scholarly journals Desensitization and resensitization of human platelets to 5-hydroxytryptamine at the level of signal transduction

1995 ◽  
Vol 307 (3) ◽  
pp. 775-782 ◽  
Author(s):  
P Roevens ◽  
D de Chaffoy de Courcelles
Keyword(s):  
Signal Transduction ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Time Course ◽  
Late Phase ◽  
Human Platelets ◽  
Uptake System ◽  
Kinase C ◽  
5Ht2 Receptor

Desensitization of platelets to 5-hydroxytryptamine (5HT) (1 microM), during active removal of the agonist by the platelet 5HT-uptake system, was studied at the level of signal transduction. Desensitization to 5HT was dose-dependent and homologous. Without occupation of the 5HT2 receptor, neither an increase in cytosolic [Ca2+] (30 nM ionomycin), nor a separate or simultaneous activation of protein kinase C (by 10 microM 1-oleoyl-2-acetylglycerol), could induce desensitization to 5HT (1 microM). During the early phase of desensitization, the 5HT2 receptor was coupled to phospholipase C, whereas during the late phase of desensitization this coupling was disconnected. However, after disappearance of the agonist, the coupling in the resting platelet recovered quickly, and was nearly complete (82%) after 30 min. During this resensitization, the 5HT-inducibility of activation of phospholipase C, of the increase in cytosolic [Ca2+] and of stimulation of protein kinase C were restored in parallel. The time course for resensitization of the 5HT-induced increase in cytosolic [Ca2+] was independent of the presence of extracellular Ca2+. It is concluded that, after dissociation of 5HT from the platelet 5HT2-receptor, 5HT-induced responses rapidly resensitize. Because of its short duration and the parallelism in recovery between the different ‘down-stream phospholipase C’ intracellular transduction signals, it is considered that desensitization arises from a reversible change in the transduction mechanism at a step up to or including the activation of phospholipase C. Neither desensitization nor resensitization to 5HT is dependent on the presence of extracellular Ca2+.

Differential megakaryocytic desensitization to platelet agonists

1992 ◽  
Vol 263 (4) ◽  
pp. C864-C872 ◽  
Author(s):  
G. W. Dorn ◽  
M. G. Davis
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Calcium Signaling ◽  
Phospholipase C ◽  
Platelet Activating Factor ◽  
Dienoic Acid ◽  
Cytosolic Calcium ◽  
Peripheral Circulation ◽  
Human Platelets ◽  
Kinase 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.

scholarly journals Activation of human platelets by peroxovanadate is associated with tyrosine phosphorylation of phospholipase Cγ and formation of inositol phosphates

1993 ◽  
Vol 290 (2) ◽  
pp. 471-475 ◽  
Author(s):  
R A Blake ◽  
T R Walker ◽  
S P Watson
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Inositol Phosphates ◽  
Human Platelets ◽  
Functional Responses ◽  
Phosphorylation Of Proteins ◽  
Kinase C

Vanadate ions in the presence of H2O2 (peroxovanadate) induce a marked increase in the degree of tyrosine phosphorylation of proteins in human platelets. This increase preceded the onset of platelet shape change and aggregation, and is associated with activation of phospholipase C and increased [32P]phosphorylation of proteins of 47 kDa, a substrate for protein kinase C, and 20 kDa, a substrate for both myosin light-chain kinase and protein kinase C. The non-selective inhibitor of protein kinases, staurosporine, inhibits the increase in tyrosine phosphorylation of nearly all proteins and inhibits completely all other functional responses, suggesting that these events may be linked. In support of this, peroxovanadate stimulates tyrosine phosphorylation of phospholipase C gamma 1, suggesting that this may underlie its mechanism of platelet activation. Staurosporine also inhibited activation of phospholipase C by collagen, suggesting that tyrosine phosphorylation has an important role in the early stages of collagen-induced platelet activation.

scholarly journals Stimulus-response coupling in human platelets activated by monoclonal antibodies to the CD9 antigen, a 24 kDa surface-membrane glycoprotein

