# Extracellular Calcium-Dependent Modulation of Endothelium Relaxation in Rat Mesenteric Small Artery: The Role of Potassium Signaling

Authors: Lise Hangaard; Peter B. Jessen; Dmitrii Kamaev; Christian Aalkjaer; Vladimir V. Matchkov
Journal: BioMed Research International
 (2015)

---

## Abstract

The nature of NO- and COX-independent endothelial hyperpolarization (EDH) is not fully understood but activation of small- and intermittent-conductance Ca2+-activated K+ channels (SK
Ca and IK
Ca) is important. Previous studies have suggested that the significance of IK
Ca depends on Ca
2
+
out. Also it has been suggested that K+ is important through localized K
+
out signaling causing activation of the Na+,K+-ATPase and inward-rectifying K+ channels (K
ir). Here we tested the hypothesis that the modulating effect of Ca
2
+
out on the EDH-like response depends on K
+
out. We addressed this possibility using isometric myography of rat mesenteric small arteries. When K
+
out was 4.2 mM, relaxation to acetylcholine (ACh) was stronger at 2.5 mM Ca
2
+
out than at 1 mM Ca
2
+
out. Inhibition of IK
Ca with TRAM34 suppressed the relaxations but did not change the relation between the relaxations at the low and high Ca
2
+
out. This Ca
2
+
out-dependence disappeared at 5.9 mM K
+
out and in the presence of ouabain or BaCl2. Our results suggest that IK
Ca are involved in the localized K
+
out signaling which acts through the Na+,K+-ATPase and K
ir channels and that the significance of this endothelium-dependent pathway is modulated by Ca
2
+
out.


---

## Body

## 1. Introduction

The importance of the arterial endothelium for controlling vascular resistance is well-established [1]. Several factors are released from the endothelium and relax the adjacent smooth muscle cells. In addition to NO and prostaglandins, the endothelium is able to produce vasodilatation through a third pathway, which is particularly important in small arteries and arterioles [2]. The third pathway is the endothelium-dependent hyperpolarization (EDH) of smooth muscles, which is present after the inhibition of NO and prostaglandin production [3, 4]. The mechanisms proposed for EDH are direct spreading of hyperpolarizing current via myoendothelial gap junctions or release of diffusible factors(s) (EDHF(s)). The relative contribution of these two pathways and the nature of the EDHFs vary among species and vascular beds, blood vessel caliber, ageing, and diseases, as well as between different experimental conditions and laboratories [3, 5]. It is unlikely that a single factor accounts for EDHF and multiple diffusible factors, including K
+, NO, HNO, epoxyeicosatrienoic acids, H2S, H2O2, and vasoactive peptides, have been suggested.A key element in the EDH pathway supported by most studies is activation of endothelial small- and intermittent-conductanceC
a
2
+-activated K
+ channels (S
K
C
a and I
K
C
a, resp.) upon agonist- or shear stress-induced increase of endothelial cell calcium (C
a
2
+
i
n) [6]. Although activation of both S
K
C
a and I
K
C
a channels can lead to vasodilation, their contribution to the EDH is different [3–5]. S
K
C
a channels are distributed homogeneously over the endothelial cell membrane and respond to increase of C
a
2
+
i
n [7, 8]. Hyperpolarization produced by the S
K
C
a channels is believed to spread via myoendothelial gap junctions to smooth muscle cells. This original hypothesis is, however, modified [5, 9] based on the observation that S
K
C
a-dependent hyperpolarization can be blocked not only by apamin, a S
K
C
a channel inhibitor, but also by B
a
2
+ in concentrations specifically blocking the inward-rectifying K
+ channels (K
i
r) [10]. It has been suggested that K
+ efflux through the opened S
K
C
a channels might increase the local K
+
o
u
t and consequently open K
i
r channels. In this model the localized K
+
o
u
t increase amplifies endothelial hyperpolarization generated by S
K
C
a channel opening [5].In contrast, theI
K
C
a channels are localized in endothelial cell projections near the adjacent smooth muscle cells [11] where K
+ efflux through these channels increases K
+
o
u
t by about 6 mM [12], which has been called a “K
+ cloud” [13]. The “K
+ cloud” may hyperpolarize smooth muscle cells via activation of the N
a
+,K
+-ATPase and K
i
r channels [11, 14]. This hypothesis is supported by ouabain- and B
a
2
+-sensitive hyperpolarization of smooth muscles in endothelium-denuded arteries with few mM elevation of K
+
o
u
t [12]. Both these K
+ sensors, that is, the Na,K-ATPase and the K
i
r channels, are expressed in smooth muscle cells [11, 15] although their importance varies between vascular beds [5].How endothelial excitation leads to differentiated activation ofS
K
C
a
/
I
K
C
a channels remains unclear but this may be modulated by C
a
2
+
o
u
t via the C
a
2
+-sensing receptor (CaSR) [16]. Thus, changes in C
a
2
+
o
u
t may switch the EDH signaling between being predominantly S
K
C
a-/myoendothelial gap junction-dependent and being I
K
C
a-/“K
+ cloud” dependent pathways.Based on these considerations we hypothesized that modulation of the EDH-like response byC
a
2
+
o
u
t is dependent on K
+
o
u
t and tested the importance of I
K
C
a, N
a
+,K
+-ATPase, and K
i
r channels for these effects of C
a
2
+
o
u
t and K
+
o
u
t.


