Results
Identification of KV7 channels within MA ECs
Initial investigation sought to identify KV7.4 and
KV7.5 channel transcript and protein within MA ECs, as
these subtypes are implicated in vascular reactivity (Barrese et
al , 2018). Transcript levels for EC and VSMC markers were determined
in cell lysates from MA with and without EC removal by mechanical
abrasion (see Methods). In cell suspensions from EC-denuded vessels
there was a reduction in EC markers (Pecam/Vwbf ) and an increase
in VSMC markers (Acta2/Myh11 ) compared to lysates from unabraded
MA (Figure 1). This was associated with a 1.8- and 2.1-fold decrease in
relative transcript abundance of Kcnq4 and Kcnq5, (Figure
1) showing that a considerable component of these transcripts were
derived from EC cells. This data suggests expression of Kcnq4 andKcnq5 within EC marker expressing cells (Figure 1).
KV7.4 was KV7.5 was detected in isolated
ECs by immunodetection (Figure 2A,C). KV7.4 had a
punctate distribution in isolated ECs (Figure 2A) whereas
KV7.5 label appeared to be predominantly cytoplasmic
with some diffuse label around the nucleus (Figure 2C). Similar to
previous reports (Zhong et al. , 2010; Oliveras et al. ,
2014; Mills et al. , 2015; Morales-Cano et al. , 2015,
Barrese et al., 2018) KV7.4 and 7.5 were identified in
isolated MA VSMCs (Figure 2B,D). Notably, KV7.4 and
KV7.5 were also detected in both ECs and VSMCs inen face whole-mount tissue (Figure 2.E-H). KV7.4
was also detected in EC by immunoelectron microscopy (supplemental
figure 1). Further, both KV7.4 and 7.5 were expressed at
a proportion of IEL hole sites at an apparently higher level than the
associated EC membrane label (Figure 3).
Removal of ECs modulates KV7.2-5 activator efficacy
A comprehensive pharmacological analysis was undertaken to determine if
KV7 channels have a functional role in MA ECs. Effects
of KV7 channel modulators were examined in MAs where the
endothelium was intact or had been removed. S-1 and ML213 are
structurally dissimilar activators of KV7.2-7.5 that
interact with the same pharmacophore centered around a tryptophan in the
S5 domain (Bentzen et al., 2006; Lyubov I. Brueggemann, Haick, Cribbs,
& Byron, 2014; T. A. Jepps et al., 2014; Schenzer, 2005). ML277 is a
potent activator of KV7.1 (Yu, 2013) with a 100-fold
increase in selectivity for KV7.1 compared to
KV7.2-5 (Yu, 2013). Consistent with previous findings
(Chadha et al., 2012; T. A. Jepps et al., 2014), S-1- and ML213-mediated
vasorelaxation was ablated by pre-incubation with 10
µmol-L-1 pan-KV7 channel inhibitor
linopirdine (Schnee & Brown, 1998); Figure 4.A,B). Relaxations produced
by 10 to 300 nmol-L-1 ML277 were also prevented by
incubation with linopirdine (Figure 4C). However, relaxations produced
by concentrations >1 µmol-L-1 ML277 were
not attenuated by linopirdine and are therefore not mediated by Kv7.1
activation.
EC removal for the following experiments was confirmed by ablation of
vasorelaxation in response to 10 µmol-L-1 CCh (Figure
5A). Removing the endothelium by mechanical abrasion has no impact on
the peak contraction produced by 300 nmol-L-1 U46619
(Figure 4B), but significantly attenuated the potency of S-1 mediated
vasorelaxation increasing EC50 from 2±0.2
µmol-L-1 to 3±0.7 µmol-L-1 (Figure
5C,D). The potency of ML213 was also impaired by endothelial removal
(EC(+) EC50 = 1±0.2 µmol-L-1 vs EC(-)
EC50 = 3±0.2 µmol-L-1; Figure 5E). The
linopirdine-sensitive relaxation produced by ML277 was not affected by
endothelial removal (Figure 5F). The accumulated findings reveal that
ECs express KV7.4 and KV7.5 and the
presence of the endothelium enhances the sensitivity of two structurally
different KV7.2-7.5 activators.
