Respiratory functions involved in the induction of puffs in Drosophila salivary glands

Printed in Sweden Copyright Q 1975 by Acadnnic Press, Inc. All riphts of reproduction in any form reserved

Experimental

RESPIRATORY PUFFS

FUNCTIONS IN

Cell Research 91 (1975) 119-124

INVOLVED

DROSOPHILA J. BEHNEL

I. Zoologisches

Institut,

IN

SALIVARY

THE

INDUCTION

OF

GLANDS

and L. RENSING

Universitiit

Go’ttingen,

034

Giittingen,

BRD

SUMMARY Salivary glands from third instar larvae of Drosophila melanogaster were incubated in vitro with various substances affecting oxidative phosphorylation. After an incubation time of l-3 h changes in puff size and in cellular ATP level were registered. IO-” M trinactin, 1O-5 or 1O-4 M oligomycin both induce puff 63BC together with some other puffs and reduce the cellular ATP level by about 80-90 %. The trinactin-dependent puff induction can be inhibited, if the medium is supplemented with 1O-3 M ATP or 1O-3 M ITP or 1OW M antimycin or 1O-2 M KCN. The effect of exogenous ATP is prevented by adding 1O-B M oligomycin to the incubation mixture; 1O-B M oligomycin alone, however, has no inductive effect on 63BC. The presence of exogenous ITP, furthermore, prevents the ATP level from being reduced by trinactin. 10M4M atractyloside lowers the ATP level by about 75 %, whereas a puff induction cannot be observed. The same is true for various concentrations of KCN. It is concluded that ATP itself is not involved directly in the regulation of puff activity but that it acts on a phosphorylating reaction that can be inhibited by oligomycin.

Treatment of salivary glands of Drosophila larvae with heat or uncoupling agentsresulted in an induction of a certain group of puffs [l-5]. Furthermore, these puffs were inducible also by the ionophorous antibiotics valinomycin and dinactin [Sj. Since, in all these cases, oxidative phosphorylation and ATPase activity seem to be affected, and since the puff induction could be prevented by exogenous ATP supply [4], it seemed worthwhile to consider the level of ATP to be involved in the regulating mechanism of gene activity. Therefore, the role of the cellular ATP level in relation to the puff induction is analysed in this paper. Puff induction is studied by using several substances such as trinactin, oligomycin, antimycin, atractyloside, and KCN which influence oxidative phosphorylation in different ways. The changes in ATP level are observed simultaneously.

While this manuscript was being revised, Leenders et al. [21] published results and conclusions that are corroborated and extended in our experiments. MATERIALS

AND METHODS

Salivary glands and puffs Excised salivary glands of late third instar larvae of Drosophila melanogaster Meigen were used in all experiments [6]. The two sister glands of larva were prepared, separated and incubated in an artificial culture medium [7]. One of the sister glands was treated with the substances dissolved in the medium while the other sister gland was kept in normal medium as a control. After the incubation time (l-3 h at 25°C) the cells were stained, squashed, and the puff size was measured. The calculation of the Duff quotient (PQ) was made as described by School & Rensing [8]: the width of a certain puff and the width of a reference band were determined by using a camera lucida and the first values divided by the latter values.

ATP content In order to determine the ATP content of the cells ten pairs of salivary glands were needed. After the Exptl

Cell Res 91 (1975)

120 Behnel and Rensing experimental treatment the glands were rapidly washed twice in a buffer solution (glycine buffer, 0.01 M glycine, 0.02 M MgS04.7H,0, pH 10.0) in order to eliminate remnants of the incubation medium. Then ATP was extracted by homogenizing the glands for 3 min at 8&95”C in 1 ml of the same buffer according to Kalbhen & Koch [9]. The homogenate was centrifuged and the supernate frozen until the ATP content was determined. In order to determine the ATP content the bioluminescence reaction of the luciferin-luciferase system (Serva) with ATP was used. The enzyme was solved in an arsenate buffer (0.1 M Na,HAsO,, 0.02 M MgS04.7HZ0, pH 7.8) and after 2 h at 4°C the result of the reaction measured for 2 set in a Packard Scintillation Counter (Tri-Carb 3320); a sample containing 0.5 ml ATP extract and 4.5 ml enzyme solution [lo]. The protein quantity of the homogenate was determined according to Lowry [ll], the ATP content calculated in nM ATP/mg protein.

