the utilization of adenosine triphosphate in rat mast cells during histamine release induced by...

Naunyn-Schmiedeberg's Arch. Pharmaeol. 288, 243--260 (1975) �9 by Springer-Verlag 1975

The Utilization of Adenosine Triphosphate in Rat Mast Cells during Histamine Release Induced

by Anaphylactic Reaction and Compound 48]80

Torben J o h a n s e n a n d N i r m a l C h a k r a v a r t y

Department of Pharmacology, Odense University

Received October 17, 1974 / Accepted January 28, 1975

Summary. The ATP content of rat peritoneal mast cells has been studied in relation to histamine release induced by compound 48/80 and antigen-antibody (anaphylactic) reaction in vitro. When the ATP content of actively sensitized mast cells was reduced to different levels by oligomycin, a good correlation was obtained between the ATP levels and the amounts of histamine released by the anaphylaetic reaction.A similar linear relation has previously been demonstrated between the ATP levels of mast cells and histamine release induced by compound 48]80. The ATP content of mast cells was also studied at different intervals after the exposure of the cells to antigen or compound 48/80. No significant change in the ATP content was observed in untreated mast cells during the short period when histamine release occurs. If, however, the mast cells were preincubated with oligomycin or 2-deoxyglucose to reduce the rate of ATP synthesis while a large part of the histamine release remained tmaffected--a decrease in the ATP content could be demonstrated in close time relation to both anaphylactic and compound 48/80-induced histamine release. The observations indicate an increased utilization of ATP in mast cells during the release process.

Key words: Histamine - - Mast Cells - - ATP -- Anaphylactie Reaction -- Compound 48/80.

I n a p rev ious c o m m u n i c a t i o n we r e p o r t e d t h a t the re was a l inear r e l a t ion be tween the a m o u n t of h i s t amine re leased b y compound 48/80 a n d the A T P con ten t of the m a s t cells ( Johansen a n d C h a k r a v a r t y , 1972). I n t he p r e sen t s t u d y a s imi lar r e l a t ion be tween the A T P con ten t of m a s t cells a n d the h i s t amine re lease is shown using anaphy lae t i o r eac t ion in vitro in ac t ive ly sens i t ized m a s t cells. Moreover , the A T P levels of the m a s t cells have been fol lowed a t d i f ferent t ime in t e rva l s a f te r the exposure of t he cells to an t igen and c o m p o u n d 48/80. B y p a r t i a l l y inh ib i t ing the syn thes i s of A T P i t has been shown t h a t t he u t i l i za t ion

Send el/print requests to: Nirmal Chakravarty, Odense Universitet, Niels Bohrs All6, DK-5000 Odense, Denmark.

244 T. Johansen and N. Chakravarty

of A T P is increased dur ing anaphylac t ie and compound 48/80-induced h is tamine release. Using the same exper imenta l procedure i t has been possible to s t udy the t ime course of the reduc t ion in the A T P level of mas t cells in re la t ion to the h is tamine release.

Materials und Methods

Male Wistar rats, 200--385 g, or Sprague-Dawley rats, 380--500 g, were used for the experiments. Wistar rats were sensitized by injecting 1 ml pertussis vaccine (1.5 • 101~ bacilli) with 25 mg egg albumin subcutaneously on the first day followed by booster doses of 12.5 mg egg albumin on the second and the third day. The rats were used for the experiments 10--20 days after the first injection. Sprague-Dawley rats were used in experiments in which histamine release was induced by compound 48/80.

Mast cells were isolated by differential centrifugation in concentrated human serum albumin by a slight modification of the method described previously (Chakra- ra t ty and Zeuthen, 1965; Chakravarty, 1965). Rats were killed by bleeding from the carotid arteries under light ether anaesthesia. Mixed peritoneal cells were collected by injecting Krebs-Ringer solution 1 containing 50 ~g/ml heparin into the abdominal cavity through a small incision. After differential centrifugation the mast cell fraction was washed twice to remove the excess albumin and suspended in Krebs-Ringer solution containing human serum albumin, 1 mg[ml, final pH 7.0. The purity of the mast cells was 96.6~177 1.48 (s.e.m.). i~ast cells pooled from 2--10 rats were divided into samples usually containing 80000--150000 cells in a final volume of 0.5 ml both for the determination of the ATP content and of histamine release. These were run as parallel experiments under identical conditions. The cell suspensions were prewarmed in a 37~ bath for 10 rain and the incubation continued thereafter with the releaser (antigen or compound 48/80) for 5 see to 70 rain. Samples without the releasers and other appropriate controls were included. In those experiments in which oligomyein or 2-deoxyglucose (2-DG) were used the samples were incubated with the inhibitors for 10 rain prior to the addition of the releaser. The result of each experiment presented in the tables and figures represents the mean value from 2--4 samples both for ATP and histamine. The spontaneous histamine release varying from 1--10 ~ (mean 3 D/0 ) has been deducted from the data presented in the tables and figures. The spontaneous release was however higher (average 12 ~ ) when the cells were incubated for the second time after washing (Fig. 7). These are also deducted from the data presented.

