1082 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 1079–1089
microtube containing 40 µ
l of 3X SDS gel sample buffer, heated to 100
° C for 5 min and subjected to SDS–
PAGE and autoradiography. 2.8. Two-dimensional PAGE 2D-PAGE analysis of
multiple S6 phosphorylation 2D-PAGE, as described previously Song and Gilbert,
1995, was used to analyze the multiple-site phosphoryl- ation of S6 stimulated by rPTTH. For purification of the
80S ribosomal proteins for 2D-PAGE analysis, 40 glands at a time were prelabeled for 1 h in 0.8 ml of fresh phos-
phate-free Grace’s medium to which carrier-free [
32
P]O
4
100 µ
Ci0.8 ml was added, followed by rPTTH challenge 0.25 nggland for 1 h. A total of 120 labeled
glands were collected for ribosomal protein purification as described previously Song and Gilbert, 1995. The
purified 80S ribosomal proteins were subjected to 2D- PAGE and autoradiography.
3. Results and discussion
The Results and discussion sections have been com- bined to facilitate comparison of the responses of the
Manduca prothoracic glands to brain extracts containing
PTTH and rPTTH. The physiological interpretation of these data is not fully reiterated here since it has been
discussed in depth in the original publications cited that utilized brain extract or semi-purified brain extract.
Thus, for each series of experiments, we determine if the brain extract and rPTTH elicit identical, similar or
different responses using a variety of critical and repro- ducible assays.
3.1. In vitro assay of rPTTH activity To investigate if rPTTH can stimulate ecdysteroidog-
enesis in the prothoracic glands in vitro as does brain extract, individual glands from V
6
larvae were prepared and incubated for 1 h in 50
µ l of Grace’s medium con-
taining the indicated doses of rPTTH or brain extract. The medium was then collected at the end of the incu-
bation period and the ecdysteroid content quantified by RIA. The data revealed an ED
50
effective dose yielding a 50 positive effect for V
6
glands stimulated by rPTTH of approximately 0.125 nggland Fig. 1. At a
dose of 0.25 nggland, rPTTH elicited a maximum five- fold increase in ecdysteroid production by the stimulated
glands when compared with control glands, a result simi- lar to that of glands stimulated by brain extract
0.25 brain equivalentgland Fig. 1. It should be noted that at the dose of 1 brain equivalentgland which is four
times the dose necessary for maximum stimulation, brain extract showed an inhibitory effect on ecdystero-
idogenesis in the V
6
gland Fig. 1 while rPTTH showed
Fig. 1. Ecdysteroidogenesis by V
6
prothoracic glands in response to varying doses of rPTTH or brain extract. The data represent the
mean ±
standard error of the mean SEM; N =
10 glandsdose. The amount of ecdysteroids produced by matched-pair control glands has
been subtracted from the data.
no inhibitory effect on ecdysteroidogenesis at a similar dose i.e., four times that necessary for maximum
stimulation. A time-course study, using the predetermined dose of
rPTTH 0.25 nggland and brain extract 0.25 brain equivalentgland, was then conducted with V
6
glands. rPTTH and brain extract showed a similar stimulatory
effect Fig. 2. A significant increase in ecdysteroid syn- thesis was observed 15 min after stimulation by both
rPTTH and brain extract. The rate of ecdysteroidogen- esis increased rapidly at 30 min and 1 h. The medium
reached a peak accumulation at 2 h, before decreasing slightly at 3 h.
To test the effect of rPTTH on ecdysteroidogenesis by glands from different developmental stages, glands from
Fig. 2. Time course of rPTTH- or brain-extract-stimulated ecdystero-
idogenesis by V
6
prothoracic glands. The data represent the mean
± SEM; N
= 10 glandstreatment. The amount of ecdysteroids pro-
duced by matched-pair control glands has been subtracted from the data.
