Convulxin induces platelet shape change through myosin light chain kinase and Rho kinase Silvia Riondino, Pier P. Gazzaniga and Fabio M. Pulcinelli Department of Experimental Medicine and Pathology, Universita ´ La Sapienza, Rome, Italy Once platelets are activated, the first event to occur is a rapid change in shape, associated with Ca 2+ /calmodulin- dependent myosin light chain (MLC) phosphorylation and with Rho kinase activation. The purpose of this study was to investigate which is the biochemical pathway that leads to platelet shape change in response to convulxin, a selective GpVI activator, and to verify whether MLC phosphoryla- tion is essential for this process. The inhibition of the Ca 2+ - dependent pathway by means of the Ca 2+ chelator BAPTA, the Ca 2+ /calmodulin inhibitor W-7 or the cAMP enhancing drug iloprost reduced about 50% of platelet shape change in response to convulxin. The treatment with either the Rho kinase inhibitors Y27632 or HA 1077 had no effect on platelet shape change induced by convulxin. When both Ca 2+ /calmodulin-dependent and Rho kinase-dependent pathways were concomitantly inhibited by the combined use of Y27632 plus BAPTA, W-7 or iloprost, platelet shape change was completely abolished. Our findings suggest that convulxin-induced platelet shape change occurs via both pathways, the Ca 2+ /calmodulin-dependent, which appears to be more important, and the Rho kinase-dependent one. The pattern of MLC phosphorylation was not modified by Rho kinase inhibitors. Conversely, the inhibition of the Ca 2+ -dependent pathway caused a strong reduction of MLC phosphorylation in BAPTA-treated platelets, and a total inhibition in W-7 or iloprost-treated platelets. Our results demonstrate that following Rho kinase-dependent pathway platelet shape change can occur without the involvement of MLC phosphorylation. Keywords: convulxin; cyclic AMP; myosin light chain kin- ase; platelet shape change; Rho kinase. One of the first events to follow platelet functional response to various stimuli is the rearrangement of cytoskeletal proteins, such as actin and myosin, with the consequent change from the discoid to the spheroid shape and the production of pseudopodia [1]. It has been demonstrated that a crucial event in triggering shape change is myosin light chain (MLC) phosphorylation [2]. The signal origin- ating from agonist-induced activation can lead to MLC phosphorylation through two distinct pathways. One is the Ca 2+ /calmodulin-dependent pathway, subsequent to Ca 2+ mobilization and mediated by MLC kinase [3], and the other is Ca 2+ -independent, subsequent to the small GTP- binding protein RhoA activation, and mediated by Rho kinase [4–6]. The Ca 2+ /calmodulin-dependent pathway which leads to MLC phosphorylation depends upon the stimulation of a G q -coupled receptor and phospholipase C (PLC) activation. This datum has also been supported by experiments on G aq -deficient mice platelets [7]. On the other hand, the Ca 2+ -independent pathway follows the stimula- tion of a G 12 /G 13 coupled receptor [4]. The second messenger cAMP also exerts an efficacious action on cytoskeletal rearrangements, by inhibiting actin assembly and focal adhesion in many cell types [8,9] through the decreased phosphorylation of various proteins [10,11] including MLC [12]. In a previous study of our group, it was found that it was possible to by-pass the inhibitory effect of cAMP on the multistep cascade of biochemical and morphological events that result in platelet aggregation, provided a G q -coupled receptor and a G i -coupled receptor were activated [12]. In the same study it was demonstrated that the activation of PLC was sufficient to elicit full aggregation, in spite of elevated intracellular concentrations of cAMP, in the presence of an activated G i protein. The activation of a PLC isoform, PLC c2, might be achieved using the snake venom toxin convulxin. Convulxin activates platelets through the collagen receptor complex glycoprotein VI (GpVI)/Fc receptor c-chain, leading to tyrosine phosphorylation and activation of syk and PLCc2 [13,14]. Following activation of GpVI by convulxin, several adapter molecules are tyrosine phosphorylated