## 2. Methods

All experiments were approved by and conducted with permission from the Animal Experiments Inspectorate of the Danish Ministry of Food, Agriculture and Fisheries. Rats were euthanized byC
O
2-inhalation.In vitro functional experiments were performed on rat mesenteric small artery. Rat mesentery was dissected and placed in ice-cold physiological salt solution (PSS). Third-order branches of the rat mesenteric small artery were dissected. The cleaned arterial segments were mounted in an isometric wire myograph (Danish Myo Technology A/S, Denmark) as described previously [17]. The myograph chamber was heated to 37°C, while the PSS was constantly aerated with 5% CO2 in air. Force was recorded with a PowerLab 4/25-Chart7 acquisition system (ADInstruments Ltd., New Zealand) and converted to wall tension by dividing force with double segment length. Contractile concentration-response relationships were constructed by cumulative noradrenaline concentrations (NA: 0.1–30 μM). The relaxation concentration-response relationships were constructed by cumulative addition of acetylcholine (ACh) (0.01–10 μM) to the arteries preconstricted with 6 μM noradrenaline. A maximum of three concentration-response relaxations were made on one artery.The 4.2 mM[
K
+
]-containing PSS composition was (in mM) 119.00 NaCl, 3.0 KCl, 1.18 KH2PO4, 1.17 MgCl2, 25.0 NaHCO3, 0.026 EDTA, and 5.5 glucose, gassed with 5% CO2 in air and adjusted to pH 7.4. The 5.9 mM [
K
+
]-containing PSS composition was (in mM) 119.00 NaCl, 4.7 KCl, 1.18 KH2PO4, 1.17 MgSO4, 25.0 NaHCO3, 0.026 EDTA, and 5.5 glucose, gassed with 5% CO2 in air and adjusted to pH 7.4. Either 1 mM or 2.5 mM CaCl2 was added to the PSS as indicated. All chemicals were obtained from Sigma-Aldrich (Brondby, Denmark). Drugs were applied 15 minutes before experiment.Statistical analyses were performed using GraphPad Prism 5 (Graph Pad Software Inc., USA). Data are expressed as mean values ± SEM. Concentration-response curves were fitted to experimental data using four-parameter, nonlinear regression curve fitting. From these curves, pD2 (the concentration required to produce a half-maximal response) and maximal response were derived and compared using an extra sum-of-squares F test. t-test was used where appropriate. P
<
0.05 was considered significant. n refers to number of rats.


