Interactions between endothelium‐derived relaxing factors in the rat hepatic artery: focus on regulation of EDHF
Authors: Zygmunt Peter M. , Plane Frances , Paulsson Marie , Garland Christopher J. , Högestätt Edward D. 
Published: January 2005

In rat isolated hepatic arteries contracted with phenylephrine, acetylcholine and the calcium ionophore A23187 each elicit endothelium‐dependent relaxations, which involve both nitric oxide (NO) and endothelium‐derived hyperpolarizing factor (EDHF). However, the contribution of prostanoids to these responses, and the potential interaction between EDHF and other endothelium‐derived relaxing factors have not been examined.

In the presence of the NO synthase inhibitor NG‐nitro‐L‐arginine (L‐NOARG, 0.3 mM) and a mixture of charybdotoxin (0.3 μM) and apamin (0.3 μM), inhibitors of the target potassium (K) channel(s) for EDHF, acetylcholine and A23187 each induced a concentration‐dependent and almost complete relaxation, which was abolished in the additional presence of indomethacin (10 μM). Thus, in addition to EDHF and NO, a relaxing factor(s) generated by cyclo‐oxygenase (COX) contributes to endothelium‐dependent relaxation in the rat hepatic artery.

The resting membrane potentials of endothelium‐intact and endothelium‐denuded vascular segments were −57 mV and −52 mV, respectively (P>0.05). In intact arteries, the resting membrane potential was not affected by L‐NOARG plus indomethacin, but reduced to −47 mV in the presence of charybdotoxin plus apamin. Acetylcholine and A23187 (10 μM each) elicited a hyperpolarization of 13 mV and 15 mV, respectively. The hyperpolarization induced by these agents was not affected by L‐NOARG plus indomethacin (12 mV and 14 mV, respectively), but reduced in the presence of charybdotoxin plus apamin (7 mV and 10 mV, respectively), and abolished in the combined presence of charybdotoxin, apamin and indomethacin.

The NO donor 3‐morpholino‐sydnonimine (SIN‐1) induced a concentration‐dependent relaxation, which was unaffected by charybdotoxin plus apamin, but abolished by the selective soluble guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxaline‐1‐one (ODQ, 10 μM). SIN‐1 (10 μM) did not alter the resting membrane potential in endothelium‐denuded vascular segments.

The COX‐dependent relaxation induced by acetylcholine was abolished following exposure to 30 mM KCl, but unaffected by glibenclamide (10 μM). The prostacyclin analogue iloprost induced a concentration‐dependent relaxation, which was also abolished in 30 mM KCl and unaffected by the combined treatment with glibenclamide, charybdotoxin and apamin. Iloprost (10 μM) induced a glibenclamide‐resistant hyperpolarization (8 mV with and 9 mV without glibenclamide) in endothelium‐denuded vascular segments.

Exposure to SIN‐1 or iloprost did not affect the EDHF‐mediated relaxation induced by acetylcholine (i.e. in the presence of L‐NOARG and indomethacin). Replacement of L‐NOARG with the NO scavenger oxyhaemoglobin (10 μM) or the soluble guanylate cyclase inhibitor ODQ (10 μM) or methylene blue (10 μM), which all significantly inhibited responses to endothelium‐derived NO, did not affect the acetylcholine‐induced relaxation in the presence of indomethacin, indicating that endogenous NO also does not suppress EDHF‐mediated responses.

These results show that, in addition to EDHF and NO, an endothelium‐derived hyperpolarizing factor(s) generated by COX contributes significantly to endothelium‐dependent relaxation in the rat heptic artery. Neither this factor nor NO seems to regulate EDHF‐mediated responses. Thus, EDHF does not serve simply as a ‘back‐up’ system for NO and prostacyclin in this artery. However, whether EDHF modulates the NO and COX pathways remains to be determined.