Protein kinase C activates non-capacitative calcium entry in human platelets

Abstract

1. In many non-excitable cells Ca2+ influx is mainly controlled by the filling state of the intracellular Ca2+ stores. It has been suggested that this store-mediated or capacitative Ca2+ entry is brought about by a physical and reversible coupling of the endoplasmic reticulum with the plasma membrane. Here we provide evidence for an additional, non-capacitative Ca2+ entry mechanism in human platelets. 2. Changes in cytosolic Ca2+ and Sr2+ were measured in human platelets loaded with the fluorescent indicator fura-2. 3. Depletion of the internal Ca2+ stores with thapsigargin plus a low concentration of ionomycin stimulated store-mediated cation entry, as demonstrated upon Ca2+ or Sr2+ addition. Subsequent treatment with thrombin stimulated further divalent cation entry in a concentration-dependent manner. 4. Direct activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol also stimulated divalent cation entry, without evoking the release of Ca2+ from intracellular stores. Cation entry evoked by thrombin or activators of PKC was abolished by the PKC inhibitor Ro-31-8220. 5. Unlike store-mediated Ca2+ entry, jasplakinolide, which reorganises actin filaments into a tight cortical layer adjacent to the plasma membrane, did not inhibit divalent cation influx evoked by thrombin when applied after Ca2+ store depletion, or by activators of PKC. Thrombin also activated Ca2+ entry in platelets in which the release from intracellular stores and store-mediated Ca2+ entry were blocked by xestospongin C. 6. These results indicate that the non-capacitative divalent cation entry pathway is regulated independently of store-mediated entry and does not require coupling of the endoplasmic reticulum and the plasma membrane. These results support the existence of a mechanism for receptor-evoked Ca2+ entry in human platelets that is independent of Ca2+ store depletion. This Ca2+ entry mechanism may be activated by occupation of G-protein-coupled receptors, which activate PKC, or by direct activation of PKC, thus generating non-capacitative Ca2+ entry alongside that evoked following the release of Ca2+ from the intracellular stores.

Figure 1. Capacitative and non-capacitative cation entry pathways in human platelets

A, fura-2-loaded human platelets were stimulated with TG (1 μm) and IONO (50 nM) in Ca²⁺-free HBS (100 μm EGTA added) and 5 min later thrombin (10 U ml⁻¹) was added (n = 5). B, cells were stimulated with TG and IONO or the vehicles alone (Control) in Ca²⁺-free HBS (100 μm EGTA added) and 3.5 min later SrCl₂ (300 μm) was added (n = 7). C, cells were stimulated with TG and IONO in Ca²⁺-free HBS (100 μm EGTA added); 3.5 min later SrCl₂ (300 μm) was added to initiate Sr²⁺ entry followed by thrombin (1 or 10 U ml⁻¹; n = 7). D, cells were stimulated with TG and IONO in Ca²⁺-free HBS (100 μm EGTA added); 3.5 min later CaCl₂ (300 μm) was added to initiate Ca²⁺ entry followed by thrombin (10 U ml⁻¹; n = 6).

Figure 2. Effect of P_2x1 receptor desensitisation on thrombin-evoked non-capacitative Sr²⁺ entry in human platelets

Fura-2-loaded human platelets were resuspended in HBS containing 1 mm CaCl₂ (A) or in Ca²⁺-free HBS (100 μm EGTA added; B) and then α,β-methylene ATP (α,β-MeATP; 10 μm) was added. A second addition of 10 μmα,β-methylene ATP in the presence of 1 mm external Ca²⁺ demonstrates desensitisation of the P_{2 x1} receptor (n = 4; A). Addition of 1 mm CaCl₂ after 10 μmα,β-methylene ATP and a second addition of 10 μmα,β-methylene ATP were without effect, indicating that desensitisation occurs independently of Ca²⁺ entry (n = 4; B). C and D, platelets were treated in Ca²⁺-free HBS (100 μm EGTA added) with α,β-methylene ATP (10 μm; D) or the vehicle (HBS; C) prior to stimulation with TG (1 μm) and IONO (50 nM); 3.5 min later SrCl₂ (300 μm) was added followed by thrombin (10 U ml⁻¹; n = 4).