1083 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 1079–1089
V
3
, V
6
and P animals were prepared and challenged for
1 h with rPTTH 0.25 nggland. The data revealed that rPTTH induced an increase of approximately three times
basal ecdysteroid production by the glands from V
3
and P
animals and a fourfold increase by V
6
glands Fig. 3. Thus, rPTTH and brain extract elicited similar, if not
identical, stimulatory effects on the prothoracic gland in vitro in terms of dose response, time course and develop-
mental studies. The maximum activation ratio for ecdys- teroid biosynthesis by rPTTH-stimulated glands versus
control non-stimulated glands was approximately four to six times at all stages examined Fig. 3, as obtained
also with brain extract. The inhibitory effect on ecdys- teroidogenesis in glands stimulated by a high dose of
brain extract 1 brain equivalentgland, about four times that necessary for maximum stimulation is in contrast
to our analogous studies with rPTTH that show no inhi- bition at the equivalent dose. This suggests the presence
of an inhibitory factor in the brain extract as was found for Bombyx mori by Hua et al. 1999, who suggested
that such a factor is a PTTH antagonist.
Using the in vivo assay of Gibbs and Riddiford 1977, the rPTTH stimulation of ecdysteroidogenesis
was also basically identical to brain extract stimulation M. Shionoya, H. Matsubayashi, S. Nagata, H. Kuni-
yoshi, M. Asahina, L.M. Riddiford and H. Kataoka, in preparation. Knowing that the effects of PTTH and
brain extract on ecdysteroidogenesis both in vivo and in vitro are identical, we proceeded to examine the action
of rPTTH on other parameters such as cAMP levels, general and specific protein synthesis, etc., since the
original observations utilized crude and semi-pure prep- arations of PTTH.
Fig. 3. rPTTH-stimulated ecdysteroidogenesis by the prothoracic
glands in vitro from V
3
, V
6
and P animals. The data represent the
mean ±
SEM; N =
10 glandsgroup.
3.2. Effects of PTTH on cAMP levels Native forms of Manduca PTTH preparations stimu-
late cAMP synthesis prior to enhancing ecdysteroid syn- thesis and secretion Smith et al., 1984. To determine
whether rPTTH has a similar effect on glandular cAMP, prothoracic glands were removed from V
3
larvae or P pupae, and challenged with a maximally effective dose
of rPTTH 1.6 nM for 5 min. As shown in Fig. 4, rPTTH alone causes a significant increase in cAMP syn-
thesis in larval prothoracic glands. In pupal glands, cAMP synthesis cannot be detected unless cAMP break-
down is concomitantly inhibited with IBMX isobutyl methyl xanthine Fig. 4, stippled bar. Even in the pres-
ence of IBMX, cAMP synthesis is stimulated more strongly in larval than in pupal glands.
The stimulation of cAMP synthesis by rPTTH in M. sexta
prothoracic glands is similar, if not identical, to that previously observed for brain extract and semi-pure
native PTTH big or small Smith et al. 1984, 1986; Smith and Pasquarello, 1989; Watson et al., 1993. As
would be expected for a relevant second messenger, cAMP levels increase within 5 min, with peak levels pre-
ceding a detectable increase in ecdysteroid secretion Smith et al., 1984. Cyclic AMP phosphodiesterase lev-
els in M. sexta prothoracic glands are elevated at the end of the fifth larval stage and beginning of the pupal stage.
Thus, in order to measure PTTH-stimulated changes in cAMP synthesis in early pupal glands, a phosphodiester-
ase inhibitor IBMX must be added. Similarly, both crude and recombinant B. mori PTTH stimulate cAMP
synthesis, and this effect is seen most readily in the pres- ence of IBMX Gu et al. 1996, 1998; Dedos et al., 1999.
Mean levels of cAMP synthesis stimulated by rPTTH
Fig. 4. rPTTH-stimulated synthesis of cAMP by prothoracic glands
in vitro. Glands were removed from day 3 fifth-stage larvae or day 0 pupae and incubated individually for 5 min in 0.03 ml of Grace’s
medium containing: no hormone black bars or 1.6 nM rPTTH white bars. Additional pupal glands were incubated in 0.1 mM IBMX
slashed bar or 0.1 mM IBMX +
1.6 nM rPTTH stippled bar. Secreted ecdysone was measured by radioimmunoassay. Each group represents
the mean ±
SEM of nine to 16 glands.
1084 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 1079–1089
in larval glands 10 pmol are less than the mean levels elicited by big PTTH 20–30 pmol Smith et al., 1984;
Smith and Pasquarello, 1989. The native form of the hormone may stimulate cAMP production more effec-
tively than the recombinant hormone as a result of yet unidentified post-translational modifications such as gly-
cosylation. Alternatively, additional factors present in PTTH preparations used in earlier studies may have aug-
mented cAMP synthesis independently of PTTH.
