Biochimica et Biophysics Acts, 451 ( 1 9 7 6 ) 1--19 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 28069

METABOLIC ASPECTS OF THE SECRETION OF STORED COMPOUNDS FROM BLOOD PLATELETS V. EFFECT OF IONOPHORE A23187 ON WASHED PLATELETS *

E.H. M U R E R , K. D A V E N P O R T , M.A. R A U S C H and H.J. DAY

SCOR Thrombosis Center, Temple University Health Sciences Center, Philadelphia, Pa. 19140 (U.S.A.) ( R e c e i v e d March 24th, 1976)

Summary 1. The A23187-induced secretion of preabsorbed serotonin from human blood platelets at 37°C is studied. Preincubation at the same temperature before the addition of ionophore is necessary for maximal release induction. When total incubation time is kept constant, longer time with ionophore results in a smaller decrease in the level of metabolic ATP and increase in metabolic IMP. This coincides with the reduction in secretion, but statistical treatment of the results suggests that the reduced secretion only partially explains the reduced drop in metabolic ATP, and that therefore a resynthesis of metabolic ATP from IMP may have taken place. 2. In some experiments induction of secretion takes place over a very narrow range of ionophore concentration. 3. When K ÷ substitutes for Na + in the extracellular medium, the need for preincubation for maximal secretion becomes less evident, and at times is abolished, while there is still a significant increase in the metabolic ATP level by prolonged incubation with ionophore. 4. A reduction in secretion is observed with metabolic blockers when the ionophore is added after preincubation, but to a much lesser degree than when secretion is induced by thrombin, in spite of a great reduction in the level of metabolic ATP. This may partly be explained by the increase in secretion induction by A23187 in the presence of inhibitors when the ionophore is added in the cold, suggesting that the inhibitor may cause a "weakening" of the platelets' "resistance" to induction of secretion by ionophore. 5. When the effect of Ca 2÷ and of Mg 2+ on the level of intermediates of the * Preliminary reports of this s t u d y w e r e p r e s e n t e d at the 59th Annual Meeting of the F e d e r a t i o n of American S o c i e t i e s for Experimental Biology, Atlantic City, N.J., April 13--18, 1975 ( A b s t r a c t Fed. Proc. (1975) 34, 289) and the 5 t h Congress o f the I n t e r n a t i o n a l S o c i e t y o n T h r o m b o s i s and Haemostasis, Paris, France, July 21--26, 1975 (Abstract No. 169).

ATP -~ h y p o x a n t h i n e conversion is studied, it is evident that the addition of Ca 2÷ causes enhanced IMP accumulation and a reduction in the level of inosine plus hypoxanthine, while Mg 2+ has the opposite effect. This suggests that the two metals affect the enzymes of the IMP -~ h y p o x a n t h i n e conversion differently. 6. Indomethacin inhibits secretion induced by A23187, suggesting that prostaglandin intermediates may amplify the ionophore-induced release. The adenine nucleotide metabolism is n o t affected. 7. The results indicate that there is an indirect, rather than a direct, link between the major metabolic changes and the secretion induced by A23187, but that the ionophore may cause intracellular changes which are not connected to its effect as release inducer. Introduction In a number of cells, Ca :+ has been indicated as the transmitter of the secret o r y impulse (i.e. the second messenger) [1--3]. This has been difficult to demonstrate in cells where the plasma membrane was impermeable to Ca 2., so that the source of the postulated transmitter had to be the cell itself. Blood platelets belong to this category [4]. Recently, ionophores have been discovered which facilitate the translocation of divalent cations across cell membranes, whether or not such a translocation can occur naturally [5--7]. When applied to blood platelets these agents were shown to induce platelet secretion either specifically with A23187 [8--12] or in combination with a monoamine-specific translocation of serotonin (with X537A) [9,13,14]. Platelet secretion induced by A23187 was independent of external Ca 2÷ [8, 12], although it has been reported t h a t the presence of chelators may affect the strength of secretion [9]. When Ca 2÷ was present extracellularly, A23187 caused calcium uptake by platelets, but this seemed to follow after the induction of secretion, indicating that the suggested increased level of free intracellular Ca :+ does indeed originate from intracellular storage sites [8,12]. When uptake of larger amounts of Ca 2÷ was permitted, so that the cytoplasmic [Ca 2+] exceeded the physiologic level, the platelets underwent vast ultrastrucrural changes and lost the ability to retain their cytoplasmic c o n t e n t [12]. The effect of metabolic inhibitors on the ionophorednduced secretion is unclear, as the secretion can take place without a decreased metabolic ATP level [15]. When A23187 was added in the cold, its effectiveness as inducer of secretion was impaired, as compared with its effect when added at 37 ° C, even if the total incubation time with ionophore was longer. There was also a smaller drop in the metabolic ATP level when the ionophore was added in the cold than at 37°C. This latter p h e n o m e n o n might be a re-synthesis of ATP from IMP [12]. These points have been further investigated in the present study together with others which might help elucidate how A23187 exerts its specific and general effects on platelet function and metabolism. Materials and Methods Radiochemicals were [3H]serotonin-binoxalate (4.3 Ci/mmol) from New England Nuclear, Boston, Mass. and [U-~4C]adenine (281 Ci/mol) from Amer-

