Changes in the cytosolic free Ca2+ concentration following cell surface receptor activation have been proposed to mediate a wide variety of cellular responses. Using the specific Ca2+ chelator quin2 as a fluorescent intracellular probe, we measured the Ca2+ levels in the cytosol of clonal rat pituitary cells, GH3 cells. We demonstrate that thyrotropin-releasing hormone (TRH) at nanomolar concentrations leads to a rapid and transient increase in cytosolic Ca2+. This increase was found to occur in Ca2+-free media in the presence of EGTA, thus at extracellular Ca2+ levels that are below the cytosolic concentrations, and was not prevented by verapamil, a Ca2+ channel blocker. Depolarization of GH3 cells with K+, which can mimic the action of TRH on prolactin release, increased cytosolic Ca2+ levels only in the presence of free extracellular Ca2+, and this increase could be blocked by verapamil. These data show that the mobilization of intracellular Ca2+ due to TRH action that has been proposed by previous studies actually leads to an increase in cytosolic free Ca2+. The kinetic features of this response emphasize the key role of cytosolic free Ca2+ in stimulus-secretion coupling.

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