Chronic treatment of male rats with daidzein and 17 β ‐oestradiol induces the contribution of EDHF to endothelium‐dependent relaxation

We investigated the effect of chronic (7 days) treatment of male rats with the isoflavone daidzein (0.2 mg kg −1 sc per day) or 17 β ‐oestradiol (0.1 mg kg −1 sc per day) on the contribution of nitric oxide (NO), prostaglandins and endothelium‐derived hyperpolarising factor (EDHF) to endothelium‐dependent relaxation of isolated aortic rings. The sensitivity and maximum relaxation to acetylcholine (ACh) were significantly greater in aortic rings from rats treated with daidzein or 17 β ‐oestradiol, in comparison to vehicle‐treated rats. Inhibition of nitric oxide synthase with N ‐nitro‐ L ‐arginine ( L ‐NOARG) abolished ACh‐induced relaxation in the aortae from vehicle‐treated rats, but only attenuated relaxation in aortae from daidzein or 17 β ‐oestradiol‐treated rats. The presence of haemoglobin in addition to L ‐NOARG did not cause any further inhibition of relaxation. The cyclooxygenase inhibitor indomethacin had no effect on endothelium‐dependent relaxation in aortae from any treatment group. Charybdotoxin (ChTX), which blocks large‐conductance calcium‐activated potassium channels (BK Ca ) and intermediate‐conductance calcium‐activated potassium channels (IK Ca ), plus apamin, which blocks small‐conductance calcium‐activated potassium channels (SK Ca ), but not iberiotoxin, which only blocks BK Ca , attenuated endothelium‐dependent relaxation of aortae from daidzein or 17 β ‐oestradiol‐treated rats. Blockade of K Ca channels had no effect on the responses to ACh in aortae from vehicle‐treated rats. In aortae from daidzein‐ or 17 β ‐oestradiol‐treated rats, endothelium‐dependent relaxation was also attenuated by inhibition of cytochrome P 450 (CY P 450) epoxygenase with 6‐(2‐propargylloxyphenyl)hexanoic acid (PPOH) or inhibition of K IR channels and Na + /K + ‐ATPase with barium and oubain, respectively. This study demonstrates that endothelium‐dependent relaxation of male rat aorta is normally entirely mediated by NO, whereas treatment with daidzein or 17 β ‐oestradiol stimulates a contribution from a non‐NO, nonprostaglandin factor acting through the opening of SK Ca and IK Ca channels, and involving activation of Na/K‐ATPase, K IR and CY P 450 epoxygenase. This pattern of sensitivity to the tested inhibitors is consistent with the contribution of EDHF to relaxation. Thus, EDHF contributes to the enhanced endothelium‐dependent relaxation that is observed after chronic treatment with the phytoestrogen daidzein or with 17 β ‐oestradiol.British Journal of Pharmacology (2004) 141 , 322–328. doi: 10.1038/sj.bjp.0705603