scholarly journals Liberation of [3H]arachidonic acid and changes in cytosolic free calcium in fura-2-loaded human platelets stimulated by ionomycin and collagen

1986 ◽  
Vol 235 (3) ◽  
pp. 869-877 ◽  
Author(s):  
W K Pollock ◽  
T J Rink ◽  
R F Irvine
Keyword(s):  
Fold Increase ◽  
Human Platelets ◽  
Free Calcium ◽  
Fura 2 ◽  
Kinase C ◽  
20 Nm ◽  
Indicator Dye ◽  
Arachidonate Release ◽  
Instantaneous Concentration ◽  
Stimulation Of

Cytosolic Ca2+ levels and arachidonate liberation were investigated in platelets loaded with the fluorescent Ca2+ indicator dye fura-2, and labelled with [3H]arachidonate. Fura-2 was used in preference to quin2 because the latter interfered with [3H]arachidonate labelling of phospholipids. From a resting free Ca2+ level of around 100 nM, ionomycin (10-200 nM) evoked an instantaneous, concentration-dependent increase in cytosolic Ca2+ that only resulted in [3H]arachidonate liberation (up to 4-fold over control) at Ca2+ levels greater than 1 microM. Addition of collagen (10 micrograms/ml) evoked an elevation in Ca2+ up to 461 +/- 133 nM. These changes in Ca2+ were accompanied by a 2-4-fold elevation in [3H]arachidonate with depletion of [3H]phosphatidylcholine by 17 +/- 4% and [3H]phosphatidylinositol by 41 +/- 7%. Indomethacin (10 microM) reduced the elevation in Ca2+ by collagen to 115 +/- 18 nM but did not significantly inhibit the 2-4-fold increase in [3H]arachidonate. [3H]Phosphatidylcholine and [3H]phosphatidylinositol were decreased by 9 +/- 7% and 10 +/- 6%, respectively, with collagen in the presence of indomethacin. Stimulation of phosphoinositide turnover by collagen in the presence and absence of indomethacin was indicated by [32P]phosphatidate formation in cells prelabelled with [32P]Pi. This phosphatidate formation was decreased (75%) by the presence of indomethacin. In the presence of indomethacin, phorbol myristate acetate (20 nM) alone or in combination with ionomycin (30 nM) failed to stimulate arachidonate liberation despite a marked stimulation of aggregation. These results indicate that, whereas ionomycin requires Ca2+ in the microM range for arachidonate liberation, collagen, notably in the presence of indomethacin, does so at basal Ca2+ levels. The mechanisms underlying the regulation of arachidonate release by collagen are not clear, but do not appear to involve activation of protein kinase C, or an elevation of cytosolic free Ca2+.

scholarly journals A novel tumour promoter, thapsigargin, transiently increases cytoplasmic free Ca2+ without generation of inositol phosphates in NG115-401L neuronal cells

1988 ◽  
Vol 253 (1) ◽  
pp. 81-86 ◽  
Author(s):  
T R Jackson ◽  
S I Patterson ◽  
O Thastrup ◽  
M R Hanley
Keyword(s):  
Inositol Phosphates ◽  
Fluorescent Indicator ◽  
Intracellular Store ◽  
Non Invasive ◽  
Kinase C ◽  
Tumour Promoters ◽  
20 Nm ◽  
Indicator Dye ◽  
Hydrolysis Of ◽  
Stimulation Of

Thapsigargin, a sesquiterpene lactone with potent irritant and tumour-promoting activities, stimulates a rapid (within 15 s) transient increase in intracellular [Ca2+] in the NG115-401L neural cell line, as measured by the fluorescent indicator dye fura-2. This increase in cytoplasmic free [Ca2+] is concentration-dependent (ED50 around 20 nM) and occurs in the absence of extracellular Ca2+. Activation of NG115-401L cells by the inflammatory peptide bradykinin generates inositol phosphates, which parallel increases in intracellular [Ca2+]. However, the rise in cytoplasmic [Ca2+] stimulated by thapsigargin occurs in the absence of detectable production of inositol phosphates. Thapsigargin is unlike phorboid tumour promoters in that it has no action on two non-invasive indicators of phorbol stimulation of these cells, i.e. [3H]choline metabolite production and rise in intracellular pH. These data suggest that thapsigargin releases Ca2+ from an intracellular store by a novel mechanism, independent of the hydrolysis of phosphoinositides and concomitant activation of protein kinase C. Thus thapsigargin may provide a valuable tool for the analysis of intracellular signalling mechanisms.