IKCa/SKCa inhibitors have no impact on
KV7 activator mediated relaxation
Having identified that the endothelium modulates responses to Kv7
activators, experiments were performed to identify the mechanism/s
involved. Endothelial IKCa and SKCachannels contribute to relaxation responses in rat MA (Crane, Gallagher,
Dora, & Garland, 2003). Thus, it is feasible that KV7
channels interact with other key endothelial potassium channels;
particularly in microdomains (Sandow et al., 2009). However, in
agreement with previous reports (T. A. Jepps, Olesen, Greenwood, &
Dalsgaard, 2016), pre-incubation with a combination of
IKCa inhibitor TRAM-34 (1 µmol-L-1;
(Wulff et al., 2000) and SKCa inhibitor apamin (100
nmol-L-1; (Spoerri, Jentsch, & Glees, 1975) had no
effect on KV7 activator mediated vasorelaxation (Figure
6A-C).
EC Kir channels modulate KV7.2-5
activator sensitivity
Within MAs of a EC-KIR2.1-/- murine model,
endothelial KCNJ2-encoded KIR2.1 channels have been
identified as ‘signal boosters’ that enhance EC-derived relaxation
(Sonkusare et al., 2016). Comparatively, the current literature
regarding rat mesenteric KIR2 channels is
limited; with KIR2.1 expression being demonstrated in EC
of whole mount rat MA (Dora, Gallagher, McNeish, & Garland, 2008),
where inwardly rectifying Ba2+ sensitive channels are
apparently restricted to the endothelial layer (Crane, Walker, Dora, &
Garland, 2003), and KIR channels contribute to
acetylcholine-mediated responses (Goto et al., 2004). We propose that
like mice, rat mesenteric ECs express functional KIR2
channels that propagate EC signals in a similar process. Therefore, we
performed a series of studies investigating the effect of two well
characterized KIR2 blockers, BaCl2(Hagiwara, Miyazaki, Moody, & Patlak, 1978) and ML133 (Wang et al.,
2011; Wu et al., 2010) on KV7 activator-mediated
vasorelaxation.
In arteries with a functional endothelium, KIR2
blockers, BaCl (100 µmol-L-1) and ML133 (20
µmol-L-1), significantly impaired vasorelaxation by
S-1 (Figure 7A, EC50= DMSO, 1.89±0.2
µmol-L-1 / BaCl, 2.3±0.31 µmol-L-1;
Figure 7B, EC50= DMSO, 0.52±0.12
µmol-L-1 / ML133, 3.1±1.5 µmol-L-1,
and ML213, Figure 7C,D, EC50= DMSO, 0.9±0.3
µmol/L-1 / BaCl, 2.2±0.5 µmol/L-1 /
ML133, 2.5±0.25 µmol-L-1) when compared to DMSO
solvent control (Figure 7A-D). No attenuation of the response to ML277
was observed consistent with EC removal data. In arteries where the
endothelium had been removed neither ML133 nor BaCl had any effect on
ML213 mediated vasorelaxation (Figure 7G,H). These data show that the
endothelium-dependent increase in potency to the KV7
activators involves endothelial KIR channels, but not
IKCa or SKCa channels.
KV7 channels contribute to CCh evoked vasorelaxation
The expression of functional KV7 channels within ECs
begs the question - Do they contribute to EC-derived responses?
Acetylcholine produces endothelium-dependent relaxations through NO-,
EDH- and prostanoid-dependent mechanisms in rat MA (Parsons, Hill,
Waldron, Plane, & Garland, 1994; Peredo, Feleder, & Adler-Graschinsky,
1997; Shimokawa et al., 1996).
