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.     

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.     

Downregulation of the cAMP signal transduction cascade in the prothoracic glands is responsible for the fenoxycarb-mediated induction of permanent 5th instar larvae in Bombyx mori

Fenoxycarb application at 48 h (day 2) of the 5th instar of Bombyx mori induced permanent larvae with prothoracic glands (PGs) exhibiting weak ecdysteroidogenic activity. Although glands from control and fenoxycarb-treated larvae exhibited similar responses to dibutyl cAMP and forskolin on day 2, forskolin could not stimulate ecdysteroid secretion from PGs of fenoxycarb-treated larvae on day 3. Glands from control larvae incubated with cholera toxin (CTX) on day 3 had increased cAMP content and enhanced ecdysteroid secretion. Cholera toxin did not stimulate ecdysteroid secretion and marginally increased cAMP content in day 3 PGs of fenoxycarb-treated larvae. After application of fenoxycarb on day 2, crude brain extracts (cBRAIN) could not increase the glandular cAMP content throughout the rest of the 5th instar of the treated larvae. Fenoxycarb did not affect the basal or cBRAIN-stimulated cAMP accumulation in control PGs on day 2 and day 3 in vitro. Application of fenoxycarb on day 2 did not affect the recombinant PTTH (rPTTH)-stimulated ecdysteroid secretion on day 3, but reduced the cBRAIN-stimulated ecdysteroid secretion on day 3 to levels similar to that of rPTTH. The combined results suggest that the cAMP signalling cascade in the PGs of B. mori becomes nonfunctional after fenoxycarb application on day 2 of the 5th instar.     

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

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.     

Ecdysteroidogenic action of Bombyx prothoracicotropic hormone and bombyxin on the prothoracic glands of Rhodnius prolixus in vitro

An in-vitro assay for ecdysteroid synthesis by the prothoracic glands (PGs) of fifth instar Rhodnius prolixus has been employed to evaluate the actions of prothoracicotropic neuropeptides from the silkmoth, Bombyx mori. Crude prothoracicotropic hormone (PTTH) extracts of recently emerged adult brain complexes of Bombyx induced a dose-dependent stimulation of ecdysteroid synthesis by Rhodnius PGs, which was similar to that obtained using crude Rhodnius PTTH. In both cases, maximum stimulation was obtained with one brain equivalent. Rhodnius PGs were then challenged with incremental doses of recombinant Bombyx PTTH and synthetic bombyxin-II. Dose-response curves for the action of both peptides on Rhodnius PGs were very similar to those obtained for their action on the pupal PGs of Bombyx in vitro. Bombyx PTTH stimulated the PGs of Rhodnius at concentrations comparable to those effective on Bombyx. The curve for Bombyx PTTH showed a steep ascending region from 3 to 8ng/ml and a sharp peak. For bombyxin, concentrations 40-fold higher were required to elicit the same amount of stimulation as obtained using Bombyx PTTH. Therefore, Rhodnius PGs possess recognition sites for both Bombyx PTTH and bombyxin. This is the first study of the ecdysteroidogenic properties of the Bombyx peptides on a heterologous species. It is suggested that the function and conformation of PTTH may be conserved between distantly related insect groups.     

Prothoracicotropic activity of SBRPs, the insulin-like peptides of the saturniid silkworm Samia cynthia ricini

Synthesis and secretion of the insect molting hormone ecdysteroid in the prothoracic glands (PGs) are stimulated by the prothoracicotropic hormone (PTTH) secreted by the brain. Bombyxins, insulin-like peptides of the silkworm Bombyx mori, show prothoracicotropic activity when administered to the saturniid silkworm Samia cynthia ricini, but they are inactive to B. mori itself. Recently, the genes for the bombyxin homologs of S. cynthia ricini (referred to as Samia bombyxin-related peptides, SBRPs) were cloned. To examine the prothoracicotropic activity of SBRPs on S. cynthia ricini, we synthesized two representative molecules, SBRP-A1 and -B1. They promoted pupa-to-adult development with ED(50) of 50 and 10 ng/pupa (EC(50) of 5 and 1 nM), respectively.     

