Endothelium-derived hyperpolarizing factor but not NO reduces smooth muscle Ca2+ during acetylcholine-induced dilation of microvessels

1 We hypothesized that nitric oxide (NO) and the endothelium-dependent hyperpolarizing factor (EDHF) may dilate microvessels by different cellular mechanisms, namely Ca2+-desensitization versus decrease in intracellular free calcium. 2 Effects of acetylcholine (ACh) and the NO donors sodium nitroprusside (SNP, 0.1 - 10 mu mol l(-1)) and S-Nitroso-N-acetyl-D,L-penicillamine (SNAP, 0.01-10 mu mol l(-1)) on intracellular calcium ([Ca2+](i), fura 2) and vascular diameter (videomicroscopy) were studied in isolated resistance arteries from hamster gracilis muscle (194+/-12 pm) pretreated with indomethacin and norepinephrine. Membrane potential changes were determined using 1,3-dibutylbarbituric acid trimethineoxonol (DiBAC(4)(3)). 3 ACh (0.1 and 1 mu mol l(-1))-induced dilations were associated with a [Ca2+](i) decrease (by 13 +/- 3 and 32 +/- 4%) and hyperpolarization of vascular smooth muscle (VSM, by 12 +/- 1% at 1 mu mol l(-1) ACh). N-omega-nitro-L-arginine (L-NA, 30 mu mol l(-1)) partially inhibited the dilation but did not affect VSM [Ca2+](i) decreases or hyperpolarization. In contrast, the K-Ca channel inhibitors tetrabutylammonium (TBA, 1 mmol l(-1)) and charybdotoxin (ChTX, 1 mu mol l(-1)) abolished the ACh-induced [Ca2+](i) decrease and the hyperpolarization in VSM while a significant dilation remained (25 and 40%). This remaining dilation was abolished by L-NB. ChTX did not affect [Ca2+](i) increase and hyperpolarization in endothelial cells. SNP- or SNAP-induced dilations were not associated with decreases in VSM [Ca2+](i) or hyperpolarization although minor transient decreases in VSM [Ca2+](i) were observed at high concentrations. 4 These data suggest that ACh-induced dilations in microvessels are predominantly mediated by a factor different from NO and PGI(2), presumably EDHF. EDHF exerts dilation by activation of K-Ca channels and a subsequent decrease in VSM [Ca2+](i), No dilates the microvessels in a calcium-independent manner.