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

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 (SKCa and IKCa) is important. Previous studies have suggested that the significance of IKCa depends on [Ca2+]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 (Kir). Here we tested the hypothesis that the modulating effect of [Ca2+]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 [Ca2+]out than at 1 mM [Ca2+]out. Inhibition of IKCa with TRAM34 suppressed the relaxations but did not change the relation between the relaxations at the low and high [Ca2+]out. This [Ca2+]out-dependence disappeared at 5.9 mM [K+]out and in the presence of ouabain or BaCl2. Our results suggest that IKCa are involved in the localized [K+]out signaling which acts through the Na+,K+-ATPase and Kir channels and that the significance of this endothelium-dependent pathway is modulated by [Ca2+]out.


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 endotheliumdependent 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, H 2 S, H 2 O 2 , and vasoactive peptides, have been suggested.
A key element in the EDH pathway supported by most studies is activation of endothelial small-and intermittentconductance Ca 2+ -activated K + channels (SK Ca and IK Ca , resp.) upon agonist-or shear stress-induced increase of endothelial cell calcium ([Ca 2+ ] in ) [6]. Although activation of both SK Ca and IK Ca channels can lead to vasodilation, their contribution to the EDH is different [3][4][5]. SK Ca channels are distributed homogeneously over the endothelial cell membrane and respond to increase of [Ca 2+ ] in [7,8]. Hyperpolarization produced by the SK Ca channels is believed 2 BioMed Research International to spread via myoendothelial gap junctions to smooth muscle cells. This original hypothesis is, however, modified [5,9] based on the observation that SK Ca -dependent hyperpolarization can be blocked not only by apamin, a SK Ca channel inhibitor, but also by Ba 2+ in concentrations specifically blocking the inward-rectifying K + channels (K ir ) [10]. It has been suggested that K + efflux through the opened SK Ca channels might increase the local [K + ] out and consequently open K ir channels. In this model the localized [K + ] out increase amplifies endothelial hyperpolarization generated by SK Ca channel opening [5].
In contrast, the IK Ca channels are localized in endothelial cell projections near the adjacent smooth muscle cells [11] where K + efflux through these channels increases [K + ] out 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 Na + ,K + -ATPase and K ir channels [11,14]. This hypothesis is supported by ouabainand Ba 2+ -sensitive hyperpolarization of smooth muscles in endothelium-denuded arteries with few mM elevation of [K + ] out [12]. Both these K + sensors, that is, the Na,K-ATPase and the K ir channels, are expressed in smooth muscle cells [11,15] although their importance varies between vascular beds [5].
How endothelial excitation leads to differentiated activation of SK Ca /IK Ca channels remains unclear but this may be modulated by [Ca 2+ ] out via the Ca 2+ -sensing receptor (CaSR) [16]. Thus, changes in [Ca 2+ ] out may switch the EDH signaling between being predominantly SK Ca -/myoendothelial gap junction-dependent and being IK Ca -/"K + cloud" dependent pathways.
Based on these considerations we hypothesized that modulation of the EDH-like response by [Ca 2+ ] out is dependent on [K + ] out and tested the importance of IK Ca , Na + ,K + -ATPase, and K ir channels for these effects of [Ca 2+ ] out and [K + ] out .

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 by CO 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). Thirdorder 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% CO 2 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 concentrationresponse relationships were constructed by cumulative noradrenaline concentrations (NA: 0.1-30 M). The relaxation concentration-response relationships were constructed by cumulative addition of acetylcholine (ACh) (0.01-10 M) to the arteries preconstricted with 6 M 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 KH 2 PO 4 , 1.17 MgCl 2 , 25.0 NaHCO 3 , 0.026 EDTA, and 5.5 glucose, gassed with 5% CO 2 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 KH 2 PO 4 , 1.17 MgSO 4 , 25.0 NaHCO 3 , 0.026 EDTA, and 5.5 glucose, gassed with 5% CO 2 in air and adjusted to pH 7.4. Either 1 mM or 2.5 mM CaCl 2 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, pD 2 (the concentration required to produce a half-maximal response) and maximal response were derived and compared using an extra sum-of-squares test. -test was used where appropriate. < 0.05 was considered significant. refers to number of rats.

