ATP-induced apoptosis of thymocytes is mediated by activation of P2 X 7 receptor and involves de novo ceramide synthesis and mitochondria.

Thymocytes were reported to undergo apoptosis in the presence of extracellular ATP through the activation of the purinergic receptors P2 X 1R, P2 X 7R or both. We investigated the identity of the P2 X R and the signaling pathways involved in ATP-mediated apoptosis. Apoptosis elicited by ATP was prevented by inhibition of P2 X 7R, or in thymocytes bearing a mutated P2 X 7R, and reproduced with a P2 X 7R agonist, but not with a P2 X 1R agonist. Stimulation of thymocytes with either ATP or a P2 X 7R agonist was found to stimulate a late de novo ceramide synthesis and mitochondrial alterations. Inhibition of either processes attenuated apoptosis. Interestingly, stimulation with either ATP or a P2 X 1R agonist induced an early ceramide accumulation and a weak caspases-3/7 activation that did not lead to apoptosis. In conclusion, de novo ceramide generation and mitochondrial alterations, both resulting from P2 X 7R activation, were implicated in ATP-induced thymocyte apoptosis.

Flavonoid inhibition of platelet procoagulant activity and phosphoinositide synthesis.

Dietary flavonoids are known for their antiplatelet activity resulting in cardiovascular protection. Phosphatidylinositol 4,5-bisphosphate (PIP2) was previously reported to play a direct role in phosphatidylserine (PS) exposure, as a Ca2+ target. Thrombin formation and platelet procoagulant activity are dependent on PS exposure. As flavonoids can inhibit phosphoinositide (PPI) kinases, we examined whether changes in PPI metabolism in flavonoid-treated platelets could be involved in their antiplatelet effects. Treatment with the flavonoids quercetin or catechin reduced PS exposure, thrombin formation, PIP2 level and resynthesis after platelet activation with collagen, thrombin or calcium ionophore. Flavonoids also prevented [Ca2+]i increase induced by collagen, but not by the ionophore. The ability of flavonoids to decrease PS exposure induced by ionophore treatment could result from the diminution of PIP2 levels, whereas PS exposure induced by collagen could also be diminished by flavonoids' effects on calcium signaling dependent on PIP2 hydrolysis. These data favor a role for PIP2 in the antiplatelet effects of flavonoids.

Involvement of phosphatidylinositol 4,5-bisphosphate in phosphatidylserine exposure in platelets: use of a permeant phosphoinositide-binding peptide.

During platelet activation, phosphatidylserine (PS) exposure on the extracellular face of the plasma membrane is associated with increased procoagulant activity. PS externalization is generally attributed to an increase in intracellular Ca(2+). Various phospholipid transporters, such as specific scramblases or proteins from the family of multidrug resistance proteins, and cofactors such as phosphatidylinositol 4,5-bisphosphate (PIP2) have been proposed to participate in this process. In this study, we used a membrane-permeant polycationic peptide (RhB-QRLFQVKGRR), derived from the PIP2-binding site of gelsolin (GS 160-169) and linked to rhodamine B, to investigate the role of PIP2 in PS externalization in whole platelets. The peptide penetrated rapidly into the platelets, specifically bound to PIP2, and induced PS exposure to a similar extent as thrombin or collagen, but independently of changes in intracellular Ca(2+) or phosphoinositide 3-kinase activity. A pretreatment of platelets with quercetin, an inhibitor of phosphoinositide metabolism, drastically decreased PS exposure induced by agonists or peptide. In large unilamellar vesicles (LUVs), the presence of PIP2 was strictly required for the induction of scrambling of NBD-labeled phospholipids (PC and PS) by the peptide. In inside-out vesicles from erythrocytes (IOVs), the peptide also induced redistribution of PC and PS. Our data suggest that, in intact platelets, PIP2 acts as a target of polycationic effectors, including Ca(2+), to promote PS exposure. The use of a membrane-permeant and fluorescent peptide which binds to PIP2 is a promising tool to investigate the role of PIP2 in various cellular processes.