Ca(2+) as second messenger in PTTH-stimulated prothoracic glands of the silkworm,Bombyx mori

Measurements of Ca²⁺ influx and [Ca²⁺]_i changes in Fura-2/AM-loaded prothoracic glands (PGs) of the silkworm, Bombyx mori, were used to identify Ca²⁺ as the actual second messenger of the prothoracicotropic hormone (PTTH) of this insect. Dose-dependent increases of [Ca²⁺]_i in PG cells were recorded in the presence of recombinant PTTH (rPTTH) within 5 minutes. The rPTTH-mediated increases of [Ca²⁺]_i levels were dependent on extracellular Ca²⁺. They were not blocked by the dihydropyridine derivative, nitrendipine, an antagonist of high-voltage-activated (HVA) Ca²⁺ channels, and by bepridil, an antagonist of low-voltage-activated (LVA) Ca²⁺ channels. The trivalent cation La³⁺, a non-specific blocker of plasma membrane Ca²⁺ channels, eliminated the rPTTH-stimulated increase of [Ca²⁺]_i levels in PG cells and so did amiloride, an inhibitor of T-type Ca²⁺ channels. Incubation of PG cells with thapsigargin resulted in an increase of [Ca²⁺]_i levels, which was also dependent on extracellular Ca²⁺ and was quenched by amiloride, suggesting the existence of store-operated plasma membrane Ca²⁺ channels, which can also be inhibited by amiloride. Thapsigargin and rPTTH did not operate independently in stimulating increases of [Ca²⁺]_i levels and one agent’s mediated increase of [Ca²⁺]_i was eliminated in the presence of the other. TMB-8, an inhibitor of intracellular Ca²⁺ release from inositol 1,4,5 trisphosphate (IP₃)-sensitive Ca²⁺ stores, blocked the rPTTH-stimulated increases of [Ca²⁺]_i levels, suggesting an involvement of IP₃ in the initiation of the rPTTH signaling cascade, whereas ryanodine did not influence the rPTTH-stimulated increases of [Ca²⁺]_i levels. The combined results indicate the presence of a cross-talk mechanism between the [Ca²⁺]_i levels, filling state of IP₃-sensitive intracellular Ca²⁺ stores and the PTTH-receptor’s-mediated Ca²⁺ influx.     

Ca as second messenger in PTTH-stimulated prothoracic glands of the silkworm, Bombyx mori

Measurements of Ca²⁺ influx and [Ca²⁺]_i changes in Fura-2/AM-loaded prothoracic glands (PGs) of the silkworm, Bombyx mori, were used to identify Ca²⁺ as the actual second messenger of the prothoracicotropic hormone (PTTH) of this insect. Dose-dependent increases of [Ca²⁺]_i in PG cells were recorded in the presence of recombinant PTTH (rPTTH) within 5 minutes. The rPTTH-mediated increases of [Ca²⁺]_i levels were dependent on extracellular Ca²⁺. They were not blocked by the dihydropyridine derivative, nitrendipine, an antagonist of high-voltage-activated (HVA) Ca²⁺ channels, and by bepridil, an antagonist of low-voltage-activated (LVA) Ca²⁺ channels. The trivalent cation La³⁺, a non-specific blocker of plasma membrane Ca²⁺ channels, eliminated the rPTTH-stimulated increase of [Ca²⁺]_i levels in PG cells and so did amiloride, an inhibitor of T-type Ca²⁺ channels. Incubation of PG cells with thapsigargin resulted in an increase of [Ca²⁺]_i levels, which was also dependent on extracellular Ca²⁺ and was quenched by amiloride, suggesting the existence of store-operated plasma membrane Ca²⁺ channels, which can also be inhibited by amiloride. Thapsigargin and rPTTH did not operate independently in stimulating increases of [Ca²⁺]_i levels and one agent’s mediated increase of [Ca²⁺]_i was eliminated in the presence of the other. TMB-8, an inhibitor of intracellular Ca²⁺ release from inositol 1,4,5 trisphosphate (IP₃)-sensitive Ca²⁺ stores, blocked the rPTTH-stimulated increases of [Ca²⁺]_i levels, suggesting an involvement of IP₃ in the initiation of the rPTTH signaling cascade, whereas ryanodine did not influence the rPTTH-stimulated increases of [Ca²⁺]_i levels. The combined results indicate the presence of a cross-talk mechanism between the [Ca²⁺]_i levels, filling state of IP₃-sensitive intracellular Ca²⁺ stores and the PTTH-receptor’s-mediated Ca²⁺ influx.     

