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A kinetic analysis of the action of the insect prothoracicotropic hormone
21
Molecular and Cellular Endocrinology, 32 (1983) 27-46 Elsevier Scientific Publishers Ireland, Ltd.
MCE 01036
A KINETIC ANALYSIS OF THE ACTION OF THE INSECT PROTHORACICOTROPIC HORMONE Walter
E. BOLLENBACHER
KATAHIRA
Department of Biology, 27514 (U.S.A.) Received
*, Martha
and Lawrence
30 March
Wilson Hall 046A,
1983; accepted
A. O’BRIEN,
Eva J.
I. Gilbert
29 April
University
of North Carolina,
Chapel Hill, NC
1983
A modified in vitro assay was used to assess the kinetics of activation and the decay of activation of the prothoracic glands (PG) by the prothoracicotropic hormone (PTTH) in Manduca sexta. Time-courses of ecdysone synthesis by PTTH-activated day 3 larval and day 0 pupal PG were comparable both quantitatively and temporally, but dose-responses of PTTH activation revealed that larval glands were 1.8 times more sensitive to the neurohormone. The exposure time necessary for maximal activation of the PG by PTTH was the same for both glands, with half-maximal activation in - 0.5 min. Once PTTH was removed the rate of ecdysone synthesis by larval and pupal PG remained constant for about 2 h, after which the activated response for both glands decayed rapidly. reaching the unactivated basal synthesis rate within 45 min. These kinetics data suggest that PG activation by PTTH in vitro occurs in a mailner indicative of activation in situ and, thus, that this in vitro system is suitable for probing the molecular mechanism by which PTTH activates the PG. Keywords:
prothoracic gland; tropic hormone.
Manduca
sexta;
ecdysone
synthesis:
prothoracico-
Molting in insects occurs in response to a cascade of neuroendocrine and endocrine events that originate in the brain with the release of the prothoracicotropic hormone (PITH) and culminate in the response of target tissue to 20-hydroxyecdysone, the molting hormone (see Granger and Bollenbacher, 1981). Although this process is understood in general terms, the exact biochemical mechanisms involved have not yet been defined. This is particularly true for one of the primary events in the molting process, activation of the prothoracic glands (PG) by PITH which leads to synthesis and secretion of the hormone, ecdysone. Measurements
of CAMP levels and PG activity
* To whom all correspondence
0303-7207/83/$03.00
have suggested
should be addressed.
0 1983 Elsevier Scientific
Publishers
Ireland,
Ltd.
that
28
W. E. Bollenbacher
et al.
PTTH activation of the PG occurs via CAMP-dependent and CAMP-independent mechanisms in response to the gated release of the neurohormone at specific stages of development (Vedeckis et al., 1976) but an unequivocal causal relationship remains to be established. Recently, an in vitro system was developed for monitoring PTTH activation of the PG directly (Bollenbacher et al., 1979, 1980) making it possible to explore PTTH activation of the PG at the biochemical level. However, in order to conduct such studies the kinetics of PG activation and the kinetics of activation decay must first be determined. This report details an investigation of the kinetics of PTTH activation of PG from larvae and pupae of the tobacco hornworm, Manduca sexta, utilizing a modified in vitro assay for this neurohormone. The data are discussed in the context of: possible mechanisms of PTTH action; the relative responsiveness of PG from different stages of development; and the nature of PTTH release during the larval-pupal development of a holometabolous insect.
MATERIALS
AND
METHODS
Animal rearing and donor selection Larvae and pupae of the tobacco hornworm, Munduca sexta, were reared individually on an artificial diet at 25°C under nondiapausing conditions (LD 16 : 8) and staged and gated as described previously (Vince and Gilbert, 1977; Wielgus and Gilbert, 1978). Since the majority of the gate II animals molt at 2 p.m., day 0 of the last larval instar and of the pupal period was set at 2 p.m. on the day of ecdysis. Brains of day 1 pupae were used as the source of a crude preparation of PTTH because the titer of PTTH is highest on day 1, approx. 3 times that of fifth instar larval brains (Bollenbacher and Gilbert, 1981). Donors for larval and pupal PG were chosen at stages of development when glands were presumably competent to respond to PTTH. One such stage during larval development occurs on day 3 of the last instar, since the gated release of PTTH defining the first head critical period of this instar occurs during the night of day 3 (Truman and Riddiford, 1974). Subsequent PG activation, wandering behavior and pupal commitment occur during day 4. Competent pupal PG were obtained from O-12 h pupae, approx. 2 days prior to the beginning of the head critical period for pupal-adult development (Bowen et al., 1983). Organ dissections and extraction procedure for PTTH Pairs of PG from individual animals were dissected in lepidopteran saline (Weevers, 1966) and placed individually in wells of multiwell
Kinetics of prothoracicotropic hormone action
29
culture plates containing Grace’s tissue culture medium. Dissection time for an in vitro assay for PTTH was held to less than 1 h to maximize gland responsiveness. For a crude preparation of PTTH, day 1 pupal brains were placed in Grace’s medium, freed of contaminating tissues, washed and then homogenized in fresh Grace’s medium at a concentration of 2 brains/O.025 ml medium. Immediately after homogenization, brain extracts were heat-treated at 100°C for 2 min to minimize proteolysis of PTTH and centrifuged at 9000 x g for 10 min. Since heat-treated Grace’s medium did not alter PG activity in vitro, the resulting supernatant could be used as the PTTH source for the in vitro assay without further purification. The reproducibility of the extraction protocol and the stability of the PTTH activity in the extract have been documented previously (Bollenbather et al., 1979; Bollenbacher and Gilbert, 1981). In vitro assay for PTTH The assay used to assess the kinetics of PTTH activation of ecdysone synthesis by the PG was essentially that described previously, with the exception that the incubation medium was analyzed for ecdysone directly rather than first extracting the medium with methanol. Therefore, the ecdysone RIA used to quantify synthesis had to be modified to permit assay of the incubation medium. In the modified assay, 0.01 ml aliquots of the 0.025 ml standing drop in which the PG were incubated were subjected to the standard ecdysone RIA protocol (Bollenbacher et al., 1979). An equivalent volume of Grace’s medium (0.01 ml) was added to each tube comprising the ecdysone standard curve to preserve the same assay volume and thus the same competitive binding conditions. The specific activity of the [23,24,3H]ecdysone stock solution used in the RIA was adjusted to 4 Ci/mmole and the concentration of the H-3 antiserum stock solution (Gilbert et al., 1977) to 0.15%; it then bound approx. 40% of the labeled ligand. Ecdysone was used as the competing, unlabeled ligand in the assay, and under these conditions a linear standard curve was obtained with concentrations of ecdysone ranging from 0.25 to 32 ng. Since the conditions of this assay resulted in a standard curve with a greater and broader response range, the modified RIA was termed a ‘macro’ RIA, in contrast to the standard ‘micro’ RIA with a response range of 0.05 to 2.0 ng. Termination and counting of the macro RIA was as previously described for the micro RIA (Borst and O’Connor, 1972), and the data were analyzed with a Wang PCS II computer using a log-logit plot.
30
W. E. Bollenbacher
et al.
Time-course of ecdysone synthesis and dose-response of PG activation by PTTH Time-courses of ecdysone synthesis for larval and pupal PG were obtained using the right glands from a population of animals for determining a basal, control rate of synthesis, and the left glands from this same population for determining the increased rate of synthesis in the presence of PTTH. This approach was employed because the basal activity of each member of a pair of PG is comparable (Bollenbacher et al., 1979). Six precisely staged larvae or pupae comprised a standard test population, and control and experimental PG from each animal were incubated separately in 0.025 ml of Grace’s medium, with and without amounts of PTTH sufficient for maximal activation of the glands (0.5 brain equivalents/assay). At designated times, 0.01 ml aliquots of medium were taken from each incubate with the immediate addition of 0.01 ml of fresh medium, with or without PTTH, to maintain the volume of the incubation at 0.025 ml for the duration of the time-course (6 h). The ecdysone present in each aliquot was quantified by the macro RIA. To generate a dose-response curve of PTTH activation for either larval or pupal PG, activation was determined by comparing the quantities of ecdysone synthesized by an experimental gland to that synthesized by the contralateral control gland for each concentration of PTTH. Activation was then expressed as an activation ratio (A,), which is the quantity of ecdysone synthesized by the experimental gland ( + PTTH) divided by the amount synthesized by the control gland ( - PTTH). The relative responsiveness of different PG to a crude PTTH preparation was assessed by determining the amount of PTTH, expressed in brain equivalents, necessary to obtain maximum activation (A,,,) in a dose-response. The amount of PTTH (ED,,) needed to achieve half-maximal activation (As,,) of the PG was then compared. The ED,, has proven to be an accurate means of quantifying relative PTTH activity in extracts of tissue (Agui et al., 1979, 1980; Bollenbacher and Gilbert, 198 1) and should be equally effective in quantifying the relative sensitivities to PTTH of PC from different developmental stages. For these kinetics studies PTTH activity was expressed in units, a unit being equivalent to the PTTH activity present in a day 1 pupal brain. This value was derived from the reciprocal of the number of day 1 pupal brain equivalents (ED,,) necessary to achieve half-maximal activation of day 0 pupal PG (A,,). For day 1 pupal brains, the ED,, was found to be 0.06 brain equivalents, the reciprocal of which is 16.7. Thus, there is sufficient PTTH in this brain to half-maximally activate 16.7 day 0 pupal PH under the established in vitro assay conditions.
