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Ethacrynic acid inhibition of histamine release from rat mast cells: effect on cellular ATP levels and thiol groups
European Journal of Pharmacology, 92 (1983) 181 - 189
181
Elsevier
ETHACRYNIC ACID I N H I B I T I O N OF H I S T A M I N E RELEASE F R O M RAT M A S T CELLS: EFFECT O N CELLULAR ATP LEVELS AND T H I O L G R O U P S TORBEN JOHANSEN
Department of Pharmacology, Odense University, J.B. Winslows Vej 19, DK-5000 Odense C, Denmark Received 11 January 1983, revised MS received 3 May 1983, accepted 26 May 1983
T. JOHANSEN, Ethacrynic acid inhibition of histamine release from rat mast cells: effect on cellular A TP levels and thiol groups, European J. Pharmacol. 92 (1983) 181-189. The experiments concerned the effect of ethacrynic acid (0.5 raM) on the adenosine triphosphate (ATP) content of rat mast cells and the effect on histamine release induced by the ionophore A23187 (10 # M). Ethacrynic acid decreased the ATP level of the cells in presence of antimycin A and glucose as well as in presence of 2-deoxyglucose. A23187-induced histamine release was inhibited by ethacrynic acid, and this inhibition was completely reversed by dithiothreitol. These observations may indicate that ethacrynic acid inhibits glycolytic and respiratory energy production in rat mast cells. Furthermore, the inhibition of histamine release occurred mainly through interaction with thiol groups, although inhibition of cellular energy production may have been an additional mechanism. Energy metabolism
Ethacrynic acid
Histamine release
I. Introduction The antibiotic A23187 specifically forms complexes with calcium and magnesium and transport these ions across a variety of membranes (Reed and Lardy, 1972). In the presence of calcium, A23187 acts as a histamine-releasing agent on isolated rat mast cells (Foreman et al., 1973). The mechanism of A23187-induced histamine release seems to be similar to that activated in rat mast cells by the antigen-antibody reaction in vitro (Johansen, 1978). Results of previous studies of the relation between mast cell ATP content and anaphylactic (Johansen and Chakravarty, 1975; Johansen, 1979b) as well as A23187-induced histamine release (Johansen, 1979a) are consistent with the view that energy-requiring processes are involved in the release mechanism. Anaphylactic histamine release and A23187-induced histamine release are completely blocked by ethacrynic acid (Magro, 1977; Chakravarty, 1979). It has been demonstrated that ethacrynic acid inhibits both glycolysis and respiration of Ehrlich ascites tumour cells and slices of kidney (Jones 0014-2999/83/$03.00 © 1983 Elsevier Science Publishers B.V.
Ionophore A23187
Mast cells
and Landon, 1967; Gordon, 1968; Gordon and Hartog, 1969). At the subcellular level ethacrynic acid interferes with a number of mitochondrial processes (Goldschmidt et al., 1976; Manuel and Weiner, 1976; Passarella and Quagliariello, 1979) and with glycolytic enzymes (Gordon and De Hartog, 1971). Ethacrynic acid is known to react with thiol groups (Komorn and Cafruny, 1965; Koechel and Cafruny, 1973; Goldschmidt et al., 1976; Manuel and Weiner, 1976), which may be involved in the metabolic reactions above as well as in secretory processes (Nemeth and Douglas, 1978). In the present investigation the effect of ethacrynic acid on A23187-induced histamine release and mast cell ATP content was examined in order to study the relation between the effect of ethacrynic acid on cellular energy metabolism and its inhibitory action on the histamine release process. It is demonstrated that ethacrynic acid, in addition to its inhibitory effect on cellular energy production, inhibits A23187-induced histamine release through interaction with thiol groups.
182 2. Materials
and methods
2.1. Isolation o f rat mast cells M a l e S p r a g u e - D a w l e y rats, 350-570 g, w e r e used for the e x p e r i m e n t s , M a s t cells were i s o l a t e d as d e s c r i b e d p r e v i o u s l y ( J o h a n s e n , 1979a) a n d susp e n d e d in K r e b s - R i n g e r s o l u t i o n c o n t a i n i n g hum a n s e r u m a l b u m i n , 1 m g / m l , final p H 7.0 to 7.1. M a s t cells: 96.4 _4- 0.7% ( m e a n + S.E.M.; n = 40) o f cell p o p u l a t i o n .
2.2. Incubation procedure (fig. 1) M a s t cell s u s p e n s i o n s p o o l e d f r o m 1 to 4 rats w e r e d i v i d e d i n t o a l i q u o t s w i t h the s a m e cell d e n sity in a final v o l u m e o f 0.5 to 0.6 ml. T h e s e w e r e
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used for d e t e r m i n a t i o n of the A T P c o n t e n t a n d for the h i s t a m i n e release e x p e r i m e n t s . T h e cell d e n s i t y in the v a r i o u s e x p e r i m e n t s v a r i e d f r o m 0.45 × 105 to 3.76 × 10S c e l l s / m l . T h e cell s u s p e n s i o n s were e q u i l i b r a t e d in a 3 7 ° C b a t h for 10 min in a c a l c i u m - f r e e K r e b s - R i n g e r solution. In the experim e n t s in fig. 2 the cells w e r e t h e n t r e a t e d with A 2 3 1 8 7 (10 # M ) for 10-26 m i n in the a b s e n c e of c a l c i u m , the last 15 s to 16 m i n with e t h a c r y n i c a c i d (0.5 m M ) p r e s e n t in the m e d i u m . H i s t a m i n e release was i n i t i a t e d by e x p o s u r e of the cells to c a l c i u m (5 m M ) for m o r e t h a n 45 s, w h i c h is k n o w n to be a s u f f i c i e n t t i m e p e r i o d for the h i s t a m i n e release o f be c o m p l e t e d ( J o h a n s e n , 1978). T h e high c o n c e n t r a t i o n of c a l c i u m was used in o r d e r to get a fast release o f h i s t a m i n e ( J o h a n s e n ,
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Fig. 1. Incubation procedure (temperature equilibration omitted). A: Antimycin A (1 ~M), Ca: Calcium (5 mM), D: Dithiothreitol (1 mM), 2-DG: 2-deoxyglucose (5 mM), E: Ethacrynic acid (0.5 mM), G: Glucose (1 raM), I: A23187 (10 /*M), O: Oligomycin (1 tzg/ml), S: Pyruvate (1 raM) or Succinate (5 mM). Abscissa: Incubation time in min. Dashed lines represent variable time periods.
Fig, 2. Time course of inhibition of histamine release and decrease in cellular ATP content by ethacrynic acid. The cells were incubated with ethacrynic acid for various time intervals in a calcium-free medium in presence of A23187 in the absence (©,D) or presence (~,a) of glucose. Cellular ATP content was then determined (r7 ,,,) or the cells were incubated with calcium in order to initiate histamine release (O,~). Abscissa: Time of incubation with ethacrynic acid. Ordinate: Percent histamine release and ATP content of the cells. Control histamine release from A23187-treated mast cells incubated in the absence of ethacrynic acid with glucose: 69.8%±3.1% (mean ±S.E.M, n = 5), or without glucose: 49.7%±3.7% (mean ±S.E.M., n = 10). 100 on the ordinate represents the normal ATP content from mast cells incubated in the absence of ethacrynic acid and A23187 with (1.46_+0.06 pmol/103 cells, mean _+S.E.M., n = 6) or without glucose ( 1.52 + 0.02 pmol/103 cells, mean ± S.E.M, n = 3). A23187dnduced histamine release in the absence of calcium and in the absence (mean 7.4%) and presence of glucose (mean 10.3%) subtracted. Spontaneous histamine release in the absence of A23187 and ethacrynic acid was 3.2% (mean value). Mean values from two (@ at 13 and 16 rain) and three to seven experiments; vertical lines show S.E.M.
