Selective blockade of endothelial Ca²⁺‐activated small‐ and intermediate‐conductance K⁺‐channels suppresses EDHF‐mediated vasodilation

Abstract

1 Activation of Ca 2+ -activated K + -channels (K Ca ) has been suggested to play a key role in endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilation. However, due to the low selectivity of commonly used K Ca -channel blockers it is still elusive which endothelial K Casubtypes mediate hyperpolarization and thus initiate EDHF-mediated vasodilation. 2 Using the non-cytochrome P450 blocking clotrimazole-derivatives, 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) and 2-(2-chlorophenyl)-2,2-diphenylacetonitrile (TRAM-39) as highly selective IK1-inhibitors, we investigated the role of the intermediate-conductance K Ca (rIK1) in endothelial hyperpolarization and EDHF-mediated vasodilation. 3 Expression and function of rIK1 and small-conductance K Ca (rSK3) were demonstrated in situ in single endothelial cells of rat carotid arteries (CA). rIK1-currents were blocked by TRAM-34 or TRAM-39, while rSK3 was blocked by apamin. In current-clamp experiments, endothelial hyperpolarization in response to acetylcholine was abolished by the combination of apamin and TRAM-34. 4 In phenylephrine-preconstricted CA, acetylcholine-induced NO and prostacyclin-independent vasodilation was almost completely blocked by ChTX, CLT, TRAM-34, or TRAM-39 in combination with the SK3-blocker apamin. Apamin, TRAM-34, and CLT alone or sulphaphenzole, a blocker of the cytochrome P450 isoform 2C9, were ine ective in blocking the EDHF-response. 5 In experiments without blocking NO and prostacyclin synthesis, the combined blockade of SK3 and IK1 reduced endothelium-dependent vasodilation. 6 In conclusion, the use of selective IK1-inhibitors together with the SK3-blocker apamin revealed that activation of both K Ca , rIK1 and rSK3 is crucial in mediating endothelial hyperpolarization and generation of the EDHF-signal while the cytochrome P450 pathway seems to play a minor or no role in rat CA.

Thromboxane receptor stimulation associated with loss of SKCa activity and reduced EDHF responses in the rat isolated mesenteric artery

“…Riluzole is an opener of SK Ca channels, although it has other effects, such as inhibition of Na þ channels and twin-pore domain K channels (Taylor & Meldrum, 1995;Song et al, 1997;Duprat et al, 2000). The SK Ca channels are restricted to the endothelium, as they are in other arteries (Marchenko & Sage, 1996;Edwards et al, 1998;Mistry & Garland, 1998a, b;Burnham et al, 2002;Eichler et al, 2003).…”

Reduced EDHF responses and connexin activity in mesenteric arteries from the insulin-resistant obese Zucker rat

“…Furthermore, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), a specific inhibitor of IK Ca channels, has been used in combination with apamin to confirm that IK Ca and SK Ca channels are involved in EDHFmediated vasodilation. The combination of TRAM-34 and apamin has been used to block EDHF responses in rat caudal [5], saphenous [6], middle cerebral [7] and mesenteric arteries [8] and guinea pig carotid arteries [9].…”

Recycling of the Ca2+-activated K+ Channel, KCa2.3, Is Dependent upon RME-1, Rab35/EPI64C, and an N-terminal Domain

“…It is now clearly established that the intermediate (KCa3.1) and small (KCa2.3) conductance Ca 2ϩ -activated K ϩ channels play a critical role in this endothelium-dependent relaxation. Initially, the involvement of KCa2.3 and KCa3.1 was demonstrated using the combination of apamin, a selective KCa2.x blocker, and charybdotoxin (an inhibitor of KCa3.1 (2-4)), although recent studies have employed the more specific KCa3.1 inhibitor TRAM-34 (5,6). Definitive evidence for the involvement of KCa2.3 in the regulation of vascular tone and hence blood pressure came from the work of Nelson and co-workers (7) in which it was demonstrated, using transgenic mice, that KCa2.3 was responsible for the sustained hyperpolarization that is communicated to the vascular smooth muscle, and suppression of KCa2.3 expression resulted in an elevation of blood pressure.…”