The Effects of Inositol Metabolism in Pregnant Women on Offspring in the North and South of China.

BACKGROUND Inositol is an essential nutrient for cell growth, survival and embryonic development. Myo-inositol is the predominant form in natural. To investigate the correlation between inositol metabolism and embryonic development, we assessed the metabolic characteristics of myo-inositol, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) of pregnant women in the North China (Yangquan and Weihai) and South China (Nanchang and Haikou) China. MATERIAL AND METHODS All data were collected by face-to-face interview during pregnant women health visits using a questionnaire. Plasma levels of myo-inositol, PI(4,5)P2 and PI(3,4,5)P3 from 89 randomly collected pregnant women were detected by gas chromatography-mass spectrometry and enzyme linked immunosorbent assay. RESULTS A total of 400 pregnant women were included in this survey. The plasma levels of myo-inositol and PI(4,5)P2 in the North China group of pregnant women were significantly higher than that in the South China group (P<0.01). The birth weight of fetuses in the North China group was heavier than that in the South China group (P<0.01). The birth length of fetuses in Yangquan was the longest among the 4 cities (P<0.01). The incidence rate of birth defects was 3.05% in the North China group, and 0.0% in the South China group. In bivariate linear correlation analysis, the body weight correlated with myo-inositol (r=0.5044, P<0.0001), PI(4,5)P2 (r=0.5950, P<0.0001) and PI(3,4,5)P3 (r=0.4710, P<0.0001), the body length was correlated with PI(4,5)P2 (r=0.3114, P=0.0035) and PI(3,4,5)P3 (r=0.2638, P<0.0130). CONCLUSIONS The plasma levels of myo-inositol and PI(4,5)P2 in pregnant women had significant difference between the North and the South of China, which might be correlated with fetal development and birth defects.

TRPM7 Kinase Controls Calcium Responses in Arterial Thrombosis and Stroke in Mice.

OBJECTIVE: TRPM7 (transient receptor potential cation channel, subfamily M, member 7) is a ubiquitously expressed bifunctional protein comprising a transient receptor potential channel segment linked to a cytosolic α-type serine/threonine protein kinase domain. TRPM7 forms a constitutively active Mg2+ and Ca2+ permeable channel, which regulates diverse cellular processes in both healthy and diseased conditions, but the physiological role of TRPM7 kinase remains largely unknown. APPROACH AND RESULTS: Here we show that point mutation in TRPM7 kinase domain deleting the kinase activity in mice (Trpm7R/R ) causes a marked signaling defect in platelets. Trpm7R/R platelets showed an impaired PIP2 (phosphatidylinositol-4,5-bisphosphate) metabolism and consequently reduced Ca2+ mobilization in response to stimulation of the major platelet receptors GPVI (glycoprotein VI), CLEC-2 (C-type lectin-like receptor), and PAR (protease-activated receptor). Altered phosphorylation of Syk (spleen tyrosine kinase) and phospholipase C γ2 and ß3 accounted for these global platelet activation defects. In addition, direct activation of STIM1 (stromal interaction molecule 1) with thapsigargin revealed a defective store-operated Ca2+ entry mechanism in the mutant platelets. These defects translated into an impaired platelet aggregate formation under flow and protection of the mice from arterial thrombosis and ischemic stroke in vivo. CONCLUSIONS: Our results identify TRPM7 kinase as a key modulator of phospholipase C signaling and store-operated Ca2+ entry in platelets. The protection of Trpm7R/R mice from acute ischemic disease without developing intracranial hemorrhage indicates that TRPM7 kinase might be a promising antithrombotic target.

Involvement of the Na+/H+ exchanger in membrane phosphatidylserine exposure during human platelet activation.

Platelet membrane phosphatidylserine (PS) exposure that regulates the production of thrombin represents an important link between platelet activation and the coagulation cascade. Here, we have evaluated the involvement of the Na+/H+ exchanger (NHE) in this process in human platelets. PS exposure induced in human platelets by thrombin, TRAP, collagen or TRAP+ collagen was abolished in a Na+ -free medium. Inhibition of the Na+/H+ exchanger (NHE) by 5-(N-Ethyl-N-Isopropyl) Amiloride (EIPA) reduced significantly PS exposure, whereas monensin or nigericin, which mimic or cause activation of NHE, respectively, reproduced the agonist effect. These data suggest a role for Na+ influx through NHE activation in the mechanism of PS exposure. This newly identified pathway does not discount a role for Ca2+, whose cytosolic concentration varies together with that of Na+ after agonist stimulation. Ca2+ deprivation from the incubation medium only attenuated PS exposure induced by thrombin, measured from the uptake of FM1-43 (a marker of phospholipid scrambling independent of external Ca2+). Surprisingly, removal of external Ca2+ partially reduced FM1-43 uptake induced by A23187, known as a Ca2+ ionophore. The residual effect can be attributed to an increase in [Na+]i mediated by the ionophore due to a lack of its specificity. Finally, phosphatidylinositol 4,5-bisphosphate (PIP2), previously reported as a target for Ca2+ in the induction of phospholipid scrambling, was involved in PS exposure through a regulation of NHE activity. All these results would indicate that the mechanism that results in PS exposure uses redundant pathways inextricably linked to the physio-pathological requirements of this process.

