Bioassay of endothelium-derived hyperpolarizing factor with abolishment of nitric oxide and the role of gap junctions in the porcine coronary circulation

Previous bioassays of endothelium-derived hyperpolarizing factor (EDHF) were partially related to the residual nitric oxide (NO) resistant to NO synthase inhibitors. Further, the role of gap junctions in EDHF is controversial. We performed a bioassay of EDHF with abolishment of NO production by using the NOS inhibitor N^G-nitro-L-arginine (L-NNA) plus the NO scavenger hemoglobin (Hb) and examined the role of gap junctions/K⁺ channels related to EDHF in porcine coronary large and microarteries in an organ chamber/myograph. Bradykinin (BK)-induced EDHF-mediated relaxation and hyperpolarization (by a microelectrode) were studied with indomethacin (7 μM), L-NNA (300 μM), and Hb (20 μM). NO concentrations ([NO]) were measured electrochemically. In large coronary arteries, BK increased [NO] (9.3±1.7 nM vs. 166.7±18.3 nM, P<0.01) that was significantly reduced by L-NNA (49.3±7.8 nM, P<0.01) and eliminated by Hb, and hyperpolarized the downstream endothelium-denuded artery by 9.0±1.4 mV (P<0.01) that was reduced but not abolished by L-NNA and Hb (5.6±0.7 mV, P<0.01). In microarteries, elimination of NO decreased but did not abolish the hyperpolarization (-63.8±1.5 mV vs. -56.9±1.6 mV, P=0.01) and relaxation (70.2±5.7% vs. 42.3±4.4%, P<0.01). Charybdotoxin (0.1 μM) and apamin (0.5 μM), but neither glybenclimide (3 μM) nor gap junction inhibitors (18α-glycyrrhetinic acid, 1-heptanol, and gap 27) reduced the EDHF-mediated response. We conclude that in porcine coronary arteries, with the abolishment of NO by NOS inhibitors plus the NO scavenger Hb, the non-NO EDHF exists and is transferable from an endothelium-intact artery to an endothelium-denuded artery. Compared with the significant involvement of calcium-activated K⁺ channels, the gap junctions may only play a minimal role in the EDHF-mediated response even in the microcirculation of the porcine coronary artery.