Signalling from parathyroid hormone.

PTH (parathyroid hormone), acting via type 1 PTH receptors, is a major regulator of plasma [Ca(2+)]. The G-protein, G(s), is an essential component of the sequence linking PTH to plasma Ca(2+) regulation, but the relative importance of intracellular signals, including Ca(2+) and cAMP, that lie downstream of G(s) is not resolved.

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.

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.

Induction of dauer pupae by fenoxycarb in the silkworm, Bombyx mori.

Topical application of fenoxycarb (1 &mgr;g per animal) at 129 or 132 h of the fifth instar larvae of the silkworm, Bombyx mori, did not induce morphological abnormalities in the pupal stage, but these animals became dauer (permanent) pupae. This condition of B. mori and the endocrine events leading to permanent pupae are discussed in this work. Application of fenoxycarb at 132 h of the fifth instar elicited a high ecdysteroid titre in the pharate pupal stage and a steadily high ecdysteroid titre in the pupal stage. The fenoxycarb-induced permanent pupae had non-degenerating prothoracic glands that secreted low amounts of ecdysteroid and did not respond to recombinant prothoracicotropic hormone (rPTTH) late in the pupal stage. The Bombyx PTTH titre in the haemolymph, determined by a time-resolved fluoroimmunoassay, was lower than that of controls at the time of pupal ecdysis, but higher than controls later in the pupal stage in fenoxycarb-treated animals. After application of fenoxycarb, its haemolymph level, measured by ELISA, reached a peak at pupal ecdysis, then remained low. These results suggest that the fenoxycarb-mediated induction of permanent pupae is only partially a brain-centred phenomenon. It also involves alterations in the hormonal interplay that govern both the initiation of pupal-adult differentiation and changes in the steroidogenic pathway of the prothoracic glands of B. mori.

Action kinetics of a prothoracicostatic peptide from Bombyx mori and its possible signaling pathway.

A prothoracicostatic peptide (PTSP), purified from the brains of Bombyx mori, was found to inhibit ecdysteroidogenesis in the prothoracic glands (PGs) of this insect. This peptide was active at inhibiting ecdysteroidogenesis in the PGs at concentrations higher than 23 nM and glands incubated in 230 nM PTSP in vitro exhibited maximum inhibition of ecdysteroid production. By incubating PGs in vitro at different incubation periods it was observed that the first statistically significant inhibitory effect occurred after 30 min incubation in the presence of PTSP. Transferral of PGs from a medium with PTSP to a medium without PTSP resulted in the resumption of ecdysteroid production. Statistically significant inhibition of ecdysteroid production by PTSP was observed only in day 6 and in day 3 PGs of the 5th instar. The extracts of day 6 glands incubated in the presence of PTSP did not contain elevated amounts of ecdysteroid relative to controls after the incubations, indicating that PTSP does not inhibit the secretion, rather the synthesis, of ecdysteroid in the PGs. The presence of PTSP completely blocked the increased ecdysteroid production via L-type Ca(2+) channel activation by S(-)*Bay K 8644. There was no inhibition of ecdysteroid production by PTSP with glands incubated in Ca(2+)-free medium. The combined results suggest that PTSP regulates ecdysteroid synthesis only during specific stages of the 5th instar through a mechanism that likely involves the blocking of Ca(2+) influx through voltage-sensitive Ca(2+) channels in the PG cells of B. mori.

Involvement of calcium, inositol-1,4,5 trisphosphate and diacylglycerol in the prothoracicotropic hormone-stimulated ecdysteroid synthesis and secretion in the prothoracic glands of Bombyx mori.

