Possible role of a cAMP-dependent phosphorylation in the calcium release mediated by inositol 1,4,5-trisphosphate in human platelet membrane vesicles

76

Biochimica et Biophvsica Acta

928 (1987) 76-82 Elsevier

BBA 11918

Possible role of a cAMP-dependent phosphorylation in the calcium release mediated by inositol 1,4,5-trisphosphate in human platelet membrane vesicles Jocelyne E n o u f a, Fran~oise G i r a u d b R a y m o n d e Bredoux a N a t h a l i e B o u r d e a u a and Sylviane Levy-Toledano a " U-150 INSERM, UA CNRS 334, Paris, and b Phvsiologie de la Nutrition, UA CNRS 646, Universit~ Paris-Sud, Orsav (France)

(Received 30 July 1986) (Revised manuscript received 27 November 1986)

Key words: Cyclic AMP; Phosphorylation; Ca 2+ release; Inositol trisphosphate; (Human platelet)

The addition of inositol 1,4,5-trisphosphate (IP3) to a 4SCa-preloaded human platelet membrane fraction (dense tubular system) induced a transient release of Ca 2+. When the vesicle fraction was loaded with 4SCa2+ to isotopic equilibrium in the presence of the catalytic subunit of the cAMP-dependent protein kinase, the level of Ca 2÷ uptake was increased and the subsequent IP3-induced Ca 2÷ release was enhanced. The stimulation was observed regardless of the IP3 concentration used, and was maximal with an enzyme concentration of 5 p g / m l . The addition of the protein kinase inhibitor prevented the stimulatory effect of the catalytic subunit on IP3-induced calcium release, and also abolished the calcium release detected in the absence of added enzyme. It is concluded that a cAMP-dependent protein phosphorylation may be involved in the regulation of the IP3-induced Ca 2 ÷ release in human platelets.

Introduction The polyphosphoinositide metabolite IP 3 is now considered to be a novel second messenger in cellular signal transduction [1] by mobilizing intracellular calcium. In blood platelets, thrombin stimulation induces a rapid turnover of the polyphosphoinositides (PtdIns (4,5)P2) [2,3] which is responsible for IP 3 formation [4]. This effect which can occur in response to various primary stimuli is accompanied by an increase of [Ca 2+ ]i in intact cells [5]. In recent years, the hypothesis suggesting IP 3 as a Ca 2+ mobilizing agent has been tested in Abbreviations: ptdlns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5-trisphosphate; Hepes, 4-(2-hydroxymethyl)-1-piperazineethanesulphonic acid. Correspondence: J. Enouf, H6pital Lariboisiere, INSERMUnite 150, 6 Rue Guy Patin, 75010, Paris Cedex, France.

the platelet system in two different ways. Firstly, IP3 was shown to induce a release of calcium from Ca 2+ transporting m e m b r a n e vesicles enriched in intracellular membranes i.e., the dense tubular system [6-8]. Secondly, addition of IP 3 to permeabilized platelets triggered Ca 2+ release from a non-mitochondrial pool [9] and increased the phosphorylation of the 20- and 40-kDa proteins [10]. Since these proteins are substrates for Ca 2+calmodulin myosin light chain kinase and Ca 2~phospholipid protein kinase respectively, the phosphorylation mediated by IP 3 was suggested to be a result of the increase in the Ca 2+ concentration. However, the mechanism of action of IP3-induced calcium release is not yet clearly defined. The presence of IP 3 receptors has been recently demonstrated in bovine adrenal cortex [11], rat liver microsomes [12], hepatocytes and neutrophils

0167-4889/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

77 [13]. A role for protein phosphorylation has been proposed in the regulation of the permeability of the sarcoplasmic reticulum to calcium [14,15] and recently a role for cAMP-dependent protein phosphorylation in the function of calcium channels has been demonstrated using a purified system [16]. In platelets cAMP has been shown to regulate calcium transport by isolated membrane vesicles [17-19]. This effect is correlated with the phosphorylation of a 23-kDa protein. This led us to investigate whether cAMP could also play a role in the mechanism of IP3-induced calcium release. Materials and Methods

Materials Bovine serum albumin, Hepes, ATP (sodium salt) the catalytic subunit of the cAMP protein kinase (P 2645) the protein kinase inhibitor (type II) P 8140, L-myo-inositol trisphosphate potassium salt from bovine brain were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 45CAC12 and [y-32p]ATP were purchased from Amersham International and New England Nuclear, respectively. Products used for electrophoresis were from Bio-Rad (Richmond, CA): glycine, tris(hydroxymethyl)aminomethane, sodium dodecyl sulfate (SDS), acrylamide, bisacrylamide, ammonium persulfate, 2-mercaptoethanol, T E M E D and Coomassie blue R-250. All other chemicals were of reagent grade.

