The transmembrane potential of Drosophila salivary gland cells is largely decreased (by 78% within 120 min) in response to the application of 5 mM-chloramphenicol (CAP), with an initial slope of 0 ·5 mV min−1. This depolarization is reversed immediately after the CAP concentration is reduced to 0 ·06 mM by step-wise dilution with normal medium. At the same concentration, thiamphenicol (TAP) induces only a small reversible depolarization by less than 30% within 120 min. These results are in agreement with the different effects of CAP and TAP on respiration and induction of the heat-shock genes, as known from previous data.

The extent of induced membrane depolarization decreases with the number of repeated applications of CAP to the same cell, alternating with 75-min periods of recovery. Moreover, reduced sensitivity to CAP is also observed in cells recovering from a transient heat shock (30 min, 36°C) 45 min prior to the addition of CAP. This phenomenon is inhibited by cycloheximide (0 ·lmM), which suggests an involvement of heat-induced proteins in the stabilization of certain membrane functions.

The activation of certain heat shock (hs) genes is a universal response of cells to various kinds of physiological and metabolic stress in most organisms, ranging from bacteria to man (Schlesinger et al. 1982). In salivary glands of Drosophila the hs gene activation can first be recognized by a rapid formation of puffs in the chromosomal hs loci, hs puffs are induced by elevated temperature and recovery of anaerobiosis, as well as by a variety of agents, most of which interfere with the energy-conserving metabolism in mitochondria (for a review, see Ashbumer & Bonner, 1979).

As known from a previous study, millimolar concentrations of chloramphenicol (CAP) added to cultured salivary glands cause hs puff formation with a coincident decrease in protein synthesis activity. In contrast to the induction by hyperthermia, the puffs do not regress until the antibiotic is removed from the medium. Both puff regression and increased synthesis of hs proteins occur only after recovery of the cells in CAP-free medium (Behnel, 1982). Besides the specific effects on the 70 S ribosomal translation, CAP also inhibits respiration and oxidative phosphorylation in eukaryotic cells at concentrations exceeding 1 mM (Freeman & Haidar, 1968; Freeman, 1970; Haidar & Freeman, 1968; Abou-Khalil et al. 1980). Although direct evidence is lacking, the CAP-induced activation of the hs loci has been attributed to disturbance of energy-providing reactions, by comparison with the well-known induction of hs genes by inhibitors of mitochondrial respiration.

The present study was attempted with the aim of determining whether a CAP-dependent energy deprivation in salivary glands may be achieved by alterations in the membrane potential, as is observed with uncouplers of oxidative phosphorylation (Behnel & Seydewitz, 1980). The following results not only corroborate this anticipation but also reveal an acquisition of increased tolerance of CAP by the cells.

Salivary glands used in this study were excised from third instar larvae of Drosophila melanogaster Meigen and cultured in a chemically defined medium according to Rensing &Fischer (1975). In this medium the glands have been found to survive for a few days (unpublished results).

Intracellular recordings of the transmembrane potential were performed essentially as described previously (Behnel & Seydewitz, 1980). Briefly, glands were incubated in a moist chamber consisting of a 6 mm diameter gasket glued in the middle of a 4 cm Petri dish, filled with 60 μl medium, and surrounded by a wet filter paper ring. The whole was covered by a centrally bored polystyrene disk in order to minimize evaporation. Using a micromanipulator cells were impaled by glass capillary microelectrodes filled with 3 M-KC1 and connected, together with an Ag ·AgCl reference electrode, to a differential high-impedance preamplifier. The output signal was fed into a pen chart recorder and the electric potential with respect to the bath was recorded continuously.

D-Threo-chloramphenicol (CAP) and cycloheximide, purchased from Serva (Heidelberg) and D-threo-thiamphenicol (TAP; Sigma) were dissolved directly in the culture medium.

For medium exchange 40 μl were always substituted three times by an equivalent volume of medium containing 5 ·2 mM of either CAP or TAP, giving a final concentration of 5 ·0 mM. Rinsing out was performed by the same procedure using normal medium for replacement, which resulted in a dilution to 0 ·06 mM of the antibiotics after the fourth step of medium exchange. The 40-μl volumes were removed and added by means of a calibrated micropipette providing a circular stream in the bath that resulted in effective mixing.

