Calcium-activated potassium channels and endothelial dysfunction: therapeutic options?

Abstract

The three subtypes of calcium-activated potassium channels (K(Ca)) of large, intermediate and small conductance (BK(Ca), IK(Ca) and SK(Ca)) are present in the vascular wall. In healthy arteries, BK(Ca) channels are preferentially expressed in vascular smooth muscle cells, while IK(Ca) and SK(Ca) are preferentially located in endothelial cells. The activation of endothelial IK(Ca) and SK(Ca) contributes to nitric oxide (NO) generation and is required to elicit endothelium-dependent hyperpolarizations. In the latter responses, the hyperpolarization of the smooth muscle cells is evoked either via electrical coupling through myo-endothelial gap junctions or by potassium ions, which by accumulating in the intercellular space activate the inwardly rectifying potassium channel Kir2.1 and/or the Na(+)/K(+)-ATPase. Additionally, endothelium-derived factors such as cytochrome P450-derived epoxyeicosatrienoic acids and under some circumstances NO, prostacyclin, lipoxygenase products and hydrogen peroxide (H(2)O(2)) hyperpolarize and relax the underlying smooth muscle cells by activating BK(Ca). In contrast, cytochrome P450-derived 20-hydroxyeicosatetraenoic acid and various endothelium-derived contracting factors inhibit BK(Ca). Aging and cardiovascular diseases are associated with endothelial dysfunctions that can involve a decrease in NO bioavailability, alterations of EDHF-mediated responses and/or enhanced production of endothelium-derived contracting factors. Because potassium channels are involved in these endothelium-dependent responses, activation of endothelial and/or smooth muscle K(Ca) could prevent the occurrence of endothelial dysfunction. Therefore, direct activators of these potassium channels or compounds that regulate their activity or their expression may be of some therapeutic interest. Conversely, blockers of IK(Ca) may prevent restenosis and that of BK(Ca) channels sepsis-dependent hypotension.

Figure 1

Potassium channels in smooth muscle cells of the guinea pig carotid artery. Effect of the combination of different inhibitors of potassium channels in freshly isolated smooth muscle cells of the guinea pig carotid artery (whole cell configuration of the patch-clamp technique). (A) Large global currents observed in the presence of intracellular calcium for a ramp depolarization from −100 to +80 mV. This current is partially inhibited by iberiotoxin (IbTx), indicating the presence of calcium-activated potassium channels of large conductance (BK_Ca). The addition of charybdotoxin (ChTx) does not produce any further inhibition, suggesting that neither the calcium-activated potassium channels of intermediate conductance (IK_Ca)-dependent current nor the A-type rapidly inactivating transient outward current (K_TO) is activated. The addition of apamin (Apa) produces a further inhibition, indicating the presence of a calcium-activated potassium channels of small conductance ‘(SK_Ca)-like’ current. The subsequent addition of 4-aminopyridine (4-AP) blocked the remaining global outward current demonstrating the contribution of voltage-activated potassium channels (K_V). (B) Large outward current observed for a step depolarization from −100 to +60 mV, confirming the contribution of BK_Ca, ‘SK_Ca-like’ and K_V channels in the recorded current. The effect of 4-AP is not shown for the sake of clarity. Modified from Quignard et al. (Br J Pharmacol 2000b).

Figure 2

Spatial distribution and functions of calcium-activated potassium channels in the vascular wall. SK_Ca (SK3) and IK_Ca (IK1) are preferentially expressed in ECs. SK_Ca are preferentially located in caveolin-rich domains, at sites of homocellular endothelial gap junctions. A global increase in endothelial [Ca²⁺]_i preferentially activates SK_Ca and an EDHF-mediated response involving a Cx-dependent pathway (Dora et al., 2008). IK_Ca are preferentially localized at the sites of endothelial projections towards the underlying smooth muscle cells. Co-localized sarcoplasmic reticulum elements and associated local calcium release (calcium pulsar) regulate IK1 activation and vascular tone via potassium efflux and subsequent activation of smooth muscle Na⁺/K⁺-ATPase (Ledoux et al., 2008b). The activation of these two channels also favours calcium entry, amplifying NO production (Stankevicius et al., 2006). When BK_Ca channels are expressed in the ECs they can also contribute to this latter mechanism (Brakemeier et al., 2003). BK_Ca are preferentially expressed in smooth muscle cells and are often clustered in large groups. They are activated by calcium sparks or following a global increase in smooth muscle [Ca²⁺]_i. They generate STOCs, leading to arterial smooth muscle hyperpolarization and relaxation (Perez et al., 1999). IK_Ca are expressed in VSMCs undergoing de-differentiation and are involved in their proliferation (Neylon et al., 1999). BK_Ca, calcium-activated potassium channels of large conductance; Cx, connexin; EC, endothelial cell; EDCF, endothelium-derived contracting factor; eNOS, endothelial nitric oxide synthase; IK_Ca, calcium-activated potassium channels of intermediate conductance; IP₃R, IP₃ recepteur; Kir, inwardly rectifying potassium channel; NO, nitric oxide; RyR, ryanodine receptor; SK_Ca, calcium-activated potassium channels of small conductance; STOCs, spontaneous transient outward currents; TRPV4, vanilloid transient receptor potential channel 4; VSMC, vascular smooth muscle cell.

