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Prostaglandin biosynthesis by fat body from true armyworms, Pseudaletia unipuncta
Insect Biochemistry and Molecular Biology 31 (2001) 435–444 www.elsevier.com/locate/ibmb
Prostaglandin biosynthesis by fat body from true armyworms, Pseudaletia unipuncta Hasan Tunaz, Russell A. Jurenka 1, David W. Stanley
*
Insect Biochemical Physiology Laboratory, University of Nebraska – Lincoln, Lincoln, NE 68583-0816, USA Received 1 September 1999; received in revised form 4 August 2000; accepted 9 August 2000
Abstract We describe prostaglandin (PG) biosynthesis by microsomal-enriched fractions of fat body prepared from true armyworms, Pseudaletia unipuncta. PG biosynthesis was sensitive to experimental conditions, including incubation time, temperature, pH, substrate and protein concentration. Optimal PG biosynthesis conditions included 1 mg of microsomal-enriched protein, incubated at 28°C for 7.5 min at pH 8. These preparations yielded four major PGs: PGA2, PGE2, PGD2 and PGF2α. PGA2 and PGE2 were the predominant eicosanoids produced under these conditions. Two non-steroidal anti-inflammatory drugs, indomethacin and naproxen, effectively inhibited PG biosynthesis. Unlike other invertebrate PG biosynthetic systems studied so far, the true armyworm system appeared to be independent of the usual exogenous co-factors required by mammalian and other invertebrate systems. These findings are discussed with respect to PG biosynthesis in other invertebrate and vertebrate systems. 2001 Published by Elsevier Science Ltd. Keywords: True armyworm; Pseudaletia unipuncta; Prostaglandins; Anti-inflammatory drugs; Insect immunity
1. Introduction Insects respond to bacterial infections with a complex immune system. We have been developing the hypothesis that eicosanoids are responsible for mediating insect immune reactions to bacterial infections. Eicosanoid is a collective term for all biologically active, oxygenated metabolites of 20:4n-6 and two other C20 polyunsaturated fatty acids (Fig. 1). Most experiments designed to test this hypothesis are based on selective inhibition of eicosanoid biosynthetic pathways, done by treating experimental animals with a variety of anti-inflammatory drugs. We began our work with tobacco hornworms, Manduca sexta, as an experimental model. Nodule formation is thought to be the predominant mechanism of clearing bacterial cells from hemolymph (Horohov and Dunn, 1983) and, using pharmaceutical inhibitors of eicosanoid biosynthesis, we found that microaggregation (an early phase of nodule formation) and nodule forma* Corresponding author. Tel.: +1-402-472-8710; fax: +1-402-4724687. E-mail address: [email protected] (D.W. Stanley). 1 Present address: Department of Entomology, Iowa State University, 411 Science II, Ames, IA 50011-3222, USA. 0965-1748/01/$ - see front matter 2001 Published by Elsevier Science Ltd. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 1 3 8 - 7
tion were severely impaired in inhibitor-treated hornworms. On the basis of these results we suggested that eicosanoids are responsible for mediating nodulation, a specific cellular reaction to bacterial infections (Miller et al., 1994). We carried out a series of similar exercises on other insect species, with a view to testing the idea that eicosanoids act in immunity of most, if not all, insect species. So far, we have obtained supporting results with larvae of a beetle, Zophobas atratus (Miller et al., 1996), and several lepidopterans, the silkworm, Bombyx mori (Stanley-Samuelson et al., 1997), cutworms, Agrotis ipsilon, and true armyworms, P. unipuncta (Jurenka et al., 1997) and larvae of the butterfly, Colias eurytheme (Stanley et al., 1999). We also tested our idea with adults of three hemimetabolous insect species, the cricket Gryllus assimilis (Miller et al., 1999) and two 17-year periodical cicadas, Magicicada septendecim and M. cassini (Tunaz et al., 1999). In addition, Downer’s group reported that two distinct phases of nodulation, cell spreading and prophenyloxidase activation, were inhibited in waxmoths, Galleria mellonella, that had been treated with eicosanoid biosynthesis inhibitors (Mandato et al., 1997). They also suggested that eicosanoids mediate phagocytosis, another independent cellu-
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Fig. 1. An overview of 20:4n-6 metabolism as understood from the mammalian background. Three polyunsaturated fatty acids, 20:3n-6, 20:4n-6 and 20:5n-3 are potential substrates for eicosanoid biosynthesis. Of these, metabolism of 20:4n-6 is most well studied. Chemical structures are denoted by numerals. 1=cellular phospholipid, 2=hydrolyzed 20:4n-6, 3=PGE2, 4=5-hydroperoxyeicosatetraenoic acid, 5=leukotriene B4, 6=11,12-epoxyeicosatrienoic acid, 7=lipoxin A. Capital letters indicate major enzyme systems responsible for eicosanoid biosynthesis. A=phospholipase A2; B=cyclooxygenase and associated enzyme steps; C=cytochrome P450 epoxygenase; D=lipoxygenase.
lar reaction to bacterial infections. Appreciation of the significance of eicosanoids in signal transduction in insect immunity was considerably broadened by Morishima et al. (1997). Based on their work with antiinflammatory drugs, this group showed that eicosanoids also mediate expression of two silkmoth fat body genes for antibacterial proteins, cecropin and lysozyme. We infer from the work with all these systems that eicosanoids are important elements of insect immunity (Stanley, 2000). The hypothesis that eicosanoids act in insect immunity would rest on firmer grounds if it were supported by results of experiments which do not solely rely on the actions of pharmaceutical products. We generated one line of work by investigating the biochemistry of eicosanoid systems in immune tissues from tobacco hornworms, M. sexta. We documented the presence of three elements of eicosanoid biosynthesis. First,
hornworm tissues maintain eicosanoid precursor polyunsaturated fatty acids, albeit in low proportions, in tissue lipids (Ogg and Stanley-Samuelson, 1992). Second, we described an intracellular phospholipase A2 in fat body and hemocytes that can release 20:4n-6 from the sn-2 position of phospholipids (Uscian and Stanley-Samuelson, 1993; Schleusener and Stanley-Samuelson, 1996). Third, we characterized eicosanoid biosynthesis in fat body and hemocytes (Stanley-Samuelson and Ogg, 1994; Gadelhak et al., 1995). These biochemical findings are important because they lie at the foundation of the biological significance of eicosanoids in immunity or any other physiological arena. Nonetheless, additional evidence more directly focused on immune reactions would provide a better test of the hypothesis that eicosanoids mediate insect immunity. We reasoned that bacterial infections stimulate biosynthesis and secretion of PGs and other eicosanoids by fat body and hemocytes. These infection-stimulated eicosanoids subsequently mediate cellular reactions to the infections. If this were so, then bacterial infections should result in measurable increases in eicosanoid titers in insect hemolymph. Returning to true armyworms, we tested this idea by injecting experimental larvae with heat-killed bacteria and injecting control larvae with saline (Jurenka et al., 1999). At 30 min post-infection, hemolymph samples were withdrawn, and eicosanoids were extracted. The eicosanoids were derivatized, then analyzed on fluorescence–HPLC. Relative to controls we recovered about four-fold more PGF2α from hemolymph of infected insects (Jurenka et al., 1999). These data showed that bacterial infections stimulate increased eicosanoid biosynthesis in true armyworms, which adds considerable support to our hypothesis. Complementary to this work on PG production, we began investigating eicosanoid biosynthesis by immune tissues prepared from true armyworms. In this paper we report on PG biosynthesis by true armyworm fat body. We describe an optimized in vitro enzyme assay for the characterization of eicosanoid biosynthesis and discuss these findings in relation to other invertebrate and vertebrate systems.
2. Materials and methods 2.1. Insects True armyworms, P. unipuncta, reared on standard culture medium, were provided by the Corn Insect Research Unit (USDA, Ames, IA). Larvae were shipped to Lincoln, NE, where they were maintained at 28°C until the sixth instar. Last instars were used throughout this study, except for a few experiments with pupae, as noted in Results.