RESULTS Incubation of the salivary glands in a medium containing the ionophorous molecule PQ I

0.9 I 0

1

2

3h

Fk. I. Abscissa: incubation time (hours):,, ordinate: puff quotient. Induction of 63 BC bv trinactin (1O-B M) and the effect of exogenous ATP. a, medium+ trinactin; b, medium + trinactin + ATP (10-a M); c, medium + trinactin + ATP (1O-4 M). Data represent means +S.E. Exptl CelI Res 91 (1975)

ATP

Fig. 2. Abscissa: incubation time (hours); ordinate: ATP content (nM/mg protein). Effect of trinactin (1O-B M) alone and the effect of exogenous ITP (1O-8 M) on the cellular ATP level. a, medium + trinactin; b, medium; c, medium + trinactin + ITP. Data represent means i SE.

trinactin (1O-6 M) induces the group of heatsensitive chromosomal regions, analogous with the findings on the effect of dinactin [5] and valinomycin [12]. In the following experiments, region 63 BC has been chosen to represent this group of puffs. The induction of puff 63 BC by trinactin is prevented by the presence of 1O-3 M ATP and delayed by 1O-4 M ATP in the medium (fig. 1). Parallel with the puff induction one can observe a decrease in the ATP content after incubation in 1O-g M trinactin. This decrease does not occur, however, if the medium is supplemented with 1O-3 M ITP (fig. 2). ITP was used in this experiment in order to minimize an interference of the exogenous nucleoside triphosphate with the luciferinluciferase system. The results of these experiments suggest that a low titre of ATP within the cells may be a decisive factor in the activation process of the heat-sensitive group of puffs. In order to analyse the relation between ATP level and puff induction in greater detail the experiments were repeat-

Respiration

and puff induction in Drosophila salivary gland

121

Table 1. The effect of oligomycin (10e6 M) and ouabain (1O-4 M) on the inhibiting influence of ITP on trinactin

inducedpuffing

and decrease of ATP level after 1 h of incubation

Puff quotient (PQ) with S.E.

Trinactin ATP (nM/mg protein)

PQ

22.0 1.5010.08

Trinactin f ITP

Trinactin +ITP + oligomycin

Trinactin + ITP + ouabain

63.0

61.5

62.5

1.09*0.1

1.75kO.07

1.0810.1

ed with oligomycin instead of trinactin. Incubation of the glands in a medium containing oligomycin in a concentration of 1O-6 M does not result in puff induction or measurable decrease of the ATP level. Oligomycin concentrations of 1O-5 M and 1O-4 M do affect both puff size and ATP content (fig. 3). Puff induction in this case could not be blocked by exogenous ATP. Moreover, the inhibition of the trinactin-dependent puff induction by exogenous ATP is prevented, if oligomycin is present in a concentration of ATP 701

PQ I

60.

1O-s M-a concentration which is unable to bring about an induction by itself (table 1). It is shown, furthermore, that in the presence: of1oligomycin the trinactin-induced puff is active even though the ATP content is kept at a normal level by exogenous ITP supply. Ouabain, instead of oligomycin, is not effective in inhibiting the blocking influence of exogenous ITP on puff induction even though it prevents the cellular ATP level from being reduced. Together with other recent findings [21] these results indicate that ATP itself is not the direct signal for the induction of heat-sensitive puffs, but that it probably plays a role in a process which is inhibited by oligomycin. The assumption of an indirect effect of ATP on the regulation of puffing activity in the heat-sensitive chromosomal regions is supported further by experiments with atractyloside. This alkaloid inhibits the adenine

Table 2. The effect of atractyloside (1O-4 M)

1 bI a

on puff size of region 63 BC and on the ATP level after 1 h of incubation

1. 10-G

10-S

10-4M

Fig. 3. Abscissa: oligomycin cont. (moles); ordinate: (inner) puff quotient (PQ); (outer) ATP content (nM/ mg protein). Effect of various concentrations of oligomycin on (6) puff size; (a) cellular ATP level. Data represent means &S.E.

The values in parentheses indicate the decrease in % of the in vivo ATP level. Puff quotient (PQ) with S.E.

ATP (nM/mg protein)

PQ

Atractyloside

Control

13.2 (75%) 0.98 +0.05

37.1 (30%) 1.00+0.08

Exptl Cell Res 91 (1975)

122 Behnel and Rensing Table 3. The effect of different concentrations of incubation

of KCN on the puff size of region 63 BC after 2 h

Puff quotient (PQ) with S.E.