Kitxtics
ofprothoracicotrapic
hormone action
31
Kinetics of activation and deactivation of PG The time necessary for PTTH to maximally activate the PG was determined using a modified left vs. right gland protocol, in which one member of a gland pair was preincubated for a specific period of time in Grace’s medium containing 0.05 units of PTTH and the contralateral gland in Grace’s medium alone. After preincubation, glands were transferred to, and incubated in, equivalent volumes of Grace’s medium (0.025 ml) without PT’lH. At the end of 2 h, a 0.01 ml aliquot of each incubate was assayed for ecdysone synthesis by the macro RIA and an A, was computed. The A, for each time point was the average of the A, values for 6-9 gland pairs. To assess the kinetics of deactivation of the PG, paired glands from larvae or pupae were preincubated separately for 30 min 0.025 ml of Grace’s medium containing a saturating amount of PTTH (0.5 units). After preincubation, one member of the gland pair was transferred to a standing drop of Grace’s medium containing the same concentration of PTTH and the other gland to a drop of Grace’s medium only. Timecourses of ecdysone synthesis were then determined using the previously described time-course protocol, with a total incubation time not exceeding the duration of a linear rate of ecdysone synthesis by the control gland.
RESULTS
The in vitro PG assay developed several years ago for assessing PTTH activity (Bollenbacher et al., 1979) was a substantial improvement over the biological assays previously used to quantify the activity of this neurohormone (see Granger and Bollenbacher, 198 1). Nevertheless, certain aspects of the assay were inefficient and somewhat tedious, and ii became apparent that in order to use if effectively for analyzing the kinetics of PTTH activation of the PG, the assay would have to be simplified. In the original in vitro assay, ecdysone synthesized by the PH was quantified by RIA of methanol extracts of incubation medium, a timeconsuming step which could be eliminated by direct RIA of incubation medium. However, since large quantities of ecdysone (20-30 ng) are synthesized by PG activated by PTTH in vitro, the micro RIA could only be used for a direct assay of incubation medium if the aliquots assayed did not exceed l-2 ~1, amounts which are difficult to manipulat reproducibly. To permit accurate quantification of larger amounts of ecdy-
W. E. Bollenbacher
32
et al.
sone, a macro RIA was developed by decreasing the specific activity of the radiolabeled ligand and increasing the antibody concentration (see Materials and Methods). A log-logit plot of a macro RIA competition curve derived with ecdysone as the competing ligand illustrates the broad linear range of this assay (0.25532 ng ecdysone) and, thus, its usefulness for assaying medium directly (Fig. 1). The validity of the macro RIA for quantifying ecdysone synthesis by PG in vitro was confirmed by demonstrating that the addition of 0.01 ml of Grace’s medium to the standard assay volume of 0.2 ml borate buffer did not significantly alter the binding equilibrium of the assay. Furthermore, a comparison of the results of a macro RIA of incubation medium with those of a micro RIA of methanol extracts of incubation medium revealed a quantitative variation between the two methods of less than 10%. A second possible way in which the in vitro assay could be simplified was to reduce the incubation time, which had previously been 6 h (Bollenbacher et al., 1979). A careful reassessment of the time-course of ecdysone synthesis by activated and nonactivated PC from both day 3 larvae and day 0 pupae revealed that by 2 h the rate of ecdysone synthesis by the PTTH-activated glands was approx. 4 times that of the
3.0
Ecdysone,
ng
Fig. 1. Log-logit plot of the macro ecdysone radioimmunoassay standard curve. The standard competing ligand was unlabeled ecdysone and linear competition was observed from 0.25 ng to 32 ng.
Kinetrcs of prothoracicotropic
hormone action
33
contralateral control glands (day 3 larval PG, 15.5 ng vs. 3.5 ng; day 0 pupal PG, 17.5 ng vs. 4.5 ng) (Figs. 2 and 3). Since this dramatic response of the PG to PTTH by 2 h was reproducible, the incubation time of the assay was reduced to 2 h. The potentially most significant change in the in vitro assay would be the elimination of the dose-response protocol. However, it was observed during the course of this study that although the rates of ecdysone synthesis were comparable between members of a gland pair, rates of synthesis by larval and pupal PG varied considerably within a population, particularly when the glands were ativated by PTTH (see Fig. 2). This consistently observed variability in PG activity between different animals and the variation in the sensitivity of the glands to P’TTH from experiment to experiment, both in absolute amounts of ecdysone synthesized and in A, values (Figs. 4 and 5), precluded any approach other than the right vs. left dose-response protocol for assessing activation. This conclusion was reinforced by the fact that the day 3 larval and day 0 pupal PC were activated over a very narrow range of hormone concentration, 2.6- and 3.2-fold, respectively. This is in marked contrast to the results of a similar study in which day 3 larval brains activated day 3 larval PG with a dynamic range of response (neurohormone concentration) covering at least two orders of magnitude (Carrow et al., 1981). The dramatic nature of the PG response to the PTTH in the day 0 pupal brian thus necessitates quantification of PTTH activity by a dose-response protocol, regardless of the glands used in the assay.
The initial step in investigating the mechanism of PTTH activation of the PG was to determine the time-course kinetics of this activation. Time-courses of ecdysone synthesis by activated and nonactivated glands were defined to establish an optimal incubation time for other kinetics analyses of PG activation. Day 3 larval and day 0 pupal PG were used for the time-course studies, since these stages of development in ~an~~ca immediately precede a period of PTTH release, i.e. a head critical period, which is the time interval during development when the head is required for molting (see Granger and Bollenbacher, 1981), and thus these PG should be competent to respond to the neurohormone. The time-course of synthesis for precisely staged day 3 larval PG revealed that the nonactivated (control) PG exhibited a basal rate of ecdysone synthesis of 1.2 ng per hour per gland that was roughly linear for 6 h, with total synthesis in 6 h being 7.7 1L:1.1 ng per gland (Fig. 2). The contralateral PTTH-activated glands exhibited the expected increase in the rate of ecdysone synthesis of 7.7 ng per hour per gland that was
W.E. Bollenbacher et al.
34
b-
I-
i-
I-
i-
1 I
2
3
4
5
6
Time, hr Fig. 2. Time-course of ecdysone synthesis for PTTH-activated (0) and nonactivated (0) gate II day 3 last instar larval PC of Manduca sexta. Each datum point represents the mean ( f SEM) of 6 separate determinations.
linear for approx. 2.5 h. The synthesis rate decreased after this time, reaching an apparent plateau by 6 h, at which time 25 + 2.4 ng ecdysone per gland was synthesized. The greatest difference in the rates of linear synthesis between activated and nonactivated glands occurred at 2 h when an A, of 4.6 was established. The time-courses of synthesis for activated and nonactivated day 0 pupal PG (Fig. 3) was similar to those for the larval glands (Fig. 2) and to time-courses previously reported for these glands (Bollenbacher et al., 1979, 1980). The basal rate of ecdysone synthesis for these PG was 1.5 ng per hour per gland and synthesis was linear over the 6 h incubation period, with total synthesis at 6 h being 11.4 k 1.6 ng per gland. The contralateral, PTTH-activated glands exhibited a linear increase in ecdy-
Kmetics
ofprothoracicotropic
35
hormone action
Time,
hr
Fig. 3. Time-course of ecdysone synthesis for PTTH-activated (0) and nonactivated (0) day 0 pupal PG of Manduca sex@. Each datum point represents the mean ( f SEM) of 6 separate determinations.
sone synthesis for - 2.0 h at an average rate of 9.7 ng per hour per gland. From 2 to 6 ht the rate of synthesis decreased rapidly before reaching an apparent plateau; total ecdysone synthesis was 24.8 It 1.8 ng per gland. As with the larval PC, the greatest difference in rates of synthesis between activated and nonactivated pupal glands occurred at 2 hr, yielding an A, of 4. This occurred at the end of the period of linear synthesis by the activated pupal glands. Thus, the basal and activated time-courses of ecdysone synthesis by activated and nonactivated glands are comparable for day 3 larvae and day 0 pupae. Dose-response of activation Although the biosynthetic
capacity
of the PG and the rate kinetics
by
W. E. Bollenbacher
36
et al.
which this capacity is expressed are important biological properties of the gland, the sensitivity of the PG to PTTH could be of equal importance, especially if this sensitivity varied substantially during development. In the absence of differences in the kinetics of biosynthesis, the responsiveness of different glands to hormone would become the most critical aspect of gland activation. These differences in responsiveness would be expressed both in terms of the amount of PTTH (EDzO) necessary to elicit a half-maximal response (A,,) from the PG and the concentration range of PITH required for a linear increase in the A,. Thus, the defining both of these parameters for larval and pupal PG should indicate whether PC do indeed change in their sensitivity to PTTH during development. In addition, such studies would help define the optimal in vitro conditions for investigating the mechanism by which gland activation occurs. To assess the nature of the response of these larval and pupal PG to PTTH, dose-responses of PTTH activation of day 3 larval and day 0 pupal PG were generated (Fig. 4). The profiles of the A, values generated by dose-response were essentially the same for both pupal and larval glands, but full activation of the larval glands required less PTTH, suggesting that these glands were more sensitive to PTTH. Day 0 pupal
1 9-
?-
I
5-
I
3-
1 II 0.1
I 0.01 PTTH,
units
Fig. 4. Dose-response of PTTH activation of gate II day 3 last instar larval PC (0) and day 0 pupal PG (0) of Manduca senta. Each datum point represents the mean (+ SEM) of the A, values for 4-6 separate determinations.