## 3. Results

The sensitivities (pD2) and maximal responses to noradrenaline were the same with all four experimental solutions, that is, 1 mM and 2.5 mM C
a
2
+
o
u
t and 4.2 mM or 5.9 mM K
+
o
u
t (Figure 1(a)). Thus, the preconstriction levels in relaxation experiments were the same in all experimental conditions. Inhibition of the I
K
C
a channels with 10 μM TRAM34 did not affect the concentration-response relationship to noradrenaline (Figure 1(b)). Neither pD2 nor maximal contractile responses were significantly affected by TRAM34.Figure 1
Concentration-dependent contractions to noradrenaline (NA) of rat mesenteric small arteries were not different in the presence of 1 mM and 2.5 mMC
a
2
+
o
u
t. The contractions were also the same in solutions with 4.2 mM K
+
o
u
t and with 5.9 mM K
+
o
u
t. Preincubation with 10 μM TRAM34 was without significant effect on the contractile responses ((a) versus (b)). n
=
6.
(a)
(b)Arteries preconstricted with 6μM noradrenaline were relaxed with cumulative addition of ACh (Figure 2). The preconstriction remained stable in time-control experiments where only vehicle was applied (Figures 2(a) and 2(b)).Figure 2
The concentration-dependent relaxation to ACh in the bath solutions containing either 4.2 mM ((a)n
=
8) or 5.9 mM ((b) n
=
6) K
+
o
u
t. In the time-control experiments only the vehicle was supplied (n
=
2
–
4). Responses were compared under control conditions and after preincubation with 100 μM L-NAME and 3 μM indomethacin. (c) and (d) show the areas under curve (AUC) for the concentration-responses shown in (a) and (b). ++ and +++ indicate P
<
0.01 and 0.001 for responses in the presence of 1 mM C
a
2
+
o
u
t versus 2.5 mM C
a
2
+
o
u
t. ∗
∗ and ∗
∗
∗ indicate P
<
0.01 and 0.001 for responses before and after application of L-NAME and indomethacin.
(a)
(b)
(c)
(d)In the 4.2 mMK
+
o
u
t containing solution the relaxation was more pronounced when C
a
2
+
o
u
t was 2.5 mM in comparison with 1 mM C
a
2
+
o
u
t (Figures 2(a) and 2(c)). This was associated with a higher sensitivity to ACh, pD2
7.16
±
0.07 versus 6.45
±
0.09 (P
<
0.01, n
=
8). When K
+
o
u
t in the bath solution was elevated to 5.9 mM, no difference between concentration-response curves at 1 mM and 2.5 mM C
a
2
+
o
u
t was observed (Figures 2(b) and 2(d)); pD2 were 7.06
±
0.08 and 7.23
±
0.05 (n
=
6), respectively.Preincubation of arteries with 100μM L-NAME and 3 μM indomethacin significantly suppressed the relaxation to ACh in the presence of both 4.2 mM and 5.9 mM K
+
o
u
t. Importantly, these inhibitors shifted the concentration-response curves to the right (Figure 2). However, when the bath solution contained 4.2 mM K
+
o
u
t the relaxation was still more pronounced in the presence of 2.5 mM compared to 1 mM C
a
2
+
o
u
t (Figures 2(a) and 2(c)). When K
+
o
u
t was 5.9 mM, no difference in the relaxations at different C
a
2
+
o
u
t was seen (Figures 2(b) and 2(d)).Repeated relaxations to cumulative addition of ACh in the presence of L-NAME and indomethacin were similar (Figure3), demonstrating that there was no time-dependent effect on relaxation to ACh.Figure 3
Repeated ACh concentration-relaxation curves in the presence of 100μM L-NAME and 3 μM indomethacin (n
=
5). Relaxations were first performed in the presence of either 1 mM or 2.5 mM C
a
2
+
o
u
t; the bath solution was then replaced with either 2.5 mM or 1 mM C
a
2
+
o
u
t. This protocol was repeated once more. ∗ indicates P
<
0.05 for 1 mM versus 2.5 mM C
a
2
+
o
u
t for the 1st stimulations; ++ indicates P
<
0.05 for 1 mM versus 2.5 mM C
a
2
+
o
u
t for the 2nd stimulations.To assess the importance ofI
K
C
a for the effect of C
a
2
+
o
u
t we repeated the experiments in the presence of 1 μM TRAM34. TRAM34 suppressed the relaxations to ACh in the presence of L-NAME and indomethacin (Figures 4(a) and 4(b)). In the 4.2 mM K
+
o
u
t containing solution the relaxations in the presence of TRAM34 were still stronger at 2.5 mM than at 1 mM C
a
2
+
o
u
t. When K
+
o
u
t was 5.9 mM no difference in relaxations at 1 mM and 2.5 mM C
a
2
+
o
u
t was seen in the presence of TRAM34 (Figure 4(c)).Figure 4
The concentration-dependent relaxation to ACh in the bath solutions containing 4.2 mM ((a)n
=
8) and 5.9 mM ((b) n
=
8) K
+
o
u
