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Effects of adenosine 3′,5′-cyclic monophosphate on membrane potential, nuclear volume, and puff size in Drosophila salivary gland in vitro
Copyright All rights
Q I972 by Academic Press, Inc. reproduction in my form reserved
of
Experimental
EFFECTS OF ADENOSINE MEMBRANE
POTENTIAL,
IN DROSOPHILA
Cell Research 73 (1972) 31l-318
3’,5’-CYCLIC
MONOPHOSPHATE
NUCLEAR
VOLUME,
SALIVARY
GLAND
ON
AND PUFF SIZE IN VITRO
L. RENSING and R. HARDELAND I. Zoologisches
Institut,
Universitiit
Gdttingen, D-34 Giittingen,
BRD
SUMMARY Salivary glands were isolated from third instar larvae of Drosophila melunoguster and kept in a culture medium. The effects of NB,02’-dibutvryl adenosine 3’.5’-cvclic monoohosohate (DBcAMP) were studied in the isolated salivary glands. 1O-8M D& increased-the &embrane potential of the salivary gland cells; this hyperpolarization was maxima1 about 6 h after addition of the agent. A significant enlargement of the nuclei could be observed 34 h after exposure to 1O-6 M DBcAMP. The latter effect could not be mimicked by theophylline, according to the lack of activators of adenyl cyclase in the incubation medium. On the contrary, theophylline caused even a decrease in nuclear volume, indicating an action of theophylline independent of cyclic AMP. Various other substances such as serotonin, histamine, and ethanol did not affect the size of the nuclei. The influence of DBcAMP on puffing was studied in different developmental stages of the third instar larvae. Depending on the age of the larvae, DBcAMP was to a more or less extent effective on different puffs. On the 3Lchromosome, the puffs 62E and 61D became enlarged by treatment with 1O-8M DBcAMP. On the X chromosome, the cyclic nucleotide caused an increase in size of puff 3C and a decrease in size of puff 2B. Accordingly, these two puffs, which are negatively correlated in the normal development, showed characteristic temporal differences in their responses to DBcAMP.
Adenosine 3’, 5’-cyclic monophosphate (CAMP) is well-known as a mediator of hormonal actions (reviews: ref. [9, 241). It plays the role of a so-called ‘second messenger’ [27], being responsible for the transmission of hormonal stimuli within the cell or within compartments of a cell. Since its mode of action is tissue-specific, CAMP exerts a great variety of different effects in different organs and organisms. At the molecular level, CAMP has been shown to activate various protein kinases [13], which in turn are able to regulate the activities of numerous other enzymes. On the other hand, it is known that CAMP does not solely act via activation mechanisms, but also influences the activities
of genes [14, 20, 291. In this paper, we shall present further evidence for the involvement of CAMP in the modulation of gene activities within an eucaryote system. The use of isolated Drosophila salivary glands allowed us to analyse the effects of the cyclic nucleotide by means of a microscope technique. Moreover, we investigated the influence of CAMP on the membrane potentials of the salivary gland cells, since it is known that CAMP takes part in the control of permeability of membranes. For instance, it has been demonstrated that CAMP leads to hyperpolarization in rat liver cells [S], and in cerebellar Purkinje cells [26]. As alterations in membrane diffusion rates are thought to Exptl Cell Res 73 (1972)
312
L. Rensing & R. Hardeland
Table 1. Classification of late third instar larvae of Drosophila
melanogaster
MiP, migration period; LMiP, late migration period; ESeP, early secretion period; LSeP, late secretion period MiP
LMiP
Crawling up and Crawling in the down upper regions ~. Outer cell membrane of each cell convex
ESeP
LSeP
Position fixed, shortening of the body Smooth surface of cell membranes Small amount of saliva present in squash preparation -
influence the puffing in dipterean polytene chromosomes [ll, 121, the effects on membrane potential could be of interest for the understanding of the actions of CAMP on the genetic level. MATERIALS
AND METHODS
Animals Third instar larvae of an inbred stock of Drosophila melanogaster Meigen were used in these experiments. They were kept at 25°C under a 12 : 12 h light-dark rCgime. The culture medium and other details have been described earlier [21]. An exact estimate of the age of each larva is very difficult: the stage when larvae crawl on the walls of the culture bottle lasts up to 12 h. Only the age of eary prepupae can be determined with an accuracy of 0.5 h. The prepupal stages, however, turned out to be not suitable for in vitro experiments with salivary glands so that we had to search for some behavioural properties of the larvae and some morphological criteria of the salivary glands to arrive at a qualitative classification of the third instar larvae (table I). Still, the borderline between these classes, especially of the migration period, cannot be defined exactly.
Large amount
used. They were connected, through a high-impedance preamplifier, to an oscilloscope and a strip chart recorder. Guiding the electrodes by a micromanipulator under visual control (magnification up to 60-fold), different cells of salivary glands were impaled and the potential of the impaling electrode with respect to the bath was recorded continuously. From the records, the differences of potential before and during each impalement were measured. Freshly excised glands of MiP larvae were used exclusively. They were supplied with a few drops of medium on a glass slide, contained by a paraffin ring, After each successful impalement, the electrode was withdrawn usually within 30 sec. It was then rapidly pointed, more or less randomly, at another cell in the vicinity and advanced again. Second or multiple re-impalements of a single cell cannot be excluded by this procedure. After having recorded l&20 control values in the normal medium at the start of an experiment, the fluid on the slide was drawn off and replaced by medium containing lO-3 M DBcAMP. Additional series of membrane potential measurements were then taken immediately following the administration of the agent, and after intervals as shown in fig. 1.
Nuclear volume
The salivary glands were prepared and kept in an artificial medium following a method which has been used in an earlier investigation [22]; we changed, however, the components of the medium [18] and adjusted the pH to 6.3. The main criterion for the living conditions of the glands in vitro was the appearance under phase contrast: if the whole gland appears dark and if the nucleus and the chromosomes are clearly visible, all functions of the cell tested so far seem to be normal [22].