Kinetics of prothoracicotropic
hormone action
31
PG were activated over a narrow dynamic range (0.04 to 0.13 units of PTTH), resulting in an ED,, of 0.076 units and an A,,, of - 7. The same PTTH preparation elicited a similar narrow dynamic range of response from the day 3 larval PG (0.025 to 0.065 units of PTTH) with an A,,, of - 6.5. However, the ED,, for the larval PG was 0.042 PTTH units, indicating that the larval glands used in this study are - 1.8 times more sensitive to a crude PTTH extract than are pupal glands. Repeated dose-response analyses of the larval and pupal glands with PTTH from day 1 pupal brains demonstrated that this difference was not only significant, but frequently even greater. Since these PG respond differently to PTTH, the sensitivity of the PG to the neurohormone apparently makes a substantial contribution to the developmental changes in gland activity. In addition to demonstrating basic differences in PG responsiveness, these dose-response data also defined the units of PTTH to be used in eliciting maximal activation of the PG for in vitro studies, the exposure time critical for PTTH activation, and the decay of the PTTH-activated response. Exposure
time for activation and decay of activated response
With the in vitro conditions defined from the dose-response data, it was possible to analyze both the kinetics of PG activation in terms of the time of exposure to PTTH necessary for activation, as well as the kinetics of the decay of activation. Defining these parameters could help elucidate the type of mechanism by which activation occurs, and may permit some speculation about the type, or mode, of PTTH release (e.g. pulsatile) that occurs in situ (Bollenbacher and Gilbert, 1981). To determine the critical period of time for which PG must be exposed to PTTH to achieve maximal activation, a pulsed preincubation protocol was used in which one gland of a pair was exposed to PTTH for a given period of time while the contralateral gland was preincubated in medium lacking PITH. Synthesis by each gland was then determined after a 2 h incubation in Grace’s medium. For day 3 larval PG, exposure to PTTH for increasing time intervals revealed an essentially immediate response, since an A, of 4 was attained after only a 0.5 min exposure (Fig. 5). This sharp increase in gland activity was followed by a more gradual increase in activation in response to longer exposure times, which plateaued at - 20 min with an A, of - 8. Since for this study the A,,, was 8, an A, of 3.5 represents the A,,, and thus 0.5 min represented the exposure time necessary for half-maximal activation of the larval PG. The immediate response of the PG to PTTH was also evident from the time-course of synthesis (Fig. 2) since a significant level of synthesis was reached very
W.E. Bollenbacher et al.
38
quickly with no lag phase apparent in the response. Day 0 pupal PG exhibited an acute response to PTTH similar to that of the larval gland, with an A, of - 4 at 0.5 min (Fig. 5). This initial response was followed by the same gradual increase in the A, with the same plateau at 20 min and an A,,, of - 8. The exposure time of the PG to PTTH necessary to half-maximally activate the pupal PG was 0.5 min, the same time required for a half-maximal response from larval glands. The A,,, of 8 observed for larval and pupal PG was greater than that noted in the dose-responses of activation for these PC (Fig. 4). This may reflect the inherent variability in activation by changing A,,, different crude preparations of PTTH and/or different lots of Grace’s medium. Thus, for each series of experiments the same Grace’s medium preparation of PTTH was used. There are many possible explanations for this variability, but since the absolute A,,, value is only important within a given set of experiments, this variability between experimental sets is not a problem. It is, however, acceptable to use the A,,, as a means of determining the number of PTTH units necessary to reach the half-maximal response (A,,) in these experiments, and then to compare
II -
9-
7-
a!
. 5-
3-
I 1
0
I
I
I
15 Exposure
I
I
I
I
to PTTH,
I
45
30 min
Fig. 5. Effect of varying times of exposure to PTTH on gate II day 3 last instar larval PG (0) and day 0 pupal PG (0) of Manduca sexta. Each datum point represents the mean (+ SEM) of the A, values for 6-9 separate determinations.
Kinetics of prothoracicotropic
39
hormone action
these ED,, values between different experiments. These comparisons are valid because the ED,, remains relatively constant with a specific experimental regimen irrespective of the A,,,. An analysis of the decay of PG activation by PTTH was made by determining changes in the rates of ecdysone synthesis by the glands after maximal activation was attained by preincubation with 0.5 units of PTTH for 20 min. For approx. 2 h after this preincubation, the rate of ecdysone synthesis by a population of right, day 3 larval PG postincubated in the absence of PTTH (- PTTH) appeared less than that for
25
I I I I
I I
I I I I I I I
I
I
I
I
I
2
Time,
hr
Fig. 6. Time-course of the decay of PTTH activation of gate II day 3 last instar larval PG of Manduca sexta. Dashed line denotes the time at which both control (right) and experimental (left) PG were preincubaied with PTTH. Control glands (0) were post incubated with PTTH while experimental glands (0) were postincubated without PTTH. Each datum point is the mean ( f SEM) of 6-9 separate determinations.
W.E. Bollenbacher et al.
40
the contralateral left glands still incubated in the presence of PTTH (+ PTTH) (Fig. 6), but this difference was not significant. The rates of synthesis in - PTTH and + PTTH PG incubations was 6 ng per hour per gland and 7.5 ng per hour per gland, respectively. Just after 2 h, however, the rate of synthesis by the - PTTH glands decreased dramatically, reaching an apparent plateau at - 3 h, while the +PTTH glands
20 -
15 -
z _ 2 :: w” 2
IO-
5-
1
2
Time,
hr
Fig. 7. Time-course of the decay of PTTH activation of day 0 pupal PG of Man&a sextu. Dashed line denotes the time at which both control (right) and experimental (left) PG were preincubated with PTTH. Control glands (0) were postincubated with PTTH while experimental glands (0) were postincubated without PTTH. Each datum point is the mean (2 SEM) of 6-9 separate determinations.
Kinetics
of prothoracicotropic
hormone
action
41
continued to synthesize ecdysone at the activated rate (7.5 ng per hour per gland). The rate kinetics for the decay of PTTH activation of day 0 pupal PG were similar to those for day 3 larval glands (Fig. 7). Ecdysone synthesis for pupal glands +PTTH and - PTTH was quantitatively indistinguishable for the first 1.6 h of the incubation period, with an average rate of 12.5 ng per hour per gland. After this time, the synthesis rate for PG in the absence of PTTH decreased sharply, reaching an apparent plateau at 3.0 h, while ecdysone synthesis by the glands +PTTH continued at a rate of 12.5 ng per hour per gland. In summary, studies determining the critical time for activation and decay of activation indicate that day 3 larval and day 0 pupal PG are activated immediately upon exposure to PTTH and rapidly reach maximal activation. Maximal activation persists for only about 1.5-2 h and shortly thereafter the glands return to a basal rate of ecdysone synthesis. Unfortunately, the kinetics of deactivation were such that a half-life could not be extrapolated. However, the fact that maximally activated larval and pupal PG become essentially inactive within 2.5 h in the absence of PTTH, with the actual decay in the activated response occurring in - 40 min, suggests that in order for PG to remain activated fro long periods of time in situ, the glands must be exposed either continuously or frequently enough to maintain the activated state.
DISCUSSION Kinetic parameters have been defined for an in vitro system suitable for investigating the mechanism of action of the insect prothoracicotropic hormone. This system is well suited for such an investigation because PTTH activates the prothoracic glands directly. Since this activation occurs in vitro, it can be assessed kinetically. The in vitro assay for PG activation by PTTH was substantially refined in the present study by simplifying the standard ecdysone RIA used to quantify ecdysone synthesis in vitro and by reducing the assay incubation time. These changes considerably improve the usefulness of this assay for conducting mechanistic studies. With this PG assay it was possible to obtain the information required for an investigation of the mechanism of action of PTTH: (1) there are specific critical times during which the PG must be exposed to PTTH for activation to occur; (2) the activated response decays at a specific rate; and (3) the PG from different developmental stages differ in their sensitivity to PTTH. Without knowledge of the above, it would not be