PQ

1O-5 M

1O-4 M

1O-3 M

1O-2 M

Control

1.25 kO.08

1.17*0.04

1.42kO.10

1.02 * 0.03

1.18&0.03

Table 4. The effect of KCN (10m2 M) and antimycin (10e6 M) on trinactin (1O-6 M) induced puffing of 63 BC after I h of incubation Puff quotient (PQ) with S.E.

PQ

Trinactin

Trinactin + KCN

Trinactin

Trinactin + antimycin

1.45 * 0.05

1.07 * 0.04

1.54kO.06

1.11*0.04

nucleotide translocation through the mitochondrial membrane [13, 141. An incubation of the salivary glands in a medium containing 1O-4 M atractyloside is followed by a marked decrease of the ATP level but does not result in puff induction (table 2). No significant increase in puff size can be registered even after incubation of the salivary glands in various concentrations of KCN (table 3), even though a considerable decreasein the ATP content of the cells must be the result of these treatments [21]. Since the function of trinactin as K+ translocating molecule is energy-dependent, one should expect an inhibition of the puff induction on the other hand by blocking the respiratory chain with KCN or antimycin. This inhibition is actually observed after incubation with trinactin together with 1O-2 M KCN or with 1O-6M antimycin A (table 4). DISCUSSION Trinactin is one of the four homologous macrotetrolides which differ in the number of methyl substituents. These antibiotics show ion-specific carrier properties by forming Exptl

Cell Res 91 (1975)

complexes with high affinity to K+-ions similar to other ionophorous substancessuch as valinomycin [151. The trinactin-K+-complex can be described as a 32-membered ring resembling the seam of a tennis ball with K+-ion in the centre and with the methyl substituents and methylene groups on the outside, resulting in membrane-lipid solubility [16]. The physiological effects described by Graven [17] are accumulation of K+-ions by active transport within the mitochondria, corresponding efflux of protons, swelling of the mitochondria, stimulation of respiration, induction of ATPase activity, and uncoupling of oxidative phosphorylation [18]. All these effects are thought to be caused by the energy dissipating primary accumulation of K+ions. The energy for this process may be taken either from ATP hydrolysis or from electron transport itself. In the first case oligomycin, in the latter case antimycin, is inhibiting. It has been concluded that the energy needed for cation accumulation is taken from an intermediate product between the electron transport chain and ADP phosphorylation [ 151.

Respiration and puff induction in Drosophila At first sight the results of this paper showing a trinactin-induced decrease of the ATP level together with an activation of heatsensitive puffs that is prevented by simultaneous supply with exogenous ATP or ITP suggest a role of the ATP level in puff induction. This conclusion seems to be supported by the experiments with oligomycin: only concentrations which are able to reduce the ATP level effect also puff formation. There is, however, a decisive difference: while the trinactin-induced puffs are repressed by a supply with high energy compounds such as ATP, the activation of puffs by oligomycin cannot be blocked by exogenous ATP. Moreover, the inhibitory effect of ATP on trinactin-induced puff formation is prevented, if oligomycin is present in the medium in concentrations ineffective in puff induction by itself. These effects may be explained by the assumption that the process leading to puff formation can be inhibited by ATP or ITP only, if the resulting reversal of the ADP phosphorylation is not inhibited by oligomycin. It is suggested, therefore, that not ATP itself but an energy-rich intermediate between respiration chain and final phosphorylation [19] is more directly involved in gene control. In accordance with this hypothesis and with the results of Leenders et al. [21] are the observed effects of atractyloside and KCN on puff size and ATP level. The primary effect of atractyloside is the inhibition of the membrane-bound adenine nucleotide translocase. This is followed by a decrease of the ATP level within the cytoplasm and inhibition of respiration caused by the low ADP/ ATP ratio and high energy pressure in the mitochondria. The effect of KCN is the well known inhibition of the cytochrome a3 mediated transfer of electrons to oxygen that results in blocking the respiratory chain. To-

salivary gland

123

gether with the findings on puff induction by puromycin and fusidic acid [8] and the results on phosphorylation of mitochondrial proteins energized by an intermediate product [20] one might arrive at the following conclusion: the energy-rich intermediate may be involved in energizing of the phosphorylation of certain repressor or activator proteins. Since respiratory chain enzymes are present also in the nuclear membrane it is tempting to assume that the regulatory processes are located in this membrane. Apart from the phosphorylation K+-transport into the nucleus may also affect the puffing activity via conformational changes of control proteins.

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Exptl

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21. Leenders, H J, Kemp, A, Koninkx, J F J G & Rosing, J, Exptl cell res 86 (1974) 25. Received May 29, 1974 Revised version received September 12, 1974