scholarly journals cADP-ribose formation by blood platelets is not responsible for intracellular calcium mobilization

1998 ◽  
Vol 331 (2) ◽  
pp. 431-436 ◽  
Author(s):  
Philippe OHLMANN ◽  
Claude LERAY ◽  
Catherine RAVANAT ◽  
Adel HALLIA ◽  
Dominique CASSEL ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Plasma Membranes ◽  
Calcium Release ◽  
Blood Platelets ◽  
Human Platelets ◽  
Cyclase Activity ◽  
Dependent Manner ◽  
Intact Cells ◽  
Adp Ribosyl Cyclase ◽  
Intracellular Calcium Mobilization

Human platelet CD38 is a multifunctional ectoenzyme catalysing the synthesis and hydrolysis of cADP-ribose (cADPR), a recently identified calcium-mobilizing agent that acts independently of d-myo-inositol 1,4,5-trisphosphate and is known to be expressed by human platelets. The present work shows that ADP-ribosyl cyclase activity is exclusively a membrane activity, of which the major part is located in plasma membranes and a small part in internal membranes. In broken cells, cyclase activity was insensitive to the presence of calcium and was not modulated by agonists such as thrombin or ADP, whereas in intact cells thrombin increased cADPR formation by 30%, an effect due to fusion of granules with the plasma membrane. In order to assess the role of cADPR as a calcium-mobilizing agent, vesicles were prepared from internal membranes and loaded with 45CaCl2. These vesicles were efficiently discharged by IP3 in a dose-dependent manner, but were not responsive to cADPR or ryanodine in the presence or absence of calmodulin. Thus cADPR is unlikely to play a role in intracellular calcium release in human blood platelets.

The Role Of Calcium In Platelet Microtubule Assembly- Disassembly

1981 ◽  
Author(s):  
M Kikuchi ◽  
Y Ikeda ◽  
M Handa ◽  
S Matsuda ◽  
H Muraki ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Dynamic Equilibrium ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Microtubule Assembly ◽  
Base Line ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Transient Decrease

Microtubules exist in a dynamic equilibrium between polymerized and depolymerized forms in human platelets, playing a major role to maintain the discoid shape of platelets. It has been previously shown that the interaction of aggregating agents with platelets leads to a rapid but transient disassembly of microtubules. ( Steiner and Ikeda, J.Clin. Invest. 63:443,1979 ) In this paper, the role of calcium in the equilibrium between assembled and disassembled microtubules was investigated. The respective pools of soluble and polymerized tubulin were “frozen” by addition of a glycerol-dimethyl sulfoxide-containing medium to platelet rich plasma, preincubated with 2 µM A23187 for various time intervals. The two pools of tubulin were estimated by measuring the colchicine binding activities of total and polymerized tubulin according to the method of Wilson.Resting platelets were found to contain 56.2 ± 2.7 µg tubulin per 109 platelets, of which 56.7 % was in polymerized form. Addition of A23187 to platelet rich plasma produced a transient decrease in the pool of polymerized tubulin within 30 sec., followed by a return to base-line values within 2 min.. TMB-8, a known intracellular calcium antagonist, abolished this transient decrease in polymerized tubulin induced by A23187 in a concentration dependent manner, while indomethacin or acetylsalycylic acid did not.These findings may indicate the important role of intracellular calcium in microtubule assembly-disassembly.

Thrombin Mediated Tyrosine Phosphorylation of PKCδ in Human Platelets Occurs in a Calcium- and Src Family Tyrosine Kinase- Dependent Manner.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3541-3541
Author(s):  
Swaminathan Murugappan ◽  
Haripriya Shankar ◽  
Satya Kunapuli
Keyword(s):  
Tyrosine Kinase ◽  
Intracellular Calcium ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Human Platelets ◽  
Dependent Manner ◽  
Protein Signaling ◽  
Glycoprotein Vi ◽  
Pkc Isoforms

