EDHF is defined as the non-nitric oxide (NO) and non-prostacyclin (PGI₂) substance that mediates endothelium-dependent hyper-polarization (EDH) of vascular smooth muscle cells (VSMC). Many candidates have been proposed to be EDHF according to the type of tissue and species studied. The aim of the present work was to characterise the mechanism of action of EDHF in the mouse gracilis artery and to reveal its possible alterations in a hypercholesterolemic (HC) mouse model.

Experiments were conducted on pressurised (80 mmHg) isolated gracilis arteries of 3 m/o C57BL/6 wild type (WT) mice (27±1g) and HC LDLR-null mice expressing the human apolipoprotein B-100 (31±1g) (Sanan et al., 1998) using a method previously described (Nguyen et al., 1999). Dilation to acetylcholine (ACh; 1nM to 30µM) was tested on vessels pre-contracted with phenylephrine (1 to 10µM). In all experiments, L-NNA (100µM) and indomethacin (10µM) were present in the oxygenated physiological salt solution (PSS; 37°C, pH 7.4) to prevent NO and prostanoid formation. When used, Ba²⁺ (30µM), ouabain (Oua, 1mM), apamin (Apa, 1µM), charybdotoxin (Chtx; 100 nM) and 17-octadecynoic acid (17-ODYA; 10µM) were added to the bath 30 minutes before the start of the experiment. Dilations to Ach were expressed as % of the maximal dilation obtained in Ca²⁺-free PSS. Results are mean±SEM. P<0.05 was accepted as significant (ANOVA, Fisher's F test).

In HC gracilis arteries, the maximal dilation (95±2%) to ACh was increased (P<0.05) when compared to WT arteries (86±3%). In both groups, the maximum dilation was partially diminished (P<0.05) either by Apa (WT: 73±4%; HC: 55±12%), a small conductance Ca²⁺-dependent K⁺ channel (SK_Ca) blocker, or Chtx (WT: 54±4%; HC: 74±10%), an intermediate conductance Ca²⁺-dependent K⁺ channel (IK_Ca) blocker. Only the combination of Apa plus Chtx blocked (P<0.05) EDHF dilation (WT: 22±4%; HC: 25±5%). Thus, activation of both endothelial SK_Ca and IK_Ca channels are essential to EDHF-dependent dilation. In WT mice arteries, the EDHF dilation was also blocked (P<0.05) by Oua (19±7%), a Na⁺/K⁺ ATPase pump inhibitor. Ba²⁺, an inward rectifier K⁺ channel (K_IR) blocker, had no effect and did not further improve the block induced by Oua alone. In contrast, EDHF dilation was partially blocked by Ba²⁺ (73±9%; P<0.05), Oua (75±10%; P<0.05) or their combination (54±11%; P<0.05) in HC mice arteries. In addition, the cytochrome (Cyt) P450 inhibitor 17-ODYA diminished ACh-induced EDHF-dependent dilation (42%±11%; P<0.05), while its combination with Ba²⁺ and Oua prevented ACh-induced dilation of isolated HC mice arteries (5±3%; P<0.05). 17-ODYA had no effect in WT mice.

In both groups, ACh stimulates endothelial SK_Ca and IK_Ca channels; this increases smooth muscle Na⁺/K⁺ ATPase pump activity, which solely accounts for EDHF-dependent dilation in WT mice. In HC mice, Na⁺/K⁺ ATPase pump, K_IRand a Cyt P450 metabolite contribute to EDHF-mediated dilation.

Nguyen et al. (1999). Cardiovasc Res, 43, 755-761.
Sanan et al. (1998). Proc Natl Acad Sci USA, 95, 4544-4549.