European Journal of Pharmacology, 58 (1979) 107--115 © Elsevier/North-Holland Biomedical Press
107
ADENOSINE TRIPHOSPHATE LEVELS DURING ANAPHYLACTIC HISTAMINE RELEASE IN R A T M A S T C E L L S IN V I T R O . E F F E C T S O F G L Y C O L Y T I C AND R E S P I R A T O R Y INHIBITORS TORBEN JOHANSEN Department of Pharmacology, Odense University, J.B. Winsl~ws Vej 19, DK-5000 Odense C, Denmark
Received 30 November 1978, revised MS received 17 April 1979, accepted 4 June 1979
T. JOHANSEN, Adenosine triphosphate levels during anaphylactic histamine release in rat mast cells in vitro. Effects of glycolytic and respiratory inhibitors, European J. Pharmacol. 58 (1979) 107--115. The adenosine triphosphate (ATP) content of rat mast cells was studied during and after anaphylactic histamine release. The almost identical time course of ATP decrease from mast cells treated with either glycolytic or respiratory inhibitors supports the view that the ATP depletion was largely related to the histamine release process and not to an uncoupling of oxidative phosphorylation by an increased concentration of cytosol Ca 2÷. The ATP content of the cells was not restored within the 2 h of observation. No inhibition of lactate production from mast cells exposed to antigen in the presence of respiratory inhibitors and glucose was observed. Based on the lactate production from mast cells, the turnover time of ATP was calculated to be about 3/4 min. ATP
Histamine release
Anaphylaxis
Mast cells
Calcium
1. I n t r o d u c t i o n A n a p h y l a c t i c histamine release in guinea pig and rat tissues is d e p e n d e n t o n o x y g e n (Parrot, 1 9 4 2 ; Mongar and Schild, 1 9 5 7 ; C h a k r a v a r t y , 1 9 6 0 ; Moussatch~ and ProuvostD a n o n . 1 9 6 0 ; Yamasaki et al., 1 9 6 0 ) . T h e inhibition o f a n a p h y l a c t i c h i s t a m i n e release b y a n o x i a is c o u n t e r a c t e d b y glycose (Chakrav a r t y , 1 9 6 2 ; D i a m a n t , 1 9 6 2 ) . T h e synthesis o f energy-rich p h o s p h a t e in a d e n o s i n e triphosp h a t e (ATP) is associated with o x i d a t i v e as well as g l y c o l y t i c e n e r g y m e t a b o l i s m . T h e d e p e n d e n c e o f a n a p h y l a c t i c h i s t a m i n e release o n the A T P c o n t e n t o f rat mast cells has b e e n d e m o n s t r a t e d , and a decrease o f t h e A T P cont e n t was observed during the t i m e c o u r s e o f histamine release ( J o h a n s e n and C h a k r a v a r t y , 1975}. It has been p r o p o s e d b y F o r e m a n and Mongar ( 1 9 7 3 ) t h a t a n a p h y l a c t i c h i s t a m i n e release results f r o m a m o v e m e n t o f calcium ions into t h e mast cells. Isolated m i t o c h o n d r i a f r o m m o s t e u k a r y o t i c cells a c c u m u l a t e cal-
cium b y an e n e r g y < t e p e n d e n t m e c h a n i s m ( L e h n i n g e r et al., 1 9 6 7 ; Lehninger, 1 9 7 0 ; Carafoli and Lehninger, 1 9 7 1 ) . An increase in c y t o s o l i c calcium c o n c e n t r a t i o n m a y possibly u n c o u p l e o x i d a t i v e p h o s p h o r y l a t i o n in t h e mast cell a n d cause t h e A T P decrease observed d u r i n g t h e a n a p h y l a c t i c release o f histamine. In t h e p r e s e n t investigation changes in t h e ATP c o n t e n t o f t h e mast cells w e r e studied during and a f t e r t h e a n a p h y l a c t i c h i s t a m i n e release process. T h e mast cells were either i n c u b a t e d w i t h 2 < t e o x y g l u c o s e in o r d e r t o b l o c k glycolysis o r i n c u b a t e d with antim y c i n A and o l i g o m y c i n which b l o c k e n e r g y p r o d u c t i o n b y t h e m i t o c h o n d r i a and m i t o chondrial calcium a c c u m u l a t i o n . T h e t i m e course o f A T P decrease in r e l a t i o n t o a n a p h y l a c t i c h i s t a m i n e release was a l m o s t identical w h e t h e r t h e mast cells d e p e n d e d on glycolytic or on mitochondrial energy
108
T. JOHANSEN
Male Wistar rats were sensitized by injecting 1 ml pertussis vaccine 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 15--30 days after the first injection.
