Two distinct Ca2+ compartments show differential sensitivity to thrombin, ADP and vasopressin in human platelets

Cellular Signalling 18 (2006) 373 – 381 www.elsevier.com/locate/cellsig

Two distinct Ca2+ compartments show differential sensitivity to thrombin, ADP and vasopressin in human platelets Jose J. Lo´pez, Pedro C. Redondo, Gine´s M. Salido, Jose A. Pariente, Juan A. Rosado* Department of Physiology, University of Extremadura, Av. Universidad s/n, Ca´ceres 10071, Spain Received 19 April 2005; accepted 6 May 2005 Available online 10 August 2005

Abstract Recent studies propose the existence of two distinct Ca2+ compartments in human platelets based on the expression of different SERCA isoforms with distinct sensitivity to thapsigargin and 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Using fura-2-loaded human platelets we have found that depletion of the TBHQ sensitive store reduces thrombin—but not ADP—or vasopressin (AVP)-induced Ca2+ release. Redistribution of cytosolic Ca2+ after thrombin stimulation resulted in overloading of the TBHQ-sensitive store. This phenomenon was not observed with ADP or AVP. We found that NAADP decreases the Ca2+ concentration into the stores in permeabilized platelets, which is prevented by depletion of the TBHQ-sensitive store. Nimodipine, an inhibitor of the NAADP receptor, reduced thrombin-induced Ca2+ release from the TBHQ-sensitive stores, without having any effect on the responses elicited by ADP or AVP. Finally, the phospholipase C inhibitor, U-73122, abolished ADP- and AVP-induced Ca2+ release, suggesting that their responses are entirely dependent on IP3 generation. In contrast, treatment with both U-73122 and nimodipine was required to abolish thrombin-induced Ca2+ release. We suggest that thrombin evokes Ca2+ release from TBHQ-sensitive and insensitive stores, which requires both NAADP and IP3, respectively, while ADP and AVP exert an IP3-dependent release of Ca2+ from the TBHQ-insensitive compartment in human platelets. D 2005 Elsevier Inc. All rights reserved. Keywords: Platelets; Ca2+ stores; Thrombin; ADP; Vasopressin; NAADP

1. Introduction Cytosolic Ca2+ concentration ([Ca2+]c) is a very important factor and perhaps the most widely used means to regulate a large number of cellular function, ranging from short-term responses, such as muscle contraction [1], secretion [2] or platelet aggregation [3] to long-term processes like cell growth [4] and proliferation [5]. Many cellular agonists increase [Ca2+]c by releasing compartmentalised Ca2+ from intracellular stores or by activating the entry of extracellular Abbreviations: [Ca2+]c, Cytosolic-free calcium concentration; [Ca2+]s, Free calcium concentration in the stores; TG, Thapsigargin; Iono, Ionomycin; PBS, Phosphate-buffered saline; HBS, HEPES-buffered saline; TBHQ, 2,5-di-(tert-butyl)-1,4-hydroquinone; AVP, Vasopressin; PMCA, Plasma membrane Ca2+-ATPase; SERCA, Sarcoendoplasmic reticulum Ca2+-ATPase; NAADP, Nicotinic acid adenine dinucleotide phosphate; cADPR, Cyclic ADP ribose. * Corresponding author. Tel./fax: +34 927 257154. E-mail address: [email protected] (J.A. Rosado). 0898-6568/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2005.05.006

Ca2+ across the plasma membrane [6]. When agonist stimulation ceases a number of mechanisms, mainly pumps and exchangers, remove Ca2+ from the cytosol to restore the resting [Ca2+]c. Among the Ca2+ removal mechanisms the most relevant are Ca2+ reuptake into the intracellular stores by the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) and Ca2+ extrusion carried out by to different transporters, the plasma membrane Ca2+-ATPase (PMCA) and the Na+/Ca2+ exchanger [7]. At least two different isoforms of SERCA, with molecular masses of 100 and 97 kDa, have been found in human platelets [8]. The 100 kDa isoform has been identified as SERCA 2b, and is inhibited by low concentrations of thapsigargin (TG). In contrast, the 97 kDa isoform, which has been identified as SERCA 3 [9,10], is inhibited only by high concentrations of TG and, unlike SERCA 2b, is sensitive to 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ) [11]. Pharmacological studies suggest that the different SERCA isoforms are distributed separately in two distinct

