful deletion of Cx40 in endothelial cells. In these animals staining cannot
be detected in smooth muscle cells suggesting that Cx40 is near exclusively expressed in the
endothelium. Brightfield (A, D) and fluorescence images (B, C, E, F) are shown, the inner edge of the
vessel (endothelium) is labelled by arrows. Bar is 20 µm.