Activation of an extracellular signal-regulated kinase (ERK) by the insect prothoracicotropic hormone.

Ecdysteroid hormones are crucial in controlling the growth, molting and metamorphosis of insects. The predominant source of ecdysteroids in pre-adult insects is the prothoracic gland, which is under the acute control of the neuropeptide hormone prothoracicotropic hormone (PTTH). Previous studies using the tobacco hornworm, Manduca sexta, have shown that PTTH stimulates ecdysteroid synthesis via a series of events, including the activation of protein kinase A and the 70 kDa S6 kinase (p70(S6k)). In this study, PTTH was shown to stimulate also mitogen-activated protein kinase (MAPK) phosphorylation and activity in the Manduca prothoracic gland. The MAPK involved appears to be an extracellular signal-regulated kinase (ERK) homologue. The ERK phosphorylation inhibitors PD 98059 and UO 126 blocked basal and PTTH-stimulated ERK phosphorylation and ecdysteroid synthesis. PTTH-stimulated ERK activity may be important for both rapid regulation of ecdysteroid synthesis and for longer-term changes in the size and function of prothoracic gland cells.     

Prothoracicotropic hormone stimulated extracellular signal-regulated kinase (ERK) activity: the changing roles of Ca(2+)- and cAMP-dependent mechanisms in the insect prothoracic glands during metamorphosis

The synthesis of ecdysteroids by the lepidopteran prothoracic gland is regulated by a brain neuropeptide hormone, prothoracicotropic hormone (PTTH). In Manduca sexta glands, PTTH stimulates several events including Ca(2+) influx, Ca(2+)-dependent cAMP generation and the activation of several protein kinases. In the present study, the path by which PTTH stimulates extracellular signal-activated regulated kinase (ERK) phosphorylation was investigated using PTTH and second messenger analogs. The results indicate that Ca(2+)-dependent processes, other than cAMP generation, play the major role in PTTH stimulation of ERK phosphorylation in larval prothoracic glands, that cAMP-dependent events increase in importance during later development and that PTTH-stimulated ERK phosphorylation is highest in larval glands. The decline in PTTH-stimulated ERK phosphorylation associated with metamorphosis results from decreased ERK levels and an increased basal rate of ERK phosphorylation. The data suggest that the role or importance of components of the PTTH signal transduction cascade are not fixed and can change during development.     

Developmental changes in phosphodiesterase activity and hormonal response in the prothoracic glands of Manduca sexta

Prothoracicotropic hormone (PTTH) stimulates ecdysteroid secretion by the prothoracic glands of Manduca sexta in a cAMP-dependent manner. However, larval and pupal glands differ markedly in the degree to which PTTH stimulates cAMP accumulation, suggesting a stage-specific difference in phosphodiesterase activity. The present study was designed to determine if and when such a difference arose during development, and its effect on PTTH-stimulated ecdysteroid secretion. The results reveal that soluble phosphodiesterase activity in the prothoracic glands changes significantly during the course of the fifth (last) larval instar, with a marked increase in activity occurring at the onset of prepupal development. Phosphodiesterase activity, particularly in the soluble cell fraction, is inversely correlated with PTTH-stimulated cAMP accumulation. Hormone-stimulated ecdysteroid secretion does not require cAMP accumulation, but does appear to require detectable cAMP synthesis as measured in the presence of phosphodiesterase inhibitors. The amount of ecdysteroid secreted, however, is not proportional to the amount of cAMP synthesized but rather is more closely correlated with developmental changes in glandular protein content.     

Inhibition of tyrosine phosphorylation blocks hormone-stimulated calcium influx in an insect steroidogenic gland

In the tobacco hornworm Manduca sexta (M. sexta) as in other insects, ecdysone synthesis occurs in the prothoracic glands and is stimulated by the brain neuropeptide prothoracicotropic hormone (PTTH). PTTH activates the prothoracic glands through the second messenger cAMP, the synthesis of which is stimulated by calcium. We previously found that the Src kinase inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-D]-pyrimidine (PP1) inhibits PTTH-stimulated cAMP synthesis and ecdysone secretion. In the present study, we show that by contrast, PP1 does not block cAMP synthesis stimulated by the calcium ionophore A23187, and that PP1 augments A23187-stimulated ecdysone secretion. Hence, once glandular levels of calcium are elevated, Src family kinase activity is no longer needed for, and may actually inhibit, steroidogenesis. PP1 blocks calcium influx in PTTH-stimulated prothoracic glands, indicating that tyrosine phosphorylation by a member of the Src kinase family is required for calcium influx. These results suggest that prothoracic gland calcium channels are regulated either directly or indirectly by tyrosine phosphorylation.     

