Ž . European Journal of Pharmacology 402 2000 119–128 www.elsevier.nlrlocaterejphar Role of gap junctions in endothelium-derived hyperpolarizing factor responses and mechanisms of K q -relaxation David Harris a , Patricia E.M. Martin b , W. Howard Evans b , David A. Kendall a , Tudor M. Griffith b , Michael D. Randall a, ) a School of Biomedical Sciences, E-Floor, UniÕersity of Nottingham Medical School, Queen’s Medical Centre, Nottingham NG7 2UH, UK b Departments of Diagnostic Radiology and Medical Biochemistry, CardioÕascular Sciences Group, UniÕersity of Wales College of Medicine, Cardiff, CF2 4XN, UK Received 10 February 2000; received in revised form 12 July 2000; accepted 17 July 2000 Abstract Ž . Ž . Ž . We have examined the effects of ouabain 1 mM , the gap junction inhibitors, 18 a-glycyrrhetinic acid 100 mM, N- piperidin-1-yl - Ž . Ž . Ž . 5- 4-chlorophenyl -1- 2,4-dichlorophenyl -4-methyl-1 H-pyrazole-3-carboxamide hydrochloride SR141716A; 10 mM and palmitoleic Ž . Ž . Ž . q acid 50 mM , and clotrimazole 10 mM against endothelium-derived hyperpolarizing factor EDHF -mediated and K -induced Ž . G Ž . vasorelaxations in the rat mesentery. In the presence of indomethacin 10 mM and 300-mM N nitro-L-arginine methyl ester L-NAME , Ž . carbachol caused EDHF-mediated relaxations R s85.3 "4.0% . In the presence of ouabain, these responses were substantially max Ž . reduced R s11.0 "2.3% . 18 a-glycyrrhetinic acid, SR141716A, palmitoleic acid and clotrimazole also significantly inhibited these max q q Ž . EDHF-mediated responses. K caused vasorelaxation of preparations perfused with K -free buffer R s73.7 "2.4% , which were max Ž . q reduced by 10-mM indomethacin R s56.4 "6.2% . K vasorelaxation was essentially abolished by endothelial denudation. Both max ouabain and 18 a-glycyrrhetinic acid opposed K q relaxations, however, neither SR141716A, clotrimazole nor palmitoleic acid had any Ž. effect. Direct cell–cell coupling via gap junctions was attenuated by ouabain, clotrimazole and palmitoleic acid. We conclude that: i that Ž. q gap junctional communication plays a major role in EDHF-mediated relaxations, ii that K -vasorelaxation is endothelium-dependent Ž q . Ž . thus, K is unlikely to represent an EDHF , and iii that the inhibitory actions of ouabain and clotrimazole on gap junctions might contribute towards their effects against EDHF. q 2000 Elsevier Science B.V. All rights reserved. Ž . q Keywords: Endothelium-derived hyperpolarizing factor EDHF ; Ouabain; 18a-Glycyrrhetinic acid; Gap junctions; K ; Endothelium 1. Introduction The identity of the endothelium-derived hyperpolarizing Ž factor EDHF; Feletou and Vanhoutte, 1988; Taylor and . Ž Weston, 1988; Garland et al., 1995 is controversial see . Mombouli and Vanhoutte, 1997 . There is now evidence that EDHF-type relaxations involve the transfer of a medi- ator from the endothelium to smooth muscle via myoen- Ž . dothelial gap junctions Chaytor et al., 1998 . EDHF-type responses are thus attenuated by connexin–mimetic pep- Ž tides Chaytor et al., 1998; Hutcheson et al., 1999; Dora et . al., 1999 , and by 18-a and 18-b glycyrrhetinic acids, ) Corresponding author. Tel.: q 44-115-9709484; fax: q 44-115- 9709259. Ž . E-mail address: michael.randall@nottingham.ac.uk M.D. Randall . Ž which inhibit gap junctions Davidson et al., 1986; Taylor . et al., 1998; Yamamoto et al., 1999 . Ž . Edwards et al. 1998 have also proposed that EDHF- type relaxations are explained by endothelial K q efflux Ž . Gordon and Martin, 1983 through apamin and charybdo- q Ž toxin-sensitive K channels Marchenko and Sage, 1996; . q Ž Doughty et al., 1999 , while vasorelaxation to K Katz and Linder, 1938; Bonaccorsi et al., 1977; Webb and Bohr, . q q 1978 is thought to be due to activation of Na rK- ATPases and opening of inward rectifier K q channels Ž Chen et al., 1972; Hirst and van Helden, 1982; Edwards and Hirst, 1988; McCarron and Halpern, 1990; Knot et al., . q 1996 . In support of their contention that K may repre- Ž . sent an EDHF, Edwards et al. 1998 reported that EDHF release was associated with endothelial K q efflux, which was sensitive to apamin and charybdotoxin. In addition, K q caused hyperpolarization via an ouabain-sensitive Na q rK q -ATPase and Ba 2q -sensitive inward rectifier K q 0014-2999r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. Ž . PII: S0014-2999 00 00512-4