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Studies of programmed salivary gland regression during larval-pupal transformation in Chironomus thummi
Printed in Swden Copyright Q 1973 by Academic Press, Inc. All ?ightp of wprodurtion in any form wsrroed
Experimental
Cell Research 82
STUDIES OF PROGRAMMED DURING
(1973) 335-340
SALIVARY
LARVAL-PUPAL
GLAND
REGRESSION
TRANSFORMATION
CHIRONOMUS
IN
THUMMP
I. Acid Hydrolase Actioity KISSU SCHIN and H. LAUFER Department of Biological Sciences, State University of New York, College of Arts and Science, Plattsburgh, N. Y. 12901, USA
SUMMARY As the salivary gland breaks down during larval-pupal transformation it displays a sharp and significant increase in the activity of p-nitrophenylphosphatase and /I-glycerophosphatase. These increases are not due to concurrent enzyme synthesis, but to the activation of pre-existing enzyme or enzymes. These events parallel the breakdown of lysosomes.
The processesleading to breakdown of certain larval tissues and the formation of new adult tissues are the most characteristic events of post-embryonic development in many dipteran insects [l-3]. These events are believed to take place under the influence of the insect molting hormone, ecdysone [4]. The salivary glands of Chironomus (midges) are programmed for death, and function in the production of secretions only until the end of the prepupal period. They finally break down, and dissolution ultimately occurs some time after the termination of the larval-pupal molt [3]. In salivary gland cells of Chironomus one of the primary effects of ecdysone is considered to be the activation of specific gene loci, as indicated by changes in chromosomal puffing patterns [5]. Since puffing is known to_ be..- an indication of differential gene ac1 This paper is dedicated to the memory of my former teacher and colleague dr U. Clever who kindly reviewed this manuscript shortly before his untimely death. 23 -731808
tivity [6, 71, the salivary gland is well suited for studies of nuclear-cytoplasmic interactions, such as genetic control of programmed cell death in the salivary gland itself. Our efforts are focused on seeking the molecular basis for glandular regression. We have found that the glandular breakdown is accompanied by changes in the activity of acid hydrolases that are presumed to be lysosomal enzymes [3, 8, 9, lo]. Although lysosomes are known to contain many other, relatively specific acid hydrolases [l 1, 12’1,our present study deals only with one of the rather unspecific enzymes, acid phosphatase. In Chironomus, this is the only acid hydrolase that can be demonstrated and traced both within and outside of the lysosomes. Further, it can be determined quantitatively by using identical substrate molecules [2,13]. In the studies reported here, our primary interest was to determine whether any quantitative change in enzyme activity preceded Exptl Cell Res 82 (1973)
336 K. Schin & H. Laufer
or followed developmental alterations such as changes in the lysosomes or the onset of tissue breakdown, and to what extent genetic activity might be involved. MATERIALS AND METHODS Chironomus thummi were reared in the laboratory under the conditions described previously [14]. Thk criteria used for the classification by developmental stage were external and internal morphology [15]. For acid phosphatase assays and protein determinations a largenumber01salivaryglands were collected in a cold 0.9 % NaCl solution and then were rapidly homogenized in a glass homogenizer. The resulting homogenate was used directly for phosphatase assays and protein determinations [16]. A determination of phosphatase activity was made on the homogenate by incubating 0.2 ml of homogenate for 1 h at 38°C in a reaction mixture containing 8 mg ofp-nitrophenylphosphate dissolved in 1 ml of 0.05 M citrate buffer (pH 4.6). The reaction was terminated by the addition of 4 ml of 0.1 N NaOH, and the amount of p-nitrophenol released from thdp-nitrophenylphosphate was measured at 410 nm in a Beckman DU spectrophotometer. In addition to p-nitrophenylphosphate we also used /3-glycerophosphate as a substrate in order to determine if p-nitrophenylphosphatase was identical with ,!Lglycerophospharase. In rhis case the incubation mixture consisted of 0.5 ml of homogenate (equivalent to 20 salivary glands) and 0.5 ml of 0.1 M acetate buffer (pH 4.6) containing 0.2 moles of /&glycerophosphate. After 1 or 2 h of incubation the reaction was stopped by the addition of 1 ml of 10% TCA. This mixture was centrifuged for 10 min at 1 000 g and a phosphorus determination was carried out on the re&iig supernatant spectrophotometrically at 660 nm, to measure the /I-glycerophosphate digested [17]. Controls consisted of incubation systems to which NaOH or TCA were added prior to adding the homogenate. Experiments involying the inhibitor of acid phosphatase activity, sodium fluoride, were carried out on animals of different stages in a medium identical to the control medium except that 0.1 ml of 1 M NaF was added to 1 ml of incubation mixture. In other experiments involving the inhibition cf protein synthesis, animals in selected developmental stages were cultured for the desired length of time, before their glands were removed, on anormal diet to which had been added 20 pg/ml of cycloheximide. This concentration of cycloheximide was sufficient to inhibit completely the glandular protein synthesis within a few hours. In our previous work on the kinetics of enzyme activity in Chironomus tentans, the concentration of substrate p-nitrophenylphosphate required for each pair of glands to achieve V,,, was determined using only late prepupal glands or other larval glands but not early pupal glands. In the present studies we
found that pupal glandsrequireda substratecon-
centration at least four times greater than that for a pair of prepupal glands. The amount of p-nitrophenylphosphate used previously was sufficient to achieve Exptl
Cell
Res 82 (1973)
V,,, for the glandsof earlier stages,but was not enoughto giveV,,, for pupal glands. Therefore, the substrate concentration required for young pupal glands must be slightly higher than pr&io&ly reported [3, lo]. As a result of this finding, 8 mg/ml of p-nitrophenylphosphate was chosen for each incubation mixture containing a pair of glands. Under our experimental conditions enzyme activity increased linearly with increased enzyme cont. and incubation time, at least up to 3 h.