The histamine release experiments were performed using the same general procedure as described previously for mixed peritoneal cells (Chakravarty, 1968 a). After incubation with the releaser the reaction was stopped by adding 7 times higher volume of ice-chilled Krebs-Ringer solution containing 1 mg/ml human serum albumin. The samples were centrifuged and the supernatant collected for the determination of the amount of histamine released. The cell deposit was boiled for 3 rain in 0.9 ~ NaCl for the determination of the residual histamine. Histamine was determined by a modification of the fiuorometric method described by Shore et al. (1959).

For the determination of ATP the reaction after incubation of the samples was stopped either by adding 11 times higher volume of boiling redistilled water

1 NaCI 139.8 raM, KC1 4.7 raM, MgS04 1.2 raM, CaCl~ 2.5raM, Na2HPO 4 2.5 raM, KH~PO a 0.6 raM.

ATP in Mast Cells during Histamine Release 245

as described previously (Johansen and Chakravarty, 1972) or by chilled perehlorie acid (PCA). To the cell suspension double the volume of ice-chilled PCA was added quickly giving a final PCA concentration of 0.33 M. The mixing of cells with the totM volume of PCA took 1 see. Immediately after this the centrifuge tubes were briefly placed on the whirl mixer and thereafter kept in an ice-bath for 5--15 rain. They were then centrifuged for 10 rain at 2000 g at 2--3~ and the supernatant neutralized with a chilled mixture of KOH solution and triethanolamine buffer to give a final pH 7.4. The samples were then kept at -- 85~ until tested for ATP content, usually within a week. Control experiments showed that there was no loss of activity in the samples after storage for 2 months at --85~ The extraction of ATP from mast cells after disrupting the cells by sonication gave the same result as direct extraction with PCA. Reextraction with PCA gave negligible ATP values.

ATP was determined by the biohiminiscence technique using luciferin-hiciferase from firefly lanterns. In a few earlier experiments the scintillation spectrometer was used to measure the luminescence following in general the method of Addanki et al. (1966). In most of the experiments the peak of the initial flash was used for the ATP assay and measured by a specially constructed photometer as described by l~asmussen and Nielsen (1968). Luciferin-lueiferase was extracted from firefly tails (lanterns) which were ground in 0,I M glycylglycine buffer, pH 7.7 containing 5 mM MgSO~ and i miV[ disodium EDTA. The superuatent after centrifugation was run through a G-25 fine sephadex (Pharmaeia) column. Elution with glycine (50 mM)-arsenate (10 raM) buffer, pH 7.7 with 1 mM EDTA yielded luciferase and luciferin fractions giving yellow and green fluorescance as described by Nielsen and Rasmussen (1968). Luciferase and hiciferin were mixed and diluted with the same buffer, which was used for eluting, with added MgS04 5 mM giving a final concentration corresponding to ca. 1 nag firefly tail per ml. The enzyme mixture was allowed to stand at room temperature 2--4 hrs to reduce the remaining amount of ATP which was present in the crude extract and then filtered through a millipore filter to remove contaminating bacteria. About 0.8 ml of this enzyme preparation was added mechanically to 0.1 ml of the neutralized samples prepared from mast cell extract.

Both ATP standard in water and internal standard from ATP added to mast cells gave linear curves. After correction for the inhibition (ca. 55 ~ ) caused by KClOa the standard ATP and internal standard curves were essentially identical. ATP standards were checked by spectrophotometry (Lamprecht and Trautschold, 1965).

Desiccated firefly tails (lanterns), ATP, 2-deoxyglucose and oligomycin (15~ oligomycin A and 85 ~ oligomycin ]3) were purchased from Sigma Chemical Com- pany. Compound 48/80 was obtained through the courtesy of AB Leo, ttElsingborg, Sweden, and pertussis vaccine was kindly supplied by Statens Seruminstitut, Copenhagen, ]Denmark.

Results

Correlation between the A T P Levels o/ Mast Cells and Anaphylactic Histamine Release

Table 1 shows the reduc t ion of the A T P con ten t of Wis ta r r a t mas t cells as a res~flt of i ncuba t ion with different concent ra t ions of ol igomycin for 10 rain. The inh ib i t ion of anaphylac t ic h i s tamine release by vary ing concent ra t ions of ol igomycin is shown in Table 2. By p lo t t ing the da ta

246 T. Johanscn and 1~. Chakravarty

Table 1. Effect of oligomycin on the ATP content of Wistar rat mast cells

Oligomycin ATP content pmoles/103 cells ~ Reduction Without With of ATP

ng/ml oligomycin oligomycin

20.4 1.76 0.64 64 18.5 1.58 0.48 70 17.0 2.01 0.94 53 14.2 1.20 0.64 47 10.4 1.59 1.28 19 9.4 1.50 1.00 33 8.0 0.99 0.64 35 7.1 1.17 1.32 0 6.2 1.29 1.07 17 5.0 0.99 0.93 6