Phosphatidylinositol 4,5-bisphosphate domain inducers promote phospholipid transverse redistribution in biological membranes.

Transmembrane phospholipid redistribution (scrambling), leading to exposure of phosphatidylserine on the cell surface, plays a physiological role to induce platelet procoagulant activity and clearance of injured or apoptotic cells. Scrambling is generally attributed to an increase in intracellular Ca(2+) and would be mediated by a protein (scramblase), whose activity could be modulated by cofactors. We reported previously that phosphatidylinositol 4,5-bisphosphate (PIP(2)) is a positive regulator of Ca(2+)-induced scrambling. We show here, using inside-out vesicles from erythrocyte membranes, that a pleckstrin homology (PH) domain, which interacts with high affinity with PIP(2), inhibited Ca(2+)-induced scrambling, confirming the role of PIP(2). As Ca(2+) is known to interact with PIP(2) and to promote the formation of lateral domains of acidic phospholipids in membranes, we investigated whether PIP(2) domain formation could be involved in scrambling. Spermine, polylysine, and MARCKS (151-175) peptide caused scrambling in parallel to their reported ability to form domains of acidic phospholipids, including PIP(2). Similarly, neomycine, another PIP(2)-interacting polycation, induced scrambling. A PIP(2) antibody was also found to induce scrambling, presumably by a similar mechanism, since phospholipid antibodies are known to promote phospholipid capping. In conclusion, Ca(2+) is not the sole inducer of scrambling, and formation of PIP(2) domains could play a critical role in this process.

Calcium induces phospholipid redistribution and microvesicle release in human erythrocyte membranes by independent pathways.

The increase in intracellular Ca2+ concentration in erythrocytes and platelets results in simultaneous phospholipid scrambling and microvesicle shedding. Microvesicle formation involves membrane fusion events which were proposed either to be tightly linked to phospholipid transversal redistribution or to occur by a separate mechanism. We report here that in erythrocytes incubated in high K+ medium, or in resealed ghosts, phospholipid scrambling can be fully induced by intracellular Ca2+ without microvesicle formation. Furthermore, in ghosts resealed in the presence of spermine, intracellular Ca2+, at low concentration, was able to induce microvesicles, whereas scrambling was drastically inhibited. Surprisingly, in spermine-containing ghosts prepared from erythrocytes of a patient with a bleeding disorder, due to a lack of Ca2+-induced phospholipid scrambling and vesicle shedding (characterized as a Scott syndrome), Ca2+ also promoted microvesicle release. Data show that phospholipid scrambling and microvesicle production, although closely regulated, proceed by independent pathways.

Drug-induced transmembrane lipid scrambling in erythrocytes and in liposomes requires the presence of polyanionic phospholipids.

The asymmetric transmembrane distribution of phospholipids between the two bilayer halves of erythrocyte can be modified upon addition of cationic amphiphilic drugs, such as chlorpromazine or verapamil. We studied this phenomenon in erythrocytes and in lipid vesicles using spin-labelled analogues of the endogenous phospholipids. The extent of the rapid disappearance of the analogues from the erythrocyte outer leaflet depended on the concentration of the drug. Up to 40% of spin-labelled sphingomyelin moved to the inner erythrocyte leaflet in 10 min in the presence of 1.5 mm chlorpromazine. Verapamil or vinblastine gave similar results. On the other hand, the inside-outside movement of the aminophospholipid analogues was less evident, and did not exceed 10%. This apparent discrepancy between inward and outward movements could result from the formation of an endovesicle which is known to occur upon drug addition at high concentration. A fraction of lipids would be trapped in the intravesicular leaflet, corresponding to the cell outer leaflet, and be inaccessible both from the cytoplasm and the extracellular medium. In cells submitted to a metabolic depletion of cellular ATP the intensity of the scrambling induced by the amphipaths was drastically lowered. We attribute this effect to the important reduction of the membrane content in phosphatidylinositol-4,5-bisphosphate (PIP2). The involvement of the latter lipid in triggering scrambling was partly confirmed by experiments carried out with artificial membranes. Indeed, in large unilamellar vesicles PIP2 is required in order to obtain a rapid redistribution of phospholipids between the two leaflets upon addition of drugs. However, the extent of phospholipid redistribution was limited to 15-20%. This redistribution was also induced when the vesicle membrane contained di-anionic phospholipids (phosphatidylinositol-4-monophosphate or diphosphatidylglycerol), but did not occur when it contained mono-anionic phospholipids (phosphatidylserine or phosphatidylinositol). Some drugs such as methochlorpromazine, active in artificial membranes, were ineffective in erythrocyte membranes, probably because they could not cross the membrane and reach PIP2 molecules at the cytoplasmic leaflet.