The objective of this study was to determine which intracellular second messenger systems are activated by prothoracicotropic hormone in the prothoracic glands (PGs) of Bombyx mori. Recombinant prothoracicotropic hormone (rPTTH) could stimulate ecdysteroid synthesis and secretion from day 6 PGs of the 5th instar of Bombyx mori within 30 min of in vitro incubation. However, rPTTH did not stimulate any increases in the glandular content of inositol 1,4,5-trisphosphate and cAMP during this short incubation period. Extracellular Ca2+ influenced the basal and rPTTH-stimulated ecdysteroid synthesis and release in a dose-dependent manner. The L-type Ca2+ channel antagonist, nitrendipine, inhibited the rPTTH-stimulated ecdysteroid synthesis and secretion (IC50-28 microM). The phospholipase C inhibitor, 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate, inhibited the rPTTH-stimulated ecdysteroid synthesis (IC50-19 microM). The protein kinase C inhibitor, chelerythrine chloride, inhibited the rPTTH-stimulated ecdysteroid synthesis (IC50-14 microM). The protein kinase C activator, phorbol-12-myristate 13-acetate (PMA), could stimulate basal ecdysteroid synthesis and secretion (EC50-1 microM) and its inactive alpha-isomer (4 alpha-PMA) was ineffective. The combined results suggest that the PTTH-stimulated ecdysteroid synthesis and release in the PGs of Bombyx is dependent on extracellular Ca2+ and the bifurcating second messenger signalling cascade of inositol 1,4,5-triphosphate and diacylglycerol.

Interactions between Ca2+ and cAMP in ecdysteroid secretion from the prothoracic glands of Bombyx mori.

The interaction between Ca2+ and cAMP in the mediation of ecdysteroid secretion from prothoracic glands (PGs) of Bombyx mori was investigated in vitro. Omission of Ca2+ from the PGs' incubation medium decreased basal ecdysteroid secretion from day 3 until day 6. On day 6, the ability of forskolin or 3-isobutyl-1-methylxanthine (IBMX) to stimulate ecdysteroid secretion was affected by the omission of Ca2+ from the medium. The cAMP agonist Sp-adenosine 3',5'-cyclic monophosphothioate (Sp-cAMPS) and the cAMP analogue dibutyryl cyclic AMP (dbcAMP) stimulated ecdysteroid secretion even in the absence of Ca2+ from the medium. The Sp-cAMPS-stimulated ecdysteroid secretion was inhibited by the cAMP antagonist Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS) and the L-type Ca2+ channel blocker verapamil. Both the Ca2+ ionophore A23187 and the L-type Ca2+ channel agonist S(-) Bay K 8644 could stimulate ecdysteroid secretion. The A23187-induced ecdysteroid secretion was partially inhibited by Rp-cAMPS. The combined results indicate that Ca2+ and cAMP signaling pathways can cooperatively, as well as independently, stimulate ecdysteroid secretion from the PGs.

Induction of dauer larvae by application of fenoxycarb early in the 5th instar of the silkworm, Bombyx mori.

Fenoxycarb application from 0 h until 60 h of the 5th instar of Bombyx mori induced 100% dauer larvae. Between the 60 and 78 h, the ratio of fenoxycarb-induced dauer larvae decreased, and the ratio of supernumerary instar moulting larvae increased. After application of fenoxycarb at the 48 h of the 5th instar, the haemolymph prothoracicotropic hormone (PTTH) titer was higher in fenoxycarb-treated larvae than in control larvae. Furthermore, brain-corpora cardiaca-corpora allata (Br-CC-CA) complexes from fenoxycarb-treated larvae released higher amounts of PTTH in vitro than the Br-CC-CA complexes of control larvae. Prothoracic glands (PGs) of fenoxycarb-treated larvae at the 48 h of the 5th instar exhibited basal and PTTH-stimulated secretory activities similar to that of control PGs until the 72 h of the 5th instar. After that time point, both basal and PTTH-stimulated secretory activity of PGs from fenoxycarb-treated larvae significantly decreased and remained low for the rest of the investigated period. The combined results suggest that the application of fenoxycarb affects the ability of the PGs to be stimulated by PTTH and the induction of dauer larvae in Bombyx mori is not due to inhibition of PTTH release from Br-CC-CA complexes.