183 1978). Samples were included for d e t e r m i n a t i o n of c a l c i u m - i n d u c e d h i s t a m i n e release from mast cells pretreated with A23187 alone, as well as samples for d e t e r m i n a t i o n of histamine release i n d u c e d by A23187 in the absence of calcium and ethacrynic acid. Three types of experiments were done to study the effect of ethacrynic acid on the A T P c o n t e n t of the mast cells. I n all three cases the cells were temperature-equilibrated in a calcium-free m e d i u m as described above. Firstly (fig. 3), the cells were i n c u b a t e d for 10 to 30 min, either with ethacrynic acid (0.5 m M ) alone or with a c o m b i n a t i o n of ethacrynic acid a n d glucose (1 m M ) or a c o m b i n a tion of ethacrynic acid a n d respiratory inhibitors ( a n t i m y c i n A, 1 /~M, a n d oligomycin, 1 /~g/ml). In the last case A23187 (10/~M) was also present in the m e d i u m in a d d i t i o n to the respiratory inhibitors and ethacrynic acid, and glucose (1 raM) was used as substrate for the glycolysis. Secondly (fig. 4), the cells were p r e i n c u b a t e d for 20 m i n with a respiratory i n h i b i t o r ( a n t i m y c i n A, 1 /~M) or an i n h i b i t o r of glycolysis (2-deoxyglucose (2DG), 5 raM). In the first case glucose (1 raM) was used as
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Fig. 4. Effect of ethacrynic acid on the ATP content of mast cells. The cells were incubated with (n) or without (O) ethacrynic acid (0.5 mM) for 20 rain as indicated on the abscissa. Ordinate: ATP content of the cells in percent of control value from untreated cells: 1.24_+0.05 pmol/103 cells (mean _+S.E.M., n = 3). Mean values from three experiments; vertical lines show S.E.M.
substrate for glycolysis. I n the last case the cells were pretreated with 2 - D G with or without a respiratory substrate (pyruvate, 1 mM, or succinate, 5 raM) present in the medium. The cells were then i n c u b a t e d with ethacrynic acid (0.5 raM) for 20 min. T h i r d l y (fig. 5), the mast cells were pretreated with 2 - D G (5 mM) for 20 rain, the last 5 m i n with ethacrynic acid (0.5 raM) present in the
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Fig. 5. Effect of ethacrynic acid on the rate of ATP depletion of mast cells. The glycolytic pathway in the cells was blocked by 2-DG. Abscissa: Time of incubation with respiratory inhibitors with (zx) or without (O) ethacrynic acid. Ordinate: ATP content of the mast cells in percent of control value from untreated cells: 1.27_+0.13 pmol/l03 cells (mean + S.E.M.). The last 5 min of preincubation with ethacrynic acid decreased the ATP content by 20 percent. Mean values from three experiments; vertical lines show S.E.M.
184
medium. A combination of antimycin A (1 ffM) and oligomycin (1 ffg/ml) was then added to the cell suspension. The cells were incubated with the respiratory inhibitors for 15 to 60 s. All three types of experiments were performed in a calcium-free medium. Samples for determination of the normal value of the cellular ATP content in the absence of the metabolic inhibitors, A23187 and ethacrynic acid, were included. In the experiments in fig. 6 the cells were temperature-equilibrated at 37°C in a calcium-free medium for 10 min in the presence or absence of dithiothreitol (1 mM) and glucose (1 mM). A23187 (10 ffM) with or without ethacrynic acid (0.5 mM) was added to the cell suspension and the incubation continued for 10 min in a calcium-free medium. The incubation was then stopped with perchloric acid (see below) for determination of cellular ATP, or calcium (5 raM) was added for more than 45 s in order to induce histamine release. The cells in the experiments in fig. 7 were temperature-equilibrated at 37°C in a calcium-free medium then preincubated for 20 rain with either antimycin A (1 ~M) and glucose (1 mM) or with 2 - D G (5 raM). In both cases the preincubation continued for a further 20 min with
A23187 and ethacrynic acid added to the cell suspension. Histamine release was then initiated by adding calcium (5 raM) to the cell suspension. Cellular ATP content was determined after the 40-rain preincubation period. Dithiothreitol (1 raM) was present in half the samples during the whole preincubation period and during incubation with calcium (as indicated in fig. 7). 2.3. Determination of histamine release and the A TP content of the mast cells
The histamine release experiments were performed as described previously (Johansen and Chakravarty, 1975). Histamine release was determined by a fluorometric method (Shore et al.. 1959) including the extraction procedure with butanol and heptane. Since it was found that dithiothreitol 0.4 mM and ethacrynic acid 0.1 ruM did not influence the histamine determination, the extraction procedure was omitted when possible. The release of histamine was calculated as a percentage of the total histamine content of the mast cells. For the determination of ATP, the reaction after incubation of the samples was stopped with
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Fig. 6. Effect of dithiothreitol on the inhibition of histamine release and the decrease of cellular ATP content caused by ethacrynic acid. The cells were incubated with the reagents indicated on the abscissa. Ordinate, left: ATP content of the cells (1~). 100 on the ordinate represents the ATP content of mast cells incubated in presence (1.55_+ 0.12 pmol/103 cells, mean + S.E.M.) and absence (1.49_+0.11 pmol/103 cells, mean + S.E.M.) of glucose. Ordinate, right: percent histamine release (E3). Appropriate spontaneous release subtracted. Spontaneous histamine release (i.e. no drug present) was 2.6% (mean value). Mean value from three experiments; vertical lines show S.E.M.
185
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Fig. 7. Effect of dithiothreitol on the inhibition of histamine release and the decrease of cellular ATP content caused by ethacrynic acid. The cells were preincubated with eiiher antimycin A and glucose (ea) or 2-DG alone (~) then with A23187 and ethacrynic acid (see Method section). Histamine release (fig. 6a) was initiated by incubation of the cells with calcium. Cellular ATP content (fig. 6b) was determined at the time of initiation of histamine release. Ordinate (fig. 6a): Percent histamine release. In control samples (0) the mast cells were pretreated with A23187 alone for 20 min then incubated with calcium. Appropriate spontaneous histamine release induced by A23187 from mast cells incubated in calcium-free medium with the metabolic inhibitors, glucose and ethacrynic acid subtracted. Spontaneous histamine release with no drugs present was 1.5% (mean value). Ordinate (fig. 6b): ATP content of the cells in percent of control value from mast cells incubated without any reagent or substrate: 1.47+0.05 pmol/103 ceils (mean + S.E.M.). Mean value from four experiments; vertical lines show S.E.M.
