Nuclear phosphoinositide kinases and inositol phospholipids

The presence of inositol phospholipids in the nuclei of mammalian cells has by now been well established, as has the presence of the enzymes responsible for their metabolism. However, our understanding of the role of these nuclear phosphoinositides in regulating cellular events has lagged far behind that for its cytosolic counterpart. It is clear, though, that the nuclear phosphoinositide pool is independent of the cytosolic pool and is, therefore, likely to be regulating a unique set of cellular events. As with its cytosolic phosphoinositides, many nuclear phosphoinositides and their metabolic enzymes are located at distinct sub-cellular structures. This arrangement spatially limits the production and activity of inositol phospholipids and is believed to be a major mechanism for regulating their function. Here, we will introduce the components of nuclear inositol phospholipid signal transduction and discuss how their spatial arrangement may dictate which nuclear functions they are modulating.     

Changing phosphoinositides “on the fly”: how trafficking vesicles avoid an identity crisis

Joining an antagonistic phosphoinositide (PtdInsP) kinase and phosphatase into a single protein complex may regulate rapid and local PtdInsP changes. This may be important for processes such as membrane fission that require a specific PtdInsP and that are innately local and rapid. Such a complex could couple vesicle formation, with erasing of the identity of the donor organelle from the vesicle prior to its fusion with target organelles, thus preventing organelle identity intermixing. Coordinating signals are postulated to switch the relative activities of the kinase and phosphatase in a spatio‐temporal manner that matches membrane fission events. The discovery of two such complexes supports this hypothesis. One regulates the interconversion of phosphatidylinositol and PtdIns(3)P by joining the Vps34 PtdIns 3‐kinase and the myotubularin 3‐phosphatases. The other regulates the interconversion between PtdIns(3)P and PtdIns(3,5)P₂ through the Fab1/PIKfyve kinase and the Fig4/mFig4 phosphatase. These lipids are essential components of the endosomal identity code.     

A phosphoinositide-linked peptide response in astrocytes: Evidence for regional heterogeneity

A phosphoinositide-linked peptide response in cultured rat astrocytes was studied by measuring the accumulation of [³H]inositol phosphates in the presence of lithium. Cultures derived from cortex, cerebellum and spinal cord each showed a unique pattern or degree of stimulation to a panel of neuropeptides. Cortical and cerebellar astrocytes were similar, responding to bradykinin, oxytocin, vasopressin, eledoisin and neurokinin , whereas spinal cord astrocytes were stimulated by substance P, bradykinin, eledoisin, and neurokinins and . These observations are evidence in favour of regional specialisations of astrocytes which may respond uniquely to peptidesreleased by particular populations of neurons.     

Potassium ions potentiate the muscarinic receptor-stimulated phosphoinositide metabolism in cerebral cortex slices: A comparison of neonatal and adult rats

Activation of cholinergic muscarinic receptors results in an increased turnover of membrane inositol phospholipids. In rat cerebral cortex slices, carbachol- and acetylcholine-induced inositol phosphates ([³H]InsPs) accumulation is maximal in 7 day-old rats and lowest in adults, while the density of muscarinic binding sites increases gradually with age, suggesting the presence of a more effective receptor-effector coupling during neonatal life. In the process of investigating the nature of such differential stimulation, we have studied the effects of potassium ions on muscarinic receptor-stimulated phosphoinositide metabolism during development. Increasing the concentration of K⁺ from 6 to 12 mM potentiated the stimulating effect of carbachol by 80–100% in adult animals, as previously shown, but only 10–20% in 7 day-old animals, without altering its EC₅₀ values. The differential potentiation by K⁺ at these two ages was specific for muscarinic receptors, since norepinephrine-stimulated accumulation was potentiated only 18% and 12% in adult and 7 day-old rats, respectively. Two other monovalent cations, rubidium and cesium, had the same effect as K⁺ on carbachol-stimulated [³H]-InsPs accumulation. The effect of K⁺ was not antagonized by the K⁺ channel blocker 4-aminopyridine, but was antagonized by tetraethylammonium (TEA). TEA, however, also interacted with muscarinic binding sites. Omission of calcium from the incubation medium did not influence the potentiating effect of 12 mM K⁺. However, when EDTA (1 mM) was added, the stimulating effect of carbachol alone or carbachol + K⁺ was almost completely prevented. The potentiating effect of K⁺ during development was inversely proportional to the stimulation of phosphoinositide metabolism induced by carbachol. These results suggest that the mechanism responsible for the potentiating effect of K⁺ in adult rats might be already operating in neonatal animals.     

