The microtubule network enables Src kinase interaction with the Na,K-ATPase to generate Ca2+ flashes in smooth muscle cells

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The microtubule network enables Src kinase interaction with the Na,K-ATPase to generate Ca2+ flashes in smooth muscle cells. / Rognant, Salomé; Kravtsova, Violetta V.; Bouzinova, Elena V.; Melnikova, Elizaveta V.; Krivoi, Igor I.; Pierre, Sandrine V.; Aalkjaer, Christian; Jepps, Thomas A.; Matchkov, Vladimir V.

In: Frontiers in Physiology, Vol. 13, 1007340, 2022.

Research output: Contribution to journal › Journal article › Research › peer-review

Harvard

Rognant, S, Kravtsova, VV, Bouzinova, EV, Melnikova, EV, Krivoi, II, Pierre, SV, Aalkjaer, C, Jepps, TA & Matchkov, VV 2022, 'The microtubule network enables Src kinase interaction with the Na,K-ATPase to generate Ca2+ flashes in smooth muscle cells', Frontiers in Physiology, vol. 13, 1007340. https://doi.org/10.3389/fphys.2022.1007340

APA

Rognant, S., Kravtsova, V. V., Bouzinova, E. V., Melnikova, E. V., Krivoi, I. I., Pierre, S. V., Aalkjaer, C., Jepps, T. A., & Matchkov, V. V. (2022). The microtubule network enables Src kinase interaction with the Na,K-ATPase to generate Ca2+ flashes in smooth muscle cells. Frontiers in Physiology, 13, [1007340]. https://doi.org/10.3389/fphys.2022.1007340

Vancouver

Rognant S, Kravtsova VV, Bouzinova EV, Melnikova EV, Krivoi II, Pierre SV et al. The microtubule network enables Src kinase interaction with the Na,K-ATPase to generate Ca2+ flashes in smooth muscle cells. Frontiers in Physiology. 2022;13. 1007340. https://doi.org/10.3389/fphys.2022.1007340

Author

Rognant, Salomé ; Kravtsova, Violetta V. ; Bouzinova, Elena V. ; Melnikova, Elizaveta V. ; Krivoi, Igor I. ; Pierre, Sandrine V. ; Aalkjaer, Christian ; Jepps, Thomas A. ; Matchkov, Vladimir V. / The microtubule network enables Src kinase interaction with the Na,K-ATPase to generate Ca2+ flashes in smooth muscle cells. In: Frontiers in Physiology. 2022 ; Vol. 13.

Bibtex

@article{fab349447ae844dab0960e866fc4f6eb,
title = "The microtubule network enables Src kinase interaction with the Na,K-ATPase to generate Ca2+ flashes in smooth muscle cells",
abstract = "Background: Several local Ca2+ events are characterized in smooth muscle cells. We have previously shown that an inhibitor of the Na,K-ATPase, ouabain induces spatially restricted intracellular Ca2+ 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 “Ca2+ flashes” 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 Ca2+ flashes. Methods: We characterized Ca2+ flashes in cultured smooth muscle cells, A7r5, and freshly isolated smooth muscle cells from rat mesenteric artery. Cells were loaded with Ca2+-sensitive fluorescent dyes, Calcium Green-1/AM and Fura Red/AM, for ratiometric measurements of intracellular Ca2+. The Na,K-ATPase α2 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. Results: The Ca2+ flashes were induced by micromolar concentrations of ouabain. Knockdown of the α2 isoform Na,K-ATPase abolished Ca2+ 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 Ca2+ flashes were associated with Src kinase activation by phosphorylation. The α2 isoform Na,K-ATPase and Src kinase colocalized in the cells. Disruption of microtubule with nocodazole inhibited Ca2+ flashes, reduced Na,K-ATPase/Src interaction and Src activation. Conclusion: We demonstrate that the Na,K-ATPase-dependent Ca2+ flashes in smooth muscle cells require an interaction between the α2 isoform Na, K-ATPase and Src kinase, which is maintained by the microtubule network.",
keywords = "Ca flashes, intracellular Ca signaling, microtubule network, Na,K-ATPase, Src kinase",
author = "Salom{\'e} Rognant and Kravtsova, {Violetta V.} and Bouzinova, {Elena V.} and Melnikova, {Elizaveta V.} and Krivoi, {Igor I.} and Pierre, {Sandrine V.} and Christian Aalkjaer and Jepps, {Thomas A.} and Matchkov, {Vladimir V.}",
note = "Publisher Copyright: Copyright {\textcopyright} 2022 Rognant, Kravtsova, Bouzinova, Melnikova, Krivoi, Pierre, Aalkjaer, Jepps and Matchkov.",
year = "2022",
doi = "10.3389/fphys.2022.1007340",
language = "English",
volume = "13",
journal = "Frontiers in Physiology",
issn = "1664-042X",
publisher = "Frontiers Media S.A.",

