The activation of intermediate (IK_Ca) and small conductance-calcium activated potassium channels (SK_Ca) in endothelial cells is a fundamental early step in the release of endothelium-derived hyperpolarizing factor (EDHF) (Busse et al. 2002). We have used selective inhibitors for these channels to investigate if the endothelial cell hyperpolarization, which is caused by their activation, is a crucial determinant of intracellular calcium concentration in the endothelium.

Male Wistar rats (200-250 g) were killed humanely and segments of a third order branch of the superior mesenteric artery (i.d. 250-300 µm) removed and mounted in a pressure myograph at 50 mmHg, perfused with MOPS at 37°C and visualized by confocal microscopy. Dilatation to acetylcholine (ACh, 1 nM-3 µM) was measured in the presence of 100 µM L-NAME, to block NO synthase, and phenylephrine to stimulate tone (PE, 3 µM). 1-[(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34, 1 µM: Wulff et al. 2000) and apamin (50 nM) were used individually and in combination to block I_KCa and SK_Ca, respectively. To assess Ca²⁺ levels, endothelial cells were selectively loaded with fluo-4 AM and stimulated with 300 nM ACh, in the presence of L-NAME alone or in combination with TRAM-34 and apamin. The change in fluorescence in individual endothelial cells was then monitored for 2 min.

Increasing concentrations of ACh caused concentration-dependent dilatation (EC₅₀ 234 nM, n = 6). The concentration response curve to ACh was shifted to the right in the presence of either TRAM-34 (EC₅₀ 613 nM, n = 3) or apamin (EC₅₀ 616 nM, n = 3), while in combination these blockers caused a marked rightward shift (EC₅₀ 4.03 µM, n = 6) and depressed the maximum dilatation (to 300 nM ACh) from 62.4 ± 7.1 % to 2.9 ± 1.3 % (P < 0.05, n = 6, Student’s unpaired t test, mean ± S.E.M.). In the imaging experiments, 300 nM ACh stimulated a sustained rise in endothelial cell Ca²⁺ (increase at t = 10s: 38 ± 13% increase at t = 120s: 28 ± 9 %, n = 4, average of 16 cells/experiment). The time course and magnitude of these increases in endothelial cell Ca²⁺ was unaltered in the combined presence of TRAM-34 and apamin (n = 3), or in the presence of raised extracellular K⁺ (35 mM, n = 3).

In conclusion, apamin and TRAM-34 inhibited EDHF-mediated vasodilatation in the mesenteric artery. However, these blockers did not modify the sustained increase in endothelial cell Ca²⁺ evoked with ACh. These results suggest that endothelial cell hyperpolarization is not essential for driving Ca²⁺ entry in these cells.

This work was supported by the British Heart Foundation and the Wellcome Trust