Heterogeneous Populations of Adrenal Fasciculata Cells Examined with Ca²⁺ Signaling and lmmunostaining: Single Cell Imaging

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When primary cultures of bovine adrenal fasciculata cells were loaded with Calcium Green-1/AM, only 5～10% of cells were labeled. We have observed with fluorescence microscopic imaging that 1 pM adrenocorticotropin (ACTH) and 45 μM ATP induced a single giant Ca²⁺ spike followed by a plateau phase in around 3% and 80% of labeled cells, respectively. The ATP-responsive cells include the ACTH-responsive cells as a subgroup.
  Immuno- and histocytochemical staining revealed that Ca²⁺ signaling cells with ATP stimulation did not contain essential steroidogenic enzymes such as cytochromes P450scc, P450c21, and 3β-hydroxysteroid dehydrogenase. On the other hand, apparent ATP-insensitive cells were rich in these three enzymes. We assigned, therefore, ATP-insensitive cells as steroidogenic cells. Moreover, we have found that steroidogenic cells did not show Ca²⁺ signaling because of insufficient loading with Calcium Green-1/AM. It should be noted that with the presence of only 0.01% Triton X-100 we achieved sufficient labeling for more than 90% of total cells with Calcium Green-1/AM. In these preparations, 1 pM ACTH induced Ca²⁺ oscillations, step-like increase in Ca²⁺ and Ca²⁺ oscillations superimposed on a steplike increase for major population of cells (～90%) in addition to a single giant Ca²⁺ spike (Kimoto, Ohta and Kawato, 1996).
  In cell population measurements with a spectrofluorometer, the observed single Ca²⁺ spike would therefore be caused by non-steroidogenic cells upon hormonal stimulation, when cells are loaded with a Ca²⁺ indicator in the absence of Triton X-100.