Introduction Directory UMM :Data Elmu:jurnal:I:Insect Biochemistry and Molecular Biology:Vol30.Issue5.2000:

Insect Biochemistry and Molecular Biology 30 2000 377–386 www.elsevier.comlocateibmb Characterization and developmental regulation of tyramine- β - hydroxylase in the CNS of the moth, Manduca sexta Herman K. Lehman a, , Cristina M. Murgiuc b , John G. Hildebrand b a Department of Biological Sciences, Hamilton College, Clinton, NY 13323, USA b Arizona Research Laboratories, Division of Neurobiology, University of Arizona, Tucson, AZ 85721-0077, USA Abstract Octopamine OA is present in insect nervous tissue, but little is known about its biosynthesis. In the CNS of Manduca sexta, OA levels increase markedly during postembryonic adult development. To study this increase, we developed an assay for tyramine- β -hydroxylase, the putatively rate-limiting enzyme for OA biosynthesis. Tyramine- β -hydroxylase activity in extracts of M. sexta CNS tissue: 1 was time- and protein-dependent, and with protein concentrations up to 2 µ g µ l, was linear for 20 min; 2 had a pH optimum of 7.0 for conversion of tyramine to OA; 3 required ascorbate, copper, and catalase; and 4 had an apparent K M, tyramine of 0.22 ± 0.04 mM. These characteristics resemble those of the mammalian enzyme dopamine- β -hydroxylase, suggesting that these two enzymes are functionally related. During adult development, tyramine- β -hydroxylase activity increased 11-fold in the brain and 9-fold in the abdominal ganglia, paralleling increases in OA levels in those CNS structures during metamorphosis. The apparent kinetic constants of tyramine- β -hydroxylase suggested that the amount of this enzyme present in the tissues increases. The increase in OA levels during adult development thus appears to be due to an increase in the level of enzyme available for OA synthesis and may reflect an increase in the number of octopaminergic neurons.  2000 Published by Elsevier Science Ltd. All rights reserved. Keywords: Insect; Biogenic amines; Tyramine; Neurotransmitter synthesis; Tyramine- β -hydroxylase; Dopamine- β -hydroxylase

1. Introduction

Neuromodulation of CNS function plays fundamental roles in the behavior of insects and other animals. Octo- pamine OA is one of the most abundant neuromodula- tors in the arthropod nervous system, and its high levels reflect a wide distribution and broad spectrum of actions Bodnaryk, 1980; Na¨ssel and Laxmyr, 1983; Evans, 1985; Davenport and Wright, 1986; Sloley and Okikaza, 1988; Nagao and Tanimura, 1988; Linn and Roelofs, 1993. In insects, OA has been shown to be involved in various peripheral functions, including control of the heart and visceral muscle contractions, control of phero- Abbreviations: D β H, dopamine- β -hydroxylase; DUM, dorsal unpaired median; ETH, ecdysis-triggering hormone; OA, octopamine; PNMT, phenylethanolamine-N-methyl transferase; SAM, S-adenosyl-l-meth- ionine; T β H, tyrosine- β -hydroxylase; TES, N-tris[hydroxymethyl]me- thyl-2-aminoethanesulfonic acid; VUM, ventral unpaired midline. Corresponding author. Tel.: + 1-315-859-4298; fax: + 1-315-859- 4807. E-mail address: hlehmanhamilton.edu H.K. Lehman 0965-174800 - see front matter  2000 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 0 0 0 0 0 1 1 - 4 mone biosynthesis and perception, modulation of flight- muscle activity, and control of the release of neurohor- mones reviewed by: David and Coulon, 1985; Evans, 1985; Orchard et al., 1993; Homberg, 1994. The actions of OA within the CNS are similarly diverse, and this amine has been implicated in such functions as olfactory learning and dishabituation Dudai et al., 1987; Bacon et al., 1995. The levels of OA in the CNS are not constant, however, but increase during metamorphosis Bodnaryk, 1980. In holometabolous insects, this increase in the level of OA may be related to the increased behavioral activity that is characteristic of adult insects. Postembryonic remodeling of the insect nervous sys- tem during pupal stages of development is a remarkable phenomenon that includes neurogenesis, apoptosis, mor- phogenic movements, changes in neuron–glial interac- tions, and structural and functional modifications of per- sistent neurons reviewed by Levine et al., 1995. Although many of these aspects of postembryonic devel- opment have been used as models for studies of basic developmental mechanisms, little is known about the mechanisms responsible for the long-term changes in the 378 H.K. Lehman et al. Insect Biochemistry and Molecular Biology 30 2000 377–386 levels of neurotransmitters such as OA. In order to understand the mechanisms controlling the levels of this amine, we have investigated its biosynthesis in the sphinx moth, Manduca sexta. OA is biosynthesized from tyrosine in the insect ner- vous system, and the studies of Livingstone and Temple 1983 suggested that OA biosynthesis requires two enzymatic steps: decarboxylation of tyrosine by tyrosine decarboxylase to form tyramine, followed by hydroxyl- ation of tyramine by tyramine- β -hydroxylase T β H to produce OA. Little information is available about either of these enzymes in insects. Tyrosine decarboxylase activity has not been characterized or identified in any arthropod, but it appears to be distinct from DOPA decarboxylase, a well-studied enzyme required for the conversion of l-DOPA to dopamine in the insect ner- vous system and cuticle Livingstone and Temple, 1983; Hirsh, 1989. In the only biochemical characterization of T β H, Wallace 1976 showed that extracts of nervous tissue from the American lobster, Homarus americanus, can hydroxlyate tyramine to produce OA, and that this conversion is dependent upon pH, ascorbic acid, copper, and catalase. These properties suggested that T β H is similar to dopamine- β -hydroxylase D β H, which in mammals is required for the hydroxylation of dopamine to form norepinephrine. Indeed, Wallace 1976 demon- strated that purified lobster T β H can produce norepi- nephrine via the β -hydroxlyation of dopamine. More- over, D β H exhibits relatively broad substrate specificity and can hydroxylate tyramine to form OA Creveling et al., 1962; Goldstein and Contrera, 1962; reviewed by Kaufman and Friedman, 1965. These results indicate that lobster T β H is functionally related to mammalian D β H. In this study we focused on T β H because it is the last and putatively rate-limiting enzyme in the biosynthetic pathway leading to OA. We document that, as in Mames- tra configurata Bodnaryk, 1980, levels of OA increase during metamorphosis in M. sexta. To determine if this OA increase is associated with an increased rate of syn- thesis, we characterized T β H activity in extracts of M. sexta CNS tissue and showed that during metamor- phosis, there is a direct correlation between increased levels of T β H activity and levels of OA. Based upon apparent T β H kinetic constants measured in extracts of late-larval and adult CNS tissue, we conclude that increased levels of T β H protein account for the increase in T β H activity during metamorphosis. The possible cellular and molecular mechanisms that may be respon- sible for a long-term increase in T β H are discussed.

2. Materials and methods