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2.2. Isolation of fat body and preparation of microsomal-enriched fractions These experiments followed protocols developed for the tobacco hornworm fat body (Stanley-Samuelson and Ogg, 1994). The larvae were anesthetized by chilling on ice, then fat body tissue was dissected in ice-cold phosphate buffer (0.05 M potassium phosphate, pH 8.0). The fat body tissues, in 1 ml Eppendorf tubes, were sonicated for 10 s at 30 W using a VibraCell sonicator (VibraCell, Danbury, CT). This preparation was centrifuged for 10 min at 735g, and the supernatant was centrifuged for another 20 min at 16,000g, both steps at 4°C. The 16,000g supernatants were microsomal-enriched preparations used in all experiments. Protein concentrations in these preparations were determined in microtiter format using the bicinchoninic acid reagent (Pierce, Rockford, IL), against bovine serum albumin as quantitative standard. The microtiter plates were read on a BioTek microtiter plate reader at 562 nm. Radioactive arachidonic acid (5,6,8,9,11,12,14,15– 3 H–20:4, 60–100 Ci/mmol) was purchased from New England Nuclear (Boston, MA). The incubation buffer was 0.05 M KH2PO4, pH 8.0, amended with a standard co-factor cocktail (2.4 mM reduced glutathione, 0.25 mM hydroquinone and 25 µg hemoglobin; StanleySamuelson and Ogg, 1994). For each PG biosynthesis reaction (unless indicated otherwise), 0.4 µCi of labeled 20:4n-6 was dispensed into reaction tubes and the solvent was evaporated. The reactions were carried out in 0.5 ml total volume. The experiments were preceded by a 10 min pre-incubation at 28°C with all reaction components, except the protein source. The reactions were stopped by acidification to pH 3.5–4.0 by addition of 0.2 ml 0.1 N HCl. Reaction products were extracted from the acidified reaction mixture three times with 0.5 ml ethyl acetate. The combined extracts, containing PGs and possible lipoxygenase products, were evaporated under N2. A mixture of appropriate unlabeled eicosanoid standards was added to each sample, then samples were applied to TLC plates (20×20 cm Silica Gel G, 0.25 mm thick, Sigma Chemical Co., St Louis, MO). The plates were developed in the A9 solvent system (Hurst et al., 1987) and fractions observed by exposure to iodine vapors. Bands corresponding to selected authentic eicosanoid standards and to free fatty acids were transferred to liquid scintillation vials. Radioactivity in each fraction was determined by adding 5 ml scintillation cocktail (ICN Biomedicals, Irvine, CA) and counting on a LKB Wallac 1209 Rackbeta Liquid Scintillation Counter (Pharmacia, Turku, Finland) at 50% counting efficiency for 3H. Eicosanoid biosynthesis was calculated from the liquid scintillation data. In control experiments, microsomal-enriched preparations were heated in boiling water for 15 min before the experiments, and processed as just described. The results of these control experiments were
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used to correct values from biosynthesis experiments as previously described (Stanley-Samuelson and Ogg, 1994). 2.3. Determining PGA2 by gas–chromatography/mass spectrometry (GC–MS) Microsomal-enriched preparations from 18 last-stage larvae were added to reaction tubes containing 10 µM arachidonic acid without added co-factors. After 7.5 min at 32°C the reactions were stopped with the addition of 0.2 ml 0.1 N HCl. Products were extracted three times with 0.5 ml ethyl acetate. The ethyl acetate extracts were combined and dried under a stream of N2. The extract was then purified and analyzed as described previously (Jurenka et al., 1999). Briefly, the extract was cleaned up on silicic acid chromatography and the acetonitrile:methanol fraction was treated with 100 µl diazomethane in diethyl ether. The resulting methyl esters were then treated with N,O-bis(trimethylsilyl)trifluoracetamide containing 1% trimethylchlorosilane and heated at 60°C for 20 min. The reaction was dried under N2 and reconstituted with methylene chloride for analysis on GC–MS. Analyses were conducted by capillary GC–MS using a Hewlett–Packard 5890 GC equipped with a DB-1 column (0.25 mm×30 m). The GC was interfaced with a Hewlett–Packard 5972 Mass Selective Detector operated in scan mode. Separations were conducted in splitless mode with temperature programming at 80°C for 1 min, then 10°/min to 320°C. 2.4. Statistical analysis Data were analyzed using the General Linear Models procedure, and mean comparisons were made using Least Significant Different (LSD) test (SAS Institute Inc., 1989).
3. Results Microsomal-enriched fractions of fat bodies prepared from last-instar true armyworms were competent to biosynthesize PGs. The fat body preparations produced four major products (PGA2, PGD2, PGE2 and PGF2α). PGA2 was the major product under most experimental conditions, although our data indicate that changes in reaction conditions influenced the overall profile of PG biosynthesis. Because PGA2 was the major PG produced by the fat body preparations we confirmed the identification of PGA2 by GC–MS. Fig. 2 shows a total ion scan of the methyl ester trimethylsilyl derivative of PGA2 obtained from incubation of true armyworm fat body preparations with arachidonic acid. The influence of reaction conditions on PG biosynthesis are reported in the following paragraphs.
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Fig. 2. A total ion scan of the methyl ester trimethylsilyl derivative of PGA2 obtained from the incubation of true armyworm fat body with arachidonic acid.
3.1. Influence of radioactive substrate concentration on PG biosynthesis The influence of four concentrations of radioactive substrate on PG biosynthesis is shown in Fig. 3. Total PG biosynthesis increased from ca 2 nmol/mg protein/h in the presence of 0.2 µCi of substrate to ⬎13 nmol/mg protein/h with 1.6 µCi of substrate. Except for 0.8 µCi of substrate, PG biosynthesis increased in an approximately linear way with increasing substrate. Highest PG biosynthesis was obtained in the presence of 1.6 µCi of substrate (significantly higher than other concentrations), however, we used 0.4 µCi of substrate per reaction to balance optimal use of radioactive material with a reasonable level of PG biosynthesis. We note that substrate concentrations influenced the profiles of radioactive PG products. At 0.4 µCi of substrate PGA2 was the predominant product, while all four PGs were produced in similar levels at 1.6 µCi of radioactive substrate. 3.2. Influence of protein concentration on PG biosynthesis Fig. 4 shows the relationship between fat body microsomal-enriched protein concentration and PG
Fig. 3. The influence of four concentrations of radioactive 20:4n-6 on PG biosynthesis by microsomal-enriched preparations of fat body from true armyworm larvae. The 0.5 ml reaction mixtures containing 0.2, 0.4, 0.8 and 1.6 µCi 3H–20:4n-6, 1 mg of microsomal-enriched protein, and the co-factor in 50 mM potassium phosphate buffer, pH 8.0. The reactions were incubated at 28°C. After 7.5 min incubations, the reactions were stopped, and the reaction products were extracted and separated as described in Materials and Methods. The histogram displays the biosynthesis of individual PGs, and the line represents total PG biosynthesis. Each point represents the mean of three separate experiments at each substrate concentration. The error bars, where visible, indicate 1 SEM.
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Fig. 4. The influence of fat body microsomal-enriched protein concentration on PG biosynthesis. The reactions were conducted and the products were extracted and separated as described in Materials and Methods. The histogram displays the biosynthesis of individual PGs, and the line represents total PG biosynthesis. Each point represents the mean of three separate experiments at each protein concentration. The error bars, where visible, indicate 1 SEM.
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Fig. 5. The influence of incubation time on PG biosynthesis by microsomal-enriched preparations of fat body from true armyworm larvae. The reactions were conducted and the products were extracted and separated as described in Materials and Methods. The histogram displays the biosynthesis of individual PGs, and the line represents total PG biosynthesis. Each point represents the mean of three separate experiments at each different incubation time. The error bars, where visible, indicate 1 SEM.
biosynthesis. The optimal protein concentration was 1 mg/reaction, which yielded significantly higher product formation. These findings provided the information for the use of 1 mg protein/reaction in subsequent experiments. We also observed the influence of protein concentration on the overall profiles of PG biosynthesis. PGA2 was the major product at 1 mg protein/reaction while PGE2 was the major product in experiments with other protein concentrations. All four major PG products were formed at all protein concentrations. 3.3. Influence of reaction time on PG biosynthesis The influence of reaction time on PG biosynthesis is shown in Fig. 5. Total PG biosynthesis increased from about 1.0 nmol/mg protein at 2 min to a high of approximately 4 nmol/mg protein at 7.5 min. Total PG production was diminished in longer incubations, to ⬍0.5 nmol/mg protein at 15 min. The data indicated highest total PG biosynthesis obtained at 7.5 min, which is statistically similar to results at 5 and 10 min. We used 7.5 min incubations in subsequent experiments. The 7.5 min optimum is unusually long for PG biosynthesis reactions. To investigate this point in a little more detail, we carried out a similar experiment using fat body from pupae. Fig. 6 shows that the optimal reaction time for the pupal preparations was 2 min (statistically significant), which accords with other insect PG systems.
Fig. 6. The influence of incubation time on PG biosynthesis by microsomal-enriched preparations of fat body from true armyworm pupae. The reactions were conducted and the products were extracted and separated as described in Materials and Methods. The histogram displays the biosynthesis of individual PGs, and the line represents total PG biosynthesis. Each point represents the mean of three separate experiments at each different incubation time. The error bars, where visible, indicate 1 SEM.
3.4. Influence of reaction temperature on PG biosynthesis Total PG biosynthesis increased significantly from 1 nmol/mg protein/h at 16°C to a high of 4 nmol/mg
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by adjusting the buffer to the indicated pH, the fat body preparations yielded all four PGs. Significantly higher PG biosynthesis, approximately 5 nmol/mg protein/h, was obtained at pH 8. We note that pH conditions influenced the profile of PG biosynthesis. While PGA2 was the major product under most experimental conditions, PGE2 emerged as the predominant product at pH 8. 3.6. True armyworm PG biosynthesis is independent of exogenous co-factors
Fig. 7. The influence of incubation temperature on PG biosynthesis by microsomal-enriched preparations of fat body from true armyworm larvae. The histogram displays the biosynthesis of individual PGs, and the line represents total PG biosynthesis. Each point represents the mean of three separate experiments at each different temperature. The error bars, where visible, indicate 1 SEM.
protein/h at 28°C (Fig. 7). The results from incubations at 24°C and 32°C were statistically similar to the results at 28°C. Higher temperatures yielded similar or lower amounts of product. PGA2 was the major product at all temperatures except for 28°C, at which PGE2 was the major product. 3.5. Influence of reaction pH on PG biosynthesis
We investigated the possibility that the true armyworm fat body preparation does not depend on exogenous co-factors that are routinely used in mammalian and invertebrate PG biosynthesis reactions (Stanley-Samuelson and Ogg, 1994). Microsomal-enriched fat body preparations were incubated with radioactive 20:4n-6 under the usual conditions, with the exception that the co-factor cocktail was left out of the reaction. At the end of 7.5 min incubations, the products were extracted and separated as usual. The absence of co-factors did not influence total PG biosynthesis nor the pattern of PG biosynthesis (Fig. 9). 3.7. The fat body preparation is sensitive to two nonsteroidal anti-inflammatory drugs Total fat body PG biosynthesis was inhibited in reactions conducted in the presence of indomethacin and
Fig. 8 shows the influence of reaction pH on PG biosynthesis. Under each of the pH conditions, obtained
Fig. 8. The influence of buffer pH on PG biosynthesis by microsomal-enriched preparations of fat body from true armyworm larvae. The reactions were conducted and the products were extracted and separated as described in Materials and Methods. The histogram displays the biosynthesis of individual PGs, and the line represents total PG biosynthesis. Each point represents the mean of three separate experiments at each different buffer pH. The error bars, where visible, indicate 1 SEM.