Involvement of cyclic AMP-dependent protein kinase in prothoracicotropic hormone-stimulated ecdysone synthesis

Prothoracicotropic hormone (PTTH) is a brain neuropeptide that stimulates the prothoracic glands to synthesize ecdysone, an event that leads to insect molting. Both cyclic AMP (cAMP) and calcium have been implicated in PTTH action, with current evidence favoring cAMP as the messenger directly regulating ecdysone synthesis. To further define the role of cAMP in PTTH action, the activity of cAMP-dependent protein kinase (cAMP-PK) was examined in prothoracic glands from two developmental stages of the tobacco hornworm, Manduca sexta (day 3 fifth instar larvae and day 0 pupae). Prothoracic glands at each of these stages of development possess two forms of cAMP-PK which resemble the vertebrate type I and type II isozymes, with the latter being the predominant form (greater than 90%). Marked developmental differences exist in the degree of activation of soluble cAMP-PK following in vitro exposure of the prothoracic glands to PTTH. In larval glands, soluble cAMP-PK is activated within 3-10 min of initial exposure to doses of PTTH that stimulate ecdysone synthesis. By contrast, activation of soluble cAMP-PK in pupal glands occurs only when PTTH is administered in the presence of a phosphodiesterase inhibitor. Developmental differences in the activation of cAMP-PK by PTTH were qualitatively identical to previously observed differences in PTTH-stimulated accumulation of intracellular cAMP. The results suggest an involvement of soluble cAMP-PK in the response of day 3 fifth instar larval prothoracic glands to PTTH, but indicate a difference in the nature, intracellular location, or time course of activation, of hormone-sensitive protein kinase in day 0 pupal glands.     

A deficiency in prothoracicotropic hormone transduction pathway during the early last larval instar of Bombyx mori

The prothoracicotropic hormone (PTTH) is an insect cerebral peptide that stimulates the prothoracic glands to produce ecdysteroids that initiate moulting and metamorphosis. During the last larval instar of holometabolous insects, a reduction in the hemolymph juvenile hormone (JH) levels is a necessary step in initiating larval-pupal transformation. Recently we have demonstrated that very low ecdysteroid levels in the early last larval instar of Bombyx mori initiate the complete inactivation of corpora allata (CA). Results presented here further indicate that PTTH signal transduction pathways undergo specific developmental changes, with a deficiency in transduction in prothoracic gland cells occurring during the early last instar. Glands from the early last instar showed no increase in either cAMP levels or steroidogenesis to the stimulation of PTTH, indicating the absence of the PTTH receptors in gland cells. We propose that this absence of PTTH receptors plays a critical role in directing larval-pupal transformation.     

Developmental changes in cyclic AMP-dependent protein kinase associated with increased secretory capacity of Manduca sexta prothoracic glands

In Manduca sexta, basal and PTTH-stimulated secretion of ecdysteroids by prothoracic glands in vitro increases from days 1 to 4 of the fifth larval stage. Glandular content of cAMP-dependent protein kinase was analyzed to determine if the enzyme changes in concert with increased secretory response. Photoaffinity labeling with [³²P]8-N₃ cAMP revealed a 55-kDa cAMP-binding protein characteristic of the regulatory subunit of type-II cAMP-dependent protein kinase (RII). It appears that RII is one of a limited number of cellular proteins that is phosphorylated in the presence of [γ-³⁵S]ATP; the thiophosphorylated protein and the photoaffinity-labeled regulatory subunit possess the same M_r and pI, and thiophosphorylation is blocked by mammalian cAMP-dependent protein kinase inhibitor. From days 1 to 4 of the fifth instar, glandular content of RII increases in conjunction with increased ecdysteroid secretory capacity. Application of JH analog on day 1 significantly inhibits the observed increase in RII. Catalytic subunit activity does not change from days 1 to 4 of the fifth instar, nor does cellular content of a 34-kDa protein previously shown to be phosphorylated in response to PTTH. While it is unlikely that increased content of RII is solely responsible for enhanced ecdysteroid secretion by the prothoracic glands, it may serve as a convenient marker for investigating the mechanism by which steroidogenic capacity is regulated.     

