In rat mesenteric arteries, ACh increases endothelial cell intracellular calcium concentration ([Ca²⁺]_i) causing membrane hyperpolarization through activation of calcium-activated K⁺ channels (K_Ca). In intact arteries, endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation, evoked by ACh, is abolished by a combination of inhibitors for intermediate- and small-conductance K_Ca (IK_Ca, SK_Ca). Furthermore, in the absence of any tone smooth muscle hyperpolarization to ACh is abolished by apamin (50nM) alone (Crane et al., 2003). To investigate more clearly the role of the endothelium in these responses, ACh-stimulated changes in [Ca²⁺]_i) and membrane potential were measured in freshly isolated sheets of endothelial cells (ECs) in the presence of the IK_Ca and SK_Ca inhibitors, TRAM-34 and apamin, respectively.

Male Wistar rats (200-250g) were killed by cervical dislocation. Small mesenteric arteries were isolated and cut longitudinally. ECs were isolated using an enzyme mix containing papain and following a digestion protocol specifically designed to leave intact sheets of endothelial cells (Takano et al., 2004). These cells could be clearly differentiated from the single, elongated smooth muscle cells. Membrane potential recordings were then obtained from the freshly isolated ECs, using the perforated-patch configuration of the patch-clamp technique. Changes in [Ca²⁺]_i) were monitored by loading the cells with the Ca²⁺-sensitive dye fluo-4/AM and visualized using a confocal fluorescence microscope. Values are expressed as fluorescence intensity / baseline fluorescence intensity (F/F0). All data of the paired experiments are presented as mean ± SEM . The averaged data of one EC-sheet from one animal is considered n=1.

In sheets of ECs, ACh stimulated a reproducible hyperpolarization ( Em=38±4 mV, n=3). Apamin alone fully blocked the hyperpolarization, and under these conditions ACh stimulated depolarization. ACh also stimulated a rise in endothelial cell Ca²⁺ in the isolated sheets of ECs (F/F 0: Peak=2.48±0.44; at 2min=1.45±0.11; n=5), which was not altered in the presence of apamin (50nM) and TRAM-34 (1µM) (F/F₀: Peak=1.79±0.14, at 2min=1.27±0.10; n=5). The observation that membrane hyperpolarization did not drive endothelial cell Ca²⁺ influx was supported by the absence of any change in [Ca²⁺]_i) with high K⁺ (35mM) in unstimulated cells.

Together, these data show that in isolated ECs of the rat mesenteric resistance artery, ACh-mediated membrane hyperpolarization is evoked by activation of SK_Ca, but the driving force for Ca²⁺ influx into the ECs is unaltered by up to 40 mV change in membrane potential.

Crane, C.J. et al., (2003) J. Physiol., 553.1, 183-189
Takano, H. et al., (2004) J. Physiol., 556.3, 887-903

Supported by the Wellcome Trust