Role of EDHF in the vasodilatory effect of loop diuretics in guinea‐pig mesenteric resistance arteries

Relaxing effect of loop diuretics, piretanide and furosemide in comparison with acetylcholine (ACh) was investigated in guinea‐pig isolated mesenteric resistance arteries. Concentration‐response curves to ACh (0.001–10 μ M ) and diuretics (0.0001–1 μ M ) were constructed in noradrenaline (10–30 μ M )‐precontracted arteries incubated either in normal physiological salt solution (PSS) or in 30 m M KCl PSS (K‐PSS). In PSS, maximal relaxations (R max ) and pD 2 to ACh were 87±2% and 7.1±0.1 ( n =10). L ‐N G ‐nitro‐arginine methyl ester ( L ‐NAME, 100 μ M ) reduced R max by 20% ( P <0.01, n =7) and pD 2 by 10% ( P <0.01). In contrast, indomethacin (10 μ M ) increased R max by 19% ( P <0.01, n =8) and pD 2 by 10% ( P <0.05). Combination of L ‐NAME+indomethacin reversed the effect observed with either of these inhibitors used alone. In K‐PSS, R max was attenuated by 40% ( P <0.001, n =6) compared to PSS. L ‐NAME reduced R max by 65% ( P <0.01, n =5) and increased pD 2 by 15 fold. L ‐NAME+indomethacin suppressed the resistant relaxation. In PSS+ L ‐NAME+indomethacin, inhibitors of small (SK Ca ; apamin, 0.1 μ M ) and large (BK Ca ; iberiotoxin and charybdotoxin, 0.1 μ M ) conductance Ca 2+ ‐sensitive K − ‐channels used alone had little effect on the ACh‐response. Combination of apamin+iberiotoxin reduced R max by 40% ( P <0.05, n =7) while apamin+charybdotoxin fully abolished the resistant relaxation. In PSS, piretanide and furosemide induced relaxation with R max : 89±3% vs 84±5% and pD 2 : 8.5±0.1 vs 7.7±0.2 ( P <0.01) for piretanide ( n =11) and furosemide ( n =10), respectively. Endothelial abrasion suppressed relaxation to diuretics. L ‐NAME and indomethacin used alone or in combination did not significantly modify the response to diuretics. In K‐PSS, piretanide‐induced relaxation was abolished whereas that to furosemide was reduced by 70% ( P <0.001, n =9) compared to PSS and was suppressed by L ‐NAME+indomethacin. In PSS+ L ‐NAME+indomethacin, apamin slightly reduced relaxation to diuretics whereas charybdotoxin or iberiotoxin abolished the response. These results indicate that ACh‐evoked relaxation is mediated by both NO/PGl 2 ‐dependent and ‐independent mechanisms. The EDHF‐dependent component relies on activation of Ca 2+ ‐activated K + channels, is sensitive to a combination of apamin+charybdotoxin and to a smaller degree to a combination of apamin+iberiotoxin. Loop diuretic‐induced relaxation is endothelium‐dependent, appears to be mediated by NO, PGl 2 and EDHF for furosemide and EDHF only for piretanide. For the two diuretics, opening of BK Ca channels may be involved in the relaxation.British Journal of Pharmacology (2000) 131 , 1211–1219; doi: 10.1038/sj.bjp.0703693