Table 2. Effect of oligomycin on anaphylaetic histamine release

Oligomycin ~ Histamine release

Without With ng/ml otigomyein oligomyein

~ Inhibition

20.4 19.2 5.6 71 18.5 15.4 1.7 89 17.0 12.1 1.6 87 14.0 20.3 7.1 65 10.4 17.5 10.6 39 9.3 19.7 10.8 45 8.0 17.9 5.9 67 7.0 15.0 13.2 12 6.2 23.5 18.8 20 5.0 17.9 17.5 2

Antigen: egg albumin, 0.2 mg/ml. Total histamine content: 21--36 ng base/10 a cells.

12 ng /ml o l igomycin was found to cause 50 ~ r educ t ion of the A T P con ten t while the concen t ra t ion causing 50 ~ inh ib i t ion of h i s t amine

release was 10.5 ng/ml . The A T P con ten t of the m a s t cells a t t he t ime of the exposure of the

cells to the an t igen showed a good cor re la t ion to the a m o u n t of h i s t amine r e l e a s e d - - t h e cor re la t ion coefficient be ing 0.82 (Fig. 1). This r e la t ionsh ip exis ts for h i s t amine release values va ry ing f rom a lmos t un inh ib i t ed to 90 ~ inh ib i t ed release. A 50 ~ inh ib i t ion of the h i s t amine release was assoc ia ted wi th 3 5 - - 4 0 ~ r educ t ion of the A T P conten t . I t m a y be seen t h a t t he cor re la t ion curve crosses t he o rd ina t e a t a b o u t 10--15~

ATP in Mast Cells during ttistamine Release 247

LU (.~

tin _J ILl

LLI Z

U~

I

0

Z 0 F-

-T Z

100

75

50

25

go @ �9 �9

o l �9

I f ! 25 50 75

% REDUCTION IN ATP CONTFNT

Fig. 1. Correlation between the ATP content of the mast cells and anaphylaetic histamine release, Correlation coefficient = 0.822 (P < 0.001). The ATP content of the untreated mast cells was 1.35 -t- 0.1 (s. e. m.) pmole/10 a cells, the reduction caused by oligomycin, 4.3--14.2 ng/ml, is shown on the abscissa. The histamine release (with 0.2 mg/ml egg albumin used as antigen) in the absence of oligomycin

varied from 12 ~ to 35 ~ Each point represents one experiment

inhibition of histamine release thus suggesting tha t oligomycin may have another mild inhibitory effect on anaphy]actie histamine release apar t from the inhibition caused by the lowering of ATP.

Changes in the ATP Content o~ Mast Cells Following Incubation with Compound 48/80 or Antigen/or Di//erent Periods

The ATP content of the mast cells after exposure to compound 48/80 is shown in Fig. 2. A slight reduction of the ATP content was observed after incubation for 1/2--1 rain with compound 48]80 in all except one experiment but the result is not statistically significant. An appreciable reduction of the ATP content (average 23 ~ ) was however observed first after incubation for 3 rain with the releaser. The reduction was thereafter somewhat less pronounced but tended to persist up to 70 rain, which was the longest period of observation. Fig. 3 shows changes in ATP in relation to anaphylactie histamine release. No significant change occurred during the first 3 rain but there was a reduction (average 14 to 15~ after incubation with the antigen for 5--10 rain, followed b y a tendency to recovery up to 30 rain. Control experiments with mast cells f rom non-sensitized rats showed no detectable histamine release and no


Z

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l~ig.2. Changes in the ATP conten$ of mast cells after incubation for 1/2--70 mia with compound 48/80. Ai1 tho values are from experiments withou~ glucose except those (at 50 and 70 rain) represented by A and o. i00 on the ordinate represent ATP contents in cells incubated without glucose and without the releaser: 1.48

0.06 (s. e.m.) pmole/103 mast cells. There was a significant reduction of ATP content at 3, 5, 10, and 30 rain (all without glucose) and at 50 min (with glucose}: P < 0.05, < 0.02, < 0.001, < 0.01, and < 0.05 respectively by paired "t" test. In the experiments represented by ~ and o glucose, 5 mM was added at 30 rain. A: mean ATP content of mast cells incubated without the releaser, o: mean ATP content of mast cells incubated with the releaser. �9 represents individual experiments. A and o are mean values from 3 experiments. Histamine release by com-

pound 48/80, 0.5--1 ~g/mh 33.0 ~: 2.19% (mean ~: s. e. m.)

