α2A-Adrenergic Receptor Stimulation Potentiates Calcium Release in Platelets by Modulating cAMP Levels
2000; Elsevier BV; Volume: 275; Issue: 3 Linguagem: Inglês
10.1074/jbc.275.3.1763
ISSN1083-351X
AutoresI. Keularts, Roosje M.A. van Gorp, Marion A.H. Feijge, Wim M.J. Vuist, Johan W. M. Heemskerk,
Tópico(s)Blood disorders and treatments
Resumoα2A-Adrenergic receptor-mediated Ca2+ signaling and integrin αIIbβ3 exposure were investigated in human platelets under conditions where indirect, thromboxane- or ADP-mediated effects were absent. The α2-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 InsP3generation (thimerosal or thapsigargin). In addition, α2-adrenergic stimulation resulted in a 20% lowering in the cytosolic cAMP level. In platelets treated with Gsα-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 αIIbβ3 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 Giα-mediated decrease in cAMP level appears to play a major role in the platelet-activating effects of α2A-adrenergic receptor stimulation. Thus, in platelets, unlike other cell types, occupation of the Giα-coupled α2A-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. α2A-Adrenergic receptor-mediated Ca2+ signaling and integrin αIIbβ3 exposure were investigated in human platelets under conditions where indirect, thromboxane- or ADP-mediated effects were absent. The α2-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 InsP3generation (thimerosal or thapsigargin). In addition, α2-adrenergic stimulation resulted in a 20% lowering in the cytosolic cAMP level. In platelets treated with Gsα-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 αIIbβ3 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 Giα-mediated decrease in cAMP level appears to play a major role in the platelet-activating effects of α2A-adrenergic receptor stimulation. Thus, in platelets, unlike other cell types, occupation of the Giα-coupled α2A-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. epinephrine Ca2+-induced Ca2+ release inositol 1,4,5-trisphosphate prostaglandin E1 cyclic adenosine monophosphorothioate In most cell types, the α2A-adrenergic receptor is linked to a Gi protein, and thus receptor occupation inhibits adenylate cyclase activity in a pertussis toxin-sensitive manner. In human platelets, containing various isoforms of both α- and β-adrenergic receptors, it appears to be mainly the α2A-receptor type that is responsible for the platelet-activating effect of epinephrine (EPI)1 and other catecholamines (1.Thomas D.P. Nature. 1967; 215: 298-299Crossref PubMed Scopus (42) Google Scholar, 2.Kobilka B.K. Matsui H. Kobilka T.S. Yang-Feng T.L. Francke U. Caron M.G. Lefkowitz R.J. Regan J.W. 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Davidson M.M.L. Davies T. Lynham J.A. McClenaghan M.D. Adv. Cycl. Nucl. Res. 1978; 9: 533-552PubMed Google Scholar). In addition, EPI evokes a range of functional platelet responses, such as activation of encrypted integrin αIIbβ3 (fibrinogen) receptors followed by platelet aggregation and, in the presence of other platelet agonists, increased exocytosis (10.Peerschke E.I. Blood. 1982; 60: 71-77Crossref PubMed Google Scholar, 11.Shattil S.J. Budzynski A. Scrutton M.C. Blood. 1989; 73: 150-158Crossref PubMed Google Scholar, 12.Huang M.M. Lipfert L. Cunningham M. Brugge J.S. Ginsberg M.H. Shattil S.J. J. Cell Biol. 1993; 122: 473-483Crossref PubMed Scopus (157) Google Scholar, 13.Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Crossref PubMed Google Scholar). However, which signaling events, putatively downstream of Gi, 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.Haslam R.J. Taylor A. Biochem. J. 1971; 125: 377-379Crossref PubMed Scopus (35) Google Scholar, 15.Haslam R.J. Davidson M.M.L. Desjardins J.V. Biochem. J. 1978; 176: 83-95Crossref PubMed Scopus (174) Google Scholar, 16.Thompson N.T. Scrutton M.C. Wallis R.B. Eur. J. Biochem. 1986; 161: 399-408Crossref PubMed Scopus (30) Google Scholar), implying that EPI may activate other, Gi-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.Siess W. Physiol. Rev. 1989; 69: 58-178Crossref PubMed Scopus (791) Google Scholar, 17.Ware J.A. Johnson P.C. Smith M. Salzman E.W. J. Clin. Invest. 