title: Humic acid induces the endothelial nitric oxide synthase phosphorylation at Ser1177 and Thr495 Via Hsp90α and Hsp90β upregulation in human umbilical vein endothelial cells.
abstract: Humic acid (HA) has been implicated as a contributory factor for blackfoot disease, which is an endemic peripheral vascular disease. 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)90α, Hsp90β, eNOS phosphorylation at Ser1177, and eNOS phosphorylation at Thr495, as compared to that in the control. In contrast, treatment with BAPTA, an intracellular Ca(2+) chelator, inhibited upregulation of these proteins induced by HA. This study demonstrates that HA treatment leads to increases in both Hsp90α and Hsp90β proteins and indicates that Hsp90α leads to eNOS phosphorylation at Ser1177 and that Hsp90β leads to eNOS phosphorylation at Thr495, respectively. Upregulation of eNOS, Hsp90α, and Hsp90β in HUVECs is regulated by intracellular Ca(2+) 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.
authors.affiliation: Laboratory of Environmental Health Sciences, Graduate School of Health Sciences, Hokkaido University, Sapporo, 060-0812, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Tanaka
authors.forename: Miki
authors.initials: M
authors.lastname: Miyajima
authors.forename: Naoko
authors.initials: N
authors.lastname: Hishioka
authors.forename: Ryo
authors.initials: R
authors.lastname: Nishimura
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Kihara
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Hosokawa
authors.forename: Masaaki
authors.initials: M
authors.lastname: Kurasaki
authors.forename: Shunitz
authors.initials: S
authors.lastname: Tanaka
authors.forename: Takeshi
authors.initials: T
authors.lastname: Saito
mesh_terms: D000328:Adult; D002118:Calcium / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D018841:HSP90 Heat-Shock Proteins / Q000096:biosynthesis*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D006812:Humic Substances / Q000032:analysis / Q000633:toxicity*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000378:metabolism; D010766:Phosphorylation; D011247:Pregnancy; D012694:Serine / Q000378:metabolism*; D012987:Soil / Q000737:chemistry; D013912:Threonine / Q000378:metabolism*
chemical_list: D002614:Chelating Agents; D018841:HSP90 Heat-Shock Proteins; C533585:HSP90AA2P protein, human; C493165:HSP90AB1 protein, human; D006812:Humic Substances; D012987:Soil; D030421:Peroxynitrous Acid; D013912:Threonine; D012694:Serine; D004533:Egtazic Acid; D007770:L-Lactate Dehydrogenase; D052250:Nitric Oxide Synthase Type III; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium
keywords: Ca2+; eNOS; eNOS phosphorylation; heat shock protein 90; humic acid