Enrichment of ion-specific adenosine triphosphatase activities by thyroxine in different tissues of the silkworm, Bombyx mori L. During development

Insect Biochem. Molee. Biol. Vol. 24, No. 3, pp. 243-248, 1994 Copyright © 1994 Elsevier Science Ltd

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Enrichment of Ion-specific Adenosine Triphosphatase Activities by Thyroxine in Different Tissues of the Silkworm. Bombyx mori L. During Development K. D A M O D A R R E D D Y , * A. B. C H A U D H U R I , t K U M U D A SUKUMAR*';~ Received 24 February 1993; revised and accepted 29 July 1993

Ion-dependent ATPase activities in gonads and silk glands of Bombyx mori L. were determined from the ffth larval stage to adult emergence after administration of different doses of mammalian thyroxine to 1 h old, fifth-instar larvae. In normal insects, both tissues exhibited maximum enzyme activity before spinning (day 10). Na ÷- K ÷- and MgZ+-ATPase activities in ovaries and silk glands markedly declined after pupation, more so on the day of adult emergence. The level of ATPase activities in the testis did not exhibit any decline. Except for the lowest dose in the case of the silk glands, all doses of thyroxine tested (0.5-2.0 p g/g) on fifth-instar larvae (1 h-old) significantly enhanced Na +- K ÷- and MgZ+-ATPase activities in the larval and pupal stages in both gonads and silk glands. In adult testes, the enhancement was restricted to Na ÷ and K ÷ ATPases. Irrespective of sex, the adult silk glands did not exhibit any enhancement in the activities of the ATPase following thyroxine treatment. The adult ovaries also did not show any increase in ATPase. The tissue ATPases of gonads and silk glands were influenced by thyroxine in larvae and pupae in a dose-dependent manner, and the results indicate that thyroxine exerts a regulatory influence on the ATPase system in the silkworm. Bombyx mori

Thyroxine Gonads

Silk gland ATPases

INTRODUCTION Ion-dependent ATPases, widely distributed in different arthropod tissues, perform important functions mainly through membrane transport (Towle, 1984). Definite ATPase activity has been reported in B o m b y x mori, and it has been suggested that ATPases may be involved in the secretions of silk proteins into the lumen or reabsorption of water from the lumen of the gland (Peacock, 1981; Akai, 1970). The protein synthesis and storage mechanisms are also energy consuming phenomena, and the supply of ATP is considered to be an important factor for these processes. Previous reports have provided evidence for the responsiveness of silkworm tissues to thyroid hormone with respect to changes in protein, nucleic acids, glycogen and cholesterol metabolism (Medda et al., 1980; Chaudhuri and Medda, 1986, 1987a, b, 1992, 1994;

Chaudhuri et al., 1987a). There are also reports that exogenous thyroxine induces ecdysteroid titre increases in the silkworm haemolymph (Thyagaraja et al., 1991). In vertebrates, some specific enzymes like the hexose monophosphatase pathway, ~-glycerophosphate dehydrogenase, NADP-dependent malic enzyme and glucose6-phosphatase activities have been reported to be activated by the application of exogenous thyroid hormone (Hochachka, 1962; Paul and Medda, 1985; Ghosh et al., 1987; De et al., 1988). It was our interest to understand the effect of thyroxine on the silkworm enzyme systems, and the present study was intended to investigate the changes in the activities of ion-specific ATPases (Na ÷- K +- and Mg:+-dependent) in certain tissues of B. mori. MATERIALS AND METHODS Animals

*Indian Institute of Chemical Technology, Biology Division, Hyderabad-500 007, A.P., India. tBose Institute, Department of Animal Physiology, Calcutta, West Bengal-700 054, India. :~Author for correspondence.

An indigenous polyvoltine Nistari strain of silkworm, B. mori was used for experiment. The larvae were reared on fresh leaves of mulberry, Morus alba at 25-26°C, 12 light: 12 dark photoperiod with a suitable 243

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K. D A M O D A R

humidity (70-90%). Synchronous populations of male and female fifth instars (1 h-old) were selected at random for thyroxine treatment. Thyroxine treatment

L-thyroxine-sodium-pentahydrate (T4) purchased from Sigma Chemical Co., U.S.A. was dissolved with a minimum amount of 0.1 M N a O H solution and the required volume was made with 0.65% saline. Different doses (0.5, 1.0, 1.5 and 2.0/~g/g) of thyroxine 5/A/larvae were injected through the 2nd abdominal leg of 1 h-old fifth-instar larvae. The same volume of 0.65% alkaline saline was injected into fifth-instar (1 h-old) silkworms for control experiments. In all experiments 30 larvae were used. Tissue preparation