1986; 77: 878-886Crossref PubMed Scopus (70) Google Scholar). In addition, EPI potentiates phosphoinositide hydrolysis evoked by other receptor agonists, due to a stimulation of ADP release and/or thromboxane A2 formation (18.Siess W. Weber P.C. Lapetina E.G. J. Biol. Chem. 1984; 259: 8286-8292Abstract Full Text PDF PubMed Google Scholar, 19.Banga H.S. Simons E.R. Brass L.F. Rittenhouse S.E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9197-9201Crossref PubMed Scopus (203) Google Scholar, 20.Steen V.M. Tysnes O.B. Holmsen H. Eur. J. Biochem. 1988; 253: 581-586Crossref Scopus (23) Google Scholar), or by enhancing the coupling of the other receptors with phospholipase C (16.Thompson N.T. Scrutton M.C. Wallis R.B. Eur. J. Biochem. 1986; 161: 399-408Crossref PubMed Scopus (30) Google Scholar,21.Crouch M.F. Lapetina E.G. J. Biol. Chem. 1988; 263: 3363-3371Abstract Full Text PDF PubMed Google Scholar). Another early proposal is that EPI may act through stimulation of the Na+/H+ exchanger in the plasma membrane (22.Sweatt J.D. Blair I.A. Cragoe E.J. Limbird L.E. J. Biol. Chem. 1986; 261: 8660-8666Abstract Full Text PDF PubMed Google Scholar), although this effect could later be ascribed to an involvement of protein kinase C (23.Nieuwland R. Siffert W. Akkerman J.W.N. Biochim. Biophys. Acta. 1993; 1148: 185-190Crossref PubMed Scopus (8) Google Scholar). A final suggestion is that EPI may act by inhibiting the GTPase-activating protein, Rap1B-GAP (24.Marti K.B. Lapetina E.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2784-2788Crossref PubMed Scopus (14) Google Scholar). Intriguingly, however, most or all of these EPI effects are also under control of the cAMP concentration (7.Siess W. Physiol. Rev. 1989; 69: 58-178Crossref PubMed Scopus (791) Google Scholar, 24.Marti K.B. Lapetina E.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2784-2788Crossref PubMed Scopus (14) Google Scholar), which may point to a possible Gi-mediated effect. In a variety of cell types other than platelets, α2A-adrenergic receptor activation leads to a potent increase in cytosolic [Ca2+]i, which is mediated by Gi activation. In erythroleukemia cells equipped with endogenous α2A-adrenergic receptors and also in cell lines expressing transfected α2A-receptors, this Ca2+ signal is a consequence of activation of phospholipase Cβ via Gβγ subunits that are released upon receptor-Gi coupling (25.Michel M.C. Brass L.F. Williams A. Bokoch G.M. LaMorte V.J. Motulsky H.J. J. Biol. Chem. 1989; 264: 4986-4991Abstract Full Text PDF PubMed Google Scholar, 26.Dorn G.W. Oswald K.J. McCluskey T.C. Kuhel D.G. Liggett S.B. Biochemistry. 1997; 36: 6415-6423Crossref PubMed Scopus (82) Google Scholar). Since also in platelets Gβγ subunits can activate phospholipase Cβ2 and β3 isoforms (27.Banno Y. Asano T. Nozawa Y. Thromb. Haemostasis. 1998; 79: 1008-1013Crossref PubMed Scopus (16) Google Scholar, 28.Rhee S.G. Bae Y.S. J. Biol. Chem. 1997; 272: 15045-15048Abstract Full Text Full Text PDF PubMed Scopus (815) Google Scholar), the question arises whether this Gβγ signaling pathway contributes to the effects of α2A-receptor stimulation in platelets. Here, we examined this subject using aspirin-treated human platelets. It appeared that, similarly to EPI, the specific α2-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 α2A-receptor stimulation activates the platelets, at least in part, through modulation of cytosolic cAMP and cAMP-dependent protein kinase. 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 pisomer of 8-(4-chloro-phenylthio) adenosine-3′,5′-monophosphorothioate ((R p)-8-CPT-cAMPS); S pisomer 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. 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.Heemskerk J.W.M. Feijge M.A.H. Henneman L. Rosing J. Hemker H.C. Eur. J. Biochem. 1997; 249: 547-555Crossref PubMed Scopus (86) Google Scholar). Where indicated, platelets were loaded with Fura-2 acetoxymethyl ester, as described elsewhere (30.Heemskerk J.W.M. Vis P. Feijge M.A.H. Hoyland J. Mason W.T. Sage S.O. J. Biol. Chem. 1993; 268 (353): 356Abstract Full Text PDF PubMed Google Scholar). 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.Cavallini L. Coassin M. Borean A. Alexandre A. J. Biol. Chem. 1996; 271: 5545-5551Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Platelets (6 × 108/2 ml) were freshly suspended in calcium-free Hepes/KCl buffer, pH 7.4, composed of 100 mmKCl, 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 andF min values, respectively. The level of [Ca2+] in the medium was calculated from the binding equation [Ca2+] = K d·β (F −F min)/(F max −F). Ultrapure, calcium-free water was used for preparation of buffers, supplements, and agonists. 