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Physiol."],"abstract":"<jats:p><jats:bold>Background:<\/jats:bold> Several local Ca<jats:sup>2+<\/jats:sup> events are characterized in smooth muscle cells. We have previously shown that an inhibitor of the Na,K-ATPase, ouabain induces spatially restricted intracellular Ca<jats:sup>2+<\/jats:sup> transients near the plasma membrane, and suggested the importance of this signaling for regulation of intercellular coupling and smooth muscle cell contraction. The mechanism behind these Na,K-ATPase-dependent \u201cCa<jats:sup>2+<\/jats:sup> flashes\u201d remains to be elucidated. In addition to its conventional ion transport function, the Na,K-ATPase is proposed to contribute to intracellular pathways, including Src kinase activation. The microtubule network is important for intracellular signaling, but its role in the Na,K-ATPase-Src kinase interaction is not known. We hypothesized the microtubule network was responsible for maintaining the Na,K-ATPase-Src kinase interaction, which enables Ca<jats:sup>2+<\/jats:sup> flashes.<\/jats:p><jats:p><jats:bold>Methods:<\/jats:bold> We characterized Ca<jats:sup>2+<\/jats:sup> flashes in cultured smooth muscle cells, A7r5, and freshly isolated smooth muscle cells from rat mesenteric artery. Cells were loaded with Ca<jats:sup>2+<\/jats:sup>-sensitive fluorescent dyes, Calcium Green-1\/AM and Fura Red\/AM, for ratiometric measurements of intracellular Ca<jats:sup>2+<\/jats:sup>. The Na,K-ATPase \u03b12 isoform was knocked down with siRNA and the microtubule network was disrupted with nocodazole. An involvement of the Src signaling was tested pharmacologically and with Western blot. Protein interactions were validated with proximity ligation assays.<\/jats:p><jats:p><jats:bold>Results:<\/jats:bold> The Ca<jats:sup>2+<\/jats:sup> flashes were induced by micromolar concentrations of ouabain. Knockdown of the \u03b12 isoform Na,K-ATPase abolished Ca<jats:sup>2+<\/jats:sup> flashes, as did inhibition of tyrosine phosphorylation with genistein and PP2, and the inhibitor of the Na,K-ATPase-dependent Src activation, pNaKtide. Ouabain-induced Ca<jats:sup>2+<\/jats:sup> flashes were associated with Src kinase activation by phosphorylation. The \u03b12 isoform Na,K-ATPase and Src kinase colocalized in the cells. Disruption of microtubule with nocodazole inhibited Ca<jats:sup>2+<\/jats:sup> flashes, reduced Na,K-ATPase\/Src interaction and Src activation.<\/jats:p><jats:p><jats:bold>Conclusion:<\/jats:bold> We demonstrate that the Na,K-ATPase-dependent Ca<jats:sup>2+<\/jats:sup> flashes in smooth muscle cells require an interaction between the \u03b12 isoform Na, K-ATPase and Src kinase, which is maintained by the microtubule network.<\/jats:p>","DOI":"10.3389\/fphys.2022.1007340","type":"journal-article","created":{"date-parts":[[2022,9,23]],"date-time":"2022-09-23T10:59:21Z","timestamp":1663930761000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":7,"title":["The microtubule network enables Src kinase interaction with the Na,K-ATPase to generate Ca2+ flashes in smooth muscle cells"],"prefix":"10.3389","volume":"13","author":[{"given":"Salom\u00e9","family":"Rognant","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Violetta V.","family":"Kravtsova","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elena V.","family":"Bouzinova","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elizaveta V.","family":"Melnikova","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Igor I.","family":"Krivoi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sandrine V.","family":"Pierre","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christian","family":"Aalkjaer","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Thomas A.","family":"Jepps","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vladimir V.","family":"Matchkov","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2022,9,23]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"215","DOI":"10.1113\/jphysiol.1985.sp015672","article-title":"Effect of ouabain on tone, membrane potential and sodium efflux compared with [3H]ouabain binding in rat resistance vessels","volume":"362","author":"Aalkjaer","year":"1985","journal-title":"J. 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