t. Responses were compared after preincubation with 100 μM L-NAME and 3 μM indomethacin (grey curves) and after preincubation with 100 μM L-NAME, 3 μM indomethacin, and 1 μM TRAM34 (black curves). (c) shows the areas under curve (AUC) for the concentration-response curves shown in (a) and (b). (d) shows a shift in sensitivities to ACh (pD2) after addition of TRAM34 in the experiments shown in (a) and (b). (e) shows changes in the maximal relaxations (to 10 μM ACh) for the experiments shown in (a) and (b). +, ++, and +++ indicate P
<
0.05, 0.01, and 0.001 for responses in the presence of 1 mM versus 2.5 mM C
a
2
+
o
u
t. ∗, ∗
∗, and ∗
∗
∗ indicate P
<
0.05, 0.01, and 0.001 for responses before and after incubation with TRAM34.
(a)
(b)
(c)
(d)
(e)In spite of similar changes in the areas under the curve, TRAM34 affected the concentration-response curves differently at 1 mM and 2.5 mMC
a
2
+
o
u
t when K
+
o
u
t was 4.2 mM. TRAM34 reduced the sensitivity to ACh at 2.5 mM C
a
2
+
o
u
t (Figure 4(d)) but suppressed the maximal relaxation at 1 mM C
a
2
+
o
u
t (Figure 4(e)).At 5.9 mMK
+
o
u
t TRAM34 had the same effect on pD2 to ACh in the presence of 1 mM and 2.5 mM C
a
2
+
o
u
t (Figure 4(d)). No effect of TRAM34 on maximal relaxation was seen in the presence of 5.9 mM K
+
o
u
t (Figure 4(e)).Preincubation of arteries with L-NAME, indomethacin, TRAM34, and apamin completely inhibited the ACh-dependent relaxations independently ofK
+
o
u
t and C
a
2
+
o
u
t (not shown, n
=
6
–
8).To assess the importance of theN
a
+,K
+-ATPase for the effect of C
a
2
+
o
u
t we repeated the experiments in the presence of 10 μM ouabain. Ouabain suppressed the ACh-induced relaxation in the presence of 100 μM L-NAME and 3 μM indomethacin (Figure 5). Ouabain suppressed the relaxation more in the presence of 4.2 mM K
+
o
u
t than in the presence of 5.9 mM K
+
o
u
t (Figure 5(c)). In the presence of ouabain there was no difference between the areas under curves in the presence of 1 mM and 2.5 mM C
a
2
+
o
u
t at any K
+
o
u
t. Importantly, the effects of ouabain on the sensitivity to ACh and the maximal relaxations were independent of C
a
2
+
o
u
t (Figures 5(d) and 5(e)). However, ouabain suppressed the maximal relaxations at 4.2 mM K
+
o
u
t more than in the presence of 5.9 mM K
+
o
u
t (Figure 5(e)).Figure 5
Incubation with 10μM ouabain in the presence of 100 μM L-NAME and 3 μM indomethacin suppressed ACh-dependent relaxations. The concentration-response curves were constructed for the experiments in the bath solutions containing 4.2 mM ((a) n
=
8) and 5.9 mM ((b) n
=
8) K
+
o
u
t. (c) shows the areas under curves (AUC) from (a) and (b). The changes in sensitivities to ACh (pD2) before and after incubation are shown in (d). The changes in maximal relaxations (to 10 μM ACh) before and after the treatment with ouabain are shown in (e). ∗
∗ and ∗
∗
∗ indicate P
<
0.01 and 0.001 for the responses before and after incubation with ouabain. ## and ### indicate P
<
0.01 and 0.001 for 4.2 mM versus 5.9 mM K
+
o
u
t.
(a)
(b)
(c)
(d)
(e)To assess the importance ofK
i
r for the effect of C
a
2
+
o
u
t we repeated the experiments in the presence of 30 μM BaCl2. BaCl2 inhibited the ACh-dependent relaxation only when the bath solution contained 4.2 mM K
+
o
u
t (Figure 6(a)). In the presence of BaCl2 no differences between the areas under the curve at 1 mM and 2.5 mM C
a
2
+
o
u
t were seen (Figure 6(c)). No effect of BaCl2 on the relaxations at 5.9 mM K
+
o
u
t was seen.Figure 6
Addition of 30μM BaCl2 to the solution containing 100 μM L-NAME and 3 μM indomethacin suppressed ACh-dependent relaxations. The concentration-response curves were constructed in the bath solutions with 4.2 mM ((a) n
=
6) and with 5.9 mM ((b) n
=
6) K
+
o
u
t. (c) shows the areas under curves (AUC) as in (a) and (b). Changes in the sensitivities to ACh (pD2) after addition of BaCl2 are shown in (d). The effects of BaCl2 on the maximal relaxations are shown in (e). ∗
∗ indicates P
<
0.01 versus 1 mM C
a
2
+
o
u
t.
(a)
(b)
(c)
(d)
(e)The effect of BaCl2 at 4.2 mM K
+
o
u
t was dependent on C
a
2
+
o
u
t. BaCl2 suppressed the sensitivity to ACh only when C
a
2
+
o
u
t was 2.5 mM (Figure 6(d)). In contrast, the maximal relaxations were similarly suppressed in the presence of both 1 mM and 2.5 mM C
a
2
+
o
u
t (Figure 6(e)).


