Naunyn-Schmiedeberg's Arch. Pharmacol. 275, 457--463 (1972) �9 by Springer-Verlag 1972

Dependence of Histamine Release from Rat Mast Cells on Adenosine Triphosphate

Torben Johansen and Nh'mal Chakravarty

Dept. of Pharmacology, Odense University, Odense, Denmark

]~eceived June 9, 1972

Summary. Using pure populations of rat mast cells, the relation of the ATP content of the cells to histamine release induced by compound 48/80 has been studied. Variable ATP levels in the mast cells have been produced by incubation with appro- priate concentrations of oligomycin.

The dose-response curves of oligomycin for the inhibition of histamine release and for the reduction in the ATP content of the mast cells are similar. The concen- tration required for 500/0 inhibition of histamine release is, however, higher than that for 50o/0 reduction of the ATP level.

Comparative study of the reduction of the ATP content and the inhibition of histamine release in samples of the same suspension of mast cells shows a linear relation between 10 to 90 ~ inhibition of histamine release and 40 to 95 ~ inhibition of ATP synthesis.

The observations support the hypothesis that ATP is involved in the process of histamine release from mast cells induced by compound 48/80.

Key words: Histamine -- Mast Cells -- ATP -- 01igomyein -- Compound 48/80.

Previous observat ions indica te t h a t anaphy lac t i c h i s tamine release is dependen t on ox ida t ive or g lycolyt ic metabo l i sm (Parrot , 1942; Mongar and Schild, 1957; Chak rava r ty , 1960, 1962a; D iaman t , 1962). The release of h i s tamine f rom eat and r a t t issues b y the po lymer ic amine compound 48/80 has the same character is t ics (Chakrava r ty et al., 1959; Wes te rho lm, 1960; Rothsch i ld et al., 1961). I t is now genera l ly assumed t h a t the process of h i s tamine release induced by ant igen-ant i - b o d y reac t ion in several species and b y compound 48/80 in cat and r a t t issues requires energy, which m a y be p rov ided b y the ox ida t ive or the g lycolyt ic p a t h w a y . Appa ren t ly , the energy-r ich compound adenosine-5 ' - t r i phospha t e ( A T P ) - - s y n t h e s i z e d b y bo th the r e a c t i o n s - - m a y be l inked wi th the process t h a t leads to h i s tamine release.

Oligomycin is known to specifical ly block the u t i l iza t ion of ox ida t ive energy for the synthesis of A T P (Lardy et al., 1958). I n the p resen t inves t iga t ion ol igomyein has been used to reduce the A T P synthesis in pure popula t ions of r a t mas t cells. I t has thus been possible to correlate the A T P con ten t of the mas t cells to the i r ab i l i t y to release h is tamine in

458 T. Johansen and N. Chakravarty:

response to compound 48/80. I t is shown in the following repor t t ha t

h is tamine release is ve ry l i t t le affected with 25--300/0 reduct ion in the

A T P content of the mas t cells. Wi th greater reduct ion in the A T P levels,

a l inear relat ionship exists between the A T P content of the mas t ceils

and the release of his tamine induced by compound 48/80.

Materials and Methods

Male Sprague-Dawley rats, 400--480 g, were used for the experiments. Pure populations of mast cells were obtained by differential centrifugation of mixed peritoneal ceils over layers of concentrated human serum albumin solution, as described previously (Chakravarty and Zeuthen, 1965; Chakravarty, 1965). The purity of the mast cell suspensions was 96--100~ (mean 98.2~ The cells were suspended in Krebs-Ringer solution with phosphate buffer containing 1 mg/ml human serum albumin (final pI:[: 7.0--7.1). Mast cells were pooled from 3--4 rats for each experiment and about 10 ~ cells in 0.4 ml solution were used for each sample. Oligomycin, first dissolved in ethyl alcohol, was diluted with the Krebs-Ringer solution to the final concentrations (1--120 ng/ml) employed in the experiments. The minute quantities of alcohol corresponding to these oligomycin concentrations had no effect on the ATP content and the histamine release.

Three types of experiments were performed:

1. Histamine Release ]rein Mast Cells. Isolated mast cells were incubated with different concentrations of oligomyein at 37~ for 10 rain. Compound 48/80 was then added to the cells (final conc. 0.5--1.2 ~g/ml) and the incubation continued for another 10 rain. Control samples without oligomycin and for spontaneous histamine release were always included. The remaining experimental procedure has been essentially the same as described earlier (Chakravarty, 1968). Histamine was, how- ever, determined in the present experiments by the fluorometric method (Shore et al., 1959). Spontaneous histamine release (1--5~ has been subtracted from the values presented. In each experiment the effect of 2--5 concentrations of oligomycin on histamine release was studied, using duplicate or triplicate samples for each concen- tration. Usually the same concentration of oligomycin was studied in 2--3 experi- ments. The mean values are presented in Table 1.

