Ecdysteroid receptors in the neuroendocrine-endocrine axis of a moth

With a combination of thaw-mount autoradiography using a tritiated 20-hydroxyecdysone agonist, ponasterone A, and immunocytochemistry with a monoclonal antibody to 29 K-prothoracicotropic hormone, high affinity binding sites for ecdysteroids were identified in the tissues of the neuroendocrine-endocrine axis inManduca sexta larvae. At specific times during larval-pupal development in fifth stadium larvae, nuclear ecdysteroid binding sites were present in the cerebral prothoracicotropes, the corpora allata and prothoracic glands, the main axis for the regulation and production of ecdysteroids. A stage-specific appearance of ecdysteroid receptors also occurred in cells of fat body, midgut and Malpighian tubules, tissues which convert ecdysone into 20-hydroxyecdysone. Our data identify new target tissues for ecdysteroids and suggest that ecdysteroids could affect their own production at the genomic level via long and short feedback loops.     

Antiecdysteroid activity of brassinosteroids

Summary We report the discovery of the first antiecdysteroids which belong to a comparatively new class of plant growth regulators, the brassinosteroids. These compounds bind competitively to ecdysteroid receptors partially purified from larvae of the blowflyCalliphora vicina and inhibit biological responses to 20-hydroxyecdysone, the active form of the molting hormone.     

Insect hormones in vertebrates: Anabolic effects of 20-hydroxyecdysone in Japanese quail

Ecdysteroids are hormones controlling cell proliferation, growth and the developmental cycles of insects and other invertebrates¹. They are occasionally present in various unrelated plants for no apparent reason; no phytohormonal function has yet been identified. In certain cases, ecdysteroids are accumulated to high levels in leaves, roots or seeds. Some ecdysteroid-containing plants have been known as medicinal plants for centuries. One of them,Leuzea carthamoides Iljin (Asteraceae), growing in Central Asia, contains 0.4% ecdysteroid in dry roots and 2% in seeds. A pharmacological preparation from this plant, Ecdisten, is already available as a commercial preparation for its anabolic, tonic and other effects, for medical use (review²). It remained problematic, however, whether ecdysteroids were truly responsible for these effects, becauseLeuzea contains a number of other biologically active compounds in addition to ecdysteroids. We extracted and purified ecdysteroids from the seeds ofLeuzea. With 6 g of 96% 20-hydroxyecdysone (20E), we made a large-scale feeding assay with Japanese quail to find out whether ecdysteroid alone could duplicate the anabolic effects of the seeds. We found that the 96% ecdysteroid increased the mass of the developing quails in a dose-dependent manner, with the rate of increase proportional to the ecdysteroid content in the seeds; there was a 115% increase in living mass with 100 mg kg^–1 of pure 20E compared with 109.5% increase with 100–180 mg kg^–1 20E equivalents in the seeds. We conclude that the plethora of growth-promoting, vitamin-like effects induced in vertebrates byLeuzea is mediated by ecdysteroids.     

Assessment of natural products in the Drosophila melanogaster BII cell bioassay for ecdysteroid agonist and antagonist activities

Ecdysteroid agonist and antagonist activities can be detected and quantified with the Drosophila melanogaster B(II) cell bioassay. This bioassay is convenient, sensitive and robust. We report the assessment with this bioassay of the activities of a wide range of compounds representing a number of classes of natural products. Many compounds were inactive over a wide concentration range (10(-8) to 10(-4) or 10(-3) M) or cytotoxic at high concentrations. However, antagonisitic activity was associated with several classes of compounds: cucurbitacins and withanolides (extending previous findings) and phenylalkanoids and certain alkaloids (described for the first time). A withanolide (withaperuvin D) is identified which possesses agonistic activity. Brassinosteroids, which have been ascribed (ant)agonistic properties in the past, were not found to be active in the B(II) bioassay, either as agonists or antagonists. Possible reasons for the prevalence of antagonists and for the low potency of the majority of them are discussed.     

Ecdysteroids inLimulus larvae

Summary Free ecdysteroids were extracted from intermolt and premolt larvae ofLimulus polyphemus in the first posthatch stage, purified by TLC and HRLC, and assayed by RIA and theLimulus bioassay 20-hydroxyecdysone appears to be a principal ecdysteroid and occurs at least 3 times higher a concentration in premolt vs intermolt animals.Supported by NSF grant GB-40620.Acknowledgments. We are indebted to Prof. G. R. Wyatt for the antibody, Prof. K. Nakanishi for use of his laboratory for HRLC separations, and to Dr David King for the³H-ecdysone and for performing radio-TLC analyses.     

