Activation of endothelial cells (ECs) is linked to increases in intracellular Ca²⁺ and membrane hyperpolarization (Carter et al., 1994). EC membrane hyperpolarization increases the electrical gradient for Ca²⁺ entry, thus has been hypothesized to control Ca²⁺ entry in these cells. In rat mesenteric arteries EC hyperpolarization to acetylcholine (ACh) is blocked by combined inhibition of intermediate- and small- conductance Ca²⁺ activated K⁺ channels (Hinton et al., 2003). The current study examined Ca²⁺ handling following EC activation with ACh and investigated the mechanisms that generate this Ca²⁺ profile.

The role of membrane potential was investigated through EC depolarisation with 35mM K⁺, and also the combined blockade of intermediate- and small- conductance Ca²⁺ activated K⁺ channels (inhibited by TRAM-34 (Wulff et al., 2000) and apamin respectively). The roles of intra- and extra-cellular calcium were investigated by blocking uptake of Ca²⁺ to intracellular stores (with cyclopiazonic acid (CPA)) and the removal of extracellular Ca²⁺. The role of protein kinase C was investigated through its inhibition by bisindolylmaleimide (BIS).

Male Wistar rats (200-250g) were killed by cervical dislocation. Sections of third order mesenteric arteries were cannulated and mounted in a pressure myograph, pressurised to 50mmHg and perfused with MOPS at 37 oC. ECs were selectively loaded with the Ca²⁺-sensitive dye, fluo-4 AM, and visualized using a confocal fluorescence microscope. ECs were activated by 300nM ACh, in the presence of either 1μM TRAM-34 and 50nM apamin, 35mM K⁺, 10μM CPA, 100nM BIS, or the removal of extracellular Ca²⁺, and the changes in EC Ca²⁺ monitored for 100s. All experiments were performed in the presence of 100μM L-NAME. Statistical comparisons were performed using one way ANOVA, with Bonferroni’s post-test.

In control experiments (n=7) 300nM ACh caused a sustained increase in EC Ca²⁺ (data shown as mean ±s.e.mean, 20s following ACh administration, relative intensity =1.50 ±0.05, 100s following ACh administration, relative intensity =1.31 ±0.03), and also caused unsynchronised oscillations in EC Ca²⁺. Removal of extracellular Ca²⁺ (n=7) decreased EC Ca²⁺, 100s following ACh administration relative intensity =1.04 ±0.03, and reduced Ca²⁺ oscillations (p<0.001) in the sustained phase of the Ca²⁺ profile, whereas the intial Ca²⁺ profile was unaffected. The EC Ca²⁺ profile was not altered by incubation with, 1μM TRAM-34 and 50nM apamin in combination (n=5), 35mM K⁺ (n=4) or 100nM BIS (n=4). Incubation with 10μM CPA blocked EC Ca²⁺ oscillations (n=5, p<0.001) and initial elevations of EC Ca²⁺ (p<0.001)

The Ca²⁺ profile in the sustained phase of activation is dependent on extracellular Ca²⁺. Oscillations in EC Ca²⁺ are dependent on a functional intracellular Ca²⁺ store, and on the presence of extracellular Ca²⁺ in the sustained phase. EC membrane potential did not affect the Ca²⁺ profile.

Carter, T. et al. (1994) Pflugers Arch., 428, 476-484
Hinton, J. et al. (2003) Br. J. Pharmacol., 138, 1031-1035
Wulff, H. et al. (2000) Proc. Nat. Acad. Sci. USA, 97, 8151-8156

This work was supported by the British Heart Foundation