scholarly journals Agonist-induced Ca2+ influx into human platelets is secondary to the emptying of intracellular Ca2+ stores

1991 ◽  
Vol 280 (3) ◽  
pp. 783-789 ◽  
Author(s):  
M T Alonso ◽  
J Alvarez ◽  
M Montero ◽  
A Sanchez ◽  
J García-Sancho
Keyword(s):  
Plasma Membrane ◽  
Platelet Activating Factor ◽  
Human Platelets ◽  
Temperature Dependent ◽  
Plasma Membrane Permeability ◽  
Low Concentrations ◽  
Cytochrome P 450 ◽  
Filling State ◽  
Ca2 Channels ◽  
Stimulation Of

We have studied the relation between the filling state of the intracellular Ca2+ stores and the plasma-membrane permeability to Mn2+, used here as a Ca2+ surrogate for Ca2+ channels. Emptying of the intracellular Ca2+ stores either by incubation in Ca(2+)-free medium or by treatment with low concentrations of the Ca2+ ionophore ionomycin accelerated the influx of Mn2+. Refilling of the Ca2+ stores by incubation in Ca(2+)-containing medium restores low Mn2+ permeability. This Ca(2+)-store-regulated permeability was inhibited by Ni2+ and by cytochrome P-450 inhibitors. Stimulation of platelets with thrombin produced Ca2+ release from the intracellular stores, which was followed, after a temperature-dependent lag (2 s at 37 degrees C; 5 s at 18 degrees C), by an acceleration of Mn2+ influx. Cytochrome P-450 inhibitors prevented the thrombin-induced Mn2+ influx, with little effect on the Ca2+ mobilization from the intracellular stores. Ki values were similar to those estimated for inhibition of the store-regulated permeability in non-stimulated platelets. Similar results were found in platelets stimulated by platelet-activating factor or by ADP. We propose that agonist-induced Ca2+ (Mn2+) influx in platelets is secondary to the emptying of the intracellular Ca2+ stores. The activation of the plasma-membrane Ca2+ (Mn2+) pathway may take place by a mechanism involving microsomal cytochrome P-450, similar to that described previously in thymocytes [Alvarez, Montero & García-Sancho (1991) Biochem. J. 274, 193-197] and neutrophils [Montero, Alvarez & García-Sancho (1991) Biochem. J. 277, 73-79].

scholarly journals Control of Ca2+ entry into HL60 and U937 human leukaemia cells by the filling state of the intracellular Ca2+ stores

1993 ◽  
Vol 289 (3) ◽  
pp. 761-766 ◽  
Author(s):  
S R Alonso-Torre ◽  
J Alvarez ◽  
M Montero ◽  
A Sanchez ◽  
J García-Sancho
Keyword(s):  
Plasma Membrane ◽  
Inositol Phosphate ◽  
Platelet Activating Factor ◽  
Membrane Receptors ◽  
Plasma Membrane Permeability ◽  
Prolonged Incubation ◽  
Differentiated Cells ◽  
Cytochrome P 450 ◽  
Filling State ◽  
Ca2 Channels

Differentiation of HL60 cells by treatment with dimethyl sulphoxide induces the expression of membrane receptors for N-formylmethionyl-leucyl-phenylalanine (fMLP) and for platelet-activating factor (PAF). In these cells both agonists produced an increase in the cytosolic Ca2+ concentration ([Ca2+]i) by release of Ca2+ from the intracellular stores, followed shortly by an acceleration of the entry of Ca2+ or Mn2+, used here as a Ca2+ surrogate for Ca2+ channels. Cytochrome P-450 inhibitors blocked the agonist-induced entry of Ca2+ or Mn2+ with no modification of Ca2+ release from the stores. Emptying the intracellular Ca2+ stores either by treatments inducing no inositol phosphate production, such as prolonged incubation in Ca(2+)-free medium or treatment with the Ca2+ ionophore ionomycin, increased the plasma-membrane permeability to Ca2+ and Mn2+. This Ca(2+)-store-regulated Mn2+ entry was inhibited by Ni2+ and by cytochrome P-450 inhibitors. Refilling of the Ca2+ stores by incubation in Ca(2+)-containing medium restored low Mn2+ permeability. The same mechanism is present and functional in non-differentiated cells, before expression of membrane receptors for fMLP and PAF. These results suggest that agonist-induced Ca2+ (Mn2+) entry is secondary to the emptying of the intracellular Ca2+ stores, which in turn activates plasma-membrane channels by a mechanism involving cytochrome P-450.

