Signal Transduction in Eclosion Hormone-induced Secretion of Ecdysis-triggering Hormone

Timothy G Kingan,Richard A Cardullo,Michael E Adams

doi:10.1074/jbc.m102421200

Timothy G Kingan, Richard A Cardullo + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m102421200

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Abstract

Inka cells of insect epitracheal glands (EGs) secrete preecdysis and ecdysis-triggering hormones (PETH and ETH) at the end of each developmental stage. Both peptides act in the central nervous system to evoke the ecdysis behavioral sequence, a stereotype behavior during which old cuticle is shed. Secretion of ETH is stimulated by a brain neuropeptide, eclosion hormone (EH). EH evokes accumulation of cGMP followed by release of ETH from Inka cells, and exogenous cGMP evokes secretion of ETH. The secretory responses to EH and cGMP are inhibited by the broad-spectrum kinase inhibitor staurosporine, and the response to EH is potentiated by the phosphatase inhibitor calyculin A. Staurosporine did not inhibit EH-evoked accumulation of cGMP. Changes in cytoplasmic Ca2+ in Inka cells during EH signaling were monitored via fluorescence ratioing with fura-2-loaded EGs. Cytoplasmic Ca2+ increases within 30-120 s after addition of EH to EGs, and it remains elevated for at least 10 min, corresponding with the time course of secretion. Secretion is increased in dose-dependent manner by the Ca2+-ATPase inhibitor thapsigargin, a treatment that does not elevate glandular cGMP above basal levels. The secretory response to EH is partially inhibited in glands loaded with EGTA, while cGMP levels are unaffected. These findings suggest that EH activates second messenger cascades leading to cGMP accumulation and Ca2+ mobilization and/or influx and that both pathways are required for a full secretory response. cGMP activates a staurosporine-inhibitable protein kinase. We propose that Ca2+ acts via a parallel cascade with a time course that is similar to that for cGMP activation of a cGMP-dependent protein kinase.

Highlights

Growth and differentiation of tissues during development of insects is orchestrated by polyhydroxylated steroids, the ecdysones, and their interplay with the sesquiterpene juvenile hormones, acting through nuclear receptors to direct transcription
To address the possibility that cGMP acts by regulating the activity of a protein kinase and the phosphorylation state of a protein in the transduction cascade, we tested the action of protein kinase and phosphatase inhibitors in basal and stimulated secretion
We have shown that release of ETH from Inka cells of insect epitracheal glands requires participation of a protein kinase that likely is activated by cGMP during eclosion hormone (EH) signaling

Summary

Introduction

Growth and differentiation of tissues during development of insects is orchestrated by polyhydroxylated steroids, the ecdysones, and their interplay with the sesquiterpene juvenile hormones, acting through nuclear receptors to direct transcription. These findings suggest that EH activates second messenger cascades leading to cGMP accumulation and Ca2؉ mobilization and/or influx and that both pathways are required for a full secretory response. We report the results of pharmacological studies with epitracheal glands showing that cGMP is likely to act in secretion via activation of a protein kinase.

Results

Conclusion