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J Biol Chem, Vol. 275, Issue 3, 1763-1772, January 21, 2000

_2A-Adrenergic Receptor Stimulation Potentiates Calcium Release in Platelets by Modulating cAMP Levels^*

Irene M. L. W. Keularts, Roosje M. A. van Gorp, Marion A. H. Feijge, Wim M. J. Vuist, and Johan W. M. Heemskerk

From the Departments of Biochemistry and Human Biology, University of Maastricht, P.O. Box 616, 6200 MD Maastricht, The Netherlands

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

_2A-Adrenergic receptor-mediated Ca²⁺ signaling and integrin _IIb3 exposure were investigated in human platelets under conditions where indirect, thromboxane- or ADP-mediated effects were absent. The ₂-adrenergic receptor agonists, UK14304 and epinephrine (EPI), were unable to raise cytosolic levels of inositol 1,4,5-trisphosphate (InsP₃) or Ca²⁺ but potentiated the [Ca²⁺]_i rises evoked by other agonists that act through stimulation of phospholipase C (thrombin or platelet-activating factor) or stimulation of Ca²⁺-induced Ca²⁺ release (CICR) in the absence of InsP₃ generation (thimerosal or thapsigargin). In addition, ₂-adrenergic stimulation resulted in a 20% lowering in the cytosolic cAMP level. In platelets treated with G_s-stimulating prostaglandin E₁, EPI increased the Ca²⁺ signal evoked by either phospholipase C- or CICR-stimulating agonists mainly through modulation of the cAMP level. The stimulating effects of UK14304 and EPI on platelet Ca²⁺ responses, and also on integrin _IIb3 exposure and platelet aggregation, were abolished by pharmacological stimulation of cAMP-dependent protein kinase, and these effects were mimicked by inhibition of this activity. In permeabilized platelets, UK14304 and EPI potentiated InsP₃-induced, CICR-mediated mobilization of Ca²⁺ from internal stores in a similar way as did inhibition of cAMP-dependent protein kinase. In summary, a G_i-mediated decrease in cAMP level appears to play a major role in the platelet-activating effects of _2A-adrenergic receptor stimulation. Thus, in platelets, unlike other cell types, occupation of the G_i-coupled _2A-adrenergic receptors does not result in phospholipase C activation but rather in modulation of the Ca²⁺ response by relieving cAMP-mediated suppression of InsP₃-dependent CICR.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In most cell types, the _2A-adrenergic receptor is linked to a G_i protein, and thus receptor occupation inhibits adenylate cyclase activity in a pertussis toxin-sensitive manner. In human platelets, containing various isoforms of both - and -adrenergic receptors, it appears to be mainly the _2A-receptor type that is responsible for the platelet-activating effect of epinephrine (EPI)¹ and other catecholamines (1-4). Thus, in platelets, EPI causes activation of G_i2 followed by adenylate cyclase inhibition (5, 6). Consequently, EPI efficiently antagonizes the cAMP-elevating effect of G_s-stimulating agents like prostacyclin and prostaglandin E₁ (PGE₁) (7-9). In addition, EPI evokes a range of functional platelet responses, such as activation of encrypted integrin _IIb3 (fibrinogen) receptors followed by platelet aggregation and, in the presence of other platelet agonists, increased exocytosis (10-13). However, which signaling events, putatively downstream of G_i, underlie these platelet reactions has long remained unclear.

Earlier data from the literature suggest that a fall in cAMP level as such is insufficient to activate platelets (14-16), implying that EPI may activate other, G_i-independent pathways. For instance, EPI can induce a transient increase in cytosolic [Ca²⁺]_i in platelets charged with the Ca²⁺-sensitive photoprotein, aequorin, although this is not the case for platelets loaded with the Ca²⁺ probes Quin-2 or Fura-2 (7, 17). In addition, EPI potentiates phosphoinositide hydrolysis evoked by other receptor agonists, due to a stimulation of ADP release and/or thromboxane A₂ formation (18-20), or by enhancing the coupling of the other receptors with phospholipase C (16, 21). Another early proposal is that EPI may act through stimulation of the Na⁺/H⁺ exchanger in the plasma membrane (22), although this effect could later be ascribed to an involvement of protein kinase C (23). A final suggestion is that EPI may act by inhibiting the GTPase-activating protein, Rap1B-GAP (24). Intriguingly, however, most or all of these EPI effects are also under control of the cAMP concentration (7, 24), which may point to a possible G_i-mediated effect.