scholarly journals Cytochrome P-450 may link intracellular Ca2+ stores with plasma membrane Ca2+ influx

1991 ◽  
Vol 274 (1) ◽  
pp. 193-197 ◽  
Author(s):  
J Alvarez ◽  
M Montero ◽  
J García-Sancho
Keyword(s):  
Plasma Membrane ◽  
Aryl Hydrocarbon Hydroxylase ◽  
Plasma Membrane Permeability ◽  
Aryl Hydrocarbon ◽  
Oxygen Scavenging ◽  
Aryl Hydrocarbon Hydroxylase Activity ◽  
Cytochrome P 450 ◽  
Degree Of Filling ◽  
Ca2 Channels ◽  
Dependent Mechanism

We have studied the mechanism of the regulation of plasma membrane Ca2+ permeability by the degree of filling of the intracellular Ca2+ stores. Using Mn2+ as a Ca2+ surrogate for plasma membrane Ca2+ channels, we found that Mn2+ uptake by rat thymocytes is inversely related to the degree of filling of the intracellular Ca2+ stores. This store-dependent plasma membrane permeability is inhibited by oxygen scavenging, CO, imidazole antimycotics and other cytochrome P-450 inhibitors. The pattern of inhibition is similar to that reported previously for the inhibition of microsomal cytochrome P-450-mediated aryl hydrocarbon hydroxylase activity of lymphocytes. Several calmodulin antagonists, both phenothiazinic (trifluoperazine, fluphenazine and chlorpromazine) and dibenzodiazepinic (clozapine), accelerate Mn2+ uptake by cells with Ca2(+)-filled stores, and this effect is prevented by imidazole antimycotics. Our results suggest that cytochrome P-450 may be the link between the stores and the plasma membrane Ca2+ pathway. We propose a model in which this cytochrome, sited at the stores, stimulates plasma membrane Ca2+ influx. This stimulatory effect is, in turn, prevented by the presence of Ca2+ inside the stores, possibly via a calmodulin-dependent mechanism.

scholarly journals Control of plasma-membrane Ca2+ entry by the intracellular Ca2+ stores. Kinetic evidence for a short-lived mediator

1992 ◽  
Vol 288 (2) ◽  
pp. 519-525 ◽  
Author(s):  
M Montero ◽  
J Alvarez ◽  
J García-Sancho
Keyword(s):  
Plasma Membrane ◽  
Time Lag ◽  
Human Neutrophils ◽  
Half Time ◽  
Plasma Membrane Permeability ◽  
Steady State Levels ◽  
Clearance Kinetics ◽  
Cytochrome P 450 ◽  
Degree Of Filling ◽  
Ca2 Channels

We have studied the correlation between the degree of filling of the intracellular Ca2+ stores and the plasma-membrane permeability to Mn2+, a Ca2+ surrogate for plasma-membrane Ca2+ channels, in human neutrophils loaded with fura-2. Refilling of the stores of cells previously depleted of Ca2+ decreased the entry of Mn2+, but the magnitude of this effect depended on the refilling protocol. When refilling was allowed to proceed to steady-state levels by a 3 min incubation with different external Ca2+ concentrations (0.05-1 mM), almost complete inhibition of Mn2+ entry was observed at 40% of maximum refilling. In contrast, when different degrees of store refilling were attained by incubation with 1 mM-Ca2+ for short periods (10-40 s), inhibition of Mn2+ entry was smaller at comparable degrees of refilling. When quick refilling was allowed to proceed up to 40% (about 20 s at 37 degrees C) and then stopped at this level by removal of external Ca2+, the rate of Mn2+ uptake was high just after refilling and then decreased with time within the next few seconds (half-times approximately 7 s at 37 degrees C and approximately 20 s at 25 degrees C). We have proposed previously that the Ca2+ stores, when emptied of Ca2+, may generate a second messenger able to open the plasma-membrane Ca2+ channels by a mechanism involving cytochrome P-450. The results here are consistent with the existence of such a messenger and suggest that it is cleared from the cytoplasm with a half-time of about 7 s at 37 degrees C. In addition, inhibition of Mn2+ entry in cells with empty Ca2+ stores by cytochrome P-450 inhibitors showed a time lag consistent with the clearance kinetics proposed above.