scholarly journals Desensitization and antagonism of vasopressin-induced phosphoinositide metabolism and elevation of cytosolic free calcium concentration in human platelets

1986 ◽  
Vol 234 (1) ◽  
pp. 67-73 ◽  
Author(s):  
W K Pollock ◽  
D E MacIntyre
Keyword(s):  
Platelet Activation ◽  
Human Platelet ◽  
Receptor Occupancy ◽  
Human Platelets ◽  
Free Calcium ◽  
Phosphoinositide Metabolism ◽  
Subsequent Challenge ◽  
Free Calcium Concentration ◽  
20 Nm ◽  
Stimulation Of

The receptor mechanisms underlying vasopressin-induced human platelet activation were investigated with respect to stimulation of phosphoinositide metabolism and changes in the cytosolic free Ca2+ concentration ([Ca2+]i). Vasopressin stimulated phosphoinositide metabolism, as indicated by the early formation of [32P]phosphatidic acid ([32P]PtdA) and later accumulation of [32P]phosphatidylinositol ([32P]PtdIns). In addition, vasopressin elicited a transient depletion of [glycerol-3H]PtdIns and accumulation of [glycerol-3H]PtdA. The effects of vasopressin on phosphoinositide metabolism were concentration-dependent, with half maximal [32P]PtdA formation occurring at 30 +/- 15 nM-vasopressin. In the presence of 1 mM extracellular free Ca2+, vasopressin induced a rapid, concentration-dependent elevation of [Ca2+]i in quin2-loaded platelets: half-maximal stimulation was observed at 53 +/- 20 nM-vasopressin. The V1-receptor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine,8-arginine]-vasopressin selectively inhibited vasopressin (100 nM)-induced [32P]PtdA formation [I50 (concn. giving 50% inhibition) = 5.7 +/- 2.4 nM] and elevation of [Ca2+]i (I50 = 3 +/- 1.5 nM). Prior exposure of platelets to vasopressin rendered them unresponsive, in terms of [32P]PtdA formation and elevation of [Ca2+]i, to a subsequent challenge with vasopressin, but responsive to a subsequent challenge with U44069, a thromboxane-A2 mimetic. These results indicate that vasopressin-induced human platelet activation is initiated by combination with specific V1 receptors on the platelet, and that the sequelae of receptor occupancy (stimulation of phosphoinositide metabolism and elevation of [Ca2+]i) are equally susceptible to inhibition by receptor antagonists and by receptor desensitization.

Increased Aggregation and Secretion Responses of Human Platelets when Loaded with the Calcium Fluorescent Probes Quin2 and Fura-2

1987 ◽  
Vol 58 (02) ◽  
pp. 737-743 ◽  
Author(s):  
Frarnçois Lanza ◽  
Alain Beretz ◽  
Martial Kubina ◽  
Jean-Pierre Cazenave
Keyword(s):  
Intracellular Calcium ◽  
Shape Change ◽  
Calcium Ionophore ◽  
Human Platelets ◽  
Arachidonic Acid Metabolism ◽  
Free Calcium ◽  
Membrane Bilayer ◽  
Fluorescent Indicators ◽  
Fura 2 ◽  
Low Concentrations

SummaryIncorporation into human platelets of the calcium fluorescent indicators quin2 or fura-2 at low concentrations used to measure intracellular free calcium leads to the potentiation of the effects of agonists on platelets. This was shown by increased aggregatory and secretory responses of quin2 or fura-2 loaded platelets after stimulation with ADP, PAP and with low concentrations of thrombin, collagen, the endoperoxide analog U-46619 and the calcium ionophore A 23187. Quin2 and fura-2 mediated platelet sensitisation could be due to altered arachidonic acid metabolism since it was inhibited by prior treatment with the cydooxygenase inhibitor acetylsalicylate. In contrast, platelets loaded with higher concentrations of calcium chelators exhibited diminished aggregation responses to all aggregating agents. This latter effect was accompanied by increased fluidity of the platelet plasma membrane bilayer and by the exposure of a new pool of membranes to the outer surface of platelets, as monitored with trimethylammonium- diphenylhexatriene (TMA-DPH) in platelets loaded with the non-fluorescent calcium probe analog MAPT. In contrast, low concentrations of quin2 did not potentiate shape change of platelets activated with ADP. Thus, shape change and aggregation can be influenced separately by intracellular Ca2+ chelators. We conclude that platelet responses are altered by the incorporation of intracellular calcium chelators at concentrations used to monitor intracellular calcium changes.