change in the A T P con ten t when i ncuba t ed wi th 0.4 m g / m l ant igen . Our obse rva t ions thus differ somewha t f rom those of D i a m a n t (1967), who r e p o r t e d no s ignif icant decrease in the A T P con ten t of m a s t cells a f te r

i ncuba t ion wi th compound 48/80. There was no apprec iab le r educ t i on in A T P in the cont ro l samples

even a f te r i ncuba t ion for 1 h. However , one canno t rule ou t the possi- b i l i t y t h a t , a t an increased r a t e of u t i l i za t ion of A T P assoc ia ted wi th h i s t amine release, the endogenous subs t r a t e s o f the m a s t cell m a y be i n a d e q u a t e to m a i n t a i n the A T P con ten t a t the or ig inal level. W e have there fore a d d e d glucose (5 raM) a t 30 rain in some of our expe r imen t s wi th c o m p o u n d 48/80 (see F ig .2) , in which changes in the A T P c o n t e n t was s t ud i ed a t 50 a n d 70 rain, t h a t is 20 a n d 40 rain a f t e r t he a d d i t i o n of glucose. I t m a y be seen f rom F i g . 2 t h a t the presence of glucose d id n o t make a n y essent ia l difference in the A T P con ten t of the mas t cells i n c u b a t e d w i thou t or w i th c o m p o u n d 48/80, as compared to the va lues

w i thou t glucose.


100

Z uJ l-- Z

' ' ' , '0 ' i'0 ~,~1 3 5 20

INCUBATION WITH ANTIGEN, MIN.

Fig.3. Changes in the ATP content of sensitized mast cells following incubation with antigen. 100 represent ATP content in cells incubated without antigen: 1.38 • 0.05 (s. e.m.) pmole/10 a cells. There was an appreciable reduction in the ATP content at 5 and 10 min (P < 0.05 at 5 rain and < 0.005 at 10 min by "t" t~st for paired data). Each point represents one experiment. Histamine release by the

antigen, egg albumin 0.2 mg/ml: 16.7 ~= 1.16% (mean =]= s. e. m.)

Changes in the ATP Content o~ Oligomycin-Treated Mast Cells during Compound 48/80.Induced and A~u~phylactie Histamine Release

The absence of a significant reduction of ATP within a minute after the initiation of histamine release from mas t cells, reported above, could be due to an enhanced ATP regeneration during histamine release (see discussion). We have therefore preincubated the cells in this series of experiments with oligomycin to reduce the synthesis of ATP. Oligomycin in adequate concentration blocks histamine release (Johansen and Chakravarty, 1972). But when a suitable oligomycin concentration is chosen, sufficient ATP is present in the cells to allow a substantial fraction of the normal histamine release to occur in response to antigen or compound 48]80.

As shown in Table 3 there was a variable reduction in the ATP content of the mas t cell in most of the experiments b y preincubation with oligomycin, 9.4--15.9 ng/ml, the average redaction being 30 ~ In these oligomycin-pretreated cells the ATP level was further reduced after incubation for 1 rain with compound 48/80 in 7 out of 10 experiments. Paired comparison of the results of the 10 experiments showed a statistically significant reduction (mean : 18 ~ reduction). 10 to 52 ~ (mean 34 ~ ) of the histamine content of the mas t cells were released by corn-

Expt.

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INCUBATION WITH COMP. 48180, SECONDS

~ig.4. Changes in the ATP content of mast cells pretreated with 2-deoxyglucose in relation to histamine release induced by compound 48/80, 1 ~g/ml. 100 on the ordinate on the left side represent the ATP content in presence of 2-deoxyglucose (5--10 raM), but without the releaser; this value was on the average 360]0 less than the ATP level of untreated cells (1.48 =]: 0.05 (s. e.m.) pmole/10 a cells). The filled circles represent the ATP levels after exposure to compound 48/80. Each point represents one experiment. P for the reduction of ATP by paired " t " test: 10 see:

< 0.05, 30 see: < 0.025, 60 see: <0.005

pound 48/80 in presence of oligomycin during the same period.The average release in the absence of oligomycin was 44 ~ The results were similar in anaphylactie reaction which caused on the average 15o/0 histamine release in the absence of an inhibitor and 11 ~ release in presence of oligomycin. The ATP content of the mast cells preincubated with oligomycin was further decreased in all the experiments following incubation with the antigen for 1 min (mean decrease: 19 ~ ).

Changes in the ATP Content o/ 2-Deoxyglucose-Treated Mast Cells during Compound 48/80-Induced and Anaphylacti~ Histamine Release

2-Deoxyglueose (2-DG) in low concentrations have been shown to have little or no effect on histamine release (Chakravarty, 1968 a). In the present experiments the mast cells were therefore preincubated with 2-DG to reduce the ATP synthesis. After preincubation with 2-DG histamine release induced by compound 48/80 was ~ssociated with a reduction of the ATP level as shown in Fig. 4.2-Deoxyglucose (5--10 mM) itself in absence of the releaser caused 30 to 43 ~ (mean 36 ~ ) decrease

Tab

le 4

. C

hang

es i

n A

TP

co

nte

nt

of

2-de

oxyg

luco

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ast

cells

, in

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for

1 ra

in w

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42

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23

17

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81

0.58

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47

19

6 23

29

21

0.