cells per ml. For the lactate experiments the cell density was 2--10 times higher. The cell suspensions were equilibrated in a 37°C bath for 10 min. Then either 2~teoxyglucose (2-DG) or antimycin A and oligomycin was added and antigen was added 20 min later. In the experiments with antimycin A and oligomycin, glucose was used as substrate for glycolysis. No substrate was present in the cell suspension in the experiments with 2-DG. In parallel experiments the mast cells were incubated with the metabolic inhibitors in order to study the effect on the ATP content. Samples without the metabolic inhibitors were included for determination of the normal ATP content of the cells as were other samples for measuring spontaneous and control histamine release.
2.2. Isolation o f rat mast cells
2.4. Determination o f lactate production
Wistar rats, 260--450 g, were used for the experiments. Mast cells were isolated by differential centrifugation in concentrated human serum albumin (Chakravarty, 1965; Chakravarty and Zeuthen, 1965). Rats were killed by bleeding from the carotid arteries under light ether anaesthesia. Mixed peritoneal cells were collected by injecting KrebsRinger solution containing 50 pg/ml heparin through a small incision into the abdominal cavity. After differential centrifugation the mast cell fraction was washed twice to remove excess albumin and suspended in Krebs-Ringer solution containing h u m a n serum albumin, 1 mg/ml, final pH 7.0--7.1. Mast cells: 95.3%--99.0% of cell population.
The cells were equilibrated in a 37°C water bath for 10 min then incubated with antimycin A (1 pM), oligomycin (1 pg/ml), and glucose (5 mM) in the presence and absence of calcium (2.54 mM). Antigen was added to the cell suspensions 20 min later. After 30 min incubation with antigen the reaction was stopped by quickly adding chilled perchloric acid (PCA} to the cell suspension giving a final PCA concentration of 330 mmole/1. Immediately after this, the centrifuge tubes were placed on the whirl mixer and thereafter kept in an ice-bath for 1 0 - 2 0 min. They were then centrifuged for 10 min, 1800 g at 2--3°C, and the supernatant neutralized with a chilled mixture of KOH solution and 2-amino-2methyl-propanol buffer to give a final pH 9.8--10.0. The samples were kept at --85°C until tested for lactate content, usually within a week. Internal standards and blanks were carried through the whole procedure omitting the incubation at 37 ° C. The internal standards consisted of mast cells, metabolic inhibitors, antigen and lactate standard. Lactate was measured according to the m e t h o d described by Lowry and Passonneau (1972). 500/~l of standards, blanks or samples
production. The observations support the view that the ATP decrease during anaphylactic histamine release is largely related to the release mechanism and not to uncoupling of oxidative phosphorylation.
2. Materials and methods
2.1. Sensitization o f the rats
2.3. Incubation procedure Mast cell suspensions from 4--9 rats were pooled and divided into aliquots with the same cell density in a final volume of 0.5 ml. These were used for determination of the ATP content and lactate production of the mast cells and for the histamine release experiments. The cell density in various experiments varied from 4 × 10" to 27 × 104
HISTAMINE RELEASE AND MAST CELL ATP CONTENT
109
were added to tubes containing 491 pl of a reagent solution containing NAD+ (600 pM), glutamate (2.5mM) and glutamic-pyruvic transaminase (5 U) in 2-amino-2-methylpropanol buffer (100 mM, pH 9.9), and t h e reaction was started by adding beef heart lactic dehydrogenase (11 U) to the tubes so as to give a final volume of 1.0 ml. The amount of lactate in the samples was expressed as pmole of lactate per 103 mast cells.
The Krebs--Ringer solution contained (in mM) NaC1 139.8, KC1 4.7, MgSO4 1.2, CaCl: 2.5, Na:HPO4 2.5, KH:PO4 0.6.