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IP3-sensitive Ca2+ stores [12]. According to this, TBHQ or high concentrations of TG release Ca2+ from one Ca2+ compartment (TBHQ-sensitive store with low affinity for TG) while a low concentrations of TG allows the depletion of a different Ca2+ pool (TBHQ-insensitive store with high affinity for TG) [12]. These findings were confirmed by immunolocalization studies, which suggest that these SERCA isoforms are distributed over different platelet membranes and have been found to be associated with motile organelles [13]. These phenomenon might be explained by the properties of the de novo conformational coupling model for store-mediated Ca2+ entry in human platelets, which is based on the transport of portions of the Ca2+ stores towards the plasma membrane to facilitate the coupling between IP3 receptors in the stores and hTRP1 channels in the plasma membrane [14 – 17]. Interestingly, we have recently reported the coexistence of two different mechanisms for storeoperated Ca2+ entry in platelets differentially regulated by the two different IP3-sensitive Ca2+ stores [18]; however, at present no attempts to elucidate the differential release of Ca2+ from both compartments stimulated by platelet agonists has been performed. In the present study, we have proceeded to investigate the differential Ca2+ release from these compartments stimulated by the physiological agonists thrombin, ADP and vasopressin (AVP) and the molecular mechanisms associated to these events.

2. Material and methods 2.1. Materials Fura-2 acetoxymethyl ester (fura-2/AM), fura-FF/AM, pluronic F-127 and calcein were from Molecular Probes (Leiden, The Netherlands). Apyrase (grade VII), aspirin, ADP, vasopressin (AVP), bovine serum albumin, thrombin, streptolysin O, nicotinic acid adenine dinucleotide phosphate (NAADP), nimodipine, thapsigargin (TG) and ionomycin (Iono) were from Sigma (Madrid, Spain). U-73122 was from Calbiochem (Nottingham, UK). TBHQ was from Alexis (Nottingham, UK). All other reagents were purchased from Panreac (Barcelona, Spain). 2.2. Platelet preparation Fura-2-loaded human platelets were prepared as described previously [14], as approved by Local Ethical Committees. Briefly, blood was obtained from healthy volunteers and mixed with one-sixth volume of acid/citrate dextrose anticoagulant containing (in mM): 85 sodium citrate, 78 citric acid and 111 d-glucose. Platelet-rich plasma was then prepared by centrifugation for 5 min at 700 g and aspirin (100 AM) and apyrase (40 Ag/mL) added. Platelet-rich plasma was incubated at 37 -C with 2 AM fura-2/AM for 45 min. Cells were then collected by centrifugation at 350 g for 20