Abstract Protein kinase C (PKC)-δ is a novel PKC that has been shown to be tyrosine phosphorylated upon stimulation with agonists in platelets. Tyrosine phosphorylation of PKCδ has been shown to occur in a Fyn-dependent manner downstream of glycoprotein VI (GPVI) signaling in platelets. Although thrombin causes tyrosine phosphorylation of PKCδ in platelets, the mechanism of this event is not elucidated. In this study, we investigated whether G-protein signaling pathways utilize similar pathways as GPVI in tyrosine phosphorylation of PKCδ. Protease activated receptor (PAR) -1 selective peptide, SFLLRN and PAR - 4 selective peptide, AYPGKF caused a time- and concentration-dependent increase in tyrosine phosphorylation of PKCδ in human platelets. However, AYPGKF failed to cause tyrosine phosphorylation of PKCδ in Gq-deficient mouse platelets. Both U73122, a phospholipase C (PLC) inhibitor, and dimethyl-BAPTA, an intracellular calcium chelator, inhibited the tyrosine phosphorylation of PKCδ downstream of the PAR activation suggesting a role for Gq/PLC pathways and intracellular calcium in mediating this event. Inhibition of PKC isoforms using GF109203X potentiated the tyrosine phosphorylation of PKCδ. The Src family tyrosine kinase inhibitors, PP1 and PP2 inhibited the tyrosine phosphorylation of PKCδ suggesting a role for Src family tyrosine kinase members in this event. We also found that both Lyn and Src are physically associated with PKCδ in a constitutive manner in platelets. Finally we found that there was a time-dependent activation of Src following activation of platelets with thrombin. Thus, the precomplexed Src and Lyn tyrosine kinases get activated following PAR stimulation resulting in the tyrosine phosphorylation of PKCδ. All these data indicate that tyrosine phosphorylation of PKCδ downstream of thrombin occurs in a calcium- and Src-family kinase dependent manner in human platelets.

Carnosine is a novel peptide modulator of intracellular calcium and contractility in cardiac cells

1997 ◽  
Vol 272 (1) ◽  
pp. H462-H468 ◽  
Author(s):  
G. P. Zaloga ◽  
P. R. Roberts ◽  
K. W. Black ◽  
M. Lin ◽  
G. Zapata-Sudo ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Calcium Release ◽  
Cardiac Contractility ◽  
Cardiac Cells ◽  
Dependent Manner ◽  
Calcium Release Channel ◽  
Release Channel ◽  
Contractile Failure

Myocardial contractile failure is a common cause of morbidity and mortality in patients with ischemic heart disease and systemic inflammatory states such as sepsis. Accumulating evidence indicates that contractile failure is associated with dysregulation of myoplasmic calcium levels. In a search for biochemical causes for contractile dysfunction, we found that the dipeptide carnosine improves cardiac contractility and tested the possibility that carnosine plays a role in the regulation of intracellular calcium. Carnosine increased contractility in a dose-dependent manner (1-10 mM) in isolated perfused rat hearts. and it also increased free intracellular calcium levels in isolated myocytes. Carnosine increased myocyte tension via calcium release from the ryanodine receptor calcium release channel in skinned myocardial fibers and increased open-state probability and dwell time of the isolated ryanodine receptor calcium release channel in lipid bilayers. In addition. we report that carnosine sensitizes the contractile proteins so calcium. These results suggest a novel role for carnosine as a modulator of intracellular calcium and contractility in cardiac tissue.

Intracellular Calcium Release from Human Platelets: Different Messengers for Multiple Stores

2008 ◽  
Vol 18 (2) ◽  
pp. 57-61 ◽  
Author(s):  
Isaac Jardín ◽  
José J. López ◽  
José A. Pariente ◽  
Ginés M. Salido ◽  
Juan A. Rosado
Keyword(s):  
Intracellular Calcium ◽  
Calcium Release ◽  
Human Platelets ◽  
Intracellular Calcium Release

Adenylate cyclase activity in Y1 mouse adrenocortical tumor cells: some properties of the enzyme associated with purified plasma membrane fractions

1983 ◽  
Vol 61 (7) ◽  
pp. 547-552 ◽  
Author(s):  
Bernard P. Schimmer
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Tumor Cell Line ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Adenylate Cyclase System ◽  
Adrenocortical Tumor ◽  
Intact Cells ◽  
Order Of Magnitude

Fractions enriched in plasma membranes were prepared from the Y1 mouse adrenocortical tumor cell line and were characterized with respect to adenylate cyclase activity. Optimal requirements of the adenylate cyclase system for guanyl nucleotides, Mg2+, ATP, and corticotropin (ACTH) were determined. The sensitivity of the adenylate cyclase system to ACTH1–24 in plasma membrane fractions was comparable with that observed in isolated intact cells. Polycations such as poly-L-arginine and histone competitively inhibited the action of ACTH1–24, supporting the view that the affinity of ACTH for the adenylate cyclase system is determined by the basic core of amino acids at residues 15–18. ACTH1–24 was at least one order of magnitude more potent than ACTH1–39 in stimulating adenylate cyclase activity in plasma membrane fractions.