Pretreatment of Human Platelets with Plasmin Inhibits Responses toThrombin, but Potentiates Responses to Low Concentrations of Aggregating Agents, Including the Thrombin Receptor Activating Peptide, SFLLRN

1997 ◽  
Vol 77 (04) ◽  
pp. 741-747 ◽  
Author(s):  
R L Kinlough-Rathbone ◽  
D W Perry ◽  
M L Rand ◽  
M A Packham
Keyword(s):  
Arachidonic Acid ◽  
Platelet Activating Factor ◽  
Human Platelets ◽  
Thrombin Receptor ◽  
Fibrinolytic Agents ◽  
Albumin Solution ◽  
Fibrinogen Receptor ◽  
Low Concentrations ◽  
Induced Aggregation

SummaryEffects of plasmin on platelets, that influence subsequent responses to aggregating agents, are relevant to attempts to prevent rethrombosis following administration of fibrinolytic agents. We describe plasmin-induced inhibition of platelet responses to thrombin, but potentiation of responses to other aggregating agents. Washed human platelets were labeled with 14C-serotonin, treated for 30 min at 37° C with 0, 0.1 or 0.2 CU/ml of plasmin, followed by aprotinin, washed and resuspended in a Tyrode-albumin solution with apyrase. Incubation with 0.2 CU/ml of plasmin almost completely inhibited thrombin-induced (0.1 U/ml) aggregation, release of 14C-serotonin, and increase in cytosolic [Ca2+]. In contrast, with plasmin-pretreated platelets, aggregation and release of 14C-serotonin were strongly potentiated in response to low concentrations of the thrombin receptor-activating peptide SFLLRN, ADP, platelet-activating factor, collagen, arachidonic acid, the thromboxane mimetic U46619, and the calcium ionophores A23187 and ionomycin. Aspirin or RGDS partially inhibited potentiation. Plasmin-pretreated platelets resuspended in plasma anticoagulated with FPRCH2C1 (PPACK) also showed enhanced responses to aggregating agents other than thrombin. The contrasting effects on responses to thrombin and SFLLRN are noteworthy. Plasmin cleaves GPIIb/IIIa so that it becomes a competent fibrinogen receptor, and binding of 125I-fibrinogen during ADP-induced aggregation was greatly potentiated within 10 s. Potentiation of aggregation by other agonists may be due to increased binding of released fibrinogen. Thus, platelets freed from a thrombus may have increased responsiveness to low concentrations of aggregating agents other than thrombin. These results provide further support for the use of inhibitors of platelet reactions in conjunction with administration of fibrinolytic agents.

Stimulation of calcium influx by platelet activating factor in Dictyostelium

1995 ◽  
Vol 108 (4) ◽  
pp. 1597-1603
Author(s):  
R. Schaloske ◽  
C. Sordano ◽  
S. Bozzaro ◽  
D. Malchow
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Dictyostelium Discoideum ◽  
Calcium Influx ◽  
Platelet Activating Factor ◽  
Inactive Compound ◽  
Time Period ◽  
Dependent Pathway ◽  
Stimulation Of

Platelet activating factor (PAF) induces Ca2+ influx in Dictyostelium discoideum. In this investigation we used this activity to analyze the mechanism of PAF action. We found that PAF activity was confined to the period of spike-shaped oscillations and suggest that the role of PAF is to augment cAMP relay. PAF seems to act only a few times during this time period of two hours, since Ca2+ entry adapted to a subsequent stimulus for about 30 minutes. PAF showed a reduced response in the G protein beta- strain LW14 and was unable to induce Ca2+ influx in the G alpha 2- strains HC85 and JM1. The latter expresses the cAMP receptors cAR1 constitutively, and exhibits cAMP-induced Ca2+ influx, albeit at a reduced level. In order to decide whether the inability of PAF to elicit a Ca2+ response in JM1 cells was due to the lack of differentiation and/or the lack of G alpha 2, we inhibited the IP3-dependent pathway with compound U73122 and found that Ca2+ entry was blocked, whereas a closely related inactive compound, U73343, did not alter the response. In agreement with this, NBD-Cl, an inhibitor of Ca2+ uptake into the IP3-sensitive store in Dictyostelium, also abolished PAF activity. The latter was not inhibited by the plasma membrane antagonists BN-52021 or WEB 2170. Therefore PAF seems to operate intracellularly via the IP3-signalling pathway at or upstream of the IP3-sensitive store.

scholarly journals Econazole inhibits thapsigargin-induced platelet calcium influx by mechanisms other than cytochrome P-450 inhibition

