Hypoosmotic Shock Induces Increases in Cytosolic Ca2+ in Tobacco Suspension-Culture Cells |
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| Authors: | Takahashi K Isobe M Knight M R Trewavas A J Muto S |
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| Affiliation: | Graduate School of Agricultural Sciences (K.T., M.I., S.M.), School of Agricultural Sciences (M.I.), Nagoya University Bioscience Center (S.M.), Nagoya University, Chikusa, Nagoya, 464-01, Japan. |
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| Abstract: | Hypoosmotic shock treatment increased cytosolic Ca2+ ion concentration (Ca2+]cyt) in tobacco (Nicotiana tabacum) suspension-culture cells. Ca2+]cyt measurements were made by genetically transforming these cells to express apoaequorin and by reconstituting the Ca2+-dependent photoprotein, aequorin, in the cytosol by incubation with chemically synthesized coelenterazine. Measurement of Ca2+-dependent luminescence output thus allowed the direct monitoring of Ca2+]cyt changes. When cells were added to a hypoosmotic medium, a biphasic increase in Ca2+]cyt was observed; an immediate small elevation (phase 1) was observed first, followed by a rapid, large elevation (phase 2). Phase 1 Ca2+]cyt was stimulated by the V-type ATPase inhibitor bafilomycin A1. Phase 2 was inhibited by the protein kinase inhibitor K-252a and required the continued presence of the hypoosmotic stimulus to maintain it. Although Ca2+ in the medium was needed to produce phase 2, it was not needed to render the cells competent to the hypoosmotic stimulus. If cells were subject to hypoosmotic shock in Ca2+- depleted medium, increases in luminescence could be induced up to 20 min after the shock by adding Ca2+ to the medium. These data suggest that hypoosmotic shock-induced Ca2+]cyt elevation results from the activity of a Ca2+ channel in the plasma membrane or associated hypoosmotic sensing components that require Ca2+- independent phosphorylation and a continued stimulus to maintain full activity. |
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