Reversible juvenile hormone inhibition of ecdysteroid and juvenile hormone synthesis by the ring gland of Drosophila melanogaster

Juvenile hormone bisepoxide (JHB3) and juvenile hormone III (JH III) both inhibited the in vitro production of ecdysteroids by ring glands and brain-ring gland complexes from third instar post-feeding larvae of Drosophila melanogaster in a reversible manner, although JHB3 had greater efficacy. The JH III and JHB3 precursor, methyl farnesoate, did not affect ecdysteroid production. The in vitro synthesis of total detectable JH (JHB3 + JH III + methyl farnesoate) by the corpus allatum portion of the isolated ring gland was also inhibited reversibly in the presence of exogenous JHB3 and JH III, but not by methyl farnesoate. These data indicating negative feedback are in agreement with the accepted dogma of endocrine gland regulation.     

Reversible juvenile hormone inhibition of ecdysteroid and juvenile hormone synthesis by the ring gland ofDrosophila melanogaster

Juvenile hormone bisepoxide (JHB₃) and juvenile hormone III (JH III) both inhibited the in vitro production of ecdysteroids by ring glands and brain-ring gland complexes from third instar post-feeding larvae ofDrosophila melanogaster in a reversible manner, although JHB₃ had greater efficacy. The JH III and JHB₃ precursor, methyl farnesoate, did not affect ecdysteroid production. The in vitro synthesis of total detectable JH (JHB₃+JH III+methyl farnesoate) by the corpus allatum portion of the isolated ring gland was also inhibited reversibly in the presence of exogenous JHB₃ and JH III, but not by methyl farnesoate. These data indicating negative feedback are in agreement with the accepted dogma of endocrine gland regulation.     

Variations des taux d'ecdystéroïdes au cours du développement deBombyx mori; rapport entre ces variations et les phases de croissance et de morphogenèse

Summary During the development ofBombyx mori (monovoltin race) ecdysteroid levels were determined in oocytes, eggs, and haemolymph of larvae, and in the haemolymph of pupae. In haemolymph, the only RIA reactive materials are ecdysone and ecdysterone. In oocytes and eggs, other ecdysteroids are also detected. During larval instars, the ecdysteroid levels are low whereas they are very high during morphogenetic periods. During embryonic diapause, the ecdysone titer decreases during the cessation of morphogenesis.     

Prothoracic gland synthesis of 3-dehydroecdysone and its hemolymph 3 beta-reductase mediated conversion to ecdysone in representative insects

The prothoracic glands of a variety of insects were tested for their ability to synthesize ecdysteroids in vitro. More specifically, they were evaluated for their ability to produce 3-dehydroecdysone and ecdysone using both radioimmunoassay and reverse phase high performance liquid chromatography. Three categories of insect prothoracic glands were noted: a) those producing much more 3-dehydroecdysone than ecdysone; b) glands synthesizing almost equivalent amounts of each of these two ecdysteroids; c) prothoracic glands that yielded more ecdysone than 3-dehydroecdysone. In addition, the 3-oxoecdysteroid 3 beta-reductase activity of the hemolymph of these insects was evaluated for its ability to convert 3-dehydroecdysone to ecdysone. The lepidopteran species tested yielded the most potent enzyme activity, although activity was demonstrated in members of other orders. These data indicate that the dehydroecdysone-ecdysone axis is not restricted to moths and butterflies.     

Exogenous makisterone A accelerates early embryonic development in the milkweed bugOncopeltus fasciatus

Summary Reproducing females ofOncopeltus fasciatus which were treated with exogenous makisterone A and 20-hydroxyecdysone laid eggs with considerably elevated ecdysteroid contents. The early embryonic development was markedly accelerated when the mother was treated with makisterone A, whereas 20-hydroxyecdysone had no influence.Supported by grants of the Deutsche Forschungsgemeinschaft (Do 163/7) to A. D.The technical assistance of Ms Heike Bender is acknowledged. We thank Dr K.-D. Spindler for providing us with antiserum for the RIA.     

