scholarly journals 4,4′-Di-isothiocyanatostilbene-2,2′-disulphonic acid (‘DIDS’) activates protein kinase C and Na+/H+ exchange in human platelets via α2A-adrenergic receptors

1993 ◽  
Vol 293 (2) ◽  
pp. 523-530 ◽  
Author(s):  
R Nieuwland ◽  
G Van Willigen ◽  
J W Akkerman
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Binding Sites ◽  
Adrenergic Receptors ◽  
Adrenergic Receptor ◽  
Early Event ◽  
Phosphoinositide Metabolism ◽  
Kinase C ◽  
Fibrinogen Binding ◽  
Disulphonic Acid

Most agonists stimulate platelets by inducing Ca2+ mobilization, Ca2+ influx and protein kinase C (PKC) activation leading to Na+/H+ exchange, exposure of fibrinogen-binding sites and aggregation. In contrast, previous studies showed that adrenaline induces exposure of fibrinogen-binding sites and aggregation without appreciable changes in cystolic Ca2+ content or PKC activity. In the present study we investigated platelet responses mediated via alpha 2A-adrenergic receptors, using 4,4′-di-isothiocyanatostilbene-2,2′-disulphonic acid (DIDS), which is known to bind to this type of receptor. Addition of DIDS (2-20 microM) induced (i) a rise in cytosolic pH of 0.23 +/- 0.05 pH unit (n = 5) as detected by BCECF fluorescence, due to activation of the Na+/H+ exchanger, (ii) a 3.5-4-fold increase in the phosphorylation of the 47 kDa protein, a major substrate of PKC, (iii) exposure of 81,072 +/- 7293 (n = 3) binding sites for 125I-fibrinogen per platelet, and (iv) irreversible aggregation. These responses occurred without changes in cytosolic [Ca2+], secretion of dense-granule contents and enhanced phosphoinositide metabolism, and were not affected by inhibition of thromboxane A2 generation (30 microM indomethacin). The alpha 2A-adrenergic-receptor antagonists oxymetazoline (500 microM) and yohimbine (1 mM) completely abolished DIDS-induced responses. Inhibition of PKC (1 microM staurosporine) prevented phosphorylation of the 47 kDa protein, the increase in Na+/H+ exchange and exposure of fibrinogen-binding sites. Thus our present data suggest that activation of PKC is an early event in DIDS-induced platelet activation via the alpha 2A-adrenergic receptor, which precedes any of the other known signal-transducing sequences.

scholarly journals Stimulation of phosphoinositide metabolism in hamster brown adipocytes exposed to α1-adrenergic agents and its inhibition with phorbol esters

1986 ◽  
Vol 236 (3) ◽  
pp. 757-764 ◽  
Author(s):  
R J Schimmel ◽  
D Dzierzanowski ◽  
M E Elliott ◽  
T W Honeyman
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Time Course ◽  
Early Event ◽  
Phosphoinositide Metabolism ◽  
Adrenergic Stimulation ◽  
Brown Adipocytes ◽  
Adrenergic Agents ◽  
Kinase C ◽  
Stimulation Of

The present experiments were undertaken to investigate the role of the phosphoinositides phosphatidylinositol 4-phosphate (PtdIns-4-P) and phosphatidylinositol 4,5-biphosphate (PtdIns-4,5-P2) in the alpha 1-adrenergic stimulation of respiration in isolated hamster brown adipocytes. Exposure of isolated brown adipocytes to the alpha-adrenergic-receptor agonist phenylephrine provoked a breakdown of 30-50% of the PtdIns-4-P and PtdIns-4,5-P2 after prelabelling of the cells with [32P]Pi. Coincident with the breakdown of phosphoinositides was an accumulation of labelled phosphatidic acid, which continued for the duration of the cell incubation. The time course of phosphoinositide breakdown was defined more precisely by pulse-chase experiments. Under these conditions, phenylephrine caused radioactivity in phosphatidylinositol, PtdIns-4-P and PtdIns-4,5-P2 to fall by more than 50% within 30 s and to remain at the depressed value for the duration of the incubation (10 min). This phospholipid response to alpha-adrenergic stimulation was blocked by exposure of the cells to phorbol 12-myristate 13-acetate (PMA); likewise phenylephrine stimulation of respiration was prevented by PMA. beta-Adrenergic stimulation of respiration and inhibition of respiration by 2-chloroadenosine and insulin were, however, unaffected by treatment with PMA. On the assumption that PMA is acting in these cells as an activator of protein kinase C, these results suggest the selective interruption of alpha-adrenergic actions in brown adipocytes by activated protein kinase C. These findings suggest that breakdown of phosphoinositides is an early event in alpha-adrenergic stimulation of brown adipocytes which may be important for the subsequent stimulation of respiration. The results from the pulse-chase studies also suggest, however, that phenylephrine-stimulated breakdown of inositol phospholipids is a short-lived event which does not appear to persist for the entire period of exposure to the alpha 1-adrenergic ligand.