87

0.66

0.

52

21

P <

0.

05

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24

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87

0.54

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43

19

P fr

om "

t" t

est

for

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ed d

ata.

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deox

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cose

: 5

-- 1

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M.

An

tig

en:

egg

albu

min

, 0.

2 m

g/m

l.


in the ATP content after incubation for 10--11 min. When these cells were exposed to compound 48/80 histamine release was accompanied by a further reduction in the ATP content in all the exper iments - - the average reduction being 11~ at 10 sec, 80/0 at 30 see, 220/0 at 60 see (P for paired data : <0.05, <0.025 and <0.005 respectively). The average histamine release during these periods varied from 31 to 45 ~ I t has been shown earlier tha t histamine release at 37~ occurs very fas t - - in 10 see- -a f te r exposure to compound 48/80 (Bloom et al., 1967 ; Bloom and Chakravarty, 1970). Thus the decrease in ATP content occurs in good t ime relation to histamine release. The experiments in Fig.4 also show tha t the histamine release was essentially complete within 10 see. Peterson and Diamant have also recently shown a decrease in the ATP content after incubation with compound 48/80 for 20--25 see in mast cells pretreated with antimyein A (Peterson, 1974).

In our studies on anaphylactie histamine release we have determined the ATP content of mast cells 5 to 60 see after exposure to antigen. The experiments, shown in Table 4, were carried out with mast cell preincubated with 2-DG. After exposure to the antigen for 1 rain on the average 17 ~ of the histamine content was released, amounting to 71 ~ of the release in the absence of 2-deoxyglueose. The normal ATP content of the mast cells was on the average reduced 38 ~ by preineubation with 2-DG. Exposure to antigen caused a further reduct ion--average 19 ~ the ATP level (P for paired data < 0.05).

The increased utilization of ATP shown in Table 4 occurs within 1 rain while histamine release is completed within 30--40 see (see below). The utilization is therefore likely to be related to the release process. In order to further explore the need for ATP, the changes in the ATP levels of mast cells were correlated to the t ime course of histamine release from its initiation. Since phosphatidyl serine has been added to the medium in these experiments and a prolongation of the t ime course of histamine release in presence of phosphatidyl serine has been reported (Mongar and Svec, 1972), we have studied the t ime course of histamine release in presence of phosphatidyl serine and 2-deoxyglueose. As shown in Fig. 5, ~he anaphylaetie histamine release was initiated in about 10 see and was completed in 30--40 see. The t ime taken to reach the peak histamine release was thus 10 see longer in the present preparat ion than tha t reported earlier in mixed peritoneal cells (Bloom and Chakravarty, 1970). There was no essential difference between the time course of anaphylactie histamine release with phosphatidyl serine alone and tha t with phosphatidyl serine d~ 2-deoxyglueose.

Fig.6 shows the changes in the ATP content of 2-deoxyglueose- pretreated mast cells for 5- -45 see after exposure to antigen. There was a significant reduction of the ATP content not only at 30 and 45 see,

17 Naunyn-Schmtedeberg's Arch. Pharmacol., Vol. 288


4 0 '

u~ 30 �9

gJ z

,,~ 2 0 ,

- r

10"

o 30

1 210 I i i I 0 30 40 60 120

INCUBATION WITH ANTIGEN, SECONDS

8,

Ld J 20 �84 Ld (3C.

::Z 10

,/&

/

20 30 40 i

'0 120

INCUBATION WITH ANTIGEN, ,SECONDS

b

Fig.Sa and b. Time course of anaphy]actic histamine release in 4 experiments. Antigen: egg albumin 0.4 mg/ml. Each of the four experiments is represented by a different symbol. (a) Phosphatidyl serine 50 txg/ml added to the medium. (b)

Phosphatidyl serine 50 txg/ml and 2-deoxyglueose 5 mM added to the medium

when histamine release was pract ical ly complete, bu t also a t 15 sec, when the release had just commenced. I t m a y also be seen t h a t the A T P content was slightly reduced in all the 4 experiments a t 5 sec when there was no detectable histamine release. I t appears likely tha t a reduct ion in A T P precedes the release.

E//ect o] Antigen-Antibody Reaction on the A T P Content o] Mast Cells in the Absence of Histamine Release

Ethano l v~as used to block his tamine release (Chakravarty, 1960). 2 e/~ (v/v) e thaaol a lmost completely inhibited anaphylaet ic histamine release, as shown in Table 5. W h e n his tamine release was thus inhibited by ethanol there was no decrease in the A T P content after exposure to ant igen in contras t to the reduct ion of A T P associated with his tamine release. E thano l by itself did no t influence the A T P content . Al though

Tab

le 5

. E

ffec

t of

ant

igen

-ant

ibod

y re

acti

on o

n th

e A

TP

con

tent

of

mas

t ce

lls

whe

n hi

stam

ine

rele

ase

is i

nhib

ited

by

etha

nol

(2 ~

v/

v)

Ex

it.