All stages of late third instar larvae were used to measure the nuclear size. Salivary glands were excised together with the mouth parts and fat body, whereas the brain was removed. They were pipetted with several drops of medium on a slide with a paraffin ring (1 cm @) which had an opening on two sides and was afterwards covered by a coverslip. Shortly after this procedure, the longest and shortest diameters of individual nuclei were determined by means of phase contrast lenses (Zeiss) and a camera lucida. Both values were multiplied with each other to give an estimate of the nuclear size proportional to the true volume. Medium containing various agents was added at one side of the coverslip, the old medium removed at the other side.
Membrane potential
Puff size
Capillary microelectrodes filled with 3 M KC1 and having a resistance of about lo6 to 10’ ohms were
Larvae of different developmental stages were prepared and the salivary glands excised. In these ex-
Salivary glands
Exptl Cell Res 73 (1972)
Effects of cyclic AMP on Drosophila salivary gland
313
periments, however, each pair of salivary glands was separated, one half was treated with various agents especially DBcAMP, the other untreated half served as control. After an incubation of about 24 h, both halves were squashed in a mixture of orcein, lactic and acetic acids [19]. The puff size was determined by means of an oil immersion phase-contrast lens and a camera lucida, taking the width of the puff and of a certain reference band as a measure to calculate the puff ratio. The puff ratio [23] is defined as the ratio of a certain puff size to a certain band size and was chosen in order to eliminate differences due to different degrees of polyteny.
Chemicals The following chemical agents have been used: N”, 02’-dibutyryl adenosine 3’, 5’-cyclic monophosphate (“DBcAMP”) (CalBiochem); histamine (Fluka); serotonin hydrogenoxalate (Schuchardt); theophylline and ethanol (Merck).
Statistics For all points of the curves presented in the figures, the 3-fold standard error, or when required by the distribution of values, the 99% confidence interval is drawn in order to allow a quick estimate on reproducibility and significance.
RESULTS Membrane potential
After the addition of medium containing 1O-3 M N6, 02’-dibutyryl adenosine 3’,5’cyclic monophosphate (DBcAMP), the membrane potential of isolated salivary glands from MiP larvae changes significantly (fig. 1): 20-25 min after application of the agent a marked increase can be observed and a maximum is reached after 30-40 min. A decrease follows in the next 30-80 min. The hyperpolarization cannot be due to the change from living to in vitro conditions, because the potential was measured at different intervals from the excision of the gland (1-4 h). Incubation without DBcAMP did not result in any changes of membrane potential, neither shortly after excision nor after even 24 h. Nuclear volume
During the first hour of incubation with 10m5M DBcAMP, no changes in size of the
II,.
.., 010M30409607080
I
I.
.I
110 ml"
Fig. 1. Abscissa: time after addition of DBcAMP (min); ordinate: membrane potential (mV); left upper ordinate for (a), right ordinate for (b), left lower ordinate for (c). Effect of 1O-3 M DBcAMP on the membrane potential of salivary gland cells. (u-c) Three different glands. Data are expressed as means i. 3 S.E.
salivary gland nuclei can be detected; then a steady increase up to 140% can be observed until a maximum is reached after about 4 h (fig. 2). It is noteworthy, however, that a considerable variation occurs in the reaction to DBcAMP (table 2): cells of the same gland and even adjacent cells may differ very markedly in their relative response. Exptl Cell Res 73 (1972)
314 L. Rensing & R. Hardeland
Table 2. Effect of 1O-5 M DBcAMP on nuclear size in adjoining cells
1.30.
(A) shows an example of great differences, (B) an example of good conformity in the reactions of adjacent nuclei. Data expressed as nuclear size indices
1.20.
hours after addition of DBcAMP Nucleus
0
1
2
3
4
A
1.00 1.00 1.00 1.00 1.00 1.00
1.10 1.08 0.95 0.93 0.93 0.99
1.12 1.01 0.81 1.05 1.08 1.09
1.39 1.07 0.82 1.24 1.21 1.17
1.23 1.23 0.93 1.30 1.37 1.53
B
1 2 3 1 2 3
1.10 -
1.00.
0.90.
As a whole, the effect of 1O-5M DBcAMP on the nuclear size is considerable and significant. This raised the question whether or not lower and higher concentrations would be also effective. In our experiments, 1O-6 M DBcAMP did not increase the nuclear size significantly within 4 h. Higher concentrations (1O-4 M) produced somewhat greater effects than obtained by 1O-5M DBcAMP. Attempts were made to mimic the wellreproducible action of DBcAMP by treatment with 1O-3 M theophylline. This drug, which is known to inhibit a cyclic nucleotide degrading phosphodiesterase [5, 71, elevates the intracellular level of CAMP in the presence of active adenyl cyclase. However, administration of theophylline resulted in no enlargement of the nuclei, but rather in a sudden decrease of the nuclear volume. A small relative increase compared with the minimum value can be observed 2-3 h after addition of the drug (fig. 2). Other substances such as serotonin, histamine, and ethanol did not change the nuclear size significantly (table 3). Puff size
Apart from the effects on membrane potential and nuclear volume, the question whether Exptl Cell Res 73 (1972)
o.so-
0.70.
4---_ ----. /---------.___ --__ 1 Tr I I Y-4 0
1
2
3
L
5
Fig. 2. Abscissa: time after addition of the agents (hours); ordinate: nuclear size index (initial values of individual nuclei were set 1.0; all following values are referred to the initial values). Effects of 1O-6 M DBcAMP and 1O-s M theophylline on nuclear size in salivary glands. O--O, controls; O-O, 1O-5 M DBcAMP; A-A, 1O-3 M theophylline. The data represent the medians and the 99 % confidence intervals.