Preparation of inositol 1,4,5-trisphosphate IP3 was prepared from human erythrocyte 32p_ labelled membranes essentially by the method of Downes et al. [20] and modified either as described in Ref. [21] or by using an alternative procedure to remove ammonium ions. The column eluate was mixed with AG1-X8 (chloride form and IP3 was eluted with 1 M LiCI, and lyophilized. LiC1 was then removed by anhydrous ethanol [22]. A blank consisting of the elution buffer which was used for eluting IP3 from the AG1-X8 column (formate form) and treated as described above was always prepared in each experiment. IP3 purity was ascertained by paper chromatography [23] and HPLC. The concentration of the final solution of IP3 was 150-200 ~M as calculated from its

radioactivity and the specific radioactivity of PtdIns(4,5)P 2. This alternative procedure permitted a reduction in the amount of contaminating Ca z+ in the preparation of both Ip~ and the blank to about 100-300/~M as measured by atomic absorption instead of 700 t~M in the procedure described in Ref. [21]. This Ca 2~ concentration was buffered with E G T A / C a 2~ = 1.24) prior to the assays.

Preparation of platelet membrane fraction The calcium accumulating membrane vesicles were prepared as previously described [24]. Briefly, fresh platelets were isolated and washed in a Tyrode's modified buffer. The cells were then disrupted by controlled ultrasonication and centrifuged at 19000 x g to eliminate unlysed platelets, mitochondria, and granules. The supernatant was centrifuged at 100000 x g and the pellet was used as a source of membrane vesicles. The characterization of this fraction has been described previously [25]. The specific activity of the endoplasmic reticulum marker enzyme, the antimycin-insensitive NADH-cytochrome c reductase was enriched by a factor of 9, but plasma membranes were also present.

Measurement of calcium uptake and release Calcium uptake [24-26] and calcium release [27] in this membrane fraction have been characterized and are mediated by independent transport systems. Calcium uptake by the membrane vesicles was measured on fresh material by incubating 0.1 ml of the 100000 × g membrane fraction (300 /~g of membrane protein) in a final reaction mixture of 1 ml containing 20 mM Hepes buffer (pH 7.4)/100 mM KC1/10 mM potassium oxalate/5 mM Mg A T P / 0 . 5 mM 45CAC12/0.62 mM EGTA. The final free Ca 2+ concentration was 1 ~M as calculated according to the method of Fabiato and Fabiato [28]. The reaction was started by adding the membrane suspension to the incubation medium and stirring at 3 7 ° C for different time intervals. Aliquots of 50 /d were withdrawn and filtered through Millipore H A W P filters (0.45 /~M pore size) previously soaked in a 2 m g / m l bovine serum albumin solution. The filters were washed with 5 ml of 0.1 M CaC12 and counted in 10 ml of

78 U n i s o l v e Scintillation liquid. In calcium release e x p e r i m e n t s , after the calciu m u p t a k e h a d reached a s t e a d y state, Ip~ was a d d e d a n d i m m e d i a t e l y m i x e d with the r e a c t i o n m e d i u m . C a l c i u m release started from this time. A l i q u o t s were filtered at the times i n d i c a t e d in the figures a n d 45Ca 2+ r e m a i n i n g in the m e m b r a n e vesicles was d e t e r m i n e d as d e s c r i b e d above.

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IP~-induced Ca 2 + release from platelet membrane uesicles Fig. 1A shows that the a d d i t i o n of I ~ (our p r e p a r a t i o n ) to 45 Ca 2 + - p r e l o a d e d m e m b r a n e vesicles in a m e d i u m c o n t a i n i n g 1 /aM free C a 2+ c o n c e n t r a t i o n i n d u c e d a transient release of C a 2 + a n d achieved a m a x i m u m effect after 1 min. T h e C a 2+ c o n t e n t r e t u r n e d to its initial s t e a d y state level within 10 min. T h e b l a n k (see M e t h o d s ) h a d no effect on the Ca 2+ c o n t e n t of the vesicles. Because of the presence of the C a 2 + - E G T A b u f f e r this release did n o t m o d i f y the free Ca 2+ conc e n t r a t i o n of the m e d i u m . T h e c o n c e n t r a t i o n dep e n d e n c e o f the I ~ - i n d u c e d Ca 2 + release is shown in Fig. l B . T h e m a x i m u m release was r e a c h e d at