and/or recruited into intracellular signalling complexes; many of the adapter proteins associate with the transmembrane adapter LAT [15]. Francischetti and his coworkers demonstrated that cAMP did not inhibit the early onset of convulxin-induced tyrosine-phosphorylation of PLCc2, which also occurs in the presence of the aIIb/b3 antagonist, peptide RGDS, or actin polymerization inhibitors, such as cytochalasin D [16]. The purpose of the present study was to investigate the biochemical pathways followed by convulxin in inducing Correspondence to P. Gazzaniga, Department of Experimental Medicine and Pathology, Universita ´ La Sapienza, Viale Regina Elena 324, 00161 Rome, Italy. Fax: + 39 064452955, Tel.: + 39 064454820, E-mail: pierpaolo.gazzaniga@uniroma1.it Abbreviations: A3P5P, adenosine 3¢-phosphate 5¢-phosphate; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid; CP/CPK, creatine phosphate/creatine kinase; HA, 1077 (1-(5-isoquinolinesulfonyl)homopiperazine); MLC, myosin light chain; MLCK, myosin light chain kinase; PLC, phospholipase C; W-7, N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide. (Received 3 January 2002, revised 8 October 2002, accepted 11 October 2002) Eur. J. Biochem. 269, 5878–5884 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03305.x platelet shape change. In particular, the roles of Rho kinase and of MLC kinase have been studied by means of specific inhibitors in order to asses whether the Rho/Rho kinase pathway is an additional route in mediating platelet shape change in response to convulxin, independently of the Ca 2+ -dependent pathway. MATERIALS AND METHODS Platelet preparation Blood samples were obtained from informed healthy volunteers who denied having taken any drugs in the two weeks before blood sampling and collected in citric acid/ citrate/dextrose-containing tubes [17]. Platelet rich plasma was obtained after centrifugation (180 g for 15 min) and further centrifuged (800 g for 20 min) to concentrate the platelets (6 · 10 8 plateletsÆmL )1 ). The concentrated platelets were incubated for 15 min at 37 °Cwith1m M aspirin (Sigma Chemicals Co., St. Louis, MO, USA) then washed twice in Tyrode’s buffer (137 m M NaCl, 2.68 m M KCl, 0.42 m M NaH 2 PO 4 ,1.7m M MgCl 2 ) containing 10 m M Hepes (pH 6.5) and resuspended in Tyrode’s buffer containing 0.2% (w/v) albumin (bovine serum fraction V-BSA), 0.1% (w/v) glucose and 10 m M Hepes (pH 7.35) (all from Sigma Chemicals). To enhance cAMP levels, platelets were treated with iloprost (3 l M for 2 min at 37 °C) (generous gift of Italfarmaco S.p.a., Italy). This platelet preparation was further incubated with the ADP scavenger system creatine phosphate/creatine kinase (CP/CPK) (4 m M and 10 UÆmL )1 , respectively, Sigma Chemicals) before the addi- tion of the agonist. Samples were treated with N-(6-amino- hexyl)-5-chloro-1-naphtalenesulfonamide (W-7) (90 l M )to inhibit the Ca 2+ /calmodulin pathway [18,19] (Sigma Chem- icals). HA 1077(1-(5-isoquinolinesulfonyl)homopiperazine) (10 l M )[20,21](BioMolRes.Laboratory,Inc.,PA,USA) and Y27632 (10–30 l M ) (Calbiochem-Novabiochem San Diego, CA, USA) were used as Rho-kinase inhibitors. Platelet stimulation was achieved by means of convulxin (5 ngÆmL )1 ) (Latoxan, Rosans, France), purified according to Polgar et al. [13]. Platelet shape change In vitro platelet shape change was evaluated in a four sample PACKS-4 (Helena Laboratories, Beaumont, TX, USA) aggregometer using siliconized glass cuvettes at 37 °C under continuous stirring. Once the full function of the platelet preparation was tested in response to convulxin (5 ngÆmL )1 ), in order to prevent platelet aggregation and allow shape change to occur, platelets were pretreated with RGDS (120 lgÆmL )1 ) which inhibits fibrinogen binding to the integrin a IIb b 3 . The magnitude of the shape change was detected by measuring the maximum decrease in light transmission and the slope value. The last parameter was obtained from the tangent to the curve and was measured in millimetres per minute. Changes in intracellular calcium mobilization The fluorescent changes in intracellular calcium mobiliza- tion were studied in Fura-2 (Molecular Probes, Eugene, OR, USA) loaded