42
W. E. Bollenbacher et al.
possible to study the cascade of events that occur in response to PTTH activation. The sensitivity to PTTH of PG from day 3 larvae and day 0 pupae of Munduca was assessed from the kinetics of PG activation by this cerebral neurohormone. Glands from day 3 of the last larval instar and day 0 of the pupal period are developmentally competent to respond to PTTH and exhibited similar cativation kinetics in the time course studies with respect to both basal and activated rates of ecdysone synthesis. However, although dose-response profiles of activation were also similar for these glands, the sensitivity of the glands to PTTH was different, i.e. day 3 larval PG responded more acutely than day 0 pupal PG. This finding suggests that the interaction between neurohormone and gland may vary during postembryonic development and that the stage-specific sensitivity of the PG to PTTH may affect the progress of development. This suggestion is supported by the observation that the PG from diapausing Manduca pupae are refractory to PTTH (Bowen et al., 1983). That larval glands were more sensitive to PTTH extract than pupal PG initially suggested that the former might be more suitable for the in vitro assay (see Bollenbacher and Bowen, 1983). However, two major problems prevent the routine use of these glands: it is laborious to stage sufficient day 3 gate II last instar Manduca larvae for the large quantities of glands needed for most experiments; and, more important, the larval glands can be activated nonspecifically (Bollenbacher, unpublished observation). For example, a broad range of concentrations of heat-denatured proteolytic enzymes such as trypsin stimulate the level of ecdysone synthesis by these glands 2-3 times over the basal rate and appear to do so in a dose-dependent manner. Since proteolytic enzymes are present in all tissues and exhibit varying degrees of heat stability, heating of a tissue extract before assay would not necessarily prevent their nonspecific activation of the PG. Thus, larval PG could only be used in a study of PTTH activation if the activity in a sample had been previously demonstrated to be the neurohormone. It has recently been reported that PTTH extract from brains of Manduca larvae activates larval PG with a dynamic concentration range spanning more than two orders of magnitude (Carrow et al., 1981). This extremely broad response range contrasts markedly with that determined for larval PG in this study, as well as with the type of response observed with other insect neurohormones (e.g. Copenhaver and Truman, 1982; Taghert and Truman, 1982). On the basis of our observations, it is possible that the broad response range reported in this earlier study is a reflection of nonspecific activation of the larval PG. One attempt to elucidate the mechanism by which PTTH activates the
Kinetics of prorhoracicotropic
hormone action
43
PC in Manduca utilized an indirect and pharmacological approach. Nevertheless, the resulting data did suggest that the PG were activated by PTTH via CAMP (Vedeckis et al., 1976; Bollenbacher et al., 1980). In this study, a CAMP titer generated for the PG of Manduca during the last larval instar revealed a dramatic increase in CAMP during the first head critical period (HCP) of the instar, i.e. during the initial period of PTTH release on day 3 plus 16 h to day 4 plus 6 h, which is responsible for a small ecdysteroid surge and ultimately for wandering and commitment. Incubation of larval PG with CAMP or with dibutyryl CAMP in the presence of a phosphodiesterase inhibitor resulted in a dose-dependent stimulation of ecdysone synthesis, supporting the concept that CAMP is involved in the activation process. Further indirect evidence for cyclic nucleotide involvement in PTTH activation of the PG comes from the observations that phosphodiesterase inhibitors can effect an increase in the amount of ecdysone synthesized by larval PG in vitro, and that adenylate cyclase activity is present in PG, providing an enzymatic basis for the involvement of cyclic nucleotides (Vedeckis and Gilbert, 1973). However, the lack of a quantitative assay which could directly measure PG activation was in part responsible for the failure to demonstrate a direct causal relationship between PTTH activation of the PG and an increased content of cyclic nucleotide in these glands. With the in vitro assay for PTTH as defined in this study and with the availability of purified and partially purified PTTH (see below), these critical experiments have now been initiated. The intriguing .observation that PG from two different stages of development responded differently to PTTH extract (Bollenbacher and Gilbert, 1981, and present data) is potentially of great significance. The fact that day 3 larval glands are more sensitive to PTTH than the pupal glands suggests that there may be something different about the PTT’H receptors of these PG, e.g. more receptors resulting in greater sensitivity or mixed populations with different affinities. In addition to gland sensitivity, the chemical nature of PTTH may also contribute to the response of the PC to this neurohormone. With the purification of Manduca PTTH nearly complete, it appears that there are two molecular forms of the neurohormone (Bollenbacher and Gilbert, 1981; Bollenbather and Bowen, 1983). Each moiety activates PG in vitro in a dose-response manner similar to that obtained with crude PTTH, but the two forms have different physical properties, e.g. different molecular weights, isoelectric points and chemical stabilities (Bollenbacher and Gilbert, unpublished observations). Furthermore, different amounts of a particular form of PTTH may be necessary to activate PG from different development stages. While the large form of PTTH (M, - 29 000) elicits
44
W. E. Bollenbacher
et al.
similar dose-responses of activation and ED,, values from day 3 larval and day 0 pupal PG, the small form of the hormone (M, - 7000) more effectively activates larval glands, i.e. lo-20 times less hormone is needed to maximally activate these PG. Thus, the greater sensitivity of the larval PG to the small form of PTTH could explain their more acute response to a crude PTTH preparation. The biological significance of two PTTHs and of developmentalspecific PG sensitivity to these two neurohormones is conjectural at present. It is possible that the large and small PTTHs are released differentially during development or are released in precise ratios to elicit a certain level of response from the PG, resulting in a specific ecdysteroid titer that is critical for the promotion of a particular developmental process (Bollenbacher and Bowen, 1983). Further, the differential activation of the PG by these two PTTHs could imply that the two forms operate via different mechanisms, e.g. CAMP-dependent and CAMP-independent (Vedeckis et al., 1976). The in vitro system defined in this study should permit an investigation of such possibilities. The data on the kinetics of PG activation by PTTH and the decay of that activation also provide a hypothesis concerning the manner in which PTTH is released in situ. In Manduca, the head critical periods (HCP), i.e. periods of PTTH release, vary during larval and larval-pupal development, ranging from a few hours for the single HCP before a larval molt, to 20 h for the first of the two HCP in the last larval instar, to several days for the HCP before pupal-adult development (Truman, 1972, Truman and Riddiford, 1974; Bollenbacher, unpublished observation). PTTH release during these HCP could occur either in a rapid, continuous manner or as bursts of release, the latter being the mode characteristic of neurosecretory cells (see Maddrell and Nordmann, 1979). Regardless of the mode, the duration of release would be determined by select external and/or internal cues. The fact that activation of the PG by PTTH in vitro is immediate (0.5 min to achieve half-maximal activation) and is followed by a decay of that response in less than 3 h, suggests that in order for PC activity to be sustained in situ for periods from a few hours to several days, PTTH must be released from its neurohemal site throughout the HCP. If PTTH release occurs in bursts, it may involve only a single burst of approx. 1 h during the fourth instar, or pulsatile releases covering many hours or days during the first HCP of the fifth instar and the pupal HCP (Bollenbacher and Gilbert, 1981). This has been thoroughly investigated for the fourth instar HCP and the first HCP of the last instar and the resulting data reveal that PTTH release occurs in bursts: a single burst for the temporally short HCP of the fourth instar, and repeated bursting, i.e. pulsatile release comprising 3
Kinems
ofprothoracicotropic hormone action
45
bursts, for the long HCP of the fifth (last) instar (Bollenbacher and Gilbert, 1981; Bollenbacher and Bowen, 1983; Bollenbacher et al., unpublished observation). These initial findings on the modes of PTTH release are consistent with the present data on the exposure time critical for PTTH activation of the PG in vitro and the duration of the activated response. The occurrence of unique patterns of release at specific stages of development could also represent the release of one or the other molecular forms of PTTH, or some combination of the two forms. In summary, an in vitro system has been established that appears to simulate PG activation in situ and therefore can be used to probe the basic endocrinology of PTTH(s), particularly the mechanism(s) by which this neurohormone(s) controls the PG.
ACKNOWLEDGEMENTS This research was supported by NIH grants NS-18791 and AM-3 1642 and USDA grant 59-2176 to W.E. Bollenbacher and NIH grant AM-301 I8 to L.I. Gilbert.
REFERENCES Agui, N., Granger, N.A., Gilbert, L.I. and Bollenbacher, W.E. (1979) Proc. Natl. Acad. Sci. (U.S.A.) 76, 5694-5698. Bollenbacher, W.E. andBowen, M.F. (1983) In: Endocrinology of Insects, Eds.: H. Laufer and R.G.H. Downer (A.R. Liss, New York) (in press). Bollenbacher, W.E. and Gilbert, L.I. (1981) In: Neurosecretion: Molecules, Cells, Systems, Eds.: D.S. Farner and K. Lederis (Plenum, New York) pp. 361-370. Bollenbacher, W.E., Agui, N., Granger, N.A. and Gilbert, L.I. (1979) Proc. Natl. Acad. Sci. (U.S.A.) 76, 5148-5152. Bollenbacher, W.E., Agui, N., Granger, N.A. and Gilbert, L.I. (1980) In: Invertebrate Systems In Vitro, Eds.: E. Kurstak, K. Maramorosch and A. Dubendorfer (Elsevier/North-Holland, Amsterdam) pp. 253-271. Borst, D.W. and O’Connor, J.D. (1972) Sicence 178, 418-419. Bowen, M.F., Bollenbacher, W.E. and Gilbert, L.I. (1983) (submitted for publication). Carrow, G.M., Calabrese, R.L. and Williams, C.M. (1981) Proc. Natl. Acad. Sci. (U.S.A.) 78, 5866-5870. Copenhaver, P.F. and Truman, J.W. (1982) J. Insect Physiol. 28, 695-701. Gilbert, L.I., Goodman, W. and Bollenbacher, W.E. (1977) In: International Review of Biochemistry: Biochemistry of Lipids II, Vol. 14, Ed.: T.W. Goodman (University Park Press, Baltimore) pp. l-50. Granger, N.A. and Bollenbacher, W.E. (1981) In: Metamorphis, 2nd Edn. Eds.: L.I. Gilbert and E. Frieden (Plenum, New York) pp. 105-137. Maddrell, S.H.P. and Nordmann, J.J. (1979) Neurosecretion (Halsted Press, New York). Taghert, P.H. and Truman, J.W. (1982) J. Exp. Biol. 98, 373-383.
46
W. E. Bollenbacher ei al.
Truman, J.W. and Riddiford, L.M. (1974) J. Exp. Biol. 60, 371-382. Vedeckis, W.V. and Gilbert, L.I. (1973) J. Insect Physiol. 19, 2445-2457. Vedeckis, W.V., Bollenbacher, W.E. and Gilbert. L.I. (1976) Mol. Cell. Endocrinol. 81-88. Vince, R. and Gilbert, L.I. (1977) Insect Biochem. 7, 115-120. Weevers, R. De G. (1966) J. Exp. Biol. 44, 163-175. Wielgus, J.J. and Gilbert, L.I. (1978) J. Insect Physiol. 24, 629-637.