x

%0/ 150

Phosphorylation assays T h e 100000 × g m e m b r a n e vesicle fraction was i n c u b a t e d at 37 ° C for 10 min in a reaction mixture c o n t a i n i n g 10 m M M g S O 4, 100 m M N a F , 6 m M [7-32p]ATP (7.4 n C i / n m o l ) , 25 m M s o d i u m p h o s p h a t e b u f f e r ( p H 7.0), in the presence or a b s e n c e of the c a t a l y t i c subunit of the c A M P - d e p e n d e n t p r o t e i n kinase, ( e n z y m e / s u b s t r a t e ratio 0.01). T h e p h o s p h o r y l a t i o n reactions were initiated b y the a d d i t i o n of A T P a n d s t o p p e d b y the a d d i t i o n of the s a m p l e buffer ( v / v ) at a final c o n c e n t r a t i o n of 0.05 M Tris ( p H 6.8), 2.5% s o d i u m d o d e c y l sulfate, 0.01% v / v b r o m o p h e n o l blue, 5% v / v sucrose, 5% v / v f l - m e r c a p t o e t h a n o l . A f t e r d i s s o c i a t i o n for 10 min in boiling water, s a m p l e s were a p p l i e d to s o d i u m d o d e c y l sulfate-polya c r y l a m i d e g r a d i e n t gels 5 20% [29]. Electrop h o r e s i s was p e r f o r m e d at 30 volts overnight. T h e gels were stained with C o o m a s s i e brilliant blue a n d dried. T h e y were then a u t o r a d i o g r a p h e d using K o d a k X O M A T A R films at - 8 0 o C with intensifying screens.

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Fig. 1. Effects of IP3 on 45Ca content of loaded platelet membrane vesicles. Membranes vesicles were incubated in the presence of 45Ca and ATE After calcium uptake had reached steady state (75-90 min) IP3 or blank (our preparations) were added to the incubation medium. A: Time-course of the IP3-induced Ca 2~ release, x, blank (10/zl); II, 0.8 ~M IP3 (5/tl); O, 1.6 ~M Ipa (10 fib. The data presented were obtained in one experiment, four others gave similar results. B: Concentration dependence of IP3-induced calcium release. The calcium released from the platelet membrane 1 rain after IP3 addition is expressed as a percentage of the calcium content before addition of IP3. O and • refer to two different experiments.

1.5 /aM IP 3 a n d the half m a x i m u m effective conc e n t r a t i o n was a b o u t 0.7 /aM. Using a c o m m e r cially available p r e p a r a t i o n of IP 3 (Sigma), similar time-course a n d m a x i m u m a m p l i t u d e of Ca 2 ~ release were observed. However, higher doses (20 /aM) of this p r e p a r a t i o n were required to o b t a i n a m a x i m u m release of Ca 2+. This might be due to differences in estimating the IP 3 c o n c e n t r a t i o n (see M e t h o d s ) b u t also to the c o n t a m i n a t i o n of the c o m m e r c i a l p r e p a r a t i o n b y the inactive i s o m e r : inositol 2,4,5-trisphosphate. T h e results also showed that the m a x i m u m

79

amount of C a 2+ released did not exceed 12-15% of the sequestered Ca 2+ in membrane vesicles, when 10 m M potassium oxalate was used as anionic precipitating agent. However, the percentage of Ca 2+ released by IP3 was not modified by the omission of potassium oxalate in the incubation medium. Under these latter conditions, 95% of the Ca 2+ taken up by the membrane vesicles could be mobilized by the Ca 2+ ionophore A23187 whereas in the presence of oxalate only 30% was released by the ionophore addition (data not shown).