Actions of inositol phosphates on Ca2+ pools in guinea-pig hepatocytes.

In permeabilized hepatocytes, inositol 1,4,5-trisphosphate, inositol 2,4,5-trisphosphate and inositol 4,5-bisphosphate induced rapid release of Ca2+ from an ATP-dependent, non-mitochondrial vesicular pool, probably endoplasmic reticulum. The order of potency was inositol 1,4,5-trisphosphate greater than inositol 2,4,5-trisphosphate greater than inositol 4,5-bisphosphate. The Ca2+-releasing action of inositol 1,4,5-trisphosphate is not inhibited by high [Ca2+], nor is it dependent on [ATP] in the range of 50 microM-1.5 mM. These results suggest a role for inositol 1,4,5-trisphosphate as a second messenger in hormone-induced Ca2+ mobilisation, and that a specific receptor is involved in the Ca2+-release mechanism.     

Quantitative changes in inositol 1,4,5-trisphosphate in chemoattractant-stimulated neutrophils

myo-Inositol 1,4,5-trisphosphate is an intracellular second messenger generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C. In the present study, we have used the abilities of inositol 1,4,5-trisphosphate to inhibit inositol 1,4,5-tris[32P]phosphate binding and to stimulate release of sequestered stores of 45Ca2+ to assay the mass of inositol 1,4,5-trisphosphate in extracts derived from [3H]inositol-prelabeled chemoattractant-stimulated neutrophils. These assays are specific for inositol 1,4,5-trisphosphate since the relative capacity of the extracts to compete with inositol 1,4,5-tris[32P]phosphate binding and to release 45Ca2+ correlated well with the [3H]inositol 1,4,5-trisphosphate content of the extract as determined by high pressure liquid chromatography. No correlation of these activities was observed with the content in the extract of either [3H]inositol 1,3,4-trisphosphate or [3H]inositol 1,3,4,5-tetrakisphosphate, whose formation exhibited kinetics distinct from [3H]inositol 1,4,5-trisphosphate. Thus, within 10 s of stimulation with 10 nM formyl-methionyl-leucyl-phenylalanine, the inositol 1,4,5-trisphosphate content of the extract increased from 0.05 to 0.55 pmol/10(6) cells, equivalent to a change in intracellular concentration from 100 nM to 1.1 microM. These studies demonstrate that neutrophils produce sufficient quantities of inositol 1,4,5-trisphosphate to mobilize Ca2+ from intracellular stores.     

Release of calcium from the endoplasmic reticulum by bile acids in rat liver cells

The effects of four bile acids on cell Ca2+ were examined in suspensions of isolated rat hepatocytes. Taurolithocholate and lithocholate which inhibit bile secretion increased the cytosolic Ca2+ concentration (ED50, 25 microM), as measured by the fluorescent indicator quin2, and promoted a net loss of Ca2+ from the cells. This effect resulted from rapid mobilization of Ca2+ from an intracellular Ca2+ store. This store corresponds to the one that is permeabilized by the inositol (1,4,5)trisphosphate-dependent hormone vasopressin. However, taurolithocholate and lithocholate, unlike the hormone, did not induce a significant accumulation of inositol trisphosphate fraction in isolated hepatocytes. In addition, these agents did not alter the cell and the mitochondria membrane permeability to ions. When applied to saponin-permeabilized cells, taurolithocholate and lithocholate released Ca2+ (ED50, 20 microM) from an ATP-dependent, nonmitochondrial pool which is sensitive to inositol (1,4,5)trisphosphate. In contrast, the bile acids taurocholate and cholate, which increase bile secretion, had no effect on cell Ca2+ in intact hepatocytes or in saponin-permeabilized hepatocytes. It is suggested that taurolithocholate and lithocholate permeabilize the endoplasmic reticulum to Ca2+ and that the resulting permeabilization of this compartment may be involved in the inhibition of bile secretion in mammalian liver.     