3.3. Effects of rPTTH in calcium-free saline Ecdysteroid secretion stimulated by rPTTH clearly
depends upon the presence of external Ca
2 +
. As seen in Fig.
5, rPTTH
does not
stimulate ecdysteroid
synthesissecretion in Ca
2 +
-free saline, although basal rates are unaffected. Overall rates of ecdysteroid syn-
thesis are lower in saline than in Grace’s medium. How- ever, the results are otherwise identical to previous find-
ings in which PTTH-stimulated ecdysteroid secretion was strongly dependent upon extracellular calcium
Smith et al., 1985; Girgenrath and Smith, 1996. See also data on M. sexta small PTTH Hayes et al., 1995,
G. mellonella
PTTH Birkenbeil, 1996 and B. mori recombinant PTTH Gu et al., 1998; Dedos et al., 1999.
An increase in intracellular calcium has been measured in the prothoracic glands of Manduca in response to
crude PTTH Birkenbeil, 1998. The prothoracic glands of Manduca possess a Ca
2 +
-sensitive adenylyl cyclase, and external calcium is required in order for PTTH to
stimulate cAMP synthesis Smith et al., 1984; Meller et al., 1988. Calcium dependence of cAMP synthesis is
also seen for B. mori in response to the recombinant form of PTTH for this species Gu et al., 1998; Dedos
et al., 1999.
Fig. 5. Effects of rPTTH on ecdysteroid secretion in calcium-free
medium. Individual prothoracic glands were incubated for 90 min in calcium-free saline black bar, calcium-free saline containing 1.6 nM
rPTTH white bar, saline containing 1 mM calcium slashed bar, or saline containing 1 mM calcium and 1.6 nM rPTTH stippled bar. The
medium was then assayed for ecdysone by RIA. Each group represents the mean
± SEM of six glands.
3.4. Effects of rPTTH on general and specific protein synthesis
The data revealed that rPTTH stimulated a progressive increase in protein synthesis as measured by TCA-pre-
cipitable radioactivity Fig. 6. In addition, SDS–PAGE indicated that rPTTH induced a temporally varying pat-
tern of changes in synthesis andor turnover of specific proteins including
β -tubulin and heat shock cognate 70
HSC protein Figs. 7 and 8. Thus, the rPTTH stimu- lates rapid increases in general and specific protein syn-
thesis in the prothoracic gland. Analysis of newly syn- thesized, radiolabeled proteins via SDS–PAGE and
autoradiography indicates that the pattern of increases in specific translation andor protein accumulation matches
that observed using brain extract and dibutyryl cAMP at several time points during the fifth instar and early
pupal–adult development Rybczynski and Gilbert, 1994; Rybczynski and Gilbert, 1995a,b; see also Gilbert
et al., 1997. Protein synthesis using Ca
2 +
ionophore has not been explored sufficiently at multiple developmental
time points to make a comparison, although the data from early fifth instar prothoracic glands match the
rPTTH observations. Previous work indicated that PTTH-containing brain extracts stimulated the overall
rate of translation in prothoracic glands in a way that was independent of ecdysteroid synthesis, i.e., that
PTTH could function as a “growth factor” Rybczynski and Gilbert, 1994. Thus, the results obtained with
rPTTH confirm that this cytokine-like activity resides in the PTTH molecule and not in another, unidentified pro-
Fig. 6. Time course of rPTTH-stimulated protein synthesis, as meas-
ured by TCA-precipitated,
35
S-methionine-labeled proteins see Ryb- czynski and Gilbert, 1994.
1085 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 1079–1089
Fig. 7. The stimulation of the synthesis of specific prothoracic gland proteins by rPTTH and visualization by SDS–PAGE of
35
S-methionine- labeled proteins. Newly synthesized prothoracic gland proteins were labeled with
35
S-methionine in the presence or absence of rPTTH during in vitro incubations, as described previously Rybczynski and Gilbert, 1994. The amount of protein loaded per gel lane was adjusted to equalize the
amount of
35
S-methionine per lane, as determined in TCA precipitations.