RESULTS AND DISCUSSION Acid phosphatase activity which is present in the salivary gland cell has been used as a marker in studies of possible correlations between cell death and changes in lysosomal activity. Phosphatase, with other hydrolytic enzymes [9, 10, 151,occurs in salivary glands throughout larval development until completion of glandular breakdown in early pupae (fig. 1). This enzyme is most active in the pH range between 4.2 and 4.6 and is almost inactive under neutral or alkaline conditions. Under optimal experimental conditions, the salivary gland exhibits the lowest hydrolase activity at the red head (RH) stage (l-day-old IVth instar) and the highest in early pupae (fig. 1). The increase in enzyme activity is
gL 8 l65 4 3
,,’
,’
,’
/l” :
Fin. 1. Abscissa: developmental stages, RH. Red-Head stage (earliest IVth in&r); ML, M;d-IVth~instar; LL, late IVth instar; EPP, early prepupa, LPP, late prepupa, EP, early pupa; ordinate: (rig,&)protein content in a pair of glands (pg); (left) amount of p-nitrophenol (PM x lo+) released from p-nitrophenylphosphate in a pair of glands incubated for 1 h at 38°C. Acid phosphatase activity and protein content per pair of salivary glands during larval-pupal transformation. O-O, phosphatase activity; O--O, protein content.
Programmed salivary gland regression. I
I /I i\
:‘-
before lxval pupal transformation lmin
Ibr 2~ ..A--A-1 3 4 after larval~pupal transformation
5
2. Ordinate: amount of p-nitrophenol (OD units x 10-l) released from p-nitrophenylphosphate in a pair of glands incubated for 1 h at 38°C. Acid phosphatase activity in early pupal glands after larval-pupal transformation.
Fig.
gradual and relatively small up to the early prepupal stage. Changes in phosphatase activity that occur during or at the transitional period from late prepupa to early are sharp and significant (fig. l), for enzyme activity increases more than six-fold as the prepupa completes its molt to become a young pupa. The increase in phosphatase activity is much more pronounced when compared to protein content, since during this period the amount of protein/gland falls sharply and significantly [3]. To examine whether the sharp increase of hydrolase activity in the pupa reflected an increased de novo synthesis of enzyme at the time of its activity increase, we carried out experiments to establish a precise timetable of the activity increase. Very old prepupae were selected and were allowed to develop toward pupation. At selected times following the larval-pupal molt, a pair of young pupal glands was collected and assayed for enzyme activity. Fig. 2 shows that within 1 min following pupation each pair of glands exhibited phosphatase activity nearly equivalent to that
337
of glands of 5 h old pupa. These results indicate that the increase in phosphatase activity previously observed in degenerating cells really occurred at the time of pupal ecdysis. In another attempt to establish a precise timetable for activity increase in phosphatase we collected 200 pairs of late prepupal glands with the expectation that if the increase nccurred during the molt some of the very old prepupal glands might display levels of phosphatase activity approximating that of pupal glands. In none of these experiments did late prepupal glands display phosphatase activity as high as that of early pupal glands. Even prepupae that would normally become pupa within 1 h exhibited phosphatase activity far less than that of pupal glands. On the basis of these observations we conclude that the increase in enzyme activity occurred almost concurrently with the instant of pupation. Experiments involving the use of cycloheximide, a potent inhibitor of protein synthesis, showed that the increase in acid hydrolase activity at pupation did not depend upon protein synthesis (table 1, c). These experiments were carried out on very late prepupae which would normally pupate within 1 to 6 h. ‘The prepupae were kept in 20 pug/ml of cycloheximide solution under constant aeration. Under these conditions less than 20% of late prepupae could complete pupation. Glands of very old late prepupae that did pupate, and that had been treated with cycloheximide for more than 2 h prior to pupation exhibited about a 5-fold increase in phosphatase activity after pupation, showing only a 17~, loss as compared with untreated pupal glands. Autoradiography showed that protein synthesis had virtually ceased in these treated glands. We also found that in many cycloheximide affected glands from very old prepupae that would have normally become pupae 4 to 6 h the level of phosphatase activity was nearly identical to that of untreated, Exptl Cell Res X2 (1973)
