Role of arylalkylamine <Emphasis Type="Italic">N</Emphasis>-acetyltransferase in regulation of biogenic amines levels by gonadotropins in <Emphasis Type="Italic">Drosophila</Emphasis>

The effect of 20-hydroxyecdysone (20E) and the juvenile hormone (JH) on the activity of the arylalkylamine N-acetyltransferase (AANAT) was studied in young females of wild-type D. virilis and D. melanogaster. 20E feeding of the flies led to a decrease in AANAT activity in both species when dopamine (DA) was used as substrate, but did not affect the enzyme activity when octopamine (OA) was used as substrate. JH application increased AANAT activity with DA as substrate in both species, but did not change it with OA as substrate. AANAT activity was also measured in young females of a JH-deficient strain of D. melanogaster, apterous ^56f . A decrease in the enzyme activity was observed in the mutant females as compared to wild-type. Mechanisms of regulation of DA level by gonadotropins in Drosophila are discussed.     

Ecdysone 20-monooxygenase activity in <Emphasis Type="Italic">Drosophila virilis</Emphasis> strains varying in ecdysteroid response to heat stress

The effect of various duration of heat stress (38°C) on the activity of ecdysone 20-monooxygenase converting ecdysone into 20-hydroxyecdysone has been studied in D. virilis of wild type and mutant strain females, which differ by the mode of heat stress response of ecdysone and 20-hydroxyecdysone. We are the first to show that heat stress induces activity of ecdysone 20-monooxygenase in Drosophila females and enzyme activity correlates with the level of 20-hydroxyecdysone.     

Effects of 20‐hydroxyecdysone and juvenile hormone on octopamine metabolism in females of Drosophila

The effect of exogenous 20‐hydroxyecdysone (20E) and juvenile hormone (JH) on the activities of the tyrosine decarboxylase (TDC), the first enzyme in octopamine (OA) synthesis, has been studied in young females of wild type D. virilis and D. melanogaster under normal and heat stress (38°C) conditions. Flies fed 20E expressed increased TDC activity in both species. JH application decreased TDC activity in both species. A rise in JH and 20E levels did not prevent a TDC response to heat stress, but changed the response intensity. A long‐term increase in JH titre had no effect on the activity of main OA catabolyzing enzyme, arylalkylamine N‐acetyltransferase, in females of both species. A possible mechanism of regulation of OA levels by 20E and JH in Drosophila females is discussed. © 2009 Wiley Periodicals, Inc.     

On the intracellular localization of the ecdysone 20-monooxygenase in Musca domestica. L

The cytochrome P-450-dependent 20-monooxygenation of ecdysone is catalyzed both by mitochondria and microsomes isolated from Musca domestica (L.) larvae; however, about 50% of the activity is associated with mitochondria, and 37% is associated with microsomes. Pretreatment of larvae with ecdysone results in an increase in Vmax and a decrease in Km values in mitochondria but not in microsomes. Phenobarbital, a known cytochrome P-450 inducer, increases the cytochrome P-450 levels in microsomes without affecting the 20-monooxygenase activity, but both the cytochrome P-450 levels and monooxygenase activity are depressed in mitochondria from phenobarbital-pretreated larvae. The ecdysone 20-monooxygenase activity is equally distributed between mitochondria and microsomes in adult insects. Pretreatment of the insects with ecdysone does not significantly modify the 20-monooxygenase activity of either mitochondrial or microsomal fractions, but the cytochrome P-450 levels are reduced in mitochondria. Phenobarbital also depresses the mitochondrial cytochrome P-450 levels while markedly increasing the microsomal cytochrome P-450 levels. However, no significant changes in ecdysone 20-monooxygenase activity are produced by phenobarbital pretreatment. The effects of ecdysone on adult cytochrome P-450 are mostly evidenced in mitochondria isolated from females, whereas in males the changes are not statistically significant. It is concluded that the mitochondrial ecdysone 20-monooxygenase is under regulatory control by ecdysone in the larval stage, which suggests that only the mitochondrial activity has a physiological role during insect development in M. domestica. In adults, both the mitochondrial and microsomal ecdysone 20-monooxygenase activities are not responsive to ecdysone, which, coupled to their high Km values, indicates that the reaction may not be of physiological importance in adult insects and that the mitochondrial cytochrome P-450 species being depressed by ecdysone in females are possibly not involved in ecdysone metabolism.     