2. A T P Content o/Mast Cells. Mast cell suspensions were prepared in the same way as described above. The samples were incubated with varying concentrations of oligomyein for 10 rain at 37~ Samples without oligomycin and appropriate controls were included. The reaction was stopped by adding 11 times higher volume of boiling redistilled water to the samples and transferring them immediately to a boiling water bath for 10 rain. The samples were cooled and centrifuged at 0--4~ and the supernatant kept at --80~ for ATP determination the following day. In each experiment the effect of two concentrations of oligomycin was studied, using triplicate samples for each concentration. Usually the same concentration of oligo- mycin was studied in 2--3 experiments. The mean values are presented in Table 2.

ATP was determined by the lueiferin-luciferase bioluminiscence method as described by Addanki et al. (1966) with some modifications. The luminiscence was measured by a Beckman liquid scintillation spectrometer, using the optimal tritium setting.

3. Correlation between A T P Content o/Mast Cells and Compound 48~80-Induced Histamine Release. In these experiments samples of the same suspension of mast cells were incubated for 10 rain with the same concentration of oligomycin both for the

Dependence of Histamine Release from Mast Cells on ATP 459

determination of histamine release and the ATP content. The experimental procedure otherwise was the same as described above under 1. and 2.

Firefly lantern extract (Sigma FLE 50) was used as the luciferin-lucfferase enzyme system. ATP and oligomycin (consisting of 15~ oligomycin A and 850/0 oligomycin B) were also purchased from Sigma Chemical Co. Human serum albumin was purchased from Kabi, Stockholm. Compound 48/80 (condensation product of p-methoxyphenethylmethylamine with formaldehyde) was obtained through the courtesy of AB Leo, H~lsingborg, Sweden.

Results

Inhibition o[ Histamine Release [tom Mast Cells by Oligomycin

Table 1 shows the f rac t ion of the t o t a l h i s tamine con ten t of the mas t cells re leased b y compound 48/80 (0.5--1.2 txg/ml) in the absence and the presence of ol igomycin. The degree of inh ib i t ion of h i s t amine release b y the different concent ra t ions of o l igomycin has been p resen ted in the l as t co lumn of the Table. B y p lo t t ing the d a t a t he concen t ra t ion of o l igomycin requi red for 50~ inh ib i t ion of h i s t amine release was found to be 18 ng/ml.

Table 1 Inhibition of histamine release by oligomycin

Oligomycin Compound Total ~ Histamine release ~ Inhibition 48/80 histamine of histamine

confent Without With release (base) of oligomycin oligomycin mast cells (a) (b)

ng/ml ~g/ml ng/108 cells (a--b) 100/a

117 0.7 29 28.3 3.5 88 97.5 0.5 39 14.8 2.7 82 58.8 0.6 33 21.8 1.9 91 41.1 1.0 30 57.6 10.9 81 29.4 0.8 33 39.2 17.2 56 26.1 1.0 33 27.8 7.9 72 23.5 1.0 29 57.6 23.6 59 19.1 0.9 26 26.6 11.7 56 14.9 0.8 31 35.0 23.2 34 11.8 1.0 28 48.9 33.3 32 10.0 1.0 26 35.2 27.5 22 8.0 1.2 23 41.6 32.8 21 7.3 0.8 31 25.2 20.3 19 6.5 0.5 38 14.8 17.7 0 4.9 0.5 36 14.8 18.8 0

32 l~aunyn-$chmiedeberg's Arch. Pharmacol., u 275

460 T. Johansen and N. Chakravarty:

Table 2 Effect of incubation of mast cells with oligomycin on their ATP content

Oligomycin ATP content, pM/10 s cells 0/0 Reduction of ATP

Without With ng/ml oligomycin (a) oligomycin (b) (a--b) 100/a

117 1.86 0.16 91 26.1 1.86 0.50 73 19.6 2.09 0.50 76 15.7 2.40 0.65 73 11.8 1.79 1.20 33 9.9 1.95 0.90 54 7.6 2.16 1.61 26 5.2 1.92 2.05 0 2.6 1.92 2.05 0

Reduction in A TP Content o/Mast Cells by Oligomycin

The ATP content of the mast cells in 7 experiments, each containing cells pooled from 3- -4 rats, was 2.02 • 0.34 pM (mean • s.e.) per 103 mast cells.

The effect of oligomycin on the ATP content of the mast cells is shown in Table 2. The degree of reduction of the ATP content by different concentrations of oligomycin (2.6--117ng/ml) is given in the last column of the Table. I t may be seen tha t there is a good dose-response relationship between 5 and 26 ng/ml oligomycin and the reduction in the ATP content. The highest concentration of o]igomycin used, 117 ~g/ml, caused 91~ reduction of the ATP level. By plotting the data the concen- trat ion of oligomycin required for 500/0 reduction of the ATP level was found to be 12.5ng/ml--being thus lower than the concentration (18 ng/ml) required for 50 ~ inhibition of histamine release.