## 4. Discussion

We have studied theC
a
2
+
o
u
t-dependent modulation of EDH-like signaling and the role of I
K
C
a and the K
+
o
u
t sensors, that is, the N
a
+,K
+-ATPase and K
i
r channels, for this modulation.


### 4.1. [Ca2+]out and the IKCa Channels

We found that an elevation ofC
a
2
+
o
u
t from 1 to 2.5 mM increases the relaxation to ACh. This was also seen after the blockade of NO- and prostaglandin-dependent pathways. This is in accordance with the previous observations where wall tension and membrane potential were measured [11]. We did not measure membrane potential, but the sensitivity of NO- and prostaglandin-independent relaxation to TRAM34 and apamin suggests that this effect was mediated via an EDH-like pathway. The effects of two relatively low (1 mM) and high (2.5 mM) C
a
2
+
o
u
t concentrations were compared, because I
K
C
a channel-dependent relaxation has previously been shown to differ strikingly at these two concentrations of C
a
2
+ [11].Although the highC
a
2
+
i
n in activated endothelial cells activates both S
K
C
a and I
K
C
a channels, the relative importance of S
K
C
a and I
K
C
a has been suggested to depend on C
a
2
+
o
u
t in the myoendothelial space [7, 8]. It has been suggested that C
a
2
+
o
u
t is sensed by the G-protein-coupled CaSR [18] which can modulate the I
K
C
a channel-dependent EDH in the vascular wall [11, 16, 19]. It was therefore tempting to speculate that the effect of C
a
2
+
o
u
t changes seen in the present study could be explained by changes in the I
K
C
a activity. However, after inhibition of I
K
C
a with TRAM34 there was still a modulating effect of C
a
2
+
o
u
t. This suggests that the modulatory role of C
a
2
+
o
u
t cannot be limited to an effect on the I
K
C
a channels.Part of theC
a
2
+ effect may, however, be via the I
K
C
a channels since TRAM34 affected the ACh concentration-response curves differently at the two C
a
2
+
o
u
t concentrations. This contrasts with a previous report [11] where TRAM34 eliminated the modulating effect of C
a
2
+
o
u
t on ACh relaxations. However, the actual values obtained in the previous study [11] were rather similar to those found in the present study; in the presence of TRAM34 the maximal relaxation tended to be less with 1 mM compared to 2.5 mM C
a
2
+
o
u
t [11]. Thus, the effect of C
a
2
+
o
u
t-CaSR signaling on the I
K
C
a channels likely explains a part of the effect of C
a
2
+
o
u
t on EDH, possibly via modulation of C
a
2
+
i
n [20, 21].


### 4.2. [Ca2+]out and the Na+,K+-ATPse/Kir Channels

Simultaneous inhibition ofS
K
C
a channels with apamin and I
K
C
a channels with TRAM34 resulted in complete inhibition of endothelium-dependent relaxation. In contrast to TRAM34, the inhibitory effect of apamin was larger at 2.5 mM C
a
2
+
o
u
t. It is therefore possible that C
a
2
+
o
u
t may also modify the activity of the S
K
C
a channels. Another possibility is that C
a
2
+
o
u
t has an effect downstream for activation of the S
K
C
a
/
I
K
C
a channels.A downstream effect ofC
a
2
+
o
u
t might include an effect on the two K
+ sensors, the ouabain-sensitive Na+,K+-ATPase and K
i
r channels, which are also known to be involved in EDH signaling [5]. To address this possibility we assessed the effect of inhibition of the N
a
+,K
+-ATPase with ouabain [11, 22] and the effect of inhibition of the K
i
r channels with B
a
2
+ [23, 24]. Both these interventions reduced the relaxation to ACh as expected. Importantly, in the presence of both ouabain and B
a
2
+
C
a
2
+
o
u
t had no effect on the ACh response. This might suggest that the effect of C
a
2
+
o
u
t is mediated via the Na,K-ATPase and K
i
r channels.Interestingly, inhibition of either theN
a
+,K
+-ATPase or K
i
r channels caused more than 50% inhibition of the relaxation at 4.2 mM K
+
o
u
t. If these K
+ sensors act strictly in parallel, this is surprising. A possibility is that these transporters interact functionally and support the activity of each other. Thus, it has previously been shown that the N
a
+,K
+-ATPase associates functionally with some K
+ channels, for example, the ATP-dependent K
+ channels (K
A
T
P) [25]. K
+ ions leaving the cell through the K
+ channels have been shown to supply the N
a
+,K
+-ATPase with K
+ even in K
+
o
u
t-free media [26], while the N
a
+,K
+-ATPase activity provides a gradient for the ionic current through the K+ channels [25]. A similar functional interaction has previously been reported in the heart [27] and pancreas [28]. It is possible that the N
a
+,K
+-ATPase and the K
i
r channels may also interact functionally to short-circuit the membrane K
+ transport.Both theN
a
+,K
+-ATPase and K
i
r channels are expressed in the smooth muscle cells, although they may also be present in the endothelial cells [11, 19, 29]. The effect of C
a
2
+
o
u
t might therefore be mediated via either smooth muscle or endothelial cells. Some studies reported that the effect of B
a
2
+ is endothelium-dependent [14, 30–33]. These endothelial K
i
r channels can still be modulated by a “K
+ cloud” thereby amplifying endothelial hyperpolarization which then spreads through the myoendothelial gap junctions. The present study was performed on endothelium-intact arteries making it impossible to distinguish the functional localization of the N
a
+,K
+-ATPase and K
i
r channels. But regardless of their localization our results indicate that the N
a
+,K
+-ATPase or K
i
r channels or both are modulated by C
a
2
+
o
u
t and that they act as sensors for K
+
o
u
t. The localized K
+
o
u
t can act either as EDHF (in case of smooth muscle cell localization of the K
+ sensors) or by amplifying the endothelial hyperpolarization which then spreads through the myoendothelial gap junctions (in case of endothelial localization of the K
+ sensors).