Phosphatidylinositol 4,5-bisphosphate regulates activation-induced platelet microparticle formation.

While the role of the cytoskeleton in microparticle formation is well-described, the role of membrane phospholipids in regulating this process is poorly defined. PIP(2) binds many cytoskeletal proteins and may oppose microparticle formation through associations with these proteins. To determine whether PIP(2) effects microparticle formation, PIP(2) was incorporated into platelet membranes prior to activation-induced microparticle formation. Incorporation of PIP(2) into platelet membranes inhibited activation-induced microparticle formation by >or=90%. Inhibition was dose-dependent with an IC(50) of 12-18 microM. A permeabilized platelet system was next used to assess the effect of modulation of endogenous PIP(2) levels on microparticle formation. Infusion of type IIbeta PIP kinase into permeabilized platelets inhibited microparticle formation by 75 +/- 8%. In contrast, incubation of permeabilized platelets with PI-specific phospholipase C augmented microparticle formation by greater than 3-fold. Evaluation of PIP kinases following platelet activation demonstrated that they were lost from platelets in a calpain-dependent manner during microparticle formation. Purified mu-calpain cleaved recombinant type IIbeta PIP kinase and inhibited its ability to phosphorylate PI(5)P. In permeabilized platelets, incubation of purified mu-calpain reduced PIP(2) levels, while exposure to calpeptin increased PIP(2) levels. Calpain has previously been implicated in platelet microparticle formation. These studies show that calpain may help limit PIP(2) formation following platelet activation and that PIP(2) content is an important determinant of platelet microparticle formation.

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.

Increased platelet membrane phosphatidylinositol-4,5-bisphosphate in drug-free depressed bipolar patients.

Prior investigations in bipolar disorder patients have suggested abnormalities in the cellular phosphoinositide second messenger system. This study was conducted to examine the levels of platelet membrane phosphoinositides in drug-free bipolar patients in the depressed state (n=9) and healthy controls (n=19). Bipolar patients had significantly increased levels of platelet membrane phosphatidylinositol-4,5-bisphosphate (PIP(2)) compared to healthy individuals (0.67+/-0.14 and 0.44+/-0.17%, respectively, t-test=3.71, d.f.=26, P=0.001). No significant differences in the levels of phosphatidylinositol-4-phosphate (PIP) (0.65+/-0.17 and 0.58+/-0.20%, respectively, t-test=1.02; d.f.=26; P=0.32) or phosphatidylinositol (PI) (5.92+/-1.23 and 5.56+/-1.45%, respectively, t-test=0.68; d.f.=26; P=0.51) were found. These findings provide the first demonstration of increased PIP(2) platelet levels in bipolar patients in the depressed state, and provide additional evidence that the phosphoinositide second messenger system may be a site of abnormality in bipolar disorder.

Elevation of cyclic AMP decreases phosphoinositide turnover and inhibits thrombin-induced secretion in human platelets.

