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

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Abstract
1. Relaxing effect of loop diuretics, piretanide and furosemide in comparison with acetylcholine (ACh) was investigated in guinea-pig isolated mesenteric resistance arteries. 2. Concentration-response curves to ACh (0.001 - 10 microM) and diuretics (0.0001 - 1 microM) were constructed in noradrenaline (10 - 30 microM)-precontracted arteries incubated either in normal physiological salt solution (PSS) or in 30 mM KCl PSS (K-PSS). 3. 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 microM) reduced R(max) by 20% (P<0.01, n=7) and pD(2) by 10% (P<0.01). In contrast, indomethacin (10 microM) 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. 4. In PSS+L-NAME+indomethacin, inhibitors of small (SK(Ca); apamin, 0.1 microM) and large (BK(Ca); iberiotoxin and charybdotoxin, 0.1 microM) 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. 5. 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. 6. 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. 7. 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.