The role of cyclic AMP in the regulation of ecdysone synthesis

The involvement of cAMP in the steroidogenic action of the cerebral neuropeptide, prothoracicotropic hormone (PTTH), was examined in the tobacco hornworm, Manduca sexta. PTTH stimulates ecdysone synthesis by paired organs in the thorax, the prothoracic glands. The steroidogenic effect of PTTH on prothoracic glands of day 3 fifth instar larvae and day 0 pupae is mimicked in vitro by agents that act by increasing the intracellular levels of cAMP (1-methyl-3-isobutylxanthine, cAMP analogs and forskolin). In addition, partially purified big PTTH (Mr 28500 daltons) stimulates the formation of cAMP in both stages of glands in a rapid, dose-dependent manner. However, a significant accumulation of cAMP in response to PTTH occurs only in larval prothoracic glands. In pupal glands, effects of the neuropeptide on cAMP synthesis are seen only in the presence of a phosphodiesterase inhibitor. The results indicate that cAMP is involved as a second messenger in PTTH action, but that the developmental status of the prothoracic glands influences the degree to which cAMP accumulates in response to the neurohormone.     

Calcium-cyclic AMP interactions in prothoracicotropic hormone stimulation of ecdysone synthesis

The calcium-dependence of ecdysone synthesis by the insect prothoracic glands was examined in vitro using glands from day 0 pupae of the tobacco hornworm, Manduca sexta. Stimulation of ecdysone synthesis by the cerebral neuropeptide, prothoracicotropic hormone (PTTH), requires extracellular calcium; peptide-stimulated steroidogenesis is blocked by omission of calcium or by addition of the calcium antagonist lanthanum. By contrast, basal synthesis of ecdysone is not calcium-dependent. A stimulatory, as opposed to simply a permissive, role for calcium is indicated by the ability of the calcium ionophore A23187 to mimic the steroidogenic effects of PTTH. Agents that act by increasing the intracellular levels of cAMP (dibutyryl cAMP, 1-methyl-3-isobutylxanthine, forskolin) enhance ecdysone synthesis equally well in the presence or absence of extracellular calcium, indicating that the site of action of the cation in the steroidogenic pathway occurs prior to that of cAMP. Both PTTH and A23187 enhance the formation of cAMP, as measured by the conversion of [3H]ATP to [3H]cAMP, in a manner absolutely dependent upon the presence of extracellular calcium. The results suggest sequential roles for calcium and cAMP in PTTH-stimulated steroidogenesis by the insect prothoracic glands. A model is presented in which the peptide stimulates cAMP formation in a calcium-dependent manner, with the cyclic nucleotide in turn enhancing ecdysone synthesis.     

The parasitoid Apanteles kariyai inhibits pupation of its host, Pseudaletia separata, via disruption of prothoracicotropic hormone release

When the parasitoid Apanteles kariyai laid eggs into host Pseudaletia separata larvae, before prothoracicotropic hormone (PTTH) was released in the last instar preparatory to metamorphosis, the host did not pupate and the larvae of the wasps emerged. The ecdysteroid titer of unparasitized intact larvae increased up to 1 microgram/ml 1 day before pupation, whereas the titer of parasitized larvae was maintained at a low level without the surge. Isolated prothoracic glands from intact larvae synthesized much more ecdysone than those of parasitized larvae both in vivo and in vitro. Administration of exogenous PTTH caused the activation of the prothoracic glands seen during parasitization. Injection of 20-hydroxyecdysone (20-HE) into the parasitized larvae caused by host's pupation, but did not affect the development of the wasp larvae. However, the sensitivity of the integument to 20-HE was lower in parasitized than in unparasitized larvae. Injection of a mixture of adult wasp calyx and venom fluids into last instar unparasitized larvae delayed their pupation, suggesting that calyx and venom fluids are factors contributing to disturbance of the normal function of brain-prothoracic gland system. These results show that parasitization inhibits secretion and/or synthesis of PTTH and also delays the larval-pupal commitment of the integument by keeping the ecdysteroid level low.     