THE ROLE OF IONISED INTRACELLULAR FREE CALCIUM ([Ca++]i) IN THROMBIN-INDUCED DENSE GRANULE SECRETION

1987 ◽  
Author(s):  
C T Poll ◽  
J Westwick
Keyword(s):  
Human Platelets ◽  
Dense Granule ◽  
Free Calcium ◽  
Fluorescent Properties ◽  
Fura 2 ◽  
Stimulus Response ◽  
Dense Granules ◽  
Dense Granule Secretion ◽  
Granule Secretion

Fura 2 is one of a recently-introduced family of Ca++ indicators with improved fluorescent properties compared to quin 2 (Grynkiewicz et al 1985). This study has examined the role of [Ca++]i in thrombin-induced dense granule release using prostacyclin-washed human platelets loaded with either thedense granule marker 14C-5HT (5HT) alone or with 5HT together with quin 2 ([quin2]i = 0.8mM) or fura 2 ([fura 2]i 20-30µM). In the presence of ImM extracellular calcium concentration ([Ca++]i) the [Ca++]e in quin 2 and fura 2 loaded platelets was 93±2 (n=10 experiments) and 133±0.3nM (n=12 experiments) respectively. In either quin 2 or fura 2 loaded platelets suspended in the presence of ImM [Ca++]e, thrombin (0.23-23.InM) promoted a rapid (in secs)concentration-dependent elevation of [Ca++]i from basal values to levels l-2µM, together with a parallel release of dense granules almost identical to that obtained with thrombin in non dye loaded platelets. In fura 2 loaded cells, removal of [Ca++]e inhibited the elevation of [Ca++]i induced by a sub-maximal concentration of thrombin (0.77nM) by 43+5% (n=4) but interestingly had no significant effect (p<0.05) on the rise in [Ca++]i elicited by low thrombin doses (0.231nM). Neither did lowering [Ca++]e inhibit the release of 5HT evoked by thrombin ( 0.231-23.InM) from either fura 2 loaded or non dye loaded platelets. In contrast, in quin 2 loaded platelets, removal of [Ca++]e inhibited the thrombin (0.231-23.InM) stimulated rise in [Ca++]i-by 90% and the 5HT release response to either low (0.231nM), sub-maximal (0.77nM) or maximal (23.InM) thrombin by 100% (n=4), 87+2°/o (n=6)and 2+l°/o (n=4) respectively. Fura 2 but not quin 2 loaded cells suspended in ImM [Ca++]e exhibited a Ca++ response to thrombin concentrations >2.31nM which could be separated into a rapid phasic component and a more sustained 'tonic' like component inhibitable by removal of [Ca++]e or by addition of ImM Ni++ . These data suggest the use of fura 2 rather than quin 2 for investigating stimulus response coupling in platelets, particularly when [Ca++]e is less than physiological. We thank the British Heart Foundation and Ciba-Geigy USA for financial support.

Phospholipases in human parturition

1990 ◽  
Vol 2 (5) ◽  
pp. 511 ◽  
Author(s):  
T Wilson
Keyword(s):  
Protein Kinase C ◽  
Phospholipase A2 ◽  
Main Role ◽  
Kinase C ◽  
Arachidonate Release ◽  
Synthesis Regulation ◽  
External Calcium ◽  
Human Parturition ◽  
Stimulation Of

Phospholipase A2 plays a major role in controlling PG synthesis. Regulation of the activity of this enzyme probably holds the key to the onset of labour. Both PLA2 and PLC can contribute to arachidonate release and PG production in cells, but PLA2 appears to be the main role of synthesis. Phospholipase A2 and PLC can be activated independently of each other; an influx of external calcium is required for PLA2 activation. It is suggested that PLC contributes to PG synthesis through product stimulation of protein kinase C which maintains a pool of free arachidonate by inhibiting reincorporation into the cell membrane. The regulatory role for lipocortin in phospholipase inhibition is controversial and unlikely to be relevant to the onset of labour.

scholarly journals Distinctive effects of glucagon on gluconeogenesis and ketogenesis in hepatocytes isolated from normal and biotin-deficient rats

