The possible pathway for Ca2+ entry into endothelial cells is regarded as non-specific cation channels (Nilius, 1990). subsequent application of thapsigargin (or NaF) failed to increase [Ca2+]i. In arterial rings precontracted with phenylephrine, NaF produced endothelium-dependent relaxation followed by Pitolisant sustained contraction even in the presence of L-NOARG and indomethacin. The relaxant response was abolished by high K+ or cyclopiazonic acid. These results indicate that NaF causes endothelium-dependent hyperpolarization, thereby leading to smooth muscle relaxation of rat mesenteric artery. This action appears to be mediated by the promotion of Ca2+ influx into endothelial cells that can be triggered by the emptying of intracellular Ca2+ stores, as proposed for those of thapsigargin and cyclopiazonic acid. for 10?min in M199 solution (Boehringer, Mannheim, Germany), the pellet of endothelial cells was purified from this suspension, resuspended in M199 solution with Earle’s salts, supplemented with 100?IU?ml?1 penicillin G, 100?g?ml?1 streptomycin and 20% newborn calf serum (GIBCO, New York, NY, U.S.A.), then aliquoted into polybiphenyl dishes fixed on 1010-mm glass cover slips, and incubated at 37C in 5% CO2 for 2 days. The medium was renewed every day. Cytosolic Ca2+ concentration ([Ca2+]i) in endothelial cells adhering the glass cover slips was measured as previously described (Watanabe values less than 0.05 were considered significant. Results Endothelium-dependent hyperpolarization by NaF The resting membrane potentials of vascular smooth muscle cells in rat mesenteric artery were ?52.10.3?mV (phosphatase inhibition. However, okadaic acid and calyculin A, both of which are potent and highly selective inhibitors of protein phosphatases (Takai a common mechanism. Thapsigargin and cyclopiazonic acid deplete the rapidly exchanging intracellular Ca2+ stores by blocking the refilling of Ca2+ stores, possibly due to inhibition of activity of the Ca2+-pump ATPase located on the endoplasmic reticulum (Georger et Pitolisant al., 1988; Seidler et al., 1989; Thastrup et al., 1990). Based on the hypothesis referred to as the capactitative model (Putney, 1990), depletion of intracellular Ca2+ stores is thought Pitolisant to trigger Ca2+ influx through some unknown mechanism (Jakob, 1990; Byron et al., 1992; Hoth & Penner, 1992). Thus, we have proposed that both thapsigargin and cyclopiazonic acid deplete intracellular Ca2+ stores in endothelial cells and the empty of the Ca2+ stores generates an intracellular signal to trigger Ca2+ influx from the extracellular medium, thereby leading to the production and release of EDHF (Fukao et al., 1995). In view of the notion that the actions of Pitolisant NaF are very similar to those of thapsigargin and cyclopiazonic acid, the same mechanism may operate for endothelium-dependent hyperpolarization and relaxation induced by NaF. When intracellular Ca2+ stores had been depleted with Pitolisant thapsigargin or cyclopiazonic acid, NaF-induced hyperpolarization did no longer occur. In addition, after treatment with cyclopiazonic acid, NaF failed to produce L-NOARG- and indomethacin-resistant relaxation. These findings suggest that the release of EDHF caused by NaF is dependent on the extent of filling of intracellular Ca2+ stores in endothelial cells. Nevertheless, the hyperpolarizing response to NaF was not observed in Ca2+-free medium, in which ACh generated a transient hyperpolarization associated with Ca2+ release from intracellular stores (Fukao et al., 1995; 1997a). The possible pathway for Ca2+ entry into endothelial cells is thought to be nonspecific cation channels (Nilius, 1990). It has been shown that depletion of intracellular Ca2+ stores with thapsigargin or cyclopiazonic acid activates nonspecific cation channels in human umbilical vein endothelial cells (Gericke et al., 1993; Zhang et al., 1994). In the presence of Ni2+, NaF failed to generate hyperpolarization and ACh produced only a transient hyperpolarization, findings which were the same as those obtained in Ca2+-free medium. This could be explained by assuming that Ni2+ may block the Ca2+ influx pathway through nonspecific cation channels. Therefore, we propose that NaF depletes intracellular Ca2+ stores in endothelial cells possibly due to inhibition of endoplasmic reticulum Ca2+-pump ATPase activity and the emptying of the Ca2+ stores triggers the promotion of Ca2+ influx through nonspecific cation channels. Opening of the channels could supply sufficient Ca2+ into the endothelial cells to initiate the production and release of EDHF. In conclusion, this study showed that NaF elicited endothelium-dependent hyperpolarization and relaxation in rat mesenteric artery. The hyperpolarization and the large part of the relaxation were due to release of EDHF. A striking similarity with the XCL1 effects of thapsigargin and cyclopiazonic acid suggests that the mechanism may be related to increased Ca2+ influx into endothelial cells triggered by depletion of intracellular Ca2+ stores due to inhibition of endoplasmic reticulum Ca2+-pump ATPase activity. Acknowledgments The authors wish to thank Miss Megumi Nakadate for her technical assistance. This work was supported in part by a Grant-in-Aid for Science Research from the Ministry of Education, Science,.
