A Rapid Increase in cAMP in Response to 20-Hydroxyecdysone in the Anterior Silk Glands of the Silkworm, Bombyx mori

Abstract

In the anterior silk glands (ASGs) of the silkworm, Bombyx mori , intracellular cAMP increases transiently to a very high level shortly after the hemolymph ecdysteroid peak in the prepupal period. In cultured ASGs obtained on the day of gut-purge, cAMP levels were increased by 20-hydroxyecdysone (20E), and this increase was enhanced by an inhibitor of phosphodiesterase, but was not affected by α α α α -amanitin, indicating the 20E action may not be mediated via gene expression. The increase in cAMP occurred within 30 seconds of exposure to a physiological concentration of 20E (1 µ µ µ µ M), and also by ponasterone A. Our findings indicate a nongenomic action of ecdysteroids in insects, which may be an additional mechanism by which this steroid hormone induces acute responses in tissues and cells.

The Steroid Hormone 20-Hydroxyecdysone Enhances Gene Transcription through the cAMP Response Element-binding Protein (CREB) Signaling Pathway

“…Previous studies have demonstrated that 20E triggered an increase in the intracellular cAMP concentrations within 30 s in the anterior silk glands of the silkworm (28). We observed that 20E induced PKAC1 phosphorylation and nuclear translocation, which was accompanied by a 20E-induced increase of cAMP, suggesting that there is a conserved mechanism of cAMP-induced PKAC phosphorylation and nuclear translocation in insects.…”

“…In addition to the PKC pathway, 20E binds to the dopamine/ecdysteroid receptor (DmDopEcR), which can mediate an increase of cyclic adenosine monophosphate (cAMP) in Drosophila melanogaster (27). A physiological concentration of 20E (1 M) or ponasterone increases cAMP within 30 s in the anterior silk glands of the silkworm (28). These data suggest that the cAMP-triggered pathway is also activated by 20E.…”

Solubilization of the Ecdysone Binding Protein from Anterior Silk Gland Cell Membranes of the Silkworm, Bombyx mori

“…The biologically active form of ecdysone, 20-hydroxyecdysone (20E), binds to a functional nuclear ecdysone receptor consisting of an ecdysone receptor (EcR) and its heterodimeric partner, Ultraspiracle (USP), and thereby controls the transcriptional activity of target genes (Riddiford et al, 2000). We previously provided preliminary evidence for the presence of a putative membrane ecdysone receptor (mEcR) located in the plasma membranes of B. mori ASGs, which may act in concert with the conventional nuclear EcR (Elmogy et al, 2004(Elmogy et al, , 2006. Thus, to further characterize mEcR, we tried to solubilize from ASGs a functional mEcR retaining its binding activity to ecdysteroids.…”

Production and characterization of monoclonal antibodies raised against the membrane ecdysone receptor from the anterior silk gland of the silkworm, Bombyx mori

“…(i) Animals Larvae of the silkworm, B. mori (Kinshu X Showa F1 hybrid) were reared on an artificial diet (Silkmate, NihonNosan-Kogyo, Yokohama, Japan) at 25ºC under a photoperiod cycle of 12h-light/12h-dark, as described previously [18]. ASGs were dissected on the day of gut purge or cultured separately in 0.3 mL Grace's insect culture medium (Gibco BRL) at 25ºC for 18 h with 20E followed by a culture in a hormone-free medium for a further 12 h. Because preliminary experiments showed that the binding activity in the membrane fractions prepared from the cultured ASGs was higher than that from the freshly dissected ASGs, we mainly used such ASGs unless mentioned otherwise.…”

“…These glands begin to undergo programmed cell death (PCD) in response to a high hemolymph ecdysteroid concentration, which induces pupal metamorphosis. Previous studies provided several biochemical and topological evidence for the presence of a putative membrane ecdysone receptor (mEcR) located in the plasma membranes of B. mori anterior silk glands (ASGs), which may act in concert with the conventional nuclear EcR [18,19,20]. Then after, we succeeded to identify, fully characterize and solubilize mEcR from ASGs [21].…”