Ca 2+ signaling in prothoracicotropic hormone-stimulated prothoracic gland cells of Manduca sexta: Evidence for mobilization and entry mechanisms

Abstract

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 Ca 2+. Although the importance of Ca 2+ has been long known, the mechanism(s) of PTTH-stimulated changes in cytoplasmic Ca 2+ [Ca 2+] i are not yet well understood. PGs from the fifth instar of Manduca sexta were exposed to PTTH in vitro. The resultant changes in [Ca 2+] 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 [Ca 2+] i, as well as PTTH-stimulated ecdysteroidogenesis and extracellular-signal regulated kinase phosphorylation, thus establishing a role for PLC and implicating inositol trisphosphate (IP 3) in PTTH signal transduction. Two antagonists of the IP 3 receptor, 2-APB and TMB-8, likewise blocked the [Ca 2+] i response by a mean of 92%. We describe for the first time the presence of Ca 2+ oscillations in PTTH-stimulated cells in Ca 2+-free medium. External Ca 2+ entered PG cells via at least two routes: store-operated (capacitative) Ca 2+ entry channels and L-type voltage-gated Ca 2+ channels. We propose that PTTH initiates a transductory cascade typical of many G-protein coupled receptors, involving both Ca 2+ mobilization and entry pathways.