338 K. Schin & H. Laufer Table 1. Acid phosphatase activity of the salivary glands after cycloheximide treatment during late IVth instar larvae and prepupae stages Amount of p-nitrophenol released (OD/2 glands/l h) Cycloheximide (20 pg/ml) treated for Stage
Controls
LL EPP LPP Ia LPP IIb
0.025 0.026 0.071 0.065
4h
0.045 0.326’
8h 0.033 0.032 0.044
12 h
18 h
24 h
0.028
0.026 0.020 0.040
0.020 0 022 0.012
Enzyme activity was determined as described in the text. a LPP I (late prepupae I) are those prepupae that would normally pupate approx. 10 to 15 h later. b Late prepupae II are those prepupae that would normally pupate within 4 to 6 h. ’ This represents the average enzyme activity of salivary glands of young pupae that have been treated with cycloheximide for at least 2-4 h prior to the instant of pupation. Note the change in the enzyme activity from 0.065 to 0.326 in the absence of protein synthesis.
normal, early pupal glands. Treatment of very old prepupae with cycloheximide for periods longer than 6 h proved impractical as it resulted in a very high rate of mortality. The response of younger prepupae (EPP) and intermolt IVth instar larvae (LL) to cycloheximide was somewhat different from that of very old prepupae (table 1). In these larvae the average enzyme activity did not change even after 24 h in cycloheximide. Cycloheximide treatment of younger late prepupae (LPPI) for 4 h reduced by approx. 30% the total acid phosphatase activity in comparison with untreated mid-prepupae. A further cycloheximide treatment of these larvae of up to 18 h did not reduce the enzyme activity any further. It was only after about 24 h that we observed sharp decreases in phosphatase activity to approx. 20 % of the activity of that of normal, untreated younger late (LPPI). These experiments do not indicate the time of synthesis of acid phosphatase with certainty. However, because of the higher sensitivity of phosphatase activity of younger late prepupae (LPPI) to cycloheximide, as compared to other stages of larval-pupal deExptl
Cell Res 82 (1973)
velopment, it is tempting to assume that the greater portion of phosphatase is produced during the younger late prepupal period (LPPI), and that it remains inactive until shortly before the instant of pupation. Also, as indicated before, since the increase in activity continues in the absenceof protein synthesis in the glands of the pupating insect, it seemscertain that the increased activity observed in the young pupal gland is due to the activation of a preexisting enzyme rather than to a concurrent synthesis of the enzyme. Recent studies on Chironomus tentans protease activity reported that in old pharate pupae (prepupae) an acid protease is activated by a process which does not require concurrent protein synthesis [lo]. Although the exact timetable for activation of this hydrolase, most active at pH 3.5 and thus somewhat different from ours, is unknown, much the same picture with respect to changesin activity at the time of cell breakdown is seen with both enzymes. On the assumption that pupal glands might contain a factor or factors that could activate inactive enzymes, we incubated a mixture of prepupal and pupal salivary gland homoge-
Programmed
0.15 0 10 o.ot
EP
N
3. Ordinate: (a\ amount of o-nitroohenol COD/2 glands/l h) releaseh from p-nitrophe&phosphate; (h) amount of phosphorus (OD x 10-‘/Z glands/l h) released from 8-glvceronhosuhate. p-Nitronhenylphosphatase and -&glyc&ophosphat~se act&y -in the controls and NaF-treated alands. LPP. late prepupae; EP, early pupae; C, cor?trol; N, ‘NaFtreated. Fk.
nates with the expectation that this procedure might result in an activation of prepupal phosphates. We found that pupal glands did not contain such an activator, since the pre-pupal-pupal mixture had a phosphatase activity much less than that of pupal glands. In an attempt to answer the question as to whether or not the observed increase in phosphatase activity might be due to the release of enzyme from the lysosome we used ,%glycerophosphate, a substrate commonly used for lysosomal phosphatases, as a substitute for p-nitrophenylphosphate. The results were much the same except for the sensitivity of P-glycerophosphatase to NaF. For example, ,8-glycerophosphatase activity was nearly zero in both prepupa and early pupa when the NaF concentration in the incubation mixture
salivary
gland regression.