Differences between recombinant PTTH and crude brain extracts in cAMP-mediated ecdysteroid secretion from the prothoracic glands of the silkworm, Bombyx mori.

The ability of recombinant prothoracicotropic hormone (rPTTH) or crude brain extract (cBRAIN) of Bombyx mori to stimulate ecdysteroid secretion from prothoracic glands (PGs) was investigated throughout the fifth instar and the first day of the pupal stage. Crude brain extracts could stimulate much higher ecdysteroid secretion than rPTTH during a 2h incubation. Recombinant PTTH did not increase the level of glandular cyclic AMP, except on days 4 and 5 of the fifth instar. Glandular cAMP levels were increased by cBRAIN from day 0 until day 5 of the fifth instar with the highest increase on day 3. On this day, rPTTH could not stimulate any increase of ecdysteroid secretion from the PGs during a 30min incubation. On the contrary, PGs incubated with cBRAIN for 30min showed increased secretory activity. Furthermore, on day 3 and in the absence of extracellular Ca(2+), rPTTH did not increase the glandular cAMP levels but cBRAIN did. Recombinant PTTH-stimulated ecdysteroid secretion from day 3 PGs was dependent on extracellular Ca(2+) in a dose-dependent manner. However, cBRAIN could stimulate ecdysteroid secretion even in the absence of extracellular Ca(2+). Taken together, the results of these experiments suggest the presence of a previously unknown cerebral prothoracicotropic factor that can stimulate glandular cAMP levels and ecdysteroid secretion from the PGs of Bombyx mori.

Disturbance of adult eclosion by fenoxycarb in the silkworm, Bombyx mori.

Fenoxycarb treatment before and after pupal ecdysis of Bombyx mori disturbed adult eclosion and the animals were unable to escape from the pupal exuviae. This effect of fenoxycarb was dose and time dependent with the highest sensitivity around the pupal ecdysis. The sensitivity rapidly diminished within 20 hours of pupal ecdysis. Twenty-hydroxyecdysone (20E) produced similar effects. Fenoxycarb injection at the pupal ecdysis induced higher ecdysteroid production by the prothoracic glands and higher PTTH-secretory activity in the brain-corpora cardiaca-corpora allata complexes. As a result, the fenoxycarb treated pupae contained higher ecdysteroid titres in the haemolymph. Both the fenoxycarb and the 20E treatments resulted in the lack of development of the rectum in pharate adults. This was the main cause of high ecdysteroid titres in the pharate adult stage. This effect was mimicked by either removal of the rectum early in the pharate adult stage or a surgical extirpation of the hindgut at the time of pupal ecdysis. These results suggest that the disturbance of adult eclosion by fenoxycarb is due in part to the inability of the formation of the rectum in the pharate adult stage.

Effects of fenoxycarb on the secretory activity of the prothoracic glands in the fifth instar of the silkworm, Bombyx mori.

Effects of fenoxycarb on the secretory activity of the prothoracic glands (PGs) of the silkworm, Bombyx mori, were investigated. Fenoxycarb inhibited the secretory activity of PGs in vitro throughout the fifth instar. Incubation of PGs in the presence of 1 microgram of fenoxycarb resulted in a approximately 50% inhibition of their secretory activity. This inhibition of fenoxycarb was dose dependent. Stimulation of the PGs by recombinant prothoracicotropic hormone (rPTTH) reached a high activation ratio of 15.9. Simultaneous presence of fenoxycarb and 1 ng rPTTH significantly decreased the stimulatory effect of rPTTH on the PGs, and the activation ratio never exceeded 3.65. Early in the fifth instar, fenoxycarb strongly inhibited the secretory activity of the PGs and induced a developmental arrest as well. After the onset of pupal commitment, its action toward the PG secretory activity was inhibitory. However, fenoxycarb treatment was not able to induce a developmental arrest in intact animals after the pupal commitment although it could do so in head-ligated animals. On the contrary, application of fenoxycarb after the wandering behavior resulted in a significantly increased secretory activity by the PGs of both intact and head-ligated larvae.