Isolation Of Dense Bodies From Human Blood Platelets On Metrizamide Gradient

1981 ◽  
Author(s):  
F Rendu ◽  
M Lebret ◽  
J P Caen
Keyword(s):  
Plasma Membranes ◽  
Blood Platelets ◽  
Human Platelets ◽  
Release Mechanism ◽  
Inherited Disorders ◽  
Platelet Factor ◽  
Functional Studies ◽  
Dense Bodies ◽  
Serotonin Uptake And Release

In view of the prominent role of dense bodies in platelet activation suggested by the platelet dysfunctions observed in storage pool diseases, we have developed a method for the isolation of human platelet dense bodies, using mepa- crine to follow the purification.Each step of the purification (washing procedures, lysis and subcellular separation) has been controlled in order to obtain the minimum release of these granules. Platelet lysates were centrifuged on a short two step discontinuous metrizamide gradient which allowed the attainment of a high density pellet. This pellet consisted of isolated mepacrine fluorescent granules which showed the typical appearance of dense bodies by electron microscopy. The granule pellet was relatively free from plasma membranes as estimated by the remaining (3H) -concanavalin A or 125I after labelling the whole platelets before the fractionation. The low contamination by other granule populations was estimated by the different assayed markers, β-glucuronidase, monoamine oxidase and platelet factor 4. The method is simple, reproducible and allows the highest enrichment in dense bodies obtained until now with human platelets(x 170 enrichment in calcium and x 110 enrichment in (14C) 5-HT after labelling the whole platelets as compared to the homogenate). Functional studies performed with the isolated granules showed a rapid accumulation of (14C)-5-HT, and the initial uptake was inhibited by reserpine but remained insensitive to imipramine.The technique can be applied to the study of inherited disorders where the serotonin uptake and release mechanism has to be clarified.

scholarly journals Nongenomic signaling of the retinoid X receptor through binding and inhibiting Gq in human platelets

Blood ◽  
2007 ◽  
Vol 109 (9) ◽  
pp. 3741-3744 ◽  
Author(s):  
Leonardo A. Moraes ◽  
Karen E. Swales ◽  
Jessica A. Wray ◽  
Amilcar Damazo ◽  
Jonathan M. Gibbins ◽  
...  
Keyword(s):  
G Protein ◽  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Hormone Receptors ◽  
Calcium Release ◽  
Human Platelets ◽  
Peroxisome Proliferator ◽  
Dependent Manner ◽  
Peroxisome Proliferator Activated Receptor ◽  
X Receptors

Abstract Retinoid X receptors (RXRs) are important transcriptional nuclear hormone receptors, acting as either homodimers or the binding partner for at least one fourth of all the known human nuclear receptors. Functional nongenomic effects of nuclear receptors are poorly understood; however, recently peroxisome proliferator-activated receptor (PPAR) \#947;, PPAR\#946;, and the glucocorticoid receptor have all been found active in human platelets. Human platelets express RXR\#945; and RXR\#946;. RXR ligands inhibit platelet aggregation and TXA2 release to ADP and the TXA2 receptors, but only weakly to collagen. ADP and TXA2 both signal via the G protein, Gq. RXR rapidly binds Gq but not Gi/z/o/t/gust in a ligand-dependent manner and inhibits Gq-induced Rac activation and intracellular calcium release. We propose that RXR ligands may have beneficial clinical actions through inhibition of platelet activation. Furthermore, our results demonstrate a novel nongenomic mode for nuclear receptor action and a functional cross-talk between G-protein and nuclear receptor signaling families.

scholarly journals The Amino Terminus of Herpes Simplex Virus 1 Glycoprotein K (gK) Is Required for gB Binding to Akt, Release of Intracellular Calcium, and Fusion of the Viral Envelope with Plasma Membranes

2018 ◽  
Vol 92 (6) ◽  
pp. e01842-17 ◽  
Author(s):  
Farhana Musarrat ◽  
Nithya Jambunathan ◽  
Paul J. F. Rider ◽  
V. N. Chouljenko ◽  
K. G. Kousoulas
Keyword(s):  
Intracellular Calcium ◽  
Plasma Membranes ◽  
Calcium Release ◽  
Virus Entry ◽  
Viral Envelope ◽  
Amino Terminus ◽  
Herpes Simplex Virus 1 ◽  
Amino Terminal ◽  
Glycoprotein K ◽  
Hsv 1