I
3 39
was 100 mM, whereas ca 24% of prepupal and 22 % of pupal phosphatase activity \fas insensitive to NaF when p-nitrophenylphosphate was the substrate. It appears that the gross increase in p-nitrophenylphosphatase activity does not only represent an increase in lysosomal phosphatas- activity, but also represents an increase in both NaF-sensitive and NaF-resistant phosphatase activity (f’ig. 3). Similar observations were reported TOI wax moths. where the p-nitrophenylphosphatases participating in the breakdown of \ilk glands were found to consist of at least two fractions; a NaF-sensitive, particle-bound phosphatase and a NaF-resistant. soluble phosphatase [18]. in the Chironomus pupal salivary glands the average net increase in NaF-sensitive phosphatase activity was 5.4fold and the average increase in NaF-resistant enzyme activity was 4.5fold. Since the increase in activity of NaF-sensitive phosphatase is close to that of ,!I-glycerophosphatase. and since the latter is highly fluoride-sensitive, it is possible that the NaF-sensitive /I-nitrophenylphosphatase is identical with the Iysosomal acid phosphatase. /+glycerophosphatase. Lysosomal phosphatase has previously been found to participate actively in the degeneration of salivary glands [2, 121. The free distribution of acid phosphalase activity in degenerating cytoplasmic areas, and the concurrent absence of lysosomes from such areas. was interpreted to mean that the relea.se of hydrolytic enzymes upon lysosomal breakdown is probably directly responsible for the lysis of salivary glands [12]. Indeed, in the silkmoth [19] and in Chironomus trntans [IO] it was reported that once cell degeneration had started, a cathepsin-like protease activity is no longer sedimentable, which indicated a release of this enzyme from lysosomes. Since lysosomal rupture, diffuse phosphatase activity, and a 600 “;, increase in acid hydrolase
340 K. Shin & H. Laufer 6. Beermann. W. J exotl zoo1 157 (1964) 49. activity occur almost concurrently with cell 7. Clever, U,’ Am zoo1 6 (1966) 33: ’ death, it seems reasonable to assume that 8. Schin, K S & Laufer, H, J cell bio155 (1972) 230a. these changes may be intimately linked, espe- 9. Rodems, A E, Henrikson, P A & Clever, U, Experientia 25 (1969) 686. cially since lysosomes are notably absent 10. Henrikson, P A & Clever, U, J insect physiol 18 (1972) 1981. from degenerating or degenerated areas of 11. DeDuve, C, Lysosomes (ed DeRueck & M P these cells [2]. Cameron) Ciba found symp p. 25. Churchill, I gratefullv acknowledge the technical assistance of M; Charles Edmiston. My deep gratitude goes to Drs R. Clark at SUNY. Plattsbureh. for his advice and for critical review of the manuschpi. This work was supported by SUNY Research Foundation, Grant 029-7132.
REFERENCES 1. Clever, U, Brookhaven symposia in biology 18 (1965) 242. 2. Schin, K S & Clever, U, Z Zellforsch 86 (1968) 262. 3. Laufer, H & Schin, K S, Can entomol 103 (1971) 454. 4. Bodenstein, D, Biol bull 84 (1943) 34. 5. Clever, U & Karlson, P, Exptl cell res 20 (1960) ___ b23.
