Need for novel targets to treat cerebral vasospasm
Thus far, the only drug available to treat cerebral vasospasm is nimodipine, a selective L-type Ca2+ channel blocker, but it has limited efficacy. This may be due to the presence of heterogeneous Ca2+ channel subunits with varying sensitivities to nimodipine (Nikitina et al., 2007; Navarro-Gonzalez et al., 2009) and increased expression of nimodipine-insensitive T-type Ca2+ channels and R-type Ca2+ channels in basilar artery myocytes following SAH (Nikitina et al., 2010). Hence, a drug that inhibits the activation of all VSCC subtypes either directly or indirectly (e.g. by activation of K+ channels and prevention of the membrane depolarization that activates VSCC) would be expected to be more efficacious than nimodipine.
Kv7 channels are expressed and functional in rat basilar artery myocytes
Message transcripts of all the known KCNQ genes (KCNQ1-5, encoding Kv7.1-7.5 channels) were detected in rat basilar artery myocytes (Figure 1). Very recently, a quantitative assessment of the expression of KCNQ genes in rat basilar artery and middle cerebral artery myocytes by Zhong et al. also revealed the presence of all five KCNQ mRNAs, though the mRNAs of KCNQ2 and KCNQ3 were much less abundant than those of KCNQ1, KCNQ4 and KCNQ5 (Zhong et al., 2010). Kv7.1 channels are insensitive to drugs (e.g. flupirtine, retigabine and N-ethyl maleimide) that activate the other Kv7 channel subtypes (Gamper et al., 2005; Munro and Dalby-Brown, 2007), and Kv7.1 subunits do not form heteromers with other Kv7 channel subunits (Schwake et al., 2003). Considering that the whole cell Kv7 currents measured in basilar artery myocytes were robustly enhanced by flupirtine (Figure 2A–C), the contribution of outward K+ conductance through homomeric Kv7.1 channels is likely to be minimal. Hence, the functional Kv7 channels in basilar artery myocytes are likely to be predominantly constituted by homo- or hetero-tetramers of Kv7.4 and Kv7.5 channel subunits.
We isolated basilar artery myocyte Kv7 currents using whole cell voltage-clamp techniques (Figure 2). The KV currents recorded at test voltages between −60 mV and −20 mV can reasonably be attributed to Kv7 channel activity based on several observations: (i) the currents measured under our recording conditions were robustly enhanced by 10 µM flupirtine, a selective Kv7 channel activator; (ii) the currents were completely inhibited by 10 µM XE991, a selective Kv7 channel blocker; and (iii) the currents had electrophysiological characteristics expected of Kv7 currents (non-inactivating, voltage-dependent with a very negative threshold for activation and half-maximal activation (V0.5) of ∼−34 mV (Adams et al., 1982; Wickenden et al., 2001; Brueggemann et al., 2007; Mackie et al., 2008). Currents recorded at voltages more positive than −20 mV were not completely blocked by XE991, suggesting that at these more depolarized voltages we were recording a mix of currents, with contributions likely from other K+ channels, such as 4-AP-sensitive Kv channels and Ca2+-activated K+ channels (KCa) channels.
Kv7 channels are critical contributors to resting membrane voltage and basilar artery contractile status
The resting membrane voltage of basilar artery myocytes measured in the current study was −57.5 ± 6.3 mV, within the range reported in previous studies for basilar artery myocytes (Allen et al., 2002; Chrissobolis and Sobey, 2002; Jahromi et al., 2008a). The maximal density of K+ currents through Kv7 channels is small (<0.3 pA/pF, Figure 2C) compared with maximal current densities reported for 4-AP-sensitive-KV (36.9 pA/pF) and KCa channels (∼140 pA/pF) (Jahromi et al., 2008a,b). However, Kv7 channels are likely to play an important role in determining the resting membrane voltage, more so than KV and KCa channels, as they are activated at more negative membrane voltages than 4-AP-sensitive KV and KCa channels [V0.5 for Kv7 currents is −34 mV (Figure 2D), compared with V0.5 of −1.3 mV for 4-AP-sensitive-KV currents (Jahromi et al., 2008a) and +86.8 mV for large-conductance KCa currents at 200 nM [Ca2+]cyt (Jahromi et al., 2008b)]. This is supported by the evidence that blockade of Kv7 channels with XE991 significantly depolarized the cell membrane (Figure 3A, B). This brought the membrane voltage from −58 mV, at which L-type VSCC have very low activity, to −35 mV, which is in the range of membrane voltage where VSCC activity increases in a steeply voltage-dependent manner (Figure 5B). Our results shown in Figure 3C support this hypothetical mechanism: XE991 robustly constricted the basilar artery and this effect was reversed by the L-type VSCC blocker nimodipine. Kv7 channels have a well-established role in stabilizing resting membrane voltages, and suppression of their activity is a common depolarizing stimulus in neurons and arterial myocytes (Adams et al., 1982; Mackie et al., 2008; Joshi et al., 2009). It remains to be determined whether suppression of Kv7 channel activity contributes substantially to the development of cerebral vasospasm after SAH.
Kv7 channel activators as candidates to treat cerebral vasospasm
The Kv7 channel activator flupirtine was able to reverse the constriction produced by the spasmogens involved in cerebral vasospasm, 5-HT, ET-1 and AVP (Figure 6). Furthermore, addition of flupirtine in the presence of nimodipine produced additional dilation. We speculate that flupirtine produced an additive vasodilatory effect by opposing the membrane depolarization produced by the spasmogens, and thereby preventing the activation of both nimodipine-sensitive and nimodipine-insensitive VSCC. Our results shown in Figure 5 are consistent with this idea in that we can detect Ba2+ currents in basilar artery myocytes that are activated by membrane depolarization, but not fully blocked by 2 µM nimodipine.