}

RIS

TY - JOUR

T1 - The microtubule network enables Src kinase interaction with the Na,K-ATPase to generate Ca2+ flashes in smooth muscle cells

AU - Rognant, Salomé

AU - Kravtsova, Violetta V.

AU - Bouzinova, Elena V.

AU - Melnikova, Elizaveta V.

AU - Krivoi, Igor I.

AU - Pierre, Sandrine V.

AU - Aalkjaer, Christian

AU - Jepps, Thomas A.

AU - Matchkov, Vladimir V.

N1 - Publisher Copyright: Copyright © 2022 Rognant, Kravtsova, Bouzinova, Melnikova, Krivoi, Pierre, Aalkjaer, Jepps and Matchkov.

PY - 2022

Y1 - 2022

N2 - Background: Several local Ca2+ events are characterized in smooth muscle cells. We have previously shown that an inhibitor of the Na,K-ATPase, ouabain induces spatially restricted intracellular Ca2+ 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 “Ca2+ flashes” 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 Ca2+ flashes. Methods: We characterized Ca2+ flashes in cultured smooth muscle cells, A7r5, and freshly isolated smooth muscle cells from rat mesenteric artery. Cells were loaded with Ca2+-sensitive fluorescent dyes, Calcium Green-1/AM and Fura Red/AM, for ratiometric measurements of intracellular Ca2+. The Na,K-ATPase α2 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. Results: The Ca2+ flashes were induced by micromolar concentrations of ouabain. Knockdown of the α2 isoform Na,K-ATPase abolished Ca2+ 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 Ca2+ flashes were associated with Src kinase activation by phosphorylation. The α2 isoform Na,K-ATPase and Src kinase colocalized in the cells. Disruption of microtubule with nocodazole inhibited Ca2+ flashes, reduced Na,K-ATPase/Src interaction and Src activation. Conclusion: We demonstrate that the Na,K-ATPase-dependent Ca2+ flashes in smooth muscle cells require an interaction between the α2 isoform Na, K-ATPase and Src kinase, which is maintained by the microtubule network.

AB - Background: Several local Ca2+ events are characterized in smooth muscle cells. We have previously shown that an inhibitor of the Na,K-ATPase, ouabain induces spatially restricted intracellular Ca2+ 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 “Ca2+ flashes” 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 Ca2+ flashes. Methods: We characterized Ca2+ flashes in cultured smooth muscle cells, A7r5, and freshly isolated smooth muscle cells from rat mesenteric artery. Cells were loaded with Ca2+-sensitive fluorescent dyes, Calcium Green-1/AM and Fura Red/AM, for ratiometric measurements of intracellular Ca2+. The Na,K-ATPase α2 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. Results: The Ca2+ flashes were induced by micromolar concentrations of ouabain. Knockdown of the α2 isoform Na,K-ATPase abolished Ca2+ 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 Ca2+ flashes were associated with Src kinase activation by phosphorylation. The α2 isoform Na,K-ATPase and Src kinase colocalized in the cells. Disruption of microtubule with nocodazole inhibited Ca2+ flashes, reduced Na,K-ATPase/Src interaction and Src activation. Conclusion: We demonstrate that the Na,K-ATPase-dependent Ca2+ flashes in smooth muscle cells require an interaction between the α2 isoform Na, K-ATPase and Src kinase, which is maintained by the microtubule network.

KW - Ca flashes

KW - intracellular Ca signaling

KW - microtubule network

KW - Na,K-ATPase

KW - Src kinase

UR - http://www.scopus.com/inward/record.url?scp=85139851212&partnerID=8YFLogxK

U2 - 10.3389/fphys.2022.1007340

DO - 10.3389/fphys.2022.1007340

M3 - Journal article

C2 - 36213229

AN - SCOPUS:85139851212

VL - 13

JO - Frontiers in Physiology

JF - Frontiers in Physiology

SN - 1664-042X

M1 - 1007340

ER -

ID: 323849003