[Various functions of human erythrocyte membrane lipids]. / Róznorodne funkcje lipidów blony plazmatycznej erytrocytów czlowieka.

The major phospholipid classes of the human red blood cell membrane are: phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin. These phospholipids are distributed asymmetrically across the two halves of the lipid bilayer. This asymmetry appears to be generated and maintained by an ATP-dependent translocation of aminophospholipids from outer to inner leaflet, and by the interaction of phospholipids with skeletal proteins. The phosphoinositides account for 3-4% of total erythrocyte membrane phospholipid. They play an important role in signal transduction and are involved in other various membrane functions.

Antagonist effects of Ca2+ and spermine on phosphatidylinositol 4,5-bisphosphate-mediated transmembrane redistribution of phospholipids in large unilamellar vesicles and in erythrocytes.

We have previously suggested the involvement of a Ca(2+)-phosphatidylinositol 4,5-bisphosphate (PIP2) complex in the phospholipid transmembrane redistribution triggered by cytosolic Ca2+ in erythrocytes. Indeed, the lipid scrambling was induced by extracellular Ca2+ in erythrocytes loaded with PIP2 and was abolished in inside-out vesicles prepared from PIP2-depleted erythrocytes (Sulpice, J.C., Zachowski, A., Devaux, P.F., & Giraud, F. (1994) J. Biol. Chem. 269, 6347-6354). Here, we show that Ca2+ triggers a partial redistribution of spin-labeled phospholipids in protein-free large unilamellar vesicles (LUVs), only when they contain PIP2. Spermine, a polyamine known to interact with PIP2 and reported to inhibit lipid scrambling in resealed ghosts, was found to inhibit also the Ca(2+)-induced scrambling in LUVs and in PIP2-loaded erythrocytes, presumably by interacting with PIP2 and preventing the formation of Ca(2+)-PIP2 complexes. A similar mechanism can account for spermine inhibition in natural membranes, confirming the role of PIP2 in the scrambling process without excluding the participation of proteins. In erythrocytes, activation of the phosphoinositide phospholipase C (PLC) or a 20 h ATP depletion, which both led to a reduction in the PIP2 content by 40-60%, did not affect Ca(2+)-induced phospholipid scrambling. In contrast, longer ATP depletion, resulting in a 80% reduction in the PIP2 content, did induce a significant decrease in lipid scrambling, suggesting that only the PIP2 pool resistant to the PLC was involved. Spermine was able to inhibit hydrolysis of this pool by an exogenous PLA2. It is thus likely that spermine antagonized the Ca(2+)-induced scrambling in resealed ghosts by interacting with the PLC-resistant pool of PIP2.

Requirement for phosphatidylinositol 4,5-bisphosphate in the Ca(2+)-induced phospholipid redistribution in the human erythrocyte membrane.

