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040P LOW CALCIUM UNMASKS A PREFERENTIAL INVOLVEMENT OF IKCA
IN ENDOTHELIUM-DEPENDENT HYPERPOLARIZATION OF THE
GUINEA-PIG CAROTID ARTERY TO ACETYLCHOLINE
1
G. Edwards,2P. Gluais, 1A. H. Weston, 3P. M. Vanhoutte and 2M. Félétou. 1School of
Biological Sciences, University of Manchester, Manchester, UK. 2Institut de
Recherches Servier, Suresnes, France. 3Department of Pharmacology, Faculty of
Medicine, Hong Kong, China.
The combination of apamin (a specific inhibitor of small conductance calcium-activated
K+ channels: SKCa) and charybdotoxin (a non-specific inhibitor of intermediate
conductance calcium-activated K+ channels: IKCa) or of apamin and the non-peptide
IKCa inhibitor (TRAM-34, Wulff et al., 2000), is required to abolish endotheliumdependent hyperpolarizations (EDHF; Busse et al., 2002; Gluais et al., 2004). The
absolute requirement for inhibitors of SKCa and IKCa could be explained either by the
simultaneous activation of the two populations of KCa channels during endothelial
stimulation or by the expression of an heterotetramer composed of SK and IK alpha
subunits. This study was designed to determine whether or not the expression of such an
heterotetramer in native endothelial cells is a likely hypothesis.
Guinea-pigs were euthanized with a lethal dose of pentobarbital and the internal carotid
arteries were dissected free and mounted in an organ bath. The smooth muscle cell
membrane potential was recorded using sharp micro-electrodes. All the experiments
were performed at 37°C in the presence of Nω-nitro-L-arginine (100 µM) and
indomethacin (5 µM), respectively.
In control conditions (Krebs solution containing 2.5 mM Ca2+), acetylcholine (10 nM10 µM) induced a concentration- and endothelium-dependent hyperpolarization.
Apamin (0.5 µM) induced a partial inhibition of the hyperpolarization to acetylcholine
(p<0.05) whereas charybdotoxin (0.1 µM) and TRAM-34 (10 µM) were completely
ineffective. However, apamin + charybdotoxin, or apamin + TRAM-34 virtually
abolished EDHF-mediated responses. In 0.5 mM Ca2+, the hyperpolarization to 1 µM
acetylcholine (–14.6 ± 1.3 mV, n=7) was significantly lower than in 2.5 mM Ca2+ (-20.3
± 1.1 mV, n=6, p < 0.05). Furthermore, the EDHF-mediated responses were
predominantly sensitive to charybdotoxin or TRAM-34 but resistant to apamin
(charybdotoxin: -21.2 ± 1.2 and –7.2 ± 1.1 mV, n=6, p < 0.05; TRAM-34: -17.5 ± 3.2
and –9.5 ± 1.4 mV, n=4 and 5, p < 0.05; apamin: -12.1. ± 2.6 and –10.7 ± 1.0 mV, n=5
and 6, p>0.05, in 2.5 and 0.5 mM calcium, respectively). In 0.5 mM calcium, the
combination of the two toxins abolished the hyperpolarization to acetylcholine.
Thus, in the quiescent carotid artery of the guinea pig under control conditions both
IKCa and SKCa are activated during the acetylcholine-induced EDHF response and
activation of SKCa plays a predominant role (possibly because of a higher level of
expression of this channel in the guinea-pig endothelial cells). However, under lowcalcium conditions (0.5 mM Ca2+), IKCa channels are preferentially activated. This is
consistent with the reported two- to five-fold higher calcium sensitivity for IKCa when
compared with the SKCa channels (www.iuphar.org). The results do not support the
existence of SKCa and IKCa heterotetramers in native endothelial cells. Instead, the
EDHF response is triggered via the combined opening of homotetrameric IKCa and
SKCa channels.
Busse, R., et al., (2002) Trends Pharmacol Sci. 23, 374-380.
Gluais, P., et al., (2004) Br. J. Pharmacol. Proc. Suppl., www.pa2online.org/
Vol1Issue4abst027P.
Wulff, H., et al., (2000) Proc. Natl. Acad. Sci. USA. 97, 8151-8158.