DBcAMP would also act on gene activities was of particular interest. In order to tackle this problem we measured the size of certain puffs on the 3L-chromosome and the X chromosome. The puff size is known to be correlated with RNA synthesis [3, 161. From our first experiments we learned that the developmental agewas of great importance for the effect of DBcAMP. Following the classification of developmental stages as defined in table 1, we determined the developmental age of the larvae tested. The puffs 3C, 2B, 63E, 62E, and 61D were found to react differently depending on the age of the larvae. When analysed by measuring the puff ratio (fig. 3), puff 63E
Effects of cyclic AMP on Drosophila salivary gland
315
Table 3. Nuclear size in salivary gland cells after treatment with various agents Data are expressed as nuclear size indices. The time intervals between addition of the agents and the measurements are represented by numbers in parentheses which are equivalent to hours
1.6
Treatment Serotonin 10m6M Serotonin 1O-5M Serotonin lo-& M + Theophylline 1O-4M Histamine 1O-5M Ethanol 0.2 M
w 62 E
1.01 (1) 0.96 (2) 0.97 (3) 1.3
0.94 (1) 0.98 (4) 0.94 (5) 1.2
0.86 (1) 0.88 (2) 0.88 (3) 0.85 (5)
1.05 (1) 0.98 (4) 1.01 (1) 1.03 (2) 1.03 (3)
1.6 1.5
did not increase in size significantly after treatment with DBcAMP. This may be due to the fact that the first two classes of larvae are too large to separate developmental stages of rather short duration. Puff 62E (fig. 3) is susceptible to DBcAMP mainly in an earlier developmental stage (MiP), where the only significant increase with respect to the controls can be registered. Puff 61D (fig. 3) is inducible during all developmental stages, somewhat more, however, during the later periods. Puff 3C (fig. 4) is significantly inducible during MiP, but not in all the later stages. Puff 2B (fig. 4), a development specific puff [2], which is inducible by ecdysone, behaves differently in comparison to the other puffs so far analysed: the control values are always larger than the DBcAMP values. It can be seen that the repression is largest, when the control values are high. This is true for the LSeP larvae and may also be true for a certain stage of MiP larvae following the release of ecdysone. This stage, however, may have been obscured by other stages of the same period.
1.4 1.3 1.2 MiP
LMiP
ESeP
LSeP
3. Abscissa: developmental stages; ordinate: puff ratio, as defined under Materials and Methods. Effects of 1O-3M DBcAMP on the size of the puffs 63E, 62E, and 61D in different developmental stages of the third instar larvae. 0 - 0, DBcAMP, 0 -0 , controls. Data represent means 1-3 S.E.
Fig.
After having demonstrated an influence of DBcAMP on puffing in different developmental stages, the dynamics of the increase or decrease in puff size seemed to be of interest. Salivary glands of MiP larvae were treated with 10~~ M DBcAMP and the puff size of 3C and 2B was measured after different periods of incubation (fig. 5). Maximum induction of puff size in 3C was achieved after 2 h-or, in some experiments, even after 0.75 h. By contrast with 3C, puff 2B, which gave only slight reactions in MiP larvae, did not show immediate effects of DBcAMP. Exptl Cell Res 73 (1972)
316 L. Rensing & R. Hardeland 1.6
1.2 1.1 1.0 1.6
28
1.5 14 1.3 1.2 1.1
:-:\‘: MiP
LMiP
ESeP
LSeP
Fig. 4. Abscissa: developmental stages; ordinate: puff ratio. Effects of 1O-3M DBcAMP on the size of the puffs 3C and 2B in different developmental stages of the third instar larvae. 0 - 0, DBcAMP; 0 - 0 , controls. Data represent means + 3 SE.
and CAMP show the same effects in the salivary gland of adult Calliphora. As the activator of adenyl cyclase has not yet been identified, it has not been possible to simulate the effect of CAMP by use of theophylline. Most likely, the enzyme is no longer active in the salivary gland kept in vitro, since adenyl cyclase activity usually varies within a few minutes after alterations in hormone concentration; therefore, the lack of activators in the incubation medium should result in a rapid decrease in activity of this enzyme. Consequently, theophylline, which is able to elevate intracellular CAMP levels by inhibiting a cyclic nucleotide degrading phosphodiesterase [5, 71, can only be effective if adenyl cyclase is in the active state; otherwise, treatment with theophylline should not result in any accumulation of CAMP. Although it can be easily understood that theophylline does not mimic the effects of CAMP in our experiments, this gives no explanation for why theophylline
DISCUSSION The present results indicate that CAMP plays an important role in the regulation of cellular processes in the larval salivary gland of Drosophila. However, it is still unknown what kind of agent could be responsible for the activation of adenyl cyclase and, hence, for the control of the intracellular CAMP level in this organ. The fact that the ecdysonspecific puff 2B is reduced in size after treatment with DBcAMP makes it very unlikely that the cyclic nucleotide could be a mediator of one of the actions of ecdysone.The further possibility that the CAMP level could be under the control of serotonin seems to be also excluded, since the biogenic amine did not show the effects observed with DBcAMP. This result differs from the finding of Berridge & Pate1[4] that serotonin Exptl Cell Res 73 (1972)
I 1
2
3
4
Fig. 5. Abscissa: time after addition (hours); ordinate: puff ratio.
5
6
of DBcAMP
Effects of 1O-s M DBcAMP on puff size of 3C and 2B in MiP larvae as a function of time. 0 - 0, DBcAMP; o -0 , controls. Data are expressed as means i3 S.E.