In a variety of cell types other than platelets, _2A-adrenergic receptor activation leads to a potent increase in cytosolic [Ca²⁺]_i, which is mediated by G_i activation. In erythroleukemia cells equipped with endogenous _2A-adrenergic receptors and also in cell lines expressing transfected _2A-receptors, this Ca²⁺ signal is a consequence of activation of phospholipase C via G subunits that are released upon receptor-G_i coupling (25, 26). Since also in platelets G subunits can activate phospholipase C2 and 3 isoforms (27, 28), the question arises whether this G signaling pathway contributes to the effects of _2A-receptor stimulation in platelets.

Here, we examined this subject using aspirin-treated human platelets. It appeared that, similarly to EPI, the specific ₂-adrenergic agonist UK14304 was unable to elicit detectable increases in cytosolic InsP₃ or Ca²⁺ concentration, whereas both compounds potentiated the release of Ca²⁺ induced by other agonists and caused aggregation of platelets, even in the absence of phospholipase C activation. Using a variety of pharmacological agents, we were able to show that _2A-receptor stimulation activates the platelets, at least in part, through modulation of cytosolic cAMP and cAMP-dependent protein kinase.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- L-()-EPI was obtained from Serva (Heidelberg, Germany); PGE₁ and sodium ethylmercurithiosalicylate (thimerosal) were from Janssen (Beersse, Belgium); KT5720 was obtained from Alexis (Läufelfingen, Switzerland); 2',5'-dideoxyadenosine, 9-(tetrahydro-2-furyl)adenine (SQ22536) and Ro-318220 were from Biomol (Plymouth Meeting, PA); fluorescent Ca²⁺ probes were from Molecular Probes (Leiden, The Netherlands). Yohimbine hydrochloride, RX-821002, and UK14304 were bought from RBI (Natick, MA). Compounds obtained from Biolog (Bremen, Germany) were as follows: R_p isomer of adenosine-3',5'-monophosphorothiorate acetoxymethyl ester ((R_p)-cAMPS-AM); R_p isomer of 8-(4-chloro-phenylthio) adenosine-3',5'-monophosphorothioate ((R_p)-8-CPT-cAMPS); S_p isomer of adenosine-3',5'-monophosphorothioate ((S_p)-cAMPS). Monoclonal mouse 4G10 antibody was purchased from Upstate Biotechnology (Lake Placid, NY), and monoclonal fluorescein-labeled PAC1 antibody was a kind gift of Dr. S. J. Shattil (Scripps, La Jolla, CA). Other reagents were of purest grade available and came from Sigma.

Platelet Preparation and Ca²⁺ Measurements-- Blood was freshly collected from healthy volunteers, who had not taken medication in at least 2 weeks. Platelets were treated with 100 µM lysine acetyl salicylate (aspirin), isolated, and then resuspended in buffer A (pH 7.45), containing 136 mM NaCl, 10 mM glucose, 5 mM Hepes, 5 mM KCl, 2 mM MgCl₂, 0.1% (w/v) bovine serum albumin, and apyrase (0.2 units of ADPase/ml) (29). Where indicated, platelets were loaded with Fura-2 acetoxymethyl ester, as described elsewhere (30). In stirred suspensions of Fura-2-loaded platelets (usually 1 × 10⁸/ml), changes in cytosolic [Ca²⁺]_i were continuously measured at 37 °C by ratio fluorometry. Because of the rapid desensitization of EPI effects, the platelets were used within 60-90 min after isolation.

Free Ca²⁺ concentrations were measured in saponin-permeabilized platelets, basically as described elsewhere (31). Platelets (6 × 10⁸/2 ml) were freshly suspended in calcium-free Hepes/KCl buffer, pH 7.4, composed of 100 mM KCl, 100 mM sucrose, 20 mM Hepes, 1.4 mM MgCl₂, 1.25 mM NaN₃, 7.5 mM phosphocreatine, 1 mM ATP, 1 mM KH₂PO₄, 30 µg/ml creatine kinase, 0.6 µg/ml oligomycin, and 1 µM Fluo-3. The platelets were treated with EPI or UK14304, if indicated, and then permeabilized by a 10-min incubation with 30-40 µg of saponin. The [Ca²⁺] was then adjusted to 120 nM by stepwise additions from a concentrated CaCl₂ solution, after which InsP₃ was added. Fluorescence intensities (F) were continuously recorded at 488-nm excitation and 526-nm emission wavelengths (slits of 4 nm), using an SLM-Aminco DMX-1100 spectrofluorometer (Rochester, NY). Calibrations were performed by the addition of excess amounts of CaCl₂ or EGTA/Tris (1:1, mol/mol) to obtain F_max and F_min values, respectively. The level of [Ca²⁺] in the medium was calculated from the binding equation [Ca²⁺] = K_d· (F  F_min)/(F_max  F). Ultrapure, calcium-free water was used for preparation of buffers, supplements, and agonists.