ABSTRACTPreviously, we have shown that the amino terminus of glycoprotein K (gK) binds to the amino terminus of gB and that deletion of the amino-terminal 38 amino acids of gK prevents herpes simplex virus 1 (HSV-1) infection of mouse trigeminal ganglia after ocular infection and virus entry into neuronal axons. Recently, it has been shown that gB binds to Akt during virus entry and induces Akt phosphorylation and intracellular calcium release. Proximity ligation and two-way immunoprecipitation assays using monoclonal antibodies against gB and Akt-1 phosphorylated at S473 [Akt-1(S473)] confirmed that HSV-1(McKrae) gB interacted with Akt-1(S473) during virus entry into human neuroblastoma (SK-N-SH) cells and induced the release of intracellular calcium. In contrast, the gB specified by HSV-1(McKrae) gKΔ31-68, lacking the amino-terminal 38 amino acids of gK, failed to interact with Akt-1(S473) and induce intracellular calcium release. The Akt inhibitor miltefosine inhibited the entry of McKrae but not the gKΔ31-68 mutant into SK-N-SH cells. Importantly, the entry of the gKΔ31-68 mutant but not McKrae into SK-N-SH cells treated with the endocytosis inhibitors pitstop-2 and dynasore hydrate was significantly inhibited, indicating that McKrae gKΔ31-68 entered via endocytosis. These results suggest that the amino terminus of gK functions to regulate the fusion of the viral envelope with cellular plasma membranes.IMPORTANCEHSV-1 glycoprotein B (gB) functions in the fusion of the viral envelope with cellular membranes during virus entry. Herein, we show that a deletion in the amino terminus of glycoprotein K (gK) inhibits gB binding to Akt-1(S473), the release of intracellular calcium, and virus entry via fusion of the viral envelope with cellular plasma membranes.

scholarly journals p120c-cbl is present in human blood platelets and is differentially involved in signaling by thrombopoietin and thrombin

Blood ◽  
1996 ◽  
Vol 88 (4) ◽  
pp. 1330-1338 ◽  
Author(s):  
A Oda ◽  
K Ozaki ◽  
BJ Druker ◽  
Y Miyakawa ◽  
H Miyazaki ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Blood Platelets ◽  
Apparent Molecular Weight ◽  
Human Platelets ◽  
Genetically Engineered ◽  
Jurkat Cells ◽  
Dependent Manner ◽  
Endogenous Substrate ◽  
Tyrosine Phosphorylated Protein ◽  
Triton X

To investigate the signaling processes induced by recombinant thrombopoietin, we used human platelets to recently show that thrombopoietin induces rapid tyrosine phosphorylation of Jak2, Tyk2, Shc, Stat3, Stat5, and other proteins in human platelets. Because the apparent molecular weight of a major tyrosine-phosphorylated protein in platelets stimulated by thrombopoietin is approximately 120 kD, we examined the possibility that this could be p120c-cbl, a protein known to be involved in signaling by many growth factors. Specific antisera against p120c-cbl recognized the same 120-kD protein in lysates of Jurkat cells, which are known to express p120c-cbl, and platelets, indicating that platelets have p120c-cbl. Thrombopoietin induced rapid tyrosine phosphorylation of p120c-cbl in platelets. Thrombopoietin also induced tyrosine phosphorylation of p120c-cbl in FDCP cells genetically engineered to express the thrombopoietin receptor, c-Mpl. Interestingly, FDCP cells, expressing a truncated c-Mpl devoid of the box-2 domain, proliferate in response to thrombopoietin. However, no increase in tyrosine phosphorylation of p120c-cbl was observed upon treatment of these cells with thrombopoietin, indicating that in this system tyrosine phosphorylation of p120c-cbl may not be essential for cell proliferation. This suggests that tyrosine phosphorylation of p120c-cbl may be required for nonmitogenic responses induced by thrombopoietin in postmitotic cells such as platelets. On the other hand, p120c-cbl was not significantly tyrosine-phosphorylated upon treatment of platelets with thrombin. However, it became incorporated into the Triton X-100-insoluble, 10,000g-sedimentable residue in an aggregation-dependent manner, suggesting that it may have a regulatory role in platelet cytoskeletal processes. p120c-cbl was constitutively associated with a 28-kD adapter protein, Grb2, that was also incorporated into the Triton X-100-insoluble, sedimentable residue dependent on aggregation. Further, we found that p120c-cbl is an endogenous substrate for calpain, a protease that may play a role in postaggregation signaling processes. Our data suggest that p120c-cbl may be involved in signal transduction following ligand binding to c- Mpl through its inducible tyrosine phosphorylation, and it may also be involved in signaling during platelet aggregation by its redistribution to the cytoskeleton.

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