Age-Related Alterations In Pre-Synaptic and Receptor-Mediated Cholinergic Functions In Rat Brain

Fractional [³H]acetylcholine (ACh) release and regulation of release process by muscarinic receptors were studied in corpus striatum of young and aged rat brains. [³H] Quinuclidinyl benzilate (QNB) binding and carbachol stimulated phosphoinositide turnover, on the other hand, were compared in striatal, hippocampal and cortical tissues. High potassium (10 mM)-induced fractional [³H]ACh release from striatal slices was reduced by aging. Although inhibition of acetylcholinesterase with eserine (20 M) significantly decreased stimulation-induced fractional [³H]ACh release in two groups of rats, this inhibition slightly lessened with aging. Incubation of striatal slices with muscarinic antagonists reversed eserine-induced inhibition in fractional [³H]ACh release with a similar order of potency (atropine = 4-DAMP > AF-DX 116 > pirenzepine) in young and aged rat striatum, but age-induced difference in stimulated ACh release was not abolish by muscarinic antagonists. These results suggested that fractional [³H]ACh release from striatum of both age groups is modulated mainly by M₃ muscarinic receptor subtype. Although both muscarinic receptor density and labeling of inositol lipids with [myo-³H]inositol decreased with aging, carbachol-stimulated [³H]myo inositol-1-fosfat (IP₁) accumulation was found similar in striatal, cortical and hippocampal slices.     

Ethanol inhibits muscarinic receptor-stimulated phosphoinositide metabolism and calcium mobilization in rat primary cortical cultures

In recent years, it has been hypothesized that muscarinic receptor-stimulated phosphoinositide (PI) metabolism may represent a relevant target for the developmental neurotoxicity of ethanol. Age-, brain region-, and receptor-specific inhibitory effects of ethanol on this system have been found, both in vitro and after in vivo administration. As a direct consequence of this action, alterations of calcium homeostasis would be expected, through alterations of inositol trisphosphate formation, which mediates intracellular calcium mobilization. In the present study, the effects of ethanol (50–500 mM) on carbachol-stimulated PI metabolism and free intracellular calcium levels were investigated in rat primary cortical cultures, by measuring release of inositol phosphates and utilizing the two calcium probes fluo-3 and indo-1 on an ACAS (Adherent Cell Analysis and Sorting) Laser Cytometer. Ethanol exerted a concentration-dependent inhibition of carbachol-stimulated PI metabolism. In addition, ethanol's inhibitory effect paralleled the temporal development of the muscarinic receptor signal transduction system, with the strongest inhibition (25–50%) occurring when maximal stimulation by carbachol occurs (days 5–7). Ethanol also exerted a concentration-dependent decrease in free intracellular calcium levels following carbachol stimulation. Both initial calcium spike amplitude, seen in all responsive cells, as well as the total number of cells responding to carbachol, were decreased by ethanol. The inhibitory effects of ethanol seemed dependent upon preincubation time, in that a longer preincubation (30 min) with the lowest dose (50 mM), showed almost the same decrease in responding cell number and reduction in spike amplitude in responding cells, as a shorter incubation (10 min) with the highest ethanol dose (500 mM). The specificity of the response to carbachol was demonstrated by blocking the response with 10 M atropine. Moreover, experiments with carbachol in calcium-free buffer with 1 mM EGTA indicated that the initial calcium spike was due to intracellular calcium mobilization from intracellular stores. Since calcium is believed to play important roles in cell proliferation and differentiation, these results support the hypothesis that this intracellular signal-transduction pathway may be a target for ethanol, contributing to its developmental neurotoxicity.     