Biogenic amines regulate the reproductive function in <Emphasis Type="Italic">Drosophila</Emphasis> as neurohormones

We studied the influence of experimental increase in the octopamine and dopamine content on the level of juvenile hormone degradation, oogenesis, and fertility in wild type Drosophila virilis flies. Feeding of flies on octopamine led to a significantly decreased level of juvenile hormone degradation (increased titer) in young and sexually mature females, rather than in males, markedly decreased the number of vitellogenic (stages 8–10) and mature (stage 14) oocytes), and sharply reduced fertility. Feeding of flies on dopamine decreased the juvenile hormone degradation (increased titer) in young wild type females and increased it (lowered the juvenile hormone titer) in sexually mature females, as well as decreased the fertility of wild type females to a level characteristic for D. virilis line with a mutation doubling the endogenous dopamine level. A possible mechanism of the influence of these amines on the reproductive function in Drosophila as neurohormones is discussed and a conclusion is drawn that the reduced fertility of females at an increased level of amines appears to be related to an increased level of ecdysteroids, which is caused by an increased, as a result of decreased degradation, juvenile hormone titer.     

Ecdysone 20-monooxygenase activity in Drosophila virilis strains varying in ecdysteroid response to heat stress

The effect of various duration of heat stress (38 degrees C) on the activity of ecdysone 20-monooxygenase converting ecdysone into 20-hydroxyecdysone has been studied in D. virilis of wild type and mutant strain females, which differ by the mode of heat stress response of ecdysone and 20-hydroxyecdysone. We are the first to show that heat stress induces activity of ecdysone 20-monooxygenase in Drosophila females and enzyme activity correlates with the level of 20-hydroxyecdysone.     

Allelochemical stimulation of ecdysone 20-monooxygenase in fall armyworm larvae

The influence of dietary allelochemical on ecdysone 20-monooxygenase activity was studied in the fall armyworm, Spodoptera frugiperda (J.E. Smith). Feeding the indoles (indole-3-carbinol, indole-3-acetonitrile), flavonoids (flavone, β-naphthoflavone), monoterpenes (menthol, menthone, peppermint oil), and a coumarin (xanthotoxin) to the larvae stimulated midgut microsomal ecdysone 20-monooxygenase activity from 28 to 200% as compared with the controls. β-Naphthoflavone was the most potent inducer among those tested. Phenobarbital, a well-known cytochrome P450 inducer, also caused a 2-fold increase in the microsomal ecdysone 20-monooxygenase activity. Ecdysone 20-monooxygenase activity was 2.7-fold higher in the microsomal fraction than in the mitochondrial fraction isolated from larval midguts. Microsomal ecdysone 20-monooxygenase activity was highest in the fat body, followed by the midgut and Malpighian tubules. Tissue localization and enzyme inducibility were different between ecdysone 20-monooxygenase and xenobiotic-metabolizing cytochrome P450 monooxygenases, including aldrin epoxidase, biphenyl hydroxylase, methoxyresorufin O-demethylase, 7-ethoxycoumarin O-deethylase, p-chloro-N-methylaniline N-demethylase, and phorate sulfoxidase in fall armyworm larvae. © 1995 Wiley-Liss, Inc.     

Mechanisms of age-specific regulation of dopamine metabolism by juvenile hormone and 20-hydroxyecdysone in <Emphasis Type="Italic">Drosophila</Emphasis> females

20-hydroxyecdysone (20E) and the juvenile hormone (JH) have an age-specific effect on total dopamine (DA) content in Drosophila (Gruntenko and Rauschenbach 2008). Earlier we studied the mechanism of influence of 20E and JH on DA metabolism in young females (Rauschenbach et al. in J Insect Physiol 53:587–591, 2007a: Arch Insect Biochem Physiol 65:95–102, 2008a; Gruntenko et al. in Arch Insect Biochem Physiol 72:263–269, 2009). Here we investigate the effects of 20E and JH on the activities of the alkaline phosphatase (ALP), tyrosine hydroxylase (TH) and DA-dependent arylalkylamine N-acetyltransferase (AANAT) in mature females of wild type D. virilis under normal conditions and under heat stress (38°C). 20E feeding of the flies led to a substantial decrease in ALP and TH activities and to an increase in AANAT activity in mature females. JH application resulted in an increasing of ALP and TH activities, but did not influence AANAT activity in mature females. A rise in JH and 20E levels was found to change ALP and TH stress reactivities. Mechanisms of age-specific regulation of DA level by 20E and JH in Drosophila females are discussed.     