scholarly journals α1B-Adrenergic receptor phosphorylation and desensitization induced by transforming growth factor-β

2002 ◽  
Vol 368 (2) ◽  
pp. 581-587 ◽  
Author(s):  
M. Teresa ROMERO-ÁVILA ◽  
C. Fabián FLORES-JASSO ◽  
J. Adolfo GARCÍA-SÁINZ
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Adrenergic Receptors ◽  
Adrenergic Receptor ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Receptor Phosphorylation ◽  
Kinase C ◽  
Phosphoinositide 3 Kinase

Transforming growth factor-β (TGF-β) induced α1B-adrenergic receptor phosphorylation in Rat-1 fibroblasts stably expressing these adrenoceptors. This effect of TGF-β was rapid, reaching a maximum within 30min and decreasing thereafter, and concentration-dependent (EC50 0.3pM). The phosphoinositide 3-kinase inhibitors wortmannin and LY294002, and the protein kinase C inhibitors staurosporine, Ro 318220 and bisindolylmaleimide, blocked the effect of this growth factor. α1B-Adrenergic receptor phosphorylation was associated with desensitization, as indicated by a reduction in the adrenergic-mediated production of [3H]inositol phosphates. Phosphorylation of α1B-adrenergic receptors by TGF-β was also observed in Cos-1 cells transfected with the receptor. Co-transfection of the dominant-negative mutant of the regulatory subunit of phosphoinositide 3-kinase (Δp85) inhibited the phosphorylation of α1B-adrenergic receptors induced by TGF-β. Our results indicate that activation of TGF-β receptors induces α1B-adrenergic receptor phosphorylation and desensitization. The data suggest that phosphoinositide 3-kinase and protein kinase C play key roles in this effect of TGF-β.

scholarly journals Protein kinase C and cyclic AMP regulate reversible exposure of binding sites for fibrinogen on the glycoprotein IIB-IIIA complex of human platelets

1991 ◽  
Vol 273 (1) ◽  
pp. 115-120 ◽  
Author(s):  
G van Willigen ◽  
J W N Akkerman
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Binding Sites ◽  
Platelet Activating Factor ◽  
Human Platelets ◽  
Common Mechanism ◽  
Tight Coupling ◽  
Kinase C ◽  
Fibrinogen Binding

Platelet aggregation is mediated via binding of fibrinogen to sites on the membrane glycoprotein IIB-IIIA complex which become exposed when the cells are stimulated. We report here evidence of a dynamic and reversible exposure of binding sites for fibrinogen. In the absence of fibrinogen, exposed sites (B*) gradually lose their capacity to bind fibrinogen and close (Bo). On stimulation with platelet-activating factor (PAF, 500 nM) at 22 degrees C, closing of B* is enhanced by agents that raise cyclic AMP levels (10 ng of prostaglandin I2/ml; 5 mM-theophylline), inhibit protein kinase C (PKC; 25 microM-sphingosine; 1 microM-staurosporine), or disrupt the energy supply (30 mM-2-deoxy-D-glucose + 1 mM-CN-), or by raising the temperature to 37 degrees C. Conversely, activation of PKC 1 microM-1,2-dioctanoyl-sn-glycerol; 55 nM-phorbol 12-myristate 13-acetate) and an increase in intracellular [Ca2+] (100 nM-ionomycin + extracellular Ca2+) oppose the disappearance of B*. Phosphorylation of the 47 kDa protein illustrates the tight coupling between PKC and B* under all conditions tested, except when the cyclic AMP level is raised, and B* is converted to Bo without affecting PKC activity. Although the increase in PKC activity is much smaller with ADP or even absent upon stimulation with adrenaline, the control of B* is equally sensitive to modulation of cyclic AMP and PKC activity. We conclude that PAF, ADP and adrenaline regulate exposure of fibrinogen binding sites through a common mechanism consisting of two independent pathways, one dominated by PKC and the other by an as yet unidentified cyclic AMP-sensitive step.

scholarly journals Regulation of hepatocyte plasma membrane α1-adrenergic receptors by 4β-phorbol 12-myristate 13-acetate

1995 ◽  
Vol 305 (1) ◽  
pp. 73-79 ◽  
Author(s):  
J F Beeler ◽  
R H Cooper
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Adrenergic Receptors ◽  
Adrenergic Receptor ◽  
Plasma Membranes ◽  
Complete Recovery ◽  
Maximal Inhibition ◽  
Kinase C