Figure 3. Effect of PMA on Ca²⁺ and Sr²⁺ entry in human platelets

A and B, platelets were treated with 1 μm PMA in HBS containing 1 mm Ca²⁺ (A) or 1 mm Sr²⁺ (B; n = 6). The same experiments were performed in the absence of external Ca²⁺ (A) or Sr²⁺ (B; n = 6). C and D, cells were treated with TG (200 nM) in Ca²⁺-free HBS (100 μm EGTA added) and 3 min later SrCl₂ (final concentration, 300 μm) was added followed by PMA (1 μm, D) or the vehicle (DMSO) alone (C; n = 6).

Figure 4. Time-dependent effect of PKC activation on cation entry

A, platelets were preincubated at 37 °C for 5 or 30 min in the absence (Control) or presence of PMA (1 μm). Cells were then treated with TG (200 nM) and 3 min later SrCl₂ (final concentration, 300 μm) was added (n = 6). B, platelets were preincubated at 37 °C for 5 or 30 min in the absence (Control) or presence of OAG (100 μm). Cells were then treated with TG (200 nM) and 3 min later SrCl₂ (final concentration, 300 μm) was added (n = 6).

Figure 5. Desensitisation of PKC inhibits non-capacitative Sr²⁺ entry in human platelets

Cells were pretreated for 30 min at 37 °C with 1 μm PMA (B) or the vehicle (DMSO) alone (Control; A). The platelets were then stimulated with TG (1 μm) and IONO (50 nM) in Ca²⁺-free HBS (100 μm EGTA added) and 3.5 min later SrCl₂ (300 μm) was added followed by thrombin (10 U ml⁻¹; n = 6).

Figure 6. Effect of PKC inhibition on non-capacitative Sr²⁺ entry in human platelets

A, cells were pretreated for 5 min at 37 °C with 3 μm Ro-31-8220 or the vehicle (DMSO) alone (Control). The platelets were then treated with 1 μm PMA in HBS containing 1 mm Sr²⁺(n = 6). B, cells were pretreated for 5 min at 37 °C with 3 μm Ro-31-8220 or the vehicle (Control). The platelets were then stimulated with TG (1 μm) and IONO (50 nM) in Ca²⁺-free HBS (100 μm EGTA added) and 3.5 min later SrCl₂ (300 μm) was added followed by thrombin (10 U ml⁻¹; n = 6).

Figure 7. Effect of jasplakinolide on non-capacitative cation entry in human platelets

A-D, platelets were pretreated for 30 min at 37 °C with 10 μm JP (C and D; n = 6) or the vehicle (ethanol; A and B; n = 6). Cells were stimulated with PMA (1 μm) in HBS containing 1 mm Sr²⁺. B and D show, respectively, confocal images of F-actin labelled with FITC-conjugated phalloidin in control or JP-treated human platelets. Scale bars represent 1 μm. E-H, cells were pretreated at 37 °C with 10 μm JP (F and H) or the vehicle (ethanol; E and G) for 30 min. The platelets were then treated with TG (1 μm) and IONO (50 nM) in Ca²⁺-free HBS (100 μm EGTA added) and 3 min later SrCl₂ (final concentration, 300 μm; E and F) or CaCl₂ (final concentration 300 μm; G and H) was added followed by thrombin (10 U ml⁻¹; n = 7).

Figure 8. Thrombin-induced non-capacitative Ca²⁺ entry is independent of store depletion

Fura-2-loaded human platelets were pretreated for 30 min at 37 °C with 20 μm Xest C (B-D) or the vehicle (DMSO) alone (A). In A and B, cells were then stimulated with TG (1 μm) and IONO (50 nM) in Ca²⁺-free HBS (100 μm EGTA added) and 3 min later CaCl₂ (final concentration, 200 μm) was added to initiate Ca²⁺ entry (n = 4). C, cells were stimulated with 10 U ml⁻¹ thrombin in Ca²⁺-free HBS (100 μm EGTA added) and 3.5 min later TG (1 μm) and IONO (50 nM) were added (n = 5). D, cells were stimulated with 10 U ml⁻¹ thrombin in the presence of 1 mm external Ca²⁺ (n = 6).