The insect neuropeptide prothoracicotropic hormone is released with a daily rhythm: re-evaluation of its role in development

Prothoracicotropic hormone (PTTH) is the central cerebral neurohormone in insect development. Its release has been believed for decades to be confined to one (or two) critical moments early in each developmental stage at which time it triggers prolonged activation of the prothoracic glands to synthesize and release the steroid molting hormones (ecdysteroids), which elicit developmental responses in target tissues. We used an in vitro assay for PTTH released from excised brains of the bug Rhodnius prolixus and report that release of PTTH does occur at the expected time on day 6, but that this release is merely the first in a daily rhythm of release that continues throughout most of the 21 days of larval-adult development. This finding, together with reports of circadian control of ecdysteroid synthesis and titer throughout this time, raises significant challenges to several features of the current understanding of the hormonal control of insect development. New questions are raised concerning the function(s) of PTTH, its relationship with the prothoracic glands, and the significance of circadian rhythmicity throughout this endocrine axis. The significance of the reported observations derives from the set of entirely new questions they raise concerning the regulation of insect development.     

Polyamines modulate multiple protein phosphorylation pathways in the insect prothoracic gland.

Multiple endogenous substrates phosphorylated by four distinct protein kinases were identified in particulate and cytosolic fractions from the larval prothoracic gland of the tobacco hornworm, Manduca sexta. Three prominent particulate-associated phosphoprotein substrates (19, 21, and 34 kDa) were of particular interest. The in vitro phosphorylation of the 19 and 21 kDa peptides was markedly enhanced by cAMP, Ca2+/calmodulin, as well as Ca2+/phospholipids, presumably via cAMP-dependent protein kinase (cAMP-PK), Ca2+/calmodulin-dependent protein kinase (Ca2+/CaM-PK), and protein kinase C (PKC), respectively. The polyamine spermine markedly inhibits both PKC- and cAMP-PK-mediated phosphorylation of the 19 and 21 kDa peptides but had no effect on the Ca2+/CaMP-PK-mediated phosphorylation. Spermine also inhibits the phosphorylation of the 34 kDa peptide via cAMP-PK but does not affect PKC-promoted phosphorylation. In contrast to this differential inhibition of phosphorylation by a polyamine, four cytosolic and three particulate-associated peptides from the prothoracic glands undergo enhanced phosphorylation in the presence of spermine, presumably by stimulating casein kinase II activity. Therefore, polyamines appear to have multiple effects on protein phosphorylation pathways in this important endocrine gland, perhaps representing an important new regulatory control mechanism.     

Announcement

The relationship between hemolymph ecdysteroid titer, ring gland (RG) activity, and prothoracicotropic hormone (PTTH) activation of RG in vitro has been examined during the postfeeding larval, prepupal, and pupal stages of Sarcophaga bullata. Using the ecdysteroid radioimmunoassay (RIA), two significant peaks were recorded during the red spiracular stage and during the first few hours after the formation of the white prepupa and a third large peak 9 hr later. It is postulated that these increases in ecdysteroid titer are involved in the processes of pupariation, puparial tanning, and pupation, respectively. Ring glands isolated from Sarcophaga of known ages were incubated in vitro and the secreted ecdysone was quantified by RIA. Ring glands from early red spiracular stage larvae proved to be the most active and subsequent secretory activity of the RG oscillated every 4 hr with the oscillations gradually decreasing in amplitude. RG activity returned to a basal level 24 hr after formation of the white prepupa, about the time that the hemolymph ecdysteroid titer fell to its basal level. To demonstrate PTTH activity in vitro, brains from 3- to 4-hr prepupae were chosen to activate ring glands from postfeeding larvae. Using a graded series of dilutions of PTTH extract it was shown that a dose-response relationship could be obtained for Sarcophaga similar to that demonstrated for the Manduca sexta PTTH-prothoracic gland system. In Sarcophaga maximal activation resulted in a 10-fold increase in ecdysone synthesis and secretion by ring glands stimulated with 0.5 brain eq. Half-maximal stimulation was attained with 0.2 brain eq of PTTH extract.     