Elevation of cyclic AMP (cAMP) in platelets inhibits agonist-induced, G protein-mediated responses and activation of polyphosphoinositide-specific phospholipase C (PLC) by ill-defined mechanism(s). Signal transduction steps downstream of PLC are inhibited by elevated cAMP, suggesting an inhibitory effect of cAMP, via protein kinase A, on PLC. In [32P]i-prelabeled platelets, forskolin increased intracellular cAMP (104 nmol/1011 cells at 10-5 M forskolin) and [32P]phosphatidylinositol 4-phosphate (Delta[32P]PIP) (30% at 10-7-10-6 M forskolin). The thrombin-induced (0.1 U/ml) increase in production of [32P]PA, 'overshoots' in [32P]PIP and [32P]PIP2 ([32P]phosphatidylinositol 4,5-bisphosphate), and the increase in [32P]PI and secretion of ADP+ATP were abolished by forskolin (10-7 M). Forskolin stimulated total [32P]Pi uptake in resting platelets (48%), increased 32P incorporation into PIP (110%), and inhibited 32P incorporation into PI (50%). The latter inhibition was most likely considerably greater due to the forskolin-induced stimulation of [32P]Pi uptake. The changes in radioactive PA, PIP and PIP2 are regarded as being proportional with their masses in the prelabeled platelets, while the increase in PI (phosphatidylinositol) is regarded as a change in specific radioactivity, and hence in its synthesis. The results suggest that cAMP elevation inhibits the flux in the polyphosphoinositide cycle through both inhibition of PIP 5-kinase and PI synthesis. The inverse relation between forskolin-produced DeltaPIP and [32P]PA production suggests that the PLC reaction is inhibited by elevated cAMP through reduction of substrate (PIP2) resynthesis, and not by inhibition of the PLC enzyme.

Effects of phosphatidylinositol diphosphate on phospholipid asymmetry in the human erythrocyte membrane.

While phospholipid asymmetry has been well characterized in red blood cells (RBCs), controversy exists as to what role PIP2 plays in cation-induced phosphatidylserine (PS) exposure. We report that PIP2 can redistribute intracellular cations and thereby lead to a loss of phospholipid asymmetry. Flow cytometry was employed to monitor intracellular cation levels by using the fluorophore Fluo-3 and exposure of PS on the outer surface of the RBC bilayer by using fluorescently labeled annexin V. The addition of PIP2 to RBCs led to a concentration-dependent increase in cytosolic cations and PS exposure. IF RBCs were preincubated with 25 microM neomycin sulfate, an inhibitor of phosphoinositide metabolism, PIP2-induced PS exposure decreased dramatically. If the RBC buffer system contained 2.5 mM EGTA, PS exposure also decreased significantly, suggesting a competition between intracellular Fluo-3 and extracellular EGTA. Together, these data indicate that (1) PS exposure was found in RBCs that exhibited an increased cytosolic cation concentration available for the fluorophore. Fluo-3, (2) both the level of intracellular cations and the movement of PS from the inner to the outer monolayer were affected by the level of PIP2 in the bilayer, (3) the cleavage of PIP2 by a phosphoinositide-specific phospholipase lead to the redistribution of intracellular cations and to an increase in the amount of PS exposed on the outer leaflet of the bilayer, and (4) a transient channel could be formed during the interaction of PIP2 with the RBC membrane which would then allow the transbilayer movement of phospholipids and cations.

Platelet membrane phosphatidylinositol-4,5-bisphosphate alterations in bipolar disorder--evidence from a single case study.

Abnormalities in the cellular phosphatidylinositol (PI) pathway have been proposed to be implicated in the pathophysiology of bipolar disorder. A platelet model was used to study phosphatidylinositol-4,5-bisphosphate (PIP2) membrane values in a bipolar disorder patient in different mood states, in a single case study. The patient was studied unmedicated, initially in the euthymic and later in the manic states, and subsequently on lithium after remission of manic symptoms. The relative percentage of PIP2 in the platelet membranes increased with cycling from the euthymic into the manic state. After lithium treatment, PIP2 decreased, and was similar to the euthymic state. This study further demonstrates the feasibility of this method, as well as its applicability to longitudinal studies in bipolar disorder, and suggests promising directions for future research in this area.

The lipid transfer activity of phosphatidylinositol transfer protein is sufficient to account for enhanced phospholipase C activity in turkey erythrocyte ghosts.