ice-chilled perchloric acid. After neutralization of the supernatant, the A T P content was determined by the bioluminescence technique using luciferinluciferase from firefly tails as described earlier (Johansen and Chakravarty, 1975). Internal standards were used for correction of any inhibition of light emission caused by potassium perchlorate or drugs in the reaction medium. N o n e of the drugs interfered with the A T P determination. 2.4. Cell viabifity D y e exclusion by the cells, with T r y p a n Blue as the dye, was used as a measure of cell viabililty. After 40-min exposure of the cells to either of the following drugs and chemicals, 94% or more of the cells excluded the dye: ethacrynic acid, antimycin A and oligomycin, 2-DG, Ca 2÷, A23187 or glucose. In presence of dithiothreitol, 90% of the cells
excluded the dye. In the absence of drugs and chemicals, 98% of the cells excluded the dye. Thus, none of the drugs and chemicals seemed to do any serious harm to the cells in the experiments in the present study. In parallel experiments, spontaneous histamine release (without the combination of calcium and A23187 present in the cell suspension) was less than 3.5%. In presence of A23187 alone, histamine release was 5.1%. In addition, cellular glycolysis, stimulated by incubation of the cells with respiratory inhibitors and glucose, was very active in presence of ethacrynic acid and ionophore A23187 (Johansen, 1980b), which supports the above conclusion. 2.5. Materials H u m a n serum albumin was supplied by AB K A B I ( S t o c k h o l m , Sweden), a n t i m y c i n A, oligomycin and dithiothreitoi by Sigma Chemical C o m p a n y (St. Louis, USA). The ionophore A23187 was a gift from Eli Lilly and Co. (Indiana, USA). A23187 was dissolved in ethanol 96% v / v and diluted in Krebs-Ringer solution for the experiments. The final concentration of ethanol during the incubation never exceeded 0.3% v / v . This concentration of ethanol had no effect on histamine release or on the A T P content of the mast cell (Johansen and Chakravarty, 1975; Johansen, 1978). All other chemicals were of analytical grade. Ethacrynic acid, which was supplied by Merck, Sharp and D o h m e International (New York, USA), was dissolved in 0.9% NaC1 at p H 11-12. After neutralization the final p H in Krebs-Ringer solution with h u m a n serum albumin, 1 m g / m l , was 7.0-7.1. The Krebs-Ringer solution had the following composition (raM): NaC1 143.3, KC1 4.7, MgSO 4 1.2, N a 2 H P O 4 2.5, K H 2 P O 4 0.6.
3. Results
3.1. Effect of ethacrynic acid on histamine release and cellular A TP content Exposure of mast cells to ethacrynic acid for short time intervals (0-2 min) caused an inhibition
186
of histamine release of about 50% (fig. 2). This was partly counteracted by adding glucose to the incubation medium. However, after 10-16-rain exposure of the cells to ethacrynic acid, histamine release was blocked, and glucose had almost no effect on the release process. There was a time-dependent ATP decrease in the presence as well as in the absence of glucose. The ATP level in the presence of glucose was 15-25% higher than that in the absence of glucose (fig. 2). In both cases the maximal ATP decrease was found after 10-rain incubation of the cells with the reagents without further changes in cellular ATP up to 16 min of incubation, which was the longest period of observation. After a lag period of 5 min, there was a timedependent decrease of the ATP content of mast cells incubated with ethacrynic acid (fig. 3). This was partly counteracted by glucose. However, even in the presence of glucose a low rate of ATP decrease was observed. Incubation of the mast cells for 10-30 min with respiratory inhibitors (antimycin A and oligomycin) and ionophore A23187 in addition to ethacrynic acid and glucose in calcium-free Krebs-Ringer solution resulted in a steady state level of ATP of 46-49% of the normal value. In presence of 2-DG, cellular ATP synthesis was largely dependent on respiratory energy production (Johansen, 1980a). In this case ethacrynic acid caused a drastic ATP decrease whether or not the respiratory substrates, pyruvate (Diamant et al., 1974) or succinate (Chakravarty and Zeuthen, 1965), were present in the medium (P < 0.0025 or less by t-test for two groups of data) (fig. 4). By contrast in presence of antimycin A and glucose ATP synthesis was largely dependent on glycolytic energy production, and the ATP decrease caused by ethacrynic acid (P < 0.0025) was less than half of that observed in the 2-DG-treated cells. Fig. 5 shows that the rate of ATP depletion of mast cells pretreated with 2-DG then incubated with respiratory inhibitors (antimycin A and oligomycin) seems to follow a first order reaction. The rate of ATP depletion tended to be less in the presence than in the absence of ethacrynic acid.
3. 2. Effect of dithiothreitol In the absence of glucose, ethacrynic acid completely blocked the histamine release (fig. 6a) as also demonstrated above (fig. 2). This was partly counteracted by dithiothreitol (b). The histamine release in presence of dithiothreitol was 50% of the control value (f). A similar effect of dithiothreitol on histamine release was observed in presence of glucose (compare c and d). Cellular ATP content was determined at the time of initiation of histamine release by calcium. The decrease in cellular ATP content caused by ethacrynic acid (a), was partly counteracted by dithiothreitol (b). In the absence and presence of dithiothreitol, the ATP levels were 67% and 82%. respectively, of the control values (e and f, respectively) (P < 0.05 by t-test for two groups of data). However, with glucose present, no significant increase in cellular ATP content by dithiothreitol was found (compare c and d). In control samples, dithiothreitol had a small potentiating effect on histamine release in the absence (e, f, P < 0.05), but not in the presence of glucose (g, h, P > 0.05). There was no effect of dithiothreitol on cellular ATP content (compare e, f ( P > 0.05) and g, h (P > 0.10)). Furthermore, dithiothreitol had no effect on the ATP content of mast cells incubated in the absence of ethacrynic acid and A23187 whether or not glucose was present in the medium (i, j). Dithiothreitol had no effect on the spontaneous histamine release from untreated cells whether or not calcium was present, and dithiothreitol also had no effect on histamine release from mast cells incubated in calcium-free medium with either A23187 alone or with both A23187 and ethacrynic acid unless, in the last case, glucose was present. In this case a small potentiating effect of dithiothreitol was found (P < 0.05 by t-test for two groups of data). Ethacrynic acid had a small potentiating effect on A23187-induced release from mast cells incubated in calcium-free medium with glucose. This was observed in the presence as well as in the absence of dithiothreitol (P < 0.05, P < 0.0125 by t-test for two groups of data). No effect was found in the absence of glucose. When the respiratory energy supply of the cells was blocked by antimycin A, dithiothreitol com-
187 pletely reversed the inhibition of histamine release by ethacrynic acid (P < 0.0005) (fig. 7). Glucose was used as substrate for glycolysis in these experiments. This effect was associated with a small decrease of the cellular ATP content (P < 0.005). In contrast, when 2-DG was used to block mast cells glycolysis, dithiothreitol caused only a small increase in histamine release (P < 0.05) and cellular ATP level (P < 0.0005). In parallel control experiments without ionophore there were similar changes in cellular ATP values in response to dithiothreitol (data not shown).
