Biomedical subjects
Publications and source records attributed to S G Dedos.
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.
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.
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.
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.
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.
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.