Purification and characterization of the prothoracicotropic hormone of Drosophila melanogaster

The prothoracicotropic hormone (PTTH) of Drosophila melanogaster is a modulator of ecdysteroid (molting hormone) synthesis and was isolated and characterized from extracts of whole larvae (≈4 × 10⁵ larvae). The purification protocol included delipidation, salt-extraction, heat treatment, conventional column chromatography, and HPLC, and yielded about 50 μg of pure hormone. Biological activity was followed using a ring gland in vitro assay in which ecdysteroidogenesis by control ring glands as measured by radioimmunoassay was compared with ring gland incubations containing active fractions. The molecular weight of the purified PTTH was 45 kDa and N-terminal amino acid sequence analysis indicated that those analyzed sequences displayed no significant homology with known peptides or peptide hormones, including PTTH from the silkmoth, Bombyx mori. Western blot analysis indicated that the native form of Drosophila PTTH was a single 66-kDa polypeptide with N-linked carbohydrate chains and intrachain disulfide bonds. The purified 45-kDa peptide is the deglycosylated form, a result of glycosidase activity present during preparation of the PTTH extract. The deglycosylated form shows heterogeneity, presumably as a result of varying degrees of deglycosylation at the N terminus.     

Juvenile hormone inhibits ecdysone secretion and responsiveness to prothoracicotropic hormone in prothoracic glands of Bombyx mori

The role of juvenile hormone (JH) in the regulation of prothoracic gland activity was investigated during the early days of the last (fifth) larval instar of Bombyx mori. Allatectomy on the day of larval ecdysis into the fifth instar or 1 day before ecdysis shortened the time between larval ecdysis and gut purge. Prothoracic glands of the freshly ecdysed fifth instar larvae were inactive and did not respond to the prothoracicotropic hormone (PTTH), whereas those larvae that were allatectomized 1 day before ecdysis exhibited secretory activity in vitro and were capable of responding to PTTH. When corpora allata were removed from freshly ecdysed fifth instar larvae, the prothoracic glands became competent to respond to PTTH in 6 hr and exhibited secretory activity in vitro 9 hr after the allatectomy. Treatment of allatectomized larvae with a JH analog resulted in the recovery of the normal inactive state of the glands. These data suggest that JH acts during the early stages of the instar to suppress both the secretory activity of prothoracic glands and also the acquisition of competence to respond to PTTH.     

Cell-signaling evidence for adenosine stimulation of coronary smooth muscle proliferation via the A1 adenosine receptor

For decades, it has been thought that adenosine is exclusively antimitogenic on vascular smooth muscles via the A2-type adenosine receptor. Recently, we have demonstrated that adenosine stimulates proliferation of porcine coronary artery smooth muscle cells (CASMC) through the A1 adenosine receptor. However, the cell-signaling mechanisms underlying A1 receptor-mediated CASMC proliferation in response to adenosine have not been defined. Here, we show that in cultured CASMC, adenosine stimulates phosphorylation of extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), and AKT in a concentration- and time-dependent manner. This effect is fully mimicked by NECA (nonselective agonist), largely mimicked by CCPA (A1-selective agonist), weakly mimicked by 2-Cl-IB-MECA (A3-selective agonist), but not by CGS21680 (A2A-selective agonist), indicating that adenosine signals strongly via the A1 receptor to these mitogenic signaling pathways. This interpretation is supported by the finding that adenosine- and CCPA-induced phosphorylation of ERK, JNK, and AKT are inhibited by pertussis toxin (inactivator of Gi proteins) and by DPCPX (A1-selective antagonist), but not by SCH58261, MRS1706, and VUF5574 (A2A-, A2B-, and A3-selective antagonists, respectively). In addition, adenosine- and CCPA-induced phosphorylation of ERK, JNK, and AKT is inhibited, respectively, by U0126, PD98059 (mitogen-activated protein kinase kinase inhibitors), SP600125 (JNK kinase inhibitor), and wortmannin (phosphatidylinositol 3-kinase inhibitor). Furthermore, these kinase inhibitors abolish or diminish adenosine- and CCPA-induced increases in the rate of cellular DNA synthesis, bromodeoxyuridine incorporation, protein synthesis, and cell number. We conclude that adenosine activates the ERK, JNK, and phosphatidylinositol 3-kinase/AKT pathways primarily through the A1 receptor, leading to CASMC mitogenesis.     