Stimulation of the Ipcinduced C a 2 + release by the catalytic subunit of the cAMP-dependent protein kinase The incubation of platelet membrane vesicles with the catalytic subunit of the cAMP-dependent protein kinase resulted in a phosphate incorporation into a 23-kDa protein (Fig. 2, compare lane 2 to the autophosphorylation in lane 1). The phosphorylation occurred to a lesser extent when cAMP was used and was totally suppressed by the addition of the protein kinase inhibitor [25]. The intensity of the 23-kDa protein phosphorylation increased up to 2 0 / ~ g / m l of the catalytic subunit of the cAMP-dependent protein kinase and the

time course of the phosphorylation reached a plateau value within 10 min [30]. The phosphorylation of the membrane vesicles remained maximal for 60 to 90 min. Neither the addition of E G T A (124 /~M), nor that of 100 ffM Ca 2+, nor both in a ratio C a 2 + / E G T A = 1.24 (giving a free Ca 2+ concentration of 1 ffM) modified the phosphorylation of the 23-kDa protein (not shown). We have tested the effect of the addition of the catalytic subunit on the IP3-induced Ca 2 ÷ release. The membrane vesicles were incubated at time 0 of the calcium uptake in the presence or absence of the catalytic subunit of the protein kinase. After 60 to 90 min of incubation, the maximal Ca 2+ uptake was increased as a function of the dose of the enzyme (Fig. 3). The addition of IP 3 (Sigma) at a concentration (1 /~M) that did not induce any detectable Ca 2+ liberation in nonphosphorylated vesicles produced a 7% Ca 2+ loss from the phosphorylated membrane vesicles (Fig. 4A). In Fig. 4B, the percentage of Ca 2+ released by the addition of 20 ffM IP3 was increased by 2-fold in the presence of the catalytic subunit of the cAMP-dependent protein kinase. The maximal percentage of releasable calcium reached about 20-25% of the Ca 2+ content in the membrane

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Fig. 2. Protein phosphorylation of the membrane vesicles. Shown are the autoradiographs of the phosphoproteins separated by 5-20% gradient polyacrylamide gel. Membrane vesicles were incubated for 10 rain with [-/-32p]ATP in the absence (lane 1) or the presence of the catalytic subunit of the cAMP-dependent protein kinase (15 # g / m l ) (lane 2).

Fig. 3. Effect of the concentration of the catalytic subunit of the cAMP-dependent protein kinase on the maximal 45Ca uptake and on the stimulation of the calcium release induced by IP 3. The membrane vesicles were incubated in the absence of potassium oxalate with the different enzyme concentrations as explained in the legends of Fig. 1. The maximal 45Ca uptake ( O ) and the percentage of '*5Ca released (O) 1 min after the addition of 2 0 / * M IP 3 (Sigma) in the presence or the absence of the enzyme are expressed as percent of their values in the absence of the enzyme. The data presented were obtained in one experiment, three others gave similar results.

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vesicles after phosphorylation. This result was obtained using 4 /ag/ml of the catalytic subunit of the cAMP-dependent protein

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Fig. 4. Effect of the catalytic subunit of the cAMP-dependent protein kina~e on the IP3-induced calcium release. Membrane vesicles were incubated as explained in the legend of Fig. I, in the absence of the enzyme ((3) or in the presence of the enzyme (4 `ag/ml) (O). When the steady state of 45Ca was reached, IP3 (Sigma) was added to the incubation medium (arrow). The data presented were obtained in one experiment, three others gave similar results. (A): 1 ,aM IP3, B: 20 ~M IP~.

kinase. Fig. 3 shows the effect of various concentrations of the active enzyme on the calcium release induced by a maximal dose (20/~M) of IP 3 (Sigma). The stimulation of the calcium release (expressed as a percentage of the release obtained in the absence of the catalytic subunit) appeared to reach a maximum value at about 5 /~g/ml of the enzyme. As already mentioned and shown in Fig. 4, Fig. 3 also shows that the active enzyme increased the steady-state level of the Ca z+ uptake. However, this increase was not parallel to the increase in the stimulation of the Ca 2~ release. The dose response for the increase in the level of the Ca 2÷ uptake was linear between 0 and 15/~g/ml of the catalytic subunit, whereas the stimulation of the Ca 2+ release was maximal at about 5 / a g / m l . The lack of correlation between the level of Ca 2+ uptake and IP3 response can be deduced from the experiments in which membrane vesicles were incubated with the enzyme in the presence or absence of potassium oxalate. The amount of 45Ca2+ incorporated in the presence of the precipitating anion was about 200 n m o l / m g and was increased only by 20% after the addition of the catalytic subunit (5 /~g/ml) (Fig. 4). The amount of 45Ca2÷ incorporated in the absence of potassium oxalate was smaller (about 60 nmol/ml), but was increased by 80% by the catalytic subunit (Fig. 3). However, in both cases, the percentage of the IP3-induced calcium release (20 ~M IP3) was about 10% in the absence of the catalytic subunit of the cAMP-dependent protein kinase and reached 20% in the presence of the enzyme. The following experiment also suggested that the increase in IP3-mediated Ca 2+ response did not result from a larger cAMP-mediated Ca 2+ uptake: the addition of the enzyme under experimental conditions which did not change the steady-state level (low concentration, 10 rain before the addition of IP3) also resulted in a stimulation of the IP3-induced calcium release (data not shown). To test the hypothesis that the 23-kDa protein phosphorylation may be involved in the Ip~-induced calcium release, we have used the protein kinase inhibitor of the cAMP-dependent protein kinase to abolish the 23-kDa protein phosphorylation [25]. Fig. 5 shows the effect of the addition of

