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 Ca
2+ 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 Ca
2+ 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 Ca
2+ 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 Ca
2+ oscillations, step-like increase
in Ca
2+ and Ca
2+ oscillations superimposed on a steplike
increase for major population of cells (~90%)
in addition to a single giant Ca
2+ spike (Kimoto,
Ohta and Kawato, 1996).
In cell population measurements with a spectrofluorometer,
the observed single Ca
2+ spike would
therefore be caused by non-steroidogenic cells upon
hormonal stimulation, when cells are loaded with a
Ca
2+ indicator in the absence of Triton X-100.
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