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

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 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...

Neuroendocrine Control of Larval Ecdysis Behavior inDrosophila: Complex Regulation by Partially Redundant Neuropeptides

“…Although no significant changes in EH-IR were detectable at tracheal collapse, Inka cells showed increases in cGMP-IR at this time. Because EH is believed to act via cGMP (Truman et al, 1979;Ewer et al, 1997;Kingan et al, 1997Kingan et al, , 2001) (for review, see Ewer and Reynolds, 2002) and increases in cGMP-IR in Inka cells did not occur in the absence of EH neurons (see below), the contemporaneous presence of cGMPimmunopositive Inka cells suggests that EH had also started to be released at this time. A fall in EH-IR was readily detectable 5 min after tracheal air filling (Air ϩ 5Ј) and showed no additional change until the onset of ecdysis.…”

“…1 Ae). Because cGMP is believed to mediate the actions of EH (Truman et al, 1979;Ewer et al, 1997;Kingan et al, 1997Kingan et al, , 2001) (for review, see Ewer and Reynolds, 2002), this increase in cGMP-IR suggests that EH was present in the hemolymph at this time. By the onset of ecdysis behavior, ETH-IR had reached its lowest level ( Fig.…”

“…3D, 4A). To distinguish between these possibilities, we injected EH KO larvae with suprathreshold amounts of ETH1 and ETH2 or with a membrane-permeable analog of cGMP (8-Br-cGMP), which mimics the action of EH (Kingan et al, 1997(Kingan et al, , 2001Husain and Ewer, 2004) (for review, see Ewer and Reynolds, 2002). We then examined the timing of air filling, pre-ecdysis, and ecdysis, as well as that of ETH release and (in control larvae) that of EH release monitored via the presence of cGMP-IR in the Inka cells.…”

Receptor guanylyl cyclases in Inka cells targeted by eclosion hormone

“…The release of ETH from Inka cells is mediated by brain neuropeptides, which mobilize cytoplasmic calcium and cGMP (11,24). In Manduca, corazonin mediates low rates of ETH release during pre-ecdysis before EH-induced cGMP elevation.…”

“…However, these cyclases are tightly regulated in the presence of GC-activating proteins (GCAPs) (30)(31)(32)(33)(34). Since EH is known to elevate Ca 2ϩ in Inka cells (24,35), the possibility that EH action on this cyclase could operate via Ca 2ϩ regulation must be seriously considered. Finally, it is also possible that BdmGC-1〉 is involved with signal transduction within the CNS, where EH levels are expected to be higher (7).…”