### 4.3. The Localized [K+]out Signaling

The importance ofC
a
2
+
o
u
t was studied at two concentrations of K
+
o
u
t. We chose values close to physiological values for rats based on the observation that increase in K
+
o
u
t to 5.9 mM induces BaCl2-sensitive relaxation while relaxation to K
+
o
u
t above 5.9 mM is diminishing [13, 19, 30, 34]. In addition, it has been suggested that the Na+,K+-ATPase in the vasculature is fully saturated at 5.9 mM K
+
o
u
t [24, 35]. This suggests that any relaxation in the presence of 5.9 mM K
+
o
u
t is unlikely to be caused by an increase of K
+
o
u
t.This suggestion was supported by our observation that in the presence of 5.9 mMK
+
o
u
t ouabain and particularly B
a
2
+ had a little effect on the relaxation, while at 4.2 mM K
+
o
u
t pronounced effects of these inhibitors were seen. This could be because the elevation of K
+
o
u
t to 5.9 mM saturates the N
a
+,K
+-ATPase and K
i
r channels in agonist preconstricted arteries [19]. In fact, the “K
+ cloud” can be generated not only by the endothelium but also via K
+ efflux from big-conductance C
a
2
+-activated K
+ channels which are activated in depolarized smooth muscle cells of agonist preconstricted arteries [14]. It has previously been shown that, under experimental conditions where the K
+-sensors are saturated, inhibition of the Na,K-pump and K
i
r channels has no effect on relaxation [14]. This indicates that the EDH under these conditions spreads through other mechanisms, possibly as a hyperpolarizing current via myoendothelial gap junctions.Consistent with this, the modulating effect ofC
a
2
+
o
u
t seen at 4.2 mM K
+
o
u
t was abolished at 5.9 mM K
+
o
u
t. This finding is further consistent with our suggestion that the modulatory function of C
a
2
+
o
u
t is mediated largely via the Na,K-pump and K
i
r channels. Finally, the lack of effect of TRAM34 at 5.9 mM K
+
o
u
t is consistent with the hypothesis [11] that the importance of the I
K
C
a channels for the EDH is associated with a localized increase in K
+
o
u
t [12] which acts through the Na,K-pump and K
i
r channels.While no effect ofB
a
2
+ was seen at 5.9 mM K
+
o
u
t, a small but significant inhibition of the relaxation was observed in the presence of ouabain. If at this K
+
o
u
t the Na+,K+-ATPase is completely saturated as suggested [24, 35], it is possible that the observed effect of ouabain was mediated via modulation of gap junctions. It has previously been shown that inhibition of the Na+,K+-ATPase suppresses intercellular communications, including myoendothelial gap junctions [22, 26, 36, 37]. Thus, part of the ouabain effect can be related to inhibition of EDH spreading to smooth muscles through the myoendothelial gap junctions.