4. Discussion 4.1. Cellular A TP
The cellular ATP depletion caused by ethacrynic acid may be explained by increased utilization of cellular ATP as well as by inhibition of oxidative or glycolytic energy production, both of which are associated with the synthesis of energy-rich phosphate in ATP. The possibilitY of an increased ATP utilization seemed unlikely since ethacrynic acid tended to decrease the rate of ATP depletion in mast cells without oxidative and glycolytic energy production. The ATP decrease seen in mast cells dependent on either glycolytic or oxidative energy production indicates an inhibition of both pathways by ethacrynic acid, the most pronounced effect being on oxidative ATP synthesis. The inhibition of oxidative ATP synthesis was not caused by lack of respiratory substrate, since the same ATP decrease was observed in presence as well as in the absence of respiratory substrates. The rate of ATP depletion caused by ethacrynic acid in presence of glucose was one third of that observed in the absence of glucose. This may indicate only a slight inhibition of glycolysis by ethacrynic acid. This view is supported by the observed steady state ATP level of mast cells dependent on glycolytic ATP synthesis. In these experiments the effect of ethacrynic acid on mitochondrial ATP production would not be apparent, since this was blocked by respiratory inhibitors. The rate of glycolytic activity when enhanced by extracellular glucose (Diamant and Peterson, 1974), seemed to
be sufficient for the cell to maintain a steady state ATP level of about half the normal ATP value. Previously, ethacrynic acid was found to cause a 21% decrease of the rate of lactate production by mast cells treated with respiratory inhibitors and glucose (Johansen, 1980b). It may be observed that the rate of the ATP decrease caused by ethacrynic acid alone (fig. 3) and the rate of that caused by ethacrynic acid in combination with A23187 (fig. 2) were of the same order of magnitude, about 45% and 35%, respectively, after 10 min. One may speculate that the steady state value of cellular ATP observed in fig. 2 when cells were incubated with A23187 and ethacrynic acid, but not seen in fig. 3 when the cells were incubated with ethacrynic acid alone, may be due to a protective effect of A23187 on oxidative energy metabolism, which was more depressed by ethacrynic acid than was glycolysis (fig. 4). However, additional experiments are necessary to further clarify the interaction between ethacrynic acid and A23187 on cellular energy metabolism. 4.2. Histamine release and A TP
The inhibition of histamine release by ethacrynic acid may be explained by its inhibitory effect on cellular energy production, since energy-requiring processes are involved in the release mechanism (Johansen, 1979a). However, this view was not supported by the observed time courses of inhibition of histamine release and decrease of cellular ATP content caused by ethacrynic acid (fig. 2). The drastic inhibition of histamine release after a short period of exposure of the cells to ethacrynic acid was associated with a moderate ATP decrease. There was some inhibition of histamine release even in presence of glucose, which seemed to counteract the effect of ethacrynic acid on cellular ATP. After more than 10 min incubation of the cells with ethacrynic acid, ionophore and glucose, there was a steady state ATP level of 60% of the normal level, and histamine release was almost blocked. In similar experiments with antimycin A but without ethacrynic acid, a steady state ATP level of 53% of the normal level was sufficient for 58% histamine release (Johansen, 1979a). Furthermore, there was a dissociation be-
188
tween the effect of dithiothreitol on histamine release and the ATP content of mast cells treated with ethacrynic acid (fig. 6). Whereas inhibition of histamine release was partly reversed by dithiothreitol, this effect was associated with only a small or no increase in cellular ATP content in absence and presence of glucose, respectively. It may be pointed out that the ATP levels shown in fig. 6 were higher than those in fig. 2 after 10 min incubation with ethacrynic acid and A23187. The reason was that in the last case the cells were also preincubated for 10 min with A23187. 4. 3. Histamine release and thiol groups
Ethacrynic acid is known to react with thiol groups, although it is not an absolutely specific thiol reagent (Gtinthers and Ahlers, 1976). Dithiothreitol keeps thiol groups in the reduced state (Cleland, 1964). When the energy supply of the mast cells was largely dependent on glycolysis, dithiothreitol completely reversed the inhibition of histamine release by ethacrynic acid. This was associated with a small decrease of cellular ATP content. This dissociation between the effect of dithiothreitol on histamine release and the cellular ATP content seems to indicate that inhibition of the secretory mechanism of the mast cell by ethacrynic acid is mainly through an interaction with thiol groups, which were not involved in cellular energy production. An additional mechanism of inhibition may be through inhibition of cellular energy production. One may speculate that this could involve interaction between ethacrynic acid and mitochondrial thiol groups. This view is supported by the small but significant reversal by dithiothreitol of histamine release as well as of the A T P content of mast cells mainly dependent on oxidative energy production. The different effect of dithiothreitol on the release process of cells incubated with either respiratory or glycolytic inhibitor excludes the possibility that the reversal by dithiothreitol of the ethacrynic acid-induced inhibition of histamine release was the result of inactivation of ethacrynic acid taking place before ethacrynic acid could react with cellular components.
In summary, ethacrynic acid inhibited ionophore A23187-induced histamine release and decreased cellular ATP content. The ATP decrease was due to inhibition of glycolytic and oxidative energy production. The most pronounced effect was on oxidative energy production. The inhibition of histamine release occurred mainly through interaction with thiol groups not involved in cellular energy production, although inhibition of energy production may be an additional mechanism.
Acknowledgements The technical assistance of Miss Marianne Svendsen, Mr. Torben Mikkelsen, Mrs. Annette Kragh Rasmussen and Mrs. Susanne Nielsen is gratefully acknowledged. The ionophore A23187 was kindly supplied by Eli Lilly and Co. (Indiana, USA) and ethacrynic acid by Merck, Sharp and Dohme International (New York, USA). This work was supported by the Danish Medical Research Council, 512-15082, and by funds obtained from Novo's Foundation.
References Chakravarty, N., 1979, The metabolism of mast cells and biochemical changes associated with histamine release, in: The Mast Cell - its Role in Health and Disease, eds. J. Pepys and A.M. Edwards (Pitman Medical Publishing Co. Ltd., Tunbridge Wells, Kent, England) pp. 38. Chakravarty, N. and E. Zeuthen, 1965, Respiration of peritoneal cells, J. Cell Biol. 25, 123. Cleland, W.W., 1964, Dithiothreitol, a new protective reagent for SH groups, Biochem. 3, 480. Diamant, B. and C. Peterson, 1974, Inhibitory action of exogenous adenosine-5'-triphosphate on glycolysis in isolated rat mast cells: significance for histamine release, Acta Pharmacol. Toxicol. 34, 325, Diamant, B., S. Norn, P. Felding, N. Olsen, A. Ziebell and J. Nissen, 1974, ATP level and CO 2 production of mast cells in anaphylaxis, Int. Arch. Allergy Appl. Immun. 47, 894. Foreman, J.C., J.L. Mongar and B.D. Gomperts, 1973, Calcium ionophores and movements of calcium ions following the physiological stimulus to a secretory process, Nature (Lond.) 245,249. Goldschmidt, D., Y. Gaudemer and D.C. Gautheron, 1976, Thiols related to mitochondrial ATPase and transport: unmasking upon conformational changes supported by the comparative effects of ethacrynate and dihydroethacrynate, Biochimie 58, 713. Gordon, E.E., 1968, Site of ethacrynic acid action of Ehrlich ascites tumour cells, Biochem. Pharmacol. 17, 1237. Gordon, E.E. and M. De Hartog, 1969, The relationship be-