Measurement of InsP₃ and cAMP Concentrations-- Levels of InsP₃ were determined in samples of resting or activated platelets (180 µl, 3.5 × 10⁸ cells). Cellular activity was stopped by adding 75 µl of ice-cold 10% (w/v) HClO₄. After standing on ice for 30 min and centrifuging at 2000 × g for 10 min (strictly at 4 °C), supernatants were collected and neutralized to pH 7 with a solution of 1.7 M KOH in 75 mM Hepes. After another 30-min incubation on ice, precipitated KClO₄ was removed by centrifugation at 2000 × g for 10 min (4 °C). In the supernatants, mass amounts of InsP₃ were measured using a Biotrak radioreceptor assay system (Amersham Pharmacia Biotech) with freshly dissolved InsP₃ as standard.

For determination of intracellular cAMP levels, samples of 200 µl of platelets (0.4 × 10⁸) in suspension were withdrawn from incubations of [Ca²⁺]_i or aggregation measurements, stopped with ice-cold ethanol (70 volume % final concentration), and frozen in liquid nitrogen. After thawing, the samples were centrifuged, and supernatants were used to measure cAMP, using the Biotrak cAMP enzyme immunoassay system from Amersham Pharmacia Biotech.

Platelet Aggregation and Tyrosine Phosphorylation-- Aggregation of aspirin-treated, washed platelets and platelets in plasma was measured in 500-µl portions by recording changes in light transmission at 37 °C. Affinity modulation of integrin _IIb3 was quantified in 10× diluted platelet-rich plasma using fluorescein-labeled PAC1 antibody directed against activated integrin _IIb3 (11), with a Becton Dickinson FACStar flow cytometer (Mountain View, CA).

Tyrosine phosphorylation of platelet proteins was determined in 100-µl samples taken from aggregation cuvettes. Reactions were stopped by adding 10 mM citrate, 5 mM EGTA, and 5 mM EDTA (final concentrations). The stopped incubation mixtures were centrifuged at 10,000 × g for 20 s, and the pellets dissolved into 100 µl of sample buffer, which was composed of 63 mM Tris, 4% (v/v) -mercaptoethanol, 10% (v/v) glycerol, and 3% (w/v) SDS (pH 6.8). Samples were heated at 90 °C for 5 min and subjected to electrophoresis on 8% (w/v) polyacrylamide gels. Prestained electrophoresis markers from Bio-Rad (Hertfordshire, United Kingdom) were run in the same gel. Protein tyrosine phosphorylation was detected on Western blots with immunostaining using the phosphotyrosine-specific monoclonal antibody 4G10, as described before (32).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Potentiation of Platelet Ca²⁺ Responses by ₂-Adrenergic Stimulation-- To determine the involvement of _2A-adrenergic signaling in mobilization of Ca²⁺ in the cytosol, platelets were loaded with Fura-2 and stimulated with EPI, as a general adrenergic agonist, or with UK14304, which is a specific ₂-adrenergic activator (25). Indirect effects due to endogenously released thromboxane A₂ or ADP were prevented by treating the platelets with aspirin and using ADP-degrading apyrase in the suspension medium. Under these conditions, neither EPI nor UK14304 (10-10,000 nM) was capable of inducing a detectable rise in [Ca²⁺]_i (Fig. 1A). This clearly contrasts with the situation in native erythroleukemia cells or in CHO cells transfected with _2A-adrenergic receptors, where low concentrations of either agonist (10 nM) were already sufficient to evoke significant Ca²⁺ responses (25, 26). On the other hand, in platelets, both UK14304 and EPI had a marked, increasing effect on the [Ca²⁺]_i rises induced by low doses of G_q-stimulating receptor agonists like thrombin (1 nM), platelet-activating factor (20 nM), and lysophosphatidate (1 µM) (Fig. 1A; see below). This is in agreement with earlier reports (16). The potentiating effect was seen upon application before or after thrombin (Fig. 1A). In case of preincubation, the potentiation was dose-dependent up to levels of 122 ± 4 and 124 ± 5% (mean ± S.E., n = 3), compared with the thrombin-evoked response, for 10 µM UK14304 and 10 µM EPI, respectively. Immediately after platelet isolation, the effect was already detectable at 2 nM EPI, and it had an EC₅₀ value of 75 nM. As a confirmation of the identity of the receptors involved, it appeared that the ₂-adrenergic antagonists (33) yohimbine (Fig. 1A) and RX-821002 (not shown) completely inhibited the EPI-induced potentiation of Ca²⁺ mobilization.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   2-Adrenergic agonists potentiate platelet [Ca2+]i rises. A, Fura-2-loaded, aspirin-treated platelets were activated with thrombin (1 nM) and EPI (10 µM), UK14304 (10 µM), or yohimbine (1 µM), as indicated. B, platelets were stimulated with a low dose of thapsigargin (100 nM) instead of thrombin. Incubation media contained apyrase (0.2 units of ADPase/ml) and 1 mM CaCl2. Traces show changes in cytosolic [Ca2+]i, representative of seven or more independent experiments.