A distinct rightward shift in the sensitivity to vasorelaxation in
response to CCh, a synthetic acetylcholine analogue, was produced by 100
µmol-L-1 eNOS inhibitor L-NAME when compared to DMSO
solvent control (EC50 DMSO = 0.59±0.1
µmol-L-1; L-NAME = 0.94±0.1
µmol-L-1; Figure 8A). A combination
IKCa and SKCainhibitors, 1 µmol-L-1 TRAM34 and 100
nmol-L-1 apamin, respectively produced greater
attenuation (EC50 TRAM34/apamin=1.5±0.7
µmol-L-1; Figure 8A). These results show that both NO-
and EDH-dependent signaling contributes to CCh-mediated vasodilation,
though the main contributor to endothelial-dependent vasodilation in
2nd order MA appears to be EDH, conferring with
previously published data (Shimokawa et al., 1996). Pre-incubating
vessels with 10 µmol-L-1 pan-KV7
channel inhibitor linopirdine significantly attenuated CCh-mediated
vasorelaxation when compared to DMSO control (EC50 DMSO
= 0.2±0.08 µmol-L-1; linopirdine = 0.7±0.3
µmol-L-1; Figure 8B). In contrast, pre-incubating
vessels with either the KV7.1 specific inhibitor
HMR-1556 (10 µmol-L-1; Figure 8C) or a combination of
non-specific KV channel inhibitors TEA (1
mmol-L-1; (Choi, Aldricht, Yellen, & Hughes, 1991)
and 4-AP (1 mmol-L-1 ; (Kurata & Fedida, 2006) had no
significant effect on CCh-evoked vasorelaxation. These results imply a
specific contribution of KV7.4 and KV7.5
channels to CCh-evoked relaxation in 2nd order rat MA.
Subsequent experiments explored whether linopirdine affected CCh-induced
relaxations in the presence of L-NAME compared to responses in
apamin/TRAM34. CCh-evoked relaxations were significantly attenuated in
vessels pre-incubated in TRAM34/apamin and linopirdine compared to
vessels only pre-incubated in TRAM34/apamin alone (EC50DMSO = 0.24±0.05 µmol-L-1; TRAM34/Apamin = 0.27±0.03
µmol-L-1; TRAM34/Apamin + linopirdine = 0.61±0.2
µmol-L-1; Figure 8E). In contrast, linopirdine failed
to attenuate CCh relaxations in arteries pre-incubated with L-NAME
(Figure 8F). These data suggest that KV7.4 and
KV7.5 channels contribute to the NO pathway of
CCh-mediated vasorelaxation.
KV7 channels contribute to NO-mediated vasorelaxation in
a vascular bed specific manner (Jennifer B. Stott et al., 2015). The
present data demonstrate that pre-incubation with the
pan-KV7 channel blocker linopirdine (10
µmol-L-1) has no effect on vasorelaxation produced by
the NO-donor s-nitroprusside (SNP; Figure 8G). However, in contrast with
previous reports (T. A. Jepps et al., 2016), pre-incubation with L-NAME
(100 µmol-L-1) significantly attenuated
KV7.2-5 activator mediated vasorelaxation (Figure 8H).
These data suggest that the NO axis of CCh-mediated vasorelaxation is
linopirdine sensitive, upstream of NO signaling.
Discussion
The present study identified Kcnq4 and Kcnq5 transcripts
within EC marker expressing cells as well as respective
KV7.4 and KV7.5 protein in isolated and
whole mount rat MA ECs/VSMCs, consistent with previous studies in the
endothelium (Chen, Li, Hiett, & Obukhov, 2016) and vascular smooth
muscle (e.g Zhong et al. , 2010; Oliveras et al. , 2014;
Mills et al. , 2015, summarised in Barrese et al ., 2018).
Functionally, the present study demonstrates that the relaxation
produced by two structurally different KV7.2-5
activators, but not a KV7.1 activator, were modulated by
the endothelium. These relaxations were also sensitive to
KIR2 inhibition and suggest a novel functional
interaction between KV7 channels and endothelial
KIR2.x channels. Furthermore, we have demonstrated that
KV7.4/7.5 channels contribute to the NO-mediated axis of
CCh-evoked endothelium-dependent relaxations. Thus, KV7
channels are expressed within ECs, contribute to endothelium-derived
responses and when pharmacologically upregulated, are functionally
coupled to other EC potassium channels.
