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 Ca 2؉ in Inka cells during EH signaling were monitored via fluorescence ratioing with fura-2-loaded EGs. Cytoplasmic Ca 2؉ 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 Ca 2؉ -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 Ca 2؉ 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 Ca 2؉ acts via a parallel cascade with a time course that is similar to that for cGMP activation of a cGMP-dependent protein kinase.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. An important outcome of this interplay is the production of cuticular exoskeleton. After new cuticle is produced at the end of each stadium, old cuticle must be shed. This shedding of old cuticle, termed ecdysis, occurs in a stereotype sequence of behaviors (1). These behaviors, most thoroughly studied in the lepidopterous insect Manduca sexta, are initiated and completed in 60 -70 minutes by actions of the peptide hormones preecdysis and ecdysis-triggering hormones (PETH and ETH) 1 (2, 3) and eclosion hormone (EH) (4). The findings from a number of laboratories suggest that the ecdysis sequence is set in motion following peripheral release of PETH and ETH (2, 3), and the behaviors are driven by multiple loci in the CNS (5-7).ETH is secreted by Inka cells of the segmentally distributed epitracheal glands (3) in response to EH (8). EH is secreted hormonally by peripheral neurohemal endings and centrally from axons, both of the brain-centered "ventromedial" neurosecretory cells (9, 10), probably in response to ETH (7,11). Together these findings suggest a model for endocrine even...