Exptl Cell Res 82 (1973)
London (1963). 12. DeDuve, C, Pressman, B C, Gianetto, R, Wattiaux, R & Appelmans, F, J biol them 60 (1955) 604. 13. Schin, K S & Clever, U, Science 150 (1965) 1053. 14. Laufer, H & Wilson, M, Laboratory experiences in general and comparative endocrinology (ed R E Peter & A Gorbman) p. 185. Prentice-Hall, Englewood Cliffs, N. J. (1970). 15. Laufer, H, Nakase, Y & Vanderberg, J, Dev bio19 (1964) 367. 16. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall. R J. J biol them 193 (1971) 265. 17. Fiske, d H & Subbarow, Y, J diol &em 66 (1925) 375. 18. Aidells, B, Lockshin, R A & Cullin, A, J insect physiol 17 (1971) 857. 19. Lockshin, R A, J insect physiol 15 (1969) 1505. Keceived March 30, 1973
Experimental
Cell Research 82
STUDIES OF PROGRAMMED DURING
(1973) 335-340
SALIVARY
LARVAL-PUPAL
GLAND
REGRESSION
TRANSFORMATION
CHIRONOMUS
IN
THUMMP
I. Acid Hydrolase Actioity KISSU SCHIN and H. LAUFER Department of Biological Sciences, State University of New York, College of Arts and Science, Plattsburgh, N. Y. 12901, USA
SUMMARY As the salivary gland breaks down during larval-pupal transformation it displays a sharp and significant increase in the activity of p-nitrophenylphosphatase and /I-glycerophosphatase. These increases are not due to concurrent enzyme synthesis, but to the activation of pre-existing enzyme or enzymes. These events parallel the breakdown of lysosomes.
The processesleading to breakdown of certain larval tissues and the formation of new adult tissues are the most characteristic events of post-embryonic development in many dipteran insects [l-3]. These events are believed to take place under the influence of the insect molting hormone, ecdysone [4]. The salivary glands of Chironomus (midges) are programmed for death, and function in the production of secretions only until the end of the prepupal period. They finally break down, and dissolution ultimately occurs some time after the termination of the larval-pupal molt [3]. In salivary gland cells of Chironomus one of the primary effects of ecdysone is considered to be the activation of specific gene loci, as indicated by changes in chromosomal puffing patterns [5]. Since puffing is known to_ be..- an indication of differential gene ac1 This paper is dedicated to the memory of my former teacher and colleague dr U. Clever who kindly reviewed this manuscript shortly before his untimely death. 23 -731808
tivity [6, 71, the salivary gland is well suited for studies of nuclear-cytoplasmic interactions, such as genetic control of programmed cell death in the salivary gland itself. Our efforts are focused on seeking the molecular basis for glandular regression. We have found that the glandular breakdown is accompanied by changes in the activity of acid hydrolases that are presumed to be lysosomal enzymes [3, 8, 9, lo]. Although lysosomes are known to contain many other, relatively specific acid hydrolases [l 1, 12’1,our present study deals only with one of the rather unspecific enzymes, acid phosphatase. In Chironomus, this is the only acid hydrolase that can be demonstrated and traced both within and outside of the lysosomes. Further, it can be determined quantitatively by using identical substrate molecules [2,13]. In the studies reported here, our primary interest was to determine whether any quantitative change in enzyme activity preceded Exptl Cell Res 82 (1973)
336 K. Schin & H. Laufer
or followed developmental alterations such as changes in the lysosomes or the onset of tissue breakdown, and to what extent genetic activity might be involved. MATERIALS AND METHODS Chironomus thummi were reared in the laboratory under the conditions described previously [14]. Thk criteria used for the classification by developmental stage were external and internal morphology [15]. For acid phosphatase assays and protein determinations a largenumber01salivaryglands were collected in a cold 0.9 % NaCl solution and then were rapidly homogenized in a glass homogenizer. The resulting homogenate was used directly for phosphatase assays and protein determinations [16]. A determination of phosphatase activity was made on the homogenate by incubating 0.2 ml of homogenate for 1 h at 38°C in a reaction mixture containing 8 mg ofp-nitrophenylphosphate dissolved in 1 ml of 0.05 M citrate buffer (pH 4.6). The reaction was terminated by the addition of 4 ml of 0.1 N NaOH, and the amount of p-nitrophenol released from thdp-nitrophenylphosphate was measured at 410 nm in a Beckman DU spectrophotometer. In addition to p-nitrophenylphosphate we also used /3-glycerophosphate as a substrate in order to determine if p-nitrophenylphosphatase was identical with ,!Lglycerophospharase. In rhis case the incubation mixture consisted of 0.5 ml of homogenate (equivalent to 20 salivary glands) and 0.5 ml of 0.1 M acetate buffer (pH 4.6) containing 0.2 moles of /&glycerophosphate. After 1 or 2 h of incubation the reaction was stopped by the addition of 1 ml of 10% TCA. This mixture was centrifuged for 10 min at 1 000 g and a phosphorus determination was carried out on the re&iig supernatant spectrophotometrically at 660 nm, to measure the /I-glycerophosphate digested [17]. Controls consisted of incubation systems to which NaOH or TCA were added prior to adding the homogenate. Experiments involying the inhibitor of acid phosphatase activity, sodium fluoride, were carried out on animals of different stages in a medium identical to the control medium except that 0.1 ml of 1 M NaF was added to 1 ml of incubation mixture. In other experiments involving the inhibition cf protein synthesis, animals in selected developmental stages were cultured for the desired length of time, before their glands were removed, on anormal diet to which had been added 20 pg/ml of cycloheximide. This concentration of cycloheximide was sufficient to inhibit completely the glandular protein synthesis within a few hours. In our previous work on the kinetics of enzyme activity in Chironomus tentans, the concentration of substrate p-nitrophenylphosphate required for each pair of glands to achieve V,,, was determined using only late prepupal glands or other larval glands but not early pupal glands. In the present studies we
found that pupal glandsrequireda substratecon-
centration at least four times greater than that for a pair of prepupal glands. The amount of p-nitrophenylphosphate used previously was sufficient to achieve Exptl
Cell
Res 82 (1973)
V,,, for the glandsof earlier stages,but was not enoughto giveV,,, for pupal glands. Therefore, the substrate concentration required for young pupal glands must be slightly higher than pr&io&ly reported [3, lo]. As a result of this finding, 8 mg/ml of p-nitrophenylphosphate was chosen for each incubation mixture containing a pair of glands. Under our experimental conditions enzyme activity increased linearly with increased enzyme cont. and incubation time, at least up to 3 h.