BACKGROUND: The minor membrane phospholipid phosphatidylinositol 4, 5-bisphosphate (PIP2) has been implicated in the control of a number of cellular processes. Efficient synthesis of this lipid from phosphatidylinositol has been proposed to require the presence of a phosphatidylinositol/phosphatidylcholine transfer protein (PITP), which transfers phosphatidylinositol and phosphatidylcholine between membranes, but the mechanism by which PITP exerts its effects is currently unknown. The simplest hypothesis is that PITP replenishes agonist-sensitive pools of inositol lipids by transferring phosphatidylinositol from its site of synthesis to sites of consumption. Recent cellular studies, however, led to the proposal that PITP may play a more active role as a co-factor which stimulates the activity of phosphoinositide kinases and phospholipase C (PLC) by presenting protein-bound lipid substrates to these enzymes. We have exploited turkey erythrocyte membranes as a model system in which it has proved possible to distinguish between the above hypotheses of PITP function. RESULTS: In turkey erythrocyte ghosts, agonist-stimulated PIP2 hydrolysis is initially rapid, but it declines and reaches a plateau when approximately 15% of the phosphatidylinositol has been consumed. PITP did not affect the initial rate of PIP2 hydrolysis, but greatly prolonged the linear phase of PLC activity until at least 70% of phosphatidylinositol was consumed. PITP did not enhance the initial rate of phosphatidylinositol 4-kinase activity but did increase the unstimulated steady-state levels of both phosphatidylinositol 4-phosphate and PIP2 by a catalytic mechanism, because the amount of polyphosphoinositides synthesized greatly exceeded the molar amount of PITP in the assay. Furthermore, when polyphosphoinositide synthesis was allowed to proceed in the presence of exogenous PITP, after washing ghosts to remove PITP before activation of PLC, enhanced inositol phosphate production was observed, whether or not PITP was present in the subsequent PLC assay. CONCLUSION: PITP acts by catalytically transferring phosphatidylinositol down a chemical gradient which is created as a result of the depletion of phosphatidylinositol at its site of use by the concerted actions of the phosphoinositide kinases and PLC. PITP is therefore not a co-factor for the phosphoinositide-metabolizing enzymes present in turkey erythrocyte ghosts.

Phospholipids and calcium alterations in platelets of schizophrenic patients.

Alterations in phospholipid metabolism in blood elements have been proposed as the possible biochemical marker of schizophrenia. In the present study, we investigated the composition and membrane distribution of phospholipids in platelets of drug-free schizophrenic patients and controls. We have demonstrated that platelets of drug-free schizophrenics have significantly higher cytosolic Ca2+ levels in comparison with healthy controls. Platelets of drug-free schizophrenic patients have a lower content of phosphatidylinositol (PI). After thrombin activation, PI is the target of phospholipase C instead of phosphatidylinositol 4,5-bisphosphate (PIP2), which is hydrolyzed in platelets of controls. Alterations in the distribution of phospholipids were found in the plasma membrane of platelets of schizophrenic patients. We suggest that alterations in phospholipid metabolism might be evoked by a disturbance of calcium homeostasis in schizophrenic patients.

Aggregation-dependent, integrin-mediated increases in cytoskeletally associated PtdInsP2 (4,5) levels in human platelets are controlled by translocation of PtdIns 4-P 5-kinase C to the cytoskeleton.

Thrombin-stimulated aggregation of human platelets promotes an increase in the phosphatidylinositol 4-phosphate (PtdIns 4-P) 5-kinase (PIPkin) activity in the cytoskeleton. This phenomenon is associated with translocation of PIPkin isoform C to the cytoskeleton and with an increase in the amount of phosphatidylinositol bisphosphate (PtdInsP2) bound to the cytoskeletal pellet. All three of these effects are prevented if the platelets are not stirred or if RGD-containing peptides are present, demonstrating that they require integrin activation. All three are also abolished by pretreatment with okadaic acid, which also prevents the aggregation-dependent translocation of pp60(c-src) to the cytoskeleton. The results point to the existence of a cytoskeletally associated PtdInsP2 pool under the control of integrin-mediated signals that act via PIPkin C and suggest that a common, okadaic acid-sensitive mechanism may underlie the aggregation-dependent translocation of certain signalling molecules to the platelet cytoskeleton.

Increased formation of phosphatidylinositol-4-phosphate in human platelets stimulated with lysophosphatidic acid.

Lysophosphatidic acid (LPA, 1-acyl-sn-glycerol 3-phosphate), at a concentration of 1-40 microM, was found to induce the formation of [3H]inositol-labelled phosphatidylinositol-4-phosphate (PIP) without significantly altering the levels of either phosphatidylinositol (PI) or phosphatidylinositol bisphosphate (PIP2) in washed human platelets. Preincubation of platelets with the cyclooxygenase/lipoxygenase inhibitor, BW755C at 100 microM, did not alter the LPA-induced formation of PIP. Activation of platelets with the phorbol ester, phorbol 12-myristate 13-acetate (PMA), elicited a similar response (induction of PIP formation). The specific protein kinase C (PKC) inhibitor, GF109203X (10 microM), completely blocked the effect of PMA but not the LPA-induced generation of PIP. The present results indicate that LPA can induce PIP formation via PI-4-kinase activation, through processes which are independent of the eicosanoid/TxA2 pathway and are not PKC-dependent.

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.