Increased Ca²⁺ signal generation was not only seen in combination with G_q-coupled receptor agonists, but also with Ca²⁺-mobilizing agents acting independently of phospholipase C. Using aspirin-treated platelets bathed with apyrase, ₂-adrenergic agonists evoked a strong potentiation of the Ca²⁺ signal induced by 100 nM thapsigargin (Fig. 1B), a compound inhibiting endomembrane Ca²⁺-ATPases (30). The EC₅₀ values of both UK14304 and EPI were now in the range of 75-100 nM. Potentiation was observed with EPI added either before or after thapsigargin, while yohimbine (Fig. 1B) and RX-821002 (not shown) were again completely inhibitory. Both adrenergic agents also caused a 2-fold increase in the Ca²⁺ response evoked by 10 µM thimerosal (Fig. 2). Thimerosal is a membrane-permeable sulfhydryl reagent that, independently of phospholipase C, sensitizes InsP₃ receptors and thereby stimulates the process of Ca²⁺-induced Ca²⁺ release (CICR). As checked with platelet agonists of various types (i.e. thrombin, platelet-activating factor, thapsigargin, and thimerosal), the magnitude of the EPI-mediated rise in [Ca²⁺]_i was independent of the presence or absence of extracellular CaCl₂ (Fig. 2), demonstrating that the principal effect of EPI is increase of the Ca²⁺ mobilization from intracellular stores instead of modulation of Ca²⁺ influx. On the other hand, in combination with ionomycin (5 µM) (i.e. a compound directly permeating the cellular membranes for Ca²⁺), EPI did not change the increase in [Ca²⁺]_i (102 ± 5% (mean ± S.E., n = 3)), which is in agreement with earlier findings (16). We observed some donor-to-donor variability in the magnitude of the actions of EPI and also noted desensitization of the effects within 2 h of platelet isolation (see below). Taken together, the comparable effects of UK14304 and EPI and the efficient suppression of these effects by yohimbine and RX-821002 strongly indicate that a single class of ₂-adrenergic receptors is involved in the potentiating effect of EPI on Ca²⁺ signal generation.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Potentiation of Ca2+ responses is independent of extracellular CaCl2. Fura-2-loaded, aspirin-treated platelets in the presence of apyrase (0.2 units of ADPase/ml) were incubated with vehicle or EPI (10 µM), followed by EGTA (1 mM) or CaCl2 (1 mM). Further stimulation was with thrombin (1 nM), platelet-activating factor (PAF, 20 nM), thapsigargin (100 nM), or thimerosal (10 µM), as indicated. Maximal increases in [Ca2+]i (in nM) were measured, and the levels in the absence of EPI were set at 100% per agonist and condition. Data are changes relative to the corresponding 100% control (mean values ± S.E., n = 3 or 4 experiments).

The complete lack of Ca²⁺ responses with UK14304 or EPI alone suggests that these agents are unable to activate phospholipase C isoforms in platelets. This was verified by measuring InsP₃ levels. While thrombin evoked the expected increase in InsP₃ concentration, UK14304 and EPI were completely ineffective, even when given in combination with thapsigargin (Table I). This agrees well with the earlier noted absence of phosphoinositide turnover in EPI-stimulated platelets (18-20). Apparently, platelets differ from other cells expressing _2A-adrenergic receptors, where compounds like UK14304 cause potent increases in both InsP₃ and [Ca²⁺]_i (25, 26).

Involvement of cAMP in ₂-Adrenergic Stimulation of Ca²⁺ Signaling-- The above results prompted us to re-examine effects of ₂-adrenergic stimulation on the classical G_i/adenylate cyclase pathway. Using freshly isolated, aspirin-treated platelets, 10 µM EPI caused a small, but nevertheless significant, reduction in cAMP concentration of about 20% (Table II). In comparison, a low dose of thrombin, i.e. another G_i-stimulating agonist, induced a smaller decrease in cAMP, whereas thapsigargin was without influence on the cAMP level. Thus, EPI might act by a G_i-mediated reduction in cAMP level and subsequent decrease in cAMP-dependent protein kinase activity. This possibility was tested in a number of ways.