## 4.1. [Ca2+]out and the IKCa Channels

We found that an elevation ofC
a
2
+
o
u
t from 1 to 2.5 mM increases the relaxation to ACh. This was also seen after the blockade of NO- and prostaglandin-dependent pathways. This is in accordance with the previous observations where wall tension and membrane potential were measured [11]. We did not measure membrane potential, but the sensitivity of NO- and prostaglandin-independent relaxation to TRAM34 and apamin suggests that this effect was mediated via an EDH-like pathway. The effects of two relatively low (1 mM) and high (2.5 mM) C
a
2
+
o
u
t concentrations were compared, because I
K
C
a channel-dependent relaxation has previously been shown to differ strikingly at these two concentrations of C
a
2
+ [11].Although the highC
a
2
+
i
n in activated endothelial cells activates both S
K
C
a and I
K
C
a channels, the relative importance of S
K
C
a and I
K
C
a has been suggested to depend on C
a
2
+
o
u
t in the myoendothelial space [7, 8]. It has been suggested that C
a
2
+
o
u
t is sensed by the G-protein-coupled CaSR [18] which can modulate the I
K
C
a channel-dependent EDH in the vascular wall [11, 16, 19]. It was therefore tempting to speculate that the effect of C
a
2
+
o
u
t changes seen in the present study could be explained by changes in the I
K
C
a activity. However, after inhibition of I
K
C
a with TRAM34 there was still a modulating effect of C
a
2
+
o
u
t. This suggests that the modulatory role of C
a
2
+
o
u
t cannot be limited to an effect on the I
K
C
a channels.Part of theC
a
2
+ effect may, however, be via the I
K
C
a channels since TRAM34 affected the ACh concentration-response curves differently at the two C
a
2
+
o
u
t concentrations. This contrasts with a previous report [11] where TRAM34 eliminated the modulating effect of C
a
2
+
o
u
t on ACh relaxations. However, the actual values obtained in the previous study [11] were rather similar to those found in the present study; in the presence of TRAM34 the maximal relaxation tended to be less with 1 mM compared to 2.5 mM C
a
2
+
o
u
t [11]. Thus, the effect of C
a
2
+
o
u
t-CaSR signaling on the I
K
C
a channels likely explains a part of the effect of C
a
2
+
o
u
t on EDH, possibly via modulation of C
a
2
+
i
n [20, 21].


## 4.2. [Ca2+]out and the Na+,K+-ATPse/Kir Channels

Simultaneous inhibition ofS
K
C
a channels with apamin and I
K
C
a channels with TRAM34 resulted in complete inhibition of endothelium-dependent relaxation. In contrast to TRAM34, the inhibitory effect of apamin was larger at 2.5 mM C
a
2
+
o
u
t. It is therefore possible that C
a
2
+
o
u
t may also modify the activity of the S
K
C
a channels. Another possibility is that C
a
2
+
o
u
t has an effect downstream for activation of the S
K
C
a
/
I
K
C
a channels.A downstream effect ofC
a
2
+
o
u
t might include an effect on the two K
+ sensors, the ouabain-sensitive Na+,K+-ATPase and K
i
r channels, which are also known to be involved in EDH signaling [5]. To address this possibility we assessed the effect of inhibition of the N
a
+,K
+-ATPase with ouabain [11, 22] and the effect of inhibition of the K
i
r channels with B
a
2
+ [23, 24]. Both these interventions reduced the relaxation to ACh as expected. Importantly, in the presence of both ouabain and B
a
2
+
C
a
2
+
o
u
t had no effect on the ACh response. This might suggest that the effect of C
a
2
+
o
u
t is mediated via the Na,K-ATPase and K
i
r channels.Interestingly, inhibition of either theN
a
+,K
+-ATPase or K
i
r channels caused more than 50% inhibition of the relaxation at 4.2 mM K
+
o
u
t. If these K
+ sensors act strictly in parallel, this is surprising. A possibility is that these transporters interact functionally and support the activity of each other. Thus, it has previously been shown that the N
a
+,K
+-ATPase associates functionally with some K
+ channels, for example, the ATP-dependent K
+ channels (K
A
T
P) [25]. K
+ ions leaving the cell through the K
+ channels have been shown to supply the N
a
+,K
+-ATPase with K
+ even in K
+
o
u
t-free media [26], while the N
a
+,K
+-ATPase activity provides a gradient for the ionic current through the K+ channels [25]. A similar functional interaction has previously been reported in the heart [27] and pancreas [28]. It is possible that the N
a
+,K
+-ATPase and the K
i
r channels may also interact functionally to short-circuit the membrane K
+ transport.Both theN
a
+,K
+-ATPase and K
i
r channels are expressed in the smooth muscle cells, although they may also be present in the endothelial cells [11, 19, 29]. The effect of C
a
2
+
o
u
t might therefore be mediated via either smooth muscle or endothelial cells. Some studies reported that the effect of B
a
2
+ is endothelium-dependent [14, 30–33]. These endothelial K
i
r channels can still be modulated by a “K
+ cloud” thereby amplifying endothelial hyperpolarization which then spreads through the myoendothelial gap junctions. The present study was performed on endothelium-intact arteries making it impossible to distinguish the functional localization of the N
a
+,K
+-ATPase and K
i
r channels. But regardless of their localization our results indicate that the N
a
+,K
+-ATPase or K
i
r channels or both are modulated by C
a
2
+
o
u
t and that they act as sensors for K
+
o
u
t. The localized K
+
o
u
t can act either as EDHF (in case of smooth muscle cell localization of the K
+ sensors) or by amplifying the endothelial hyperpolarization which then spreads through the myoendothelial gap junctions (in case of endothelial localization of the K
+ sensors).