1978 ◽  
Vol 172 (3) ◽  
pp. 517-521 ◽  
Author(s):  
E A Siess ◽  
D G Brocks ◽  
O H Wieland
Keyword(s):  
Dose Response ◽  
Redox State ◽  
Fold Increase ◽  
Primary Site ◽  
Hormone Action ◽  
Response Curves ◽  
Mitochondrial Redox State ◽  
Dose Response Curves ◽  
20 Nm ◽  
Stimulation Of

In hepatocytes from 48 h-starved rats identical glucagon dose-response curves were obtained for the stimulation of gluconeogenesis from lactate, for ketogenesis and for the decreasing of the C5-dicarboxylate pool. Glucagon (20 nM) caused a 5-fold increase in 3-hydroxybutyrate formation, but decreased acetoacetate production 50% of that of the control. In hepatocytes from biotin-deficient rats glucagon no longer stimulated gluconeogenesis from lactate, but still produced its effects on the mitochondrial redox state and the C5-dicarboxylate pool. The results suggest that the primary site of the hormone action on gluconeogenesis is located within the mitochondria rather than in the cytosol.

scholarly journals Caerulein and carbamoylcholine stimulate pancreatic amylase release at resting cytosolic free Ca2+

1986 ◽  
Vol 235 (1) ◽  
pp. 139-143 ◽  
Author(s):  
R Bruzzone ◽  
T Pozzan ◽  
C B Wollheim
Keyword(s):  
Fold Increase ◽  
Free Calcium ◽  
Amylase Secretion ◽  
Rapid Phase ◽  
Secretory Rate ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Significant Stimulation ◽  
Phase 0 ◽  
Stimulation Of

Cytosolic free calcium concentrations ([Ca2+]i) and amylase secretion were measured in isolated rat pancreatic acini loaded with the intracellularly trapped fluorescent indicator quin2. Both caerulein and carbamoylcholine caused a rapid increase in [Ca2+]i, with a maximal 3-fold increase at 10(-9) M-caerulein and 10(-4) M-carbamoylcholine. However, caerulein (10(-12) M and 10(-11) M) as well as carbamoylcholine (10(-7) M) caused a significant stimulation of amylase release, while not inducing any detectable rise in [Ca2+]i. Changes in [Ca2+]i after addition of either secretagogue were transient and did not last more than 2-3 min. By contrast, when amylase secretion was monitored as a function of time, two distinct secretory phases could be observed upon addition of either carbamoylcholine (10(-5) M) or caerulein (10(-10) M). An initial, rapid phase (0-5 min) which caused a 6-7-fold increase above basal, followed by a sustained (5-30 min), but less marked, secretory rate (2-3-fold above basal). Addition of atropine (10(-4) M) 5 min after carbamoylcholine (10(-5) M) (i.e. after termination of the rise in [Ca2+]i and of the first secretory phase) did not cause any significant change in [Ca2+]i, while significantly inhibiting amylase secretion from 5 to 30 min to the same rate observed in the absence of the secretagogue. These results show that caerulein and carbamoylcholine, two agents thought to activate secretion mainly through mobilization of Ca2+ from intracellular stores, are capable of eliciting amylase secretion independently of a concomitant rise in [Ca2+]i. Furthermore, with both secretagogues the rise in [Ca2+]i, when observed, was only transient, while the stimulation of amylase release was sustained.

scholarly journals Spiking in cytosolic calcium concentration in single fibrinogen-bound fura-2-loaded human platelets

1992 ◽  
Vol 283 (2) ◽  
pp. 379-383 ◽  
Author(s):  
J W Heemskerk ◽  
J Hoyland ◽  
W T Mason ◽  
S O Sage
Keyword(s):  
Calcium Concentration ◽  
Calcium Signalling ◽  
Peak Frequency ◽  
Cytosolic Calcium ◽  
Light Level ◽  
Optical Probe ◽  
Human Platelets ◽  
Free Calcium ◽  
Fura 2 ◽  
Subsequent Activation

Fura-2-loaded human platelets were immobilized on a fibrinogen-coated surface and the cytosolic free calcium concentration ([Ca2+]i) was measured in single platelets by low-light-level video-ratio image-processing of the optical probe signal. Some fibrinogen-bound platelets showed repetitive spiking in [Ca2+]i with a mean frequency of about 2/min, which increased to 5/min in the presence of ADP. Other cells showed no activity until the addition of agonist. When immobilized in the presence of prostaglandin I2 and the fibrinogen antagonist Arg-Gly-Asp-Ser, the platelets adhered less firmly to fibrinogen, and in many [Ca2+]i remained low and constant. Subsequent activation of such platelets with ADP evoked oscillations in [Ca2+]i with a peak frequency of about 5/min and which persisted for at least 5 min. These results indicate that human platelets, like many other non-excitable cells, have an elaborate system of calcium signalling involving spiking.

