期刊
AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY
卷 287, 期 5, 页码 F969-F978出版社
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajprenal.00096.2004
关键词
intracellular calcium ion; antibody; Madin-Darby canine kidney cells; fibronectin; fluorescence imaging
Because beta(1)-integrin is involved in sensing of fluid flow rate in endothelial cells, a function that in Madin-Darby canine kidney (MDCK) cells is confined to the primary cilium, we hypothesized beta(1)-integrin to be an important part of the primary ciliary mechanosensory apparatus in MDCK cells. We observed that beta(1)-integrin, alpha(3)-integrin, and perhaps alpha(5)-integrin were localized to the primary cilium of MDCK cells by combining lectin and immunofluorescence confocal microscopy. beta(1)-Integrin was also colocalized with tubulin to the primary cilia of the rat renal collecting ducts, as well as to the cilia of proximal tubules and thick ascending limbs. Immunogold-electron microscopy confirmed the presence of beta(1)-integrin on primary cilia of MDCK cells and rat collecting ducts. Intracellular Ca2+ levels, monitored by fluorescence microscopy on fluo 4-loaded MDCK cells, significantly increased on addition of fibronectin, a beta(1)-integrin ligand, to mature MDCK cells with an IC50 of 0.02 mg/l. In immature, nonciliated cells or in deciliated mature cells, the IC50 was 0.40 mg/l. Blocking the fibronectin-binding sites of beta(1)-integrin with RGD peptide prevented the Ca2+ signal. Crosslinking of beta(1)-integrins by Sambucus nigra agglutinin produced a Ca2+ response similar to the addition of fibronectin. Furthermore, the fibronectin-induced response was not dependent on flow or a flow-induced Ca2+ response. Finally, the flow-induced Ca2+ response was not prevented by the fibronectin-induced signal. Although beta(1)-integrin on the primary cilium greatly potentiates the fibronectin-induced Ca2+ signaling in MDCK cells, the flow-dependent Ca2+ signal is not mediated through activation of beta(1)-integrin.
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