1990 ◽  
Vol 266 (2) ◽  
pp. 527-535 ◽  
Author(s):  
R C Carroll ◽  
R E Worthington ◽  
C Boucheix
Keyword(s):  
Monoclonal Antibody ◽  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Surface Membrane ◽  
Human Platelets ◽  
Membrane Glycoprotein ◽  
Kinase C ◽  
Autocrine Stimulation

The CD9 molecule is a 24 kDa surface-membrane glycoprotein present on platelets and a variety of haematopoetic and non-haematopoetic tissues. In the present study we utilized specific inhibitors of thromboxane A2 (TxA2) formation (aspirin), protein kinase C [H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine]] and autocrine stimulation by secreted ADP (apyrase) to modify platelet activation by a monoclonal antibody ALB-6 to the CD9 antigen. This activation is only partially inhibited by aspirin alone but, in combination with either H-7 or apyrase, more than 50% inhibition of platelet aggregation and secretion was observed. This combination of inhibitors was also required to inhibit effectively the phosphorylation of myosin light chain and the 47 kDa substrate of protein kinase C. Intracellular Ca2+ flux monitored by the fluorescent dye fura-2 showed that this was almost completely mediated by the aspirin-sensitive TxA2 pathway. We suggest that the aspirin-insensitive pathway is primarily mediated by phospholipase C formation of diacylglycerol to activate protein kinase C. The inhibition by apyrase suggests a strong dependency on autocrine stimulation by secreted ADP to fully activate both phospholipase C and express fibrinogen-binding sites mediating platelet aggregation. This alternate pathway of phospholipase C activation by ALB-6 may be mediated by cytoplasmic alkalinization [monitored by SNARF-1 (5′(6′)-carboxy-10-bismethylamino-3-hydroxy-spiro-[7H- benzo[c]xanthine-1′,7(3H)-isobenzofuran]-3′-one) fluorescence of the dye]. Both activation pathways are dependent on intact antibodies, since F(ab′)2 fragments of SYB-1, a monoclonal antibody against the CD9 antigen with activation characteristics identical with those of ALB-6, do not elicit activation. Besides thrombin, collagen is another physiological agonist shown to induce aspirin-insensitive activation. Similarities to ALB-6 in collagen sensitivity to apyrase in combination with aspirin inhibitors were noted with respect to aggregation and secretion, as well as a complete block of Ca2+ flux by aspirin. However, it is unlikely that collagen activation is mediated by the CD9 antigen, since SYB-1 F(ab′)2 fragments had no effect on collagen activation and aspirin also completely blocked the alkalinization response to collagen, in contrast with ALB-6.

Signal transduction pathways involved in fluid flow-induced PGE2 production by cultured osteocytes

1999 ◽  
Vol 276 (1) ◽  
pp. E171-E178 ◽  
Author(s):  
N. E. Ajubi ◽  
J. Klein-Nulend ◽  
M. J. Alblas ◽  
E. H. Burger ◽  
P. J. Nijweide
Keyword(s):  
Signal Transduction ◽  
Protein Kinase C ◽  
Fluid Flow ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Phospholipase A ◽  
Signal Transduction Pathways ◽  
Prostaglandin E ◽  
Kinase C ◽  
Intracellular Ca

To maintain its structural competence, the skeleton adapts to changes in its mechanical environment. Osteocytes are generally considered the bone mechanosensory cells that translate mechanical signals into biochemical, bone metabolism-regulating stimuli necessary for the adaptive process. Prostaglandins are an important part of this mechanobiochemical signaling. We investigated the signal transduction pathways in osteocytes through which mechanical stress generates an acute release of prostaglandin E2(PGE2). Isolated chicken osteocytes were subjected to 10 min of pulsating fluid flow (PFF; 0.7 ± 0.03 Pa at 5 Hz), and PGE2release was measured. Blockers of Ca2+ entry into the cell or Ca2+ release from internal stores markedly inhibited the PFF-induced PGE2 release, as did disruption of the actin cytoskeleton by cytochalasin B. Specific inhibitors of Ca2+-activated phospholipase C, protein kinase C, and phospholipase A2 also decreased PFF-induced PGE2 release. These results are consistent with the hypothesis that PFF raises intracellular Ca2+ by an enhanced entry through mechanosensitive ion channels in combination with Ca2+- and inositol trisphosphate (the product of phospholipase C)-induced Ca2+ release from intracellular stores. Ca2+ and protein kinase C then stimulate phospholipase A2activity, arachidonic acid production, and ultimately PGE2 release.