Since both G_s- and adenylate cyclase-stimulating agents (e.g. PGE₁ and forskolin), which raise the cAMP level, are known to down-regulate the Ca²⁺ responses of platelets (31, 34), this type of intervention is expected to oppose the Ca²⁺ release-stimulating effect of _2A-adrenergic agonists. Indeed, both EPI and UK14304 (10 µM each) efficiently antagonized the inhibitory effect of PGE₁ on the [Ca²⁺]_i rises evoked by thrombin or thapsigargin, i.e. both in the presence and in the absence of phospholipase C activation (Fig. 3). Plots constructed of the thrombin-induced increase in [Ca²⁺]_i versus the cAMP concentration at the time of Ca²⁺ measurement reveal a nonlinear relationship, in which the Ca²⁺ response steeply declines with the increase in cAMP level (deflection point around 3 pmol of cAMP/10⁸ platelets). Typically, the relation between Ca²⁺ response and cAMP level was similar in the presence and absence of EPI (Fig. 4). When thapsigargin was used as co-agonist instead of thrombin, essentially the same results were obtained (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   2-Adrenergic agonists antagonize inhibitory effect of PGE1 on platelet Ca2+ responses. Fura-2-loaded, aspirin-treated platelets were pretreated with 1-5000 nM PGE1 for 4 min, followed by vehicle () or 10 µM EPI () during 2 min. A hexagon indicates the presence of UK14304 (10 µM) instead of EPI. A, platelets were stimulated with 1 nM thrombin in the presence of 1 mM CaCl2; maximal increases in [Ca2+]i were recorded. B, platelets were stimulated with 100 nM thapsigargin and 1 mM CaCl2; rises in [Ca2+]i after 2 min of activation were recorded. Data are mean values ± S.E. (n = 3 experiments).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Relation of cAMP and Ca2+ levels in EPI-stimulated platelets. Platelets were preincubated with 0 (a), 5 (b), 50 (c), 200 (d), 500 (e), 5000 (f), or 20,000 (g) nM PGE1 and then stimulated with 1 nM thrombin alone (open squares) or thrombin in combination with 10 µM EPI (filled circles), as described for Fig. 3. After maximal levels of [Ca2+]i were reached, samples were taken from the incubation mixtures to measure cAMP. The plot gives Ca2+ response as a function of the cAMP concentration at the time of measurement. Results are from one representative experiment out of three performed.

This type of experiment gave more information on the apparent time-dependent desensitization of the EPI effects. In all cases where tested, platelets that after more than 2 h of isolation did not respond to EPI by an increased Ca²⁺ signal were well able to do so when pretreated with a low dose of PGE₁ (data not shown). This suggests that the observed desensitization is not a consequence of changed receptor binding or transduction properties but, instead, of a time-dependent change in basal cAMP level. Indeed, in two experiments, basal cAMP (in the absence of agonists) was found to decrease from 3.5 to 2.6 pmol/10⁸ platelets (mean values) in a time period of 90 min.

Second, experiments were conducted in which cAMP-dependent protein kinase activity was stimulated by interfering in the signaling pathway downstream of adenylate cyclase. Platelets were therefore treated with the cAMP-dependent phosphodiesterase inhibitor, isobutyl 1-methylxanthine (400 µM), to block cAMP degradation (14). This treatment resulted in a 2-fold increase in cAMP level and diminished the thrombin- and thapsigargin-evoked rises in [Ca²⁺]_i. Moreover, it abolished the stimulating effect of EPI on the Ca²⁺ signal (Fig. 5). This suggests that resting platelets have a basal, non-zero activity of cAMP-dependent protein kinase, as indeed assessed by others (35). Platelets were also treated with the phosphodiesterase-resistant cAMP analog, (S_p)-cAMPS, which specifically activates cAMP-dependent protein kinase (36). This treatment again reduced the Ca²⁺ responses with thrombin and thapsigargin and totally inhibited the Ca²⁺-potentiating effect of EPI (Fig. 5).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Conditions activating cAMP-dependent protein kinase suppress 2-adrenergic effect. Fura-2-loaded, aspirin-treated platelets were preincubated with 200 µM (Sp)-cAMPS or 400 µM isobutyl 1-methylxanthine (IBMX) for 2 min or left untreated. Cells were then activated with 1 nM thrombin (A) or 100 nM thapsigargin (B) with or without 10 µM EPI, as described for Fig. 1. Rises in [Ca2+]i are expressed as percentages of the values obtained with only thrombin or thapsigargin (mean ± S.E., n = 3-5).