AT

P c

onte

nt p

mol

es/1

03 c

ells

H

ista

min

e N

o.

Con

trol

(w

itho

ut a

ntig

en)

Wit

h an

tige

n T

otal

~

Rel

ease

wit

h an

tige

n

Wit

hout

W

ith

Wit

hout

W

ith

hist

amin

e W

ith

ou

t W

ith

etha

nol

etha

nol

etha

nol

etha

nol

cont

ent

(bas

e)

etha

nol

etha

nol

(a)

(b)

(e)

(d)

of m

ast

ceil

s ng

/103

cel

ls

C~

1 0.

68

0.72

0.

55

0.67

21

.5

23

1.2

2 0.

70

0.74

0.

59

0.67

21

.0

26

0.8

3 0.

95

1.05

0.

75

0.93

24

.7

29

0.3

4 0.

97

0.91

0.

75

0.83

28

.5

20

1.1

5 0.

77

0.81

0.

69

0.87

2

2.2

21

0

Mea

n 0.

81

0.85

0.

67

0.79

23

.6

24

0.7

~v

Incu

bati

on t

ime:

1

rain

wit

h an

tige

n (e

gg a

lbum

in:

0.2-

-0.4

mg/

ml)

. P

hosp

hati

dyl

seri

ne (

25 ~

tg/m

l) a

nd 2

-deo

xygl

ucos

e (1

0 m

M)

wer

e ad

ded

to t

he m

ediu

m.

P by

pai

red

"t"

test

for

cha

nges

in

the

AT

P c

onte

nt p

rodu

ced

by a

ntig

en (

a an

d e)

~

0.01

and

by

anti

gen

in p

rese

nce

of e

than

ol (

b an

d d)

>

0.1;

P

for

colu

mns

a a

nd b

>

0.2.

For

evi

denc

e of

ant

igen

-ant

ibod

y re

acti

on i

n pr

esen

ce o

f et

hano

l se

e F

ig. 7

.

tx~

r e~


I00

i i I i I i |

15 30 45 INCUBATION WITH ANTIGEN, SECONDS

4O

3O o

a:

2o ~

x;

10

Fig. 6. Changes in the ATP content of mast cells preincubated with 2-deoxyglucose (5 raM) during anaphylactie histamine release. :PhosphatidyI serine 50 tzg/ml was added to the medium. The ATP content of the untreated mast cells was 1.34 • 0.07 (s. e. m.) pmole/103 cells. 100 on the ordinate on the left side represent ATP levels in prbsence of 2-deoxyglueose and phosphatidy] serine, but without antigen. 2-Deoxyglucose caused 43 ~ mean reduction in the ATP content. The mean reduction of ATP after exposure to antigen (egg albumin, 0.4 mg/ml) was 8.4~ at 5 sec (P ~ 0.1), 12.9~ at 15 sec (P ~ 0.02), 14.1~ at 30 sec (P ~ 0.01) and 13.8~

at 45 see (P ~ 0.02). P determined for paired data

ethanol blocked histamine release, it did not prevent the initial step viz. an t igen-ant ibody react ion as shown by the subsequent desensitization of the mas t cells. Desensit ization was demons t ra ted by incubat ing the cells twice at 37~ in fresh solutions, the cells being washed at 0- -4~ between the two incubat ions (Fig.7). The first two columns f rom the left side show the usual desensitization after the first incubat ion with ant igen causing histamine release. The two middle columns show tha t the effect of e thanol is reversible. Desensit ization of mas t cells after the incubat ion with ant igen in presence of e thano l - -which blocks histamine release-- is indicated by the poor release induces by ant igen during the second incubat ion wi thou t e thanol (last column).

Discussion

Using oligomycin to inhibit A T P synthesis we have previously shown a good correlation between the A T P content of the mas t cells and the amoun t of his tamine released by compound 48/80 (Johansen and Chakra-


1 0 0 - -

h i

i f )

W - . J

uJ 50 c c Z 0 0

v _ _ c " r "1-. , ~

~ C M

O - ~ ' - ~

ETHANOL O O

2% (v/v) ANTIGEN

OAmglml + +

C c" .o_ .o_

. 0 .Q

C u u o .E .-=

"O

4- 0 4- 0

0 + + +

Fig. 7. Desensitization of mast cells after incubation with antigen in presence of ethanol. The cells were washed (0--4~ between the two incubations in fresh solutions. 100 on the ordinate represent anaphylactic histamine release in the absence of ethanol. This amounted to the release of 20 to 29 ~ (mean 24 ~ ) of the histamine content of the mast cells. The first two columns from the left side show the usual desensitization after exposure to antigen. The two middle columns show that the effect of ethanol is reversible. The two last columns show substantial desensitization of the mast cells after the first exposure to antigen although histamine release was blocked by ethanol during the first incubation. The histamine release shown in the last column is 25 ~ in relation to the corresponding control value shown in the fourth column (P < 0.005). Mean vahes from 5 experiments