189 tween cell membrane potassium ion transport and glycolysis, J. Gen. Physiol. 54, 650. Gordon, E.E. and M. De Hartog, 1971, Localization and characterization of the inhibitory action of ethacrynic acid on glycolysis, Biochem. Pharmacol. 20, 2339. G~inthers, T. and J. Ahlers, 1976, Specificity of ethacrynic acid as a sulfhydryl reagent, Arzneim. Forsch. (Drug Res.) 26, 13. Johansen, T., 1978, Mechanism of histamine release from rat mast cells induced by the ionophore A23187: effects of calcium and temperature, Br. J. Pharmacol. 63, 643. Johansen, T., 1979a, Utilization of adenosine triphosphate in rat mast cells during histamine release induced by the ionophore A23187, Br. J. Pharmacol. 65, 103. Johansen, T., 1979b, Adenosine triphosphate levels during anaphylactic histamine release in rat mast cells in vitro. Effects of glycolytic and respiratory inhibitors, European J. Pharmacol. 58, 107. Johansen, T., 1980a, Adenosine triphosphate levels during histamine release induced by compound 48/80 in rat mast cells in vitro, Life Sci. 26, 61. Johansen, T., 1980b, Further observations on the utilization of adenosine triphosphate in rat mast cells during histamine release induced by the ionophore A23187, Br. J. Pharmacol. 69, 657, Johansen, T. and N. Chakravarty, 1975, The utilization of adenosine triphosphate in rat mast cells during histamine release induced by anaphylactic reaction and compound 48/80, Naunyn-Schmiedeb. Arch. Pharmacol. 288, 243. Jones, V.D. and E.J. Landon, 1967, The effect of ouabain,
meralluride and ethacrynic acid on respiration and glycolysis in kidney slices, Biochem. Pharmacol. 16, 2163. Koechel, D.A. and E.J. Cafruny, 1973, Synthesis and structure-activity relationship of some thiol adducts of ethacrynic acid, J. Med., Chem. 16, 1147. Komorn, R. and E.J. Cafruny, 1965, Effects of ethacrynic acid on renal protein-bound sulfhydryl groups, J. Pharmacol. Exp. Ther. 148, 367. Magro, A.M., 1977, Ethacrynic acid inhibitable Ca 2+ and Mg2+-activated membrane adenosine triphosphatase in rat mast cells, Clin. Exp. Immunol. 30, 160. Manuel, M.A. and M.W. Wether, 1976, Effects of ethacrynic acid and furosemide on isolated rat kidney mitochondria: inhibition of electron transport in the region of phosphorylation site IL J. Pharmacol. Exp, Ther. 198, 209. Nemeth, E.F. and W.W. Douglas, 1978, Effects of microfilament-active drugs, phalloidin and the cytochalasins A and B, on exocytosis in mast cells evoked by 48/80 or A23187, Naunyn-Schmiedeb. Arch. Pharmacol. 302, 153. Passarella, S. and E. Quagliariello, 1979, Effect of ethacrynic acid on the permeability of the mitochondrial membrane in rat liver mitochondria, Biochem. Pharmacol. 28, 2267. Reed, P.W. and H.A. Lardy, 1972, Antibiotic A23187 as a probe for the study of calcium and magnesium function in biological systems, in: The role of Membranes in Metabolic Regulation, ed. Mehlman, M.A. and R.W. Hanson (Academic Press, New York and London) pp. 11. Shore, P.A., A. Burkhalter and V.H. Cohn, 1959, A method for the fluorometric assay of histamine in tissues, J. Pharmacol. Exp. Ther. 127, 182.
181
Elsevier
ETHACRYNIC ACID I N H I B I T I O N OF H I S T A M I N E RELEASE F R O M RAT M A S T CELLS: EFFECT O N CELLULAR ATP LEVELS AND T H I O L G R O U P S TORBEN JOHANSEN
Department of Pharmacology, Odense University, J.B. Winslows Vej 19, DK-5000 Odense C, Denmark Received 11 January 1983, revised MS received 3 May 1983, accepted 26 May 1983
T. JOHANSEN, Ethacrynic acid inhibition of histamine release from rat mast cells: effect on cellular A TP levels and thiol groups, European J. Pharmacol. 92 (1983) 181-189. The experiments concerned the effect of ethacrynic acid (0.5 raM) on the adenosine triphosphate (ATP) content of rat mast cells and the effect on histamine release induced by the ionophore A23187 (10 # M). Ethacrynic acid decreased the ATP level of the cells in presence of antimycin A and glucose as well as in presence of 2-deoxyglucose. A23187-induced histamine release was inhibited by ethacrynic acid, and this inhibition was completely reversed by dithiothreitol. These observations may indicate that ethacrynic acid inhibits glycolytic and respiratory energy production in rat mast cells. Furthermore, the inhibition of histamine release occurred mainly through interaction with thiol groups, although inhibition of cellular energy production may have been an additional mechanism. Energy metabolism
Ethacrynic acid
Histamine release
I. Introduction The antibiotic A23187 specifically forms complexes with calcium and magnesium and transport these ions across a variety of membranes (Reed and Lardy, 1972). In the presence of calcium, A23187 acts as a histamine-releasing agent on isolated rat mast cells (Foreman et al., 1973). The mechanism of A23187-induced histamine release seems to be similar to that activated in rat mast cells by the antigen-antibody reaction in vitro (Johansen, 1978). Results of previous studies of the relation between mast cell ATP content and anaphylactic (Johansen and Chakravarty, 1975; Johansen, 1979b) as well as A23187-induced histamine release (Johansen, 1979a) are consistent with the view that energy-requiring processes are involved in the release mechanism. Anaphylactic histamine release and A23187-induced histamine release are completely blocked by ethacrynic acid (Magro, 1977; Chakravarty, 1979). It has been demonstrated that ethacrynic acid inhibits both glycolysis and respiration of Ehrlich ascites tumour cells and slices of kidney (Jones 0014-2999/83/$03.00 © 1983 Elsevier Science Publishers B.V.
Ionophore A23187
Mast cells
and Landon, 1967; Gordon, 1968; Gordon and Hartog, 1969). At the subcellular level ethacrynic acid interferes with a number of mitochondrial processes (Goldschmidt et al., 1976; Manuel and Weiner, 1976; Passarella and Quagliariello, 1979) and with glycolytic enzymes (Gordon and De Hartog, 1971). Ethacrynic acid is known to react with thiol groups (Komorn and Cafruny, 1965; Koechel and Cafruny, 1973; Goldschmidt et al., 1976; Manuel and Weiner, 1976), which may be involved in the metabolic reactions above as well as in secretory processes (Nemeth and Douglas, 1978). In the present investigation the effect of ethacrynic acid on A23187-induced histamine release and mast cell ATP content was examined in order to study the relation between the effect of ethacrynic acid on cellular energy metabolism and its inhibitory action on the histamine release process. It is demonstrated that ethacrynic acid, in addition to its inhibitory effect on cellular energy production, inhibits A23187-induced histamine release through interaction with thiol groups.
182 2. Materials
and methods
2.1. Isolation o f rat mast cells M a l e S p r a g u e - D a w l e y rats, 350-570 g, w e r e used for the e x p e r i m e n t s , M a s t cells were i s o l a t e d as d e s c r i b e d p r e v i o u s l y ( J o h a n s e n , 1979a) a n d susp e n d e d in K r e b s - R i n g e r s o l u t i o n c o n t a i n i n g hum a n s e r u m a l b u m i n , 1 m g / m l , final p H 7.0 to 7.1. M a s t cells: 96.4 _4- 0.7% ( m e a n + S.E.M.; n = 40) o f cell p o p u l a t i o n .
