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We investigated the effect of HA on the regulation of endothelial nitric oxide (NO) synthase (eNOS) in human umbilical vein endothelial cells (HUVECs) to evaluate the involvement of eNOS and related factors in peripheral vascular impairment with HA exposure. Treatment of HUVECs with HA induced upregulation of eNOS. This result coincides with those of previous studies. Furthermore this is the first study to report that HA induces upregulation of heat shock protein (Hsp)90\u03b1, Hsp90\u03b2, eNOS phosphorylation at Ser1177, and eNOS phosphorylation at Thr495, as compared to that in the control. In contrast, treatment with BAPTA, an intracellular Ca<jats:sup>2+<\/jats:sup> chelator, inhibited upregulation of these proteins induced by HA. This study demonstrates that HA treatment leads to increases in both Hsp90\u03b1 and Hsp90\u03b2 proteins and indicates that Hsp90\u03b1 leads to eNOS phosphorylation at Ser1177 and that Hsp90\u03b2 leads to eNOS phosphorylation at Thr495, respectively. Upregulation of eNOS, Hsp90\u03b1, and Hsp90\u03b2 in HUVECs is regulated by intracellular Ca<jats:sup>2+<\/jats:sup> accumulation induced by HA. These results suggest that upregulation of eNOS phosphorylation at Ser1177 and eNOS phosphorylation at Thr495 produce NO and superoxide anions, respectively, resulting in generation of peroxynitrite, which causes impairment of vascular endothelial cells. \u00a9 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 223\u2013231, 2015.<\/jats:p>","DOI":"10.1002\/tox.21888","type":"journal-article","created":{"date-parts":[[2013,7,8]],"date-time":"2013-07-08T13:27:03Z","timestamp":1373290023000},"page":"223-231","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Humic acid induces the endothelial nitric oxide synthase phosphorylation at Ser1177 and Thr495 Via Hsp90\u03b1 and Hsp90\u03b2 upregulation in human umbilical vein endothelial cells"],"prefix":"10.1002","volume":"30","author":[{"given":"Masato","family":"Tanaka","sequence":"first","affiliation":[{"name":"Laboratory of Environmental Health Sciences Graduate School of Health Sciences Hokkaido University Sapporo 060\u20130812 Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miki","family":"Miyajima","sequence":"additional","affiliation":[{"name":"Laboratory of Environmental Health Sciences Graduate School of Health Sciences Hokkaido 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