Fig. 8. Time course of rPTTH-stimulated
β -tubulin and HSC 70 syn-
thesis. Following SDS–PAGE and autoradiography, the intensity of the band representing radiolabeled
β -tubulin and HSC 70 was quantified
via scanning as described by Rybczynski and Gilbert 1994. The amount of protein loaded per gel lane was adjusted to equalize the
amount of
35
S-methionine per lane, as determined in TCA precipi- tations.
tein found in the brain extracts. These data are consistent with the hypothesis that rPTTH has multiple effects on
the cell biology of the prothoracic glands in addition to stimulating ecdysteroidogenesis as does brain extract
Rybczynski and Gilbert, 1994. Additional functions could include control of the growth and maintenance of
prothoracic gland cells or modulation of the feedback control of ecdysteroid synthesis.
3.5. Effect of monoclonal antibody to rPTTH on PTTH stimulation of ecdysteroidogenesis by the
prothoracic gland
When monoclonal antibody 3H3 was added to the brain extract–prothoracic gland in vitro assay system, the
activation ratio for paired glands averaged 0.85 ±
0.16, indicating that the 3H3 antibody inhibited the normal
ecdysteroidogenic activity present in the brain extract expected A
r
for 0.5 brain equivalents: 4.0–6.0 +
. The molar ratio of antibody to PTTH was 400, based on
the molecular weights of IgG 150,000 and PTTH 30,000 as a native homodimer; see Rybczynski et al.,
1996 and assuming 1 ng PTTH per brain equivalent. These data suggest strongly that the natural PTTH binds
to an antibody generated against rPTTH and therefore that the rPTTH is equivalent to the single natural PTTH
of Manduca in these extracts.
In contrast, previous work with a monoclonal antibody against Bombyx PTTH revealed binding to Manduca
PTTH in such extracts but not in a way that prevented Manduca
PTTH from activating ecdysteroidogenesis Rybczynski et al., 1996. These recent data also provide
1086 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 1079–1089
evidence that in P
1
brains, there is only one significant, active PTTH species; i.e., the anti-PTTH antibody was
able to completely block brain-extract-elicited ecdys- teroidogenesis.
Thus far, the effects of rPTTH were identical to those elicited by brain extract, but we wished to identify the
cells synthesizing this rPTTH by immunocytochemistry. 3.6. Immunocytochemistry
Fig. 9 shows the results of immunocytochemical analysis using rPTTH polyclonal antibodies. These data
are basically identical to those obtained using mono- clonal antibodies mAbs generated against partially pur-
ified Manduca PTTH O’Brien et al., 1988 and against a synthetic Bombyx PTTH fragment on Manduca Br–
CC–CA complexes Dai et al., 1994. That is, the anti- bodies stained only two pairs of lateral neurosecretory
cells of the brain the prothoracicotropes in both stages examined, and the pathway of the axons was the same
as shown in the previous studies in which other PTTH antibodies were used. The axons originating from the
lateral neurosecretory cells are fasciculated, traverse the midline of the brain to enter the contralateral lobe, pro-
ceed posteriorly toward the retrocerebral nerve, and reach the corpora allata neurohemal organs where they
form arborized endings. Between the soma and the mid-
Fig. 9. Immunocytochemical detection of rPTTH in the brain and corpus allatum. a Whole mount of the pupal P
Manduca brain showing
staining for PTTH in cell bodies and axons of prothoracicotropes. b Whole mount of arborized axons in the P corpus allatum PTTH neurohemal
organ. c Whole mount of cell bodies and axons of prothoracicotropes in the V
3
larval brain. d Section of V
3
cell bodies and dendrites arborized axons of prothoracicotropes located at the periphery of the neuropile. Scale bars
= 100
µ m.
line of the brain, many collaterals branching from the axons were observed see whole mount stain and they
were located along the periphery of the neurophile see section stain. In contrast to another report using a differ-
ent mAb Westbrook et al., 1993, no other cells were stained with our antibody.
Thus, the PTTH peptide was localized to only the lat- eral cells identified by Agui et al. 1979 as the protho-
racicotropes and in the corpus allatum identified pre- viously as the neurohemal organ for PTTH Agui et
al., 1980.