Fig. 9. Schematic representation of background corrected TLC separation of PGs biosynthesized by isolated fat body of true armyworm larvae. The reactions were conducted and the products were extracted and separated as described in Materials and Methods. The TLC plate was divided into 2 cm fractions, all of which were scraped into vials, and the radioactivity in all fractions was estimated by liquid scintillation counting. Radioactivity in fraction 10, which is associated with the substrate 3H–20:4n-6, is not shown. The reaction from which these data were generated was conducted in the absence of the standard cofactor cocktail.
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4. Discussion
Fig. 10. The influence of indomethacin on PG biosynthesis by microsomal-enriched preparations of fat body from true armyworm larvae. The reactions were conducted and the products were extracted and separated as described in Materials and Methods. Each bar represents the mean of three separate experiments at each concentration of indomethacin. The error bars, where visible, indicate 1 SEM. Bars with different fill patterns represent statistical differences.
naproxen (Figs. 10 and 11). In reactions with indomethacin (1 µM), PG biosynthesis declined significantly from about 4 nmol/mg protein/h to approximately 1 nmol/mg protein/h. Higher indomethacin concentrations did not decrease PG biosynthesis. Naproxen reduced PG biosynthesis from about 4 nmol/mg protein/h to 3 nmol/mg protein/h at 1 µM; 10µM naproxen significantly reduced PG biosynthesis to approximately 1 nmol/mg protein/h.
Fig. 11. The influence of naproxen on PG biosynthesis by microsomal-enriched preparations of fat body from true armyworm larvae. The reactions were conducted and the products were extracted and separated as described in Materials and Methods. Each bar represents the mean of three separate experiments at each concentration of naproxen. The error bars, where visible, indicate 1 SEM. Bars with different fill patterns represent statistical differences.
In this paper we document PG biosynthesis by microsomal-enriched preparations of true armyworm fat bodies. Under our experimental conditions, PGE2, PGD2, PGF2α and PGA2 were formed. While PGA2 was the quantitatively predominant product under most conditions, we recorded substantial biosynthesis of all four PGs. Identification of PGA2 by GC–MS (Fig. 2) adds considerable verisimilitude to studies of PG biosynthesis in insects. As seen in other studies of insect tissues (Stanley-Samuelson, 1994; Stanley et al., 1999), PG biosynthesis in the true armyworm preparations was sensitive to reaction conditions, including temperature, pH, incubation time, and protein concentration. In our protocols, optimal reaction conditions for PG biosynthesis include reacting 1.0 mg microsomal-enriched protein with 0.4 µCi of 3H–20:4n-6 at 28°C and pH 8.0 for 7.5 min. Also, PG biosynthesis by the true armyworm preparations was inhibited in reactions conducted in the presence of two non-steroidal anti-inflammatory drugs. We infer from these findings that the true armyworm fat body is competent to biosynthesize PGs. We have discussed most aspects of PG biosynthesis, including the influence of reaction conditions and nonsteroidal anti-inflammatory drugs on PG biosynthesis, in our work on other species (Stanley-Samuelson and Ogg, 1994; Gadelhak et al., 1995; Pedibhotla et al., 1995). Those remarks will not be repeated here. Instead, we now have enough information to begin appreciating comparative aspects of eicosanoid biosynthesis in insects. The thrust of the following remarks is recognizing the diversity of PG biosynthesis systems among insects. While the findings with true armyworm fat body preparations superficially resemble results from other insect species, we recorded important comparative differences. One departure relates to the requirement for a cocktail of co-factors, usually including hemoglobin and reduced glutathione, to support PG biosynthesis. The roles of these co-factors can be understood in terms of the enzymes responsible for PG biosynthesis (Fig. 1), which typically requires four steps (Stanley, 2000). First, a phospholipase A2 step is responsible for releasing 20:4n6 from cellular phospholipids; second, a cyclooxygenase step yields the hydroperoxide, PGG2; third, a glutathione peroxidase converts the PGG2 to PGH2; finally, a fourth enzyme is responsible for converting PGH2 into the final PG product. The cyclooxygenase and peroxidase activities are combined in a single holoenzyme called PGH synthase. The terms COX-1 and COX-2 are used in preference to PGH synthase in the literature on mammals, in which two forms of the enzyme are found (Otto and Smith, 1995). We use the term PGH synthase because the possibility that insects express two forms of this enzyme has not yet been considered. The hemoglobin
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component of the co-factor cocktail provides a heme, often in the form of iron protoporphyrin IX, which both the cyclooxygenase and peroxidase activities require for catalytic activity (Kulmacz and Lands, 1987). The reduced glutathione is required by the glutathione peroxidase which reduces the PGG2 to PGH2 and oxidizes the reduced glutathione (Kulmacz and Lands, 1987). Based on this background from studies on mammalian systems, researchers working on invertebrates conduct PG biosynthesis reactions in the presence of a more or less standard cocktail of co-factors that provide a heme group, reduced glutathione and hydroquinone. Because the point had not been specifically addressed in insect studies, we assessed the need for co-factors in tobacco hornworm fat body preparations by running PG biosynthesis reactions in the absence of co-factors (Stanley-Samuelson and Ogg, 1994). The results indicated that PG biosynthesis was completely abolished in the absence of added co-factors, from which we inferred that insect PG biosynthesizing systems require a mixture of co-factors similar to the co-factors required by mammalian PG biosynthesizing enzymes. Analogous experiments with internal-tissue preparations from the tick, Amblyomma americanum, showed the tick systems also require the added co-factor cocktail to support PG biosynthesis (Pedibhotla et al., 1995). We similarly assessed the need for co-factors in true armyworm fat body preparations. Contrary to expectation, we recorded no difference in PG biosynthesis in the presence or absence of our standard co-factor cocktail (Fig. 9). A couple of possibilities account for this new finding. Either the armyworm homogenates provide sufficient endogenous co-factors to support PG biosynthesis, or the armyworm fat body enzymes responsible for PG biosynthesis do not require the co-factors for catalysis. In either case, the armyworm preparations differ from the mammalian and the other invertebrates that have been studied on the matter of co-factor requirements for in vitro PG biosynthesis. The true armyworm fat body preparations mark another departure from the mammalian background. The mammalian cyclooxygenases are known as “suicide” enzymes, because they are spontaneously and permanently inactivated after about 1400 catalytic operations (Smith et al., 1991). For this reason, most PG biosynthesis reactions are carried out in very short incubation periods, usually 1–2 min. We found that short incubation periods were also optimal for tobacco hornworm fat body preparations (Stanley-Samuelson and Ogg, 1994) and for tick internal-tissue preparations (Pedibhotla et al., 1995). This agreed with outcomes from housefly, Musca domestica, preparations in which very little PG product accumulated in incubation times longer than 2 min (Wakayama et al., 1986). But it differed from findings with spermathecal preparations from the cricket, Teleogryllus commodus, in which PGs accumulated in a
linear way over 60 min incubations (Tobe and Loher, 1983). The optimal reaction period for true armyworm fat body preparations was 7.5 min, after which total PG product accumulation declined. The declines are probably due to the asymmetry in biosynthesis and degradation of products, discussed in detail elsewhere (Stanley-Samuelson and Ogg, 1994). Because incubations longer than 7.5 min yielded reduced product, we speculate that the armyworm fat body system also undergoes spontaneous inactivation, however, not within the short periods seen in other systems. We investigated the 7.5 min incubation periods in a little more detail by generating a similar time course study for the pupal stage of true armyworms. For pupae, we found that 2 min incubations were optimal, and PG yield declined in longer incubations. Two points are important in this exercise. First, we note the difference in optimal reaction time between two stages of the same species. Second, these differences underscore the differences noted in the larval stages of true armyworms. Insects make up the most diverse class of animals, and it may be supposed that variation in the biochemistry of PG biosynthesis among insect species and tissues will prove to be substantial. Indeed, the variation within insects may be greater than the recorded variations between insects and mammals. For a single example, let us consider the pharmaceutical product, indomethacin, a prescription analgesic which potently inhibits cyclooxygenases in mammalian and most invertebrate tissues. Contrarily, indomethacin does not inhibit PG biosynthesis in reproductive tract preparations from the house cricket, Acheta domesticus (Destephano et al., 1974). We expect that continued investigations with insects and other invertebrates will reveal many more variations on the theme of PG biosynthesis. The true armyworm preparations yielded relatively low rates of PG biosynthesis, as seen in other insects. Although the point has not been investigated in detail, insects appear to biosynthesize PGs at rates considerably below the rates recorded for some mammalian tissues. A direct comparison was provided by Jurenka et al. (1986) who assessed PG biosynthetic activity in fat body homogenates from the cockroach, P. americana, and in bovine seminal vesicles. The PG biosynthetic rates in the cockroach preparations were lower, by approximately 10-fold, than the rates recorded from the bovine seminal vesicles. Among mammalian tissues, the bovine preparations are among the most active PG producers, but the point remains that insect PG biosynthetic systems differ in production rate from their mammalian counterparts. The biological significance of PG biosynthesis in insect fat body is complex. Drawing on the mammalian background (Otto and Smith, 1995; Stanley et al., 1999), PGs are thought to mediate cellular events in a couple of ways. For one, PGs regulate events involved in day-
