Insect Biochemistry and Molecular Biology 30 2000 1079–1089 www.elsevier.comlocateibmb
Dynamic regulation of prothoracic gland ecdysteroidogenesis: Manduca sexta
recombinant prothoracicotropic hormone and brain extracts have identical effects
L.I. Gilbert
a,
, R. Rybczynski
a
, Q. Song
b
, A. Mizoguchi
c
, R. Morreale
d
, W.A. Smith
d
, H. Matubayashi
e
, M. Shionoya
e
, S. Nagata
e
, H. Kataoka
e
a
Department of Biology, Campus Box 3280, Coker Hall, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA
b
Department of Entomology, University of Missouri, Columbia, MO 65211, USA
c
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
d
Department of Biology, Mugar Hall, Northeastern University, Boston, MA 02115, USA
e
Department of Biotechnology, Graduate School of Agricultural and Life Science, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan
Received 5 January 2000; received in revised form 15 March 2000; accepted 29 March 2000
Abstract
Multiple assays were conducted in order to determine if the recently available recombinant prothoracicotropic hormone rPTTH from Manduca sexta is identical, or similar, to the natural hormone and if results from its use in a variety of assays confirm, or
are inconsistent with, previous studies over the past 20 years on PTTH action using brain extract. Brain extracts and rPTTH showed similar, if not identical, effects on the cell biology of Manduca prothoracic gland cells with the following results: increased levels of
cAMP adenosine 39:59 cyclic monophosphate synthesis; requirement for extracellular Ca
2 +
in in vitro studies; ecdysteroidogenesis stimulation in vitro; stimulation of general and specific protein synthesis; immunocytochemical identification of the two lateral cells
in each brain hemisphere as the source of PTTH the prothoracicotropes; the ability of antibodies to rPTTH to inhibit ecdysteroido- genesis stimulation in vitro; and the multiple phosphorylation of the ribosomal protein S6. The data revealed that brain extract and
rPTTH show equivalent effects in all of the assays, indicating that this rPTTH is the natural PTTH of Manduca and that the data generated with brain extracts over the past two decades are indeed relevant.
2000 Elsevier Science Ltd. All rights reserved.
Keywords: Ecdysteroidogenesis, control of; S6 phosphorylation; Cyclic AMP, stimulation of; Molting, control of; Brain hormone
1. Introduction
Research on the mechanisms by which protho- racicotropic hormone PTTH stimulates ecdysteroidog-
enesis began 27 years ago when Vedeckis and his col- leagues demonstrated a correlation between prothoracic
gland cAMP titer and ecdysteroid biosynthesis in lepi- dopteran prothoracic glands Vedeckis et al. 1974,
1976. A critical event for these and further studies was the development of the ecdysteroid radioimmunoassay
Borst and O’Connor, 1972. The research of Gibbs and
Corresponding author. Tel.: +
1-919-966-2055; fax: +
1-919-962- 1344.
E-mail address: lgilbertunc.edu L.I. Gilbert.
0965-174800 - see front matter
2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 0 0 0 0 0 8 3 - 7
Riddiford 1977 showed that brain extracts could affect ecdysteroidogenesis in vivo, and that lateral regions of
the brain possessed the PTTH activity. The second criti- cal event was the development of an in vitro assay that
took advantage of the finding that each of the two pro- thoracic glands in Manduca sexta synthesized and
secreted ecdysteroid at the same rate. Thus, a quantitat- ive activation ratio A
r
could be calculated and the mechanism of PTTH action could be investigated in a
rigorous manner Bollenbacher et al., 1979. This led to the identification of the site of PTTH synthesis as two
lateral cells in each brain hemisphere Agui et al., 1979. The in vitro assay was, therefore, the basis for studies
investigating the control of ecdysteroidogenesis.
The third critical event was the demonstration that PTTH acts through a transductory cascade which
1080 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 1079–1089
included a requirement for extracellular Ca
2 +
Smith et al., 1985, increased activity of an adenylate cyclase
Smith et al., 1984 now known to be a Ca
2 +
-calmodulin activated adenylate cyclase Meller et al. 1988, 1990,
and the subsequent activation of the cAMP protein kin- ase PKA Smith et al., 1986; see review by Gilbert et
al., 1988. Further studies revealed that the final step in the cascade ended with the multiple phosphorylation of
the ribosomal protein S6 mediated by PKA and S6 kin- ase Song and Gilbert, 1995; see review by Henrich et
al., 1999.
The fourth critical event was the availability of Mand- uca
recombinant PTTH rPTTH produced by the Kataoka laboratory M. Shionoya, H. Matsubayashi, S.
Nagata, H. Kuniyoshi, M. Asahina, L.M. Riddiford and H. Kataoka, in preparation. This was of great impor-
tance for obvious reasons. In addition to being a probe for the study of the PTTH receptor, the utilization of
rPTTH in the in vitro system could establish whether it was PTTH or one or more other molecules in brain
extracts that elicited the transductory cascade and increased ecdysteroidogenesis in the prothoracic gland.
This was important in order to establish, or cast doubt on, the conclusion that PTTH is the active component
in the crude and semi-pure brain extracts utilized in the studies summarized above. Indeed, the use of crude
extracts was viewed quite negatively by at least one granting agency and even led to negative reviews of
manuscripts. Thus, the availability of Manduca rPTTH was vital to corroborate or not 20 years of research on
PTTH action as well as for future studies. For these reasons we decided to compare the action of rPTTH and
brain extracts using a variety of critical parameters, and present the data here.
2. Materials and methods
