Release of nitric oxide is modulated by endothelial cell potassium channels in rat basilar artery

Frances Plane, Mircea Iftinca & William C. Cole, The Smooth Muscle Research Group, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, Canada.

Endothelium-dependent relaxation of rat basilar artery to acetylcholine can be fully accounted for by the release of nitric oxide (NO; Allen et al., 2002). The release of endothelium-derived NO in response to agonists is dependent on a rise in intracellular calcium levels within endothelial cells and several lines of evidence indicate that endothelial cell membrane potential plays a critical role in the regulation of calcium entry (Nilius & Droogmans, 2001). In the present study, the role of endothelial calcium-activated potassium channels in acetylcholine-evoked relaxation of basilar arteries was investigated.

Male Sprague Dawley rats (300-350 g) were killed by halothane inhalation and exsanguination. Segments of basilar artery (2 mm) were mounted in a wire myograph containing oxygenated (95% O2/5% CO2) Krebs buffer at 37 oC. Concentration-response curves for acetylcholine (0.01-10 µM), authentic NO (1-10 µmoles) and 1-ethyl-2-benzimidazolinone (1-EBIO; 1-300 µM) were determined in arterial segments pre-constricted with 5-hydroxytryptamine (5-HT; 0.1-1 µM).

Relaxations of isolated segments of rat basilar artery to acetylcholine or 1-EBIO were abolished by endothelial removal (n=5 and 4, respectively), pre-incubation with the inhibitor of NO synthase L-NG-nitroarginine methyl ester (n=10 and 5, respectively) or pre-contraction of arterial segments with KC1 (50 mM; n=4 and 4, respectively). In contrast, relaxations to authentic NO were not significantly different between tissues pre-contracted with 5-HT or KCl (n=3).

Relaxations to acetylcholine were significantly inhibited by pre-incubation with either apamin (100 nM), an inhibitor of small conductance calcium-activated potassium channels or charybdotoxin (ChTX; 100 nM) or 1-(2-chlorophenyl)(diphenyl)methyl-1H-pyrazole (TRAM-34; 1 µM), inhibitors of intermediate conductance calcium-activated potassium channels. Maximum responses to acetylcholine were reduced from 94.3 ± 0.4 %, 94.3 ± 1.4 % and 93.6 ± 1.4 % to 58.5 ± 11.9 % (n=6; P<0.01), 27.9 ± 4.5 % (n=5; P<0.01) and 54.0 ± 4.7 % (n=8; P<0.01) in the presence of apamin, ChTX and TRAM-34 respectively.

In the presence of a combination of TRAM-34 and apamin maximal responses to acetylcholine were reduced from 91.0 ± 1.9 % to 60.0 ± 6.7 % (n=5; P<0.01) and in the presence of charybdotoxin and apamin, acetylcholine-evoked relaxations were abolished (n=5). Relaxations to authentic NO were unaffected by any of the potassium channel inhibitors either alone or in combination (n=5-8; P>0.05).

These data demonstrate that endothelial cell hyperpolarisation mediated by activation of calcium-activated potassium channels plays a key role in the synthesis and/or release of endothelium-derived NO evoked by acetylcholine in the rat basilar artery.

Allen, T. et al., (2002). J.Physiol.,545, 975-986.s
Nilius, B. & Droogmans, G. (2001). Physiological. Revs., 81, 1415-59.

This study was funded by CIHR and Wellcome Trust. TRAM-34 was generously donated by Heike Wulff.