min and resuspended in HEPES-buffered saline (HBS) containing (in mM): 145 NaCl, 10 HEPES, 10 d-glucose, 5 KCl, 1 MgSO4, pH 7.45 and supplemented with 0.1% w / v bovine serum albumin and 40 Ag/mL apyrase. 2.3. Cell viability Calcein and trypan blue were used to assess cell viability. For calcein loading, resting cells, or treated with inhibitors for the times indicated, were incubated for 30 min with 5 AM calcein – AM at 37 -C, centrifuged and the pellet was resuspended in fresh HBS. Fluorescence was recorded from 2 mL aliquots using a Shimadzu Spectrophotometer (Shimadzu, Japan). Samples were excited at 494 nm and the resulting fluorescence was measured at 535 nm. The calcein fluorescence remaining in the cells after treatment with the inhibitors used was the same as in control, suggesting that under our conditions there was no cellular damage. The results obtained with calcein were confirmed using the trypan blue exclusion technique. Ninety-five percent of cells were viable after treatment with the inhibitors, similar to that observed in our resting platelet suspensions. 2.4. Measurement of cytosolic free calcium concentration ([Ca2+]c) Fluorescence was recorded from 2 mL aliquots of magnetically stirred cell suspensions (108 cells/mL) at 37 -C using a fluorescence spectrophotometer (Varian Ltd., Madrid, Spain) with excitation wavelengths of 340 and 380 nm and emission at 505 nm. Changes in [Ca2+]c were monitored using the fura-2 340 / 380 fluorescence ratio and calibrated according to the method of Grynkiewicz et al. [19]. Thrombin- or ADP- and AVP-induced Ca2+ release were estimated using the integral of the rise in [Ca2+]c for three or two min after its addition, respectively, taking a sample every second, and was expressed as nM.s as described previously [20]. 2.5. Determination of changes in calcium concentration in the stores ([Ca2+]s) Platelet-rich plasma was incubated at 37 -C with 5 AM fura-FF/AM and pluronic F-127 (0.025%) for 1 h. Cells were then collected by centrifugation at 350 g for 20 min and resuspended in modified intracellular solution containing (in mM): 20 NaCl, 10 HEPES –KOH, 120 KCl, 1.13 MgCl2, pH 7.2 and supplemented with 40 Ag/mL apyrase. Fluorescence was recorded from 2 mL aliquots of magnetically stirred cell suspensions (108 cells/mL) at 37 -C using a Fluorescence Spectrophotometer (Varian Ltd., Madrid, Spain) with excitation wavelengths of 340 and 380 nm and emission at 505 nm. Cell were permeabilized by incubation with 0.5 U/mL streptolysin O for 30 s in a Ca2+-free medium, which removes the fluorescence of the cytosolic fura-FF. Changes in [Ca2+]s were monitored using the fura-FF 340 / 380 fluorescence ratio.

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2.6. Statistical analysis Analysis of statistical significance was performed using Student’s t-test and only values with P < 0.05 were accepted as significant.

3. Results 3.1. Agonist-induced Ca2+ release from TBHQ-sensitive and TBHQ-insensitive compartments Fura-2-loaded aspirin-treated human platelets were used to assess Ca2+ release from the intracellular stores evoked by different physiological agonists. As previously reported [6], in the absence of external Ca2+, 1 U/mL thrombin evoked a transient increase in [Ca2+]c. The addition of TBHQ to the platelet suspension, to allow depletion of its sensitive store, 9 min prior to stimulation with thrombin decreased thrombin-induced Ca2+ mobilization. As shown in Fig. 1A, in a Ca2+-free medium, 1 U/mL thrombin evoked a peak [Ca2+]c rise over the resting level of 144 T 4 nM (top panel; mean T S.E.M.; n = 12). Addition of 20 AM TBHQ induced a sustained increase of [Ca2+]c of 29 T 6 nM. Under these conditions, thrombin-induced peak [Ca2+]c rise over the pre-stimulated level was only 120 T 6 nM (Fig. 1A bottom panel; P < 0.05; n = 12). The estimation of thrombin-evoked Ca2+ release (see Material and methods) was found to be greater in platelets treated with TBHQ (7850 T 444 and 7178 T 342 nM.s in the presence and absence of TBHQ, respectively; P < 0.05; n = 12), which might be explained by the inactivation of SERCA 3 by TBHQ and the subsequent effect on Ca2+ reuptake into the TBHQ-sensitive stores. Due to this fact, the peak response in [Ca2+]c induced by the different agonists will be used as the indicative of Ca2+ release throughout this study. The findings reported above suggest that thrombin releases Ca2+ from TBHQ-sensitive and TBHQ-insensitive stores. To confirm this possibility Ca2+ release was stimulated either with thrombin or thrombin plus TBHQ. Under our experimental conditions, thrombin evoked a similar peak [Ca2+]c rise when added alone or in combination with TBHQ (150 T 6 and 145 T 10 nM; Fig. 1B; P > 0.05; n = 10 –15). Subsequently cells were treated with TG (1 AM) combined with ionomycin (Iono; 50 nM) to allow rapid and complete release of the Ca2+ remaining in the stores. As expected, TBHQtreated cells accumulated a smaller amount of Ca2+ in the stores, which confirms that TBHQ effectively prevented Ca2+ reuptake into the sensitive compartment (Fig. 1B). In contrast to the results obtained with thrombin, treatment of platelets with TBHQ (20 AM) did not modify the peak [Ca2+]c elevation induced by ADP (10 AM) or AVP (0.1 AM) in a Ca2+-free medium, suggesting that Ca2+

