Delphinidin-3-rutinoside relaxes the bovine ciliary smooth muscle through activation of ET_B receptor and NO/cGMP pathway

Delphinidin-3-rutinoside (D3R) is the major anthocyanin component in blackcurrant (Ribes nigrum L.) fruits. We investigated the relaxation mechanism of D3R in bovine ciliary smooth muscle (CM). D3R at a concentration of 10 ^-5 m produced a sustained and progressive relaxation during the contraction induced by endothelin (ET)-1 in the bovine CM specimens. After the pre-treatment with D3R, the anthocyanin exerted an inhibitory effect on the ET-1-induced contraction with a concomitant increase in cyclic GMP production and decreased phosphorylation ratio of myosin light chain (RLC). The inhibitory effect of D3R was significantly attenuated in the presence of either N ^G-nitro-l-arginine (NOARG) as a nitric oxide synthase (NOS) inhibitor, carboxy-PTIO as a NO scavenger, ODQ as an inhibitor of guanylyl cyclase, or BQ788 as a selective ET_B receptor antagonist. The atteuation with NOARG was reversed by the addition of excess l-arginine. However, iberiotoxin as a Ca²⁺-activated K⁺ channel inhibitor, propranolol as a β-adrenoceptor antagonist, and indomethacin as a cyclooxygenase inhibitor failed to modify the inhibitory effect of D3R. Scatchard plot analysis revealed that the [¹²⁵I]-ET-1 binding site constituted a single population with Kd of 54.5±4.6 nm and maximum binding site (B_max) of 168.4±25.4 fmol/mg protein in the ciliary epithelium (CE), and Kd of 141.7±18.0 nm and B_max of 357.7±35.8 fmol/mg protein in CM. [¹²⁵I]-ET-1 binding was completely displaced by BQ788 with K_i values of 56.7±10.8 pm in CE and 93.4±23.3 pm in CM. Meanwhile, partial displacement (approximately 40%) was observed by BQ123 as a selective ET_A receptor antagonist in both preparations. ET_B receptor was predominant subtype in CE and CM, whereas kinetics of the binding was different in two preparations. These results suggest that D3R possibly stimulates ET_B receptors to produce/release NO, and results in an inhibition of myosin RLC phosphorylation and/or acceleration of dephosphorylation, thereby causing relaxation and producing an inhibitory effect on the ET-1-induced contraction in the bovine CM.