2.2. Incubation procedure (fig. 1) M a s t cell s u s p e n s i o n s p o o l e d f r o m 1 to 4 rats w e r e d i v i d e d i n t o a l i q u o t s w i t h the s a m e cell d e n sity in a final v o l u m e o f 0.5 to 0.6 ml. T h e s e w e r e
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used for d e t e r m i n a t i o n of the A T P c o n t e n t a n d for the h i s t a m i n e release e x p e r i m e n t s . T h e cell d e n s i t y in the v a r i o u s e x p e r i m e n t s v a r i e d f r o m 0.45 × 105 to 3.76 × 10S c e l l s / m l . T h e cell s u s p e n s i o n s were e q u i l i b r a t e d in a 3 7 ° C b a t h for 10 min in a c a l c i u m - f r e e K r e b s - R i n g e r solution. In the experim e n t s in fig. 2 the cells w e r e t h e n t r e a t e d with A 2 3 1 8 7 (10 # M ) for 10-26 m i n in the a b s e n c e of c a l c i u m , the last 15 s to 16 m i n with e t h a c r y n i c a c i d (0.5 m M ) p r e s e n t in the m e d i u m . H i s t a m i n e release was i n i t i a t e d by e x p o s u r e of the cells to c a l c i u m (5 m M ) for m o r e t h a n 45 s, w h i c h is k n o w n to be a s u f f i c i e n t t i m e p e r i o d for the h i s t a m i n e release o f be c o m p l e t e d ( J o h a n s e n , 1978). T h e high c o n c e n t r a t i o n of c a l c i u m was used in o r d e r to get a fast release o f h i s t a m i n e ( J o h a n s e n ,
Co
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Fig. 1. Incubation procedure (temperature equilibration omitted). A: Antimycin A (1 ~M), Ca: Calcium (5 mM), D: Dithiothreitol (1 mM), 2-DG: 2-deoxyglucose (5 mM), E: Ethacrynic acid (0.5 mM), G: Glucose (1 raM), I: A23187 (10 /*M), O: Oligomycin (1 tzg/ml), S: Pyruvate (1 raM) or Succinate (5 mM). Abscissa: Incubation time in min. Dashed lines represent variable time periods.
Fig, 2. Time course of inhibition of histamine release and decrease in cellular ATP content by ethacrynic acid. The cells were incubated with ethacrynic acid for various time intervals in a calcium-free medium in presence of A23187 in the absence (©,D) or presence (~,a) of glucose. Cellular ATP content was then determined (r7 ,,,) or the cells were incubated with calcium in order to initiate histamine release (O,~). Abscissa: Time of incubation with ethacrynic acid. Ordinate: Percent histamine release and ATP content of the cells. Control histamine release from A23187-treated mast cells incubated in the absence of ethacrynic acid with glucose: 69.8%±3.1% (mean ±S.E.M, n = 5), or without glucose: 49.7%±3.7% (mean ±S.E.M., n = 10). 100 on the ordinate represents the normal ATP content from mast cells incubated in the absence of ethacrynic acid and A23187 with (1.46_+0.06 pmol/103 cells, mean _+S.E.M., n = 6) or without glucose ( 1.52 + 0.02 pmol/103 cells, mean ± S.E.M, n = 3). A23187dnduced histamine release in the absence of calcium and in the absence (mean 7.4%) and presence of glucose (mean 10.3%) subtracted. Spontaneous histamine release in the absence of A23187 and ethacrynic acid was 3.2% (mean value). Mean values from two (@ at 13 and 16 rain) and three to seven experiments; vertical lines show S.E.M.
183 1978). Samples were included for d e t e r m i n a t i o n of c a l c i u m - i n d u c e d h i s t a m i n e release from mast cells pretreated with A23187 alone, as well as samples for d e t e r m i n a t i o n of histamine release i n d u c e d by A23187 in the absence of calcium and ethacrynic acid. Three types of experiments were done to study the effect of ethacrynic acid on the A T P c o n t e n t of the mast cells. I n all three cases the cells were temperature-equilibrated in a calcium-free m e d i u m as described above. Firstly (fig. 3), the cells were i n c u b a t e d for 10 to 30 min, either with ethacrynic acid (0.5 m M ) alone or with a c o m b i n a t i o n of ethacrynic acid a n d glucose (1 m M ) or a c o m b i n a tion of ethacrynic acid a n d respiratory inhibitors ( a n t i m y c i n A, 1 /~M, a n d oligomycin, 1 /~g/ml). In the last case A23187 (10/~M) was also present in the m e d i u m in a d d i t i o n to the respiratory inhibitors and ethacrynic acid, and glucose (1 raM) was used as substrate for the glycolysis. Secondly (fig. 4), the cells were p r e i n c u b a t e d for 20 m i n with a respiratory i n h i b i t o r ( a n t i m y c i n A, 1 /~M) or an i n h i b i t o r of glycolysis (2-deoxyglucose (2DG), 5 raM). In the first case glucose (1 raM) was used as
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Fig. 4. Effect of ethacrynic acid on the ATP content of mast cells. The cells were incubated with (n) or without (O) ethacrynic acid (0.5 mM) for 20 rain as indicated on the abscissa. Ordinate: ATP content of the cells in percent of control value from untreated cells: 1.24_+0.05 pmol/103 cells (mean _+S.E.M., n = 3). Mean values from three experiments; vertical lines show S.E.M.
substrate for glycolysis. I n the last case the cells were pretreated with 2 - D G with or without a respiratory substrate (pyruvate, 1 mM, or succinate, 5 raM) present in the medium. The cells were then i n c u b a t e d with ethacrynic acid (0.5 raM) for 20 min. T h i r d l y (fig. 5), the mast cells were pretreated with 2 - D G (5 mM) for 20 rain, the last 5 m i n with ethacrynic acid (0.5 raM) present in the
80 ATP (% of confrot) 80
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Min Fig, 3. Effect of ethacrynic acid on the ATP content of the mast cells. The cells were incubated in calcium-free medium with ethacrynic acid in the absence (O) or presence (O) of glucose. ,x represents the ATP content of mast cells treated with respiratory inhibitors, ionophore A23187 and ethacrynic acid. Glucose was used as substrate for glycolysis. Abscissa: Time of incubation of the mast cells with the reagents. Ordinate: ATP content in percent of control value from mast cells treated with glucose alone: 1.35_+0.02 pmol/10 3 cells (mean _+S.E.M., n = 3). Mean values from three experiments; vertical lines show S.E.M.
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Fig. 5. Effect of ethacrynic acid on the rate of ATP depletion of mast cells. The glycolytic pathway in the cells was blocked by 2-DG. Abscissa: Time of incubation with respiratory inhibitors with (zx) or without (O) ethacrynic acid. Ordinate: ATP content of the mast cells in percent of control value from untreated cells: 1.27_+0.13 pmol/l03 cells (mean + S.E.M.). The last 5 min of preincubation with ethacrynic acid decreased the ATP content by 20 percent. Mean values from three experiments; vertical lines show S.E.M.
184
medium. A combination of antimycin A (1 ffM) and oligomycin (1 ffg/ml) was then added to the cell suspension. The cells were incubated with the respiratory inhibitors for 15 to 60 s. All three types of experiments were performed in a calcium-free medium. Samples for determination of the normal value of the cellular ATP content in the absence of the metabolic inhibitors, A23187 and ethacrynic acid, were included. In the experiments in fig. 6 the cells were temperature-equilibrated at 37°C in a calcium-free medium for 10 min in the presence or absence of dithiothreitol (1 mM) and glucose (1 mM). A23187 (10 ffM) with or without ethacrynic acid (0.5 mM) was added to the cell suspension and the incubation continued for 10 min in a calcium-free medium. The incubation was then stopped with perchloric acid (see below) for determination of cellular ATP, or calcium (5 raM) was added for more than 45 s in order to induce histamine release. The cells in the experiments in fig. 7 were temperature-equilibrated at 37°C in a calcium-free medium then preincubated for 20 rain with either antimycin A (1 ~M) and glucose (1 mM) or with 2 - D G (5 raM). In both cases the preincubation continued for a further 20 min with
A23187 and ethacrynic acid added to the cell suspension. Histamine release was then initiated by adding calcium (5 raM) to the cell suspension. Cellular ATP content was determined after the 40-rain preincubation period. Dithiothreitol (1 raM) was present in half the samples during the whole preincubation period and during incubation with calcium (as indicated in fig. 7). 2.3. Determination of histamine release and the A TP content of the mast cells
The histamine release experiments were performed as described previously (Johansen and Chakravarty, 1975). Histamine release was determined by a fluorometric method (Shore et al.. 1959) including the extraction procedure with butanol and heptane. Since it was found that dithiothreitol 0.4 mM and ethacrynic acid 0.1 ruM did not influence the histamine determination, the extraction procedure was omitted when possible. The release of histamine was calculated as a percentage of the total histamine content of the mast cells. For the determination of ATP, the reaction after incubation of the samples was stopped with
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Fig. 6. Effect of dithiothreitol on the inhibition of histamine release and the decrease of cellular ATP content caused by ethacrynic acid. The cells were incubated with the reagents indicated on the abscissa. Ordinate, left: ATP content of the cells (1~). 100 on the ordinate represents the ATP content of mast cells incubated in presence (1.55_+ 0.12 pmol/103 cells, mean + S.E.M.) and absence (1.49_+0.11 pmol/103 cells, mean + S.E.M.) of glucose. Ordinate, right: percent histamine release (E3). Appropriate spontaneous release subtracted. Spontaneous histamine release (i.e. no drug present) was 2.6% (mean value). Mean value from three experiments; vertical lines show S.E.M.
