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Abstract
20-Hydroxyecdysone (20E) triggers programmed cell death (PCD) and regulates de novo
gene expression in the anterior silk glands (ASGs) of the silkworm Bombyx mori. PCD
is mediated via a nongenomic pathway that includes Ca2+ as a second messenger and
the activation of protein kinase C/caspase-3-like protease; however, the steps leading
to a concomitant buildup of intracellular Ca2+ are unknown. We employed pharmacological
tools to identify the components of this pathway. ASGs were cultured in the presence
of 1 microM 20E and one of the following inhibitors: a G-protein-coupled receptor
(GPCR) inhibitor, a phospholipase C (PLC) inhibitor, an inositol 1,4,5-trisphosphate
receptor (IP3R) antagonist, and an L- or T-type Ca2+ channel blocker. The T-type Ca2+
channel blocker inhibited 20E-induced nuclear and DNA fragmentation; in contrast,
PCD was induced by 20E in Ca2+-free medium, indicating that the source of Ca2+ is
an intracellular reservoir. The IP3R antagonist inhibited nuclear and DNA fragmentation,
suggesting that the endoplasmic reticulum may be the Ca2+ source. Finally, the GPCR
and PLC inhibitors effectively blocked nuclear and DNA fragmentation. Our results
indicate that 20E increases the intracellular level of Ca2+ by activating IP3R, and
that this effect may be brought about by the serial activation of GPCR, PLC, and IP3.