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to-day homeostasis. These are colloquially known as “housekeeping” functions. We know relatively little about the housekeeping roles of PGs in insects. We suggested that PGs regulate basal fluid secretion rates in Malpighian tubules of adult female mosquitoes and worker ants (Petzel and Stanley-Samuelson, 1992; Van Kerkhove et al., 1995). Relative to fat body, Keeley et al. (1996) suggested that some eicosanoids may act in the signal transduction pathways to influence fat body cellular responses to hypertrehalosemic hormone in the cockroach Blaberus discoidalis. In a similar line of research using trophocytes prepared from disaggregated fat bodies of another cockroach, Periplaneta americana, Ali and Steele (1997a,b) investigated the influence of three eicosanoid biosynthesis inhibitors on phosphorylase activation. Phosphorylase activation is the first step in the trehalose biosynthetic pathway, which is activated by hypertrehalosemic hormone in P. americana. Eicosanoids may down-regulate phosphorylase activation because incubations in the presence of eicosanoid biosynthesis inhibitors resulted in increased phosphorylase activation. In these instances, eicosanoids appear to act in housekeeping roles within Malpighian tubule and fat body cells. In these housekeeping roles, PGs and perhaps other eicosanoids are thought to act through an autocrine or paracrine mechanism, depicted in a general way by Stanley-Samuelson and Pedibhotla (1996). In this mechanism, PGs produced within fat body cells are transported across the cell membranes to the outside of the cells, where the PGs can interact with specific functional receptor sites located on the outer surface of the PGproducing cells or their neighbors. The receptors are thought to influence intracellular events through G-protein coupled signal transduction mechanisms. This scheme accounts for the action of most PGs, including PGD, PGE, PGF and PGI (Smith, 1992; Negishi et al., 1995a). However, it does not account for the actions of PGA2. As seen in the tobacco hornworm and true armyworm fat body, we recorded substantial biosynthesis of PGA2 by the true armyworm fat body preparations. Two PGs, ⌬12-PGJ2 and PGA2, are cyclopentenone PGs. Unlike the PGs discussed in the preceding paragraph, these PGs have no cell surface receptors. Instead, the cyclopentenone PGs are actively taken into cells, and are accumulated in the nuclei, where they influence expression of genes (Negishi et al., 1995b). Hence, while some PGs are responsible for housekeeping operations, the cyclopentenone PGs exert far more long-reaching actions. For a couple of examples, PGA2 arrested the cell cycle of cultured vascular smooth muscle cells (Sasaguri et al., 1992) and cyclopentenone prostaglandins induced heat shock protein biosynthesis in cultured L-1210 cells (Santoro et al., 1989). Stanley-Samuelson and Ogg (1994) speculated that PGA2 may be involved in reg-
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ulating gene expression in the tobacco hornworm fat body. Morishima et al. (1997) added considerable support to this speculation with their finding that eicosanoids mediate expression of the genes for two anti-bacterial proteins in the silkworm fat body. The finding that the true armyworm fat body preparation produced primarily PGE2 and PGA2 is interesting in light of our recent finding that PGF2α was the main identified PG detected in hemolymph after bacterial infections (Jurenka et al., 1999). As indicated here, however, differences in experimental conditions can influence the profiles of PGs biosynthesized by in vitro preparations. The PGF2α was found in hemolymph and could have been produced by several tissues including fat body and hemocytes. Perhaps the PGs produced by fat body act on other fat body cells in a paracrine mode, while PGF2α is released into the hemolymph by fat body and/or by hemocytes to act on circulating hemocytes.
Acknowledgements We thank Dr Ralph W. Howard (USDA/ARS, Manhattan, KS) and Dr Steve Skoda (USDA/ARS, Lincoln, NE) for helpful comments on a draft of this paper. This is paper no. 12,754, Nebraska Agricultural Research Division and contribution no. 1,047 of the Department of Entomology. This work was supported by a fellowship from KahramanMaras Sutcu Imam University to H. Tunaz and by the Agricultural Research Division, University of Nebraska (NEB-17-054).
References Ali, I., Steele, J.E., 1997a. Hypertrehalosemic hormones increase the concentration of free fatty acids in trophocytes of the cockroach (Periplaneta americana) fat body. Comp. Biochem. Physiol. 118A, 1225–1231. Ali, I., Steele, J.E., 1997b. Evidence that free fatty acids in trophocytes of Periplaneta americana fat body may be regulated by the activity of phospholipase A2 and cyclooxygenase. Insect Biochem. Molec. Biol. 27, 681–692. Destephano, D.B., Brady, U.E., Lovins, R.E., 1974. Partial characterization of prostaglandin synthesis in the reproductive tract of the male house cricket, Acheta domesticus. Prostaglandins 11, 261– 273. Gadelhak, G.G., Pedibhotla, V.K., Stanley-Samuelson, D.W., 1995. Eicosanoid biosynthesis by hemocytes from the tobacco hornworm, Manduca sexta. Insect Biochem. Molec. Biol. 25, 743–749. Horohov, D.W., Dunn, P.E., 1983. Phagocytosis and nodule formation by hemocytes of Manduca sexta larvae following injection of Pseudomonas aeruginosa. J. Invertebr. Pathol. 41, 203–213. Hurst, J.S., Flatman, S., McDonald-Gibson, R.G., 1987. Thin-layer chromatography (including radio thin-layer chromatography and autoradiography) of prostaglandins and related compounds. In: Benedetto, C., McDonald-Gibson, R.G., Nigam, S., Slater, T.F. (Eds.), Prostaglandins and Related Substances: A Practical Approach. IRL Press, Oxford, pp. 53–73. Jurenka, R.A., Howard, R.W., Blomquist, G.J., 1986. Prostaglandin
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biosynthesis inhibitors in insect defensive secretions. Naturwissenschaften 73, S735–737. Jurenka, R.A., Miller, J.S., Pedibhotla, V.K., Rana, R.L., StanleySamuelson, D.W., 1997. Eicosanoids mediate microaggregation and nodulation responses to bacterial infections in black cutworms, Agrotis ipsilon, and true armyworms, Pseudaletia unipuncta. J. Insect Physiol. 43, 125–133. Jurenka, R.A., Pedibhotla, V.K., Stanley, D.W., 1999. Prostaglandin production in response to a bacterial infection in true armyworm larvae. Arch. Insect Biochem. Phyisol. 41, 225–232. Keeley, L.L., Park, J.H., Lu, K.-H., Bradfield, J.Y., 1996. Neurohormone signal transduction for dual regulation of metabolism and gene expression in insects: Hypertrehalosemic hormone as a model. Arch. Insect Biochem. Physiol. 33, 283–301. Kulmacz, R.J., Lands, W.E.M., 1987. Cyclo-oxygenase: measurement, purification and properties. In: Benedetto, C., McDonald-Gibson, R.G., Nigam, S., Slater, T.F. (Eds.), Prostaglandins and Related Substances: A Practical Approach. IRL Press, Oxford, pp. 209–227. Mandato, C.A., Diehl-Jones, W.L., Moore, S.J., Downer, R.G.H., 1997. The effects of eicosanoid biosynthesis inhibitors on prophenoloxidase activation, phagocytosis and cell spreading in Galleria mellonella. J. Insect Physiol. 43, 1–8. Miller, J.S., Nguyen, T., Stanley-Samuelson, D.W., 1994. Eicosanoids mediate insect nodulation responses to bacterial infections. Proc. Natl. Acad. Sci. USA 91, 12418–12422. Miller, J.S., Howard, R.W., Nguyen, T., Nguyen, A., Rosario, R.M.T., Stanley-Samuelson, D.W., 1996. Eicosanoids mediate nodulation responses to bacterial infections in larvae of the tenebrionid beetle, Zophobas atratus. J. Insect Physiol. 42, 3–12. Miller, J.S., Howard, R.W., Rana, R.L., Tunaz, H., Stanley, D.W., 1999. Eicosanoids mediate nodulation reactions to bacterial infections in adults of the cricket, Gryllus assimilis. J. Insect Physiol. 45, 75–83. Morishima, I., Yamano, Y., Inoue, K., Matsuo, N., 1997. Eicosanoids mediate induction of immune genes in the fat body of the silkworm, Bombyx mori. FEBS Letters 419, 83–86. Negishi, M., Sugimoto, Y., Ichikawa, A., 1995a. Molecular mechanisms of diverse actions of prostanoid receptors. Biochim. Biophys. Acta 1259, 109–120. Negishi, M., Koizumi, T., Ichikawa, A., 1995b. Biological actions of ⌬12-prostaglandin J2. J. Lipid Mediators and Cell Signalling 12, 443–448. Ogg, C.L., Stanley-Samuelson, D.W., 1992. Phospholipid and triacylglycerol fatty acid compositions of the major life stages and selected tissues of the tobacco hornworm Manduca sexta. Comp. Biochem. Physiol. 101B, 345–351. Otto, J.C., Smith, W.L., 1995. Prostaglandin endoperoxide synthases1 and -2. J. Lipid Mediators and Cell Signalling 12, 139–156. Pedibhotla, V.K., Sarath, G., Sauer, J.R., Stanley-Samuelson, D.W., 1995. Prostaglandin biosynthesis and subcellular localization of prostaglandin H synthase activity in the lone star tick, Amblyomma americanum. Insect Biochem. Molec. Biol. 25, 1027–1039. Petzel, D.H., Stanley-Samuelson, D.W., 1992. Inhibition of eicosanoid biosynthesis modulates basal fluid secretion in the Malpighian tubules of the yellow fever mosquito (Aedes aegypti). J. Insect Phyiol. 38, 1–8.