Third, effects were determined of inhibition of platelet cAMP-dependent protein kinase. For this purpose, initially the reported adenylate cyclase inhibitors, 2',5'-dideoxyadenosine and SQ22536, were used (15, 37). However, in our hands, these compounds (even high concentrations (500 µM)) diminished the PGE₁-evoked reduction of the thrombin-induced Ca²⁺ responses with no more than 12 and 25%, respectively, which points to only moderate adenylate cyclase inhibition. Platelet treatment with SQ22536 was only of little influence on the EPI-mediated Ca²⁺ release (Fig. 6). Clearer results were obtained with selective inhibitors of cAMP-dependent protein kinase, i.e. the structurally dissimilar hydrolysis-resistant cAMP analog, (R_p)-8-CPT-cAMPS (200 µM) (38), and the kinase active-site inhibitor, KT5720 (2.5 µM) (39). Both compounds caused appreciable abrogation of the PGE₁-induced reduction of the Ca²⁺ response, with 55 and 70%, respectively. In addition, they had a marked effect on the Ca²⁺ signal in the absence of PGE₁. (R_p)-8-CPT-cAMPS and KT5720 raised the basal [Ca²⁺]_i from 45 ± 3 nM to 65 ± 4 and 70 ± 2 nM (mean ± S.E., n = 3), respectively. The compounds increased both the thrombin- and thapsigargin-evoked Ca²⁺ responses and canceled the EPI-mediated potentiation of these responses (Fig. 6). Similarly, the time curves of the [Ca²⁺]_i transients indicated that (R_p)-8-CPT-cAMPS (not shown) and KT5720 (Fig. 7A) mimicked the effects of EPI. As a comparison, we monitored effects of the protein kinase C inhibitor, Ro-318220 (3 µM), and the protein-tyrosine kinase inhibitor, genistein (100 µM) (40). However, neither of these substances altered the effect of EPI on the thrombin-evoked Ca²⁺ response (117 ± 2 and 120 ± 1% of control values, respectively, with EPI alone stimulating to a level of 120 ± 3%).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Conditions inhibiting cAMP-dependent protein kinase mimic 2-adrenergic effect. Fura-2-loaded platelets were activated with 1 nM thrombin (A) or 100 nM thapsigargin (B) with or without 10 µM EPI, as described for Fig. 1. The platelets were pretreated with vehicle (Control) or agents with a suppressing effect on cAMP-dependent protein kinase. Pretreatment was with 500 µM SQ22536 (10 min), 200 µM (Rp)-8-CPT-cAMPS (10 min), or 2.5 µM KT5720 (2 min). Where indicated, EPI was then given, followed by thrombin or thapsigargin. Maximal changes in [Ca2+]i were measured (in nM). Data (mean values ± S.E., n = 3-5) are percentages of the Ca2+ responses relative to the control condition. *, significantly different from the corresponding condition without EPI (p < 0.05, t test, one-sided).

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Stimulation of Ca2+ mobilization by EPI and KT5720. A, aspirin-treated, Fura-2-loaded platelets were stimulated with thapsigargin (TG), EPI, and/or KT5720, as indicated for Fig. 6. B, platelets in KCl/ATP medium were pretreated with EPI (10 µM) and/or KT5720 (2.5 µM), as indicated. Subsequently, the cells were permeabilized with saponin, and mobilization of Ca2+ from stores was measured with Fluo-3. After adjustment of the Ca2+ level of the medium to 110 nM, InsP3 (100 nM) was added as indicated. Data are from a representative experiment out of five or more performed.

Fifth, since the InsP₃ receptor channel in platelets is known to be cAMP-sensitive, we determined the effect of ₂-adrenergic agents on InsP₃-evoked Ca²⁺ release from intracellular stores in saponin-permeabilized platelets. As shown in Fig. 7B, prestimulation with EPI before the addition of saponin led to a potent increase in the Ca²⁺-releasing effect of InsP₃. Preincubation of the platelets with KT5720 had a similar effect. When using freshly isolated platelets, UK14304 and EPI potentiated the InsP₃-evoked Ca²⁺ mobilization to 140 ± 6 and 161 ± 8% of the control value, respectively (mean ± S.E., n = 6-8, p < 0.01). At 2 h after platelet isolation, the stimulating effect of EPI was decreased to 111 ± 5% (n = 10, p < 0.04). Taken together, these results suggest that, in freshly isolated platelets, InsP₃-induced Ca²⁺ mobilization is partially down-regulated by tonic activity of the cAMP-dependent protein kinase and that ₂-adrenergic stimulation can relieve this down-regulation.

Involvement of cAMP in ₂-Adrenergic Mediated Integrin _IIb3 Activation-- As an extension of the Ca²⁺ measurements, we monitored effects of ₂-adrenergic stimulation on the exposure of active integrin _IIb3 on the platelet surface by using fluoresceinisothiocyanate-labeled PAC1 antibody, which only binds to this activated integrin form. Confirming an earlier report (11), platelet stimulation with 10 µM EPI or UK14304 resulted in a considerable increase in fluorescein-PAC1 binding (Fig. 8). The EPI-induced integrin _IIb3 activation was completely blocked by yohimbine, isobutyl 1-methylxanthine, or PGE₁ but not by Ro-318220. Since integrin exposure causes platelet aggregation when fibrinogen is present, and extensive protein tyrosine phosphorylation (12, 13), these platelet responses were also measured. Yohimbine, isobutyl 1-methylxanthine, PGE₁, or (S_p)-cAMPS almost completely suppressed the EPI-induced aggregate formation (Fig. 9A) and tyrosine phosphorylation events (Fig. 9B).