185
[ ] Anfimycin A + glucose 80 7
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Fig. 7. Effect of dithiothreitol on the inhibition of histamine release and the decrease of cellular ATP content caused by ethacrynic acid. The cells were preincubated with eiiher antimycin A and glucose (ea) or 2-DG alone (~) then with A23187 and ethacrynic acid (see Method section). Histamine release (fig. 6a) was initiated by incubation of the cells with calcium. Cellular ATP content (fig. 6b) was determined at the time of initiation of histamine release. Ordinate (fig. 6a): Percent histamine release. In control samples (0) the mast cells were pretreated with A23187 alone for 20 min then incubated with calcium. Appropriate spontaneous histamine release induced by A23187 from mast cells incubated in calcium-free medium with the metabolic inhibitors, glucose and ethacrynic acid subtracted. Spontaneous histamine release with no drugs present was 1.5% (mean value). Ordinate (fig. 6b): ATP content of the cells in percent of control value from mast cells incubated without any reagent or substrate: 1.47+0.05 pmol/103 ceils (mean + S.E.M.). Mean value from four experiments; vertical lines show S.E.M.
ice-chilled perchloric acid. After neutralization of the supernatant, the A T P content was determined by the bioluminescence technique using luciferinluciferase from firefly tails as described earlier (Johansen and Chakravarty, 1975). Internal standards were used for correction of any inhibition of light emission caused by potassium perchlorate or drugs in the reaction medium. N o n e of the drugs interfered with the A T P determination. 2.4. Cell viabifity D y e exclusion by the cells, with T r y p a n Blue as the dye, was used as a measure of cell viabililty. After 40-min exposure of the cells to either of the following drugs and chemicals, 94% or more of the cells excluded the dye: ethacrynic acid, antimycin A and oligomycin, 2-DG, Ca 2÷, A23187 or glucose. In presence of dithiothreitol, 90% of the cells
excluded the dye. In the absence of drugs and chemicals, 98% of the cells excluded the dye. Thus, none of the drugs and chemicals seemed to do any serious harm to the cells in the experiments in the present study. In parallel experiments, spontaneous histamine release (without the combination of calcium and A23187 present in the cell suspension) was less than 3.5%. In presence of A23187 alone, histamine release was 5.1%. In addition, cellular glycolysis, stimulated by incubation of the cells with respiratory inhibitors and glucose, was very active in presence of ethacrynic acid and ionophore A23187 (Johansen, 1980b), which supports the above conclusion. 2.5. Materials H u m a n serum albumin was supplied by AB K A B I ( S t o c k h o l m , Sweden), a n t i m y c i n A, oligomycin and dithiothreitoi by Sigma Chemical C o m p a n y (St. Louis, USA). The ionophore A23187 was a gift from Eli Lilly and Co. (Indiana, USA). A23187 was dissolved in ethanol 96% v / v and diluted in Krebs-Ringer solution for the experiments. The final concentration of ethanol during the incubation never exceeded 0.3% v / v . This concentration of ethanol had no effect on histamine release or on the A T P content of the mast cell (Johansen and Chakravarty, 1975; Johansen, 1978). All other chemicals were of analytical grade. Ethacrynic acid, which was supplied by Merck, Sharp and D o h m e International (New York, USA), was dissolved in 0.9% NaC1 at p H 11-12. After neutralization the final p H in Krebs-Ringer solution with h u m a n serum albumin, 1 m g / m l , was 7.0-7.1. The Krebs-Ringer solution had the following composition (raM): NaC1 143.3, KC1 4.7, MgSO 4 1.2, N a 2 H P O 4 2.5, K H 2 P O 4 0.6.
3. Results
3.1. Effect of ethacrynic acid on histamine release and cellular A TP content Exposure of mast cells to ethacrynic acid for short time intervals (0-2 min) caused an inhibition
186
of histamine release of about 50% (fig. 2). This was partly counteracted by adding glucose to the incubation medium. However, after 10-16-rain exposure of the cells to ethacrynic acid, histamine release was blocked, and glucose had almost no effect on the release process. There was a time-dependent ATP decrease in the presence as well as in the absence of glucose. The ATP level in the presence of glucose was 15-25% higher than that in the absence of glucose (fig. 2). In both cases the maximal ATP decrease was found after 10-rain incubation of the cells with the reagents without further changes in cellular ATP up to 16 min of incubation, which was the longest period of observation. After a lag period of 5 min, there was a timedependent decrease of the ATP content of mast cells incubated with ethacrynic acid (fig. 3). This was partly counteracted by glucose. However, even in the presence of glucose a low rate of ATP decrease was observed. Incubation of the mast cells for 10-30 min with respiratory inhibitors (antimycin A and oligomycin) and ionophore A23187 in addition to ethacrynic acid and glucose in calcium-free Krebs-Ringer solution resulted in a steady state level of ATP of 46-49% of the normal value. In presence of 2-DG, cellular ATP synthesis was largely dependent on respiratory energy production (Johansen, 1980a). In this case ethacrynic acid caused a drastic ATP decrease whether or not the respiratory substrates, pyruvate (Diamant et al., 1974) or succinate (Chakravarty and Zeuthen, 1965), were present in the medium (P < 0.0025 or less by t-test for two groups of data) (fig. 4). By contrast in presence of antimycin A and glucose ATP synthesis was largely dependent on glycolytic energy production, and the ATP decrease caused by ethacrynic acid (P < 0.0025) was less than half of that observed in the 2-DG-treated cells. Fig. 5 shows that the rate of ATP depletion of mast cells pretreated with 2-DG then incubated with respiratory inhibitors (antimycin A and oligomycin) seems to follow a first order reaction. The rate of ATP depletion tended to be less in the presence than in the absence of ethacrynic acid.