5,
Molecular and Cellular Endocrinology, 32 (1983) 27-46 Elsevier Scientific Publishers Ireland, Ltd.
MCE 01036
A KINETIC ANALYSIS OF THE ACTION OF THE INSECT PROTHORACICOTROPIC HORMONE Walter
E. BOLLENBACHER
KATAHIRA
Department of Biology, 27514 (U.S.A.) Received
*, Martha
and Lawrence
30 March
Wilson Hall 046A,
1983; accepted
A. O’BRIEN,
Eva J.
I. Gilbert
29 April
University
of North Carolina,
Chapel Hill, NC
1983
A modified in vitro assay was used to assess the kinetics of activation and the decay of activation of the prothoracic glands (PG) by the prothoracicotropic hormone (PTTH) in Manduca sexta. Time-courses of ecdysone synthesis by PTTH-activated day 3 larval and day 0 pupal PG were comparable both quantitatively and temporally, but dose-responses of PTTH activation revealed that larval glands were 1.8 times more sensitive to the neurohormone. The exposure time necessary for maximal activation of the PG by PTTH was the same for both glands, with half-maximal activation in - 0.5 min. Once PTTH was removed the rate of ecdysone synthesis by larval and pupal PG remained constant for about 2 h, after which the activated response for both glands decayed rapidly. reaching the unactivated basal synthesis rate within 45 min. These kinetics data suggest that PG activation by PTTH in vitro occurs in a mailner indicative of activation in situ and, thus, that this in vitro system is suitable for probing the molecular mechanism by which PTTH activates the PG. Keywords:
prothoracic gland; tropic hormone.
Manduca
sexta;
ecdysone
synthesis:
prothoracico-
Molting in insects occurs in response to a cascade of neuroendocrine and endocrine events that originate in the brain with the release of the prothoracicotropic hormone (PITH) and culminate in the response of target tissue to 20-hydroxyecdysone, the molting hormone (see Granger and Bollenbacher, 1981). Although this process is understood in general terms, the exact biochemical mechanisms involved have not yet been defined. This is particularly true for one of the primary events in the molting process, activation of the prothoracic glands (PG) by PITH which leads to synthesis and secretion of the hormone, ecdysone. Measurements
of CAMP levels and PG activity
* To whom all correspondence
0303-7207/83/$03.00
have suggested
should be addressed.
0 1983 Elsevier Scientific
Publishers
Ireland,
Ltd.
that
28
W. E. Bollenbacher
et al.
PTTH activation of the PG occurs via CAMP-dependent and CAMP-independent mechanisms in response to the gated release of the neurohormone at specific stages of development (Vedeckis et al., 1976) but an unequivocal causal relationship remains to be established. Recently, an in vitro system was developed for monitoring PTTH activation of the PG directly (Bollenbacher et al., 1979, 1980) making it possible to explore PTTH activation of the PG at the biochemical level. However, in order to conduct such studies the kinetics of PG activation and the kinetics of activation decay must first be determined. This report details an investigation of the kinetics of PTTH activation of PG from larvae and pupae of the tobacco hornworm, Manduca sexta, utilizing a modified in vitro assay for this neurohormone. The data are discussed in the context of: possible mechanisms of PTTH action; the relative responsiveness of PG from different stages of development; and the nature of PTTH release during the larval-pupal development of a holometabolous insect.
MATERIALS
AND
METHODS
Animal rearing and donor selection Larvae and pupae of the tobacco hornworm, Munduca sexta, were reared individually on an artificial diet at 25°C under nondiapausing conditions (LD 16 : 8) and staged and gated as described previously (Vince and Gilbert, 1977; Wielgus and Gilbert, 1978). Since the majority of the gate II animals molt at 2 p.m., day 0 of the last larval instar and of the pupal period was set at 2 p.m. on the day of ecdysis. Brains of day 1 pupae were used as the source of a crude preparation of PTTH because the titer of PTTH is highest on day 1, approx. 3 times that of fifth instar larval brains (Bollenbacher and Gilbert, 1981). Donors for larval and pupal PG were chosen at stages of development when glands were presumably competent to respond to PTTH. One such stage during larval development occurs on day 3 of the last instar, since the gated release of PTTH defining the first head critical period of this instar occurs during the night of day 3 (Truman and Riddiford, 1974). Subsequent PG activation, wandering behavior and pupal commitment occur during day 4. Competent pupal PG were obtained from O-12 h pupae, approx. 2 days prior to the beginning of the head critical period for pupal-adult development (Bowen et al., 1983). Organ dissections and extraction procedure for PTTH Pairs of PG from individual animals were dissected in lepidopteran saline (Weevers, 1966) and placed individually in wells of multiwell
Kinetics of prothoracicotropic hormone action
29
culture plates containing Grace’s tissue culture medium. Dissection time for an in vitro assay for PTTH was held to less than 1 h to maximize gland responsiveness. For a crude preparation of PTTH, day 1 pupal brains were placed in Grace’s medium, freed of contaminating tissues, washed and then homogenized in fresh Grace’s medium at a concentration of 2 brains/O.025 ml medium. Immediately after homogenization, brain extracts were heat-treated at 100°C for 2 min to minimize proteolysis of PTTH and centrifuged at 9000 x g for 10 min. Since heat-treated Grace’s medium did not alter PG activity in vitro, the resulting supernatant could be used as the PTTH source for the in vitro assay without further purification. The reproducibility of the extraction protocol and the stability of the PTTH activity in the extract have been documented previously (Bollenbather et al., 1979; Bollenbacher and Gilbert, 1981). In vitro assay for PTTH The assay used to assess the kinetics of PTTH activation of ecdysone synthesis by the PG was essentially that described previously, with the exception that the incubation medium was analyzed for ecdysone directly rather than first extracting the medium with methanol. Therefore, the ecdysone RIA used to quantify synthesis had to be modified to permit assay of the incubation medium. In the modified assay, 0.01 ml aliquots of the 0.025 ml standing drop in which the PG were incubated were subjected to the standard ecdysone RIA protocol (Bollenbacher et al., 1979). An equivalent volume of Grace’s medium (0.01 ml) was added to each tube comprising the ecdysone standard curve to preserve the same assay volume and thus the same competitive binding conditions. The specific activity of the [23,24,3H]ecdysone stock solution used in the RIA was adjusted to 4 Ci/mmole and the concentration of the H-3 antiserum stock solution (Gilbert et al., 1977) to 0.15%; it then bound approx. 40% of the labeled ligand. Ecdysone was used as the competing, unlabeled ligand in the assay, and under these conditions a linear standard curve was obtained with concentrations of ecdysone ranging from 0.25 to 32 ng. Since the conditions of this assay resulted in a standard curve with a greater and broader response range, the modified RIA was termed a ‘macro’ RIA, in contrast to the standard ‘micro’ RIA with a response range of 0.05 to 2.0 ng. Termination and counting of the macro RIA was as previously described for the micro RIA (Borst and O’Connor, 1972), and the data were analyzed with a Wang PCS II computer using a log-logit plot.
30
W. E. Bollenbacher
et al.
Time-course of ecdysone synthesis and dose-response of PG activation by PTTH Time-courses of ecdysone synthesis for larval and pupal PG were obtained using the right glands from a population of animals for determining a basal, control rate of synthesis, and the left glands from this same population for determining the increased rate of synthesis in the presence of PTTH. This approach was employed because the basal activity of each member of a pair of PG is comparable (Bollenbacher et al., 1979). Six precisely staged larvae or pupae comprised a standard test population, and control and experimental PG from each animal were incubated separately in 0.025 ml of Grace’s medium, with and without amounts of PTTH sufficient for maximal activation of the glands (0.5 brain equivalents/assay). At designated times, 0.01 ml aliquots of medium were taken from each incubate with the immediate addition of 0.01 ml of fresh medium, with or without PTTH, to maintain the volume of the incubation at 0.025 ml for the duration of the time-course (6 h). The ecdysone present in each aliquot was quantified by the macro RIA. To generate a dose-response curve of PTTH activation for either larval or pupal PG, activation was determined by comparing the quantities of ecdysone synthesized by an experimental gland to that synthesized by the contralateral control gland for each concentration of PTTH. Activation was then expressed as an activation ratio (A,), which is the quantity of ecdysone synthesized by the experimental gland ( + PTTH) divided by the amount synthesized by the control gland ( - PTTH). The relative responsiveness of different PG to a crude PTTH preparation was assessed by determining the amount of PTTH, expressed in brain equivalents, necessary to obtain maximum activation (A,,,) in a dose-response. The amount of PTTH (ED,,) needed to achieve half-maximal activation (As,,) of the PG was then compared. The ED,, has proven to be an accurate means of quantifying relative PTTH activity in extracts of tissue (Agui et al., 1979, 1980; Bollenbacher and Gilbert, 198 1) and should be equally effective in quantifying the relative sensitivities to PTTH of PC from different developmental stages. For these kinetics studies PTTH activity was expressed in units, a unit being equivalent to the PTTH activity present in a day 1 pupal brain. This value was derived from the reciprocal of the number of day 1 pupal brain equivalents (ED,,) necessary to achieve half-maximal activation of day 0 pupal PG (A,,). For day 1 pupal brains, the ED,, was found to be 0.06 brain equivalents, the reciprocal of which is 16.7. Thus, there is sufficient PTTH in this brain to half-maximally activate 16.7 day 0 pupal PH under the established in vitro assay conditions.