The results presented in Tables 1 and 2 being based on different experiments for the ATP content and the histamine release are, however, less suitable for a correlation of these two data.

Correlation between A TP Content o/ Mast Cells and Histamine Release Induced by Compound 48/80

Because of the variation in the initial ATP level in different rats and some variation in the inhibition caused by oligomycin in ATP synthesis and histamine release, i t was necessary to determine both the ATP content and the histamine release in samples of the same mast cell sus- pension using the same concentration of oligomycin for the correlation of these two data. The results of such correlative studies--using 1.0 to 1.2~g/ml compound 48/80 for histamine release and 7 - -117ng/ml

Dependence of Histamine Release from Mast Cells on ATP 461

Ioo

~ 75 E

~ so g

.~ 2s

/ ~

Ooo/ I I 7,5 l 25 5O 100

% Reduction in ATP content

Fig. 1. Correlation of the reduction of ATP content in mast cells to the inhibition of histamine release. Different oligomycin concentrations (7--117 ng/ml) were used to produce variable reduction in the ATP content, and the effect on histamine release induced by compound 48/80 (1--1.2 ~zg/mt) was studied in samples of the same cell suspension with the same oligomyein and compound 48/80 concentrations. Results from 6 experiments with 2 concentrations of oligomycin in each. Correlation coef-

ficient: 0.91, P < 0.001

ol igomycin--are shown in Fig. 1. The reduction of the ATP content of the mast cells shows a linear relation to the inhibition of histamine release. The correlation coefficient is 0.91 (P < 0.001). There is thus a good correlation between histamine release and the ATP level. I t may be observed tha t 500/0 inhibition of the histamine release is associated with 65--700/0 reduction in the ATP content. On the other hand, 500/0 reduc- tion of the ATP content correlates with about 300/o inhibition of the histamine release. The lowest ATP reduction shown in the curve, viz. 420/0, corresponds to only 11~ inhibition of histamine release.

Discuss ion

The work for the last 15--20 years has pointed to an enzymatic mechanism for the anaphylactic histamine release and- - in certain species-- the release by compound 48/80. t I is tamine release has been shown to be blocked by lack of oxygen, inhibitors of the respiratory chain and inhibitors of glycolysis (Mongar and Schild, 1957, 1962 ; Chakravarty, 1960, 1962a; Diamant and Uvn~s, 1961; Moussatchd and Prouvost- Danon, 1962). Uncouplers of oxidative phosphorylation like 2-4-dinitro- phenol (Moussateh6 and Prouvost-Danon, 1958) and dicoumarol have also been shown to block histamine release. Dieoumarol (0.1 raM) inhibits histamine release from rat mas t cells induced both by compound

32*

462 T. Johansen and N. Chakravarty:

48/80 and by antigen-antibody reaction (Chakravarty, 1967). The work with the inhibitors, however, provide indirect evidence of energy requirement and of the possible role of ATP.

Using isolated pure populations of rat mast cells and oligomycin to inhibit the synthesis of ATP in the cells, it has been possible in the present investigation to vary the ATP content of mast cells and to observe its influence on histamine release induced by compound 48/80. A linear relation has been shown (Fig. 1) between about 40--950/0 reduc- tion of the ATP content of mast cells and 10--90~ inhibition of hista- mine release. I t is likely tha t there is a critical ATP level which is com- patible with normal histamine release. I t may be seen from the curve tha t about 400/0 reduction of the ATP content caused only 10~ inhibi- tion of histamine release. In other words, 60~ of the normal ATP level is sufficient for 900/0 of the histamine release induced by compound 48/80. By extrapolation it seems likely tha t the ATP content of the mast cells must be reduced to at least three-fourths of the normal level to influence histamine release. I t may be pointed out in this connection tha t in order to inhibit anaphylactic histamine release from guinea pig lung, over two-thirds of the oxygen uptake must be blocked (Chakravarty, 1962b) or the medium bubbled with less than 0.50/0 oxygen (Edman and Mongar, 1961).

Our observations provide evidence for the involvement of ATP in the histamine release process from mast cells. The linear relationship between the reduction of the ATP level and the inhibition of compound 48/80- induced histamine release over a wide range supports the hypothesis tha t ATP is required in the chain of the reactions tha t leads to histamine release.

Acknowledgment. Our thanks are due to Mrs. Lis Claudi Moller for her technical assistance and to Mr. Kurt Munch for his help with the figure.

References

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Chakravarty, N. : The mechanism of histamine release in anaphylactie reaction in guinea pig and rat. Acta physiol, scand. 48, 146--166 (1960).

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Dependence of Histamine Release from ~as t Cells on ATP 463

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Torben Johansen Nirmal Chakravarty Dept. of Pharmacology University of Odense Niels Bohrs All6 25 DK-5000 Odense Denmark