In order to investigate how calcium on the cytosolic side of human erythrocytes induces the transmembrane redistribution of phospholipids, we studied the effect of this cation on the transmembrane movements of spin-labeled phospholipids (phosphatidylserine (PS) and phosphatidylcholine (PC)) incorporated into inside-out vesicles derived from human erythrocytes. We found that the extent of the Ca(2+)-induced lipid scrambling was dependent upon the level of phosphatidylinositol 4,5-bisphosphate (PIP2) contained in the external leaflet of inside-out vesicles. The level of PIP2 in this leaflet, which normally accounts for 80% of the total membrane PIP2, was manipulated either by ATP depletion of the original erythrocytes or by incorporation of exogenous PIP2. Similarly, loading the outer monolayer of the membrane of intact erythrocytes with exogenous PIP2 caused, in a dose-dependent way, the scrambling of spin-labeled phosphatidylethanolamine, sphingomyelin, PC, and PS and in parallel the stomatocytic conversion of the cells. Both scrambling and stomatocytosis were strictly dependent on the presence of divalent cations in the medium. Mg2+ could replace Ca2+ but required a 10 times higher concentration. The effect was specific for PIP2, the other phosphoinositides being unable to induce the lipid redistribution. The shape change, but not the scrambling, required a normal ATP level. These results show that Ca2+ or Mg2+ trigger the lipid redistribution either from the internal or the external side of the membrane, provided that enough PIP2 is present on that side. Thus, no specific protein is required for this process. We infer that the ATP-dependent shape change of erythrocytes after incubation with PIP2 and Ca2+ results from the bilayer imbalance due to the activity of the aminophospholipid translocase which relocates PS and phosphatidylethanolamine to the inner monolayer without simultaneous outward diffusion of PC and sphingomyelin.

Stretch-induced inositol trisphosphate and tetrakisphosphate production in rat cardiomyocytes.

The kinetics and signal transduction of inositol phosphate production were studied after a 20% stretch of neonatal rat cardiomyocytes in culture. Inositol trisphosphate (IP3) production was increased by 41% above control after 10-20 s of cellular stretch but returned to control after 120 s of stretch. The increase in IP3 was potentiated in high K+ medium and was inhibited by pertussis toxin, suggesting the existence of a pertussis toxin-sensitive G protein in signal transduction. Ion-pair HPLC analysis of cell extracts stretched for 20 s showed an increase in both IP3 isomers, mostly 1,4,5-IP3 (+66%) with a weak increase in IP4 (+10%), whereas 120 s stretch induced an increase in IP4 (+26% above control) associated with a decrease of 1,4,5-IP3 isomer as compared with 20 s stretch. It is concluded that the progressive increase in IP4 production associated with an early rise in IP3 after stretching myocardial cells may be a factor inducing the length-dependent activation of cardiac muscle through a modulation of intracellular free calcium concentration.

Characterization of structural and functional phosphoinositide domains in human erythrocyte membranes.

In the erythrocyte membrane, only a fraction (50-60%) of phosphatidylinositol 4,5-bisphosphate (PIP2) and of phosphatidylinositol 4-phosphate (PIP) is rapidly turned over by specific kinases and phosphatases and accessible to hydrolysis by the polyphosphoinositide (PPI)-specific phospholipase C (PLC). To investigate whether the metabolic segregation of PPI resulted from preferential interactions with proteins, we have measured the accessibility of PPI to bee venom phospholipase A2 (PLA2) in native erythrocyte membranes, or after treatments designed to remove peripheral proteins and cytoplasmic domains of integral proteins. In native membranes, PPI, as well as the other major phospholipids, behaved as two distinct fractions (R1 and R2) differing by their sensitivity to PLA2. Such a behavior was not observed in PIP and PIP2 containing artificial vesicles. Evidence was provided that the highly sensitive fraction of PIP and PIP2 (R1) may be identical to the PLC-sensitive and rapidly metabolized pool. Removal of peripheral proteins, followed by proteolysis of the cytoplasmic domain of integral proteins, mainly glycophorins and band 3, led to a reduction of the R1 fraction of PIP and of PIP2. It is proposed that the rapidly metabolized pool of PIP2 and PIP, involved in the regulation of major cellular functions, would be maintained in its functional state through interactions with integral proteins.

Asymmetric distribution of phosphoinositides and phosphatidic acid in the human erythrocyte membrane.