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. 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 αIIbβ3 was quantified in 10× diluted platelet-rich plasma using fluorescein-labeled PAC1 antibody directed against activated integrin αIIbβ3(11.Shattil S.J. Budzynski A. Scrutton M.C. Blood. 1989; 73: 150-158Crossref PubMed Google Scholar), 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) β-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.Vuist W.M.J. Levy R. Maloney D.G. Blood. 1994; 83: 899-906Crossref PubMed Google Scholar). To determine the involvement of α2A-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 α2-adrenergic activator (25.Michel M.C. Brass L.F. Williams A. Bokoch G.M. LaMorte V.J. Motulsky H.J. J. Biol. Chem. 1989; 264: 4986-4991Abstract Full Text PDF PubMed Google Scholar). 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.1 A). This clearly contrasts with the situation in native erythroleukemia cells or in CHO cells transfected with α2A-adrenergic receptors, where low concentrations of either agonist (10 nm) were already sufficient to evoke significant Ca2+ responses (25.Michel M.C. Brass L.F. Williams A. Bokoch G.M. LaMorte V.J. Motulsky H.J. J. Biol. Chem. 1989; 264: 4986-4991Abstract Full Text PDF PubMed Google Scholar, 26.Dorn G.W. Oswald K.J. McCluskey T.C. Kuhel D.G. Liggett S.B. Biochemistry. 1997; 36: 6415-6423Crossref PubMed Scopus (82) Google Scholar). 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 Gq-stimulating receptor agonists like thrombin (1 nm), platelet-activating factor (20 nm), and lysophosphatidate (1 μm) (Fig. 1 A; see below). This is in agreement with earlier reports (16.Thompson N.T. Scrutton M.C. Wallis R.B. Eur. J. Biochem. 1986; 161: 399-408Crossref PubMed Scopus (30) Google Scholar). The potentiating effect was seen upon application before or after thrombin (Fig.1 A). 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 α2-adrenergic antagonists (33.Galitzky J. Senard J.M. Lafontan M. Stillings M. Montastruc J.L. Berlan M. Br. J. Pharmacol. 1990; 100: 862-866Crossref PubMed Scopus (25) Google Scholar) yohimbine (Fig.1 A) and RX-821002 (not shown) completely inhibited the EPI-induced potentiation of Ca2+ mobilization. Increased Ca2+ signal generation was not only seen in combination with Gq-coupled receptor agonists, but also with Ca2+-mobilizing agents acting independently of phospholipase C. Using aspirin-treated platelets bathed with apyrase, α2-adrenergic agonists evoked a strong potentiation of the Ca2+ signal induced by 100 nm thapsigargin (Fig. 1 B), a compound inhibiting endomembrane Ca2+-ATPases (30.Heemskerk J.W.M. Vis P. Feijge M.A.H. Hoyland J. Mason W.T. Sage S.O. J. Biol. Chem. 1993; 268 (353): 356Abstract Full Text PDF PubMed Google Scholar). 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. 1 B) 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 InsP3receptors 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.Thompson N.T. Scrutton M.C. Wallis R.B. Eur. J. Biochem. 1986; 161: 399-408Crossref PubMed Scopus (30) Google Scholar). 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 α2-adrenergic receptors is involved in the potentiating effect of EPI on Ca2+ signal generation. 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 InsP3levels. While thrombin evoked the expected increase in InsP3 concentration, UK14304 and EPI were completely ineffective, even when given in combination with thapsigargin (TableI). This agrees well with the earlier noted absence of phosphoinositide turnover in EPI-stimulated platelets (18.Siess W. Weber P.C. Lapetina E.G. J. Biol. Chem. 1984; 259: 8286-8292Abstract Full Text PDF PubMed Google Scholar, 19.Banga H.S. Simons E.R. Brass L.F. Rittenhouse S.E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9197-9201Crossref PubMed Scopus (203) Google Scholar, 20.Steen V.M. Tysnes O.B. Holmsen H. Eur. J. Biochem. 