Figure 4

Endothelial vasoactive factors and smooth muscle BK_Ca activity. 20-HETE, 20-hydroxyeicosatetraenoic acid; BK_Ca, calcium-activated potassium channels of large conductance; cAMP, cyclic-adenosine monophosphate; cGMP, cyclic-guanosine monophosphate; COX, cyclooxygenase; EC, endothelial cell; EDCFs, endothelium-derived contracting factors; EDRFs, endothelium-derived relaxing factors; EETs, epoxyeicosatrienoic acids; eNOS, endothelial nitric oxide synthase; GS, G-protein S; GSα, α subunit of G-protein S; H₂O₂, hydrogen peroxide; IP, prostcyclin receptor; NO, nitric oxide; NOX, NADPH oxidase; P450 2C-2J or 4A, cytochrome P450 monooxygenase 2C, 2J or 4A; PGI₂, prostacyclin; R?, putative receptor; ROS, reactive oxygen species; sGC, soluble guanylate cyclase; SOD, superoxide dismutase; TP, thromboxane/endoperoxide receptor; TRPV4, vanilloid transient receptor potential channel 4; VSMC, vascular smooth muscle cell; XO, xanthine oxidase.

Figure 3

Contribution of BK_Ca in NO-induced relaxation. (A) In rat aorta, acetylcholine-induced endothelium-dependent relaxation is blocked by the guanylate cyclase inhibitor ODQ and partially inhibited by IbTX, indicating that endogeneous NO induces cGMP-dependent relaxation and that the activation of BK_Ca contributes to the mechanism of this relaxation. (B) In rat aorta, a NO donor, DEA-NONOate, also produces cGMP-dependent relaxations, which involves the activation of BK_Ca. (C) In rat aorta, a NO-independent activator of soluble guanylate cyclase, BAY 58-2667, produces a slowly developing relaxation, which involves the activation of BK_Ca. (D) In freshly isolated smooth muscle cells from rabbit carotid artery SIN-1, a NO donor increases the amplitude of the BK_Ca current elicited by a step depolarization (whole cell configuration of the patch-clamp technique, left panel). The effect of SIN-1 is associated with an increase in the open probability of the channel (cell-attached configuration of the patch-clamp technique, right panel; modified from Quignard et al. (Eur J Pharmacol, 2000a). BK_Ca, calcium-activated potassium channels of large conductance; cGMP, cyclic-guanosine monophosphate; IbTX, iberiotoxin; NO, nitric oxide; sGC, soluble guanylate cyclase.

Figure 5

Potassium currents in porcine coronary arterial endothelial cells. Effect of the combination of different inhibitors of potassium channels in freshly isolated porcine coronary arterial endothelial cells (whole cell configuration of the patch-clamp technique). (A) Representative K⁺-currents elicited by 10 mV voltage steps in control, after application of iberiotoxin (IbTX), iberiotoxin + apamin (IbTX + Apamin) and iberiotoxin + apamin + charybdotoxin (IbTX + Apamin + ChTX). (B) Summary bar graph: the presence of apamin (Apa.) and charybdotoxin produced a statistically significant inhibition of the amplitude of the K⁺-currents.

Figure 6

Involvement of SK_Ca and IK_Ca in EDHF-mediated responses. Endothelium-dependent hyperpolarizations to ACh in vascular smooth muscle of the guinea pig carotid artery (in the presence of inhibitors of NOS and COX). (A) Original membrane potential recordings showing the concentration-dependent hyperpolarizing effect of ACh. (B) The concentration- and endothelium-dependent hyperpolarizations produced by ACh are not affected by the presence of ChTx, partially inhibited by that of Apa. and virtually abolished by the combination of the two toxins. (C) Similarly, TRAM-34, a non-peptidic and selective blocker of IK_Ca, does not affect the hyperpolarization elicited by ACh while UCL 1684, a selective blocker of SK_Ca, produces a partial inhibition. The combination of the two blockers prevents the hyperpolarizing effect of ACh. Modified from Gluais et al. (Br J Pharmacol, 2005). ACh, acetylcholine; Apa., apamin; ChTx, charybdotoxin; COX, cyclooxygenase; EDHF, endothelium-derived hyperpolarizing factor; IK_Ca, calcium-activated potassium channels of intermediate conductance; NOS, nitric oxide synthase; SK_Ca, calcium-activated potassium channels of small conductance.

Figure 7

NS-309 induces endothelium-dependent hyperpolarization. (A) Chemical structure of NS-309, an activator of calcium-activated potassium channels of intermediate and small conductance. (B) Original membrane potential recordings showing the effects of acetylcholine (ACh) and NS-309 in vascular smooth muscle of the guinea pig carotid artery with endothelium (in the presence of inhibitors of nitric oxide synthase and cyclooxygenase). (C) Concentration-dependent effects of NS-309. (D) Summary of the effects of endothelial removal on ACh-, NS-309- and levcromakalim (Levcrom.)-induced hyperpolarization. (E) Effects of the combinations of apamin plus charybdotoxin on ACh-, NS-309- and levcromakalim-induced hyperpolarizations. Modified from Leuranguer et al. (Naunyn Schmiedebergs Arch Pharmacol, 2008).