The distribution of phosphoinositides and phosphatidic acid (PA) between the outer and inner layers of the human erythrocyte membrane was investigated by using two complementary methodologies: hydrolysis by phospholipase A2 (PLA2) and immunofluorescence detection with monoclonal antibodies against polyphosphoinositides. The contents of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and PA were decreased by 15-20% after 60 min incubation with PLA2, while that of phosphatidylinositol (PI) was increased. Studies with 32P-labelled cells revealed that PLA2 treatment led to indirect effects on the metabolism of these phospholipids. Therefore, the asymmetric distribution of phosphoinositides and PA was inferred from the data obtained in ATP-depleted erythrocytes. In these cells with arrested phosphoinositide metabolism, the asymmetric distribution of the major phospholipids was maintained: PLA2 hydrolyzed approx. 20% of PI, PIP2 and PA (but no PIP) indicating their localization in the outer layer of the membrane. This finding was confirmed by immunofluorescence studies with antibodies specific to each phosphoinositide. External addition of anti-PIP2 but not anti-PIP gave a positive reaction both in control and in ATP-depleted erythrocytes. A pretreatment of cells with PLA2 led to a decrease in the intensity of anti-PIP2 staining. These results demonstrate that significant fractions of PIP2, PI and PA are localized on the outer surface of the erythrocyte membrane.

Mechanisms involved in platelet activation induced by a monoclonal antibody anti glycoprotein IIb-IIIa: inositol phosphate production is not the primary event.

The mechanisms involved in platelet aggregation by a monoclonal antibody (mAb) P256 specific for the GPIIb-IIIa complex was investigated following metabolic 32P labelling of platelets. When compared with thrombin, inositol phosphates (InsP) production during P256-induced activation was delayed and no apparent peak, but a small and sustained production of [32P]-Ins(1,4,5)P3 and [32P]-Ins(1,3,4,5)P4, was observed between 20 and 90 s. [32P]-Ins(1,3,4)P3 was also produced with a maximum after 90 s. Addition of the ADP scavenger creatinine phosphate/creatine phosphokinase (CP/CPK) and of the cyclooxygenase inhibitor aspirin together with P256 almost totally abolished InsP formation, whereas platelet aggregation and protein phosphorylation were partially inhibited. F(ab')2 fragments of P256 also aggregated platelets but to a smaller extent than IgG, and without any measurable InsPs. To characterize further P256-induced activation, the phosphorylation of p43, the main substrate of protein kinase C (PKC) and the phosphorylation of tyrosine protein (P-Tyr) was also studied. PKC activation was smaller with P256-IgG than with thrombin but both thrombin and P265-IgG induced a similar profile of P-Tyr involving seven major bands, whereas P256-F(ab')2 only occasionally activated PKC but always significantly phosphorylated a 64,000 molecular weight P-Tyr. The data indicate that the binding of P256 to GPIIb-IIIa, in contrast with thrombin, does not initially lead directly to the activation of the phosphoinositide phospholipase C to produce InsP's but rather involves the activation of protein kinases and also both fragments F(ab')2 and Fc play a specific role in the platelet responses to the mAb. Only the crosstalk between the two pathways evoked by F(ab')2 and Fc respectively allows the activation of all platelet activation systems.

Critical analysis of the use of 14C-acetate for measuring in vivo rat cholesterol synthesis.

