Protein kinase C-dependent and Ca2+-dependent mechanisms of secretion from streptolysin O-permeabilized platelets: effects of leakage of cytosolic proteins.
Human platelets containing dense granules labelled with 5-hydroxy[14C]tryptamine ([14C]5-HT) were permeabilized by exposure to streptolysin O (SLO) in the presence of 4 mM [gamma-32P]ATP. Addition of either 100 nM phorbol 12-myristate 13-acetate (PMA) or of Ca2+ (pCa 5) at the same time as SLO induced secretion of dense-granule [14C]5-HT and the phosphorylation of pleckstrin by protein kinase C (PKC). Ca2+ also induced phosphorylation of myosin P-light chains. Guanosine 5'-[gamma-thio]triphosphate (GTP[S], 100 microM) did not stimulate secretion from SLO-permeabilized platelets in the absence of Ca2+ (pCa>9), but greatly potentiated secretion in the presence of low PMA (10 nM) or low Ca2+ (pCa 6). However, GTP[S] did stimulate myosin P-light-chain phosphorylation in the absence of Ca2+, an effect that was associated with morphological changes, including granule centralization. Inhibition of PKC and of pleckstrin phosphorylation by Ro 31-8220 blocked secretion induced by PMA or by GTP[S] and PMA in the absence of Ca2+, but did not prevent the GTP[S]-induced phosphorylation of myosin P-light chains or secretion induced by Ca2+ at pCa 5. When the time period between exposure of platelets to SLO and challenge at pCa>9 with PMA or with GTP[S] and PMA was increased, there were rapid and parallel decreases in the secretion and pleckstrin phosphorylation responses, which were lost after 3-5 min. In contrast, the responsiveness of secretion to Ca2+ (pCa 5) or to GTP[S] and Ca2+ (pCa 6) persisted for at least 10 min after exposure of platelets to SLO, although the ability of pleckstrin to undergo phosphorylation was still lost after 3-5 min. Both PKC and pleckstrin were undetectable within platelets after 5 min exposure to SLO. The results suggest that the loss of responsiveness to PMA or to GTP[S] and PMA is attributable to the leakage of PKC (and possibly pleckstrin) from the platelets, whereas secretion stimulated by Ca2+ or by GTP[S] and Ca2+ utilizes membrane-associated Ca2+- and GTP-binding proteins and occurs independently of PKC activation.
Sloan
D C
DC
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada L8N 3Z5.
Haslam
R J
RJ
D001426:Bacterial Proteins; D001792:Blood Platelets / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D001798:Blood Proteins / Q000378:metabolism / Q000502:physiology; D002118:Calcium / Q000502:physiology*; D002250:Carbon Radioisotopes; D002463:Cell Membrane Permeability / Q000187:drug effects*; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D018994:Myosin Light Chains / Q000378:metabolism / Q000502:physiology; D010750:Phosphoproteins; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology*; D012701:Serotonin / Q000378:metabolism; D013301:Streptolysins / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors
D001426:Bacterial Proteins; D001798:Blood Proteins; D002250:Carbon Radioisotopes; D018994:Myosin Light Chains; D010750:Phosphoproteins; D013301:Streptolysins; C039077:platelet protein P47; C025964:streptolysin O; D012701:Serotonin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium