thapsigargin and edelfosine

The role of the tyrosine kinase pp60src in PAR-1-dependent Ca2+ entry was investigated in human platelets. pp60src plays a role in thapsigargin (TG)-evoked store-operated Ca2+ entry (SOCE), which is thought to be a major component of thrombin-evoked Ca2+ entry.. pp60src tyr416 phosphorylation was used to assess pp60src activation. Fura-2-loaded platelets were used to monitor intracellular Ca2+ concentration ([Ca2+]i).. Activation of PAR-1 with the specific agonist SFLLRN increased pp60src activation within 10 s. This required phospholipase C (PLC) activity, Ca2+ release and a rise in intracellular Ca2+. PP2, an inhibitor of Src-family tyrosine kinases, inhibited SFLLRN-evoked Ca2+ entry, but also inhibited Ca2+ release and the extrusion of Ca2+ by the plasma membrane Ca2+ ATPase. Actin polymerization and conventional protein kinase C (cPKC) activity were required for TG- and SFLLRN-evoked pp60src activation. Although Gö6976, an inhibitor of cPKCs, inhibited TG-evoked SOCE, it had little effect on SFLLRN- or thrombin-evoked Ca2+ entry.. These data indicate that stimulation of PAR-1 leads to activation of pp60src in human platelets, through PLC and cPKC activation, Ca2+ release and actin polymerization. However, as PKC and actin polymerization are not needed for SFLLRN-evoked Ca2+ entry, we suggest that pp60src is also not required. The apparent inhibition of SFLLRN-evoked Ca2+ entry by PP2 is likely to be secondary to reduced Ca2+ release. These data argue against a contribution of this SOCE pathway to PAR-1-dependent Ca2+ entry.

Topics: Actins; Blood Platelets; Calcium; Calcium Signaling; Carbazoles; Chelating Agents; Cytochalasin D; Cytoskeleton; Egtazic Acid; Enzyme Activation; Enzyme Inhibitors; Humans; In Vitro Techniques; Indoles; Peptide Fragments; Phospholipid Ethers; Phosphorylation; Plasma Membrane Calcium-Transporting ATPases; Protein Kinase C; Proto-Oncogene Proteins pp60(c-src); Pyrimidines; Receptor, PAR-1; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Time Factors; Type C Phospholipases

The effect of the ether lipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3) on the intracellular free Ca2+ concentration ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was studied using fura-2 as the Ca2+ probe. In Ca2+ medium, ET-18-OCH3 induced a significant rise in [Ca2+]i at concentrations between 10-100 microM with a concentration-dependent delay of 45-175 s. The [Ca2+]i signal was composed of a gradual rise and a sustained plateau. In Ca2+-free medium, ET-18-OCH3 (10-100 microM) induced a Ca2+ release from internal Ca2+ stores with a concentration-dependent delay of 45-175 s. This discharge of internal Ca2+ triggered capacitative Ca2+ entry in a concentration-dependent manner. This capacitative Ca2+ entry was not inhibited by econazole (25 microM), 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF96365; 50 microM), nifedipine (10 microM), verapamil (10 microM), diltiazem (10 microM) and cadmium (0.5 microM). Methyl 2-(phenylthio)ethyl-1,4-dihydro-2,4,6-trimethylpyridine-3,5-dicarboxylat e (PCA-4248), a platelet-activating factor (PAF) receptor antagonist, inhibited 25 microM ET-18-OCH3-induced [Ca2+]i rise in a concentration-dependent manner between 1-20 microM, with 20 microM exerting a complete block. The [Ca2+]i rise induced by ET-18-OCH3 (25 microM) was not altered when the production of inositol 1,4,5-trisphosphate (IP3) was suppressed by the phospholipase C inhibitor U73122 (2 microM), but was partly inhibited by the phospholipase D inhibitor propranolol (0.1 mM) or the phospholipase A2 inhibitor aristolochic acid (20-40 microM). In Ca2+-free medium, pretreatment with 25 microM ET-18-OCH3 completely depleted the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin-sensitive Ca2+ store. In contrast, pretreatment with thapsigargin abolished 0.1 mM ATP-induced [Ca2+]i rise without altering the ET-18-OCH3-induced [Ca2+]i rise. This suggests that ET-18-OCH3 depleted thapsigargin-sensitive Ca2+ stores and also released Ca2+ from thapsigargin-insensitive stores. The thapsigargin-insensitive stores involve mitochondria because the mitochondria uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM) induced a release of mitochondrial Ca2+ which was abolished by pretreatment with 25 microM ET-18-OCH3. ET-18-OCH3 (25 microM) induced a significant Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength confirming that ET-18-OCH3 induced capacitative Ca2+ entry. La3+

Topics: Adenosine Triphosphate; Animals; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line; Cytosol; Dihydropyridines; Dogs; Dose-Response Relationship, Drug; Enzyme Inhibitors; Estrenes; Kidney; Phosphodiesterase Inhibitors; Phospholipid Ethers; Platelet Membrane Glycoproteins; Pyrrolidinones; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Thapsigargin; Type C Phospholipases; Uncoupling Agents