1993 ◽  
Vol 295 (2) ◽  
pp. 525-529 ◽  
Author(s):  
J G Vostal ◽  
J C Fratantoni
Keyword(s):  
Plasma Membrane ◽  
Antifungal Agents ◽  
Calcium Influx ◽  
Calcium Stores ◽  
Alternative Mechanism ◽  
Resting Cells ◽  
Calcium Efflux ◽  
Plasma Membrane Permeability ◽  
Cytochrome P 450 ◽  
Internal Calcium

Cytochrome P-450 has been suggested as a mediator of the signal between depleted platelet calcium stores and an increase in plasma membrane permeability to calcium which follows depletion of the stores. This hypothesis is based on the observations that inhibitors of cytochrome P-450, such as the imidazole antifungal agents, also inhibit influx of a calcium surrogate (manganese) into calcium-depleted platelets. We tested the effects of econazole and of a cytochrome P-450 inhibitor, carbon monoxide (CO), on thapsigargin (TG)-induced platelet 45Ca2+ influx. TG specifically depletes internal calcium stores and activates store-regulated calcium influx. Econazole blocked 45Ca2+ influx when it was added before TG (IC50 11 microM). Econazole at a concentration (20 microM) that inhibited 83% of TG-induced calcium influx was not inhibitory to TG-induced calcium efflux from 45Ca(2+)-loaded platelets, and did not affect calcium fluxes in resting platelets. This econazole concentration was also inhibitory to calcium influx even when it was added after the stores had been calcium-depleted by EGTA and TG for 15 min and the signal to increase calcium influx had already been generated. Inhibition of cytochrome P-450 with CO bubbled through platelet suspensions did not change calcium influx in resting cells and potentiated TG-induced calcium influx (160% of control calcium accumulation at 20 min). This effect appeared to be concentration-dependent, such that a 5 min exposure to CO produced a greater influx potentiation than a 3 min exposure. These observations indicate that (1) cytochrome P-450 does not mediate store-regulated calcium influx, and (2) econazole probably inhibits store-regulated calcium influx by an alternative mechanism, such as interaction with plasma membrane calcium channels.

Characteristics of Thrombin-Degranulated Human Platelets: Development of a Method that Does not Use Proteolytic Enzymes for Deaggregation

1991 ◽  
Vol 65 (04) ◽  
pp. 403-410 ◽  
Author(s):  
Raelene L Kinlough-Rathbone ◽  
Marian A Packham ◽  
Maria A Guccione ◽  
Mary Richardson ◽  
Elizabeth J Harfenist ◽  
...  
Keyword(s):  
Proteolytic Enzymes ◽  
Platelet Activating Factor ◽  
Calcium Ionophore ◽  
Human Platelets ◽  
Dense Granule ◽  
Electron Microscopic ◽  
Human Fibrinogen ◽  
Platelet Surface ◽  
Low Concentrations ◽  
The Stability

SummaryA method has been developed for preparing suspensions of washed human platelets that have lost as much as 90% of their dense granule and alpha granule contents as a result of stimulation by thrombin (0.9 U/ml for 3 min at 37° C), and recovering the platelets without using a proteolytic erwyme. Glycyl-Lprolyl-Larginyl-L-proline (GPRP) was used to prevent polymerization of released fibrinogen and arginyl-glycyl-aspartyl-serine (RGDS) to block the interaction of released fibrinogen, vWf or fibronectin with the glycoprotein IIb/IIIa complex. The thrombin used to degranulate the platelets was neutralized with D-phenylalanyl-Lprolyl-L-arginine chlororhethyl ketone (FPRCH2CI) and prostaglandin E1 was added to return the platelets towards a disc shape. The degranulated platelets aggregated in response to ADR platelet activating factor, arachidonate and the thromboxane 42 mimetic, U46619 in the presence of added fibrinogen; the platelets changed shape but did not aggregate in response to collagen. Thrombin and the calcium ionophore, A23L87, caused aggregation without added fibrinogen. Synergism between pairs of aggregating agents at low concentrations was observed. Little TXB2 was formed when the platelets were reaggregated by thrombin. RGDS and F(ab’)2 fragments of an antibody to fibrinogen inhibited reaggregation induced by thrombin and A23187 indicating that small amounts of fibrinogen at the platelet surface may support aggregation by strong agonists. Adherence of thrombin-degranulated platelets to a collagen-coated surface was less than for controls, but spreading was more extensive. Electron-microscopic immunogold cytochemistry with anti-human fibrinogen IgG showed numerous gold particles in platelet vacuoles. Thrombin-degranulated platelets can be used to study pathways involved in platelet aggregation without the complicating effects of released granule contents including ADR and to study indirectly the factors released from platelets that contribute to the stability of platelet aggregates.

Evidence for two distinct modalities of CA2+ influx into pancreatic B cell

1982 ◽  
Vol 242 (1) ◽  
pp. E59-E66 ◽  
Author(s):  
P. Lebrun ◽  
W. J. Malaisse ◽  
A. Herchuelz
Keyword(s):  
B Cell ◽  
Glucose Concentration ◽  
Islet Cells ◽  
Inhibitory Effect ◽  
Low Concentrations ◽  
45Ca Efflux ◽  
Selective Blocker ◽  
Two Populations ◽  
Ca2 Channels ◽  
Stimulation Of

The pathways through which glucose stimulates Ca2+ inflow into islet cells were investigated by comparing the inhibitory effect of verapamil, a selective blocker of voltage-sensitive Ca2+ channels, on glucose- and K+-stimulated insulin release and 45Ca efflux from perifused rat pancreatic islets. The islets stimulated by K+ (20 mM) were more sensitive to verapamil than those exposed to glucose (27.8 mM). The stimulation of 45Ca efflux by a low concentration of glucose (8.3 mM) was extremely resistant to verapamil, whereas that induced by a rise in the glucose concentration from 8.3 to 27.8 mM displayed the same sensitivity towards verapamil as that characterizing the response to K+. Because the increase in 45Ca efflux evoked by glucose or K+. Because the increase in 45Ca efflux evoked by glucose or K+ reflects a stimulation of Ca2+ entry into islet cells, it is proposed that the B cell may be equipped with two populations of Ca2+ channels that differ in their sensitivity towards verapamil and possibly their voltage-dependency. Glucose apparently stimulates Ca2+ inflow through both types of channels. At low concentrations, glucose may stimulate Ca2+ inflow, in part, by gating the voltage-insensitive pathway.

scholarly journals Pharmacology of a Ca2+-influx pathway activated by emptying the intracellular Ca2+ stores in HL-60 cells: evidence that a cytochrome P-450 is not involved

1994 ◽  
Vol 302 (1) ◽  
pp. 187-190 ◽  
Author(s):  
B D Koch ◽  
G F Faurot ◽  
M V Kopanitsa ◽  
D C Swinney
Keyword(s):  
Plasma Membrane ◽  
Selective Inhibitors ◽  
High Affinity ◽  
Voltage Dependent ◽  
Influx Pathways ◽  
Subsequent Activation ◽  
Cytochrome P 450 ◽  
Haem Iron ◽  
Dependent Pathway ◽  
Ca2 Channels

In HL-60 cells, inhibition of the endoplasmic-reticular Ca2+ pump by thapsigargin leads to the emptying of this intracellular Ca2+ store and a subsequent activation of plasma-membrane Ca2+ influx through a non-voltage-dependent pathway. The elevated intracellular free Ca2+ concentration ([Ca2+]i) produced and maintained by this Ca2+ inflow was used to examine the potency of various compounds to inhibit this influx mechanism. As expected, specific blockers of known Ca2+ channels, such as nifedipine, omega-conotoxin GVIA and ryanodine were without effect. The less selective inhibitors La3+, SKF-96365 and L-651,582, which are thought to inhibit both voltage-dependent and voltage-independent Ca2+ channels, decreased [Ca2+]i back to resting levels, with pIC50 values of 5.2, 5.9 and 6.2 respectively. It has been proposed that a cytochrome P-450 is involved in activating Ca(2+)-influx pathways in thymocytes, neutrophils and platelets. Consistent with this idea, the imidazole cytochrome P-450 inhibitors miconazole, econazole, clotrimazole and ketoconazole inhibited the thapsigargin-elevated [Ca2+]i with pIC50 values of 7.1, 7.1, 7.1 and 5.8 respectively. The high affinity of imidazoles for cytochromes P-450 is due to co-ordinate binding to the haem. This interaction is greatly decreased in 2-substituted imidazoles. We examined whether the inhibition of Ca2+ influx was due to an interaction of the inhibitor imidazole nitrogen with the haem iron of the putative cytochrome P-450 by comparing the activity of two compounds, identical except that one was methylated at the imidazole 2-position. They were found to block thapsigargin-activated Ca2+ influx with equal potency. These results strongly suggest that a cytochrome P-450 is not involved in the activation of the Ca2+ influx produced by emptying the intracellular Ca2+ stores.

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