Whole mount in situ hybridization of day 1, pupal brains with a 1 kb Manduca PTTH cDNA probe labeled
with digoxigenin showed that the PTTH RNA was present only in the two lateral neurosecretory cells in
each protocerebrum M. Shionoya, H. Matsubayashi, S. Nagata, H. Kuniyoshi, M. Asahina, L.M. Riddiford and
H. Kataoka, in preparation. These cells are likely the same cells identified with the rPTTH antibody and are
in the same position as the pairs of cells found to be biologically active in stimulating ecdysteroid biosynth-
esis by the prothoracic glands Agui et al., 1979.
3.7. rPTTH-stimulated phosphorylation of S6 What appears to be the terminal step in the transduc-
tory cascade leading to increased ecdysteroidogenesis in
1087 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 1079–1089
Fig. 10. Time course of rPTTH-stimulated a or brain-extract-stimulated b S6 phosphorylation in the prothoracic glands of V
6
Manduca larvae.
The phosphoproteins were separated by 12.5 SDS–PAGE and subjected to autoradiography. The arrowheads indicate the position of the phos- phorylated S6 protein.
the prothoracic gland of Manduca is the multiple-site phosphorylation of S6, an important regulatory protein
of the 40S subunit of the ribosome Song and Gilbert 1994, 1995. To investigate the effect of rPTTH on pro-
tein phosphorylation, prothoracic glands from V
6
larvae were prelabeled with [
32
P]O
4
for 1 h, followed by rPTTH stimulation for 1 h, and the phosphoproteins were then
subjected to SDS–PAGE and autoradiography. Fig. 10 shows that similar protein phosphorylation patterns are
observed with both rPTTH- and brain-extract-stimulated glands. S6 phosphorylation was observed 15 min after
glands were challenged by rPTTH and by brain extract Fig. 11, and reached the maximum level at 1 h in both
cases. Dephosphorylation of S6 in rPTTH-stimulated glands began at 2 h after stimulation and S6 phosphoryl-
ation returned to its basal level at 3 h after stimulation Fig. 10. However, S6 dephosphorylation in brain-
Fig. 11. rPTTH- or brain-extract-stimulated S6 phosphorylation in
the prothoracic glands of V
3
, V
6
and P animals. The phosphoproteins
were separated by 15 SDS–PAGE and subjected to autoradiography. The arrowheads indicate the position of phosphorylated S6 proteins.
extract-stimulated glands did not start until 3 h after stimulation and the rate of S6 phosphorylation returned
to its basal level by 4 h. The rapid dephosphorylation of S6 in rPTTH-stimulated glands may be due to the lability
of pure rPTTH in Grace’s medium. It should also be noted that the phosphorylation of a yet uncharacterized
25 kDa protein was enhanced at 2 h, reaching a maximum level at 3 h for rPTTH-stimulated glands and
at 4 h for brain extract-stimulated glands Fig. 11. S6 phosphorylation patterns resulting from stimulation by
rPTTH in glands from V
3
, V
6
and P animals were simi-
lar to those from brain extract stimulation Fig. 11. 2D-PAGE analysis revealed that all five phosphoryl-
ation sites of S6 characterized previously were occupied in rPTTH-stimulated glands as shown in a Commassie-
blue-stained gel map [Fig. 12b] and confirmed by a corresponding autoradiograph [Fig. 12d]. Most of the
phosphorylated S6 was in the P
4–5
phosphorylation state, a result similar to that found with brain-extract-stimu-
lated glands Song and Gilbert, 1995. In contrast, only a trace amount of S6 phosphorylation was detected in
control glands as evidenced in both Commassie-blue staining [Fig. 12a] and the corresponding autoradio-
graph [Fig. 12c]. The S6 in control samples was radiol- abeled predominately in the P
1
state, indicating that a basal level of S6 phosphorylation exists. Thus, all the
data concerning the phosphorylation of S6 are consistent with the premise that the rPTTH is indeed the natural
PTTH in the Manduca sexta brain, and that it is the PTTH in brain extracts that was the principal active
agent in previous research on the subject from our lab- oratories.
1088 L.I. Gilbert et al. Insect Biochemistry and Molecular Biology 30 2000 1079–1089
Fig. 12. 2D-PAGE mini-gel maps revealing the degree of S6 phosphorylation in ribosomal proteins from control a, c and rPTTH-stimulated b,
d prothoracic glands. Panels a and b show Coomassie-blue staining. c and d are autoradiographs corresponding to a and b, respectively. The numbers indicate the phosphorylation states of the S6 protein.
4. Conclusions