Effects of cyclic AMP on Drosophila salivary gland causes a decrease in nuclear size. One could speculate whether the methyl xanthine may interfere with other pathways of the purine metabolism. Competitive inhibition of RNA synthesis, e.g., would presumably cause a decrease in nuclear size. The reactions of different salivary gland cells to DBcAMP are not completely uniform. Surprisingly, even the adjoining cells often differ very much with respect to their variations in nuclear size. It is also evident from the inspection of the puffing pattern that there is no strict synchrony between the cells of a salivary gland. Since it is not clear to what degree and in what way neighbouring cells can influence each other, the possible physiological significance of these differences remains obscure. By contrast, the temporal variations in reactivity to DBcAMP, which can be observed at the chromosomal level, seem to be of importance. Obviously they reflect development-specific alterations in regulation of gene activities. Many puffs are only inducible for a short time within the development. Since DBcAMP did not prove to prolong these periods of inducibility, but rather seemed to be effective only when puff induction normally occurs, CAMP cannot be involved in the ‘switching-on’ of gene activation itself. Instead, CAMP appears to be a modulator and amplifier of gene activation. It is worth noting that, if this interpretation is correct, CAMP plays a role in the salivary gland which is comparable to that in microorganisms. Repression of bacterial genes cannot be overcome by CAMP, but after a gene has been derepressed, CAMP takes part in the control of gene activity [6, 10, 28, 291. Findings by Leenders et al. [14] seem to support our assumption on the role of CAMP in the salivary glands: in Drosophila hydei, CAMP amplified the puff-inducing potency of ecdysterone, whereas the cyclic nucleotide
317
alone did not change the puff size significantly. In our opinion, these data would corroborate our results that CAMP is not able to induce puffing in a non-activated chromosomal region, but rather amplifies inductive effects, regardless, perhaps, of the chemical nature of the inducing agent. However, CAMP cannot be regarded as a mediator of the action of ecdysone: in the experiments of Leenders et al., CAMP did not mimic the effect of the hormone; moreover, we are now able to demonstrate that DBcAMP can counteract ecdysone in the case of puff 2B. The puffs investigated become inducible at different stages of development. The phases at which these puffs normally occur (cf [l, 21) also seem to determine the phases of inducibility by CAMP. Interestingly, there is a case of antagonism between the two puffs from the X chromosome, 3C and 2B. In the normal development, 3C is only enlarged when 2B is small and vice versa [2]. This negative correlation also becomes well documented in our induction experiments. First, the effect of DBcAMP is contrary in both puffs: 3C becomes enlarged, whereas 2B shows a reduction in size after treatment with DBcAMP. Moreover, the phases of effectiveness are different in 3C and 2B. In the migration phase, MiP, 3C can be greatly induced, but the effect on puff 2B is relatively poor. When 3C ceases to be inducible by DBcAMP, 2B becomes enlarged and the effectiveness of DBcAMP increases. Furthermore, DBcAMP leads much faster to measurable alterations in puff size in the case of 3C as in 2B. It would be of great interest to know whether there may be any functional relationship between the three effects of DBcAMP reported here, i.e., the hyperpolarization of the cell membrane, the modification in puff size, and the increase in nuclear volume. Exptl Cell Res 73 (1972)
318 L. Rensing & R. Hardeland Such a correlation has already been taken into consideration for the larval and prepupal development, since the alterations in the puffing pattern [l, 21 are accompanied by changes in membrane potential [12, 151and in nuclear volume [22] within the course of ontogeny. Although no definite conclusions can be drawn so far from our experiments, some of the possibilities should be mentioned. As Kroeger [I 1, 121has pointed out, in the salivary gland there seemsto exist a relation between ion permeability and gene activity. Since the hyperpolarization precedes the alterations in puff size, it is at least imaginable that CAMP influences the puffing via control of ion permeability. It is not clear whether the increase in nuclear volume may be due to an alteration in permeability of the nuclear membrane or may be related to another function such as, e.g., RNA content. Since ethanol, which unspecifically enhances the permeability of membranes, failed to be effective, the influence of DBcAMP on nuclear size should be expected to be rather specific. It has not been the subject of this investigation to elucidate the mechanism of action of CAMP at the molecular level. However, it would be of particular interest to know how regulation of gene activities is affected by CAMP. In this context, it may be noted that a CAMP-dependent protein kinase has been found, which is rather specific for phosphorylation of histones [17, 251. It is, however, far from clear whether phosphorylation of histones could result in selective activation of certain genes. Moreover, the possibility should not be neglected that CAMP could act on transcription in a manner analogous to that one in microorganisms.
the membranepotential.The technicalassistence of Miss U. Hofacker is greatly acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft. REFERENCES 1. Ashburner, M, Chromosoma 21 (1967) 398. 2. - Ibid 27 (1969) 47. 3. Beermann, W, Differentiation and development (ed A G Bearn & S J Farber) p. 49. Little, Brown & Co., Boston (1964). 4. Berridge, M J & Patel, N G, Science 162 (1968) 462. 5. Butcher, R W & Sutherland, E W, J biol them 237 (1962) 1244. 6. Chambers, D A & Zubay, G, Proc natl acad sci US 63 (1969) 117. 7. Cheung, W Y, Biochemistry 6 (1967) 1079. 8. Friedmann, N, Somlyo, A V & Somlyo, A P, Science 171 (1971) 400. 9. Hardeland, R, Naturwiss 24 (1971) 199. 10. Jacquet, M & Kepes, A, Biochem biophys res commun 36 (1969) 84. 11. Kroeeer. H. Nature 200 (1963) 1234. 12. Kroeger; H; Exptl cell re4 41 (1966) 64. 13. Kuo, J F & Greengard, P, Proc natl acad sci US 64 (1969) 1349. 14. Leenders, H J, Wullems, G J & Berendes, H D, Exptl cell res 63 (1970) 159. 15. Loewenstein, W R & Kanno, Y, J gen physio146 (1963) 1123. 16. MecGelke, F, Naturwiss 21 (1959) 609. 17. Miyamoto, E, Kuo, J F & Greengard, P, Science 165 (1969) 63. 18. Nagel, G. In preparation. 19. Nicoletti, B, Drosophila inf serv 33 (1959) 181. 20. Pastan, I & Perlmann, R L, J biol them 244 (1969) 2226. 21. Rensing, L, Z vergl physiol 53 (1966) 62. 22. - J insect physiol 15 (1969) 2285. 23. Rensing, L & Hardeland, R, J insect physiol 13 (1967) 1547. 24. kobison, G A, Butcher, R W &Sutherland, E W, Ann rev biochem 37 (1968) 149. 25. Shepherd, G R, Noland, BJ & Hardin, J M, Exptl cell res 67 (1971) 474. 26. Siggins, G R, Oliver, A P, Hoffer, B J & Bloom, F E, Science 171 (1971) 192. 27. Sutherland, E W, (aye, I &Butcher, R W, Recent progr hormone res 21 (1965) 623. 28. Zubay, G & Chambers, D A, Cold Spring Harbor symp quant biol 34 (1969) 753. 29. Zubay, G, Schwartz, D & Beckwith, J, Proc natl acad sci US 66 (1970) 104.