Fig. 1. Thrombin releases Ca2+ from TBHQ-sensitive and TBHQinsensitive stores. (A) Fura-2-loaded human platelets were stimulated with TBHQ (20 AM; lower panel) or the vehicle (DMSO; top panel), as indicated, in a Ca2+-free medium (100 AM EGTA was added). Thrombin (final concentration 0.5 U/mL) was added to the medium nine min later. (B) Platelets were stimulated with thrombin (0.5 U/mL) in the absence or presence of TBHQ (20 AM) in a Ca2+-free medium (100 AM EGTA was added) and 5 min later TG (1 AM) plus Iono (50 nM) were added to the platelet suspension. Elevations in [Ca2+]c were monitored using the 340 / 380 nm ratio and traces were calibrated in terms of [Ca2+]c. Traces shown are representative of twelve separate experiments.

responses induced by these two agonists do not require release from the TBHQ-sensitive store (149 T 18 vs. 149 T 16 nM for ADP and 111 T 5 vs. 123 T 7 nM for AVP in the absence or presence of TBHQ; Fig. 2A and C; P > 0.05; n = 8 – 12). Consistent with this, when platelets were stimulated with ADP or AVP in combination with TBHQ the initial peak [Ca2+]c elevation reaches a value that was approximately the result of the sum of the peak [Ca2+]c rises induced by the agonist and TBHQ separately (219 T 3 and 202 T 6 for ADP or AVP in combination with TBHQ; Fig. 2B and D). To further investigate whether ADP and AVP release Ca2+ only from the TBHQ-insensitive store we

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Fig. 2. ADP and AVP release Ca2+ from the TBHQ-insensitive Ca2+ store. (A and C) Fura-2-loaded human platelets were stimulated with TBHQ (20 AM) or the vehicle (DMSO) in a Ca2+-free medium (100 AM EGTA was added). ADP (10 AM; A) or AVP (0.1 AM; C) were added to the medium 9 min later to activate Ca2+ release. (B and D) Platelets were stimulated in a Ca2+-free medium (100 AM EGTA was added) with ADP (10 AM; B) or AVP (0.1 AM; D) in the absence or presence of TBHQ (20 AM). (E) Human platelets were stimulated in a Ca2+-free medium (100 AM EGTA was added) either with ADP (10 AM), AVP (0.1 AM) or both, at the time indicated by the thick arrow. Elevations in [Ca2+]c were monitored using the 340 / 380 nm ratio and traces were calibrated in terms of [Ca2+]c. Traces shown are representative of eight to twelve separate experiments.

stimulated platelets with both agonists separately or in combination. As shown in Fig. 2E, treatment of platelets in a Ca2+-free medium with ADP plus AVP induced a similar increase in [Ca2+]c to both agonists when added separately ( P > 0.05; n = 12).

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Fig. 3. Ca2+ loading of the TBHQ-sensitive store after treatment with agonists. (A) Fura-2-loaded human platelets were stimulated with thrombin (0.5 U/mL) or left untreated and four min later 20 AM TBHQ was added to allow depletion of the TBHQ-sensitive store. Four minutes later platelets were treated with TG (1 AM) plus Iono (50 nM) to deplete the TBHQ-insensitive store. (B and C) Human platelets were stimulated with ADP (10 AM; B) or AVP (0.1 AM; C) or the vehicle (HBS; top panels) and 4 min later cells were treated with 20 AM TBHQ. Elevations in [Ca2+]c were monitored using the 340 / 380 nm ratio and traces were calibrated in terms of [Ca2+]c. Traces shown are representative of five separate experiments.