RESULTS AND DISCUSSION Acid phosphatase activity which is present in the salivary gland cell has been used as a marker in studies of possible correlations between cell death and changes in lysosomal activity. Phosphatase, with other hydrolytic enzymes [9, 10, 151,occurs in salivary glands throughout larval development until completion of glandular breakdown in early pupae (fig. 1). This enzyme is most active in the pH range between 4.2 and 4.6 and is almost inactive under neutral or alkaline conditions. Under optimal experimental conditions, the salivary gland exhibits the lowest hydrolase activity at the red head (RH) stage (l-day-old IVth instar) and the highest in early pupae (fig. 1). The increase in enzyme activity is
gL 8 l65 4 3
,,’
,’
,’
/l” :
Fin. 1. Abscissa: developmental stages, RH. Red-Head stage (earliest IVth in&r); ML, M;d-IVth~instar; LL, late IVth instar; EPP, early prepupa, LPP, late prepupa, EP, early pupa; ordinate: (rig,&)protein content in a pair of glands (pg); (left) amount of p-nitrophenol (PM x lo+) released from p-nitrophenylphosphate in a pair of glands incubated for 1 h at 38°C. Acid phosphatase activity and protein content per pair of salivary glands during larval-pupal transformation. O-O, phosphatase activity; O--O, protein content.
Programmed salivary gland regression. I
I /I i\
:‘-
before lxval pupal transformation lmin
Ibr 2~ ..A--A-1 3 4 after larval~pupal transformation
5
2. Ordinate: amount of p-nitrophenol (OD units x 10-l) released from p-nitrophenylphosphate in a pair of glands incubated for 1 h at 38°C. Acid phosphatase activity in early pupal glands after larval-pupal transformation.
Fig.
gradual and relatively small up to the early prepupal stage. Changes in phosphatase activity that occur during or at the transitional period from late prepupa to early are sharp and significant (fig. l), for enzyme activity increases more than six-fold as the prepupa completes its molt to become a young pupa. The increase in phosphatase activity is much more pronounced when compared to protein content, since during this period the amount of protein/gland falls sharply and significantly [3]. To examine whether the sharp increase of hydrolase activity in the pupa reflected an increased de novo synthesis of enzyme at the time of its activity increase, we carried out experiments to establish a precise timetable of the activity increase. Very old prepupae were selected and were allowed to develop toward pupation. At selected times following the larval-pupal molt, a pair of young pupal glands was collected and assayed for enzyme activity. Fig. 2 shows that within 1 min following pupation each pair of glands exhibited phosphatase activity nearly equivalent to that
337
of glands of 5 h old pupa. These results indicate that the increase in phosphatase activity previously observed in degenerating cells really occurred at the time of pupal ecdysis. In another attempt to establish a precise timetable for activity increase in phosphatase we collected 200 pairs of late prepupal glands with the expectation that if the increase nccurred during the molt some of the very old prepupal glands might display levels of phosphatase activity approximating that of pupal glands. In none of these experiments did late prepupal glands display phosphatase activity as high as that of early pupal glands. Even prepupae that would normally become pupa within 1 h exhibited phosphatase activity far less than that of pupal glands. On the basis of these observations we conclude that the increase in enzyme activity occurred almost concurrently with the instant of pupation. Experiments involving the use of cycloheximide, a potent inhibitor of protein synthesis, showed that the increase in acid hydrolase activity at pupation did not depend upon protein synthesis (table 1, c). These experiments were carried out on very late prepupae which would normally pupate within 1 to 6 h. ‘The prepupae were kept in 20 pug/ml of cycloheximide solution under constant aeration. Under these conditions less than 20% of late prepupae could complete pupation. Glands of very old late prepupae that did pupate, and that had been treated with cycloheximide for more than 2 h prior to pupation exhibited about a 5-fold increase in phosphatase activity after pupation, showing only a 17~, loss as compared with untreated pupal glands. Autoradiography showed that protein synthesis had virtually ceased in these treated glands. We also found that in many cycloheximide affected glands from very old prepupae that would have normally become pupae 4 to 6 h the level of phosphatase activity was nearly identical to that of untreated, Exptl Cell Res X2 (1973)