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Suppression of EPI-evoked exposure of integrin IIb3. Aspirin-treated platelet-rich plasma, containing apyrase, was diluted 10× with Hepes buffer and preincubated for 5 min with vehicle (control), 1 µM yohimbine, 400 µM isobutyl 1-methylxanthine (IBMX), 5 µM PGE1, or 3 µM Ro-318220, as indicated. The platelets were then activated with 10 µM EPI in the presence of fluorescein isothiocyanate-labeled PAC1 antibody for 15 min, after which antibody binding was analyzed by flow cytometry.

View larger version (48K): [in this window] [in a new window]   Fig. 9.   Suppression of EPI-evoked platelet aggregation and protein tyrosine phosphorylation. Aspirin-treated platelet-rich plasma was incubated with vehicle (control), Ro-318220, PGE1, yohimbine, or isobutyl 1-methylxanthine (IBMX), as described for Fig. 8. Other platelets were preincubated with 200 µM (Sp)-cAMPS during 3 min. Cells were then activated with 10 µM EPI, as indicated. A, changes in light transmission determined with a platelet aggrometer. B, protein tyrosine phosphorylation patterns of platelet samples, withdrawn from incubations after 2 min of activation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The present results demonstrate that, in platelets, the signaling pathway upon _2A-adrenergic stimulation differs from that reported for other cells expressing this receptor, i.e. erythroleukemia cells and embryonic kidney 293 cells with native receptors (41, 42), and Chinese hamster ovary and COS-7 cells transfected with _2A-receptors (26). In Fura-2-loaded platelets, which respond to minor increases in InsP₃ level by a clear Ca²⁺ signal (30), neither UK14304 nor EPI appears to elevate InsP₃ or Ca²⁺ concentrations, which agrees well with the reported lack of EPI to induce hydrolysis of radioactive labeled phosphoinositides (18-20). This suggests that, in platelets, the G-mediated pathway of phospholipase C stimulation, as identified for the other cell types (26, 42), does not play an important role in the ₂-adrenergic signaling cascade. On the other hand, our data confirm those from others that ₂-adrenergic receptor (EPI) stimulation of platelets causes G_i activation with consequent inhibition of adenylate cyclase (1-9), ultimately leading to the exposure of integrin _IIb3 (11-13).

Compatible with a G_i-mediated action, we detected a significant decrease in basal cAMP level in the EPI-treated platelets (Table II). Various sets of experiments suggest that this cAMP change is crucial in the effect of EPI to potentiate the Ca²⁺ responses with phospholipase C-stimulating (e.g. thrombin) and phospholipase C-independent (e.g. thapsigargin) platelet agonists (Figs. 1 and 2). For instance, both UK14304 and EPI efficiently antagonize the G_s/cAMP-mediated suppression of the Ca²⁺ signal (Fig. 3). In addition, when comparing intracellular levels of Ca²⁺ and cAMP in platelets that were pretreated with various doses of PGE₁, the Ca²⁺ responses appear to decline steeply with an increase in cAMP concentrations independently of the presence or absence of EPI, with a reflection point that is close to the cAMP level of resting platelets (Fig. 4). Other evidence that cAMP and cAMP-dependent protein kinase are involved in this _2A-adrenergic response comes from the observation that agents that stimulate the kinase suppress the Ca²⁺ responsiveness and inhibit the EPI effect (Fig. 5); and also from data that inactivation of the kinase results in increased Ca²⁺ responses while blocking the EPI effect (Fig. 6). In addition, both UK14304 and EPI appear to have a highly stimulatory effect on InsP₃-induced Ca²⁺ mobilization in permeabilized platelets (Fig. 7), a reaction that is particularly sensitive to the cAMP and Ca²⁺ concentrations (31, 34).² The latter adrenergic effect can be mimicked by inhibition of cAMP-dependent protein kinase, which suggests that ₂-adrenergic agents act by relieving the partial suppression of the Ca²⁺ mobilization by the basal cAMP level. Taken together, these results suggest that _2A-adrenergic receptor stimulation in platelets influences the Ca²⁺ responses primarily by modulation of the cAMP concentration.