Results
The sensitivities (pD 2 ) and maximal responses to noradrenaline were the same with all four experimental solutions, that is, 1 mM and 2.5 mM [Ca 2+ ] out and 4.2 mM or 5.9 mM [K + ] out (Figure 1(a)). Thus, the preconstriction levels in relaxation experiments were the same in all experimental conditions. Inhibition of the IK Ca channels with 10 M TRAM34 did not affect the concentration-response relationship to noradrenaline (Figure 1(b)). Neither pD 2 nor maximal contractile responses were significantly affected by TRAM34.
Arteries preconstricted with 6 M 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)).
Preincubation of arteries with 100 M L-NAME and 3 M indomethacin significantly suppressed the relaxation to ACh in the presence of both 4.2 mM and 5.9 mM [K + ] out . Importantly, these inhibitors shifted the concentration-response curves to the right (

Discussion
We have studied the [Ca 2+ ] out -dependent modulation of EDH-like signaling and the role of IK Ca and the [K + ] out sensors, that is, the Na + ,K + -ATPase and K ir channels, for this modulation.

[Ca 2+ ]
and the IK Channels. We found that an elevation of [Ca 2+ ] out from 1 to 2.5 mM increases the relaxation to ACh. This was also seen after the blockade of NOand 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 NOand prostaglandin-independent relaxation to TRAM34 and apamin suggests that this effect was mediated via an EDHlike pathway. The effects of two relatively low (1 mM) and high (2.5 mM) [Ca 2+ ] out concentrations were compared, because IK Ca channel-dependent relaxation has previously been shown to differ strikingly at these two concentrations of Ca 2+ [11].
Although the high [Ca 2+ ] in in activated endothelial cells activates both SK Ca and IK Ca channels, the relative importance of SK Ca and IK Ca has been suggested to depend on [Ca 2+ ] out in the myoendothelial space [7,8]. It has been suggested that [Ca 2+ ] out is sensed by the G-protein-coupled CaSR [18] which can modulate the IK Ca channel-dependent EDH in the vascular wall [11,16,19]. It was therefore tempting to speculate that the effect of [Ca 2+ ] out changes seen in the present study could be explained by changes in the IK Ca activity. However, after inhibition of IK Ca with TRAM34 there was still a modulating effect of [Ca 2+ ] out . This suggests that the modulatory role of [Ca 2+ ] out cannot be limited to an effect on the IK Ca channels.
Part of the Ca 2+ effect may, however, be via the IK Ca channels since TRAM34 affected the ACh concentrationresponse curves differently at the two [Ca 2+ ] out concentrations. This contrasts with a previous report [11] where TRAM34 eliminated the modulating effect of [Ca 2+ ] out 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 [Ca 2+ ] out [11]. Thus, the effect of [Ca 2+ ] out -CaSR signaling on the IK Ca channels likely explains a part of the effect of [Ca 2+ ] out on EDH, possibly via modulation of [Ca 2+ ] in [20,21]. 2+ ] and the Na + ,K + -ATPse/K Channels. Simultaneous inhibition of SK Ca channels with apamin and IK Ca 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 [Ca 2+ ] out . It is therefore possible that [Ca 2+ ] out may also modify the activity of the SK Ca channels. Another possibility is that [Ca 2+ ] out has an effect downstream for activation of the SK Ca /IK Ca channels.