Kitxtics
ofprothoracicotrapic
hormone action
31
Kinetics of activation and deactivation of PG The time necessary for PTTH to maximally activate the PG was determined using a modified left vs. right gland protocol, in which one member of a gland pair was preincubated for a specific period of time in Grace’s medium containing 0.05 units of PTTH and the contralateral gland in Grace’s medium alone. After preincubation, glands were transferred to, and incubated in, equivalent volumes of Grace’s medium (0.025 ml) without PT’lH. At the end of 2 h, a 0.01 ml aliquot of each incubate was assayed for ecdysone synthesis by the macro RIA and an A, was computed. The A, for each time point was the average of the A, values for 6-9 gland pairs. To assess the kinetics of deactivation of the PG, paired glands from larvae or pupae were preincubated separately for 30 min 0.025 ml of Grace’s medium containing a saturating amount of PTTH (0.5 units). After preincubation, one member of the gland pair was transferred to a standing drop of Grace’s medium containing the same concentration of PTTH and the other gland to a drop of Grace’s medium only. Timecourses of ecdysone synthesis were then determined using the previously described time-course protocol, with a total incubation time not exceeding the duration of a linear rate of ecdysone synthesis by the control gland.
RESULTS
The in vitro PG assay developed several years ago for assessing PTTH activity (Bollenbacher et al., 1979) was a substantial improvement over the biological assays previously used to quantify the activity of this neurohormone (see Granger and Bollenbacher, 198 1). Nevertheless, certain aspects of the assay were inefficient and somewhat tedious, and ii became apparent that in order to use if effectively for analyzing the kinetics of PTTH activation of the PG, the assay would have to be simplified. In the original in vitro assay, ecdysone synthesized by the PH was quantified by RIA of methanol extracts of incubation medium, a timeconsuming step which could be eliminated by direct RIA of incubation medium. However, since large quantities of ecdysone (20-30 ng) are synthesized by PG activated by PTTH in vitro, the micro RIA could only be used for a direct assay of incubation medium if the aliquots assayed did not exceed l-2 ~1, amounts which are difficult to manipulat reproducibly. To permit accurate quantification of larger amounts of ecdy-
W. E. Bollenbacher
32
et al.
sone, a macro RIA was developed by decreasing the specific activity of the radiolabeled ligand and increasing the antibody concentration (see Materials and Methods). A log-logit plot of a macro RIA competition curve derived with ecdysone as the competing ligand illustrates the broad linear range of this assay (0.25532 ng ecdysone) and, thus, its usefulness for assaying medium directly (Fig. 1). The validity of the macro RIA for quantifying ecdysone synthesis by PG in vitro was confirmed by demonstrating that the addition of 0.01 ml of Grace’s medium to the standard assay volume of 0.2 ml borate buffer did not significantly alter the binding equilibrium of the assay. Furthermore, a comparison of the results of a macro RIA of incubation medium with those of a micro RIA of methanol extracts of incubation medium revealed a quantitative variation between the two methods of less than 10%. A second possible way in which the in vitro assay could be simplified was to reduce the incubation time, which had previously been 6 h (Bollenbacher et al., 1979). A careful reassessment of the time-course of ecdysone synthesis by activated and nonactivated PC from both day 3 larvae and day 0 pupae revealed that by 2 h the rate of ecdysone synthesis by the PTTH-activated glands was approx. 4 times that of the
3.0
Ecdysone,
ng
Fig. 1. Log-logit plot of the macro ecdysone radioimmunoassay standard curve. The standard competing ligand was unlabeled ecdysone and linear competition was observed from 0.25 ng to 32 ng.
Kinetrcs of prothoracicotropic
hormone action
33
contralateral control glands (day 3 larval PG, 15.5 ng vs. 3.5 ng; day 0 pupal PG, 17.5 ng vs. 4.5 ng) (Figs. 2 and 3). Since this dramatic response of the PG to PTTH by 2 h was reproducible, the incubation time of the assay was reduced to 2 h. The potentially most significant change in the in vitro assay would be the elimination of the dose-response protocol. However, it was observed during the course of this study that although the rates of ecdysone synthesis were comparable between members of a gland pair, rates of synthesis by larval and pupal PG varied considerably within a population, particularly when the glands were ativated by PTTH (see Fig. 2). This consistently observed variability in PG activity between different animals and the variation in the sensitivity of the glands to P’TTH from experiment to experiment, both in absolute amounts of ecdysone synthesized and in A, values (Figs. 4 and 5), precluded any approach other than the right vs. left dose-response protocol for assessing activation. This conclusion was reinforced by the fact that the day 3 larval and day 0 pupal PC were activated over a very narrow range of hormone concentration, 2.6- and 3.2-fold, respectively. This is in marked contrast to the results of a similar study in which day 3 larval brains activated day 3 larval PG with a dynamic range of response (neurohormone concentration) covering at least two orders of magnitude (Carrow et al., 1981). The dramatic nature of the PG response to the PTTH in the day 0 pupal brian thus necessitates quantification of PTTH activity by a dose-response protocol, regardless of the glands used in the assay.
The initial step in investigating the mechanism of PTTH activation of the PG was to determine the time-course kinetics of this activation. Time-courses of ecdysone synthesis by activated and nonactivated glands were defined to establish an optimal incubation time for other kinetics analyses of PG activation. Day 3 larval and day 0 pupal PG were used for the time-course studies, since these stages of development in ~an~~ca immediately precede a period of PTTH release, i.e. a head critical period, which is the time interval during development when the head is required for molting (see Granger and Bollenbacher, 1981), and thus these PG should be competent to respond to the neurohormone. The time-course of synthesis for precisely staged day 3 larval PG revealed that the nonactivated (control) PG exhibited a basal rate of ecdysone synthesis of 1.2 ng per hour per gland that was roughly linear for 6 h, with total synthesis in 6 h being 7.7 1L:1.1 ng per gland (Fig. 2). The contralateral PTTH-activated glands exhibited the expected increase in the rate of ecdysone synthesis of 7.7 ng per hour per gland that was
W.E. Bollenbacher et al.
34
b-
I-
i-
I-
i-
1 I
2
3
4
5
6
Time, hr Fig. 2. Time-course of ecdysone synthesis for PTTH-activated (0) and nonactivated (0) gate II day 3 last instar larval PC of Manduca sexta. Each datum point represents the mean ( f SEM) of 6 separate determinations.
linear for approx. 2.5 h. The synthesis rate decreased after this time, reaching an apparent plateau by 6 h, at which time 25 + 2.4 ng ecdysone per gland was synthesized. The greatest difference in the rates of linear synthesis between activated and nonactivated glands occurred at 2 h when an A, of 4.6 was established. The time-courses of synthesis for activated and nonactivated day 0 pupal PG (Fig. 3) was similar to those for the larval glands (Fig. 2) and to time-courses previously reported for these glands (Bollenbacher et al., 1979, 1980). The basal rate of ecdysone synthesis for these PG was 1.5 ng per hour per gland and synthesis was linear over the 6 h incubation period, with total synthesis at 6 h being 11.4 k 1.6 ng per gland. The contralateral, PTTH-activated glands exhibited a linear increase in ecdy-
Kmetics
ofprothoracicotropic
35
hormone action
Time,
hr
Fig. 3. Time-course of ecdysone synthesis for PTTH-activated (0) and nonactivated (0) day 0 pupal PG of Manduca sex@. Each datum point represents the mean ( f SEM) of 6 separate determinations.
sone synthesis for - 2.0 h at an average rate of 9.7 ng per hour per gland. From 2 to 6 ht the rate of synthesis decreased rapidly before reaching an apparent plateau; total ecdysone synthesis was 24.8 It 1.8 ng per gland. As with the larval PC, the greatest difference in rates of synthesis between activated and nonactivated pupal glands occurred at 2 hr, yielding an A, of 4. This occurred at the end of the period of linear synthesis by the activated pupal glands. Thus, the basal and activated time-courses of ecdysone synthesis by activated and nonactivated glands are comparable for day 3 larvae and day 0 pupae. Dose-response of activation Although the biosynthetic
capacity
of the PG and the rate kinetics
by
W. E. Bollenbacher
36
et al.
which this capacity is expressed are important biological properties of the gland, the sensitivity of the PG to PTTH could be of equal importance, especially if this sensitivity varied substantially during development. In the absence of differences in the kinetics of biosynthesis, the responsiveness of different glands to hormone would become the most critical aspect of gland activation. These differences in responsiveness would be expressed both in terms of the amount of PTTH (EDzO) necessary to elicit a half-maximal response (A,,) from the PG and the concentration range of PITH required for a linear increase in the A,. Thus, the defining both of these parameters for larval and pupal PG should indicate whether PC do indeed change in their sensitivity to PTTH during development. In addition, such studies would help define the optimal in vitro conditions for investigating the mechanism by which gland activation occurs. To assess the nature of the response of these larval and pupal PG to PTTH, dose-responses of PTTH activation of day 3 larval and day 0 pupal PG were generated (Fig. 4). The profiles of the A, values generated by dose-response were essentially the same for both pupal and larval glands, but full activation of the larval glands required less PTTH, suggesting that these glands were more sensitive to PTTH. Day 0 pupal
1 9-
?-
I
5-
I
3-
1 II 0.1
I 0.01 PTTH,
units
Fig. 4. Dose-response of PTTH activation of gate II day 3 last instar larval PC (0) and day 0 pupal PG (0) of Manduca senta. Each datum point represents the mean (+ SEM) of the A, values for 4-6 separate determinations.