Received December 1, 1971 Revised version received on February 16, 1972 The authors wish to thank Dr J. B. Walther, I. Zoologisches Institut, Universitlt GGttingen, who kindly helped us to carry out the measurements of Exptl
Cell Res 73 (1972)
Q I972 by Academic Press, Inc. reproduction in my form reserved
of
Experimental
EFFECTS OF ADENOSINE MEMBRANE
POTENTIAL,
IN DROSOPHILA
Cell Research 73 (1972) 31l-318
3’,5’-CYCLIC
MONOPHOSPHATE
NUCLEAR
VOLUME,
SALIVARY
GLAND
ON
AND PUFF SIZE IN VITRO
L. RENSING and R. HARDELAND I. Zoologisches
Institut,
Universitiit
Gdttingen, D-34 Giittingen,
BRD
SUMMARY Salivary glands were isolated from third instar larvae of Drosophila melunoguster and kept in a culture medium. The effects of NB,02’-dibutvryl adenosine 3’.5’-cvclic monoohosohate (DBcAMP) were studied in the isolated salivary glands. 1O-8M D& increased-the &embrane potential of the salivary gland cells; this hyperpolarization was maxima1 about 6 h after addition of the agent. A significant enlargement of the nuclei could be observed 34 h after exposure to 1O-6 M DBcAMP. The latter effect could not be mimicked by theophylline, according to the lack of activators of adenyl cyclase in the incubation medium. On the contrary, theophylline caused even a decrease in nuclear volume, indicating an action of theophylline independent of cyclic AMP. Various other substances such as serotonin, histamine, and ethanol did not affect the size of the nuclei. The influence of DBcAMP on puffing was studied in different developmental stages of the third instar larvae. Depending on the age of the larvae, DBcAMP was to a more or less extent effective on different puffs. On the 3Lchromosome, the puffs 62E and 61D became enlarged by treatment with 1O-8M DBcAMP. On the X chromosome, the cyclic nucleotide caused an increase in size of puff 3C and a decrease in size of puff 2B. Accordingly, these two puffs, which are negatively correlated in the normal development, showed characteristic temporal differences in their responses to DBcAMP.
Adenosine 3’, 5’-cyclic monophosphate (CAMP) is well-known as a mediator of hormonal actions (reviews: ref. [9, 241). It plays the role of a so-called ‘second messenger’ [27], being responsible for the transmission of hormonal stimuli within the cell or within compartments of a cell. Since its mode of action is tissue-specific, CAMP exerts a great variety of different effects in different organs and organisms. At the molecular level, CAMP has been shown to activate various protein kinases [13], which in turn are able to regulate the activities of numerous other enzymes. On the other hand, it is known that CAMP does not solely act via activation mechanisms, but also influences the activities
of genes [14, 20, 291. In this paper, we shall present further evidence for the involvement of CAMP in the modulation of gene activities within an eucaryote system. The use of isolated Drosophila salivary glands allowed us to analyse the effects of the cyclic nucleotide by means of a microscope technique. Moreover, we investigated the influence of CAMP on the membrane potentials of the salivary gland cells, since it is known that CAMP takes part in the control of permeability of membranes. For instance, it has been demonstrated that CAMP leads to hyperpolarization in rat liver cells [S], and in cerebellar Purkinje cells [26]. As alterations in membrane diffusion rates are thought to Exptl Cell Res 73 (1972)
312
L. Rensing & R. Hardeland
Table 1. Classification of late third instar larvae of Drosophila
melanogaster
MiP, migration period; LMiP, late migration period; ESeP, early secretion period; LSeP, late secretion period MiP
LMiP
Crawling up and Crawling in the down upper regions ~. Outer cell membrane of each cell convex
ESeP
LSeP
Position fixed, shortening of the body Smooth surface of cell membranes Small amount of saliva present in squash preparation -
influence the puffing in dipterean polytene chromosomes [ll, 121, the effects on membrane potential could be of interest for the understanding of the actions of CAMP on the genetic level. MATERIALS
AND METHODS
Animals Third instar larvae of an inbred stock of Drosophila melanogaster Meigen were used in these experiments. They were kept at 25°C under a 12 : 12 h light-dark rCgime. The culture medium and other details have been described earlier [21]. An exact estimate of the age of each larva is very difficult: the stage when larvae crawl on the walls of the culture bottle lasts up to 12 h. Only the age of eary prepupae can be determined with an accuracy of 0.5 h. The prepupal stages, however, turned out to be not suitable for in vitro experiments with salivary glands so that we had to search for some behavioural properties of the larvae and some morphological criteria of the salivary glands to arrive at a qualitative classification of the third instar larvae (table I). Still, the borderline between these classes, especially of the migration period, cannot be defined exactly.
Large amount
used. They were connected, through a high-impedance preamplifier, to an oscilloscope and a strip chart recorder. Guiding the electrodes by a micromanipulator under visual control (magnification up to 60-fold), different cells of salivary glands were impaled and the potential of the impaling electrode with respect to the bath was recorded continuously. From the records, the differences of potential before and during each impalement were measured. Freshly excised glands of MiP larvae were used exclusively. They were supplied with a few drops of medium on a glass slide, contained by a paraffin ring, After each successful impalement, the electrode was withdrawn usually within 30 sec. It was then rapidly pointed, more or less randomly, at another cell in the vicinity and advanced again. Second or multiple re-impalements of a single cell cannot be excluded by this procedure. After having recorded l&20 control values in the normal medium at the start of an experiment, the fluid on the slide was drawn off and replaced by medium containing lO-3 M DBcAMP. Additional series of membrane potential measurements were then taken immediately following the administration of the agent, and after intervals as shown in fig. 1.
Nuclear volume
The salivary glands were prepared and kept in an artificial medium following a method which has been used in an earlier investigation [22]; we changed, however, the components of the medium [18] and adjusted the pH to 6.3. The main criterion for the living conditions of the glands in vitro was the appearance under phase contrast: if the whole gland appears dark and if the nucleus and the chromosomes are clearly visible, all functions of the cell tested so far seem to be normal [22].