Juvenile hormone regulates the steroidogenic competence of Manduca sexta prothoracic glands

The acquisition of steroidogenic competence by prothoracic glands of last instar Manduca sexta larvae is regulated by juvenile hormone (JH). Topical treatment of pre-commitment larvae with JH I or (7S)-hydroprene (a JH analog) delays development by increasing the time to pupal commitment and wandering. Prothoracic gland competence is suppressed in JH-treated larvae: Unstimulated and prothoracicotropic hormone (PTTH)-stimulated rates of in vitro ecdysone secretion are decreased relative to rates of secretion by competent glands. (7S)-Hydroprene also suppresses the competence of glands in head-ligated pre-commitment larvae, suggesting the hormone acts directly on the glands. Two results indicate PTTH plays a role in controlling competence, and that JH regulates competence indirectly by inhibiting PTTH release: (1) head-ligation prevents the acquisition of full competence, and (2) cAMP levels are elevated in glands from JH-treated larvae. Thus, the decrease in the JH titer that precedes pupal commitment in Manduca is permissive for the acquisition by prothoracic glands of steroidogenic competence.     

Inhibition of Ca2+-dependent PKC isoforms unmasks ERK-dependent hypertrophic growth evoked by phenylephrine in adult ventricular cardiomyocytes

OBJECTIVE: The duration of extracellular signal-regulated kinase (ERK) activation and the ERK-dependency of hypertrophic growth differ between stimulation of alpha-adrenoceptors or angiotensin II receptors. As both receptor systems activate different protein kinase C (PKC) isoforms, we hypothesized that PKC isoforms contribute to the specific effect of alpha-adrenoceptor stimulation. METHODS: Isolated adult ventricular cardiomyocytes from rats were used. Different PKC isoforms were inhibited either pharmacologically by six different PKC inhibitors or specifically downregulated by antisense oligonucleotides. ERK activation was determined by phosphorylation relative to total ERK. The rate of protein synthesis was determined by 14C-phenylalanine incorporation. RESULTS: The hypertrophic response of phenylephrine was inhibited in a concentration-dependent fashion by three different inhibitors of Ca2+-independent PKC isoforms (G?6983, rottlerin, G?6850), but not by three distinct PKC inhibitors directed preferentially against Ca2+-dependent PKC isoforms (Ro32-0432, HBDDE, G?6976). Antisense oligonucleotides directed against PKC-alpha, -delta, or -epsilon downregulated their specific isoforms. Their corresponding sense oligonucleotides did not affect PKC isoform expression. The phenylephrine-induced increase in protein synthesis was blocked by antisense oligonucleotides directed against PKC-delta or PKC-epsilon but not PKC-alpha, confirming the pharmacological experiments. Inhibition of Ca2+-dependent PKC isoforms by HBDDE or G?6976 converted a transient activation of ERK by phenylephrine into a sustained response. Under these conditions, phenylephrine increased protein synthesis in an ERK-dependent way. CONCLUSION: Inhibition of Ca2+-dependent PKC isoforms converts the ERK-independent effect of phenylephrine on protein synthesis into an ERK-dependent induction of protein synthesis. We conclude that co-activation of Ca2+-dependent PKC isoforms by phenylephrine contributes to the specific effect on adult ventricular cardiomyocytes from rat.