v a r t y , 1972). A s imi lar cor re la t ion is now d e m o n s t r a t e d be tween the A T P levels of the m a s t cells and h i s t amine release induced by a n t i g e n - a n t i b o d y (anaphylac t ie ) r eac t ion in vitro. B y ex t rapo la t ion , the corre la t ion curve for the compound 48/80 exper imen t s , r e p o r t e d before, i nd ica t ed t h a t up to 25 ~ r educ t ion of the A T P con ten t was compa t ib l e wi th n o r m a l h i s t amine release. F u r t h e r expe r imen t s wi th compound 48/80 have now confi rmed t h a t h i s t amine release is n e a r l y no rma l wi th 10- -25 ~ reduc- t ion in ATP . This p a r t of the cor re la t ion curve differs f rom t h e ' c u r v e for t he a n a p h y l a c t i e h i s t amine release, because a min ima l inh ib i t ion of the anaphy l ae t i c re lease seems to occur even when the A T P con ten t is normal . This could be accoun ted for b y some o ther u n e x p l a i n e d ac t ion of o l igomyein. A n a p h y l a e t i c h i s t amine release was found to be more sensi t ive to o l igomycin t h a n compound 48/80-induced r e l e a s e - - t h e concen t ra t ion r equ i r ed for 50 ~ inh ib i t ion being 10.5 ng /ml and 18 ng /ml respec t ive ly .

These obse rva t ions show the dependence of h i s t amine release induced b y compound 48/80 and a n a p h y l a c t i c r eac t ion on adenos ine t r i phospha te .


The question remains however whether there is an increased utilization of ATP during the release process. I t has been suggested by some authors tha t energy is required to maintain the metabolic functions, and anaphylactic histamine release can only occur from such a functioning cell (Mongar and Sehild, 1962). Our observations support the hypothesis tha t energy metabolism is directly involved in the release process. Ap- parently, ATP was regenerated almost as fast as it was utilized and the steady state value showed no significant change in untreated mast cells.

In order to explore if a reduction of ATP content due to its increased utilization occurs in relation to the histamine release process, we preincubated the mast cells with oligomycin or 2-deoxyglucose (2-DG) to reduce the resynthesis of ATP. The inhibitors caused a decrease in the ATP level but a large fraction of the histamine release in response to compound 48/80 or to the specific antigen remained unaffected. In this experimental situation histamine release from oligomyein- or 2-DG-treated mast cells was associated with a decrease in the A T P content. Histamine release following incubation with compound 48/80 and antigen for 1 min after preincubation with oligomycin was associated with 18--19~ mean decrease in the ATP level. Histamine release induced by compound 48/80 and anaphytaetic reaction is however completed within t0 and 40 sec respectively. We have therefore compared the time course of the release with tha t of changes in the ATP content from 5 to 60 sec after exposure of mast cells, preincubated with 2-deoxyglucose, to the releasers. A decrease of the ATP level could be demonstrated even 10 sec after exposure to compound 48/80 tha t is directly in t ime relation with the release (Fig.4). In the experiments with sensitized cells, a decrease of the ATP level was very well correlated to the time course of histamine release. A reduction of the ATP content was observed in all the 4 experiments even a t 5 scc when histamine release did not yet commence. However this was not statistically significant probably because the decrease was relatively small. But a t 15 sec, when histamine release had just s tar ted and at 30, 45, and 60 sec (Fig.6 and Table 4), when the release rose to the peak, there was a significant decrease in the ATP contents (13 to 19~ mean reduction). From the sequence of changes in the ATP content and histamine release it appears tha t an increased utilization of ATP precedes and accompanies the release.

However, since histamine release is completed within 1 rain, the later decrease in the ATP content from 3 min onwards (see Figs.2 and 3) could be related to the recuperative and synthetic processes initiated in the cells by the changes in the mast cell associated with the release process.

The experiments with ethanol reveal tha t the activation process initiated by antigen-antibody reaction does not require energy if histamine


release is blocked (Table 5 and Fig.7). E thano l inhibits his tamine release and dogs not produce any change in the ATP content . The demons t ra t ion of substant ial desensitization in e thanol- t reated cells should be interpreted as an expression of the occurrence of ant igen-ant ibody react ion followed by a t ransient enzyme activation. E thano l apparen t ly acts at a point distal to this initial act ivation. Schild (1968) explains the desensitization of sensitized chopped lung in presence of phenol as an indication for the format ion and decay of the initial active state even when histamine release is blocked.