Santoro, M.G., Garaci, E., Amici, C., 1989. Prostaglandin with antiproliferative activity induces the synthesis of a heat shock protein in human cells. Proc. Natl. Acad. Sci. USA 86, 8407–8411. SAS Institute Inc., 1989. In:. SAS/STAT User’s Guide, Version 6, fourth ed, vol. 2. SAS Institute Inc, Cary, NC. Sasaguri, T., Masuda, J., Shimokado, K., Yokota, T., Kosaka, C., Fujishima, M., Ogata, J., 1992. Prostaglandins A and J arrest the cell cycle of cultured vascular smooth muscle cells without suppression of c-myc expression. Exp. Cell Res. 200, 351–357. Schleusener, D.R., Stanley-Samuelson, D.W., 1996. Phospholipase A2 in hemocytes of the tobacco hornworm, Manduca sexta. Arch. Insect Biochem. Phyisol. 33, 63–74. Smith, W.L., 1992. Prostanoid biosynthesis and mechanisms of action. Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32), F181– F191. Smith, W.L., Borgeat, P., Fitzpatrick, F.A., 1991. The eicosanoids: cyclooxygenase, lipoxygenase, and epoxygenase pathways. In: Vance, D.E., Vance, J.E. (Eds.), Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier, Amsterdam, pp. 297–325. Stanley, D.W., 2000. Eicosanoids in Invertebrate Signal Transduction Systems. Princeton University Press, Princeton, NJ. Stanley, D.W., Hoback, W.W., Bedick, J.C., Tunaz, H., Rana, R.L., Nor Aliza, A.R., Miller, J.S., 1999. Eicosanoids mediate nodulation reactions to bacterial infections in larvae of the butterfly, Colias eurytheme. Comp. Biochem. Physiol. C., 217–223. Stanley-Samuelson, D.W., 1994. Prostaglandins and related eicosanoids in insects. Adv. Insect Physiol. 24, 115–212. Stanley-Samuelson, D.W., Ogg, C.L., 1994. Prostaglandin biosynthesis by fat body from the tobacco hornworm, Manduca sexta. Insect Biochem. Molec. Biol. 24, 481–491. Stanley-Samuelson, D.W., Pedibhotla, V.K., 1996. What can we learn from prostaglandins and related eicosanoids in insects? Insect Biochem. Molec. Biol. 26, 223–234. Stanley-Samuelson, D.W., Pedibhotla, V.K., Rana, R.L., Nor Aliza, A.R., Hoback, W.W., Miller, J.S., 1997. Eicosanoids modulate nodulation responses to bacterial infections in larvae of the silkmoth, Bombyx mori. Comp. Biochem. Phyisol. 118A, 93–100. Tobe, S.S., Loher, W., 1983. Properties of the prostaglandin synthetase complex in the cricket Teleogryllus commodus. Insect Biochem. 13, 137–141. Tunaz, H., Bedick, J.C., Miller, J.S., Hoback, W.W., Rana, R.L., Stanley, D.W., 1999. Eicosanoids mediate nodulation reactions to bacterial infections in adults of two 17-year periodical cicadas, Magicicada septendecim and M. cassini. J. Insect Physiol. 45, 923–931. Uscian, J.M., Stanley-Samuelson, D.W., 1993. Phospholipase A2 activity in the fat body of the tobacco hornworm Manduca sexta. Arch. Insect Biochem. Physiol. 24, 187–201. Van Kerkhove, E., Pirotte, P., Petzel, D.H., Stanley-Samuelson, D.W., 1995. Eicosanoid biosynthesis inhibitors modulate basal fluid secretion rates in the Malpighian tubules of the ant, Formica polyctena. J. Insect Physiol. 41, 435–441. Wakayama, E.J., Dillwith, J.W., Blomquist, G.J., 1986. Characterization of prostaglandin synthesis in the housefly, Musca domestica (L.). Insect Biochem. 16, 903–909.
Prostaglandin biosynthesis by fat body from true armyworms, Pseudaletia unipuncta Hasan Tunaz, Russell A. Jurenka 1, David W. Stanley
*
Insect Biochemical Physiology Laboratory, University of Nebraska – Lincoln, Lincoln, NE 68583-0816, USA Received 1 September 1999; received in revised form 4 August 2000; accepted 9 August 2000
Abstract We describe prostaglandin (PG) biosynthesis by microsomal-enriched fractions of fat body prepared from true armyworms, Pseudaletia unipuncta. PG biosynthesis was sensitive to experimental conditions, including incubation time, temperature, pH, substrate and protein concentration. Optimal PG biosynthesis conditions included 1 mg of microsomal-enriched protein, incubated at 28°C for 7.5 min at pH 8. These preparations yielded four major PGs: PGA2, PGE2, PGD2 and PGF2α. PGA2 and PGE2 were the predominant eicosanoids produced under these conditions. Two non-steroidal anti-inflammatory drugs, indomethacin and naproxen, effectively inhibited PG biosynthesis. Unlike other invertebrate PG biosynthetic systems studied so far, the true armyworm system appeared to be independent of the usual exogenous co-factors required by mammalian and other invertebrate systems. These findings are discussed with respect to PG biosynthesis in other invertebrate and vertebrate systems. 2001 Published by Elsevier Science Ltd. Keywords: True armyworm; Pseudaletia unipuncta; Prostaglandins; Anti-inflammatory drugs; Insect immunity
1. Introduction Insects respond to bacterial infections with a complex immune system. We have been developing the hypothesis that eicosanoids are responsible for mediating insect immune reactions to bacterial infections. Eicosanoid is a collective term for all biologically active, oxygenated metabolites of 20:4n-6 and two other C20 polyunsaturated fatty acids (Fig. 1). Most experiments designed to test this hypothesis are based on selective inhibition of eicosanoid biosynthetic pathways, done by treating experimental animals with a variety of anti-inflammatory drugs. We began our work with tobacco hornworms, Manduca sexta, as an experimental model. Nodule formation is thought to be the predominant mechanism of clearing bacterial cells from hemolymph (Horohov and Dunn, 1983) and, using pharmaceutical inhibitors of eicosanoid biosynthesis, we found that microaggregation (an early phase of nodule formation) and nodule forma* Corresponding author. Tel.: +1-402-472-8710; fax: +1-402-4724687. E-mail address: [email protected] (D.W. Stanley). 1 Present address: Department of Entomology, Iowa State University, 411 Science II, Ames, IA 50011-3222, USA. 0965-1748/01/$ - see front matter 2001 Published by Elsevier Science Ltd. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 1 3 8 - 7
tion were severely impaired in inhibitor-treated hornworms. On the basis of these results we suggested that eicosanoids are responsible for mediating nodulation, a specific cellular reaction to bacterial infections (Miller et al., 1994). We carried out a series of similar exercises on other insect species, with a view to testing the idea that eicosanoids act in immunity of most, if not all, insect species. So far, we have obtained supporting results with larvae of a beetle, Zophobas atratus (Miller et al., 1996), and several lepidopterans, the silkworm, Bombyx mori (Stanley-Samuelson et al., 1997), cutworms, Agrotis ipsilon, and true armyworms, P. unipuncta (Jurenka et al., 1997) and larvae of the butterfly, Colias eurytheme (Stanley et al., 1999). We also tested our idea with adults of three hemimetabolous insect species, the cricket Gryllus assimilis (Miller et al., 1999) and two 17-year periodical cicadas, Magicicada septendecim and M. cassini (Tunaz et al., 1999). In addition, Downer’s group reported that two distinct phases of nodulation, cell spreading and prophenyloxidase activation, were inhibited in waxmoths, Galleria mellonella, that had been treated with eicosanoid biosynthesis inhibitors (Mandato et al., 1997). They also suggested that eicosanoids mediate phagocytosis, another independent cellu-
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Fig. 1. An overview of 20:4n-6 metabolism as understood from the mammalian background. Three polyunsaturated fatty acids, 20:3n-6, 20:4n-6 and 20:5n-3 are potential substrates for eicosanoid biosynthesis. Of these, metabolism of 20:4n-6 is most well studied. Chemical structures are denoted by numerals. 1=cellular phospholipid, 2=hydrolyzed 20:4n-6, 3=PGE2, 4=5-hydroperoxyeicosatetraenoic acid, 5=leukotriene B4, 6=11,12-epoxyeicosatrienoic acid, 7=lipoxin A. Capital letters indicate major enzyme systems responsible for eicosanoid biosynthesis. A=phospholipase A2; B=cyclooxygenase and associated enzyme steps; C=cytochrome P450 epoxygenase; D=lipoxygenase.