Ecdysone 20-Monooxygenase Activity of Cytochrome P450 in Ajuga reptans L. Plants and Cell Culture

The concentration of cytochrome P₄₅₀ and ecdysone 20-monooxygenase activity in plants and callus cell culture of carpet bugleweed Ajuga reptans L. were determined. The maximal ecdysone 20-monooxygenase activity of cytochrome P₄₅₀ was found in vegetative rosettes of intact plants. During the stage of flowering, the ecdysone 20-monooxygenase activity of cytochrome P₄₅₀ in plant leaves was higher than in other organs. It was demonstrated that the content of ecdysteroids in callus cell culture is higher than in the intact plant, with concurrent retention of a high ecdysone-20-monooxygenase activity.     

InR gene expression and octopamine metabolism in Drosophila melanogaster females

The influence of suppression of the expression of the Drosophila insulin-like receptor gene (InR) in corpus allatum (the gland-synthesizing juvenile hormone) on octopamine (OA) and juvenile hormone metabolism and on the development of the stress-reaction in Drosophila melanogaster females was studied. It was demonstrated that the suppression of InR gene expression in corpus allatum induces in D. melanogaster females an increase in the activity of the enzyme that limits the rate of octopamine synthesis (tyrosine decarboxylase), as well as in the level of juvenile hormone degradation and the intensity of the response of octopamine and juvenile hormone metabolism systems to heat stress. It was mentioned that a decrease in InR gene expression in corpus allatum does not influence the activity of OA-dependent N-acetyltransferase (the enzyme that degrades octopamine). It was established that the influence of suppression of the InR gene expression in corpus allatum on octopamine metabolism is mediated by juvenile hormone, since the treatment of flies by exogenous juvenile hormone restores the activity of tyrosine decarboxylase in flies with decreased InR expression in corpus allatum up to the normal level.     

Possible role for covalent modification in the reversible activation of ecdysone 20-monooxygenase activity

Evidence is presented for the reversible activation-inactivation of the microsomal ecdysone 20-monooxygenase from fat body of the cotton leafworm, Spodoptera littoralis, in a manner commensurate with reversible changes in its phosphorylation state. The activity of the monooxygenase was higher following preincubation with fluoride (an inhibitor of phosphoprotein phosphatases) than in its absence. Preincubation with alkaline phosphatase or with cAMP-dependent protein kinase resulted in appreciable diminution or enhancement, respectively, in monooxygenase activity. Activation of ecdysone 20-monooxygenase activity could also be effected by incubation with a cytosolic fraction in the presence of cAMP, ATP, and fluoride; this activation was prevented by a cAMP-dependent protein kinase inhibitor. Similarly, inactivation of the monooxygenase was achieved by preincubation with cytosol, the effect being enhanced by Ca²⁺-calmodulin or by Mg²⁺ ions. The combined results provide indirect evidence that the microsomal ecdysone 20-monooxygenase exists in an active phosphorylated form and an inactive dephosphorylated form, interconvertible by a cAMP-dependent protein kinase and a phosphoprotein phosphatase.     

Separation of oviposition-stimulating peptides and myotropic factors from head extracts of Galleria mellonella L.: Comparative effects of myotropic and non-myotropic factors on egg laying

Methanolic extracts from heads of the wax moth, Galleria mellonella L. contain several factors that stimulate oviposition of virgin females in vivo and spontaneous contractions of the oviduct in vitro of the cricket, Gryllus bimaculatus. The myotropic and egglaying activities behaved distinctly during all steps of purification, including fractionation on reverse-phase high-performance liquid chromatography. A novel neuropeptide was isolated from Galleria mellonella. The peptide does not increase the motility of isolated cricket oviducts. Among 13 other myotropic or non-myotropic factors studied, none were found to be as potent as the new peptide in stimulating egg-laying activity. The dose-response curves of myotropic and non-myotropic factors indicate that (i) proctolin, l-glutamate, octopamine, leucokinin-VI, leucopyrokinin, ecdysone and 20-hydroxy-ecdysone, or equivalent structures, might be involved in Galleria mellonella oviposition control, and (ii) that only the novel wax moth neuropeptide has the ability to trigger egg laying in that insect. The significance of these findings is discussed.Abbreviations AcN acetonitrile - E head equivalent - 20-HE 20-hydroxyccdysone - JH 20-hydroxyecdysone, juvenile hormone - Lem-K VI leucokinin-VI - Lem-K I leucokinin-I - Lem MS leucomyosupressin - Lem PK leukopyrokinin - TFA trifluoroacetic acid     

Effects of octopamine on reproduction, juvenile hormone metabolism, dopamine, and 20-hydroxyecdysone contents in Drosophila

The effect of an experimentally increased octopamine content (feeding flies with OA) on the levels of juvenile hormone (JH) degradation, dopamine (DA), and 20-hydroxyecdysone (20E) contents, oogenesis, and fecundity of wild type Drosophila flies has been studied. OA feeding of the flies was found to (1) cause a considerable decrease in JH degradation in females, but not males, of D. melanogaster and D. virilis; (2) have no effect on DA content in D. melanogaster and D. virilis; (3) increase 20E contents in D. virilis females; (4) decrease to a large extent the number of vitellogenic (stages 8-10) and mature (stage 14) oocytes in D. virilis; and (5) decrease the fecundity of D. melanogaster and D. virilis. A possible mechanism of action of OA as a neurohormone on the reproductive function of Drosophila is discussed.     