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[Ca2+]c is mainly mediated by two mechanisms, sequestration of Ca2+ into intracellular compartments by SERCAs and Ca2+ extrusion. We have now investigated the redistribution of cytosolic Ca2+ in both compartments after an agonist-induced response. Treatment of human platelets in a Ca2+-free medium with 0.5 U/mL thrombin induced a transient increase in [Ca2+]c. As shown in Fig. 3A, addition of TBHQ when thrombin-response is terminated resulted in a transient increase in [Ca2+]c that was significantly higher than that induced by TBHQ in nonstimulated cells (7354 T 422 nM.s in thrombin-treated cells vs. 5563 T 669 nM.s in controls; P < 0.05; n = 5). The subsequent addition of TG (1 AM) combined with Iono (50 nM) confirms that less Ca2+ was accumulated in the TBHQ-insensitive store after treatment with thrombin (Fig. 3A). In contrast, when platelets were stimulated with 10 AM ADP (Fig. 3B) or 0.1 AM AVP (Fig. 3C) the amount of Ca2+ released by TBHQ was similar to that found in non-stimulated cells. These observations suggest that cytosolic Ca2+ redistribution into the internal stores favors the TBHQ-sensitive compartment only if this pool is previously emptied.

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3.4. Ca2+ release from the TBHQ-sensitive compartment is mediated by NAADP and is sensitive to nimodipine NAADP, an intracellular Ca2+-mobilizing molecule [22], might be an alternative second messenger to release Ca2+ from the TBHQ-sensitive store. The effects on [Ca2+]s of

3.3. Ca2+ release from the TBHQ-insensitive compartment is sensitive to U-73122 The possible requirement of IP3 for agonist-induced Ca2+ release was investigated by inhibiting phospholipase C and therefore blocking the synthesis of IP3 by incubating fura-2-loaded platelets with U-73122 (30 AM) for 30 min. In control cells, in the absence of external Ca2+, the physiological agonist thrombin (0.5 U/mL) evoked a typical rise in [Ca2+]c due to the release of Ca2+ from intracellular stores (Fig. 4A). As shown previously [21], thrombin-evoked rise in [Ca2+]c was partially but significantly reduced after pretreatment with U-73122 by 79.6% (Fig. 4A; P < 0.001; n = 6), although the subsequent addition of TG (1 AM) and Iono (50 nM) was able to release Ca2+ from the stores (Fig. 4A). This confirmed that U-73122 was essentially eliminating IP3 production, while leaving the Ca2+ stores intact. By contrast, pretreatment with U-73122 abolished ADP (Fig. 4B) and AVP (Fig. 4C)-induced Ca2+ release from the intracellular stores. These findings indicate that while ADP and AVP-mediated Ca2+ release are entirely IP3dependent, an IP3-independent pathway is also activated by thrombin. Then we have investigated whether the IP3-independent pathway is responsible for Ca2+ release from the TBHQsensitive store. Resting cells or cells incubated for 30 min at 37 -C with 30 AM U-73122 were treated with TBHQ (20 AM) in a Ca2+-free medium and 9 min later stimulated with thrombin. As shown in Fig. 4D, under these conditions, U-73122 was able to abolish thrombin-induced Ca2+ release from the TBHQ-insensitive compartment indicating that this event requires IP3 production.

Fig. 4. Effect of U-73122 on agonist-induced Ca2+ release from the intracellular stores. (A) Platelets suspended in a Ca2+-free medium (100 AM EGTA was added) were incubated for 30 min at 37 -C in the absence or presence of U-73122 (30 AM) and then stimulated with thrombin (0.5 U/ mL). Platelets pretreated with U-73122 were further stimulated with TG (1 AM) plus Iono (50 nM) 5 min later. (B and C) Platelets were incubated for 30 min at 37 -C in the absence or presence of U-73122 (30 AM). Cells were then stimulated with ADP (10 AM; B) or AVP (0.1 AM; C) in a Ca2+-free medium (100 AM EGTA was added). (D) Cells were incubated for 30 min at 37 -C in the absence or presence of U-73122 (30 AM). Platelets were then treated with TBHQ (20 AM) for nine min followed by stimulation with 0.5 U/mL thrombin. Elevations in [Ca2+]c were monitored using the 340 / 380 nm ratio and traces were calibrated in terms of [Ca2+]c. Traces shown are representative of six separate experiments.

