title: NOS inhibition synchronizes calcium oscillations in human adipose tissue-derived mesenchymal stem cells by increasing gap-junctional coupling.
abstract: Adipose tissue-derived mesenchymal stem cells (ASCs) are a promising stem cell source for cell transplantation. We demonstrate that undifferentiated ASCs display robust oscillations of intracellular calcium [Ca(2+) ](i) which may be associated with stem cell maintenance since oscillations were absent in endothelial cell differentiation medium supplemented with FGF-2. [Ca(2+) ](i) oscillations were dependent on extracellular Ca(2+) and Ca(2+) release from intracellular stores since they were abolished in Ca(2+) -free medium and in the presence of the store-depleting agent thapsigargin. They were inhibited by the phospholipase C antagonist U73,122, the inositol 1,4,5-trisphosphate (InsP(3) ) receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) as well as by the gap-junction uncouplers 1-heptanol and carbenoxolone, indicating regulation by the InsP(3) pathway and dependence on gap-junctional coupling. Cells endogenously generated nitric oxide (NO), expressed NO synthase 1 (NOS 1) and connexin 43 (Cx 43). The nitric oxide NOS inhibitors NG-monomethyl-L-arginine (L-NMMA), N(G)-nitro-L-arginine methyl ester (L-NAME), 2-ethyl-2-thiopseudourea, and diphenylene iodonium as well as si-RNA-mediated down-regulation of NOS 1 synchronized [Ca(2+) ](i) oscillations between individual cells, whereas the NO-donors S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) as well as the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) were without effects. The synchronization of [Ca(2+) ](i) oscillations was due to an improvement of intracellular coupling since fluorescence recovery after photobleaching (FRAP) revealed increased reflow of fluorescent calcein into the bleached area in the presence of the NOS inhibitors DPI and L-NAME. In summary our data demonstrate that intracellular NO levels regulate synchronization of [Ca(2+) ](i) oscillations in undifferentiated ASCs by controlling gap-junctional coupling.
authors.affiliation: Department of Physiology, Justus Liebig University Giessen, Germany. heinrich.sauer@physiologie.med.uni-giessen.de
authors.forename: Heinrich
authors.initials: H
authors.lastname: Sauer
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Sharifpanah
authors.forename: Myriam
authors.initials: M
authors.lastname: Hatry
authors.forename: Paul
authors.initials: P
authors.lastname: Steffen
authors.forename: Caroline
authors.initials: C
authors.lastname: Bartsch
authors.forename: Regine
authors.initials: R
authors.lastname: Heller
authors.forename: Manju
authors.initials: M
authors.lastname: Padmasekar
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Howaldt
authors.forename: Gregor
authors.initials: G
authors.lastname: Bein
authors.forename: Maria
authors.initials: M
authors.lastname: Wartenberg
mesh_terms: D000273:Adipose Tissue / Q000166:cytology*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002450:Cell Communication / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000037:antagonists & inhibitors* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D034741:RNA, Small Interfering; D009569:Nitric Oxide; D015544:Inositol 1,4,5-Trisphosphate; C496320:NOS1 protein, human; D052248:Nitric Oxide Synthase Type I; D002118:Calcium