Effects of CAP and TAP on the membrane potential

Salivary glands excised from larvae and adapted to the artificial culture medium for 15 min maintain a membrane potential between −33 and −37 mV inside. Except for small variations, the potential of a well-punctured cell membrane is stable for at least 3h without renewal of the medium. After 20 min the impaled microelectrode is tightly surrounded by the membrane and the culture medium can be exchanged without injury to the cell.

When the medium is enriched step-wise with 5 mM-CAP cells respond with a slow but large depolarization of the plasma membrane. Values were measured after 60 min and 120 min at 13 mV and 8 mV, respectively (Fig. 1). Accordingly, a step-wise reduction of the CAP concentration to 0 ·06 mM results in a recovery of the membrane potential reaching a new equilibrium value after 40 –50 min that is usually slightly lower (1 –3 mV) than at the beginning. These results indicate that the course of CAP-induced puff activity described previously (Behnel, 1982) coincides with a large reversible depolarization of the outer cell membrane.

Fig. 1.

Typical changes in the membrane potential of salivary gland cells in response to the application of 5 mM-chloramphenicol (CAP) or 5 mM-thiamphenicol (TAP). Points represent values measured in intervals of 5 min. Arrowheads indicate changes in the culture medium; M, normal medium.

Fig. 1.

Typical changes in the membrane potential of salivary gland cells in response to the application of 5 mM-chloramphenicol (CAP) or 5 mM-thiamphenicol (TAP). Points represent values measured in intervals of 5 min. Arrowheads indicate changes in the culture medium; M, normal medium.

In comparison, TAP, the methylsulphonyl analogue of CAP, which does not induce hs puffs (except in the 93D region) (Behnel, 1982), has only a small depolarizing effect on the plasma membrane. The minimum potential measured after 120 min is rarely lower than 25 mV (Fig. 1).

Response of the membrane potential to periodic applications of CAP

After regression of puffs following the first heat treatment the chromosomal hs loci fail to respond immediately to a second hyperthermic shock (Ashbumer & Bonner, 1979; Rensing et al. 1982). This refractoriness is also observed in cells recovering from a transient CAP treatment, i.e. they cannot quite be reinduced by a second exposure to CAP. However, the normal puff regression does not occur when protein synthesis is inhibited, which suggests a negative feedback control on the puffs exerted by hs proteins (Rensing et al. 1982).

In order to analyse whether the failure of puffs to be reinduced can be correlated with a decreased sensitivity of the cells to CAP, the sequence of infusion and rinsing out of the antibiotic was repeated periodically at intervals of 120 min (45 min CAP treatment and 75 min recovery). The results depicted in Fig. 2A demonstrate that during the first application period the membrane potential declines to 35% of the initial value. After the second and third periods the depolarization decreases, so that during the fourth infusion the membrane potential does not fall below 62% of the initial value. This gradual stabilization of the potential is not observed in the presence of cycloheximide in the medium for the entire recording period (Fig. 2B).

Fig. 2.

Continuous recordings of the membrane potential of salivary gland cells during periodic applications of 5 mM-CAP: A, without, and B, in the presence of 0·1mM cycloheximide added 15 min before the first application of CAP. The upper line indicates changes in the culture medium with time; M, normal medium.

Fig. 2.

Continuous recordings of the membrane potential of salivary gland cells during periodic applications of 5 mM-CAP: A, without, and B, in the presence of 0·1mM cycloheximide added 15 min before the first application of CAP. The upper line indicates changes in the culture medium with time; M, normal medium.

These results are confirmed by the study of heat-conditioned cells, which also demonstrates a reduced sensitivity to CAP at normal temperature (25 °C) for 45 min (Fig. 3A). Again, the heat-induced CAP resistance is not acquired when protein synthesis is inhibited by 0 ·1 mM-cycloheximide (Fig. 3B).

Fig. 3.