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NAADP were therefore explored in permeabilized platelets at a concentration previously shown to be effective in several cell types [23,24]. Treatment of platelets with 100 nM NAADP caused a significant decrease in [Ca2+]s (0.36 T 0.03 arbitrary units; Fig. 5A; P < 0.05; n = 4) that was prevented by previous treatment with 20 AM TBHQ (Fig. 5B), which suggests that Ca2+ release by NAADP occurs entirely from the TBHQ-sensitive store. Treatment of platelets with Iono (100 nM), which accelerates the Ca2+ leakage from both compartments, induced a greater decrease in [Ca2+] s (0.94 T 0.05 arbitrary units; Fig. 5A). To further investigate the role of NAADP in Ca2+ release from the stores and the different effect of platelet agonists, we tested the effect of nimodipine, a L-type Ca2+ channel inhibitor previously shown to block NAADP receptors [24]. As shown in Fig. 6A, treatment of platelets for 3 min with 10 AM nimodipine reduced thrombininduced Ca2+ release by 23% ( P < 0.05; n = 6). The effect of nimodipine was entirely dependent on Ca2+ release from the TBHQ-sensitive store, since no effect of nimodipine was observed on thrombin-stimulated release in cells

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Fig. 6. Nimodipine reduces thrombin-induced Ca2+ release from intracellular stores. (A) Fura-2-loaded human platelets suspended in a Ca2+free medium (100 AM EGTA was added) were treated with nimodipine (10 AM; NIM) or the vehicle (DMSO) as control and 3 min later were stimulated with thrombin (0.5 U/mL). (B) Platelets were incubated for three min in the absence or presence of nimodipine (10 AM) and then treated with TBHQ (20 AM) in a Ca2+-free medium (100 AM EGTA was added). Platelets were stimulated with thrombin (0.5 U/mL) 9 min later. (C) Platelets were either incubated in a Ca2+-free medium (100 AM EGTA was added) for 30 min at 37 -C in the presence of U-73122 (30 AM) followed by treatment with nimodipine (10 AM) for 3 min or left untreated as control. Cells were then stimulated with thrombin (0.5 U/ mL). Elevations in [Ca2+]c were monitored using the 340 / 380 nm ratio and traces were calibrated in terms of [Ca2+]c. Traces shown are representative of six separate experiments.

Time (min) Fig. 5. NAADP releases Ca2+ from TBHQ-sensitive stores in permeabilized platelets. Human platelets loaded with fura-FF were suspended in a Ca2+free modified intracellular medium (100 AM EGTA was added) and permeabilized with streptolysin O (0.5 U/mL). Cells were then treated with NAADP (100 nM) or ionomycin (100 nM) as indicated by the thick arrow (A) or with TBHQ (20 AM) followed by NAADP (100 nM; B). Changes in [Ca2+]s were monitored using the fura-FF 340 / 380 fluorescence ratio as described under ‘‘Material and methods’’. Traces shown are representative of four separate experiments.

where the TBHQ-sensitive stores had been previously depleted by pretreatment with TBHQ (Fig. 6B; P > 0.05; n = 6). Nimodipine had no effect on TBHQ-induced Ca2+ release (Fig. 6B), which indicates that this agent did not modify the accumulation of Ca2+ into the TBHQ-sensitive stores. To further explore whether nimodipine prevents thrombin-evoked Ca2+ mobilization from the TBHQsensitive stores we tested the combined effects of U-

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73122 and nimodipine on thrombin-induced Ca2+ release. As shown in Fig. 6C, treatment of platelets with both inhibitors abolished thrombin-induced Ca2+ mobilization (n = 6). These findings suggest that thrombin induces Ca2+ release from both TBHQ-insensitive and TBHQ-sensitive compartments by IP3 and NAADP dependent pathway, respectively. We have previously shown (Fig. 4B and C) that ADP and AVP-evoked Ca2+ release is entirely dependent on IP3 generation. As expected treatment of human platelets with nimodipine had no effect on ADP or AVP-elicited responses (Fig. 7; P > 0.05; n = 6).