All stages of late third instar larvae were used to measure the nuclear size. Salivary glands were excised together with the mouth parts and fat body, whereas the brain was removed. They were pipetted with several drops of medium on a slide with a paraffin ring (1 cm @) which had an opening on two sides and was afterwards covered by a coverslip. Shortly after this procedure, the longest and shortest diameters of individual nuclei were determined by means of phase contrast lenses (Zeiss) and a camera lucida. Both values were multiplied with each other to give an estimate of the nuclear size proportional to the true volume. Medium containing various agents was added at one side of the coverslip, the old medium removed at the other side.
Membrane potential
Puff size
Capillary microelectrodes filled with 3 M KC1 and having a resistance of about lo6 to 10’ ohms were
Larvae of different developmental stages were prepared and the salivary glands excised. In these ex-
Salivary glands
Exptl Cell Res 73 (1972)
Effects of cyclic AMP on Drosophila salivary gland
313
periments, however, each pair of salivary glands was separated, one half was treated with various agents especially DBcAMP, the other untreated half served as control. After an incubation of about 24 h, both halves were squashed in a mixture of orcein, lactic and acetic acids [19]. The puff size was determined by means of an oil immersion phase-contrast lens and a camera lucida, taking the width of the puff and of a certain reference band as a measure to calculate the puff ratio. The puff ratio [23] is defined as the ratio of a certain puff size to a certain band size and was chosen in order to eliminate differences due to different degrees of polyteny.
Chemicals The following chemical agents have been used: N”, 02’-dibutyryl adenosine 3’, 5’-cyclic monophosphate (“DBcAMP”) (CalBiochem); histamine (Fluka); serotonin hydrogenoxalate (Schuchardt); theophylline and ethanol (Merck).
Statistics For all points of the curves presented in the figures, the 3-fold standard error, or when required by the distribution of values, the 99% confidence interval is drawn in order to allow a quick estimate on reproducibility and significance.
RESULTS Membrane potential
After the addition of medium containing 1O-3 M N6, 02’-dibutyryl adenosine 3’,5’cyclic monophosphate (DBcAMP), the membrane potential of isolated salivary glands from MiP larvae changes significantly (fig. 1): 20-25 min after application of the agent a marked increase can be observed and a maximum is reached after 30-40 min. A decrease follows in the next 30-80 min. The hyperpolarization cannot be due to the change from living to in vitro conditions, because the potential was measured at different intervals from the excision of the gland (1-4 h). Incubation without DBcAMP did not result in any changes of membrane potential, neither shortly after excision nor after even 24 h. Nuclear volume
During the first hour of incubation with 10m5M DBcAMP, no changes in size of the
II,.
.., 010M30409607080
I
I.
.I
110 ml"
Fig. 1. Abscissa: time after addition of DBcAMP (min); ordinate: membrane potential (mV); left upper ordinate for (a), right ordinate for (b), left lower ordinate for (c). Effect of 1O-3 M DBcAMP on the membrane potential of salivary gland cells. (u-c) Three different glands. Data are expressed as means i. 3 S.E.
salivary gland nuclei can be detected; then a steady increase up to 140% can be observed until a maximum is reached after about 4 h (fig. 2). It is noteworthy, however, that a considerable variation occurs in the reaction to DBcAMP (table 2): cells of the same gland and even adjacent cells may differ very markedly in their relative response. Exptl Cell Res 73 (1972)
314 L. Rensing & R. Hardeland
Table 2. Effect of 1O-5 M DBcAMP on nuclear size in adjoining cells
1.30.
(A) shows an example of great differences, (B) an example of good conformity in the reactions of adjacent nuclei. Data expressed as nuclear size indices
1.20.
hours after addition of DBcAMP Nucleus
0
1
2
3
4
A
1.00 1.00 1.00 1.00 1.00 1.00
1.10 1.08 0.95 0.93 0.93 0.99
1.12 1.01 0.81 1.05 1.08 1.09
1.39 1.07 0.82 1.24 1.21 1.17
1.23 1.23 0.93 1.30 1.37 1.53
B
1 2 3 1 2 3
1.10 -
1.00.
0.90.
As a whole, the effect of 1O-5M DBcAMP on the nuclear size is considerable and significant. This raised the question whether or not lower and higher concentrations would be also effective. In our experiments, 1O-6 M DBcAMP did not increase the nuclear size significantly within 4 h. Higher concentrations (1O-4 M) produced somewhat greater effects than obtained by 1O-5M DBcAMP. Attempts were made to mimic the wellreproducible action of DBcAMP by treatment with 1O-3 M theophylline. This drug, which is known to inhibit a cyclic nucleotide degrading phosphodiesterase [5, 71, elevates the intracellular level of CAMP in the presence of active adenyl cyclase. However, administration of theophylline resulted in no enlargement of the nuclei, but rather in a sudden decrease of the nuclear volume. A small relative increase compared with the minimum value can be observed 2-3 h after addition of the drug (fig. 2). Other substances such as serotonin, histamine, and ethanol did not change the nuclear size significantly (table 3). Puff size
Apart from the effects on membrane potential and nuclear volume, the question whether Exptl Cell Res 73 (1972)
o.so-
0.70.
4---_ ----. /---------.___ --__ 1 Tr I I Y-4 0
1
2
3
L
5
Fig. 2. Abscissa: time after addition of the agents (hours); ordinate: nuclear size index (initial values of individual nuclei were set 1.0; all following values are referred to the initial values). Effects of 1O-6 M DBcAMP and 1O-s M theophylline on nuclear size in salivary glands. O--O, controls; O-O, 1O-5 M DBcAMP; A-A, 1O-3 M theophylline. The data represent the medians and the 99 % confidence intervals.