Role of low ecdysteroid levels in the early last larval instar ofBombyx mori

The relationship between juvenile hormone and ecdysteroid titers during the early stage of the last larval instar of the silkworm,Bombyx mori, was examined in this study. When larvae were fed 20-hydroxyecdysone-supplemented mulberry leaves throughout the last larval instar, 100% underwent supernumerary larval molting instead of metamorphosis. The application of juvenile hormone mimic during the early last instar did not induce supernumerary larval molting, but did delay metamorphosis. Temporal and quantitative ecdysteroid titer measurements revealed that in normal larvae the titers maintained very low levels (3–12 ng/ml) during the early stage of the last instar; however, in 20-hydroxyecdysone-treated larvae, levels ranging from 24 to 45 ng/ml were detected, and a major peak (246 ng/ml) was observed on day 6. These results show that very low ecdysteroid titer levels during the early stage of the last larval instar may play an important role in initiating decreases in juvenile hormone titers as well as in directing metamorphosis.     

The biotransformation of tritiated 3-dehydroecdysone by crayfish,Procambarus clarkii

The injection of 3-dehydroecdysone (3dhE, 5 /g), the major ecdysteroid secreted by the Y-organ of crayfishProcambarus clarkii, resulted in apolysis within about 5 days. The hormonal response at the molecular level was investigated by injection of the radio-labeled compound; within 3 h of injection of [³H]3dhE, most radio-isotope was found in the extracted epidermal tissues and identified as ecdysone, 20-hydroxyecdysone (20E), and their 3-hydroxy epimers. The biotransformation was undoubtedly performed in the peripheral area of the Y-organ. Cleavage of the polar conjugates, using an enzyme fromHelix pomatia, gave all of the above ecdysteroids including 3dhE. It was also found that the biosynthetic site of 3dhE was different from that of ecdysone at the subcellular level of the Y-organ.     

Makisterone A and 24-methylenecholesterol from the ovaries of the honey bee,Apis mellifera L

Summary Makisterone A, a 28-carbon ecdysteroid (molting hormone) has been isolated from the ovaries of queen bees. Analysis by reversed-phase and silica high performance liquid chromatography (HPLC) in conjunction with a radioimmune assay (RIA) revealed about 11 ng of makisterone A present per gram of ovaries on a fresh weight basis. No C₂₇ ecdysteroids were detected. The predominant neutral sterol present was 24-methylenecholesterol.     

Prothoracic gland synthesis of 3-dehydroecdysone and its hemolymph 3β-reductase mediated conversion to ecdysone in representative insects

Summary The prothoracic glands of a variety of insects were tested for their ability to synthesize ecdysteroids in vitro. More specifically, they were evaluated for their ability to produce 3-dehydroecdysone and ecdysone using both radioimmunoassay and reverse phase high performance liquid chromatography. Three categories of insect prothoracic glands were noted: a) those producing much more 3-dehydroecdysone than ecdysone; b) glands synthesizing almost equivalent amounts of each of these two ecdysteroids; c) prothoracic glands that yielded more ecdysone than 3-dehydroecdysone. In addition, the 3-oxoecdysteroid 3-reductase activity of the hemolymph of these insects was evaluated for its ability to convert 3-dehydroecdysone to ecdysone. The lepidopteran species tested yielded the most potent enzyme activity, although activity was demonstrated in members of other orders. These data indicate that the dehydroecdysone-ecdysone axis is not restricted to moths and butterflies.     

In vitro secretion of ecdysteroids by Y-organs of the crayfish,Procambarus clarkii

It was demonstrated that excised Y-organs of the crayfish,Procambarus clarkii, synthesize in vitro 3-dehydroecdysone (3-DHE) as the major product, together with small amounts of ecdysone. Both were identified by immunological and spectroscopic methods. The increase of ecdysteroidogenesis in the Y-organs was accompanied by an increase of the major free ecdysteroid, 20-hydroxyecdysone, in the hemolymph. This suggests a physiological role of 3-DHE, the details of which are still to be elucidated.     