lar reaction to bacterial infections. Appreciation of the significance of eicosanoids in signal transduction in insect immunity was considerably broadened by Morishima et al. (1997). Based on their work with antiinflammatory drugs, this group showed that eicosanoids also mediate expression of two silkmoth fat body genes for antibacterial proteins, cecropin and lysozyme. We infer from the work with all these systems that eicosanoids are important elements of insect immunity (Stanley, 2000). The hypothesis that eicosanoids act in insect immunity would rest on firmer grounds if it were supported by results of experiments which do not solely rely on the actions of pharmaceutical products. We generated one line of work by investigating the biochemistry of eicosanoid systems in immune tissues from tobacco hornworms, M. sexta. We documented the presence of three elements of eicosanoid biosynthesis. First,
hornworm tissues maintain eicosanoid precursor polyunsaturated fatty acids, albeit in low proportions, in tissue lipids (Ogg and Stanley-Samuelson, 1992). Second, we described an intracellular phospholipase A2 in fat body and hemocytes that can release 20:4n-6 from the sn-2 position of phospholipids (Uscian and Stanley-Samuelson, 1993; Schleusener and Stanley-Samuelson, 1996). Third, we characterized eicosanoid biosynthesis in fat body and hemocytes (Stanley-Samuelson and Ogg, 1994; Gadelhak et al., 1995). These biochemical findings are important because they lie at the foundation of the biological significance of eicosanoids in immunity or any other physiological arena. Nonetheless, additional evidence more directly focused on immune reactions would provide a better test of the hypothesis that eicosanoids mediate insect immunity. We reasoned that bacterial infections stimulate biosynthesis and secretion of PGs and other eicosanoids by fat body and hemocytes. These infection-stimulated eicosanoids subsequently mediate cellular reactions to the infections. If this were so, then bacterial infections should result in measurable increases in eicosanoid titers in insect hemolymph. Returning to true armyworms, we tested this idea by injecting experimental larvae with heat-killed bacteria and injecting control larvae with saline (Jurenka et al., 1999). At 30 min post-infection, hemolymph samples were withdrawn, and eicosanoids were extracted. The eicosanoids were derivatized, then analyzed on fluorescence–HPLC. Relative to controls we recovered about four-fold more PGF2α from hemolymph of infected insects (Jurenka et al., 1999). These data showed that bacterial infections stimulate increased eicosanoid biosynthesis in true armyworms, which adds considerable support to our hypothesis. Complementary to this work on PG production, we began investigating eicosanoid biosynthesis by immune tissues prepared from true armyworms. In this paper we report on PG biosynthesis by true armyworm fat body. We describe an optimized in vitro enzyme assay for the characterization of eicosanoid biosynthesis and discuss these findings in relation to other invertebrate and vertebrate systems.
2. Materials and methods 2.1. Insects True armyworms, P. unipuncta, reared on standard culture medium, were provided by the Corn Insect Research Unit (USDA, Ames, IA). Larvae were shipped to Lincoln, NE, where they were maintained at 28°C until the sixth instar. Last instars were used throughout this study, except for a few experiments with pupae, as noted in Results.
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2.2. Isolation of fat body and preparation of microsomal-enriched fractions These experiments followed protocols developed for the tobacco hornworm fat body (Stanley-Samuelson and Ogg, 1994). The larvae were anesthetized by chilling on ice, then fat body tissue was dissected in ice-cold phosphate buffer (0.05 M potassium phosphate, pH 8.0). The fat body tissues, in 1 ml Eppendorf tubes, were sonicated for 10 s at 30 W using a VibraCell sonicator (VibraCell, Danbury, CT). This preparation was centrifuged for 10 min at 735g, and the supernatant was centrifuged for another 20 min at 16,000g, both steps at 4°C. The 16,000g supernatants were microsomal-enriched preparations used in all experiments. Protein concentrations in these preparations were determined in microtiter format using the bicinchoninic acid reagent (Pierce, Rockford, IL), against bovine serum albumin as quantitative standard. The microtiter plates were read on a BioTek microtiter plate reader at 562 nm. Radioactive arachidonic acid (5,6,8,9,11,12,14,15– 3 H–20:4, 60–100 Ci/mmol) was purchased from New England Nuclear (Boston, MA). The incubation buffer was 0.05 M KH2PO4, pH 8.0, amended with a standard co-factor cocktail (2.4 mM reduced glutathione, 0.25 mM hydroquinone and 25 µg hemoglobin; StanleySamuelson and Ogg, 1994). For each PG biosynthesis reaction (unless indicated otherwise), 0.4 µCi of labeled 20:4n-6 was dispensed into reaction tubes and the solvent was evaporated. The reactions were carried out in 0.5 ml total volume. The experiments were preceded by a 10 min pre-incubation at 28°C with all reaction components, except the protein source. The reactions were stopped by acidification to pH 3.5–4.0 by addition of 0.2 ml 0.1 N HCl. Reaction products were extracted from the acidified reaction mixture three times with 0.5 ml ethyl acetate. The combined extracts, containing PGs and possible lipoxygenase products, were evaporated under N2. A mixture of appropriate unlabeled eicosanoid standards was added to each sample, then samples were applied to TLC plates (20×20 cm Silica Gel G, 0.25 mm thick, Sigma Chemical Co., St Louis, MO). The plates were developed in the A9 solvent system (Hurst et al., 1987) and fractions observed by exposure to iodine vapors. Bands corresponding to selected authentic eicosanoid standards and to free fatty acids were transferred to liquid scintillation vials. Radioactivity in each fraction was determined by adding 5 ml scintillation cocktail (ICN Biomedicals, Irvine, CA) and counting on a LKB Wallac 1209 Rackbeta Liquid Scintillation Counter (Pharmacia, Turku, Finland) at 50% counting efficiency for 3H. Eicosanoid biosynthesis was calculated from the liquid scintillation data. In control experiments, microsomal-enriched preparations were heated in boiling water for 15 min before the experiments, and processed as just described. The results of these control experiments were
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used to correct values from biosynthesis experiments as previously described (Stanley-Samuelson and Ogg, 1994). 2.3. Determining PGA2 by gas–chromatography/mass spectrometry (GC–MS) Microsomal-enriched preparations from 18 last-stage larvae were added to reaction tubes containing 10 µM arachidonic acid without added co-factors. After 7.5 min at 32°C the reactions were stopped with the addition of 0.2 ml 0.1 N HCl. Products were extracted three times with 0.5 ml ethyl acetate. The ethyl acetate extracts were combined and dried under a stream of N2. The extract was then purified and analyzed as described previously (Jurenka et al., 1999). Briefly, the extract was cleaned up on silicic acid chromatography and the acetonitrile:methanol fraction was treated with 100 µl diazomethane in diethyl ether. The resulting methyl esters were then treated with N,O-bis(trimethylsilyl)trifluoracetamide containing 1% trimethylchlorosilane and heated at 60°C for 20 min. The reaction was dried under N2 and reconstituted with methylene chloride for analysis on GC–MS. Analyses were conducted by capillary GC–MS using a Hewlett–Packard 5890 GC equipped with a DB-1 column (0.25 mm×30 m). The GC was interfaced with a Hewlett–Packard 5972 Mass Selective Detector operated in scan mode. Separations were conducted in splitless mode with temperature programming at 80°C for 1 min, then 10°/min to 320°C. 2.4. Statistical analysis Data were analyzed using the General Linear Models procedure, and mean comparisons were made using Least Significant Different (LSD) test (SAS Institute Inc., 1989).
3. Results Microsomal-enriched fractions of fat bodies prepared from last-instar true armyworms were competent to biosynthesize PGs. The fat body preparations produced four major products (PGA2, PGD2, PGE2 and PGF2α). PGA2 was the major product under most experimental conditions, although our data indicate that changes in reaction conditions influenced the overall profile of PG biosynthesis. Because PGA2 was the major PG produced by the fat body preparations we confirmed the identification of PGA2 by GC–MS. Fig. 2 shows a total ion scan of the methyl ester trimethylsilyl derivative of PGA2 obtained from incubation of true armyworm fat body preparations with arachidonic acid. The influence of reaction conditions on PG biosynthesis are reported in the following paragraphs.
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Fig. 2. A total ion scan of the methyl ester trimethylsilyl derivative of PGA2 obtained from the incubation of true armyworm fat body with arachidonic acid.
3.1. Influence of radioactive substrate concentration on PG biosynthesis The influence of four concentrations of radioactive substrate on PG biosynthesis is shown in Fig. 3. Total PG biosynthesis increased from ca 2 nmol/mg protein/h in the presence of 0.2 µCi of substrate to ⬎13 nmol/mg protein/h with 1.6 µCi of substrate. Except for 0.8 µCi of substrate, PG biosynthesis increased in an approximately linear way with increasing substrate. Highest PG biosynthesis was obtained in the presence of 1.6 µCi of substrate (significantly higher than other concentrations), however, we used 0.4 µCi of substrate per reaction to balance optimal use of radioactive material with a reasonable level of PG biosynthesis. We note that substrate concentrations influenced the profiles of radioactive PG products. At 0.4 µCi of substrate PGA2 was the predominant product, while all four PGs were produced in similar levels at 1.6 µCi of radioactive substrate. 3.2. Influence of protein concentration on PG biosynthesis Fig. 4 shows the relationship between fat body microsomal-enriched protein concentration and PG
Fig. 3. The influence of four concentrations of radioactive 20:4n-6 on PG biosynthesis by microsomal-enriched preparations of fat body from true armyworm larvae. The 0.5 ml reaction mixtures containing 0.2, 0.4, 0.8 and 1.6 µCi 3H–20:4n-6, 1 mg of microsomal-enriched protein, and the co-factor in 50 mM potassium phosphate buffer, pH 8.0. The reactions were incubated at 28°C. After 7.5 min incubations, the reactions were stopped, and the reaction products were extracted and separated as described in Materials and Methods. The histogram displays the biosynthesis of individual PGs, and the line represents total PG biosynthesis. Each point represents the mean of three separate experiments at each substrate concentration. The error bars, where visible, indicate 1 SEM.