DBcAMP would also act on gene activities was of particular interest. In order to tackle this problem we measured the size of certain puffs on the 3L-chromosome and the X chromosome. The puff size is known to be correlated with RNA synthesis [3, 161. From our first experiments we learned that the developmental agewas of great importance for the effect of DBcAMP. Following the classification of developmental stages as defined in table 1, we determined the developmental age of the larvae tested. The puffs 3C, 2B, 63E, 62E, and 61D were found to react differently depending on the age of the larvae. When analysed by measuring the puff ratio (fig. 3), puff 63E
Effects of cyclic AMP on Drosophila salivary gland
315
Table 3. Nuclear size in salivary gland cells after treatment with various agents Data are expressed as nuclear size indices. The time intervals between addition of the agents and the measurements are represented by numbers in parentheses which are equivalent to hours
1.6
Treatment Serotonin 10m6M Serotonin 1O-5M Serotonin lo-& M + Theophylline 1O-4M Histamine 1O-5M Ethanol 0.2 M
w 62 E
1.01 (1) 0.96 (2) 0.97 (3) 1.3
0.94 (1) 0.98 (4) 0.94 (5) 1.2
0.86 (1) 0.88 (2) 0.88 (3) 0.85 (5)
1.05 (1) 0.98 (4) 1.01 (1) 1.03 (2) 1.03 (3)
1.6 1.5
did not increase in size significantly after treatment with DBcAMP. This may be due to the fact that the first two classes of larvae are too large to separate developmental stages of rather short duration. Puff 62E (fig. 3) is susceptible to DBcAMP mainly in an earlier developmental stage (MiP), where the only significant increase with respect to the controls can be registered. Puff 61D (fig. 3) is inducible during all developmental stages, somewhat more, however, during the later periods. Puff 3C (fig. 4) is significantly inducible during MiP, but not in all the later stages. Puff 2B (fig. 4), a development specific puff [2], which is inducible by ecdysone, behaves differently in comparison to the other puffs so far analysed: the control values are always larger than the DBcAMP values. It can be seen that the repression is largest, when the control values are high. This is true for the LSeP larvae and may also be true for a certain stage of MiP larvae following the release of ecdysone. This stage, however, may have been obscured by other stages of the same period.
1.4 1.3 1.2 MiP
LMiP
ESeP
LSeP
3. Abscissa: developmental stages; ordinate: puff ratio, as defined under Materials and Methods. Effects of 1O-3M DBcAMP on the size of the puffs 63E, 62E, and 61D in different developmental stages of the third instar larvae. 0 - 0, DBcAMP, 0 -0 , controls. Data represent means 1-3 S.E.
Fig.
After having demonstrated an influence of DBcAMP on puffing in different developmental stages, the dynamics of the increase or decrease in puff size seemed to be of interest. Salivary glands of MiP larvae were treated with 10~~ M DBcAMP and the puff size of 3C and 2B was measured after different periods of incubation (fig. 5). Maximum induction of puff size in 3C was achieved after 2 h-or, in some experiments, even after 0.75 h. By contrast with 3C, puff 2B, which gave only slight reactions in MiP larvae, did not show immediate effects of DBcAMP. Exptl Cell Res 73 (1972)
316 L. Rensing & R. Hardeland 1.6
1.2 1.1 1.0 1.6
28
1.5 14 1.3 1.2 1.1
:-:\‘: MiP
LMiP
ESeP
LSeP
Fig. 4. Abscissa: developmental stages; ordinate: puff ratio. Effects of 1O-3M DBcAMP on the size of the puffs 3C and 2B in different developmental stages of the third instar larvae. 0 - 0, DBcAMP; 0 - 0 , controls. Data represent means + 3 SE.
and CAMP show the same effects in the salivary gland of adult Calliphora. As the activator of adenyl cyclase has not yet been identified, it has not been possible to simulate the effect of CAMP by use of theophylline. Most likely, the enzyme is no longer active in the salivary gland kept in vitro, since adenyl cyclase activity usually varies within a few minutes after alterations in hormone concentration; therefore, the lack of activators in the incubation medium should result in a rapid decrease in activity of this enzyme. Consequently, theophylline, which is able to elevate intracellular CAMP levels by inhibiting a cyclic nucleotide degrading phosphodiesterase [5, 71, can only be effective if adenyl cyclase is in the active state; otherwise, treatment with theophylline should not result in any accumulation of CAMP. Although it can be easily understood that theophylline does not mimic the effects of CAMP in our experiments, this gives no explanation for why theophylline
DISCUSSION The present results indicate that CAMP plays an important role in the regulation of cellular processes in the larval salivary gland of Drosophila. However, it is still unknown what kind of agent could be responsible for the activation of adenyl cyclase and, hence, for the control of the intracellular CAMP level in this organ. The fact that the ecdysonspecific puff 2B is reduced in size after treatment with DBcAMP makes it very unlikely that the cyclic nucleotide could be a mediator of one of the actions of ecdysone.The further possibility that the CAMP level could be under the control of serotonin seems to be also excluded, since the biogenic amine did not show the effects observed with DBcAMP. This result differs from the finding of Berridge & Pate1[4] that serotonin Exptl Cell Res 73 (1972)
I 1
2
3
4
Fig. 5. Abscissa: time after addition (hours); ordinate: puff ratio.
5
6
of DBcAMP
Effects of 1O-s M DBcAMP on puff size of 3C and 2B in MiP larvae as a function of time. 0 - 0, DBcAMP; o -0 , controls. Data are expressed as means i3 S.E.