The silk glands, ovaries and testes were dissected out from larvae, pupae and adults at different days of interval in ice-cold buffer (0.05 M Hepes, 0.025 M Tris, 0.25 M sucrose at pH 7.4), transferred to the same buffer and homogenized using a Potter-Elvehjem all glass homogenizer at 2000 rpm (10 strokes, 1 min). The resulting homogenate was centrifuged at 7000g, for 10 min at 4°C to remove cell debris, and the supernatant was used for assay of ATPase activity. Enzyme assay

The assay of Na +- K +- and Mg2+-ATPase activities followed the method of Ismail-Beigi and Edelman (1971) with some modifications. Na +- K+-ATPase activity was measured using a medium containing 80 mM NaC1, 10 mM KC1, 2.5 mM MgClz, 2.5 mM ATP, 20 mM Tris at pH 7.4. Ouabain, a specific inhibitor of Na +- K+-AT Pase activity, was used as a concentration of 1 mM in the required tubes as a control. The difference between the amount of inorganic phosphorus liberated in the media with and without ouabain after addition of the enzyme aliquots showed the Na +- K+-ATPase activity. Mg 2+ATPase activity was similarly determined by using

R E D D Y et al.

2.5 mM MgC1 z, 2.5 mM ATP, 20 mM Tris in the presence of 1 mM ouabain. The reaction mixture was incubated at 30°C for 5 min, and the reaction was stopped by addition of 10% TCA. The tubes were then transferred to an in ice water bath and left for 10 min after which time the samples were centrifuged at 1000g for 5 min. The supernatant was saved and used for the determination inorganic phosphorus (Chen et al., 1956). The protein content was estimated following the method of Lowry et al. (1951). Statistical analysis was done by Student's t-test, and the mean value taken was the average of three replications.

RESULTS Na ÷- K ÷- and Mg2+-ATPase activities in silk glands (Figs 1 and 2), ovaries (Figs 3 and 4) of control silkworms increased gradually during the fifth-larval instar reaching a maximum on the 10th day (before spinning), then decreased sharply in 1 day old pupae remaining low after adult emergence. In the case of the testes (Figs 5 and 6), the Na +- K +- and Mg2+-ATPase activities increased to a maximum in late-fifth larvae, retaining that level in pupal and adult stages. It appears that there is a specific pattern of variation in ion-dependent ATPase activity in different tissues of silkworm during different developmental stages. In control silkworms, the ATPase activities in the gonads were much more pronounced than those in the silk glands, and between the gonads, the ovaries showed higher activity than the testes throughout the fifth larval stage. During the pupal and adult stages, however, the testes exhibited much higher activity. A single injection of various doses of T4 to the fifth-instar (0.5, 1.0, 1.5 and 2.0/~g/g) induced a significant increase by day 2 (Figs 1-6) in both the ATPase systems of all tissues, except in the silk glands where the 0.5/~g/g dose was not significantly different from controls. The activity was dose-dependent, the highest dose

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1. Effect o f t h y r o x i n e (T4) on N a +- K + - A T P a s e in the silk glands of B. mori. (Each c o l u m n in the figure indicates m e a n + SE (vertical bar) * P < 0.05; **P < 0.02; ***P < 0.01; ****P < 0.001 c o m p a r e d to control.)

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F I G U R E 2. Effect of thyroxine (T4) on Mg2+-ATPase in the silk glands of B. mori. (Each column in the figure indicates mean _ SE (vertical bar) *P < 0.05; **P < 0.02; ***P < 0.01; ****P < 0.001 compared to control.)

giving the maximum activity, and the magnitude of the T 4 response was tissue-specific. Similar responses were found in day 5 and day l0 fifth-instar larvae and day 1 pupae, although the highest dose did not always give the maximum activity. No significant differences were recorded in the ATPase activities of adult tissues following thyroxine treatment, except in the testis Na +- K +ATPase activity where an increase was observed with increasing dose.

DISCUSSION Tissue-specific, age-dependent N a +- K +- and Mg 2+ATPase activities were established in B. mori from the present investigation. The maximum activity of these ATPase enzymes on the 10th day of the fifth-larval instar (before spinning) may reflect the higher energy ,requirements for silk protein synthesis and reproductive prep250 -225 200 175

