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(Circulation Research. 2005;97:399.)
© 2005 American Heart Association, Inc.

Integrative Physiology

Rapid Endothelial Cell–Selective Loading of Connexin 40 Antibody Blocks Endothelium-Derived Hyperpolarizing Factor Dilation in Rat Small Mesenteric Arteries

Simon Mather, Kim A. Dora, Shaun L. Sandow, Polly Winter, Christopher J. Garland

From the Department of Pharmacy and Pharmacology (S.M., K.A.D., S.L.S., P.W., C.J.G.), University of Bath, UK; and the Department of Physiology and Pharmacology (S.L.S.), University of New South Wales, Sydney, Australia.

Correspondence to C.J. Garland, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 6AD, UK. E-mail c.j.garland{at}bath.ac.uk

In resistance arteries, spread of hyperpolarization from the endothelium to the adjacent smooth muscle is suggested to be a crucial component of dilation resulting from endothelium-derived hyperpolarizing factor (EDHF). To probe the role of endothelial gap junctions in EDHF-mediated dilation, we developed a method, which was originally used to load membrane impermeant molecules into cells in culture, to load connexin (Cx)-specific inhibitory molecules rapidly (15 minutes) into endothelial cells within isolated, pressurized mesenteric arteries of the rat. Validation was achieved by luminally loading cell-impermeant fluorescent dyes selectively into virtually all the arterial endothelial cells, without affecting either tissue morphology or function. The endothelial monolayer served as an effective barrier, preventing macromolecules from entering the underlying smooth muscle cells. Using this technique, endothelial cell loading either with antibodies to the intracellular carboxyl-terminal region of Cx40 (residues 340 to 358) or mimetic peptide for the cytoplasmic loop (Cx40; residues 130 to 140) each markedly depressed EDHF-mediated dilation. In contrast, multiple antibodies directed against different intracellular regions of Cx37 and Cx43, and mimetic peptide for the intracellular loop region of Cx37, were each without effect. Furthermore, simultaneous intra- and extraluminal incubation of pressurized arteries with inhibitory peptides targeted against extracellular regions of endothelial cell Cxs (⁴³Gap 26, ⁴⁰Gap 27, and ^37,43Gap 27; 300 µmol/L each) for 2 hours also failed to modify the EDHF response. High-resolution immunohistochemistry localized Cx40 to the end of endothelial cell projections at myoendothelial gap junctions. These data directly demonstrate a critical role for Cx40 in EDHF-mediated dilation of rat mesenteric arteries.

Key Words: endothelium-derived hyperpolarizing factor • myoendothelial gap junctions • endothelium-dependent dilation • acetylcholine • connexin 40

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