platelets (3 l M at 37 °C for 30 min) and monitored in a Kontron SFM 25 spectrofluorimeter thermostatically regulated at 37 °C. Excitation and emission wavelengths were 340 n M and 510 n M , respectively. Intra- cellular free calcium concentration was calibrated according to Grynkiewicz [22]. Preparation of BAPTA-loaded platelets The concentrated platelets were incubated for 30 min at 37 °Cwith1m M aspirin, 100 l M 1,2-bis(2-aminophen- oxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester (BAPTA-AM) (Molecular Probes), then washed by centri- fugation. The platelet preparation was further treated with CP/CPK (4 m M and 10 UÆmL )1 , respectively) before the addition of the agonist. If the Dn M of Ca 2+ obtained in response to agonist stimulation was higher than 10, the platelet suspension was not used. Myosin light chain phosphorylation MLC phosphorylation was analysed according to Daniel et al. [23]. Briefly, aspirinated platelets were resuspended in Tyrode’s buffer at a concentration of 2 · 10 9 cellsÆmL )1 . Aliquots of 500 lL were stirred at 37 °C in an aggrego- meter. After30 s from convulxin stimulation (5 ngÆmL )1 )the reaction was stopped by adding 6.6 M HClO 4 ; the resulting precipitate was kept for 45 min in ice. The pellets were centrifuged at 10 000 g for 2 min and washed twice with ice- cold deionized water. The proteins were further centrifuged at 10 000 g for 2 min and the resulting pellets were dissolved in 50 lL of sample buffer containing 8 M urea, 20 m M Tris, (pH 8.6), 122 m M glycine, 5 m M dithiothreitol to which 0.1% (w/v) bromophenol blue dye was added. The suspen- ded pellet was sonicated in a PBI Briansonic 220 sonication bath for 30 min. Gel electrophoresis was performed in a 10% (w/v) polyacrylamide-urea minigel apparatus (Hoefer Scientific Instruments, San Francisco, CA, UA). The running buffer used in the top chamber was composed of 20 m M Tris and 122 m M glycine at pH 8.6 containing 4 m M urea. The electrophoresis was stopped 1 h after the blue marker had migrated off the end of the gel. Gels were stained in 0.25% (w/v) Coomassie Brilliant Blue (Sigma), destained and dried in an SE 1200 Easy Breeze Air Gel Dryer (Hoefer) apparatus. The gels were then scanned using a Gel Doc 2000 (Bio-Rad Laboratories, Hercules, CA, USA) scanner and the optical densities corresponding to the phosphorylated form of MLC were analysed using the program NIH IMAGE 1.62 and reported as mean grey values. The results were expressed as the percentage of maximal MLC phosphorylation induced by convulxin. To confirm the identity of the detected band, further sample proteins were transferred to Immobilion-P (Milli- pore) membranes and identified with anti-MLC mono- clonal Ig (Sigma Chemicals), followed by horseradish peroxidase-conjugated secondary antibody and visualized with ECL chemiluminescence reaction reagent (Amersham) and Kodak X-ray film (X-OMAT AR). RESULTS To investigate the correlation between the increase in intracellular Ca 2+ concentration and shape change in Ó FEBS 2002 Convulxin-induced platelet shape change (Eur. J. Biochem. 269) 5879 convulxin-induced platelet activation, we studied platelet shape change and MLC phosphorylation in experimental conditions in which either Ca 2+ /calmodulin or Rho-kinase pathways were inhibited. The increase in the cytosolic Ca 2+ concentration, which occurs normally after convulxin (5 ngÆmL )1 ) stimulation (743.6 ± 192.2 Dn M ), was strongly inhibited in platelets treated with the cAMP-enhancing drug iloprost (3 l M ) (Fig. 1). Table 1 summarizes the variation in shape change induced by 5 ngÆmL )1 convulxin, indicating the magnitude and slope of aspirinated platelets in the presence of Ca 2+ / calmodulin or Rho-kinase inhibitors, that are also repre- sentatively shown in Fig. 2. The results shown in Fig. 2A demonstrate that when the two pathways were separately inhibited by treating the platelets with either the calmodulin inhibitor W-7 (90 l M ) or with the Rho kinase inhibitors HA 1077 (10 l M ) or Y27632 (30 l M ), we observed only a slight reduction in the rate of shape change (more pronounced when the Ca 2+ -dependent pathway was inhib- ited) as compared to