338 K. Schin & H. Laufer Table 1. Acid phosphatase activity of the salivary glands after cycloheximide treatment during late IVth instar larvae and prepupae stages Amount of p-nitrophenol released (OD/2 glands/l h) Cycloheximide (20 pg/ml) treated for Stage
Controls
LL EPP LPP Ia LPP IIb
0.025 0.026 0.071 0.065
4h
0.045 0.326’
8h 0.033 0.032 0.044
12 h
18 h
24 h
0.028
0.026 0.020 0.040
0.020 0 022 0.012
Enzyme activity was determined as described in the text. a LPP I (late prepupae I) are those prepupae that would normally pupate approx. 10 to 15 h later. b Late prepupae II are those prepupae that would normally pupate within 4 to 6 h. ’ This represents the average enzyme activity of salivary glands of young pupae that have been treated with cycloheximide for at least 2-4 h prior to the instant of pupation. Note the change in the enzyme activity from 0.065 to 0.326 in the absence of protein synthesis.
normal, early pupal glands. Treatment of very old prepupae with cycloheximide for periods longer than 6 h proved impractical as it resulted in a very high rate of mortality. The response of younger prepupae (EPP) and intermolt IVth instar larvae (LL) to cycloheximide was somewhat different from that of very old prepupae (table 1). In these larvae the average enzyme activity did not change even after 24 h in cycloheximide. Cycloheximide treatment of younger late prepupae (LPPI) for 4 h reduced by approx. 30% the total acid phosphatase activity in comparison with untreated mid-prepupae. A further cycloheximide treatment of these larvae of up to 18 h did not reduce the enzyme activity any further. It was only after about 24 h that we observed sharp decreases in phosphatase activity to approx. 20 % of the activity of that of normal, untreated younger late (LPPI). These experiments do not indicate the time of synthesis of acid phosphatase with certainty. However, because of the higher sensitivity of phosphatase activity of younger late prepupae (LPPI) to cycloheximide, as compared to other stages of larval-pupal deExptl
Cell Res 82 (1973)
velopment, it is tempting to assume that the greater portion of phosphatase is produced during the younger late prepupal period (LPPI), and that it remains inactive until shortly before the instant of pupation. Also, as indicated before, since the increase in activity continues in the absenceof protein synthesis in the glands of the pupating insect, it seemscertain that the increased activity observed in the young pupal gland is due to the activation of a preexisting enzyme rather than to a concurrent synthesis of the enzyme. Recent studies on Chironomus tentans protease activity reported that in old pharate pupae (prepupae) an acid protease is activated by a process which does not require concurrent protein synthesis [lo]. Although the exact timetable for activation of this hydrolase, most active at pH 3.5 and thus somewhat different from ours, is unknown, much the same picture with respect to changesin activity at the time of cell breakdown is seen with both enzymes. On the assumption that pupal glands might contain a factor or factors that could activate inactive enzymes, we incubated a mixture of prepupal and pupal salivary gland homoge-
Programmed
0.15 0 10 o.ot
EP
N
3. Ordinate: (a\ amount of o-nitroohenol COD/2 glands/l h) releaseh from p-nitrophe&phosphate; (h) amount of phosphorus (OD x 10-‘/Z glands/l h) released from 8-glvceronhosuhate. p-Nitronhenylphosphatase and -&glyc&ophosphat~se act&y -in the controls and NaF-treated alands. LPP. late prepupae; EP, early pupae; C, cor?trol; N, ‘NaFtreated. Fk.
nates with the expectation that this procedure might result in an activation of prepupal phosphates. We found that pupal glands did not contain such an activator, since the pre-pupal-pupal mixture had a phosphatase activity much less than that of pupal glands. In an attempt to answer the question as to whether or not the observed increase in phosphatase activity might be due to the release of enzyme from the lysosome we used ,%glycerophosphate, a substrate commonly used for lysosomal phosphatases, as a substitute for p-nitrophenylphosphate. The results were much the same except for the sensitivity of P-glycerophosphatase to NaF. For example, ,8-glycerophosphatase activity was nearly zero in both prepupa and early pupa when the NaF concentration in the incubation mixture
salivary
gland regression.