sham/Searle Corp., Chicago, Ill. 2,5-Diphenyloxazole (PPO), bis-(2(5-phenyloxazolyl))-benzene (POPOP), indomethacin, monoiodoacetate (sodium salt), antimycin A, Type III, 2-deoxy-D-glucose and bovine serum albumin, Cohn fraction V, were from Sigma Chemical Co., St. Louis, Mo. A23187 was a gift from Dr. R o b e r t L. Hamill, Eli Lilly and Co., Indianapolis, Ind. and imipramine from the Ciba-Geigy Corp., London, U.K. and Nutley, N.J. Thrombin was provided b y Parke-Davis, Chicago, Ill. Blood from healthy donors who had not used anti-inflammatory drugs during the last 7 days was collected in 11 mM sodium citrate (final concentration) of pH 7.9. The platelet-rich plasma prepared by 15 min centrifugation at 250 × g and room temperature was incubated with 3H-labeled serotonin-binoxalate (1--2 pCi/ml) and 14C-labeled adenine (250--500 nCi/ml) by shaking 20 min at 37°C in a D u b n o f f metabolic incubator (75 strokes/rain) after which the pH was adjusted to 6.0 with 0.1 M citric acid to facilitate resuspension of the platelets in the wash medium. The platelets were washed twice in 0.15 M NaCI containing 11 mM sodium citrate of pH 6.0, and finally resuspended in 0.15 M NaC1 or KC1. A mixture of the platelet suspension with 10 mM sodium citrate of pH 7.9, 25 mM Tris • HC1 of pH 7.4, 2.5 pM imipramine added to prevent reabsorption of serotonin [16] and additions to be tested (total volume 4 ml and NaC1 or KC1 concentration 0.13 M) was stored in 25-ml Erlenmeyer flasks in an ice bath until start of the experiment, which was performed by shaking at 37°C. Incubation was stopped by cooling the flask in an ice bath and aliquots removed for different tests (the total incubate referred to in the tables) after which the remainder of the incubation mixture was centrifuged and new aliquots taken from the supernatant material. To measure 14C-labeled metabolites o f ATP, aliquots from the incubation mixture and from the supernatant were mixed with an equal amount of 90% ethanol with 7.7 mM EDTA of pH 7.4. The ~4C-labeled metabolites were separated by paper strip electrophoresis [17] and counted in a Beckman LS330 scintillation counter in toluene with 0.2% PPO and 0.01% POPOP. To measure secretion 50-pl aliquots for the determination of [3H]serotonin were taken from the supernatant or incubation mixture and added directly to the scintillation fluid which had been mixed 1 : 1 with Triton X-100. The counts in the 3H-channel were corrected for spillover of 14C. Platelet factor 4 was determined by radial immunodiffusion technique by the courtesy of Dr. S. Niewiarowski [18]. The ionophore was added to the incubation medium in 5 pl volume. Stock solution of A23187 was 8 mM in ethanol/ acetone (1 : 1, v/v), which was stored at --20°C and diluted with ethanol to the desired concentration for 5-pl additions. Results

The relationship between concentrations of added A23187 and degree of secretion was not consistent, in that some preparations showed increase in secretion over a wide concentration range, others (after 9 min preincubation) over a very narrow range. Under the latter circumstances there was a striking relationship between release, drop in metabolic ATP level and increase in IMP (Fig. 1). When the total incubation time was kept constant, b u t the time with ionophore increased, a transient increase in the metabolic IMP could be seen

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F i g . 1. C o n c e n t r a t i o n dependence of A23187-induced secretion and metabolic changes. Total incubation time 10 min at 37°C. Ionophore added after 9 rain preineubation, oo, [ 3 H ] s e r o t o n i n i n s u p e r natant; o ...... o, [ 1 4 C ] A T P i n t o t a l i n c u b a t e ; X . . . . . . X, [ 1 4 C ] I M P i n t o t a l i n c u b a t e . O r d i n a t e is perc e n t o f t o t a l 3 H ( s e r o t o n i n ) o r o f t o t a l 14 C ( a d e n i n e m e t a b o l i t e s ) . M e a n o f t w o e x p e r i m e n t s .

