Concentration of Na+/K+-ATPase to holes in the internal elastic lamina underlies part of the EDHF effect in rat mesenteric arteries

N.T. Gallagher, M.M. Spitaler, K.A. Dora & C.J. Garland, Department of Pharmacy and Pharmacology, University of Bath BA2 7AY

Smooth muscle hyperpolarization and relaxation evoked by EDHF (endothelium–dependent hyperpolarizing factor) is underpinned by the activation of both SKCa and IKCa channels in the endothelium. In the rat mesenteric artery, the contribution from each channel-type can be distinguished separately (Crane et al, 2003). Such functional separation correlates with a focused concentration of IKCa within endothelial cell projections through the internal elastic lamina. These projections form myoendothelial gap junctions (MEGJs) with the adjacent smooth muscle (Mather et al, 2005;Sandow et al 2006). In the mesenteric artery, EDHF effects are mediated by K+ released from the endothelium and hyperpolarization spreading through MEGJs (Mather et al, 2005; Edwards et al, 1998). We have now investigated whether the input to EDHF responses by K+ may preferentially arise from the IKCa concentrated close to the smooth muscle.

Male Wistar rats (circa 250g) were killed (schedule 1 method, Animals (Scientific Procedures) Act 1986). Small (1-2mm long) segments of mesenteric artery were isolated and mounted in a myograph (Crane et al, 2003). All experiments were performed at 37ºC with arteries exposed continually to the NO synthase inhibitor, L-NAME (100μM). ACh evoked EDHF-relaxation in arteries stimulated sub-maximally with phenylephrine (circa 0.5μM). Immunohistochemistry was carried out on arteries isolated and pressurised to 50mmHg before fixation.

ACh (0.01-3 μM) stimulated concentration-dependent EDHF relaxation, which was unaffected by the Na+/K+-ATPase inhibitor 100μM ouabain (ED50 39nM, maximum relaxation 100±1.1%, n=4). However, in the presence of the gap-junction uncoupler carbenoxolone (100 µM), which alone had minimal effect, EDHF relaxation was abolished by ouabain. The ability to block EDHF relaxation in the presence of carbenoxolone was mimicked by the IKCa blocker, TRAM-34 (1μM) but not apamin (50nM, SKCa blocker, each n=13). Immunohistochemistry revealed a clear concentration of the Na+/K+-ATPase aligned with and within holes through the internal elastic lamina.

These data reveal discrete localization of the Na+/K+-ATPase in regions known to form MEGJs and to contain a high density of IKCa. Although normally block of EDHF-relaxation requires the simultaneous addition of SKCa and IKCa inhibitors, either TRAM-34 or ouabain alone can block EDHF relaxation, but only if a gap junction uncoupling agent is present to limit spread of hyperpolarization through MEGJs. This suggests that K+ efflux through IKCa in the region of endothelial cell projections facilitates relaxation via the Na+/K+-ATPase in the rat mesenteric artery.

Crane et al, 2003. J. Physiol. 553, 1, 183-189
Edwards et al, 1998. Nature 396, 269-272
Mather et al, 2005 Circ. Res 97, 399-407
Sandow et al, 2006 J. Anat. In the Press.

Supported by the Wellcome Trust