[Ca
A downstream effect of [Ca 2+ ] out might include an effect on the two K + sensors, the ouabain-sensitive Na + ,K + -ATPase and K ir channels, which are also known to be involved in EDH signaling [5]. To address this possibility we assessed the effect of inhibition of the Na + ,K + -ATPase with ouabain [11,22] and the effect of inhibition of the K ir channels with Ba 2+ [23,24]. Both these interventions reduced the relaxation to ACh as expected. Importantly, in the presence of both ouabain and Ba 2+ [Ca 2+ ] out had no effect on the ACh response. This might suggest that the effect of [Ca 2+ ] out is mediated via the Na,K-ATPase and K ir channels.
Interestingly, inhibition of either the Na + ,K + -ATPase or K ir channels caused more than 50% inhibition of the relaxation at 4.2 mM [K + ] out . 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 Na + ,K + -ATPase associates functionally with some K + channels, for example, the ATP-dependent K + channels (K ATP ) [25]. K + ions leaving the cell through the K + channels have been shown to supply the Na + ,K + -ATPase with K + even in [K + ] outfree media [26], while the Na + ,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 Na + ,K + -ATPase and the K ir channels may also interact functionally to short-circuit the membrane K + transport.
Both the Na + ,K + -ATPase and K ir channels are expressed in the smooth muscle cells, although they may also be present in the endothelial cells [11,19,29]. The effect of [Ca 2+ ] out might therefore be mediated via either smooth muscle or endothelial cells. Some studies reported that the effect of Ba 2+ is endothelium-dependent [14,[30][31][32][33]. These endothelial K ir 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 Na + ,K + -ATPase and K ir channels. But regardless of their localization our results indicate that the Na + ,K + -ATPase or K ir channels or both are modulated by [Ca 2+ ] out and that they act as sensors for [K + ] out . The localized [K + ] out 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).

The Localized [K + ]
Signaling. The importance of [Ca 2+ ] out was studied at two concentrations of [K + ] out . We chose values close to physiological values for rats based on the observation that increase in [K + ] out to 5.9 mM induces BaCl 2 -sensitive relaxation while relaxation to [K + ] out 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 + ] out [24,35]. This suggests that any relaxation in the presence of 5.9 mM [K + ] out is unlikely to be caused by an increase of [K + ] out . This suggestion was supported by our observation that in the presence of 5.9 mM [K + ] out ouabain and particularly Ba 2+ had a little effect on the relaxation, while at 4.2 mM [K + ] out pronounced effects of these inhibitors were seen. This could be because the elevation of [K + ] out to 5.9 mM saturates the Na + ,K + -ATPase and K ir 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 Ca 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 ir 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 of [Ca 2+ ] out seen at 4.2 mM [K + ] out was abolished at 5.9 mM [K + ] out . This finding is further consistent with our suggestion that the modulatory function of [Ca 2+ ] out is mediated largely via the Na,K-pump and K ir channels. Finally, the lack of effect of TRAM34 at 5.9 mM [K + ] out is consistent with the hypothesis [11] that the importance of the IK Ca channels for the EDH is associated with a localized increase in [K + ] out [12] which acts through the Na,K-pump and K ir channels. While no effect of Ba 2+ was seen at 5.9 mM [K + ] out , a small but significant inhibition of the relaxation was observed in the presence of ouabain. If at this [K + ] out 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.

Conclusion
The main findings of this study are as follows: (i) an elevation of [Ca 2+ ] out enhances the EDH-like relaxation to ACh, but (ii) this Ca 2+ effect disappears with an elevation of [K + ] out .
(iii) The effect of [Ca 2+ ] out is maintained after blocking the IK Ca channels, but (iv) it disappears after blockade of the Na + ,K + -ATPase and K ir channels. (v) Finally, inhibitors of the Na + ,K + -ATPase and K ir channels, ouabain and Ba 2+ , have large effect on EDH-like relaxation only when [K + ] out is low. Thus, we have suggested that the localized [K + ] out signaling acts through the Na + ,K + -ATPase and K ir channels, and we have provided strong evidence that these two K + sensors are affected by [Ca 2+ ] out .