Hypoosmotic shock induces increases in cytosolic Ca2+ in tobacco suspension-culture cells

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 A(1). 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.