4. Discussion Ca2+ release from and reuptake into intracellular Ca2+ stores play a key role in the activity of most cells,

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Fig. 7. ADP and AVP-induced Ca release from intracellular stores is not affected by nimodipine. Fura-2-loaded human platelets suspended in a Ca2+-free medium (100 AM EGTA was added) were treated with nimodipine (10 AM) or the vehicle (DMSO) as control and 3 min later were stimulated with ADP (10 AM; A) or AVP (0.1 AM; B). Elevations in [Ca2+]c were monitored using the 340 / 380 nm ratio and traces were calibrated in terms of [Ca2+]c. Traces shown are representative of six separate experiments.

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including that of excitation – aggregation coupling in platelets. The present study shows differential Ca2+ mobilization by several physiological agonists, such as thrombin, ADP and AVP, in human platelets. In these cells, two distinct Ca2+ pools have been reported on the basis of several differences in the expression of distinct SERCA isoforms [13], sensitivity to TBHQ [11], Ca2+ leakage rates [25] or even the nature of the stores, so that the TBHQsensitive store has been identified as a lysosomal-related organelle while the TBHQ-insensitive store is likely to be the dense tubular system [26]; however, at present the effect of platelet agonists on Ca2+ release from these compartments has not been explored. Our results indicate that depletion of the TBHQ-sensitive store, which has been reported to accumulate a smaller amount of Ca2+ than those insensitive to TBHQ [12,18], reduces the initial peak [Ca2+]c elevation induced in the absence of external Ca2+ by thrombin but not by ADP or AVP, which reflects that thrombin is the only agonist, of the three tested in this work, that releases Ca2+ from the TBHQsensitive store. This event was confirmed by stimulating platelets simultaneously with the agonists and TBHQ. If an agonist is able to release Ca2+ from TBHQ-sensitive and TBHQ-insensitive pools one would expect no differences between the Ca2+ elevation induced by the agonist alone or in combination with TBHQ. Consistent with this, the initial peak [Ca2+]c elevation induced by thrombin alone or combined with TBHQ was very similar. By contrast, when platelets were stimulated with ADP or AVP we observed an additional increase in [Ca2+]c over that induced by the agonists, which was similar in magnitude to that induced by TBHQ alone. When cells were stimulated with the agonists in combination with TBHQ we also found a detectable change in the rate of decay of [Ca2+]c to basal levels probably due to the inhibitory effect of TBHQ on Ca2+ reuptake by SERCA 3 [11]. To our knowledge this is the first time that an agonist is shown to release Ca2+ from two separate compartments in human platelets. In other cell types, such as pancreatic acinar cells, the physiological agonist cholecystokinin – octapeptide releases Ca2+ from two different Ca2+ pools, one of them activated by IP3 and cyclic ADP ribose (cADPR) and the second induced by the synthesis of NAADP [27,28]. In contrast, other agonists, such as ACh and bombesin, basically mobilize Ca2+ from one pool identified as the endoplasmic reticulum through the generation of IP3 and cADPR, since they are unable to stimulate NAADP synthesis [27]. The putative receptors for NAADP are probably located on lysosome-related intracellular Ca2+ stores distinct from those sensitive to IP3 [22,23,28,29]. In human platelets, where we have recently reported the existence of two distinct Ca2+ stores, the well known dense tubular system and an acidic organelle [26], our results indicate that Ca2+ release stimulated by ADP and AVP is entirely dependent of IP3 synthesis. In contrast, U-73122 was unable to abolish thrombin-induced Ca2+ release under