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aration in both sexes. The reduction in the activities of these ion-dependent ATPases in untreated animals following the lOth day correlates with the concentration of these ions in different organs during the larval, pupal and adult stages of the silkworm. The present investigation is in agreement with findings on age-related changes including post-reproductive decline of ATPases in other insects (Sohal, 1976; Bains et al., 1990). The ovary is activated in the fifth-larval instar to initiate germinal cell multiplication (Engelmann, 1970) and in the pupal stage, the uptake of haemolymph vitellogenin by the ovary helps in subsequent maturation (Ono et al., 1975). The initiation and completion of these processes may be the causes of the increase and subsequent decrease in ATPase activities in the silkworm ovary during the larval, pupal and adult stages, respectively. Maintenance of high levels of adenosine triphosphatase activities in the testes during the pupal and adult stages ~

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F I G U R E 3. Effect of thyroxine (T4) on Na ÷- K+-ATPase in the ovaries of B. mori. (Each column in the figure indicates mean + SE (vertical bar) *P < 0.05; **P < 0.02; ***P < 0.01; ****P < 0.001 compared to control.)

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F I G U R E 4. Effect of thyroxine (T4) on Mg2+-ATPase in the ovaries ofB. mori. (Each column in the figure indicates mean -t- SE (vertical bar) *P < 0.05; **P < 0.02; ***P < 0.01; ****P < 0.001 compared to control.)

may be attributed to the need for energy during spermatogenesis and sperm maturation, which continue into the adult phase (Chaudhuri and Medda, 1986). Thyroxine-induced, enhanced enzyme activity appears to be a result of an increase in enzyme mass (Murphy and Walker, 1974; Li et al., 1975). A dose-dependent response was observed in the ATPase activities in silkworm tissues after thyroxine treatment in the present investigation. A lesser sensitivity was observed in tissue metabolism of B. mori with thyroxine at doses lower than 0.5 #g/g (Chaudhuri and Medda, 1987a, b, 1994; Chaudhuri et al., 1987b). The increase in the activities of different ion-dependent ATPases in the silk gland and gonads is presumably due to an increased synthesis of these enzymes which may be a reflection of their action of T 4 primarily at the nuclear level. Binding of ~25I-T3 in 200

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the rat liver nuclear receptor and a concomitant rise in Na ÷- K+-ATPase activity show a nuclear activation of T 3 for the stimulation of Na ÷- K+-ATPase (Ismail-Beigi and Edelman, 1974). Again, increased incorporation of [3H]- or [35S]-methionine in the 0~-subunit of the Na +K+-ATPase of the kidney and a higher amount of 3H-ouabain binding in the Na ÷- K+-ATPase in the liver, muscle and kidney of the rat after administration of thyroid hormone (T3) suggests increased synthesis or decreased degradation of Na ÷- K÷-ATPase in these organs (Lo and Edelman, 1976; Lin and Akera, 1978; Lo and Lo, 1980). Thyroxine-induced alteration in silkworm Na ÷- K+-ATPase activity may be due to increased T4-induced active Na ÷ or K ÷ transport across the cell membrane as reported in rat and fish liver (Guerensy and Edelman, 1983; De et al., 1986), and thereby activation of more Na+-pump units which utilize energy

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F I G U R E 5. Effect of thyroxine (T4) on N a +- K+-ATPase in the testes of B. mori. (Each column in the figure indicates mean -I- SE (vertical bar) *P < 0.05; **P < 0.02; ***P < 0.01; ****P < 0.001 compared to control.)

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FIGURE 6. Effect of thyroxine (T4) on Mg2+-ATPase in the testes ofB. mori. (Each column in the figure indicates mean + SE (vertical bar) *P < 0.05; **P < 0.02; ***P < 0.01; ****P < 0.001 compared to control.)

from ATP to form ADP and Pi with the help of Na +K+-ATPase. Mg2+-dependent ATPase enzymes perform a variety of functions like ATP hydrolysis, muscular contraction and transport of ions across the cell and mitochondrial membranes in higher animals (Lehninger, 1986). It has been reported that Ca z+- and Mg2+-ATPase activities were enhanced after administration of thyroid hormone in rat and guinea pig liver (Tobin et al., 1979a, b). In our studies, T 4 induced increased MgZ+-ATPase activity in all of the tissues, and this may be due to increased Mg2+-ion transport inside the cells. The present investigation suggests the synthesis of specific enzyme proteins of silk glands and gonads are enhanced by thyroxine treatment in B. mori. However, the availability of co-factors and substrate in cells during treatment may affect the enzyme activity, apart from its synthesis, stability and degradation. It appears, therefore, that some silkworm tissues are thyroid hormone target organs and the ion-dependent ATPase activity of the silk gland and gonads may be used as a thyroid-hormone-sensitive parameter in B. mori to demonstrate the physiological expression of T 4 action in insects.

REFERENCES

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Acknowledgements--The authors are thankful to the Director, IICT for providing facilities and Mr M. Thandapani, Computer Programmer, CTR & TI, Ranchi for computing the data.