The main findings of the present study were that the hypoxia-induced reduction in [Ca2+]i in PGF2α-contracted arteries can be attributed to opening of one or more K channel types. The KV7.4 and KV7.5 channels, which we identified in the coronary arteries, appear to be particularly important in the hypoxia-induced vasodilatation. The hypoxic vasodilatation was inhibited by two different blockers of KV7 channels and the voltage clamp results further indicate that KV7 channels are involved in this response to hypoxia. Our findings suggest that hypoxia may act via K channel opening, which is, in part mediated by H2S.
Role of intracellular calcium and K channels in hypoxia-induced dilatation
Previous studies have shown that [Ca2+]i is reduced during hypoxia (Shimizu et al., 2000), but it has also been suggested that reduced [Ca2+]i cannot fully account for hypoxia-induced dilatation (Aalkjaer and Lombard, 1995; Shimizu et al., 2000; Thorne et al., 2001; Gu et al., 2005). We found that in PGF2α-contracted arteries, hypoxia lowered [Ca2+]i and induced dilatation. In K30PSS-contracted arteries, hypoxia did not alter [Ca2+]i but caused dilatation. In our experiments, the level of contraction induced by K30PSS or PGF2α did not differ. Since hypoxia-induced dilatation was only partially inhibited by K30PSS contraction, part of this dilatation must be caused by mechanisms independent of changes in [Ca2+]i, such as force suppression or Ca2+ desensitization. The decrease in [Ca2+]i observed in PGF2α-contracted arteries suggested the involvement of K channels, since opening of these will lead to hyperpolarization, which decreases [Ca2+]i. Blocking calcium influx may also contribute to the hypoxia-induced relaxation (Franco-Obregon and Lopez-Barneo, 1996; Smani et al., 2002), but the effect of the K channel blockers indicate that K channels are involved in the hypoxic relaxation.
Several K channels including KATP (Daut et al., 1990; Dart and Standen, 1995; Liu and Flavahan, 1997; Lee et al., 1998; Kamekura et al., 1999), KCa1.1 (BKCa;Gebremedhin et al., 1994; Nelson and Quayle, 1995; Lopez-Barneo et al., 2004) and certain KV channel subtypes KV1.5 and KV2.1 (Shimizu et al., 2000; Thorne et al., 2002) have been suggested to be involved in hypoxia-induced vasodilatation. In the present study, glibenclamide significantly inhibited the relaxations induced by the KATP opener, cromakalim, but in the same preparations only slightly inhibited the relaxations evoked by lowering oxygen. These findings agree with the observations in rat and human coronary small arteries, which also suggested that the contribution of KATP channels to hypoxic vasodilatation is negligible (Lynch et al., 2006). Although 4-AP can also inhibit KATP currents at concentrations above 0.2 mM (Beech and Bolton, 1989; Nelson and Quayle, 1995), in our experiments the concentration of 4-AP used (0.5 mM) has been shown to be relatively selective for KV channels (Nelson and Quayle, 1995). In the present study, 4-AP did not affect the cromakalim-induced relaxation, and, therefore, we believe that the reduction in hypoxic vasodilatation observed in the present study can be ascribed to inhibition of 4-AP-sensitive KV channels.
In coronary arteries with endothelium, NO release is increased and contributes to hypoxic vasodilatation (Lynch et al., 2006; Hedegaard et al., 2011) and, recently, we found that NO induces vasodilatation by activation of TEA-sensitive channels (Hedegaard et al., 2011). In the present study of porcine coronary arteries without endothelium, TEA also caused a small reduction in the hypoxia-induced vasodilatation at concentrations (10−3 M) selective for KCa1.1 (BKCa) channels (Lang and Ritchie, 1990). The observed expression of the α and β subunits of KCa1.1 (BKCa) in these arteries, and the finding that IbTX, a selective blocker of KCa1.1 channels, reduced the hypoxic vasodilatation, further indicate that smooth muscle KCa1.1 channels contribute to the hypoxic vasodilatation. The combined blockade of 4-AP-sensitive KV channels and KCa1.1 channels did not cause further inhibition of hypoxic vasodilatation. Thus, KCa1.1 channels may mediate the endothelium-dependent component, involving NO, of the hypoxic vasodilatation (Hedegaard et al., 2011). However, in porcine coronary arteries, in the absence of endothelium, our results show that KATP, 4-AP-sensitive KV and KCa1.1 channels contribute little to hypoxic vasodilatation, not enough to explain the pronounced inhibition of hypoxic vasodilatation observed in the preparations contracted by increasing the extracellular K concentration.
