Regulation of capacitative Ca²⁺ entry by prothoracicotropic hormone in the prothoracic glands of the silkworm, Bombyx mori

Abstract

Measurements of Ca2+ influx in Fura-2/AM loaded steroidogenic cells (prothoracic glands; PGs) of the silkworm, Bombyx mori showed that application of the neuropeptide prothoracicotropic hormone (PTTH) can increase the intracellular [Ca2+]i. This PTTH-mediated Ca2+ influx in PG cells had kinetic patterns and pharmacological characteristics similar to those induced by thapsigargin. Namely, it produced increases in intracellular Ca2+ levels only in the presence of extracellular Ca2+, it was blocked by Gd3+ and 2-Aminoethoxydiphenylborate (2-APB), and it was unaffected by several toxins or compounds that block voltage-activated Ca2+ channels. Moreover, the PTTH-stimulated increase of Ca2+ levels was eliminated in the presence of heparin (an IP3 receptor blocker), and by TMB-8 which also blocked any PTTH-dependent increase of ecdysteroid secretion. The PTTH-mediated increase of Ca2+ levels was not affected by the non-hydrolysable GDP analogue, GDPbetaS, an indication that a G protein is not downstream of the PTTH receptor. These results argue strongly in favor of gating by the PTTH receptor of capacitative Ca2+ entry (CCE) channels (or store-operated Ca2+ channels (SOCs)) by a mechanism that does not involve any G proteins but requires the presence of functional IP3 receptors. Because the ability of PTTH to stimulate the [Ca2+]i levels of PG cells was completely mimicked by thapsigargin and exhibited a pharmacological profile similar to CCE mechanisms, we believe that PTTH directly regulates a CCE pathway in PG cells thereby activating a plethora of downstream regulators responsible for ecdysteroid secretion by the PGs of Bombyx mori.

Insulin signaling pathways in lepidopteran ecdysone secretion

“…The PTTH receptor is known as Torso, first characterized in Drosophila embryos (Casanova and Struhl, 1989; Li, 2005; Rewitz et al, 2009). Unlike insulin, PTTH increases intracellular levels of cyclic AMP, through a PTTH-stimulated increase in intracellular calcium and the activation of a calcium-sensitive adenylyl cyclase (Smith et al, 1984, 1985; Meller et al, 1988; Dedos et al, 2005, 2007; Fellner et al, 2005). Increased intracellular calcium also results in activation of mitogen-activated protein kinases (MAPkinases) including MEK and ERK (Rybczynski and Gilbert, 2003).…”

Temperature-induced stress response in Lymantria dispar neurosecretory neurons

“…This form of PTTH has a multifunctional role in stress-protective mechanisms, stimulating ecdysone synthesis through increased Ca 2+ influx in the protothoracic gland cells (Dedos et al, 2005). Under some environmental conditions a decrease in PTTH level is adjustable, i.e.…”

Different Ca2+ signalling cascades manifested by mastoparan in the prothoracic glands of the tobacco hornworm, Manduca sexta, and the silkworm, Bombyx mori

“…We initially tested guanosine 5′-[β-thio]diphosphate (see also Dedos et al, 2005) and introduced it into the cells with the aid of the influx™ pinocytotic cell-loading reagent (Fig. 2).…”

“…Since heparin was effective in abolishing the prothoracicotropic hormone-mediated increase in [Ca 2+ ] i (Dedos et al, 2005), it seems reasonable to infer that in B. mori prothoracic gland cells the mastoparan-stimulated increase in [Ca 2+ ] i does not result from mobilization of Ca 2+ from IP 3 -sensitive intracellular stores via the activation of a G protein-coupled PLC.…”

“…This protein acts through a transduction cascade requiring extracellular Ca 2+ (Smith, 1995) and results in activation of a Ca 2+ /calmodulin dependent adenylyl cyclase (Meller et al, 1988;Chen et al, 2001) and increased cAMP levels (Smith, 1995). Although the regulatory mechanisms responsible for increases in [Ca 2+ ] i in prothoracic gland cells are not well understood, pharmacological manipulation of [Ca 2+ ] i can help identify and distinguish between the various mechanisms underlying the regulation of [Ca 2+ ] i in these cells (Fellner et al, 2005;Priester and Smith, 2005;Dedos et al, 2005). One pharmacological agent is mastoparan, which produces increases in [Ca 2+ ] i in prothoracic gland cells of M. sexta (Birkenbeil, 2000).…”