Calcium-calmodulin stimulates inositol 1,4,5-trisphosphate kinase activity from insulin-secreting RINm5F cells

In a cytosolic fraction derived from insulin-secreting RINm5F cells, the rate of conversion of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) to inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) was half-maximally stimulated by 0.8 microM Ca2+ (Biden, T. J., and Wollheim, C. B. (1986) J. Biol. Chem. 261, 11931-11934). In the present study we show that after initial purification by anion exchange chromatography, the Ins-1,4,5-P3 kinase activity responsible for that conversion is stimulated by Ca2+-calmodulin, but not by Ca2+ alone. This is almost certainly due to a specific interaction of the enzyme and its activator since kinase activity was retained on a calmodulin-linked Sepharose 6B column in the presence of Ca2+ but eluted upon chelation of the cation. After this two-step purification, Ins-1,4,5-P3 kinase activity was maximally stimulated 5-fold by 10 microM calmodulin in the presence of 10(-5) M Ca2+, and 2 1/2-fold at 10(-6) M Ca2+. Under these conditions the minimum concentrations of calmodulin needed to stimulate activity were in the 10-50 nM range. At 10(-7) M Ca2+, calmodulin (up to 30 microM) was without effect. Stimulated Ins-1,4,5-P3 kinase activity was inhibited in a dose-dependent fashion by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) although the calmodulin antagonist had no effect on the residual activity seen at 10(-7) M Ca2+. These results strongly support our previous suggestion that alterations in cytosolic free Ca2+ concentrations play an important role in regulating the levels of Ins-1,4,5-P3 and Ins-1,3,4,5-P4 during cellular stimulation.     

CP55,940 increases intracellular Ca2+ levels in Madin-Darby canine kidney cells

The effect of CP55,940, a presumed CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels ([Ca2+]i) in Madin-Darby canine kidney cells was examined by using the fluorescent dye fura-2 as a Ca2+ indicator. CP55,940 (2-50 microM) increased [Ca2+]i concentration-dependently with an EC50 of 8 microM. The [Ca2+]i signal comprised an initial rise and a sustained phase. Extracellular Ca2+ removal decreased the maximum [Ca2+]i signals by 32+/-12%. CP55,940 (20 microM)-induced [Ca2+]i signal was not altered by 5 microM of two cannabinoid receptor antagonists, AM-251 and AM-281. CP55,940 (20 microM)-induced [Ca2+]i increase in Ca2+-free medium was inhibited by 86+/-3% by pretreatment with 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 20 microM CP55,940 in Ca2+-free medium for 6 min abolished thapsigargin-induced [Ca2+]i increases. CP55,940 (20 microM)-induced intracellular Ca2+ release was not inhibited when inositol 1,4,5-trisphosphate formation was abolished by suppressing phospholipase C with 2 microM U73122. Collectively, this study shows that CP,55940 induced significant [Ca2+]i increases in canine renal tubular cells by releasing stored Ca2+ from the thapsigargin-sensitive pools in an inositol 1,4,5-trisphosphate-independent manner, and also by causing extracellular Ca2+ entry. The CP55,940's action appears to be dissociated from stimulation of cannabinoid receptors.     

Thrombin and phorbol ester stimulate inositol 1,3,4,5-tetrakisphosphate 3-phosphomonoesterase in human platelets

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which mobilizes intracellular Ca2+, is metabolized either by dephosphorylation to inositol 1,4-bisphosphate(Ins-(1,4)P2) or by phosphorylation to inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). It has been shown in vitro that Ins(1,3,4,5)P4 is also dephosphorylated by a 5-phosphomonoesterase to inositol 1,3,4-trisphosphate. However, we have found that exogenous Ins(1,3,4,5)P4 is dephosphorylated to predominantly Ins(1,4,5)P3 in saponin-permeabilized platelets in the presence of KCl (40-160 mM). This inositol polyphosphate 3-phosphomonoesterase activity is independent of Ca2+ (0.1-100 microM), and it was also observed when the ionic strength of the incubation medium was increased with Na+. The action of KCl appears to be due to activation of a 3-phosphomonoesterase as well as an inhibition of the 5-phosphomonoesterase, because the dephosphorylation of Ins(1,4,5)P3 to Ins(1,4)P2 was completely inhibited by KCl. The 3-phosphomonoesterase may be regulated by a protein kinase C, since both thrombin and phorbol dibutyrate increase 3-phosphomonoesterase activity and this is inhibited by staurosporine. The formation of Ins(1,4,5)P3 from Ins(1,3,4,5)P4 reported here provides an additional pathway for the formation of the Ca2+-mobilizing second messenger in stimulated cells.     

