Inhibition of cAMP signalling cascade-mediated Ca2+ influx by a prothoracicostatic peptide (Mas-MIP I) via dihydropyridine-sensitive Ca2+ channels in the prothoracic glands of the silkworm, Bombyx mori

Abstract Measurements of Ca 2+ influx in Fura-2/AM loaded prothoracic glands (PGs) of the silkworm, Bombyx mori , after application of forskolin or the cAMP analogue, 8-bromo-cAMP, showed a steady increase in [Ca 2+ ] i , which was of extracellular origin and was inhibited, in both cases, by the dihydropyridine (DHP) derivative, nitrendipine. Nitrendipine also inhibited the abrupt S(-)·Bay K 8644-mediated increase in [Ca 2+ ] i and its effects were mimicked by a myoinhibitory/prothoracicostatic peptide (Mas-MIP I/PTSP), which was isolated from Manduca sexta and was found to possess ecdysteroidostatic activity in Bombyx mori PGs. This peptide blocked both the forskolin and S(-)·Bay K 8644-mediated increase in [Ca 2+ ] i of PG cells. It was ineffective, however, in blocking the recombinant prothoracicotropic hormone (rPTTH)-stimulated high increase in [Ca 2+ ] i of PG cells suggesting that distinct and independently regulated Ca 2+ influx mechanisms operate in the PG cells of Bombyx mori . The dependence of DHP-sensitive Ca 2+ channels on the cAMP-signalling cascade was further corroborated by the inabilitity of nitrendipine to block the thapsigargin-stimulated high increase in [Ca 2+ ] i after depletion of Ca 2+ from the intracellular stores. This, together with the inability of thapsigargin to stimulate the cAMP levels of PG cells suggest that there is a tightly regulated cross-talk mechanism between the two signalling cascades of Ca 2+ and cAMP. The combined results suggest a cAMP-mediated regulation of the opening-state of DHP-sensitive Ca 2+ channels and stimulation of [Ca 2+ ] i increases and ecdysteroid secretion by a positive feedback mechanism. Mas-MIP I/PTSP interferes with this mechanism by blocking DHP-sensitive Ca 2+ channels. This regulatory mechanism appears to be autonomously stimulating ecdysteroidogenesis by the PGs, it is regulated by Mas-MIP I/PTSPS, and it is not involved in other Ca 2+ influx mechanisms that operate within the PG cells of Bombyx mori .