KV7 channel expression and
function within
ECs
Within smooth muscle, when active, KV7 channels
hyperpolarize the membrane potential decreasing voltage-gated calcium
channel (VGCC) open probability, therefore decreasing the influx of
extracellular calcium, with this decrease resulting in relaxation of the
cell. Within rodent models, the pharmacopeia of KV7
channel modulators has revealed KV7.4 and
KV7.5 channels; 1. contribute to resting vascular tone
via regulation of the resting membrane potential; 2. are upregulated
during cGMP and cAMP/EPAC/PKA-mediated vasodilation, and; 3. are
supressed via PKC-mediated vasoconstriction. Conjunctly, the use of
molecular techniques has revealed that
KV7.4/KV7.5 is the most predominant
heterotetramer expressed within specific rodent arteries. Comparatively,
no functional role for KV7.1 in arteries has been
identified (see (Byron & Brueggemann, 2018; Barrese et al ,
2018), for review). A caveat of these observations is a lack of
differentiation between VSMCs and ECs. However, KV7
channels were recently identified in pig coronary artery ECs (Chen et
al., 2016) where they modulate bradykinin-evoked endothelium-derived
vasorelaxation (Chen et al., 2016). The data presented here demonstrate
KV7 channel expression in rat mesenteric endothelium and
support their functional role.
In most arteries, the endothelium and smooth muscle are
electrochemically linked via MEGJs formed from connexin proteins within
heterocellular communicating microdomains (see (Sandow et al., 2012) for
review). Via these connections, it has been purported that current
injection into ECs passes into VSMCs (Sandow, Tare, Coleman, Hill, &
Parkington, 2002). As KV7 channels are expressed within
rat mesenteric ECs, the present data suggest a potential role for this
process in KV7 activator-mediated vasorelaxation. This
conjecture is supported by a significant attenuation of vasorelaxation
of pre-constricted arterial tone by two structurally different
KV7.2-5 activators in the absence of ECs. In contrast,
EC removal had no impact on KV7.1 activator-mediated
vasorelaxation, suggesting that only KV7.4 and
KV7.5 channels are functionally expressed within MA ECs.
However, Chen et al, (2016) suggest that endothelial removal had
no impact on ML213-mediated relaxation in pig coronary artery segments.
Consequently, these collective data suggest that this phenomenon is both
animal and artery bed specific, potentially being dependent on the
molecular architecture of the vessel, such as MEGJ and related
microdomain properties.
A novel functional interaction with
endothelial KIRchannels
KIR2.x channels have been characterized in a variety of
rat vascular beds including cerebral and coronary arteries (Smith et
al., 2008), where their selective inhibition by Ba2+revealed KIR channel amplification of a
K+ channel activator conductance (Smith et al., 2008).
However, there is a degree of conflict regarding the role of
KIR2.x channels within rat MAs. As above, a
Ba2+ sensitive current and KIR2.x
expression has been demonstrated in rat MA ECs (Crane, Walker, et al.,
2003; Smith et al., 2008). Furthermore, within primary MA
KIR2.x channels were purported to contribute to
KCa mediated-hyperpolarization during ACh-derived
EC-dependent responses (Goto et al., 2004). In contrast, Smith et
al. , (2008) demonstrate that within 3rd order MA
ACh-mediated K+ conductance-dependent vasorelaxation
was insensitive to Ba2+, indicating that
KIR2.x channels do not augment K+conductance in these vessels during receptor-mediated vasodilation.