## 4.3. The Localized [K+]out Signaling

The importance ofC
a
2
+
o
u
t was studied at two concentrations of K
+
o
u
t. We chose values close to physiological values for rats based on the observation that increase in K
+
o
u
t to 5.9 mM induces BaCl2-sensitive relaxation while relaxation to K
+
o
u
t above 5.9 mM is diminishing [13, 19, 30, 34]. In addition, it has been suggested that the Na+,K+-ATPase in the vasculature is fully saturated at 5.9 mM K
+
o
u
t [24, 35]. This suggests that any relaxation in the presence of 5.9 mM K
+
o
u
t is unlikely to be caused by an increase of K
+
o
u
t.This suggestion was supported by our observation that in the presence of 5.9 mMK
+
o
u
t ouabain and particularly B
a
2
+ had a little effect on the relaxation, while at 4.2 mM K
+
o
u
t pronounced effects of these inhibitors were seen. This could be because the elevation of K
+
o
u
t to 5.9 mM saturates the N
a
+,K
+-ATPase and K
i
r channels in agonist preconstricted arteries [19]. In fact, the “K
+ cloud” can be generated not only by the endothelium but also via K
+ efflux from big-conductance C
a
2
+-activated K
+ channels which are activated in depolarized smooth muscle cells of agonist preconstricted arteries [14]. It has previously been shown that, under experimental conditions where the K
+-sensors are saturated, inhibition of the Na,K-pump and K
i
r channels has no effect on relaxation [14]. This indicates that the EDH under these conditions spreads through other mechanisms, possibly as a hyperpolarizing current via myoendothelial gap junctions.Consistent with this, the modulating effect ofC
a
2
+
o
u
t seen at 4.2 mM K
+
o
u
t was abolished at 5.9 mM K
+
o
u
t. This finding is further consistent with our suggestion that the modulatory function of C
a
2
+
o
u
t is mediated largely via the Na,K-pump and K
i
r channels. Finally, the lack of effect of TRAM34 at 5.9 mM K
+
o
u
t is consistent with the hypothesis [11] that the importance of the I
K
C
a channels for the EDH is associated with a localized increase in K
+
o
u
t [12] which acts through the Na,K-pump and K
i
r channels.While no effect ofB
a
2
+ was seen at 5.9 mM K
+
o
u
t, a small but significant inhibition of the relaxation was observed in the presence of ouabain. If at this K
+
o
u
t the Na+,K+-ATPase is completely saturated as suggested [24, 35], it is possible that the observed effect of ouabain was mediated via modulation of gap junctions. It has previously been shown that inhibition of the Na+,K+-ATPase suppresses intercellular communications, including myoendothelial gap junctions [22, 26, 36, 37]. Thus, part of the ouabain effect can be related to inhibition of EDH spreading to smooth muscles through the myoendothelial gap junctions.


## 5. Conclusion

The main findings of this study are as follows: (i) an elevation ofC
a
2
+
o
u
t enhances the EDH-like relaxation to ACh, but (ii) this Ca2+ effect disappears with an elevation of K
+
o
u
t. (iii) The effect of C
a
2
+
o
u
t is maintained after blocking the I
K
C
a channels, but (iv) it disappears after blockade of the Na+,K+-ATPase and K
i
r channels. (v) Finally, inhibitors of the Na+,K+-ATPase and K
i
r channels, ouabain and Ba2+, have large effect on EDH-like relaxation only when K
+
o
u
t is low. Thus, we have suggested that the localized K
+
o
u
t signaling acts through the Na+,K+-ATPase and K
i
r channels, and we have provided strong evidence that these two K+ sensors are affected by C
a
2
+
o
u
t.



---

*Source: 758346-2015-10-04.xml*