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 μmol l^-1) and S-Nitroso-N-acetyl-D,L-penicillamine (SNAP, 0.01-10 μmol 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 μm) pretreated with indomethacin and norepinephrine. Membrane potential changes were determined using 1,3-dibutylbarbituric acid trimethineoxonol (DiBAC₄(3)). 3. ACh (0.1 and 1 μmol 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 μmol l^-1 ACh). N(ω)-nitro-L-arginine (L-NA, 30 μmol 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 μmol 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-NA. 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 PGI₂, 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.