81

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Fig. 5. Effect of the protein kinase inhibitor on the percentage of calcium release-induced by IP3. Membrane vesicles were incubated in the presence of different concentrations of the protein kinase inhibitor at time 0 of the calcium uptake and with (O) or without (©) the catalytic subunit of the cAMP-dependent protein kinase (4 # g / m l ) After 80 min, 20 /~M IP3 (Sigma) were added. The percentage of Ca 2+ released 1 min after IP3 addition is shown. The data presented were obtained in one experiment, three others gave similar results.

the inhibitor at time 0 of the calcium uptake, on the IP3-induced calcium release. These experiments were carried out in the presence or absence of the catalytic subunit of the cAMP-dependent protein kinase. An inhibition of the calcium release was detected even in the absence of the added enzyme, indicating that some of the phosphorylation processes were due to the presence of an endogenous cAMP-dependent protein kinase in the membrane preparation. Maximal inhibition was obtained with 0.16 m g / m l inhibitor corresponding to a ratio inhibitor/protein kinase of 1.5 expressed in terms of phosphorylating units. Discussion

Previous observations on IP3-induced Ca 2+ release from platelet vesicles enriched in internal membranes [6-9] are confirmed in this study. So, the role of IP3 as a C a 2+ mobilizing agent from internal stores can be now considered as well established in the platelet system. In addition, new information about the mechanism of action of IP3 is provided, i.e., a possible role of a cAMP-dependent protein phosphorylation.

(1) The preincubation of the membrane vesicles with the catalytic subunit of the cAMP-dependent protein kinase increases the magnitude of the IP3-induced Ca 2+ release. The increase in the release of Ca 2+ induced by the preincubation with the enzyme does not appear to be the simple consequence of the larger Ca 2+ uptake. This observation gains considerable support from studies recently described by two different groups. Wolf et al. [31] studied the Ca 2+ dependence of the IP3-induced Ca 2+ release in digitonine-treated pancreatic islets. They found that the Ca 2+ released (percent of control) was inversely proportional to the Ca 2+ content in the membranes. Similarly, Delfert et al. [32] showed that the extent of IP3-induced Ca 2+ release was inversely proportional to the concentration of free Ca 2+ in the assay medium. (2) The addition of the protein kinase inhibitor to the membrane vesicles preparation abolishes the Ca 2+ release induced by IP3. These conclusions do not disagree with the results reported by different authors on IP3-induced Ca 2 + release, since these effects were tested after a C a 2+ loading period in the presence of ATP, at 30 or 37 o C, conditions leading to protein phosphorylation [6-10]. Indeed, a low level of 23-kDa protein phosphorylation can be detected in autophosphorylation experiments (Fig. 2). Our observations can also explain why microsomal preparations [33,34] are poorly responsive to IP3 in comparison to permeabilized cells [35-38]. Because of the predominantly cytosolic localization of the enzyme [39], the protein phosphorylation which is possible in permeabilized cells would be much reduced in isolated membrane vesicles. Finally, these results have led us to propose a more complex role of cAMP in platelet activation. Indeed, this intracellular messenger could modulate Ca 2+ movements across the dense tubular system through influx (Ca2+-ATPase) and the IP3-sensitive component of Ca 2+ efflux. This new information is not in contradiction with the wellestablished role of cAMP as inhibitor of platelet function [40-42]. Indeed, the effect of cAMP on the IP3-induced Ca 2+ release would occur at low (basal?) enzyme concentrations and would be a transient effect. The stimulation of the C a 2 ÷ up-

82 t a k e b y c A M P w o u l d m o d i f y t h e s t e a d y - s t a t e level o f C a 2÷ a n d w o u l d t a k e p l a c e a t h i g h e r e n z y m e c o n c e n t r a t i o n s s u c h as t h o s e e x p e c t e d f r o m a g e n t s which elevated cAMP concentration.

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