Ecdysone 20-monooxygenase in a cricket,Gryllus bimaculatus (Ensifera,Gryllidae)—characterization of the microsomal midgut steroid hydroxylase in adult females

Summary Ecdysone 20-monooxygenase, the enzyme system which converts ecdysone into 20-hydroxyecdysone, was characterized in the midgut of 4-day-old female adult Gryllus bimaculatus using an in vitro radioassay. Differential centrifugation and sucrose gradient centrifugation revealed that ecdysone 20-monooxygenase activity is associated with the microsomal fractions. The 20-monooxygenase was found to be most active in potassium phosphate buffer, pH 7.8, at an osmolarity of 100 mOsm and at 39 °C assay temperature. The conversion of ecdysone into 20-hydroxyecdysone was linear over an incubation period of 12 min and with respect to a protein concentration of 3 mg·ml^–1. K⁺ and Na⁺ (10^–3–10^–1 M), Ca²⁺ (2.3 mM), and EDTA (1–5 mM) did not affect monooxygenase activity, whereas Mg²⁺ (2.3–10 mM) slightly inhibited enzyme activity. The enzyme complex has an apparent K_m for ecdysone of 3.7·10^–7 M and is competitively inhibited by its product, 20-hydroxyecdysone, with an apparent K_i of 4·10^–6 M. The cytochrome P-450 nature of the steroid hydroxylase was shown by its obligate requirement for NADPH and its inhibition by carbon monoxide, metyrapone, and p-chloromercuribenzoate, but not by cyanide. The insect systemic growth disruptor, azadirachtin, was found to inhibit ecdysone 20-monooxygenase activity with a I₅₀ of 8·10^–4 M. From the CO-difference spectrum, a cytochrome P-450 content of 285 pmol·mg protein^–1 was calculated for midgut microsomes of 4-day-old females.Abbreviations GO carbon monoxide - EDTA ethylenediamine tetraacetic acid - HPLC high performance liquid chromatography - I ₅₀ concentration for 50% inhibition - KCN potassium cyanide - K ₁ inhibition constant - K _m Michaelis-Menten constant - MOPS 3-morpholinopropanesulfonic acid - NADH/NAD ⁺ nicotinamide adenine dinucleotide reduced/oxidized - NADPH/NADP ⁺ nicotinamide adenine dinucleotide phosphate reduced/oxidized - Na ₂ S ₂ O ₄ sodium dithionite - SEM Standard error of mean - TLC thin-layer chromatography - TRIS 2-amino 2-hydroxymethyl-1,3-propanediol (trishydroxymethyl aminomethane) - V _max maximal reaction velocity     

Ecdysone 20-hydroxylation in Manduca sexta midgut: Kinetic parameters of mitochondrial and microsomal ecdysone 20-monooxygenases

The larval midgut of the tobacco hornworm, Manduca sexta, has high ecdysone 20-monooxygenase (E20MO) activity, located both in the mitochondria and in the microsomes. The apparent kinetic parameters for E20MO in mitochondria and microsomes were determined. The K_m⁵ (for ecdysone) of the mitochondrial and microsomal enzymes were 1.63 × 10⁻⁵ and 3.67 × 10⁻⁷ M, respectively. The V_max was 82.7 pmol/min/mg protein for mitochondria and 32.0 pmol/min/mg protein for microsomes. Although the mitochondrial E20MO has the higher V_max, at physiological ecdysone concentrations (10⁻⁷ − 10⁻⁸ M) it is only one-eighth to one-tenth as active as the microsomal enzyme. It is concluded that the microsomal E20MO is the primary, if not the only, enzyme involved in ecdysone 20-hydroxylation in M. sexta midgut. © 1996 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

Dopamine and octopamine regulate 20-hydroxyecdysone level in vivo in Drosophila

The effects of increased level of dopamine (DA) (feeding flies with DA precursor, L-dihydroxyphenylalanine, L-DOPA) on the level of 20-hydroxyecdysone (20E) and on juvenile hormone (JH) metabolism in young (2-day-old) wild type females (the strain wt) of Drosophila virilis have been studied. Feeding the flies with L-DOPA increased DA content by a factor of 2.5, and led to a considerable increase in 20E level and a decrease of JH degradation (an increase in JH level). We have also measured the levels of 20E in the young (1-day-old) octopamineless females of the strain Tbetah(nM18) and in wild type females, Canton S, of D. melanogaster. The absence of OA led to a considerable decrease in 20E level (earlier it was shown that in the Tbetah(nM18) females, JH degradation was sharply increased). We have studied the effects of JH application on 20E level in 2-day-old wt females of D. virilis and demonstrated that an increase in JH titre results in a steep increase of 20E level. The supposition that biogenic amines act as intermediary between JH and 20E in the control of Drosophila reproduction is discussed.     