scholarly journals Endotoxic lipid A induces intracellular Ca2+ increase in human platelets

1991 ◽  
Vol 278 (1) ◽  
pp. 75-80 ◽  
Author(s):  
M Romano ◽  
M Molino ◽  
C Cerletti
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Lipid A ◽  
Human Platelets ◽  
Free System ◽  
Fura 2 ◽  
Kinase C ◽  
Activated Platelets ◽  
A Cell ◽  
Protein Kinase C Activation

The activation of protein kinase C by endotoxic lipid A was observed with both intact platelets and in a cell-free system [Romano & Hawiger (1990) J. Biol. Chem. 265, 1765-1770]. We have now studied the action of lipid A on intracellular Ca2+ concentration ([Ca2+]i). Lipid A induced a concentration-dependent rise in [Ca2+]i in human platelets loaded with fura-2, which reached a maximum at 37.1 +/- 3.8 s (tmax). Maximum [Ca2+]i levels, observed at 30 microM lipid A, were 432 +/- 60 nM. EGTA (2 mM) or NiCl2 (1 mM) each decreased the lipid A-dependent elevation of [Ca2+]i by 50-60% without significant modification of tmax, but shortening the time for 50% recovery (t50) from greater than 400 s to 113.1 +/- 29.1 s and 54 +/- 2.1 s, respectively. Quenching of the fura-2 signal was also observed in lipid A-stimulated platelets resuspended with MnCl2 (1 mM), suggesting that both mobilization and external influx of Ca2+ occur. Intracellular Ca2+ mobilization depended on release from Ins(1,4,5)P3-sensitive stores, since Ins(1,4,5)P3 accumulation was detected in lipid A-activated platelets. Staurosporine, an inhibitor of protein kinase C, blocked the [Ca2+]i rise generated by lipid A in platelets [concn. giving 50% inhibition (IC50) = 0.1 microM], prolonging the tmax. to 54.7 +/- 5.1 s, but decreasing the t50 to 157.5 +/- 31.8 s. Staurosporine also suppressed InsP3 accumulation (IC50 = 0.15 microM). These results suggest that platelet activation by lipid A involves an interaction between [Ca2+]i elevation and protein kinase C activation.

High Density Lipoproteins Enhance the Na+/H+ Antiport in Human Platelets

1996 ◽  
Vol 75 (04) ◽  
pp. 635-641 ◽  
Author(s):  
Jerzy-Roch Nofer ◽  
Martin Tepel ◽  
Beate Kehrel ◽  
Michael Walter ◽  
Udo Seedorf ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Human Platelets ◽  
Covalent Modification ◽  
High Density ◽  
High Density Lipoproteins ◽  
Free Calcium ◽  
Sodium Propionate ◽  
Cytosolic Ph ◽  
Kinase C

SummaryIn the present study, we investigated the effect of high density lipoproteins 3 (HDL3) on Na+/H+ exchanger activity and cytosolic pH (pHi) in human platelets. HDL3 alone failed to affect pHi? but preincubation with HDL3 significantly enhanced the Na+/H+ antiport activation brought about by acidification with 100 mM sodium propionate or stimulation with 0.05 U/ml thrombin. The stimulatory effect of HDL3 was unaffected by indomethacin excluding a role for cyclooxygenase products. The HDL3 effect was not mediated by Ca2+/calmodulin-dependent protein kinase as HDL3 failed to increase cytosolic free calcium concentration. However, the potentiating effect of HDL3 was completely blocked in the presence of the protein kinase C inhibitor, bisindoylmaleimide and the phosphatidylcholine-specific phospholi-pase C inhibitor, D609. Furthermore, the effect of HDL3 was abolished after covalent modification of HDL3 with dimethylsuberimidate and was not observed in platelets from Glanzmann thrombasthenia type 1 which do not express GP IIb/IIIa, as well as in platelets preincubated with anti-GP Ilb/IIIa polyclonal antibodies. We conclude that HDL3 enhances the sodium propionate- and thrombin-induced Na+/H+ antiport activity in human platelets via binding to GP Ilb/IIIa and activation of protein kinase C and phosphatidylcholine-specific phospholipase C.

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