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Fig. 4. The influence of fat body microsomal-enriched protein concentration on PG biosynthesis. The reactions were conducted and the products were extracted and separated as described in Materials and Methods. The histogram displays the biosynthesis of individual PGs, and the line represents total PG biosynthesis. Each point represents the mean of three separate experiments at each protein concentration. The error bars, where visible, indicate 1 SEM.
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Fig. 5. The influence of incubation time on PG biosynthesis by microsomal-enriched preparations of fat body from true armyworm larvae. The reactions were conducted and the products were extracted and separated as described in Materials and Methods. The histogram displays the biosynthesis of individual PGs, and the line represents total PG biosynthesis. Each point represents the mean of three separate experiments at each different incubation time. The error bars, where visible, indicate 1 SEM.
biosynthesis. The optimal protein concentration was 1 mg/reaction, which yielded significantly higher product formation. These findings provided the information for the use of 1 mg protein/reaction in subsequent experiments. We also observed the influence of protein concentration on the overall profiles of PG biosynthesis. PGA2 was the major product at 1 mg protein/reaction while PGE2 was the major product in experiments with other protein concentrations. All four major PG products were formed at all protein concentrations. 3.3. Influence of reaction time on PG biosynthesis The influence of reaction time on PG biosynthesis is shown in Fig. 5. Total PG biosynthesis increased from about 1.0 nmol/mg protein at 2 min to a high of approximately 4 nmol/mg protein at 7.5 min. Total PG production was diminished in longer incubations, to ⬍0.5 nmol/mg protein at 15 min. The data indicated highest total PG biosynthesis obtained at 7.5 min, which is statistically similar to results at 5 and 10 min. We used 7.5 min incubations in subsequent experiments. The 7.5 min optimum is unusually long for PG biosynthesis reactions. To investigate this point in a little more detail, we carried out a similar experiment using fat body from pupae. Fig. 6 shows that the optimal reaction time for the pupal preparations was 2 min (statistically significant), which accords with other insect PG systems.
Fig. 6. The influence of incubation time on PG biosynthesis by microsomal-enriched preparations of fat body from true armyworm pupae. The reactions were conducted and the products were extracted and separated as described in Materials and Methods. The histogram displays the biosynthesis of individual PGs, and the line represents total PG biosynthesis. Each point represents the mean of three separate experiments at each different incubation time. The error bars, where visible, indicate 1 SEM.
3.4. Influence of reaction temperature on PG biosynthesis Total PG biosynthesis increased significantly from 1 nmol/mg protein/h at 16°C to a high of 4 nmol/mg
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by adjusting the buffer to the indicated pH, the fat body preparations yielded all four PGs. Significantly higher PG biosynthesis, approximately 5 nmol/mg protein/h, was obtained at pH 8. We note that pH conditions influenced the profile of PG biosynthesis. While PGA2 was the major product under most experimental conditions, PGE2 emerged as the predominant product at pH 8. 3.6. True armyworm PG biosynthesis is independent of exogenous co-factors
Fig. 7. The influence of incubation temperature on PG biosynthesis by microsomal-enriched preparations of fat body from true armyworm larvae. The histogram displays the biosynthesis of individual PGs, and the line represents total PG biosynthesis. Each point represents the mean of three separate experiments at each different temperature. The error bars, where visible, indicate 1 SEM.
protein/h at 28°C (Fig. 7). The results from incubations at 24°C and 32°C were statistically similar to the results at 28°C. Higher temperatures yielded similar or lower amounts of product. PGA2 was the major product at all temperatures except for 28°C, at which PGE2 was the major product. 3.5. Influence of reaction pH on PG biosynthesis
We investigated the possibility that the true armyworm fat body preparation does not depend on exogenous co-factors that are routinely used in mammalian and invertebrate PG biosynthesis reactions (Stanley-Samuelson and Ogg, 1994). Microsomal-enriched fat body preparations were incubated with radioactive 20:4n-6 under the usual conditions, with the exception that the co-factor cocktail was left out of the reaction. At the end of 7.5 min incubations, the products were extracted and separated as usual. The absence of co-factors did not influence total PG biosynthesis nor the pattern of PG biosynthesis (Fig. 9). 3.7. The fat body preparation is sensitive to two nonsteroidal anti-inflammatory drugs Total fat body PG biosynthesis was inhibited in reactions conducted in the presence of indomethacin and
Fig. 8 shows the influence of reaction pH on PG biosynthesis. Under each of the pH conditions, obtained
Fig. 8. The influence of buffer pH on PG biosynthesis by microsomal-enriched preparations of fat body from true armyworm larvae. The reactions were conducted and the products were extracted and separated as described in Materials and Methods. The histogram displays the biosynthesis of individual PGs, and the line represents total PG biosynthesis. Each point represents the mean of three separate experiments at each different buffer pH. The error bars, where visible, indicate 1 SEM.
Fig. 9. Schematic representation of background corrected TLC separation of PGs biosynthesized by isolated fat body of true armyworm larvae. The reactions were conducted and the products were extracted and separated as described in Materials and Methods. The TLC plate was divided into 2 cm fractions, all of which were scraped into vials, and the radioactivity in all fractions was estimated by liquid scintillation counting. Radioactivity in fraction 10, which is associated with the substrate 3H–20:4n-6, is not shown. The reaction from which these data were generated was conducted in the absence of the standard cofactor cocktail.
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4. Discussion
Fig. 10. The influence of indomethacin on PG biosynthesis by microsomal-enriched preparations of fat body from true armyworm larvae. The reactions were conducted and the products were extracted and separated as described in Materials and Methods. Each bar represents the mean of three separate experiments at each concentration of indomethacin. The error bars, where visible, indicate 1 SEM. Bars with different fill patterns represent statistical differences.
naproxen (Figs. 10 and 11). In reactions with indomethacin (1 µM), PG biosynthesis declined significantly from about 4 nmol/mg protein/h to approximately 1 nmol/mg protein/h. Higher indomethacin concentrations did not decrease PG biosynthesis. Naproxen reduced PG biosynthesis from about 4 nmol/mg protein/h to 3 nmol/mg protein/h at 1 µM; 10µM naproxen significantly reduced PG biosynthesis to approximately 1 nmol/mg protein/h.
Fig. 11. The influence of naproxen on PG biosynthesis by microsomal-enriched preparations of fat body from true armyworm larvae. The reactions were conducted and the products were extracted and separated as described in Materials and Methods. Each bar represents the mean of three separate experiments at each concentration of naproxen. The error bars, where visible, indicate 1 SEM. Bars with different fill patterns represent statistical differences.