Judging f rom the respirat ion ra te of the mas t cells the turnover ra te of A T P seems to be ra ther slow in these cells. I t is not known which endogenous subst ra te is used to sustain the respirat ion ra te of 0.29 • 10 -8 ~zl per cell per hour in a subst ra te free medium (Chakravar ty and Zeuthen, 1965). Assuming t h a t glycogen is the only subst ra te used, 0.215 • 10 -s ~zmole glucose would be oxidized per cell per hour producing 1.4 pmole ATP/103 cells/rain. The turnover ra te of A T P in un t rea ted mas t cells would thus be around 1/2--1 rain. At this turnover ra te it could just be possible to demons t ra te a decrease in A T P even in un t rea ted cells during his tamine release. A tendency in tha t direction is seen during the first minute bu t it is neither conspicuous nor significant. The reason it c anno t be demons t ra ted is no t so much the basal turnover ra te of A T P as the increased turnover ra te during his tamine release due to a metabolic stimulation. This is consistent with the findings tha t a s t imulat ion of respirat ion and of glucose metabol ism occurs in relat ion to histamine release (Chakravarty, 1968b; Chakrava r ty and S~rensen, 1974a, b).

Acknowledgements. The work was supported by a grant (project No. 512-1962 & 3096) from the Danish Medical Research Council. Our thanks are due to Mrs. An- nette Rasmussen for her skilful technical assistance and to Lektor Hans l~asmussen for the construction of the photometer.

References AddanM, S., Sotos, J. F., Rearick, P. D. : Rapid determination of picomole quanti-

tires of ATP with a liquid scintillation counter. Analyt. Biochem. 14, 261 --264 (1966)

Bloom, G. D., Chakravar~y, N. : Time course of anaphylactic histamine release and morphological changes in rat peritoneal mast cells. Acta physiol, scand. 78, 410--419 (1970)

Bloom, G.D., Fredholm, B., Haegermark, (~.: Studies on the time course of histamine release and morphological changes induced by histamine liberators in rat peritoneal mast cells. Acta physiol, scand. 71, 270--282 (1967)

Chakravarty, N. : The mechanism of histamine release in anaphylactic reaction in guinea pig and rat. Aeta physiol, scan& 48, 146--166 (1960)

Chakravarty, N. : Glycolysis in rat peritoneal mast cells. J. Cell Biol. 25, 123--128 (1965)


Chakravarty, N. : Further observations on the inhibition of histamine release by 2-deoxyglucose. Aeta physiol, scand. 72, 425--432 (1968a)

Chakravarty, N. : Respiration of rat peritoneal mast cells during histamine release induced by antigen-antibody reaction. Exp. Cell Res. 49, 160--168 (1968b)

Chakravarty, N., Sorensen, H. J. : Stimulation of glucose metabolism in rat mast cells by antigen, dextran and compound 48/80, used as histamine releasing agents. Aeta physiol, scand. 91, 339--353 (1974a)

Chakravarty, N., Sr H. J. : Effect of histamine releasing agents on the metabolism of exogenous glucose in rat mast cells. Agents and Actions 4, 196--197 (19745)

Chakravarty, N., Zeuthen, E. : Respiration of peritoneal cells. J. Cell Biol. 25, 113--121 (1965)

Diamant, B. : The effect of compound 48/80 and distilled water on the adenosine triphosphate of isolated rat mast cells. Acta physiol, scand. 71, 282--290 (1967)

Johansen, T., Chakravarty, N. : Dependence of histamine release from rat mast cells on adenosine triphosphate. Naunyn-Schmiedeberg's Arch. Pharmacol. 275, 457--463 (1972)

Lamprecht, W., Trautschold, I.: Determination of ATP with hexokinase and glucose-6-phosphate dehydrogenase. In: H.U. Bergmeyer (ed.): Methods of enzymatic analysis, pp. 543--551. Weinheim (Bergstr.): Verlag Chemie GmbH 1965

Mongar, J. L., Schild, H. O. : Cellular mechanisms in anaphylaxis. :Physiol. Rev. 42, 226--270 (1962)

Mongar, J. L., Svec, P.: The effect of phospholipids on anaphylactie histamine release. Brit. J. Pharmacol. 46, 741--752 (1972)

Nielsen, R., Rasmussen, I~I. : Fractionation of extracts of firefly tails by gel filtra- tion. Acta chem. seand. 22, 1757--1762 (1968)

Peterson, C. : Rote of energy metabolism in histamine release. Thesis at Karolinska Institute, Stockholm. Acta physiol, scand., Suppl. 413 (1974)

Rasmussen, H., Nielsen, R. : An improved analysis of adenosine triphosphate by the luciferase method. Acta chem. scan& 22, 1745--1756 (1968)

Schild, H. O. : Mechanism of anaphylactic histamine release. In: K. F. Austen and E. L. Becker (eds.): Biochemistry of the acute allergic reactions, pp. 99--118. Oxford-Edinburgh: Blackwell Scientific :Publications 1968

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the utilization of adenosine triphosphate in rat mast cells during histamine release induced by...

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