I
3 39
was 100 mM, whereas ca 24% of prepupal and 22 % of pupal phosphatase activity \fas insensitive to NaF when p-nitrophenylphosphate was the substrate. It appears that the gross increase in p-nitrophenylphosphatase activity does not only represent an increase in lysosomal phosphatas- activity, but also represents an increase in both NaF-sensitive and NaF-resistant phosphatase activity (f’ig. 3). Similar observations were reported TOI wax moths. where the p-nitrophenylphosphatases participating in the breakdown of \ilk glands were found to consist of at least two fractions; a NaF-sensitive, particle-bound phosphatase and a NaF-resistant. soluble phosphatase [18]. in the Chironomus pupal salivary glands the average net increase in NaF-sensitive phosphatase activity was 5.4fold and the average increase in NaF-resistant enzyme activity was 4.5fold. Since the increase in activity of NaF-sensitive phosphatase is close to that of ,!I-glycerophosphatase. and since the latter is highly fluoride-sensitive, it is possible that the NaF-sensitive /I-nitrophenylphosphatase is identical with the Iysosomal acid phosphatase. /+glycerophosphatase. Lysosomal phosphatase has previously been found to participate actively in the degeneration of salivary glands [2, 121. The free distribution of acid phosphalase activity in degenerating cytoplasmic areas, and the concurrent absence of lysosomes from such areas. was interpreted to mean that the relea.se of hydrolytic enzymes upon lysosomal breakdown is probably directly responsible for the lysis of salivary glands [12]. Indeed, in the silkmoth [19] and in Chironomus trntans [IO] it was reported that once cell degeneration had started, a cathepsin-like protease activity is no longer sedimentable, which indicated a release of this enzyme from lysosomes. Since lysosomal rupture, diffuse phosphatase activity, and a 600 “;, increase in acid hydrolase
340 K. Shin & H. Laufer 6. Beermann. W. J exotl zoo1 157 (1964) 49. activity occur almost concurrently with cell 7. Clever, U,’ Am zoo1 6 (1966) 33: ’ death, it seems reasonable to assume that 8. Schin, K S & Laufer, H, J cell bio155 (1972) 230a. these changes may be intimately linked, espe- 9. Rodems, A E, Henrikson, P A & Clever, U, Experientia 25 (1969) 686. cially since lysosomes are notably absent 10. Henrikson, P A & Clever, U, J insect physiol 18 (1972) 1981. from degenerating or degenerated areas of 11. DeDuve, C, Lysosomes (ed DeRueck & M P these cells [2]. Cameron) Ciba found symp p. 25. Churchill, I gratefullv acknowledge the technical assistance of M; Charles Edmiston. My deep gratitude goes to Drs R. Clark at SUNY. Plattsbureh. for his advice and for critical review of the manuschpi. This work was supported by SUNY Research Foundation, Grant 029-7132.
REFERENCES 1. Clever, U, Brookhaven symposia in biology 18 (1965) 242. 2. Schin, K S & Clever, U, Z Zellforsch 86 (1968) 262. 3. Laufer, H & Schin, K S, Can entomol 103 (1971) 454. 4. Bodenstein, D, Biol bull 84 (1943) 34. 5. Clever, U & Karlson, P, Exptl cell res 20 (1960) ___ b23.
Exptl Cell Res 82 (1973)
London (1963). 12. DeDuve, C, Pressman, B C, Gianetto, R, Wattiaux, R & Appelmans, F, J biol them 60 (1955) 604. 13. Schin, K S & Clever, U, Science 150 (1965) 1053. 14. Laufer, H & Wilson, M, Laboratory experiences in general and comparative endocrinology (ed R E Peter & A Gorbman) p. 185. Prentice-Hall, Englewood Cliffs, N. J. (1970). 15. Laufer, H, Nakase, Y & Vanderberg, J, Dev bio19 (1964) 367. 16. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall. R J. J biol them 193 (1971) 265. 17. Fiske, d H & Subbarow, Y, J diol &em 66 (1925) 375. 18. Aidells, B, Lockshin, R A & Cullin, A, J insect physiol 17 (1971) 857. 19. Lockshin, R A, J insect physiol 15 (1969) 1505. Keceived March 30, 1973