The bulk of cholesterol produced by the liver and the gut enters the mobile pool of body cholesterol. This process is called internal secretion in contrast with the fraction of biosynthesized cholesterol directly eliminated in the feces (fecal external secretion). In rats, under various conditions, a linear relationship was found between the rates of internal secretion measured by the isotope equilibrium method (range: 10-60 mg/day) and the sum of sterol radioactivities measured in liver and intestine 70 min after a [14C]-acetate pulse. In fact, a better correlation was found between the radioactivities of liver sterols and the values for internal secretion. In this new relationship, the ordinate at the origin corresponds to a minimal internal secretion of about 10 mg/day, which implies an important extrahepatic cholesterol production, probably from the gut. Indeed, in adult male rats, fed a semi-purified sucrose-rich diet, the relative contribution of this organ to the internal secretion was higher than in adult rats fed a commercial diet and higher than in young animals, whatever the circadian period. It can be concluded that some of the discrepancies observed in the literature about the relative participation of the intestine and the liver in the internal secretion of cholesterol are probably due to differences in experimental and nutritional conditions (age and sex of the animals, diet composition, time of the circadian cycle) rather than to the cholesterol precursor used (3H2O or [14C] acetate) to assess the activity of cholesterol synthesis. Indeed, a comparative study of 3H2O and [14C]acetate incorporation into sterols of enterocytes indicated the same crypt-villus radioactive gradient, regardless of the intestinal site studied (duodenum, jejunum or ileum) and thus validated the use of [14C]acetate. Other experiments however, showed evidence of some local differences in the cytosolic dilution of labeled acetyl CoA by the endogenous cholesterol precursor in rats under various conditions (control or cholestyramine-enriched diet, parenteral nutrition). After intravenous infusion of 1,2[13C]acetate, mass fragmentography of free cholesterol isolated from liver and intestine indicated different 13C-labeling patterns of newly synthesized molecules. They indicate that cholesterol is generally synthesized from acetyl CoA with a lower 13C-content in the liver than in the intestine. The local endogenous flow of acetyl CoA used for cholesterol synthesis was about 2-fold higher in the hepatocytes than in the enterocytes. This conclusion was confirmed by the results obtained with several experimental groups exhibiting a large range of both internal secretion of cholesterol and sterol radioactivities in liver and intestine after [14C]acetate injection.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional heterogeneity of polyphosphoinositides in human erythrocytes.

After labelling of erythrocytes with [32P]P1 for 23 h, the specific radioactivities of the phosphomonoester groups of PtdIns4P and of PtdIns(4,5)P2 approached equilibrium values which were close to that of the gamma-phosphate of ATP (78-85%), showing that almost all of these phosphate groups were metabolically active. Phosphoinositidase C (PIC) activation, using Ca2+ and the ionophore A23187, of 32P-prelabelled erythrocytes was used to investigate a possible functional heterogeneity of the phosphoinositides. Hydrolysis of PtdIns(4,5)P2, measured from its radioactivity, decreased as function of the time of prelabelling up to a constant value equal to that measured from its content. In contrast, hydrolysis of PtdIns4P, determined both from radioactivity and from content, was always the same. These data suggest that newly labelled molecules of PtdIns(4,5)P2, initially accessible to PIC, then moved towards a PIC-resistant pool. This was further confirmed by measuring the fraction of labelled PtdIns(4,5)P2 molecules accessible to PIC after a prelabelling period of 5 min and different times of reincubation. Hydrolysis by PIC was also measured in erythrocytes in which the phosphoinositide content had been modified by activation (Mg2+-enriched cells) or inhibition (ATP-depleted cells) of the phosphoinositide kinases. The sizes of the PIC-resistant pools of polyphosphoinositides were not affected by these treatments, indicating that the kinases (and the phosphatases) act on the PIC-sensitive pools. This was also shown by the decrease in the production of Ins(1,4,5)P3 upon PIC activation in ATP-depleted erythrocytes. A model is presented in which the PIC-sensitive pools of polyphosphoinositides are those which are accessible to the kinases and the phosphatases and are rapidly turned over.

The separation of [32P]inositol phosphates by ion-pair chromatography: optimization of the method and biological applications.

We have developed an ion-pair reverse-phase HPLC method to measure inositol phosphates in 32P-labeled cells. The different chromatographic parameters were analyzed to optimize the resolution of the 32P-labeled metabolites. Analysis of inositol phosphates in biological samples was improved by a single charcoal pretreatment which eliminated interfering nucleotides without removing inositol phosphates. The kinetics of production of inositol phosphates in calcium-activated erythrocytes, vasopressin-stimulated hepatocytes, and thrombin-activated platelets were analyzed. Original data on the activation of phosphoinositide phospholipase C were obtained in intact erythrocytes by direct measurement of inositol (1,4,5)P3. Data from agonist-stimulated hepatocytes and platelets were consistent with those from previous studies. In conclusion, this technique offers many advantages over the methodologies currently employed involving anion-exchange chromatography and [3H]inositol labeling: (i) 32P labeling is less expensive and more efficient than 3H labeling and can be used with all types of cells without permeabilization treatments and (ii) ion-pair HPLC gives good resolution of inositol phosphates from nucleotides with shorter retention times, and long reequilibration periods are not required.