Typical response of the membrane potential of salivary gland cells recovered 45 min after a heat shock (30 min at 36°C): A, without, and B, in the presence of 0 ·1 mM-cycloheximide added 15 min before heat shock. The upper line indicates temporal changes in the culture medium; M, normal medium.

Fig. 3.

Typical response of the membrane potential of salivary gland cells recovered 45 min after a heat shock (30 min at 36°C): A, without, and B, in the presence of 0 ·1 mM-cycloheximide added 15 min before heat shock. The upper line indicates temporal changes in the culture medium; M, normal medium.

It should be mentioned that the combined effects of cycloheximide and hyperthermia result in a destabilization of the plasma membrane, and many cells impaled by a microelectrode do not maintain a stable membrane potential. Those cells selected as useful for the experiments shown in Fig. 3B did not survive the first or second CAP treatment in most cases. The recovery of membrane stability after hyperthermia is clearly a different process from the recovery of membrane potential after CAP treatment in nonheat-shocked cells, since the latter are not affected by cycloheximide. Thus, unlike brief exposure to CAP, hyperthermia (Δt =11 deg. C) results in damage to physiological functions that is not repaired in the absence of protein synthesis, and is shown by the instability of the membrane potential.

At least two independent effects of CAP on mitochondrial functions have been reported: at low concentrations, both CAP and TAP inhibit specifically the 70 S ribosomal translation (over 80% at 30 μM) (Abou-Khalil et al. 1980), whereas CAP, unlike TAP, inhibits respiration and oxidative phosphorylation at much higher concentrations (50% at 1 ·5 mM and 86% at 6mM) (Freeman & Haidar, 1968; Haidar & Freeman, 1968). The latter effect of CAP, which could result from direct inhibition of the mitochondrial NADH dehydrogenase activity (Freeman & Haidar, 1968; Freeman, 1970), is thought to be responsible for the shutdown of protein synthesis in eukaryotic cells (Haidar & Freeman, 1968) and for the induction of hs puffs in Drosophila salivary glands (Behnel, 1982).

The present data now demonstrate a CAP-induced large depolarization of the outer cell membrane. Although direct alteration of membrane permeability by CAP cannot be ruled out, it seems to be unlikely because of the low rate of depolarization, which amounts to 0 ·5 mV min−1. On the other hand, the similar effects of CAP and TAP (1) in inhibiting respiration (Freeman & Haidar, 1968; Haidar & Freeman, 1968; Freeman, 1970) and (2) in affecting the membrane potential, argue for an indirect effect of CAP on the membrane, via energy deprivation. Another question that arises from the data refers to the stabilization of the membrane potential after repeated application of CAP. The gradual decrease in the depolarizations induced by a second and third infusion of CAP suggests that the cells acquire a progressive resistance to the detrimental effect produced by the antibiotic. This resistance is impaired, however, by cycloheximide, indicating that the synthesis of new protein is required for the observed stabilization of the membrane potential. Since in the presence of 5 mM-CAP translational activity is strongly reduced (Behnel, 1982), production of new protein between the first and second additions of CAP occurs in the 75-min period of recovery, when an increased synthesis of hs proteins occurs. This fact, together with the resistance to CAP acquired following heat shock, suggests an involvement of hs protein in the stabilization of membrane functions.

Although little is known about the distinct functions of the hs proteins, they are presumably involved in the generation of thermotolerance as well as in the stabilization of certain nuclear and cytoplasmic structures (Leicht et al. 1986; for a review, see Nover, 1984). Moreover, Burdon & Cutmore (1982) reported an increased activity of the membrane-bound Na+,K+-ATPase in heat-shocked and arsenite-treated cells that is impaired in the presence of actinomycin D or cycloheximide. This argues for a direct or indirect modulation of this enzyme activity, which contributes mainly to the maintenance of the steady-state potential across cellular plasma membranes. Whether the CAP resistance observed in salivary gland cells is due to a modulation of the Na+,K+-ATPase activity, altered membrane permeability, or a more complex change in the energy-conserving pathways, remains to be resolved by further studies.

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