Kinetics of prothoracicotropic
hormone action
31
PG were activated over a narrow dynamic range (0.04 to 0.13 units of PTTH), resulting in an ED,, of 0.076 units and an A,,, of - 7. The same PTTH preparation elicited a similar narrow dynamic range of response from the day 3 larval PG (0.025 to 0.065 units of PTTH) with an A,,, of - 6.5. However, the ED,, for the larval PG was 0.042 PTTH units, indicating that the larval glands used in this study are - 1.8 times more sensitive to a crude PTTH extract than are pupal glands. Repeated dose-response analyses of the larval and pupal glands with PTTH from day 1 pupal brains demonstrated that this difference was not only significant, but frequently even greater. Since these PG respond differently to PTTH, the sensitivity of the PG to the neurohormone apparently makes a substantial contribution to the developmental changes in gland activity. In addition to demonstrating basic differences in PG responsiveness, these dose-response data also defined the units of PTTH to be used in eliciting maximal activation of the PG for in vitro studies, the exposure time critical for PTTH activation, and the decay of the PTTH-activated response. Exposure
time for activation and decay of activated response
With the in vitro conditions defined from the dose-response data, it was possible to analyze both the kinetics of PG activation in terms of the time of exposure to PTTH necessary for activation, as well as the kinetics of the decay of activation. Defining these parameters could help elucidate the type of mechanism by which activation occurs, and may permit some speculation about the type, or mode, of PTTH release (e.g. pulsatile) that occurs in situ (Bollenbacher and Gilbert, 1981). To determine the critical period of time for which PG must be exposed to PTTH to achieve maximal activation, a pulsed preincubation protocol was used in which one gland of a pair was exposed to PTTH for a given period of time while the contralateral gland was preincubated in medium lacking PITH. Synthesis by each gland was then determined after a 2 h incubation in Grace’s medium. For day 3 larval PG, exposure to PTTH for increasing time intervals revealed an essentially immediate response, since an A, of 4 was attained after only a 0.5 min exposure (Fig. 5). This sharp increase in gland activity was followed by a more gradual increase in activation in response to longer exposure times, which plateaued at - 20 min with an A, of - 8. Since for this study the A,,, was 8, an A, of 3.5 represents the A,,, and thus 0.5 min represented the exposure time necessary for half-maximal activation of the larval PG. The immediate response of the PG to PTTH was also evident from the time-course of synthesis (Fig. 2) since a significant level of synthesis was reached very
W.E. Bollenbacher et al.
38
quickly with no lag phase apparent in the response. Day 0 pupal PG exhibited an acute response to PTTH similar to that of the larval gland, with an A, of - 4 at 0.5 min (Fig. 5). This initial response was followed by the same gradual increase in the A, with the same plateau at 20 min and an A,,, of - 8. The exposure time of the PG to PTTH necessary to half-maximally activate the pupal PG was 0.5 min, the same time required for a half-maximal response from larval glands. The A,,, of 8 observed for larval and pupal PG was greater than that noted in the dose-responses of activation for these PC (Fig. 4). This may reflect the inherent variability in activation by changing A,,, different crude preparations of PTTH and/or different lots of Grace’s medium. Thus, for each series of experiments the same Grace’s medium preparation of PTTH was used. There are many possible explanations for this variability, but since the absolute A,,, value is only important within a given set of experiments, this variability between experimental sets is not a problem. It is, however, acceptable to use the A,,, as a means of determining the number of PTTH units necessary to reach the half-maximal response (A,,) in these experiments, and then to compare
II -
9-
7-
a!
. 5-
3-
I 1
0
I
I
I
15 Exposure
I
I
I
I
to PTTH,
I
45
30 min
Fig. 5. Effect of varying times of exposure to PTTH on gate II day 3 last instar larval PG (0) and day 0 pupal PG (0) of Manduca sexta. Each datum point represents the mean (+ SEM) of the A, values for 6-9 separate determinations.
Kinetics of prothoracicotropic
39
hormone action
these ED,, values between different experiments. These comparisons are valid because the ED,, remains relatively constant with a specific experimental regimen irrespective of the A,,,. An analysis of the decay of PG activation by PTTH was made by determining changes in the rates of ecdysone synthesis by the glands after maximal activation was attained by preincubation with 0.5 units of PTTH for 20 min. For approx. 2 h after this preincubation, the rate of ecdysone synthesis by a population of right, day 3 larval PG postincubated in the absence of PTTH (- PTTH) appeared less than that for
25
I I I I
I I
I I I I I I I
I
I
I
I
I
2
Time,
hr
Fig. 6. Time-course of the decay of PTTH activation of gate II day 3 last instar larval PG of Manduca sexta. Dashed line denotes the time at which both control (right) and experimental (left) PG were preincubaied with PTTH. Control glands (0) were post incubated with PTTH while experimental glands (0) were postincubated without PTTH. Each datum point is the mean ( f SEM) of 6-9 separate determinations.
W.E. Bollenbacher et al.
40
the contralateral left glands still incubated in the presence of PTTH (+ PTTH) (Fig. 6), but this difference was not significant. The rates of synthesis in - PTTH and + PTTH PG incubations was 6 ng per hour per gland and 7.5 ng per hour per gland, respectively. Just after 2 h, however, the rate of synthesis by the - PTTH glands decreased dramatically, reaching an apparent plateau at - 3 h, while the +PTTH glands
20 -
15 -
z _ 2 :: w” 2
IO-
5-
1
2
Time,
hr
Fig. 7. Time-course of the decay of PTTH activation of day 0 pupal PG of Man&a sextu. Dashed line denotes the time at which both control (right) and experimental (left) PG were preincubated with PTTH. Control glands (0) were postincubated with PTTH while experimental glands (0) were postincubated without PTTH. Each datum point is the mean (2 SEM) of 6-9 separate determinations.
Kinetics
of prothoracicotropic
hormone
action
41
continued to synthesize ecdysone at the activated rate (7.5 ng per hour per gland). The rate kinetics for the decay of PTTH activation of day 0 pupal PG were similar to those for day 3 larval glands (Fig. 7). Ecdysone synthesis for pupal glands +PTTH and - PTTH was quantitatively indistinguishable for the first 1.6 h of the incubation period, with an average rate of 12.5 ng per hour per gland. After this time, the synthesis rate for PG in the absence of PTTH decreased sharply, reaching an apparent plateau at 3.0 h, while ecdysone synthesis by the glands +PTTH continued at a rate of 12.5 ng per hour per gland. In summary, studies determining the critical time for activation and decay of activation indicate that day 3 larval and day 0 pupal PG are activated immediately upon exposure to PTTH and rapidly reach maximal activation. Maximal activation persists for only about 1.5-2 h and shortly thereafter the glands return to a basal rate of ecdysone synthesis. Unfortunately, the kinetics of deactivation were such that a half-life could not be extrapolated. However, the fact that maximally activated larval and pupal PG become essentially inactive within 2.5 h in the absence of PTTH, with the actual decay in the activated response occurring in - 40 min, suggests that in order for PG to remain activated fro long periods of time in situ, the glands must be exposed either continuously or frequently enough to maintain the activated state.
DISCUSSION Kinetic parameters have been defined for an in vitro system suitable for investigating the mechanism of action of the insect prothoracicotropic hormone. This system is well suited for such an investigation because PTTH activates the prothoracic glands directly. Since this activation occurs in vitro, it can be assessed kinetically. The in vitro assay for PG activation by PTTH was substantially refined in the present study by simplifying the standard ecdysone RIA used to quantify ecdysone synthesis in vitro and by reducing the assay incubation time. These changes considerably improve the usefulness of this assay for conducting mechanistic studies. With this PG assay it was possible to obtain the information required for an investigation of the mechanism of action of PTTH: (1) there are specific critical times during which the PG must be exposed to PTTH for activation to occur; (2) the activated response decays at a specific rate; and (3) the PG from different developmental stages differ in their sensitivity to PTTH. Without knowledge of the above, it would not be