2.5. Determination o f histamine release and A T P content o f the mast cells
Glucose and antimycin A enhance the rate of lactate production by rat mast cells (Diamant and Peterson, 1971). 2
107
ADENOSINE TRIPHOSPHATE LEVELS DURING ANAPHYLACTIC HISTAMINE RELEASE IN R A T M A S T C E L L S IN V I T R O . E F F E C T S O F G L Y C O L Y T I C AND R E S P I R A T O R Y INHIBITORS TORBEN JOHANSEN Department of Pharmacology, Odense University, J.B. Winsl~ws Vej 19, DK-5000 Odense C, Denmark
Received 30 November 1978, revised MS received 17 April 1979, accepted 4 June 1979
T. JOHANSEN, Adenosine triphosphate levels during anaphylactic histamine release in rat mast cells in vitro. Effects of glycolytic and respiratory inhibitors, European J. Pharmacol. 58 (1979) 107--115. The adenosine triphosphate (ATP) content of rat mast cells was studied during and after anaphylactic histamine release. The almost identical time course of ATP decrease from mast cells treated with either glycolytic or respiratory inhibitors supports the view that the ATP depletion was largely related to the histamine release process and not to an uncoupling of oxidative phosphorylation by an increased concentration of cytosol Ca 2÷. The ATP content of the cells was not restored within the 2 h of observation. No inhibition of lactate production from mast cells exposed to antigen in the presence of respiratory inhibitors and glucose was observed. Based on the lactate production from mast cells, the turnover time of ATP was calculated to be about 3/4 min. ATP
Histamine release
Anaphylaxis
Mast cells
Calcium
1. I n t r o d u c t i o n A n a p h y l a c t i c histamine release in guinea pig and rat tissues is d e p e n d e n t o n o x y g e n (Parrot, 1 9 4 2 ; Mongar and Schild, 1 9 5 7 ; C h a k r a v a r t y , 1 9 6 0 ; Moussatch~ and ProuvostD a n o n . 1 9 6 0 ; Yamasaki et al., 1 9 6 0 ) . T h e inhibition o f a n a p h y l a c t i c h i s t a m i n e release b y a n o x i a is c o u n t e r a c t e d b y glycose (Chakrav a r t y , 1 9 6 2 ; D i a m a n t , 1 9 6 2 ) . T h e synthesis o f energy-rich p h o s p h a t e in a d e n o s i n e triphosp h a t e (ATP) is associated with o x i d a t i v e as well as g l y c o l y t i c e n e r g y m e t a b o l i s m . T h e d e p e n d e n c e o f a n a p h y l a c t i c h i s t a m i n e release o n the A T P c o n t e n t o f rat mast cells has b e e n d e m o n s t r a t e d , and a decrease o f t h e A T P cont e n t was observed during the t i m e c o u r s e o f histamine release ( J o h a n s e n and C h a k r a v a r t y , 1975}. It has been p r o p o s e d b y F o r e m a n and Mongar ( 1 9 7 3 ) t h a t a n a p h y l a c t i c h i s t a m i n e release results f r o m a m o v e m e n t o f calcium ions into t h e mast cells. Isolated m i t o c h o n d r i a f r o m m o s t e u k a r y o t i c cells a c c u m u l a t e cal-
cium b y an e n e r g y < t e p e n d e n t m e c h a n i s m ( L e h n i n g e r et al., 1 9 6 7 ; Lehninger, 1 9 7 0 ; Carafoli and Lehninger, 1 9 7 1 ) . An increase in c y t o s o l i c calcium c o n c e n t r a t i o n m a y possibly u n c o u p l e o x i d a t i v e p h o s p h o r y l a t i o n in t h e mast cell a n d cause t h e A T P decrease observed d u r i n g t h e a n a p h y l a c t i c release o f histamine. In t h e p r e s e n t investigation changes in t h e ATP c o n t e n t o f t h e mast cells w e r e studied during and a f t e r t h e a n a p h y l a c t i c h i s t a m i n e release process. T h e mast cells were either i n c u b a t e d w i t h 2 < t e o x y g l u c o s e in o r d e r t o b l o c k glycolysis o r i n c u b a t e d with antim y c i n A and o l i g o m y c i n which b l o c k e n e r g y p r o d u c t i o n b y t h e m i t o c h o n d r i a and m i t o chondrial calcium a c c u m u l a t i o n . T h e t i m e course o f A T P decrease in r e l a t i o n t o a n a p h y l a c t i c h i s t a m i n e release was a l m o s t identical w h e t h e r t h e mast cells d e p e n d e d on glycolytic or on mitochondrial energy
108
T. JOHANSEN
Male Wistar rats were sensitized by injecting 1 ml pertussis vaccine 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 15--30 days after the first injection.