control, untreated, convulxin-stimula- ted platelets. Then, in order to minimize the cytosolic concentration of calcium, we treated our platelet prepar- ation with 5,5¢-dimethyl BAPTA (Fig. 2B), and studied both the magnitude and the slope of the shape change. Under these conditions we found that both were slightly decreased, with a delay in the initiation of the process. A more pronounced reduction was observed in the presence of high concentrations of iloprost (3 l M ) (Fig. 2C). A similar degree of reduction was observed after the simultaneous treatment with calmodulin inhibitors and iloprost, suggest- ing that these agents were acting on a common pathway. However, when iloprost was added to 5,5¢-dimethyl BAPTA-loaded platelets, both the extent and the rate of shape change were significantly inhibited (Fig. 2C). A complete inhibition was obtained when both pathways, Ca 2+ -dependent and Rho-dependent, were simultaneously blocked by adding the calmodulin inhibitor W-7 or iloprost or when the increase in cytosolic Ca 2+ concentration was minimized by BAPTA, to HA 1077- or Y27632-treated platelets. Interestingly, a complete inhibition of platelet shape change was achieved by notably lowering the concentrations of HA 1077 or Y-27632 when used in combination with W-7 (Table 1). To avoid any possible interference with platelet shape change caused by released ADP on its receptor P2Y 1 ,we incubated our CP/CPK-treated platelet preparation with Fig. 1. Changes in intracellular calcium concentration in 5 ngÆmL )1 convulxin-stimulated aspirinated platelets. Stimulation in the (a) absence and (b) presence of 3 l M iloprost. The figure is representative of three experiments performed. Table 1. Effects of different inhibitory treatments on platelet shape change in response to 5 ngÆmL )1 convulxin stimulation. Treatment Magnitude Slope None 14.07 ± 3.34 6.74 ± 2.11 + HA 1077 (10 l M ) 13.36 ± 2.87 5.59 ± 2.81 + Y-27632 (30 l M ) 13.96 ± 2.31 6.09 ± 2.77 + W-7 (90 l M ) 5.87 ± 3.34* 3.38 ± 1.22** + W-7 (30 l M ) + HA 1077 (5 l M ) absent not measurable + W-7 (30 l M ) + Y-27632 (10 l M ) absent not measurable BAPTA (100 l M ) 6.75 ± 2.51* 3.21 ± 1.18** + HA 1077 (10 l M ) absent not measurable + Y-27632 (30 l M ) absent not measurable + W-7 (90 l M ) 5.99 ± 2.31* 3.76 ± 2.17** + Iloprost (3 l M ) 4.29 ± 0.76** 2.01 ± 1.14** Iloprost (3 l M ) 4.48 ± 0.96** 2.99 ± 1.25** + HA 1077 (10 l M ) absent not measurable + Y-27632 (30 l M ) absent not measurable + W-7 (90 l M ) 4.24 ± 1.18** 2.83 ± 1.75** *P < 0.05; **P < 0.01. 5880 S. Riondino et al.(Eur. J. Biochem. 269) Ó FEBS 2002 the selective P2Y 1 inhibitor adenosine 3¢-phosphate 5¢-phosphate (A3P5P) (100 l M for 1 min at 37 °C) and obtained the same results as those observed after CP/CPK treatment alone. This result suggests that the ADP secreted is promptly scavenged by CP/CPK and that the residual ADP is at too low a concentration to activate the P2Y 1 -receptor coupled pathway. Because MLC phosphorylation has been suggested to be involved in early processes during platelet activation [24] we measured the appearance of a 20-kDa band, indicating MLC phosphorylation consequent to the activation of the two pathways, Ca 2+ -dependent and Ca 2+ -independent, and determined the action exerted by the different inhibi- tors. Rho kinase inhibition by HA 1077 or Y27632 did not significantly modify the pattern of MLC phosphorylation after convulxin stimulation (63.0 ± 10.9 for HA 1077 and 66.33 ± 12.6 for Y-27632 vs. 78.6 ± 11.4 mean grey value), equal to 19.8% and 15% reduction, respectively (Fig. 3A). The identity of the 20 kDa band detected was confirmed by immunoblot analysis by means of an anti-MLC mono- clonal Ig (data not shown). Inhibition of the increase in cytosolic Ca 2+ by BAPTA (Fig. 3B) reduced by about 40% the maximum level of MLC phosphorylation (45.7 ± 9.6 mean grey value vs. 78.6 ± 11.4) induced by convulxin. It is worth noting that, since BAPTA treatment might delay the time course of MLC phosphorylation [5], we performed experiments at 60, 120, 150 and 180 s, but we did not observe any further MLC phosphorylation (data not shown). Inhibition of calmodulin by W-7 (90 l M ) completely abolished MLC phosphoryla- tion (Fig. 3A), while a certain degree of platelet shape change was still preserved although significantly reduced (Table 1). The increase in intracellular cAMP levels by iloprost completely inhibited MLC phosphorylation (4.2 ± 0.7 mean grey value) (Fig. 3C). When both the Ca 2+ -dependent and Ca 2+ -independent pathways were inhibited by the combined treatment with HA 1077 or Y27632 and W-7 or BAPTA, we did not observe any phosphorylation of MLC. DISCUSSION The initial functional response of platelets to stimuli is represented by shape change, during which phase platelets are subject to cytoskeletal reorganization consequent to the phosphorylation of several contractile proteins. It has already been demonstrated that such a phenomenon can occur both in the presence and in the absence of an increase of cytosolic Ca 2+ concentration in response to thrombin, ionomycin, the stable prostaglandin endoperoxide analogue U44069 and the thrombin receptor activating peptide [25–27]. Other authors [4] observed that the thromboxane A2 synthetic analogue, U46619, was not able to induce a detectable elevation of intracellular Ca 2+ concentration in the absence of G aq and that Rho kinase inhibitor Y-27632 and C3 exoenzyme inhibited the U46619-induced MLC phosphorylation in G aq -deficient mouse platelets. These observations led them to conclude that a Rho/Rho kinase pathway regulating MLC phosphorylation operated in platelets and that Rho kinase mediates the Ca 2+ -independ- ent pathway. The dichotomous regulation of MLC phos- phorylation and shape change by Rho kinase and calcium in human platelets was demonstrated by other studies carried out with several agonists, different from convulxin [5,6,28]. Elevated cAMP and the consequent activation of protein kinase A affects cell morphology, inducing the loss of actin stress fibres and their detachment from the underlying Fig. 2. Platelet shape change in response to 5ngÆmL )1 convulxin. (A) Shape change in the presence of the calmodulin inhibitor W-7 (90 l M ) or Rho kinase inhibitors HA 1077 (10 l M ) and Y-27632 (30 l M )inaspirinated, RGDS-treated, control platelets. The response to convulxin stimulation in the presence of the cited inhibitors of platelets loaded with 100 l M BAPTA for 30 min at 37 °C(B)ortreatedwith3l M iloprost for 2minat37°C (C). The figure is representative of four experiments performed. Ó FEBS 2002 Convulxin-induced platelet shape change (Eur. J. Biochem. 269) 5881 substratum in many cell types [9,29]. Protein kinase A, once activated, phosphorylates myosin light chain kinase (MLCK), thus inhibiting its activity and causing a decrease in MLC phosphorylation [8]. As regards the inhibition exerted by cAMP on the biochemical pathway leading to platelet shape change following convulxin activation, the results shown in this paper demonstrate that it is possible to evoke platelet shape change even in the presence of elevated intracellular concentrations of cAMP. This observation leads us to hypothesize that the inhibitory action of cAMP is not exerted at the level of Rho kinase pathway. The conclusion that shape change responses were not due to ADP release arises from two observations. First, the degranulation process was dramatically reduced in convul- xin-stimulated platelets treated with iloprost. This showed a reduction of about 86% of the intracellular ATP as compared to convulxin-stimulated platelets untreated with iloprost, as already demonstrated in a previous study from our group [12]. Second, in all preparations platelets were treated with the ADP scavenger system CP/CPK to ensure that even traces of ADP released could not interfere with the experiments. Furthermore, experiments employing the inhibitor of the ADP receptor P2Y 1 (the only receptor involved in platelet shape change induced by ADP [28,30]), A3P5P, demon- strated no additional inhibition as compared to CP/CPK alone (data not shown). Iloprost treatment strongly affected convulxin-induced MLC phosphorylation, only partially acting on the Ca 2+ - dependent pathway which, in fact, was inhibited as dem- onstrated by fluorimetric studies on intracellular Ca 2+ mobilization. This result also demonstrates that in response