Regulation of platelet phospholipase C

1988 ◽  
Vol 320 (1199) ◽  
pp. 299-311 ◽  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Adenylyl Cyclase ◽  
Phospholipase C ◽  
Phorbol Esters ◽  
Human Platelets ◽  
Direct Effects ◽  
Inhibitory Effects ◽  
Factors Affecting ◽  
Kinase C

We have investigated factors affecting the activation of phospholipase C in human platelets. Prior exposure of platelets to phorbol esters that stimulate protein kinase C inhibits the activation of phospholipase C in response to a variety of receptor-directed agonists, including x- and y-thrombin and thromboxane A 2 analogues. Such activation has been assayed by measurements of accumulated InsP 3 (including Ins(l,4,5)P 3 and Ins(l,3,4)P 3 ) and PtdOH. Inhibition is not overcome by Ca 2+ ionophores, and substances that block or mimic Na+-H + exchange neither block nor mimic these inhibitory effects. Cyclic AMP and cyclic GMP, other agents known to inhibit phospholipase C activation, do not accumulate in platelets exposed to phorbol esters. Although a portion of the effects of phorbol ester on InsP 3 accumulation may be explained by 5-phosphomonoesterase activity, it is likely that more direct effects on phospholipase C are being exerted as well, and contribute the major inhibitory route. We have examined the susceptibility of adenylyl cyclase-associated G 1 and ‘G p ’- activated phospholipase C to inhibitory ADP-ribosylation by pertussis toxin-derived enzyme (S 1 protomer) administered to saponin-permeabilized platelets. The effects of a-thrombin on adenylyl cyclase can be inhibited by up to 50% by S 1 at which point inhibition of phospholipase C is barely detectable. Thromboxane A 2 analogues, which do not affect adenylyl cyclase (G 1 ), stimulate phospholipase C; this effect is not impaired by Sr We therefore propose that the inhibitory effects of phorbol esters on the activation of phospholipase C are not mediated primarily by effects on G 1 .

scholarly journals Protein kinase C-dependent desensitization of HDL3-activated phospholipase C in human platelets.

1994 ◽  
Vol 14 (8) ◽  
pp. 1321-1326 ◽  
Author(s):  
H Nazih ◽  
F Nazih-Sanderson ◽  
V Magret ◽  
B Caron ◽  
J Goudemand ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Human Platelets ◽  
Kinase C

scholarly journals Effect of propranolol on platelet signal transduction

1995 ◽  
Vol 309 (1) ◽  
pp. 99-104 ◽  
Author(s):  
D Dash ◽  
K Rao
Keyword(s):  
Signal Transduction ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Molecular Mechanisms ◽  
Phorbol Esters ◽  
Inositol Phosphates ◽  
Major Effect ◽  
Dependent Manner ◽  
Kinase C

Propranolol inhibits platelet secondary aggregation and secretion by mechanisms unrelated to its beta-adrenergic-blocking activity. We previously reported that a major effect of the drug is perturbation of the physical microenvironment of the human platelet membrane. To explore further the molecular mechanisms underlying propranolol-mediated platelet inhibition, we studied protein kinase C activity, estimated from the phosphorylation of the substrate protein pleckstrin, in propranolol-treated human platelets. The drug inhibited activation of the enzyme in thrombin-stimulated platelets but not in platelets stimulated with phorbol esters, indicating that its site of action might be upstream of protein kinase C. It also inhibited the activity of phospholipase C, determined from the extent of generation of inositol phosphates and phosphatidic acid, in platelets stimulated with thrombin as well as the non-hydrolysable GTP analogue guanosine 5′-[beta, gamma-imido]triphosphate in a dose-dependent manner. These data suggest that propranolol inhibits signal transduction in thrombin-stimulated platelets by interacting at the level of phospholipase C and exclude interaction of the drug with the downstream effector enzyme protein kinase C.

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