The present data indicate that the channel activity of the platelet InsP₃ receptors is altered by EPI action. It seems that EPI reduces the basal, cAMP-dependent phosphorylation of InsP₃ receptors and, thereby, increases their responsiveness toward InsP₃-generating and Ca²⁺-mobilizing agonists. This explains why the ₂-adrenergic agonists potentiate the Ca²⁺ responses even in combination with thapsigargin and thimerosal, compounds that operate independently of phospholipase C stimulation. Indeed, in platelets, the process of CICR by which InsP₃ receptors mobilize Ca²⁺ in a Ca²⁺-stimulated way appears to be influenced not only by increased Ca²⁺ leakage fom stores (by thapsigargin) or sensitization of the InsP₃ receptor (by thimerosal), but also by changes in intracellular cAMP.² Although the platelet InsP₃ receptors have been recognized as major cAMP sensors in Ca²⁺ signaling (31), it is not unlikely that EPI may also influence other cAMP-sensitive steps involved in the regulation of the Ca²⁺ signal, e.g. by stimulating phosphoinositide turnover (44) or reducing plasma membrane Ca²⁺-ATPase activity (45, 46).

The current proposal that small changes in (basal) cAMP concentration significantly modulate the Ca²⁺ responses agrees well with published evidence that the estimated level of cAMP in unstimulated platelets (about 4.4 µM) is somewhat higher than the apparent cAMP dissociation constant of cAMP-dependent protein kinases (47) and only slightly lower than the intracellular concentration of cAMP-binding sites on the kinases of 6.2 µM (35, 48). Thus, cAMP-mediated protein phosphorylation in platelets can be considered as a signal transduction pathway of both high sensitivity and high capacity (48). On the other hand, it is clear from our findings and the literature that a decrease in cAMP as such is insufficient to produce full platelet activation. For instance, we found that the kinase inhibitors (R_p)-8-CPT-cAMPS and KT5720 potentiate platelet activation but do not evoke a major Ca²⁺ signal or aggregation of the platelets. This is in agreement with published evidence that the use of 2',5'-dideoxyadenosine or SQ22536 to inhibit adenylate cyclase is insufficient to induce aggregate formation (9, 15, 37). Although under our conditions these compounds were only weak adenylate cyclase inhibitors, it is likely that other signaling events than a decrease in cAMP level are needed for full platelet activation.

A typical effect of _2A-adrenergic receptor stimulation in other cell types than platelets is the G-mediated activation of a mitogen-activated protein kinase cascade (26, 42). Such a signaling route, although not yet proven, might also occur in EPI-stimulated platelets. In particular, this might be involved in the EPI-induced exposure of integrin _IIb3 and, thus, in platelet aggregation. Nevertheless, also for this ₂-adrenergic response, modulation of the cAMP level seems to play an ultimate controlling role, as apparent from the integrin- and tyrosine phosphorylation-inhibitory effects of cAMP-elevating interventions (Figs. 8 and 9).

In summary, we propose the following scheme of _2A-adrenergic receptor stimulation in platelets (Fig. 10). Receptor occupation results in G_i-mediated inhibition of adenylate cyclase. Consequent lowering in cAMP level and decreased activity of cAMP-dependent protein kinase relieves the tonic, suppressive effect of cAMP-dependent phosphorylation on the Ca²⁺ channel function of the InsP₃ receptors. These channels allow Ca²⁺ release into the cytosol in a InsP₃- and Ca²⁺-dependent way according to the CICR mechanism. In addition to this G_i-mediated effect, the cAMP level is controlled by G_s-mediated activation of adenylate cyclase and cAMP hydrolysis by phosphodiesterases. Signaling through G subunits may contribute to the platelet-activating effect of _2A-receptor agonists; however, not by activation of phospholipase C.

View larger version (18K): [in this window] [in a new window]   Fig. 10.   Proposed scheme of platelet-activating effect of 2A-adrenergic receptor stimulation. A full explanation of signaling routes is given under "Discussion." The gray arrows marked with asterisks represent inhibitory actions. 2A-R, 2A-adrenergic receptor; 2i, q, s, and , - and subunits of G-proteins; AC, adenylate cyclase; IP3-R, InsP3 receptor; PDE, phosphodiesterase; PKA, cAMP-dependent protein kinase; PLC-3, phospholipase C3.

	FOOTNOTES

^* This work was supported by Netherlands Heart Foundation Grant NHS 93.166.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Depts. of Human Biology/Biochemistry, University of Maastricht, P.O. Box 616, 6200 MD Maastricht, The Netherlands. Tel.: 31-43-3881684; Fax: 31-43-3884160; E-mail: JWM.Heemskerk@Bioch.Unimaas.nl.

² R. van Gorp, unpublished results.

	ABBREVIATIONS

The abbreviations used are: EPI, epinephrine; CICR, Ca²⁺-induced Ca²⁺ release; InsP₃, inositol 1,4,5-trisphosphate; PGE₁, prostaglandin E₁; cAMPS, cyclic adenosine monophosphorothioate.

	REFERENCES

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