3. 2. Effect of dithiothreitol In the absence of glucose, ethacrynic acid completely blocked the histamine release (fig. 6a) as also demonstrated above (fig. 2). This was partly counteracted by dithiothreitol (b). The histamine release in presence of dithiothreitol was 50% of the control value (f). A similar effect of dithiothreitol on histamine release was observed in presence of glucose (compare c and d). Cellular ATP content was determined at the time of initiation of histamine release by calcium. The decrease in cellular ATP content caused by ethacrynic acid (a), was partly counteracted by dithiothreitol (b). In the absence and presence of dithiothreitol, the ATP levels were 67% and 82%. respectively, of the control values (e and f, respectively) (P < 0.05 by t-test for two groups of data). However, with glucose present, no significant increase in cellular ATP content by dithiothreitol was found (compare c and d). In control samples, dithiothreitol had a small potentiating effect on histamine release in the absence (e, f, P < 0.05), but not in the presence of glucose (g, h, P > 0.05). There was no effect of dithiothreitol on cellular ATP content (compare e, f ( P > 0.05) and g, h (P > 0.10)). Furthermore, dithiothreitol had no effect on the ATP content of mast cells incubated in the absence of ethacrynic acid and A23187 whether or not glucose was present in the medium (i, j). Dithiothreitol had no effect on the spontaneous histamine release from untreated cells whether or not calcium was present, and dithiothreitol also had no effect on histamine release from mast cells incubated in calcium-free medium with either A23187 alone or with both A23187 and ethacrynic acid unless, in the last case, glucose was present. In this case a small potentiating effect of dithiothreitol was found (P < 0.05 by t-test for two groups of data). Ethacrynic acid had a small potentiating effect on A23187-induced release from mast cells incubated in calcium-free medium with glucose. This was observed in the presence as well as in the absence of dithiothreitol (P < 0.05, P < 0.0125 by t-test for two groups of data). No effect was found in the absence of glucose. When the respiratory energy supply of the cells was blocked by antimycin A, dithiothreitol com-
187 pletely reversed the inhibition of histamine release by ethacrynic acid (P < 0.0005) (fig. 7). Glucose was used as substrate for glycolysis in these experiments. This effect was associated with a small decrease of the cellular ATP content (P < 0.005). In contrast, when 2-DG was used to block mast cells glycolysis, dithiothreitol caused only a small increase in histamine release (P < 0.05) and cellular ATP level (P < 0.0005). In parallel control experiments without ionophore there were similar changes in cellular ATP values in response to dithiothreitol (data not shown).
4. Discussion 4.1. Cellular A TP
The cellular ATP depletion caused by ethacrynic acid may be explained by increased utilization of cellular ATP as well as by inhibition of oxidative or glycolytic energy production, both of which are associated with the synthesis of energy-rich phosphate in ATP. The possibilitY of an increased ATP utilization seemed unlikely since ethacrynic acid tended to decrease the rate of ATP depletion in mast cells without oxidative and glycolytic energy production. The ATP decrease seen in mast cells dependent on either glycolytic or oxidative energy production indicates an inhibition of both pathways by ethacrynic acid, the most pronounced effect being on oxidative ATP synthesis. The inhibition of oxidative ATP synthesis was not caused by lack of respiratory substrate, since the same ATP decrease was observed in presence as well as in the absence of respiratory substrates. The rate of ATP depletion caused by ethacrynic acid in presence of glucose was one third of that observed in the absence of glucose. This may indicate only a slight inhibition of glycolysis by ethacrynic acid. This view is supported by the observed steady state ATP level of mast cells dependent on glycolytic ATP synthesis. In these experiments the effect of ethacrynic acid on mitochondrial ATP production would not be apparent, since this was blocked by respiratory inhibitors. The rate of glycolytic activity when enhanced by extracellular glucose (Diamant and Peterson, 1974), seemed to
be sufficient for the cell to maintain a steady state ATP level of about half the normal ATP value. Previously, ethacrynic acid was found to cause a 21% decrease of the rate of lactate production by mast cells treated with respiratory inhibitors and glucose (Johansen, 1980b). It may be observed that the rate of the ATP decrease caused by ethacrynic acid alone (fig. 3) and the rate of that caused by ethacrynic acid in combination with A23187 (fig. 2) were of the same order of magnitude, about 45% and 35%, respectively, after 10 min. One may speculate that the steady state value of cellular ATP observed in fig. 2 when cells were incubated with A23187 and ethacrynic acid, but not seen in fig. 3 when the cells were incubated with ethacrynic acid alone, may be due to a protective effect of A23187 on oxidative energy metabolism, which was more depressed by ethacrynic acid than was glycolysis (fig. 4). However, additional experiments are necessary to further clarify the interaction between ethacrynic acid and A23187 on cellular energy metabolism. 4.2. Histamine release and A TP
The inhibition of histamine release by ethacrynic acid may be explained by its inhibitory effect on cellular energy production, since energy-requiring processes are involved in the release mechanism (Johansen, 1979a). However, this view was not supported by the observed time courses of inhibition of histamine release and decrease of cellular ATP content caused by ethacrynic acid (fig. 2). The drastic inhibition of histamine release after a short period of exposure of the cells to ethacrynic acid was associated with a moderate ATP decrease. There was some inhibition of histamine release even in presence of glucose, which seemed to counteract the effect of ethacrynic acid on cellular ATP. After more than 10 min incubation of the cells with ethacrynic acid, ionophore and glucose, there was a steady state ATP level of 60% of the normal level, and histamine release was almost blocked. In similar experiments with antimycin A but without ethacrynic acid, a steady state ATP level of 53% of the normal level was sufficient for 58% histamine release (Johansen, 1979a). Furthermore, there was a dissociation be-
188
tween the effect of dithiothreitol on histamine release and the ATP content of mast cells treated with ethacrynic acid (fig. 6). Whereas inhibition of histamine release was partly reversed by dithiothreitol, this effect was associated with only a small or no increase in cellular ATP content in absence and presence of glucose, respectively. It may be pointed out that the ATP levels shown in fig. 6 were higher than those in fig. 2 after 10 min incubation with ethacrynic acid and A23187. The reason was that in the last case the cells were also preincubated for 10 min with A23187. 4. 3. Histamine release and thiol groups
Ethacrynic acid is known to react with thiol groups, although it is not an absolutely specific thiol reagent (Gtinthers and Ahlers, 1976). Dithiothreitol keeps thiol groups in the reduced state (Cleland, 1964). When the energy supply of the mast cells was largely dependent on glycolysis, dithiothreitol completely reversed the inhibition of histamine release by ethacrynic acid. This was associated with a small decrease of cellular ATP content. This dissociation between the effect of dithiothreitol on histamine release and the cellular ATP content seems to indicate that inhibition of the secretory mechanism of the mast cell by ethacrynic acid is mainly through an interaction with thiol groups, which were not involved in cellular energy production. An additional mechanism of inhibition may be through inhibition of cellular energy production. One may speculate that this could involve interaction between ethacrynic acid and mitochondrial thiol groups. This view is supported by the small but significant reversal by dithiothreitol of histamine release as well as of the A T P content of mast cells mainly dependent on oxidative energy production. The different effect of dithiothreitol on the release process of cells incubated with either respiratory or glycolytic inhibitor excludes the possibility that the reversal by dithiothreitol of the ethacrynic acid-induced inhibition of histamine release was the result of inactivation of ethacrynic acid taking place before ethacrynic acid could react with cellular components.
In summary, ethacrynic acid inhibited ionophore A23187-induced histamine release and decreased cellular ATP content. The ATP decrease was due to inhibition of glycolytic and oxidative energy production. The most pronounced effect was on oxidative energy production. The inhibition of histamine release occurred mainly through interaction with thiol groups not involved in cellular energy production, although inhibition of energy production may be an additional mechanism.
Acknowledgements The technical assistance of Miss Marianne Svendsen, Mr. Torben Mikkelsen, Mrs. Annette Kragh Rasmussen and Mrs. Susanne Nielsen is gratefully acknowledged. The ionophore A23187 was kindly supplied by Eli Lilly and Co. (Indiana, USA) and ethacrynic acid by Merck, Sharp and Dohme International (New York, USA). This work was supported by the Danish Medical Research Council, 512-15082, and by funds obtained from Novo's Foundation.
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