Occurrence of 3-epi-22-deoxy-20-hydroxyecdysone and its phosphoric ester in diapause eggs of the silkworm,Bombyx mori

Ecdysteroids in diapause eggs of the silkworm,Bombyx mori, were analyzed using high-performance liquid chromatography (HPLC) combined with radioimmunoassay (RIA). A relatively large amount of an unidentified free ecdysteroid and its phosphoric ester (conjugated form) were detected. These two compounds were isolated by a combination of column chromatography on silicic acid, thin-layer chromatography (TLC), and HPLC using a reverse-phase (RP) column. The purified compounds were identified as 3-epi-22-deoxy-20-hydroxyecdysone (22d20E) and 3-epi-22-deoxy-20-hydroxyecdysone 2-phosphate (22d20E2P) by means of mass spectrometry and nuclear magnetic resonance spectroscopy. to our knowledge, this is the first report of 22d20E and 22d20E2P.     

The association of phytoecdysteroids with flowering in fat hen,Chenopodium album,and other members of the Chenopodiaceae

Very high concentrations of ecdysteroid are associated with flowering inChenopodium album. Highest concentrations are found in anthers, but significant levels are also found in the stamens, carpels and sepals. In contrast, pollen contains only low levels. The ecdysteroid profile is the same in anthers as in whole-plant extracts, with 20-hydroxyecdysone and polypodine B predominating. The results for flowers ofC. album are compared with the patterns determined for other chenopods (C. capitatum, C. polyspermum, C. anthelminticum, C. giganteum, C. quinoa andC. foliosum). The significance of these findings for plant-insect interactions and the relationship to the mode of plant pollination are discussed.     

Cellular models for the detection and evaluation of drugs that modulate human phagocyte activity

Simple testing models have been developed for the evaluation of chemical or biological compounds that modulate the activity of human phagocytes. Human neutrophils from buffy coats of donor blood are used. They are stimulated with receptor agonists, and the effects of test compounds on exocytosis of different enzymes, the generation of superoxide (respiratory burst), and cytotoxicity are quantified. All assays are performed in microtiter plates and the responses are evaluated by multi-well photometry or fluorimetry. The models are apt to detect compounds acting on phagocytes as agonists or antagonists, signal transduction activators or inhibitors and primers of agonist responses, and to assess cytotoxic effects.     

Cellular models for the detection and evaluation of drugs that modulate human phagocyte activity

Summary Simple testing models have been developed for the evaluation of chemical or biological compounds that modulate the activity of human phagocytes. Human neutrophils from buffy coats of donor blood are used. They are stimulated with receptor agonists, and the effects of test compounds on exocytosis of different enzymes, the generation of superoxide (respiratory burst), and cytotoxicity are quantified. All assays are performed in microtiter plates and the responses are evaluated by multi-well photometry or fluorimetry. The models are apt to detect compounds acting on phagocytes as agonists or antagonists, signal transduction activators or inhibitors and primers of agonist responses, and to assess cytotoxic effects.     

Agonist-induced internalization and desensitization of the human nociceptin receptor expressed in CHO cells

In this study, we examined agonist-induced internalization, recycling and signalling (measure of cAMP levels) of the cloned human nociceptin receptor (hNOP) expressed in CHO-K1 cells. Internalization was proven by a receptor-binding assay on viable cells. The agonist nociceptin/orphanin FQ (NC) promoted rapid internalization of the hNOP receptor (approximately 78% of cell surface receptors were lost after 2 min exposure to 1 microM NC) in a clathrin- and ATP-dependent manner. Internalization was more rapid and marked in CHO-K1 cells than, as we previously reported, in SK-N-BE cells. This difference may be related to higher levels of beta-arrestin isoforms detected in CHO-K1 than in SK-N-BE cells. hNOP receptor internalization was partially reversible and recycling occurred in the presence of the agonist; receptor recycling was dependent on okadaic acid-sensitive phosphatases and was blocked by monensin. Confocal microscopy analysis confirmed the internalization and the recycling back to the plasma membrane of an epitope-tagged hNOP receptor expressed in CHO-K1 cells. These receptors underwent rapid desensitization upon agonist challenge: NC efficacy in inhibiting forskolin-stimulated cAMP production was significantly reduced 10 min after exposure and correlated with the rate of receptor internalization. Moreover, we observed that blockade of hNOP receptor recycling by monensin would cause a more prolonged and relevant desensitization of this receptor. Thus, the dynamic cycle between hNOP receptor activation, internalization and recycling determines the activity of this receptor on the cell surface.