to convulxin Ca 2+ mobilization can be inhibited by elevating the levels of intracellular cAMP, as shown in studies using different agonists [31]. However, the inhibition of Ca 2+ mobilization alone is not sufficient to cause a complete inhibition of MLC phosphorylation, as demon- strated by the experiments with BAPTA, which only diminished MLC phosphorylation by about 50%. The enhanced capability of cAMP of inhibiting MLC phos- phorylation can be explained by the direct action of cAMP on MLCK [8]. Antagonizing the Ca 2+ -dependent pathway at different stages by means of the Ca 2+ /calmodulin inhibitor, W-7, or with the cytosolic Ca 2+ chelator BAPTA, we observed a good degree of platelet shape change in response to convulxin. This indicates that the inhibition exerted by these inhibitors on the Ca 2+ -dependent pathway did not affect the Rho kinase-dependent one. In fact, while the inhibition of Rho kinase by means of the inhibitors HA 1077 or Y-27632 was without any effect on platelet shape change and MLC phosphorylation induced by convulxin, a complete inhibition of platelet shape change was achieved only when both pathways were concomitantly inhibited, combining HA 1077 or Y-27632 treatment with iloprost, W-7 or BAPTA treatment. These results suggest that both pathways are involved in platelet shape change induced by convulxin and that, since Rho kinase inhibitors are less powerful in interfering with such a process, the Ca 2+ -dependent one is more effective. The fact that BAPTA treatment reduced convulxin- induced MLC phosphorylation, while the rate of inhibition of platelet shape change was similar to that observed after W-7 or iloprost treatment, might be explained by the demonstration that a weak activation of MLC phosphory- lation is not sufficient to elicit shape change [6]. Bauer and coworkers [5] suggested that the GpVI- specific agonist, collagen-related peptide, causes shape change by a mechanism that is entirely dependent on calcium, since it is not affected by the Rho kinase inhibitor Y-27632. Their conclusion delineated the exist- ence of two distinct pathways of myosin phosphorylation Fig. 3. Myosin light chain phosphorylation in response to aspirin-trea- ted, convulxin stimulated (5 ng mL )1 , for 30 s at 37 °C) platelets. (A) Phosphorylation in the presence of calmodulin inhibitor W-7 (90 l M ) or Rho kinase inhibitors HA 1077 (10 l M ) and Y-27632 (30 l M ). The effects of the same inhibitors on (B) BAPTA-treated (100 l M for 30 min at 37 °C) or (C) Iloprost-treated (3 l M for 2 min at 37 °C) platelets. The quantitative comparison of the effects of the cited inhibitors is reported. Results are shown as mean ± SEM of four experiments. The level of MLC phosphorylation of resting platelets was subtracted and the results expressed as percent of the maximal MLC phosphorylation induced by convulxin alone (78.6 ± 11.4 mean grey value) (A). 5882 S. Riondino et al.(Eur. J. Biochem. 269) Ó FEBS 2002 and platelet shape change, one mediated by Rho kinase and the other by the Ca 2+ -dependent activation of MLC kinase. These two pathways could be activated independ- ently of each other, depending on the type and concen- tration of the agonist used. The Rho kinase-dependent pathway was stimulated by the activation of thrombin and thromboxane receptors as opposed to the Ca 2+ -/ MLC kinase-dependent pathway that was stimulated by ADP and collagen receptor activation. Like collagen, convulxin has also been described as a GpVI agonist [13]. The different behaviour of this agonist that emerged in our study might be explained by the fact that it is more efficacious than collagen-related peptide [15]. Taken together our findings suggest that convulxin is able to cause shape change by activating both pathways, the Ca 2+ -dependent and Rho/Rho kinase-dependent. The former appears to be more important, while the latter seems to represent an additional route in mediating platelet shape change and becomes preferentially involved in response to stimuli that do not produce any increase in cytosolic Ca 2+ concentration, or when the Ca 2+ /calmo- dulin-dependent pathway is not operating (Fig. 4). 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