AJP - Endocrinology and Metabolism, Vol 256, Issue 3 E375-E379, Copyright © 1989 by American Physiological Society
Cytosolic free calcium in normal somatotropes: effects of forskolin and phorbol ester
R. W. Holl, M. O. Thorner and D. A. Leong
Department of Internal Medicine, University of Virginia Medical Center, Charlottesville 22908.
Digital imaging microscopy using the calcium-sensitive indicator probe
fura-2 was combined with a reverse hemolytic plaque assay (RHPA) for growth
hormone (GH) secretion. This technique allows dynamic measurements of the
cytosolic free calcium concentration ([Ca2+]i) in individual pituitary
somatotropes. Stimulation by growth hormone-releasing factor (GRF)
increases, whereas somatostatin (SRIF) reduces [Ca2+]i in this cell type.
[Ca2+]i increased in somatotropes when the cellular content of adenosine
3',5'-cyclic monophosphate (cAMP) was elevated by 1) activating cellular
adenylate cyclase with forskolin (5 microM) and 2) treatment with the
cAMP-analogues dibutyryl-cAMP (1 mM) or 8-bromo-cAMP (5 mM). The
forskolin-induced calcium rise was abolished in the absence of
extracellular calcium. This indicates that cAMP increases the influx of
calcium into the cytosol and thereby stimulates hormone release. When
forskolin was given in combination with SRIF (10 nM), [Ca2+]i decreased to
the same level reached with SRIF treatment alone, indicating a site of
action distal to the generation of cAMP. Activating protein kinase C with
the phorbol ester 12,13-phorbol dibutyrate (PDB; 100 nM) increased [Ca2+]i
as well. Again, this effect was dependent on extracellular calcium and
blocked when PDB and SRIF were applied simultaneously. Combined stimulation
with GRF plus PDB did not augment the response of [Ca2+]i over GRF
treatment alone.