In this paper we document PG biosynthesis by microsomal-enriched preparations of true armyworm fat bodies. Under our experimental conditions, PGE2, PGD2, PGF2α and PGA2 were formed. While PGA2 was the quantitatively predominant product under most conditions, we recorded substantial biosynthesis of all four PGs. Identification of PGA2 by GC–MS (Fig. 2) adds considerable verisimilitude to studies of PG biosynthesis in insects. As seen in other studies of insect tissues (Stanley-Samuelson, 1994; Stanley et al., 1999), PG biosynthesis in the true armyworm preparations was sensitive to reaction conditions, including temperature, pH, incubation time, and protein concentration. In our protocols, optimal reaction conditions for PG biosynthesis include reacting 1.0 mg microsomal-enriched protein with 0.4 µCi of 3H–20:4n-6 at 28°C and pH 8.0 for 7.5 min. Also, PG biosynthesis by the true armyworm preparations was inhibited in reactions conducted in the presence of two non-steroidal anti-inflammatory drugs. We infer from these findings that the true armyworm fat body is competent to biosynthesize PGs. We have discussed most aspects of PG biosynthesis, including the influence of reaction conditions and nonsteroidal anti-inflammatory drugs on PG biosynthesis, in our work on other species (Stanley-Samuelson and Ogg, 1994; Gadelhak et al., 1995; Pedibhotla et al., 1995). Those remarks will not be repeated here. Instead, we now have enough information to begin appreciating comparative aspects of eicosanoid biosynthesis in insects. The thrust of the following remarks is recognizing the diversity of PG biosynthesis systems among insects. While the findings with true armyworm fat body preparations superficially resemble results from other insect species, we recorded important comparative differences. One departure relates to the requirement for a cocktail of co-factors, usually including hemoglobin and reduced glutathione, to support PG biosynthesis. The roles of these co-factors can be understood in terms of the enzymes responsible for PG biosynthesis (Fig. 1), which typically requires four steps (Stanley, 2000). First, a phospholipase A2 step is responsible for releasing 20:4n6 from cellular phospholipids; second, a cyclooxygenase step yields the hydroperoxide, PGG2; third, a glutathione peroxidase converts the PGG2 to PGH2; finally, a fourth enzyme is responsible for converting PGH2 into the final PG product. The cyclooxygenase and peroxidase activities are combined in a single holoenzyme called PGH synthase. The terms COX-1 and COX-2 are used in preference to PGH synthase in the literature on mammals, in which two forms of the enzyme are found (Otto and Smith, 1995). We use the term PGH synthase because the possibility that insects express two forms of this enzyme has not yet been considered. The hemoglobin
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component of the co-factor cocktail provides a heme, often in the form of iron protoporphyrin IX, which both the cyclooxygenase and peroxidase activities require for catalytic activity (Kulmacz and Lands, 1987). The reduced glutathione is required by the glutathione peroxidase which reduces the PGG2 to PGH2 and oxidizes the reduced glutathione (Kulmacz and Lands, 1987). Based on this background from studies on mammalian systems, researchers working on invertebrates conduct PG biosynthesis reactions in the presence of a more or less standard cocktail of co-factors that provide a heme group, reduced glutathione and hydroquinone. Because the point had not been specifically addressed in insect studies, we assessed the need for co-factors in tobacco hornworm fat body preparations by running PG biosynthesis reactions in the absence of co-factors (Stanley-Samuelson and Ogg, 1994). The results indicated that PG biosynthesis was completely abolished in the absence of added co-factors, from which we inferred that insect PG biosynthesizing systems require a mixture of co-factors similar to the co-factors required by mammalian PG biosynthesizing enzymes. Analogous experiments with internal-tissue preparations from the tick, Amblyomma americanum, showed the tick systems also require the added co-factor cocktail to support PG biosynthesis (Pedibhotla et al., 1995). We similarly assessed the need for co-factors in true armyworm fat body preparations. Contrary to expectation, we recorded no difference in PG biosynthesis in the presence or absence of our standard co-factor cocktail (Fig. 9). A couple of possibilities account for this new finding. Either the armyworm homogenates provide sufficient endogenous co-factors to support PG biosynthesis, or the armyworm fat body enzymes responsible for PG biosynthesis do not require the co-factors for catalysis. In either case, the armyworm preparations differ from the mammalian and the other invertebrates that have been studied on the matter of co-factor requirements for in vitro PG biosynthesis. The true armyworm fat body preparations mark another departure from the mammalian background. The mammalian cyclooxygenases are known as “suicide” enzymes, because they are spontaneously and permanently inactivated after about 1400 catalytic operations (Smith et al., 1991). For this reason, most PG biosynthesis reactions are carried out in very short incubation periods, usually 1–2 min. We found that short incubation periods were also optimal for tobacco hornworm fat body preparations (Stanley-Samuelson and Ogg, 1994) and for tick internal-tissue preparations (Pedibhotla et al., 1995). This agreed with outcomes from housefly, Musca domestica, preparations in which very little PG product accumulated in incubation times longer than 2 min (Wakayama et al., 1986). But it differed from findings with spermathecal preparations from the cricket, Teleogryllus commodus, in which PGs accumulated in a
linear way over 60 min incubations (Tobe and Loher, 1983). The optimal reaction period for true armyworm fat body preparations was 7.5 min, after which total PG product accumulation declined. The declines are probably due to the asymmetry in biosynthesis and degradation of products, discussed in detail elsewhere (Stanley-Samuelson and Ogg, 1994). Because incubations longer than 7.5 min yielded reduced product, we speculate that the armyworm fat body system also undergoes spontaneous inactivation, however, not within the short periods seen in other systems. We investigated the 7.5 min incubation periods in a little more detail by generating a similar time course study for the pupal stage of true armyworms. For pupae, we found that 2 min incubations were optimal, and PG yield declined in longer incubations. Two points are important in this exercise. First, we note the difference in optimal reaction time between two stages of the same species. Second, these differences underscore the differences noted in the larval stages of true armyworms. Insects make up the most diverse class of animals, and it may be supposed that variation in the biochemistry of PG biosynthesis among insect species and tissues will prove to be substantial. Indeed, the variation within insects may be greater than the recorded variations between insects and mammals. For a single example, let us consider the pharmaceutical product, indomethacin, a prescription analgesic which potently inhibits cyclooxygenases in mammalian and most invertebrate tissues. Contrarily, indomethacin does not inhibit PG biosynthesis in reproductive tract preparations from the house cricket, Acheta domesticus (Destephano et al., 1974). We expect that continued investigations with insects and other invertebrates will reveal many more variations on the theme of PG biosynthesis. The true armyworm preparations yielded relatively low rates of PG biosynthesis, as seen in other insects. Although the point has not been investigated in detail, insects appear to biosynthesize PGs at rates considerably below the rates recorded for some mammalian tissues. A direct comparison was provided by Jurenka et al. (1986) who assessed PG biosynthetic activity in fat body homogenates from the cockroach, P. americana, and in bovine seminal vesicles. The PG biosynthetic rates in the cockroach preparations were lower, by approximately 10-fold, than the rates recorded from the bovine seminal vesicles. Among mammalian tissues, the bovine preparations are among the most active PG producers, but the point remains that insect PG biosynthetic systems differ in production rate from their mammalian counterparts. The biological significance of PG biosynthesis in insect fat body is complex. Drawing on the mammalian background (Otto and Smith, 1995; Stanley et al., 1999), PGs are thought to mediate cellular events in a couple of ways. For one, PGs regulate events involved in day-
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to-day homeostasis. These are colloquially known as “housekeeping” functions. We know relatively little about the housekeeping roles of PGs in insects. We suggested that PGs regulate basal fluid secretion rates in Malpighian tubules of adult female mosquitoes and worker ants (Petzel and Stanley-Samuelson, 1992; Van Kerkhove et al., 1995). Relative to fat body, Keeley et al. (1996) suggested that some eicosanoids may act in the signal transduction pathways to influence fat body cellular responses to hypertrehalosemic hormone in the cockroach Blaberus discoidalis. In a similar line of research using trophocytes prepared from disaggregated fat bodies of another cockroach, Periplaneta americana, Ali and Steele (1997a,b) investigated the influence of three eicosanoid biosynthesis inhibitors on phosphorylase activation. Phosphorylase activation is the first step in the trehalose biosynthetic pathway, which is activated by hypertrehalosemic hormone in P. americana. Eicosanoids may down-regulate phosphorylase activation because incubations in the presence of eicosanoid biosynthesis inhibitors resulted in increased phosphorylase activation. In these instances, eicosanoids appear to act in housekeeping roles within Malpighian tubule and fat body cells. In these housekeeping roles, PGs and perhaps other eicosanoids are thought to act through an autocrine or paracrine mechanism, depicted in a general way by Stanley-Samuelson and Pedibhotla (1996). In this mechanism, PGs produced within fat body cells are transported across the cell membranes to the outside of the cells, where the PGs can interact with specific functional receptor sites located on the outer surface of the PGproducing cells or their neighbors. The receptors are thought to influence intracellular events through G-protein coupled signal transduction mechanisms. This scheme accounts for the action of most PGs, including PGD, PGE, PGF and PGI (Smith, 1992; Negishi et al., 1995a). However, it does not account for the actions of PGA2. As seen in the tobacco hornworm and true armyworm fat body, we recorded substantial biosynthesis of PGA2 by the true armyworm fat body preparations. Two PGs, ⌬12-PGJ2 and PGA2, are cyclopentenone PGs. Unlike the PGs discussed in the preceding paragraph, these PGs have no cell surface receptors. Instead, the cyclopentenone PGs are actively taken into cells, and are accumulated in the nuclei, where they influence expression of genes (Negishi et al., 1995b). Hence, while some PGs are responsible for housekeeping operations, the cyclopentenone PGs exert far more long-reaching actions. For a couple of examples, PGA2 arrested the cell cycle of cultured vascular smooth muscle cells (Sasaguri et al., 1992) and cyclopentenone prostaglandins induced heat shock protein biosynthesis in cultured L-1210 cells (Santoro et al., 1989). Stanley-Samuelson and Ogg (1994) speculated that PGA2 may be involved in reg-
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ulating gene expression in the tobacco hornworm fat body. Morishima et al. (1997) added considerable support to this speculation with their finding that eicosanoids mediate expression of the genes for two anti-bacterial proteins in the silkworm fat body. The finding that the true armyworm fat body preparation produced primarily PGE2 and PGA2 is interesting in light of our recent finding that PGF2α was the main identified PG detected in hemolymph after bacterial infections (Jurenka et al., 1999). As indicated here, however, differences in experimental conditions can influence the profiles of PGs biosynthesized by in vitro preparations. The PGF2α was found in hemolymph and could have been produced by several tissues including fat body and hemocytes. Perhaps the PGs produced by fat body act on other fat body cells in a paracrine mode, while PGF2α is released into the hemolymph by fat body and/or by hemocytes to act on circulating hemocytes.
Acknowledgements We thank Dr Ralph W. Howard (USDA/ARS, Manhattan, KS) and Dr Steve Skoda (USDA/ARS, Lincoln, NE) for helpful comments on a draft of this paper. This is paper no. 12,754, Nebraska Agricultural Research Division and contribution no. 1,047 of the Department of Entomology. This work was supported by a fellowship from KahramanMaras Sutcu Imam University to H. Tunaz and by the Agricultural Research Division, University of Nebraska (NEB-17-054).
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