(Fig. 2), similar to t hat seen with NaF [19]. When, however, the i o n o p h o r e c o n c e n t r a t i o n was increased b e y o n d what would simulate " p h y s i o l o g i c " conditions, the IMP accumulation became irreversible and would trap the major part o f the radioactivity which had entered the platelet in the form of [ '4C]adenine [12]. As in the previous studies [ 12] it was found t hat with little or no preincubation the secretion induced by a particular i o n o p h o r e c o n c e n t r a t i o n was less c o m p l e t e than after longer preincubation. The degree of difference varied, but the consistent ef f ect of preincubation was proved by statistic t r e a t m e n t of 42 d if f er en t sets, where the secretion when the i o n o p h o r e was added after 0 or 1 min preincubation was c om par e d with the release when 9 min preincubation was allowed, with a P < 0.00005 for the two values in the set. There was also a consistently higher level of metabolic ATP with shorter preincubation (or longer incubation with ionophore). A question is w het her the shorter preincubation with resulting lower secretion induction was the reason for the higher ATP level or w he t he r it was caused by a resynthesis of ATP from IMP. One suggestion th a t t he latter was the case follows from a consideration of the statistical data. When the set o f values obtained by adding the i o n o p h o r e in the cold before incubation and after 9 min preincubation are com pared with the

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F i g . 2. E f f e c t o f t i m e w i t h A 2 3 1 8 7 o n m e t a b o l i c p a r a m e t e r s . T o t a l i n c u b a t i o n t i m e 1 0 r a i n a t 3 7 ° C . T h e ionophore (final concentration 5 0 n M ) a d d e d e i t h e r in the c o l d o r a f t e r 3, 5, 7 o r 9 m i n p r e i n c u b a t i o n . o ...... o, [ 1 4 C ] A T P ; • ....... • , [ 1 4 C ] A D P ; z~. . . . . . z~, [ 1 4 C ] A M P ; • o, [ 1 4 C ] I M P ; o o, [ 14C] inosine plus hypoxanthine, all i n t o t a l i n c u b a t e ; . , ., [ 3 H ] s e r o t o n i n i n s u p e r n a t a n t . M e a n o f two experiments.

set o f values obtained when the i onophore is added after 1 min and 9 min preincubation, there is in the latter case a decrease in significant difference between the ATP levels from P = 0.0028 to P = 0.0054, while the decrease for secretion is f r om P < 0.00005 to P = 0.027. In ot her words, the difference between the ATP values relative to that between secretion values changes from considerably less significant when none and 9 min preincubation are compared to five times more significant when 1 and 9 min preincubation are compared (Table I). This suggests t hat there is a real increase in the ATP level upon increased incubation with ionophore, and thus supports the proposed reversal of the ATP -* IMP conversion [12]. The effect of preincubation on secretion was directly tested by incubating a bulk o f 21 ml platelet suspension in citrated buffered saline and taking out samples after varying of preincubation times for 1-min incubations with A23187. The results show t hat there was a great increase in serotonin secretion when preincubation was longer than 3 min, and that there was no time relationship between this increase in secretion and the decrease in the level o f ['4C]ATP. There was, however, a significant j um p in the inosine plus h y p o x a n t h i n e

TABLE

I

STATISTICAL SIGNIFICANCE OF METABOLIC ATP LEVEL AND OF SECRETION, ACCORDING TO WHETHER IONOPHORE WAS ADDED IN THE COLD, AFTER 1 MIN INCUBATION OR AFTER 9 MIN INCUBATION, AND WHETHER Na ÷ OR K + WAS THE DOMINANT ION IN THE MEDIUM

T h e S t u d e n t ' s t-test for p r o b a b i l i t y o f e x p e r i m e n t a l n u m b e r s w a s p e r f o r m e d a c c o r d i n g t o F e d e r i g h i [ 2 3 ] • M e a n values w i t h standard d e v i a t i o n , t value, p r o b a b i l i t y and n u m b e r o f sets for e a c h c a l c u l a t i o n axe given. All e x p e r i m e n t s w e r e d o n e w i t h a t o t a l i n c u b a t i o n t i m e o f 1 0 rain and i o n o p h o r e added e i t h e r in t h e c o l d , a f t e r 1 rain i n c u b a t i o n or after 9 rain i n c u b a t i o n , I and II give n u m b e r o f m i n u t e s w i t h i o n o p h o r e in c o l u m n I and n o f e a c h set. Numbers of sets

All e x p e r i m e n t s M e a n value t-test P In Na + milieu M e a n value t-test P

42

21

In K + milieu M e a n value t-test P

21

All e x p e r i m e n t s M e a n value t-test P

20

All e x p e r i m e n t s M e a n value t-test P

22

rain w i t h ionophore

ATP I

I

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9 or 10

1

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9 or 10

9

10

Secretion I

ATP l!

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5 3 . 3 +- 8 . 3 43.8 ± 10.9 4.49