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conditions that blocked ADP or AVP-evoked responses, and similar results have been previously reported using this and other inhibitors of the IP3 pathway in these cells [15,21]. Therefore, we investigated whether the IP3-insensitive store might correspond to the TBHQ-sensitive pool. We found that U-73122 abolished thrombin-induced Ca2+ release from the TBHQ-insensitive store, therefore, the remaining Ca2+ source for thrombin must be the compartment sensitive to TBHQ. It remains to be elucidated the second messenger involved in the mobilization of Ca2+ from the TBHQsensitive pool. It has been previously shown that cADPR is unlikely to play an important role in Ca2+ release in human platelets [30]; therefore, we tested for the role of NAADP on Ca2+ mobilization from the TBHQ-releasable store. Since NAADP is not cell-permeant we have tested its effect on Ca2+ release in streptolysin O-permeabilized platelets. Our results indicate that NAADP is able to release Ca2+ from intracellular stores, an effect that was prevented by depletion of the TBHQ-sensitive store, suggesting that NAADP releases Ca2+ from that compartment. In addition, we have indirectly tested the role of NAADP in Ca2+ release by using nimodipine, which has been shown to inhibit NAADP receptors in see urchin eggs [23], brain microsomes [31] and the mouse pancreatic h-cell line (MIN6) [24]. Nimodipine did not alter the release of Ca2+ induced by TBHQ, suggesting that this inhibitor neither impair the accumulation of Ca2+ into this compartment nor induce Ca2+ release per se. Here we show that nimodipine prevents thrombininduced Ca2+ release from the TBHQ-sensitive store which is based on several findings: 1) Nimodipine reduced thrombin-induced response by 20%, which is consistent with the estimated relative size of the TBHQ-sensitive and TBHQ-insensitive compartments [18]. 2) Nimodipine has no effect on thrombin-stimulated Ca2+ release from the TBHQ-insensitive pool. 3) In platelets treated with the phospholipase C inhibitor U-73122, nimodipine abolished the remaining Ca2+ mobilization evoked by thrombin. In contrast, nimodipine had no effect on ADP or AVP-induced Ca2+ mobilization, which is consistent with the lack of effect of these agonists on Ca2+ mobilization from the TBHQsensitive pool, and further confirms that the entire effect of these agonists is mediated by IP3 generation. In addition, these observations clearly indicate that nimodipine did not alter the IP3-dependent intracellular signaling or the accumulation of Ca2+ in IP3-releasable compartments. Therefore, our results suggest for the first time a role for NAADP in thrombin-evoked Ca2+ release from the TBHQsensitive compartment. Once platelet stimulation ceases Ca2+ sequestration into the intracellular stores is one of the main mechanism involved in Ca2+ removal from the cytosol. Since thrombin releases Ca2+ from both stores we explored the redistribution of Ca2+ after Ca2+ reuptake into these compartments. Interestingly, when the TBHQ-sensitive stores is emptied by thrombin, redistribution of released Ca2+ into both compartments favors the pool sensitive to TBHQ, so that subsequent depletion of

this store by TBHQ induced an increase in [Ca2+]c that was found to be greater than that observed when this store was not previously depleted, i.e. after platelet stimulation with ADP or AVP or in non-stimulated cells. These findings demonstrate that SERCA 3 activity, the isoform sensitive to TBHQ, is enhanced by a low free Ca2+ concentration into the store lumen. In agreement with this, regulation of SERCA activity by the intraluminal Ca2+ concentration has been previously demonstrated in pancreatic acinar cells [32]. In addition, our results suggest that SERCA 3 is either more active than SERCA 2b or that its activity is less sensitive to high intraluminal Ca2+ concentrations, so that it can accumulate more Ca2+ into the TBHQ-sensitive store, which is supposed to be smaller in size [18]. Our observations shed new light on the mechanisms involved in agonist-induced Ca2+ mobilization in human platelets. The data presented are consistent with the existence of two distinct Ca2+ stores in these cells [12,18,26,33], where thrombin releases Ca2+ from both pools, the TBHQinsensitive dense tubular system and the TBHQ-sensitive acidic store [26], which is likely mediated by IP3 and NAADP, respectively. On the other hand, ADP and AVP only mobilize Ca2+ from the IP3-releasable TBHQ-insensitive dense tubular system [26]. The functional relevance of these differences remains unclear but it might provide an explanation for the distinct functional effects that these agonists induce in human platelets upon stimulation.

Acknowledgements We thank Mercedes Go´mez Bla´zquez for her technical assistance. This work was supported by Consejerı´a de Educacio´n, Ciencia y Tecnologı´a-Junta de Extremadura (2PR04A009), DGI-MEC Grant BFU2004-00165 and Consejerı´a de Sanidad y Consumo-Junta de Extremadura (SCSS0405). P.C.R. is supported by a DGESIC fellowship (BFI2001-0624). J.J.L. held fellowship from the Valhondo Calaff Foundation.

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