cells per ml. For the lactate experiments the cell density was 2--10 times higher. The cell suspensions were equilibrated in a 37°C bath for 10 min. Then either 2~teoxyglucose (2-DG) or antimycin A and oligomycin was added and antigen was added 20 min later. In the experiments with antimycin A and oligomycin, glucose was used as substrate for glycolysis. No substrate was present in the cell suspension in the experiments with 2-DG. In parallel experiments the mast cells were incubated with the metabolic inhibitors in order to study the effect on the ATP content. Samples without the metabolic inhibitors were included for determination of the normal ATP content of the cells as were other samples for measuring spontaneous and control histamine release.
2.2. Isolation o f rat mast cells
2.4. Determination o f lactate production
Wistar rats, 260--450 g, were used for the experiments. Mast cells were isolated by differential centrifugation in concentrated human serum albumin (Chakravarty, 1965; Chakravarty and Zeuthen, 1965). Rats were killed by bleeding from the carotid arteries under light ether anaesthesia. Mixed peritoneal cells were collected by injecting KrebsRinger solution containing 50 pg/ml heparin through a small incision into the abdominal cavity. After differential centrifugation the mast cell fraction was washed twice to remove excess albumin and suspended in Krebs-Ringer solution containing h u m a n serum albumin, 1 mg/ml, final pH 7.0--7.1. Mast cells: 95.3%--99.0% of cell population.
The cells were equilibrated in a 37°C water bath for 10 min then incubated with antimycin A (1 pM), oligomycin (1 pg/ml), and glucose (5 mM) in the presence and absence of calcium (2.54 mM). Antigen was added to the cell suspensions 20 min later. After 30 min incubation with antigen the reaction was stopped by quickly adding chilled perchloric acid (PCA} to the cell suspension giving a final PCA concentration of 330 mmole/1. Immediately after this, the centrifuge tubes were placed on the whirl mixer and thereafter kept in an ice-bath for 1 0 - 2 0 min. They were then centrifuged for 10 min, 1800 g at 2--3°C, and the supernatant neutralized with a chilled mixture of KOH solution and 2-amino-2methyl-propanol buffer to give a final pH 9.8--10.0. The samples were kept at --85°C until tested for lactate content, usually within a week. Internal standards and blanks were carried through the whole procedure omitting the incubation at 37 ° C. The internal standards consisted of mast cells, metabolic inhibitors, antigen and lactate standard. Lactate was measured according to the m e t h o d described by Lowry and Passonneau (1972). 500/~l of standards, blanks or samples
production. The observations support the view that the ATP decrease during anaphylactic histamine release is largely related to the release mechanism and not to uncoupling of oxidative phosphorylation.
2. Materials and methods
2.1. Sensitization o f the rats
2.3. Incubation procedure Mast cell suspensions from 4--9 rats were pooled and divided into aliquots with the same cell density in a final volume of 0.5 ml. These were used for determination of the ATP content and lactate production of the mast cells and for the histamine release experiments. The cell density in various experiments varied from 4 × 10" to 27 × 104
HISTAMINE RELEASE AND MAST CELL ATP CONTENT
109
were added to tubes containing 491 pl of a reagent solution containing NAD+ (600 pM), glutamate (2.5mM) and glutamic-pyruvic transaminase (5 U) in 2-amino-2-methylpropanol buffer (100 mM, pH 9.9), and t h e reaction was started by adding beef heart lactic dehydrogenase (11 U) to the tubes so as to give a final volume of 1.0 ml. The amount of lactate in the samples was expressed as pmole of lactate per 103 mast cells.
The Krebs--Ringer solution contained (in mM) NaC1 139.8, KC1 4.7, MgSO4 1.2, CaCl: 2.5, Na:HPO4 2.5, KH:PO4 0.6.
2.5. Determination o f histamine release and A T P content o f the mast cells
Glucose and antimycin A enhance the rate of lactate production by rat mast cells (Diamant and Peterson, 1971). 2