Role of KV7 channels in hypoxic vasodilatation
KV7 channels are involved in the regulation of vascular tone in a number of rodent blood vessels (Yeung and Greenwood, 2005; Joshi et al., 2006; Yeung et al., 2007; Mackie et al., 2008; Zhong et al., 2010) and it has also been suggested that they counteract changes induced by chronic hypoxia in the pulmonary circulation (Morecroft et al., 2009). Therefore, these channels may also be involved in hypoxic vasodilatation. Indeed, in the present study we found that two different blockers of KV7.1–7.5 channels XE991 and linopirdine inhibited the dilatation induced by hypoxia, adenosine and flupirtine, an opener of KV7.2–7.5 channels, whereas the KV7.1 inhibitor, chromanol 293B did not affect the dilatation induced by these agents. The effect of XE991 appears to be specific, since the SNP-induced relaxation was not changed in the presence of XE991.
Previous studies have shown that mRNA for KV7.1, KV7.4 and KV7.5 is readily detectable in mice portal vein, thoracic aorta, carotid and femoral artery smooth muscle (Yeung et al., 2007; 2008b). KV7.1 and KV7.5 transcripts have also been detected in adult rat aorta (Brueggemann et al., 2007) and KV7.1, KV7.4 and KV7.5 in rat mesenteric artery smooth muscle cells (Mackie et al., 2008). In human arteries, KV7.4 was shown to be expressed in all the arteries examined with variable contributions from KV7.1, KV7.3 and KV7.5 (Ng et al., 2011). We were able to detect mRNA transcripts from KV7.1, KV7.4 and KV7.5, but not KV7.2 and KV7.3, in the coronary artery, even though we tested four different sets of primers and obtained a positive result in control tissue from human heart. Our results agree with previous results showing that KV7.1, KV7.4 and KV7.5 are the dominantly expressed genes in the vasculature, and there is accumulating evidence indicating that KV7.4 and KV7.5 are responsible for the activity in the vasculature (Jepps et al., 2011). Immunoblotting showed that KV7.4 and KV7.5 channel proteins are also expressed in porcine coronary arteries. Taken together with the observations that XE991 inhibits KV7.1–7.4 with IC50 values ∼1–5 μmol·L−1 (Yeung et al., 2007; 2008a) and KV7.5 with an IC50∼60 μmol·L−1 (Jensen et al., 2005; Yeung et al., 2008a), and since we used 10 μM XE991, our results suggest that the effect we observed is likely to be mediated by KV7.4 channels.
The combination of IbTX, 4-AP and XE991 had a greater inhibitory effect on the hypoxic vasodilatation compared to XE991 alone and the effect of this combination was similar to that observed in preparations contracted with 30 mM potassium (e.g. Figure 2 versus Supporting Information Figure S4). These results suggest that XE991 has an effect on the KV7 channels, which can be separated from that induced by blocking other potassium channels.
In previous studies, resting membrane potential in pinned coronary arteries from pigs has been measured to be approximately −50 mV (Edwards et al., 2000) and PGF2α (2–6 μM) was found to depolarize the membrane to −40 to −35 mV (Thollon et al., 2002). Resting membrane potential is more depolarized in pressurized preparations, while agonist-induced depolarization is less (Schubert et al., 1996). This confirms that the XE991-sensitive currents activated above −40 mV are within the range of physiologically relevant membrane potentials.
Interestingly, in the voltage-clamp study, the membrane conductance was higher under hypoxic conditions. This hypoxia-induced current was sensitive to XE991 at a concentration that has previously been shown to inhibit KV7 currents (Yeung and Greenwood, 2005). Although recent studies have suggested that XE991 can also attenuate KV1.2/KV1.5 and KV2.1/KV9.3 channels, an overexpression study confirmed that its major effect is on KV7 channels (Zhong et al., 2010). We thus propose that members of the KV7 channel family (i.e. KV7.2-KV7.5 channels) are activated by hypoxia in coronary artery smooth muscle cells and are involved in the vasodilator response.
Role of KV7 channels in H2S and adenosine induced vasodilatation
A fall in oxygen availability during hypoxia decreases mitochondrial H2S oxidation resulting in an increase in biologically active H2S, and therefore, H2S has been suggested to mediate hypoxic vasodilatation (Skovgaard et al., 2011). Moreover, H2S has been shown to cause relaxation by activation of KATP channels and KV7 channels (Zhang et al., 2007; Schleifenbaum et al., 2010). Incubation of the porcine coronary arteries with inhibitors of enzymes synthesizing H2S attenuated the vasodilatation induced by lowering oxygen tension to 1% and the combination of these enzyme inhibitors with XE991 inhibited the hypoxic vasodilatation. In addition, the inhibitory effect of XE991 on the H2S-induced relaxation suggests that part of the KV7 activation in hypoxia may be attributed to H2S.
Another metabolite released from the myocardium in ischaemic conditions is adenosine (Decking et al., 1997). Adenosine increases cAMP and a study in renal arteries demonstrated that KV7 channels are regulated by cAMP-dependent processes (Chadha et al., 2012). This finding is in line with our observation that adenosine-induced relaxation was inhibited in the presence of XE991. Although further studies are required to reveal the source of the mediators, these results suggest that H2S and adenosine may lead to the activation of KV7 channels during hypoxia in porcine coronary arteries.