Phosphoinositide metabolism in resting and thrombin-stimulated human platelets: Evidence for metabolic homogeneity

Tysnes, Q-B; Verhoeven, Adrie J.M.; Aarbakke, Grethe M.; Holmsen, Holm

doi:10.1055/s-0038-1644517

Cited by 2 publications

(8 citation statements)

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“…The recovery was 100 f 9%, 102 f 11%, 93 5 6% and 56 f 5% (n = 6) for PtdIns(4,5)P2, PtdIns4P, PtdIns and X, respectively. (X is an as-yet unidentified phospholipid, that co-chromatographs with PtdIns4P in several solvent systems, but clearly separates from PtdIns4P in the solvents used here [20]. Since this compound can also be labelled in intact platelets with myo-inositol, it is tentatively assigned to the class of phosphoinositol lipids).…”

Section: P-labelled Inositol Lipidsmentioning

confidence: 99%

“…[20]). In contrast to the abovementioned solvent, this system gives good separation of PtdIns and (non-labelled) acylGroPEtn, so that acylGroPEtn does not interfere with subsequent phosphorus determinations.…”

Section: Amount Of Phosphoinositol Lipidsmentioning

confidence: 99%

“…HCI(20: 10: 1, by vol. [20]) and extraction of known amounts of these fractions together with unlabelled, gel-filtered platelets as described above. The recovery was 100 f 9%, 102 f 11%, 93 5 6% and 56 f 5% (n = 6) for PtdIns(4,5)P2, PtdIns4P, PtdIns and X, respectively.…”

Section: P-labelled Inositol Lipidsmentioning

confidence: 99%

“…Platelet-rich plasma was prepared as described previously [20], and incubated for 60min at 37°C with 1 pM 12-3H]adenine (specific radioactivity 3.5 Ci/mmol. New England Nuclear, code NET-350) and/or 0.05 mCi/ml of [32P]orthophosphate (carrier-free, Amersham International, code PBS.11).…”

Section: Platelet Isolationmentioning

confidence: 99%

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Turnover of the phosphomonoester groups of polyphospoinositol lipids in unstimulated human platelets

et al. 1987

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The metabolic activity of the polyphosphoinositol lipids in unstimulated human platelets was studied by shortterm labelling with [32P]Pi, by replacement of [32P]Pi from pre-labelled platelets with unlabelled phosphate, and by depriving the cells of metabolic ATP.1. Under short-term labelling conditions, the 4-and 5-phosphate groups of phosphatidylinositol4-phosphate (PtdIns4P) and phosphatidylinositol4,5-bisphosphate [PtdIns(4,5)Pz] had the same specific 32P radioactivity as the y-phosphate of metabolic ATP. The specific 32P radioactivity of the 1-phosphates of phosphatidylinositol, PtdIns4P and PtdIns(4,5)P2 was similar, but only 4 -13% compared to that of the ATP-y-phosphate.2. When [32P]Pi pre-labelled platelets were incubated with up to 25 mM of unlabelled phosphate, the displacement of the 32P label from PtdIns4P, PtdIns(4,5)P2 and metabolic ATP followed similar kinetics.3. Inhibition of ATP regeneration in platelets pre-labelled with [32P]Pi resulted in a rapid fall in metabolic ATP with a much slower fall in [32P]PtdIns(4,5)Pz, whereas [32P]PtdIns4P increased initially. However, ATP turnover was not abolished, as indicated by the marked (25% of the control) incorporation of extracellular [32P]Pi into PtdIns4P and PtdIns(4,5)Pz in metabolically inhibited platelets. This low phosphate turnover may explain the relative resistance of PtdIns4P and PtdIns(4,5)P2 to metabolic inhibition. We conclude that PtdIns4P and PtdIns(4,5)Pz are present as a single metabolic pool in human platelets. Turnover of the 4-and 5-phosphates of PtdIns4P and PtdIns(4,5)P2 in unstimulated platelets is as rapid as that of the y-phosphate of metabolic ATP, and accounts for about 7% of basal ATP consumption.The polyphosphoinositol lipids constitute a minor fraction of total cell phospholipids. Nonetheless, important functions are assigned to them, such as receptor-mediated transmembrane signalling (reviewed in [l]), facilitation of exocytotic processes [2, 31, the anchorage of membrane-associated proteins [4 -81 and the regulation of ion transport [9]. The polyphosphoinositol lipids are highly dynamic and undergo continuous phosphorylation and dephosphorylation. PtdIns is converted in two separate ATP-dependent reactions to PtdIns4P and PtdIns(4,5)P2. The latter are dephosphorylated again, thereby constituting 'futile' cycles in which two moles of ATP are consumed for each mole of recycled PtdIns. This cycling was estimated by Maretzki et al. [lo] to account for as much as 20% of basal ATP consumption in mature erythrocytes, but in more thorough studies Muller et al. [ll] and Dale [I21 found much lower values of 2-5% and 0.5-1 %, respectively. The relative importance of phosphoinositol lipid cycling for total ATP consumption in other cell types has not yet been quantified.In platelets, the polyphosphoinositol lipids are thought to be crucial in signal processing. Upon stimulation the steady-

