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(Ca)-subtypes 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 ineffective 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.

Figure 1

Representative whole-cell recordings of CAEC from rat carotid artery (CA) and inhibition by selective IK1 and SK3 blockers. For K_Ca-activation CAEC were dialyzed with a 140 mM K⁺ pipette solution containing 3 μM [Ca²⁺]_free. Bathing solution contained 4.5 mM K⁺. Inhibition of Ca²⁺-activated K⁺-currents by TRAM-34 (A) and TRAM-39 (B) at indicated concentrations. TRAM-34- and TRAM-39-insensitive Ca²⁺-activated K⁺-currents were inhibited by APA. (C) Concentration-dependent blockade of apamin-resistant K_Ca-currents by TRAM-34 (n=4–6) and TRAM-39 (n=4–6) determined at membrane potential of 0 mV and in the presence of 200 nM apamin. Data points were fitted to the Boltzmann equation. Values are given as mean±s.e.mean. (D-F) Representative current-clamp recordings of endothelial membrane potentials and ACh-induced hyperpolarization and its inhibition by APA (200 nM) and TRAM-34 (1 μM). (G) Representative ‘multiplex' RT–PCR analysis of single EC from rat CA in situ. Ethidium bromide-stained gels of RT–PCR products of eNOS and the K_Ca-genes, rSK3 and rIK1 from 2 EC, one medium sample as negative control and one non-template control (H₂0); right lane, molecular weight marker.

Figure 2

TRAM-34 did not affect the function of BK-, K_V-, and K_ATP-channels in CASMC. (A) Whole-cell recordings of BK-currents in the presence or absence of TRAM-34 (10 μM) and blockade of BK-currents by selective BK-blocker iberiotoxin (IbTX, 100 nM). For K_Ca-activation CASMC were dialysed with a 140 mM K⁺ pipette solution containing 3 μM [Ca²⁺]_free. Bathing solution contained 4.5 mM K⁺. The experiment shown is representative of seven experiments with similar results. (B) Whole-cell recordings of K_V-currents in the presence or absence of TRAM-34 (10 μM) and blockade of K_V-currents by the K_V-blocker 4-aminopyridine (4-AP, 2 mM). CASMC were dialysed with a 140 mM K⁺-pipette solution containing 0.1 μM [Ca²⁺]_free. The experiment shown is representative of ten experiments with similar results. Bathing solution used for measurements of BK- and K_V-currents contained 4.5 mM K⁺. (C) Levcromakalim (LCRKM, 10 μM)-induced whole-cell currents through K_ATP-channels in CASMC in absence and presence of TRAM-34 (10 μM) and blockade of K_ATP-currents by the K_ATP-blocker glibenclamide (GLIB, 10 μM). CASMC were dialysed with a 140 mM K⁺-pipette solution containing 0.1 μM [Ca²⁺]_free and 5.3 μM [ATP]_free. Bathing solution contained 50 mM K⁺. Note that under these conditions and at holding potentials more negative than −26 mV, K_ATP currents were inwardly directed. The experiment shown is representative of eight experiments with similar results.

Figure 3

Inhibition of EDHF-type vasodilations in rat carotid arteries by K_Ca-blockers. (A) Inhibition of EDHF-type vasodilator responses by combinations of K_Ca-blockers as indicated. K_Ca-blocker concentrations: 200 nM APA, 1 μM CLT, 1 μM TRAM-34, 1 μM TRAM-39. Values are given as mean±s.e.mean. ^*P<0.05, ^**P<0.01, Mann–Whitney U-test. (B) Original trace illustrating EDHF-type vasodilator responses to intraluminal application of 0.2 μM ACh and in combination with APA (200 nM) or with APA and TRAM-34 (1 μM) or with APA and TRAM-39 (1 μM). Vessels were preconstricted with 1 μM phenylephrine (PE). Endothelium-independent vasodilation and maximal vasoconstriction were induced by extraluminal application of 1 μM sodium nitroprusside (SNP) and 60 mM KCl, respectively. Boxes indicate exposure intervals followed by wash-out (right upper lane). (C) Dose-dependent EDHF-type vasodilator response in response to ACh and inhibition by the combination of APA (200 nM) and ChTX (100 nM) but not by APA (2 μM) or TRAM-34 (1 μM) alone.

Figure 4

(A) Dose-dependent EDHF-type vasodilations in rat carotid arteries in the presence of CLT (10 μM) or SPZ (10 μM). (B) Original tracings illustrating dose-dependent EDHF-type vasodilator responses in the presence of CLT (10 μM) alone and in combination with APA (200 nM). Values are given as mean±s.e.mean.

Figure 5

Inhibition of ACh-induced vasodilations by the K_Ca-blockers APA (200 nM) and TRAM-34 (1 μM) in the absence of inhibitors of NO-synthase and cyclo-oxygenase. (A) Dose-response curves of ACh-induced vasodilations in the presence (n=8) or absence (n=6) of APA (200 nM) and TRAM-34 (1 μM) or in the presence of TRAM-34 (1 μM) alone. Values are given as mean±s.e.mean. ^*P<0.05, ^**P<0.01, Mann–Whitney U-test. (B) Original trace illustrating dose-dependent vasodilations to intraluminal application of ACh alone and of ACh in combination with APA (200 nM) and TRAM-34 (1 μM).