Interaction of synthetic D-6-deoxy-myo-inositol 1,4,5-trisphosphate with the Ca2(+)-releasing D-myo-inositol 1,4,5-trisphosphate receptor, and the metabolic enzymes 5-phosphatase and 3-kinase.

The ability of D-6-deoxy-myo-inositol 1,4,5-trisphosphate [6-deoxy-Ins(1,4,5)P3], a synthetic analogue of the second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], to mobilise intracellular Ca2+ stores in permeabilised SH-SY5Y neuroblastoma cells was investigated. 6-Deoxy-Ins(1,4,5)P3 was a full agonist (EC50 = 6.4 microM), but was some 70-fold less potent than Ins (1,4,5)P3 (EC50 = 0.09 microM), indicating that the 6-hydroxyl group of Ins(1,4,5)P3 is important for receptor binding and stimulation of Ca2+ release, but is not an essential structural feature. 6-Deoxy-Ins(1,4,5)P3 was not a substrate for Ins (1,4,5)P3 5-phosphatase, but inhibited both the hydrolysis of 5-[32P]+ Ins (1,4,5)P3 (Ki 76 microM) and the phosphorylation of [3H]Ins(1,4,5)P3 (apparent Ki 5.7 microM). 6-Deoxy-Ins (1,4,5)P3 mobilized Ca2+ with different kinetics to Ins(1,4,5)P3, indicating that it is probably a substrate for Ins (1,4,5)P3 3-kinase.     

Effects of Phorbol Esters and Forskolin on Basal and Histamine-induced Accumulation of Inositol Phosphates in Cultured Bovine Adrenal Chromaffin Cells

The effect of phorbol esters and forskolin pretreatment on basal and histamine-induced accumulation of inositol phosphates and catecholamine release was examined in cultures of bovine adrenal chromaffin cells. Histamine caused a dose-dependent, Ca2+-dependent accumulation of total inositol phosphates with an EC50 at approximately 1 microM and an eight- to 10-fold increase at 100 microM within 30 min of incubation. Histamine (10 microM) also caused the release of cellular catecholamines amounting to some 2.8% of cellular stores released over a 20-min period. Both the inositol phosphate and catecholamine responses were completely blocked by the H1-antagonist mepyramine and were insensitive to the H2-antagonist cimetidine. Examination of the time course of accumulation of the individual inositol phosphates stimulated by histamine revealed an early and sustained rise in inositol 1,4-bisphosphate content but not inositol 1,4,5-trisphosphate content at 1 min and the overall largest accumulation of inositol monophosphate after 30 min of stimulation. Pretreatment with the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in a dose-dependent, time-dependent inhibition of histamine-induced inositol phosphate formation and catecholamine secretion. In this inhibitory action, PMA exhibited high potency (IC50 of approximately 0.5 nM), an effect not shared by the inactive phorbol ester 4-alpha-phorbol 12,13-didecanoate. Pretreatment with forskolin, on the other hand, only marginally inhibited the histamine-induced inositol phospholipid metabolism and catecholamine secretion. These data suggest that protein kinase C activation in chromaffin cells may mediate a negative feedback control on inositol phospholipid metabolism.     

Second Messengers and the Action of Prothoracicotropic Hormone in Manduca sexta

SYNOPSIS. The large (26 kDa) prothoracicotropic hormone of Manducasexta stimulates ecdysteroid secretion by the prothoracic glandsthrough the action of cyclic AMP (cAMP). Adenylate cyclase inthe prothoracic glands is sensitive to calcium/calmodulin, andenhancement of intracellular calcium levels may be the meansby which PTTH stimulates cAMP synthesis. The cyclic nucleotidein turn activates cAMP-dependent protein kinase and proteinphosphorylation, most notably of a 34 kDa membraneassociatedprotein. It does not appear that protein kinase C plays a rolein the acute action of PTTH, nor has the hormone been foundto stimulate formation of inositol trisphosphate undercurrentassay conditions. PTTH rapidly increases protein synthesis bythe prothoracic glands, and translation inhibitors block PTTH-stimulatedecdysteroid secretion. Connections between protein phosphorylation,protein synthesis, and ecdysone secretion remain to be clarified.     