Characterization of ecdysone 20-monooxygenase activity in wandering stage larvae of Drosophilamelanogaster: Evidence for mitochondrial and microsomal cytochrome P-450 dependent systems

Ecdysone 20-monooxygenase, the enzyme system that hydroxylates ecdysone to 20-hydroxyecdysone, was characterized in wandering stage larvae of Drosophila melanogaster using an in vitro radioassay in conjunction with analytical thin layer chromatography. 20-Hydroxyecdysone was confirmed to be the product of the enzyme radioassay system by high pressure liquid chromatography. The 20-monooxygenase was found to be most active in a 0.10 M phosphate buffer, pH 7.5, was inhibited by Ca²⁺, Mg²⁺ and Se⁴⁺ and exhibited a temperature optimum at 35°C. Differential centrifugation, sucrose step gradient centrifugation, electron microscopy and organelle-marker enzyme analysis revealed that ecdysone 20-monooxygenase activity is associated with both the mitochondrial and microsomal fractions. Substrate kinetics experiments indicated that the mitochondrial and microsomal monooxygenase systems exhibit apparent K_ms for ecdysone of 6.4 × 10⁻⁸ and 9.9 × 10⁻⁸ M, respectively, with apparent V_maxs of 4.1 and 10.2 pg 20-hydroxyecdysone formed/min per mg tissue equiv., respectively. Both monooxygenase systems were inhibited by their product 20-hydroxyecdysone. The cytochrome P-450 nature of these insect steroid hydoxylases was initially suggested by their requirement for NADPH, NADH was approximately half as effective in supporting the mitochrondrial monooxygenase activity. In addition, both monooxygenase systems were inhibited by carbon monoxide, ellipticine, p-chloromercuribenzoate, metyrapone and p-aminoglutethimide but not by cyanide. Photochemical action spectra of ecdysone 20-monooxygenase activity confirmed the cytochrome P-450 dependency of both the mitochondrial and microsomal ecdysone 20-hydroxylase systems.     

The <Emphasis Type="Italic">ecdysoneless</Emphasis><Superscript>1</Superscript> Gene Regulates Metabolism of the Juvenile Hormone and Dopamine in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The dopamine (DA) content and the level of juvenile hormone (JH) degradation were studied in females of the wild-type Canton S strain and the ecdysoneless ¹ (ecd ¹) mutant, which does not produce ecdysone at a restrictive temperature (29°C). Exposure at the restrictive temperature considerably increased the JH-hydrolyzing activity and the DA content in five-day ecd ¹ females compared with flies of both strains growing at 19°C and Canton S females exposed at 29°C. In one-day ecd ¹ females, the level of JH degradation also increased at the restrictive temperature, but the DA content was low. The effect of ecdysone deficiency on the stress reaction in Drosophila melanogaster females was studied using changes in DA content and JH degradation as the reaction indicators. The ecd ¹ mutation did not prevent the initiation of the stress reaction in females exposed at the restrictive temperature, but changed its intensity (stress reactivity). The interaction of 20-hydroxyecdysone with JH and DA in regulating Drosophila reproduction under normal conditions and in stress is discussed.     

Ecdysone 20-monooxygenase activity in land crabs

1. Ecdysone 20-monooxygenase activity has been found in hepatopancreas, gonads, epidermis and muscle of the crab Gecarcinus lateralis. Activity was assayed by measuring the in vitro conversion of [³H]-ecdysone to [³H]-20-hydroxyecdysone. Maximal activity is obtained at 30°C and pH 8.0 in sodium phosphate buffer.2. Activity from hepatopancreas is localized in a fraction which sediments at 10,000 g, probably mitochondria.3. NADPH stimulates activity and metyrapone or oxygen deprivation inhibits it, as has been observed for cytochrome P-450-dependent monooxygenases.4. Changes in ecdysone 20-monooxygenase activity at different stages of the molt cycle are not directly correlated to changes in ecdysteroid levels in the hemolymph.