The effect of phorbol 12-myristate 13-acetate (PMA) on hepatocyte alpha 1-adrenergic receptors was determined by [3H]prazosin binding to plasma membranes from control and PMA-treated hepatocytes. Membranes from hepatocytes incubated with PMA (1 microgram/ml) for 1 h exhibited a 40% decrease in alpha 1-adrenergic receptors (481 +/- 10 fmol/mg of protein; mean +/- S.E.M. for three separate experiments) relative to vehicle-treated (dimethylformamide) hepatocytes (802 +/- 91 fmol/mg of protein; n = 3), with no significant effect on the KD. The PMA-induced decrease in alpha 1-adrenergic receptors was maximal by 30 min and half-maximal inhibition of [3H]prazosin binding occurred with a PMA concentration of approx. 15 ng/ml. Pretreatment of hepatocytes with staurosporine (5 microM) blocked the effect of PMA, and 4 beta-phorbol 13-monoacetate was ineffective, suggesting the involvement of protein kinase C (PKC). Treatment of hepatocytes with primaquine (300 microM) for 15 min decreased hepatocyte plasma membrane alpha 1-adrenergic receptors by 34.0 +/- 2.4% (mean +/- S.E.M. of three experiments). Removal of primaquine allowed essentially complete recovery (98 +/- 4%; mean +/- S.E.M. for five separate experiments) of plasma membrane [3H]prazosin binding within 20 min, suggesting that the alpha 1-adrenergic receptor undergoes endocytotic recycling. Addition of PMA (1 microgram/ml) to hepatocytes immediately after removal of primaquine, completely inhibited the increase in plasma membrane alpha 1-adrenergic receptors relative to control cells, but had no effect on hepatocytes whose cell surface alpha 1-receptors remaining after primaquine treatment had been inactivated by alkylation. These observations suggested that activation of PKC may facilitate the internalization of the alpha 1-adrenergic receptor in hepatocytes.

scholarly journals Decrease in equilibrative uridine transport during monocytic differentiation of HL-60 leukaemia: involvement of protein kinase C

1994 ◽  
Vol 300 (2) ◽  
pp. 407-412 ◽  
Author(s):  
C W Lee
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Nucleoside Transport ◽  
Protective Effects ◽  
Transport Activity ◽  
Monocytic Differentiation ◽  
Response Curves ◽  
Kinase C

The dose-response curves for the inhibition of equilibrative uridine transport by dilazep, dipyridamole and nitrobenzylthioinosine (NBMPR) in undifferentiated HL-60 cells were biphasic. Some 70% of the transport activity was inhibited with IC50 values of 0.7, 1 and 7 nM respectively. No inhibition of the remaining 30% of transport activity was observed until the dilazep, dipyridamole and NBMPR concentrations exceeded 1, 0.1 and 3 microM respectively. Exposure to phorbol 12-myristate 13-acetate (PMA) for 48 h, to induce monocytic differentiation, caused a 20-fold decrease in Vmax. of both NBMPR-sensitive and NBMPR-insensitive equilibrative uridine transport. The decrease in NBMPR-sensitive uridine transport induced by PMA corresponded to a decrease in NBMPR binding sites. A 30% decrease in specific NBMPR binding sites occurred within 6 h of PMA exposure, and could be prevented by uridine and thymidine at concentrations as low as 100 microM, and by staurosporine at 40 nM. However, the protective effects of these compounds diminished with prolonged PMA exposure. No protection was observed with uracil. Exogenous protein kinase C (PKC) in the presence of ATP and PMA decreased the number of specific NBMPR-binding sites in purified HL-60 cell plasma membranes. These results suggest that a PKC-induced conformational change in substrate-binding/transporting site may be responsible for the decrease in NBMPR-sensitive nucleoside transport during PMA-induced monocytic differentiation of HL-60 cells.

Expression of histamine H1 receptors in autoimmune myocarditis mice

1993 ◽  
Vol 71 (9) ◽  
pp. 639-644 ◽  
Author(s):  
Nora Goren ◽  
Claudia Perez Leiros ◽  
Leonor Sterin-Borda ◽  
Enri Borda
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Receptor Antagonist ◽  
Binding Sites ◽  
Affinity Binding ◽  
Autoimmune Myocarditis ◽  
Kinase C ◽  
Chronotropic Effects ◽  
Histamine H1 Receptors ◽  
Two Populations

Two populations of histaminergic H1 receptors with distinct high and low affinity binding sites were characterized by the specific H1 receptor antagonist [3H]mepyramine in autoimmune myocardium. No saturable binding of the radiolabelled H1 antagonist was observed in normal myocardium. Reaction of autoimmune myocardium with specific H1 agonist (2-thiazolyl-ethylamine (ThEA)) triggered positive inotropy and negative chronotropy, which were inhibited by mepyramine. Inhibitors of phospholipase C and protein kinase C attenuated both the inotropic and chronotropic effects of ThEA, suggesting the participation of phosphoinositide hydrolysis in this phenomenon. The latter was verified by measurement of polyphosphoinositide hydrolysis in autoimmune myocardium following the reaction of ThEA with histaminergic H1 receptors. We conclude that functional H1 histaminergic receptors could involve a distinctive mechanism operating in autoimmune myocardium as a result of cardiac antigen immunization.Key words: histamine, H1 receptors, myocarditis, autoimmunity.

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