1988; 253: 581-586Crossref Scopus (23) Google Scholar). Apparently, platelets differ from other cells expressing α2A-adrenergic receptors, where compounds like UK14304 cause potent increases in both InsP3 and [Ca2+]i (25.Michel M.C. Brass L.F. Williams A. Bokoch G.M. LaMorte V.J. Motulsky H.J. J. Biol. Chem. 1989; 264: 4986-4991Abstract Full Text PDF PubMed Google Scholar, 26.Dorn G.W. Oswald K.J. McCluskey T.C. Kuhel D.G. Liggett S.B. Biochemistry. 1997; 36: 6415-6423Crossref PubMed Scopus (82) Google Scholar).Table IEffect of UK14304 and EPI on InsP3 levelsAgonistα2-Adrenergic agonistNoneUK14304EPIpmol InsP3/10 8 plateletsNone0.48 ± 0.150.35 ± 0.150.36 ± 0.15Thrombin1.41 ± 0.37NDaND, not determined.NDThapsigargin0.33 ± 0.120.45 ± 0.190.35 ± 0.14Aspirin-treated platelets in apyrase/CaCl2-containing buffer medium (5 × 108/ml) were untreated or treated with UK14304 (10 μm) or EPI (10 μm). After 2 min, the cells were activated with 3 nm thrombin or 300 nm thapsigargin. Samples were taken before activation and at times where maximal increases in [Ca2+]i were measured in parallel incubations with Fura-2-loaded platelets. Data are mean levels of InsP3 ± S.E. (n = 3–5 experiments).a ND, not determined. Open table in a new tab Aspirin-treated platelets in apyrase/CaCl2-containing buffer medium (5 × 108/ml) were untreated or treated with UK14304 (10 μm) or EPI (10 μm). After 2 min, the cells were activated with 3 nm thrombin or 300 nm thapsigargin. Samples were taken before activation and at times where maximal increases in [Ca2+]i were measured in parallel incubations with Fura-2-loaded platelets. Data are mean levels of InsP3 ± S.E. (n = 3–5 experiments). The above results prompted us to re-examine effects of α2-adrenergic stimulation on the classical Giα/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 Gi-stimulating agonist, induced a smaller decrease in cAMP, whereas thapsigargin was without influence on the cAMP level. Thus, EPI might act by a Giα-mediated reduction in cAMP level and subsequent decrease in cAMP-dependent protein kinase activity. This possibility was tested in a number of ways.Table IIEffect of EPI on cAMP levelsAgonistα2-Adrenergic agonistStatisticsnoneEPIpmol cAMP/10 8 plateletsNone3.1 ± 0.162.4 ± 0.12p < 0.01aStudent's t test (two-sided).Thrombin2.5 ± 0.392.2 ± 0.23p > 0.10Thapsigargin2.9 ± 0.242.4 ± 0.26p > 0.10Aspirin-treated platelets in apyrase/CaCl2-containing buffer medium were untreated or treated with 10 μm EPI for 2 min, and then activated with 3 nm thrombin or 300 nm thapsigargin, as indicated. Samples were taken before activation and at times where maximal increases in [Ca2+]i were measured in parallel incubations with Fura-2-loaded platelets. Data are mean levels of cAMP in the samples ± S.E. (n = 5–8 experiments).a Student's t test (two-sided). Open table in a new tab Aspirin-treated platelets in apyrase/CaCl2-containing buffer medium were untreated or treated with 10 μm EPI for 2 min, and then activated with 3 nm thrombin or 300 nm thapsigargin, as indicated. Samples were taken before activation and at times where maximal increases in [Ca2+]i were measured in parallel incubations with Fura-2-loaded platelets. Data are mean levels of cAMP in the samples ± S.E. (n = 5–8 experiments). Since both Gsα- 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.Cavallini L. Coassin M. Borean A. Alexandre A. J. Biol. Chem. 1996; 271: 5545-5551Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 34.Tertyshnikova S. Fein A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1613-1617Crossref PubMed Scopus (89) Google Scholar), this type of intervention is expected to oppose the Ca2+ release-stimulating effect of α2A-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/108platelets). 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).Figure 4Relation 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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.Haslam R.J. Taylor A. Biochem. J. 1971; 125: 377-379Crossref PubMed Scopus (35) Google Scholar). 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.Eigenthaler M. Nolte C. Halbrügge M. Walter U. Eur. J. Biochem. 1992; 205: 471-481Crossref PubMed Scopus (146) Google Scholar). Platelets were also treated with the phosphodiesterase-resistant cAMP analog, (S p)-cAMPS, which specifically activates cAMP-dependent protein kinase (36.Sandberg M. Butt E. Nolte C. Fischer L. Halbrügge M. Beltman J. Jahnsen T. Genieser H.G. Jastorff B.
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