42
W. E. Bollenbacher et al.
possible to study the cascade of events that occur in response to PTTH activation. The sensitivity to PTTH of PG from day 3 larvae and day 0 pupae of Munduca was assessed from the kinetics of PG activation by this cerebral neurohormone. Glands from day 3 of the last larval instar and day 0 of the pupal period are developmentally competent to respond to PTTH and exhibited similar cativation kinetics in the time course studies with respect to both basal and activated rates of ecdysone synthesis. However, although dose-response profiles of activation were also similar for these glands, the sensitivity of the glands to PTTH was different, i.e. day 3 larval PG responded more acutely than day 0 pupal PG. This finding suggests that the interaction between neurohormone and gland may vary during postembryonic development and that the stage-specific sensitivity of the PG to PTTH may affect the progress of development. This suggestion is supported by the observation that the PG from diapausing Manduca pupae are refractory to PTTH (Bowen et al., 1983). That larval glands were more sensitive to PTTH extract than pupal PG initially suggested that the former might be more suitable for the in vitro assay (see Bollenbacher and Bowen, 1983). However, two major problems prevent the routine use of these glands: it is laborious to stage sufficient day 3 gate II last instar Manduca larvae for the large quantities of glands needed for most experiments; and, more important, the larval glands can be activated nonspecifically (Bollenbacher, unpublished observation). For example, a broad range of concentrations of heat-denatured proteolytic enzymes such as trypsin stimulate the level of ecdysone synthesis by these glands 2-3 times over the basal rate and appear to do so in a dose-dependent manner. Since proteolytic enzymes are present in all tissues and exhibit varying degrees of heat stability, heating of a tissue extract before assay would not necessarily prevent their nonspecific activation of the PG. Thus, larval PG could only be used in a study of PTTH activation if the activity in a sample had been previously demonstrated to be the neurohormone. It has recently been reported that PTTH extract from brains of Manduca larvae activates larval PG with a dynamic concentration range spanning more than two orders of magnitude (Carrow et al., 1981). This extremely broad response range contrasts markedly with that determined for larval PG in this study, as well as with the type of response observed with other insect neurohormones (e.g. Copenhaver and Truman, 1982; Taghert and Truman, 1982). On the basis of our observations, it is possible that the broad response range reported in this earlier study is a reflection of nonspecific activation of the larval PG. One attempt to elucidate the mechanism by which PTTH activates the
Kinetics of prorhoracicotropic
hormone action
43
PC in Manduca utilized an indirect and pharmacological approach. Nevertheless, the resulting data did suggest that the PG were activated by PTTH via CAMP (Vedeckis et al., 1976; Bollenbacher et al., 1980). In this study, a CAMP titer generated for the PG of Manduca during the last larval instar revealed a dramatic increase in CAMP during the first head critical period (HCP) of the instar, i.e. during the initial period of PTTH release on day 3 plus 16 h to day 4 plus 6 h, which is responsible for a small ecdysteroid surge and ultimately for wandering and commitment. Incubation of larval PG with CAMP or with dibutyryl CAMP in the presence of a phosphodiesterase inhibitor resulted in a dose-dependent stimulation of ecdysone synthesis, supporting the concept that CAMP is involved in the activation process. Further indirect evidence for cyclic nucleotide involvement in PTTH activation of the PG comes from the observations that phosphodiesterase inhibitors can effect an increase in the amount of ecdysone synthesized by larval PG in vitro, and that adenylate cyclase activity is present in PG, providing an enzymatic basis for the involvement of cyclic nucleotides (Vedeckis and Gilbert, 1973). However, the lack of a quantitative assay which could directly measure PG activation was in part responsible for the failure to demonstrate a direct causal relationship between PTTH activation of the PG and an increased content of cyclic nucleotide in these glands. With the in vitro assay for PTTH as defined in this study and with the availability of purified and partially purified PTTH (see below), these critical experiments have now been initiated. The intriguing .observation that PG from two different stages of development responded differently to PTTH extract (Bollenbacher and Gilbert, 1981, and present data) is potentially of great significance. The fact that day 3 larval glands are more sensitive to PTTH than the pupal glands suggests that there may be something different about the PTT’H receptors of these PG, e.g. more receptors resulting in greater sensitivity or mixed populations with different affinities. In addition to gland sensitivity, the chemical nature of PTTH may also contribute to the response of the PC to this neurohormone. With the purification of Manduca PTTH nearly complete, it appears that there are two molecular forms of the neurohormone (Bollenbacher and Gilbert, 1981; Bollenbather and Bowen, 1983). Each moiety activates PG in vitro in a dose-response manner similar to that obtained with crude PTTH, but the two forms have different physical properties, e.g. different molecular weights, isoelectric points and chemical stabilities (Bollenbacher and Gilbert, unpublished observations). Furthermore, different amounts of a particular form of PTTH may be necessary to activate PG from different development stages. While the large form of PTTH (M, - 29 000) elicits
44
W. E. Bollenbacher
et al.
similar dose-responses of activation and ED,, values from day 3 larval and day 0 pupal PG, the small form of the hormone (M, - 7000) more effectively activates larval glands, i.e. lo-20 times less hormone is needed to maximally activate these PG. Thus, the greater sensitivity of the larval PG to the small form of PTTH could explain their more acute response to a crude PTTH preparation. The biological significance of two PTTHs and of developmentalspecific PG sensitivity to these two neurohormones is conjectural at present. It is possible that the large and small PTTHs are released differentially during development or are released in precise ratios to elicit a certain level of response from the PG, resulting in a specific ecdysteroid titer that is critical for the promotion of a particular developmental process (Bollenbacher and Bowen, 1983). Further, the differential activation of the PG by these two PTTHs could imply that the two forms operate via different mechanisms, e.g. CAMP-dependent and CAMP-independent (Vedeckis et al., 1976). The in vitro system defined in this study should permit an investigation of such possibilities. The data on the kinetics of PG activation by PTTH and the decay of that activation also provide a hypothesis concerning the manner in which PTTH is released in situ. In Manduca, the head critical periods (HCP), i.e. periods of PTTH release, vary during larval and larval-pupal development, ranging from a few hours for the single HCP before a larval molt, to 20 h for the first of the two HCP in the last larval instar, to several days for the HCP before pupal-adult development (Truman, 1972, Truman and Riddiford, 1974; Bollenbacher, unpublished observation). PTTH release during these HCP could occur either in a rapid, continuous manner or as bursts of release, the latter being the mode characteristic of neurosecretory cells (see Maddrell and Nordmann, 1979). Regardless of the mode, the duration of release would be determined by select external and/or internal cues. The fact that activation of the PG by PTTH in vitro is immediate (0.5 min to achieve half-maximal activation) and is followed by a decay of that response in less than 3 h, suggests that in order for PC activity to be sustained in situ for periods from a few hours to several days, PTTH must be released from its neurohemal site throughout the HCP. If PTTH release occurs in bursts, it may involve only a single burst of approx. 1 h during the fourth instar, or pulsatile releases covering many hours or days during the first HCP of the fifth instar and the pupal HCP (Bollenbacher and Gilbert, 1981). This has been thoroughly investigated for the fourth instar HCP and the first HCP of the last instar and the resulting data reveal that PTTH release occurs in bursts: a single burst for the temporally short HCP of the fourth instar, and repeated bursting, i.e. pulsatile release comprising 3
Kinems
ofprothoracicotropic hormone action
45
bursts, for the long HCP of the fifth (last) instar (Bollenbacher and Gilbert, 1981; Bollenbacher and Bowen, 1983; Bollenbacher et al., unpublished observation). These initial findings on the modes of PTTH release are consistent with the present data on the exposure time critical for PTTH activation of the PG in vitro and the duration of the activated response. The occurrence of unique patterns of release at specific stages of development could also represent the release of one or the other molecular forms of PTTH, or some combination of the two forms. In summary, an in vitro system has been established that appears to simulate PG activation in situ and therefore can be used to probe the basic endocrinology of PTTH(s), particularly the mechanism(s) by which this neurohormone(s) controls the PG.
ACKNOWLEDGEMENTS This research was supported by NIH grants NS-18791 and AM-3 1642 and USDA grant 59-2176 to W.E. Bollenbacher and NIH grant AM-301 I8 to L.I. Gilbert.
REFERENCES Agui, N., Granger, N.A., Gilbert, L.I. and Bollenbacher, W.E. (1979) Proc. Natl. Acad. Sci. (U.S.A.) 76, 5694-5698. Bollenbacher, W.E. andBowen, M.F. (1983) In: Endocrinology of Insects, Eds.: H. Laufer and R.G.H. Downer (A.R. Liss, New York) (in press). Bollenbacher, W.E. and Gilbert, L.I. (1981) In: Neurosecretion: Molecules, Cells, Systems, Eds.: D.S. Farner and K. Lederis (Plenum, New York) pp. 361-370. Bollenbacher, W.E., Agui, N., Granger, N.A. and Gilbert, L.I. (1979) Proc. Natl. Acad. Sci. (U.S.A.) 76, 5148-5152. Bollenbacher, W.E., Agui, N., Granger, N.A. and Gilbert, L.I. (1980) In: Invertebrate Systems In Vitro, Eds.: E. Kurstak, K. Maramorosch and A. Dubendorfer (Elsevier/North-Holland, Amsterdam) pp. 253-271. Borst, D.W. and O’Connor, J.D. (1972) Sicence 178, 418-419. Bowen, M.F., Bollenbacher, W.E. and Gilbert, L.I. (1983) (submitted for publication). Carrow, G.M., Calabrese, R.L. and Williams, C.M. (1981) Proc. Natl. Acad. Sci. (U.S.A.) 78, 5866-5870. Copenhaver, P.F. and Truman, J.W. (1982) J. Insect Physiol. 28, 695-701. Gilbert, L.I., Goodman, W. and Bollenbacher, W.E. (1977) In: International Review of Biochemistry: Biochemistry of Lipids II, Vol. 14, Ed.: T.W. Goodman (University Park Press, Baltimore) pp. l-50. Granger, N.A. and Bollenbacher, W.E. (1981) In: Metamorphis, 2nd Edn. Eds.: L.I. Gilbert and E. Frieden (Plenum, New York) pp. 105-137. Maddrell, S.H.P. and Nordmann, J.J. (1979) Neurosecretion (Halsted Press, New York). Taghert, P.H. and Truman, J.W. (1982) J. Exp. Biol. 98, 373-383.
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Truman, J.W. and Riddiford, L.M. (1974) J. Exp. Biol. 60, 371-382. Vedeckis, W.V. and Gilbert, L.I. (1973) J. Insect Physiol. 19, 2445-2457. Vedeckis, W.V., Bollenbacher, W.E. and Gilbert. L.I. (1976) Mol. Cell. Endocrinol. 81-88. Vince, R. and Gilbert, L.I. (1977) Insect Biochem. 7, 115-120. Weevers, R. De G. (1966) J. Exp. Biol. 44, 163-175. Wielgus, J.J. and Gilbert, L.I. (1978) J. Insect Physiol. 24, 629-637.
5,