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

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Abstract
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. 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)). 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). Nω-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 KCa 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. 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 KCa channels and a subsequent decrease in VSM [Ca2+]i, NO dilates the microvessels in a calcium-independent manner.