In the present study, significant attenuation of both S-1 and ML213
KV7.2-5 activator-mediated vasorelaxation was found
after pre-incubation in two structurally different
KIR2.x blockers ML133 (Wu et al., 2010) and
Ba2+ (Hagiwara et al., 1978). ML133 has been
identified via high-throughput and mutagenesis investigation as a novel
inhibitor of KIR2.1 channels with an
IC50 of 1.8 μmol-L-1 at pH 7.4, with
little to no selectivity against the other members of the
KIR2.x family (Wang et al., 2011; Wu et al., 2010) where
it exerts its activity via D172 and I176 within the M2 region of
KIR2.1 (Wang et al., 2011). Presently, ML133 is the most
selective inhibitor of the KIR2 family. In parallel to
the endothelium denudation experiments, no effect was seen on the
KV7.1 activator ML277-mediated vasorelaxation; implying
a specific interplay with KV7.4 and
KV7.5 channels. Furthermore, KIR2
blockers had no effect on KV7.2-7.5 activator-mediated
relaxation in EC-denuded arteries, supporting the notion that functional
KIR2 channels (in the rat MA bed) are restricted to the
endothelium (Crane, Walker, et al., 2003), and the agents do not inhibit
KV7 channels per se . The present study suggests
that pharmacological activation of endothelial KV7.4/7.5
channels also activates an endothelial KIR2 channel
increased K+ conductance, which in turn accounts for a
significant degree of the EC augmentation of KV7.2-5
activator-mediated vasorelaxation (Figure 11). However, based on the
findings described by Goto et al,. (2004) and Smith et
al,. (2008), the collective data suggest that this phenomenon is
dependent on the branch order of MA. Furthermore, it remains unclear if
this occurs during receptor mediated signaling, or that is only present
during pharmacological activation of endothelial KV7
channels.
A primary concern for identification of novel functional interactions
between ion channels using pharmacological tools is off-target effects.
However, KV7 activator-mediated vasorelaxation in
vessels pre-incubated in 10 µmol-L-1 linopirdine was
abolished. If S-1 or ML213 were activating other channels, i.e. e.g.
KIR channels, a degree of vasorelaxation would still be
observed in the presence of linopirdine. The present findings therefore
suggest that both S-1 and ML213 work exclusively via KV7
channels and that a novel functional coupling of
KV7.4/7.5 and KIR2 occurs in rat MA ECs.
The contribution of KV7
channels to CCh evoked relaxations within rat
MA
KV7 channels contribute to cGMP mediated vasorelaxation
in a vascular bed specific manner (Stott et al., 2015). Within rat
aortic, renal and pulmonary arteries, overexpression models, and native
VSMCs KV7.4/KV7.5 have been identified
as downstream targets of cGMP signaling during both isometric tension
recording and whole cell current recording (Mondéjar-Parreño et al.,
2019; Jennifer B. Stott et al., 2015). Conversely, KV7
channels do not contribute to NO donor SNP-mediated vasorelaxation
within rat renal arteries (Jennifer B. Stott et al., 2015).
In light of the significant impact of KV7 inhibition on
CCh-mediated vasorelaxation during the suppression of EDH, the present
study suggests that KV7 channels contribute to
CCh-evoked NO-cGMP mediated relaxation within rat MA. Though, similar to
renal arteries, KV7 channels do not represent downstream
targets of NO signaling in rat MA as KV7 channel
inhibition does not impair SNP-mediated relaxations. However, eNOS
inhibition does impair relaxation to a KV7 activator,
implying that KV7 channels are involved in the
production or release of NO in response to CCh. Though this appears to
be a vascular bed specific phenomenon, as L-NAME significantly impairs
ML213 relaxations in pig coronary artery (Chen et al., 2016), but not
rat penile artery (T. A. Jepps et al., 2016).
Conclusions
In conclusion, the present data identify a novel functional interaction
between mesenteric endothelial KV7.4/7.5 and
KIR2 channels and supports the proposition that
endothelial KV7 channels contribute to endogenous
endothelial-derived responses. These findings highlight the complex
nature of the vascular response to KV7 channel
upregulation and emphasize the importance of KV7
channels to vascular signaling cascades. The present data are consistent
with KV7 channels representing a novel therapeutic
target in endothelial dysfunction.