Endogenous calcium in sickle cells does not activate polyphosphoinositide phospholipase C.

Sickle-cell-anaemia erythrocytes (SS cells) are known to have a high Ca2+ content (particularly the dense cell fraction) and to take up Ca2+ on deoxygenation. It has been reported that this high Ca2+ was responsible for the activation of the Ca2+-dependent K+ loss, and of the Ca2+-sensitive polyphosphoinositide phospholipase C (PIC) in dense SS cells. We found that, either in the total population of SS cells or in the light or dense fractions, the content of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was not changed, whereas that of phosphatidylinositol 4-phosphate was increased and that of phosphatidic acid (PtdOH) was decreased compared with normal (AA) erythrocytes. Deoxygenation-induced Ca2+ entry into SS cells did not change the concentration or, in 32P-prelabelled cells, the radioactivity of polyphosphoinositides and PtdOH. It also failed to induce the formation of inositol 1,4,5-trisphosphate, the product of PtdIns(4,5)P2 hydrolysis by PIC, which was measured by an original method using ion-pair reverse-phase h.p.l.c. Thus there was no evidence of an endogenous Ca2+ effect on the PIC activity in SS cells, in agreement with the demonstration that the excess Ca2+ in SS cells is compartmentalized into internal vesicles and unavailable as free Ca2+. The 32P incorporation in polyphosphoinositides and PtdOH was markedly higher in SS than in AA cells, but this increase was the same in both dense and light SS cells. The increase in the turnover of these phospholipids in SS cells is consistent either with an activation of the lipid kinases and phosphatases or with perturbation in the metabolic compartmentation of these lipids.

Stimulation of polyphosphoinositide turnover upon activation of protein kinases in human erythrocytes.

Activation of protein kinase C in erythrocytes by 4-beta-phorbol 12-myristate 13-acetate (PMA) resulted in a parallel stimulation (time course and dose response) of the phosphorylation of both membrane proteins (heterodimers of 107 kDa and 97 kDa, protein 4.1 and 4.9, respectively) and of phosphatidylinositol 4-phosphate (PIP) and, to a lesser extent, of phosphatidylinositol 4,5-bisphosphate (PIP2). Evidence that the effect on lipid was mediated by protein kinase C activation and not by a direct action of PMA was provided by (1) the lack of effect of a phorbol ester that did not activate protein kinase C or of PMA addition on isolated membranes from control erythrocytes, (2) the reversal of the effect in the presence of protein kinase C inhibitors (alpha-cobrotoxin, H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine) or trifluoperazine). PMA treatment did not change the specific activity of ATP or the content of PIP2, but increased the content of PIP and decreased that of PI, indicating that the phosphorylation or dephosphorylation reactions linking PI and PIP were the target for the action of PMA. PMA treatment had no effect on the Ca2+-dependent PIP/PIP2 phospholipase C activity measured in isolated membranes. Mezerein, another protein kinase activator, had similar effects on both protein and lipid phosphorylation, when added with alpha-cobrotoxin. Activation of protein kinase A by cAMP also produced increases in phosphorylation, although quantitatively different from those induced by protein kinase C, in proteins and PIP. Simultaneous addition of PMA and cAMP at maximal doses resulted in only a partially additive effect on PIP labelling. These results show that inositol lipid turnover can be modulated by a protein kinase C and protein kinase A-dependent process involving the phosphorylation of a common protein. This could be PI kinase or PIP phosphatase or another protein regulating the activity of these enzymes.