Protein kinase C promotes restoration of calcium homeostasis to platelet activating factor-stimulated human neutrophils by inhibition of phospholipase C

Platelets are released into the peripheral circulation from the bone marrow where they arise as fragments of megakaryocyte cytoplasm. To characterize the effects of platelet agonists on megakaryocytes, we examined calcium signaling and desensitization to thrombin, the thromboxane A2 (TxA2) mimetic (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619), and platelet-activating factor (PAF) in cultured CHRF-288-11 megakaryocytic cells. Initially, we compared agonist-stimulated calcium transients in fura-2-loaded CHRF-288-11 cells and human platelets. The 50% effective concentration values for the agonists to increase free cytosolic calcium were as follows: thrombin (0.11 +/- 0.02 U/ml in CHRF, 0.19 +/- 0.03 U/ml in platelets), U46619 (147 +/- 33 nM in CHRF, 157 +/- 5 nM in platelets), and PAF [15 +/- 2 nM in CHRF, 16 +/- 2 nM in platelets (n = 4 each)]. CHRF-288-11 thrombin, TxA2, and PAF receptors were demonstrated to be coupled to phospholipase C because each of the agonists stimulated phosphatidylinositol hydrolysis in myo-[3H]inositol-loaded CHRF-288-11 cells and pharmacological inhibition of phospholipase C-blunted agonist-stimulated calcium signaling. CHRF-288-11 cells exposed to the three agonists for 1 h showed different patterns and extent of homologous and heterologous desensitization. Protein kinase C activation appeared to be necessary but not sufficient for desensitization because 1) activation of protein kinase C with phorbol 12-myristate 13-acetate inhibited the calcium responses to all three agonists, 2) inhibition of protein kinase C with staurosporine attenuated subsequent desensitization to each agonist, and 3) each agonist increased protein kinase C activity in CHRF-288-11 cell homogenates.

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Tyrosine phosphorylation is involved in receptor coupling to phospholipase D but not phospholipase C in the human neutrophil

Biochemical Journal ◽

10.1042/bj2810597 ◽

1992 ◽

Vol 281 (3) ◽

pp. 597-600 ◽

Cited By ~ 104

Author(s):

I J Uings ◽

N T Thompson ◽

R W Randall ◽

G D Spacey ◽

R W Bonser ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Tyrosine Kinase ◽

Phospholipase C ◽

Tyrosine Phosphorylation ◽

Phospholipase D ◽

Human Neutrophil ◽

Human Neutrophils ◽

Receptor Coupling ◽

Kinase C

The tyrosine kinase inhibitors ST271, ST638 and erbstatin inhibited phospholipase D (PLD) activity in human neutrophils stimulated by fMet-Leu-Phe, platelet-activating factor and leukotriene B4. These compounds did not inhibit phorbol ester-stimulated PLD, indicating that they do not inhibit PLD per se, but probably act at a site between the receptor and the phospholipase. In contrast, the protein kinase C inhibitor Ro-31-8220 inhibited phorbol 12,13-dibutyrate- but not fMet-Leu-Phe-stimulated PLD activity, arguing against the involvement of protein kinase C in the receptor-mediated activation of PLD. ST271 did not inhibit Ins(1,4,5)P3 generation, but did inhibit protein tyrosine phosphorylation stimulated by fMet-Leu-Phe. The phosphotyrosine phosphatase inhibitor pervanadate increased tyrosine phosphorylation and stimulated PLD. These results suggest that tyrosine kinase activity is involved in receptor coupling to PLD but not to PtdIns(4,5)P2-specific phospholipase C in the human neutrophil.

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Stimulation and priming of protein kinase C translocation by a Ca2+ transient-independent mechanism. Studies in human neutrophils challenged with platelet-activating factor and other receptor agonists.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)45785-7 ◽

1990 ◽

Vol 265 (35) ◽

pp. 21619-21623 ◽

Cited By ~ 1

Author(s):

J T O'Flaherty ◽

J F Redman ◽

D P Jacobson ◽

A G Rossi

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Platelet Activating Factor ◽

Human Neutrophils ◽

Receptor Agonists ◽

Kinase C ◽

Independent Mechanism

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Sphingosine enhances platelet aggregation through an increase in phospholipase C activity by a protein kinase C-independent mechanism

Biochemical Journal ◽

10.1042/bj2820243 ◽

1992 ◽

Vol 282 (1) ◽

pp. 243-247 ◽

Cited By ~ 18

Author(s):

T Hashizume ◽

T Sato ◽

T Fujii

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phospholipase C ◽

Platelet Activating Factor ◽

Membrane Phospholipids ◽

Stimulus Response ◽

Protein Kinase C Inhibitors ◽

Kinase C ◽

Low Concentrations ◽

Positive Modulator

Sphingosine (a potent inhibitor of protein kinase C) at 5-10 microM, which are concentrations lower than those that inhibit this enzyme activity, enhanced the aggregation of rabbit platelets induced by low concentrations of U46619, platelet-activating factor, thrombin and arachidonic acid, whereas H-7 and staurosporine, other protein kinase C inhibitors, failed to do so. Of the sphingosine analogues which also inhibit protein kinase C, psychosine and lyso-GM3 did not show such an enhancing effect. Sphingosine promoted both Ins(1,4,5)P3 formation and an increase in the cytoplasmic free Ca2+ concentration in response to all the agonists used. Furthermore, the hydrolytic action of exogenously added phospholipase C (from Clostridium perfringens) on platelet membrane phospholipids was dose-dependently enhanced by pretreatment of the platelets with sphingosine. These results imply that sphingosine, at relatively low concentrations, brings about hyperaggregability of the platelets by the agonists employed, probably owing to enhancement of the phospholipase C activity. Such an effect appears to be induced by a mechanism independent of protein kinase C inhibition. We suggest that sphingosine might act as a positive modulator for the stimulus-response coupling in the platelets.

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