Role of Heterocellular Gap Junctional Communication in Endothelium-Dependent Smooth Muscle Hyperpolarization: Inhibition by a Connexin-Mimetic Peptide Kim A. Dora,* Patricia E. M. Martin,† Andrew T. Chaytor,‡ W. Howard Evans,† Christopher J. Garland,* and Tudor M. Griffith‡ ,1 ‡Department of Radiology and †Department of Medical Biochemistry, Cardiovascular Sciences Research Group, University of Wales College of Medicine, Heath Park, Cardiff, CF4 4XN United Kingdom; and *Department of Pharmacology, University of Bristol, Bristol, BS8 1TD United Kingdom Received November 3, 1998 A synthetic connexin-mimetic peptide (Gap 27 pep- tide) was used to evaluate the contribution of gap junctional communication to smooth muscle re- sponses mediated by the endothelium-dependent ago- nist acetylcholine (ACh) in rabbit mesenteric arteries. Hyperpolarizations and relaxations to 0.1 and 1 M ACh observed in the presence of nitric oxide synthase and cyclooxygenase inhibition were markedly attenu- ated by the peptide at a concentration of 300 M, whereas the hyperpolarizing response to levcro- makalim, a K ATP channel opener, was unaffected. The peptide also attenuated intercellular transfer of Luci- fer yellow in confluent cultures of COS-7 cells, thus confirming its ability to modulate the permeability of gap junctions. The findings demonstrate that hetero- cellular gap junctional communication contributes to NO- and prostanoid-independent mechanisms of va- sorelaxation that are widely attributed to an endothelium-derived hyperpolarizing factor. © 1999 Academic Press Key Words: gap junctions; endothelium-dependent hyperpolarization; acetylcholine; levcromakalim; Lu- cifer yellow. NO- and prostanoid-independent vascular relax- ations are generally thought to be mediated by an endothelium-derived hyperpolarizing factor (EDHF) that diffuses from the endothelium, via the extracellu- lar space, to cause hyperpolarization and vasodilata- tion by opening smooth muscle K + channels (1–5). The existence of myoendothelial gap junctions in the vas- cular wall also suggests the possibility of direct elec- trical and chemical transfer of signals between the endothelium and adjacent smooth muscle cells. Indeed, recent studies indicate that heterocellular gap junc- tional communication plays a central role in NO- and prostanoid-independent relaxations induced by acetyl- choline (ACh), adenosine triphosphate (ATP) and cy- clopiazonic acid (CPA) in rabbit aorta, superior mesen- teric and iliac arteries (6, 7). In the present study, we have used intracellular recordings of smooth muscle cell membrane potential to study the effects of a short synthetic connexin- mimetic undecapeptide on the hyperpolarizing re- sponses evoked by ACh in rabbit mesenteric arteries. This peptide possesses sequence homology with a region of the second extracellular loop (Gap 27) of connexin subtypes found in the vascular wall, and is highly effective in interrupting cooperative cell-cell interactions such as the synchronous beating of em- bryonic cardiomyocytes (8), rhythmic smooth muscle contractile activity (9) and endothelium-dependent relaxations (6). It is likely that the peptide prevents accretion of free connexon hemichannels present in the cell membrane into gap junctions and/or perturbs the connexin-connexin interactions that maintain channel integrity in already-formed gap junctions (9). To assess the effect of Gap 27 peptide on direct cell-cell coupling we studied dye transfer of Lucifer yellow CH (LY), a small fluorescent tracer with a charge of -2 and molecular weight of 457 Da (10 –12) in cultured COS-7 cells, a monkey fibroblast cell line that endogenously expresses low levels of Cx43 (13, 14). To confirm the specificity of Gap 27 peptide against electrical events mediated via the endothe- lium, levcromakalim, which hyperpolarizes vascular 1 To whom correspondence should be addressed. Fax: 01222- 744726. E-mail: griffith@cardiff.ac.uk. Biochemical and Biophysical Research Communications 254, 27–31 (1999) Article ID bbrc.1998.9877, available online at http://www.idealibrary.com on 27 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.