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Section: P-labelled Inositol Lipidsmentioning

confidence: 99%

Section: Amount Of Phosphoinositol Lipidsmentioning

confidence: 99%

Section: P-labelled Inositol Lipidsmentioning

confidence: 99%

Section: Platelet Isolationmentioning

confidence: 99%

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Turnover of the phosphomonoester groups of polyphospoinositol lipids in unstimulated human platelets

et al. 1987

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“…5553) in chloroform/methanol/20% methylamine (60: 36: 10) as described previously [9]. In this system PtdOH was insufficiently separated from phosphatidylserine [9]. Therefore, PtdOH was separated by the two-dimensional chromatography system of…”

mentioning

confidence: 99%

Rates of production and consumption of phosphatidic acid upon thrombin stimulation of human platelets

Tysnes

Verhoeven

Holmsen

1988

European Journal of Biochemistry

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Human platelets were labelled with [32P]Pi and [3H]glycerol before gel filtration. In unstimulated cells, the specific 32P radioactivity in phosphatidic acid (PtdOH) was similar to that of phosphatidylinositol (PtdIns) but only 4% of that of the y-phosphate of ATP. Upon 3 min of stimulation with 0.5 U/ml of thrombin, there was a 20-fold increase in specific 32P radioactivity of PtdOH which approached that of the ATP y-phosphate. Based on constant rates of synthesis and removal, this thrombin-induced increase in specific 32P radioactivity in PtdOH allowed us to calculate the flux of phosphate through PtdOH upon stimulation. Synthesis and removal occurred at rates of 107 and 52 nmol min-'/lO" cells, respectively. The specific [3H]glycerol radioactivity was similar in PtdIns, phosphatidylinositol4-phosphate and phosphatidylinositol4,5-bisphosphate in unstimulated platelets. In PtdOH, it was 50% of that of the inositol phospholipids. Thrombin stimulation induced no changes in the specific 3H radioactivity of the inositol phospholipids whereas specific [3H]PtdOH increased to the level of these lipids. It is concluded that PtdIns, PtdInsP and PtdInsP2 exist in a metabolic homogenous pool in human platelets.It is now generally accepted that the first step in agonistinduced alterations in inositol phospholipid metabolism is the phosphodiesteratic cleavage of PtdInsP2 to yield diacylglycerol and inositol 1,4,5-trisphosphate (for review, see [l]). Whereas this inositol phosphate is believed to release Ca2+ from intracellular stores (for review, see [2, 3]), diacylglycerol serves as second messenger in the activation of protein kinase C [4] and is rapidly removed, mainly through phosphorylation to PtdOH in the diacylglycerol kinase reaction [5]. PtdOH is in turn metabolized through CDPdiacylglycerol to PtdIns which finally can be phosphorylated to PtdInsP and PtdInsP2. These reactions are generally referred to as the 'polyphosphoinositide cycle'.Whereas the agonist-induced changes in actual concentration of the metabolites of this cycle now seem well established, little is known about the flux through the system, i. e. rates of synthesis and degradation of the various metabolites. We have previously determined the rate of PtdIns production upon thrombin stimulation of platelets [6]. This was based on differences in specific radioactivity between the diester phosphate of PtdIns and the ATP y-phosphate. Upon stimulation of platelets, there is an extensive formation of PtdOH from diacylglycerol [2, 71. This production is crucial to the cell since it probably represents the major pathway to remove diacylglycerol [6,8], thereby controlling the activity of protein kinase C. In the present study we show that the monoesterified phosphate of PtdOH is far from being in metabolic equilibrium with the y-P of ATP in unstimulated platelets. This metabolic non-equilibrium situation enabled us to determine Correspondence to 0.-B. Tysnes, Biokjemisk Institutt, Universitetet i Bergen, Arstadveien 19, N-5000 Bergen, NorwayAbbreviations. PtdI...

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Phosphoinositide metabolism in resting and thrombin-stimulated human platelets: Evidence for metabolic homogeneity

Cited by 2 publications

References 0 publications

Turnover of the phosphomonoester groups of polyphospoinositol lipids in unstimulated human platelets

Turnover of the phosphomonoester groups of polyphospoinositol lipids in unstimulated human platelets

Rates of production and consumption of phosphatidic acid upon thrombin stimulation of human platelets

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