Second messenger function of inositol 1,4,5-trisphosphate. Early changes in inositol phosphates, cytosolic Ca2+, and insulin release in carbamylcholine-stimulated RINm5F cells

The second messenger function of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) was investigated in carbamylcholine-stimulated RINm5F cells by analysis of the early changes in inositol phosphates, cytosolic free Ca2+ concentration ([Ca2+]i), and insulin secretion. After a lag of 2 s, [Ca2+]i rose to a peak at 13 +/- 2 s, a response which was due mainly to mobilization from intracellular stores since it persisted even in the absence of extracellular Ca2+. The Ca2+ response had already declined toward prestimulatory levels by the time insulin secretion reached its maximal rate (2-3 min). Although the rises in inositol trisphosphate preceded those of both inositol bisphosphate and monophosphate, all three attained maximal concentrations after 1 min and remained elevated for at least 10 min. The accumulation of inositol trisphosphate was truly Ca2+-independent since it persisted under conditions in which the rise in [Ca2+]i was abolished by prior depletion of intracellular Ca2+ pools. Further analysis by high performance liquid chromatography revealed the presence of the two isomers, Ins-1,4,5-P3 and Ins-1,3,4-P3 in stimulated cells. The latter was virtually absent under nonstimulatory conditions but started to accumulate after a 5-s lag and reached maximal levels after 30 s of stimulation. Ins-1,4,5-P3 doubled within 1 s of carbamylcholine addition, reached a peak after 5 s, and, although declining thereafter, remained slightly elevated for at least 3 min. Hence, both the onset and peak of the rise of Ins-1,4,5-P3 preceded that of [Ca2+]i, which in turn preceded the peak in insulin release. These results strongly suggest that Ins-1,4,5-P3 acts as the second messenger by which carbamylcholine mobilizes intracellular Ca2+ during the initiation of insulin release.     

Modulation of intracellular Ca2+ in the parathyroid cell. Release of Ca2+ from non-mitochondrial pools by inositol trisphosphate

Stimuli which enhance secretion from parathyroid cells such as low extracellular Ca2+ or Mg2+ are associated with a decrease in the cytosolic Ca2+ concentration as measured by quin2. Current evidence suggests that increased production of inositol 1,4,5-triphosphate (IP3) releases Ca2+ from cellular stores thus increasing cytosolic Ca2+. We used saponin-permeabilized dispersed bovine parathyroid cells to study the effect of IP3 on intracellular Ca2+. IP3 released Ca2+ from these cells in a dose-dependent manner; half-maximal response occurred with 0.3 microM IP3 and maximal response with 1.2 microM IP3. Permeabilized cells incubated in the presence of the mitochondrial inhibitor antimycin A released a similar amount of Ca2+ suggesting that IP3 releases Ca2+ from a non-mitochondrial pool. These results suggest that IP3 regulates cytosolic Ca2+ in this system and may function as a second messenger controlling hormone secretion.     

The digitonin-permeabilized pancreatic islet model. Effect of myo-inositol 1,4,5-trisphosphate on Ca2+ mobilization.

Glucose-induced insulin secretion is thought to be mediated by submicromolar increases in intracellular Ca2+, although the intracellular processes are not well understood. We have used the previously characterized digitonin-permeabilized insulin-secreting pancreatic islet model to study the role of myo-inositol 1,4,5-trisphosphate (IP3), a putative second messenger for mobilization of intracellular Ca2+. Ca2+ efflux from the endoplasmic reticulum was studied with or without vanadate present to inhibit Ca2+ reuptake. IP3 (10 microM), at a free Ca2+ level of 0.06 microM, increased Ca2+ release by 30% and, when vanadate was present, by 50%. Maximal and half-maximal Ca2+ release was observed at 10 microM- and 2.5 microM-IP3, respectively. IP3 provoked a rapid release that was followed by slow reuptake. Reuptake was diminished in the presence of vanadate. Inositol 1,4-bisphosphate, inositol 1-phosphate and other phosphoinositide metabolites did not have any significant effect. Because increases in Ca2+ levels in the submicromolar range have been previously shown to induce insulin release in digitonin-permeabilized islets, our results are consistent with the concept of IP3 serving as a second messenger for insulin secretion.