A 10-fold higher concentration of flupirtine was required in the vascular reactivity studies compared with patch-clamp studies. The discrepancy is likely, because, in intact arteries, depolarization of a small proportion of smooth muscle cells in response to addition of spasmogens leads to depolarization of the adjacent smooth muscle cells, which are connected by gap junctions. Therefore, the concentration of flupirtine required to oppose the concerted depolarization of VSMCs in a physiologically integrated arterial system is higher than in the single cell patch clamp studies. Conversely, a threefold lower concentration of spasmogen was required in vascular reactivity studies compared with patch-clamp studies (Mackie et al., 2008).
Celecoxib (Celebrex®) is marketed as a selective inhibitor of cyclooxygenase-2 (COX-2) and is widely prescribed to treat pain and inflammation. However, our present findings indicate that celecoxib is a robust Kv7 channel activator (like flupirtine) (Figure 4) as well as a VSCC blocker in basilar artery myocytes (more effective than 2 µM nimodipine; Figure 5). We recently demonstrated similar effects of celecoxib in mesenteric artery myocytes and provided evidence that these effects are apparent at concentrations of celecoxib that may be achieved with clinical therapy (Brueggemann et al., 2009). In contrast, the concentration of nimodipine (2 µM), which induced significantly less suppression of VSCC than 10 µM celecoxib did, is more than 1000-fold higher than concentrations achieved in cerebrospinal fluid after nimodipine administration in patients (Allen et al., 1983). Although the electrophysiological methods used may not reveal differences in drug effects that might occur under more physiological conditions, the pressure myography experiments are performed under much more physiological conditions and reveal a similar difference in efficacy between celecoxib and nimodipine. Though the mechanism(s) by which celecoxib modulates ion channels are not known, these effects are expected to influence the contractile status of basilar artery myocytes.
As expected from its dual ion channel effects, celecoxib very effectively reversed the constriction produced by the spasmogens (Figure 7A, B) and, like flupirtine, celecoxib was a more effective dilator of basilar artery than nimodipine (Figure 7D, E). The additional dilation produced by celecoxib could be due to two mechanisms: enhancement of Kv7 currents thereby limiting the voltage change necessary to activate all of the VSCCs, or Kv7 channel-independent inhibition of nimodipine-insensitive VSCC. Our results provide evidence for both of these mechanisms. The finding that flupirtine produces an additional dilation in the presence of nimodipine supports the former (Figure 6D), and that celecoxib produces additional inhibition of voltage-sensitive Ba2+ currents in the presence of 2 µM nimodipine supports the latter (Figure 5C).
The vasodilatory effects observed with celecoxib were independent of its ability to inhibit COX-2 as rofecoxib, a more potent inhibitor of COX-2 than celecoxib, did not induce vasodilatory responses, but dimethyl celecoxib, a structural analogue of celecoxib modified to eliminate COX-2 activity, also almost completely relaxed spasmogen pre-constricted artery segments (Supporting Information Figure S3; Brueggemann et al., 2009). We do not rule out the possibility of modulation of other ion channels or intracellular Ca2+ mobilization pathways by celecoxib that may also contribute in part to the vasodilatory responses observed here.
Direct Kv7 channel activators have already found their way to clinical trials to treat neuronal excitability disorders such as epilepsy and neuropathic pain (Miceli et al., 2008). Our findings suggest that, in addition to their direct neuroprotective effects (Boscia et al., 2006), these drugs with established safety profiles (Blackburn-Munro et al., 2005) can be readily adopted to prevent or limit the neurological deficits after SAH by reducing basilar artery vasospasm. Kv7 channel activators were also recently reported to reverse the pressure-induced constriction in resistance cerebral arteries (Zhong et al., 2010). Hence, the effect of Kv7 activators in improving the cerebral blood flow after SAH may extend beyond the ability of the drugs to dilate the conduit arteries like the basilar artery (Figure 6) that are primarily affected during the syndrome. In terms of potential utility in the treatment of SAH-induced cerebral vasospasm, celecoxib has additional effects that might also be beneficial. Celecoxib is a VSCC blocker, like nimodipine, and might therefore be expected to be more effective as a vasodilator than a drug like flupirtine that only activates Kv7 channels. It is increasingly recognized that an inflammatory component is associated with the development of cerebral vasospasm (Pradilla et al., 2010); the anti-inflammatory effects of celecoxib may therefore also be beneficial. Celecoxib could be an ideal drug to reduce the inflammation associated with SAH and simultaneously oppose the spasmogenic effects of locally elevated vasoconstrictor agonists. Preclinical trials to test the efficacy of these drugs to relieve basilar artery vasospasm in animal models of SAH are warranted.
In conclusion, this study provides the first evidence for the functional presence of Kv7 channels in basilar artery myocytes. Our findings suggest that K+ conductance through the Kv7 channels maintains hyperpolarized resting membrane voltages, preventing the activation of VSCC, and thereby regulating the contractile status of basilar artery. Flupirtine, a selective Kv7 channel activator or celecoxib, a dual Kv7 channel activator and VSCC blocker can reverse the constrictor effects of spasmogens implicated in SAH-induced cerebral vasospasm. These drugs could, therefore, be regarded as candidates for development of novel therapies for patients who have or may develop cerebral vasospasm.