Ca2+ signaling in prothoracicotropic hormone-stimulated prothoracic gland cells of Manduca sexta: evidence for mobilization and entry mechanisms

• Published in: Insect biochemistry and molecular biology 2005-04, Vol.35 (4), p.263-275
• Main Authors: Fellner, Susan K, Rybczynski, Robert, Gilbert, Lawrence I
• Format: Article
• Language: English
• Subjects: Animals; Calcium Signaling - physiology; Ecdysterone - metabolism; Extracellular Signal-Regulated MAP Kinases - metabolism; Glycoproteins - pharmacology; Insect Hormones - pharmacology; Larva - drug effects; Larva - physiology; Manduca - drug effects; Manduca - growth & development; Manduca - physiology; Signal Transduction - drug effects; Signal Transduction - physiology; Thorax - physiology
• ISSN: 0965-1748
• DOI: 10.1016/j.ibmb.2004.11.006

Prothoracicotropic hormone (PTTH) stimulates ecdysteroidogenesis in lepidopteran prothoracic glands (PGs), thus indirectly controlling molting and metamorphosis. PTTH triggers a signal transduction cascade in PGs that involves an early influx of Ca2+. Although the importance of Ca2+ has been long known, the mechanism(s) of PTTH-stimulated changes in cytoplasmic Ca2+ [Ca2+]i are not yet well understood. PGs from the fifth instar of Manduca sexta were exposed to PTTH in vitro. The resultant changes in [Ca2+]i were measured using ratiometric analysis of a fura-2 fluorescence signal in the presence and absence of inhibitors of specific cellular signaling mechanisms. The phospholipase C (PLC) inhibitor U-73122 nearly abolished the PTTH-stimulated increase in [Ca2+]i, as well as PTTH-stimulated ecdysteroidogenesis and extracellular-signal regulated kinase phosphorylation, thus establishing a role for PLC and implicating inositol trisphosphate (IP3) in PTTH signal transduction. Two antagonists of the IP3 receptor, 2-APB and TMB-8, likewise blocked the [Ca2+]i response by a mean of 92%. We describe for the first time the presence of Ca2+ oscillations in PTTH-stimulated cells in Ca2+-free medium. External Ca2+ entered PG cells via at least two routes: store-operated (capacitative) Ca2+ entry channels and L-type voltage-gated Ca2+ channels. We propose that PTTH initiates a transductory cascade typical of many G-protein coupled receptors, involving both Ca2+ mobilization and entry pathways.