Effects of cyclic AMP on Drosophila salivary gland causes a decrease in nuclear size. One could speculate whether the methyl xanthine may interfere with other pathways of the purine metabolism. Competitive inhibition of RNA synthesis, e.g., would presumably cause a decrease in nuclear size. The reactions of different salivary gland cells to DBcAMP are not completely uniform. Surprisingly, even the adjoining cells often differ very much with respect to their variations in nuclear size. It is also evident from the inspection of the puffing pattern that there is no strict synchrony between the cells of a salivary gland. Since it is not clear to what degree and in what way neighbouring cells can influence each other, the possible physiological significance of these differences remains obscure. By contrast, the temporal variations in reactivity to DBcAMP, which can be observed at the chromosomal level, seem to be of importance. Obviously they reflect development-specific alterations in regulation of gene activities. Many puffs are only inducible for a short time within the development. Since DBcAMP did not prove to prolong these periods of inducibility, but rather seemed to be effective only when puff induction normally occurs, CAMP cannot be involved in the ‘switching-on’ of gene activation itself. Instead, CAMP appears to be a modulator and amplifier of gene activation. It is worth noting that, if this interpretation is correct, CAMP plays a role in the salivary gland which is comparable to that in microorganisms. Repression of bacterial genes cannot be overcome by CAMP, but after a gene has been derepressed, CAMP takes part in the control of gene activity [6, 10, 28, 291. Findings by Leenders et al. [14] seem to support our assumption on the role of CAMP in the salivary glands: in Drosophila hydei, CAMP amplified the puff-inducing potency of ecdysterone, whereas the cyclic nucleotide
317
alone did not change the puff size significantly. In our opinion, these data would corroborate our results that CAMP is not able to induce puffing in a non-activated chromosomal region, but rather amplifies inductive effects, regardless, perhaps, of the chemical nature of the inducing agent. However, CAMP cannot be regarded as a mediator of the action of ecdysone: in the experiments of Leenders et al., CAMP did not mimic the effect of the hormone; moreover, we are now able to demonstrate that DBcAMP can counteract ecdysone in the case of puff 2B. The puffs investigated become inducible at different stages of development. The phases at which these puffs normally occur (cf [l, 21) also seem to determine the phases of inducibility by CAMP. Interestingly, there is a case of antagonism between the two puffs from the X chromosome, 3C and 2B. In the normal development, 3C is only enlarged when 2B is small and vice versa [2]. This negative correlation also becomes well documented in our induction experiments. First, the effect of DBcAMP is contrary in both puffs: 3C becomes enlarged, whereas 2B shows a reduction in size after treatment with DBcAMP. Moreover, the phases of effectiveness are different in 3C and 2B. In the migration phase, MiP, 3C can be greatly induced, but the effect on puff 2B is relatively poor. When 3C ceases to be inducible by DBcAMP, 2B becomes enlarged and the effectiveness of DBcAMP increases. Furthermore, DBcAMP leads much faster to measurable alterations in puff size in the case of 3C as in 2B. It would be of great interest to know whether there may be any functional relationship between the three effects of DBcAMP reported here, i.e., the hyperpolarization of the cell membrane, the modification in puff size, and the increase in nuclear volume. Exptl Cell Res 73 (1972)
318 L. Rensing & R. Hardeland Such a correlation has already been taken into consideration for the larval and prepupal development, since the alterations in the puffing pattern [l, 21 are accompanied by changes in membrane potential [12, 151and in nuclear volume [22] within the course of ontogeny. Although no definite conclusions can be drawn so far from our experiments, some of the possibilities should be mentioned. As Kroeger [I 1, 121has pointed out, in the salivary gland there seemsto exist a relation between ion permeability and gene activity. Since the hyperpolarization precedes the alterations in puff size, it is at least imaginable that CAMP influences the puffing via control of ion permeability. It is not clear whether the increase in nuclear volume may be due to an alteration in permeability of the nuclear membrane or may be related to another function such as, e.g., RNA content. Since ethanol, which unspecifically enhances the permeability of membranes, failed to be effective, the influence of DBcAMP on nuclear size should be expected to be rather specific. It has not been the subject of this investigation to elucidate the mechanism of action of CAMP at the molecular level. However, it would be of particular interest to know how regulation of gene activities is affected by CAMP. In this context, it may be noted that a CAMP-dependent protein kinase has been found, which is rather specific for phosphorylation of histones [17, 251. It is, however, far from clear whether phosphorylation of histones could result in selective activation of certain genes. Moreover, the possibility should not be neglected that CAMP could act on transcription in a manner analogous to that one in microorganisms.
the membranepotential.The technicalassistence of Miss U. Hofacker is greatly acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft. REFERENCES 1. Ashburner, M, Chromosoma 21 (1967) 398. 2. - Ibid 27 (1969) 47. 3. Beermann, W, Differentiation and development (ed A G Bearn & S J Farber) p. 49. Little, Brown & Co., Boston (1964). 4. Berridge, M J & Patel, N G, Science 162 (1968) 462. 5. Butcher, R W & Sutherland, E W, J biol them 237 (1962) 1244. 6. Chambers, D A & Zubay, G, Proc natl acad sci US 63 (1969) 117. 7. Cheung, W Y, Biochemistry 6 (1967) 1079. 8. Friedmann, N, Somlyo, A V & Somlyo, A P, Science 171 (1971) 400. 9. Hardeland, R, Naturwiss 24 (1971) 199. 10. Jacquet, M & Kepes, A, Biochem biophys res commun 36 (1969) 84. 11. Kroeeer. H. Nature 200 (1963) 1234. 12. Kroeger; H; Exptl cell re4 41 (1966) 64. 13. Kuo, J F & Greengard, P, Proc natl acad sci US 64 (1969) 1349. 14. Leenders, H J, Wullems, G J & Berendes, H D, Exptl cell res 63 (1970) 159. 15. Loewenstein, W R & Kanno, Y, J gen physio146 (1963) 1123. 16. MecGelke, F, Naturwiss 21 (1959) 609. 17. Miyamoto, E, Kuo, J F & Greengard, P, Science 165 (1969) 63. 18. Nagel, G. In preparation. 19. Nicoletti, B, Drosophila inf serv 33 (1959) 181. 20. Pastan, I & Perlmann, R L, J biol them 244 (1969) 2226. 21. Rensing, L, Z vergl physiol 53 (1966) 62. 22. - J insect physiol 15 (1969) 2285. 23. Rensing, L & Hardeland, R, J insect physiol 13 (1967) 1547. 24. kobison, G A, Butcher, R W &Sutherland, E W, Ann rev biochem 37 (1968) 149. 25. Shepherd, G R, Noland, BJ & Hardin, J M, Exptl cell res 67 (1971) 474. 26. Siggins, G R, Oliver, A P, Hoffer, B J & Bloom, F E, Science 171 (1971) 192. 27. Sutherland, E W, (aye, I &Butcher, R W, Recent progr hormone res 21 (1965) 623. 28. Zubay, G & Chambers, D A, Cold Spring Harbor symp quant biol 34 (1969) 753. 29. Zubay, G, Schwartz, D & Beckwith, J, Proc natl acad sci US 66 (1970) 104.
Received December 1, 1971 Revised version received on February 16, 1972 The authors wish to thank Dr J. B. Walther, I. Zoologisches Institut, Universitlt GGttingen, who kindly helped us to carry out the measurements of Exptl
Cell Res 73 (1972)