7‐Ketocholesterol impairs phagocytosis and efferocytosis via dysregulation of phosphatidylinositol 4,5‐bisphosphate

The plasma membrane is inhomogeneously organized containing both highly ordered and disordered nanodomains. 7‐Ketocholesterol (7KC), an oxysterol formed from the nonenzymatic oxidation of cholesterol, is a potent disruptor of membrane order. Importantly, 7KC is a component of oxidized low‐density lipoprotein and accumulates in macrophage and foam cells found in arterial plaques. Using a murine macrophage cell line, J774, we report that both IgG‐mediated and phosphatidylserine‐mediated phagocytic pathways are inhibited by the accumulation of 7KC. Examination of the well‐studied Fcγ receptor pathway revealed that the cell surface receptor abundance and ligand binding are unaltered while downstream signaling and activation of spleen tyrosine kinase is not affected. However, while the recruitment of phospholipase Cγ1 was unaffected its apparent enzymatic activity was compromised resulting in sustained phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P₂] levels and polymerized actin at the base of the phagocytic cup. Additionally, we found that 7KC prevented the activation of PLCβ downstream of the P2Y₆ G‐protein coupled receptor and that 7KC impaired PLCγ activity in response to a direct elevation of cytosolic calcium induced by ionomycin. Finally, we demonstrate that 7KC partly attenuates the activity of rapamycin recruitable constitutively active PLCβ3. Together, our results demonstrate that the accumulation of 7KC impairs macrophage function by altering PtdIns(4,5)P₂ catabolism and, thus, impairing actin depolymerization required for the completion of phagocytosis.      

Paladin is a phosphoinositide phosphatase regulating endosomal VEGFR2 signalling and angiogenesis

Cell signalling governs cellular behaviour and is therefore subject to tight spatiotemporal regulation. Signalling output is modulated by specialized cell membranes and vesicles which contain unique combinations of lipids and proteins. The phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P₂), an important component of the plasma membrane as well as other subcellular membranes, is involved in multiple processes, including signalling. However, which enzymes control the turnover of non‐plasma membrane PI(4,5)P₂, and their impact on cell signalling and function at the organismal level are unknown. Here, we identify Paladin as a vascular PI(4,5)P₂ phosphatase regulating VEGFR2 endosomal signalling and angiogenesis. Paladin is localized to endosomal and Golgi compartments and interacts with vascular endothelial growth factor receptor 2 (VEGFR2) in vitro and in vivo. Loss of Paladin results in increased internalization of VEGFR2, over‐activation of extracellular regulated kinase 1/2, and hypersprouting of endothelial cells in the developing retina of mice. These findings suggest that inhibition of Paladin, or other endosomal PI(4,5)P₂ phosphatases, could be exploited to modulate VEGFR2 signalling and angiogenesis, when direct and full inhibition of the receptor is undesirable.     

Allosteric Activation of PI3Kα Results in Dynamic Access to Catalytically Competent Conformations

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Guanine nucleotide- and muscarinic agonist-dependent phosphoinositide metabolism in synaptoneurosomes from cerebral cortex of immature rats

Guanine nucleotide-, neurotransmitter-, and fluoride-stimulated accumulation of [³H]inositol phosphates ([³H]InsPs) was measured in [³H]inositol-labeled synaptoneurosomes from cerebral cortex of immature (7-day-old) and adult rats, in order to clarify the role of GTP-binding proteins (G-proteins) in modulating phosphoinositide (PtdIns) metabolism during brain development. GTP(S) [Guanosine 5-O-(3-thio)triphosphate] time- and concentration-dependently stimulated PtdIns hydrolysis. Its effect was potentiated by full (carbachol, metacholine) and partial (oxotremorine) cholinergic agonists through activation of muscarinic receptors. The presence of deoxycholate was required to demonstrate agonist protentiation of the guanine nucleotide effect. The response to GTP(S) was higher in adult than in immature rats, while the effect of cholinergic agonists was similar at the two ages examined. At both ages, histamine potentiated the effect of GTP(S), while norepinephrine was ineffective. At both ages, guanosine 5-O-(2-thio)diphosphate [GDP(S)] and pertussis toxin significantly decreased GTP(S)-induced [³H]InsPs formation. The phorbol ester phorbol 12-myristate 13-acetate (PMA), on the other hand, did not inhibit the guanine nucleotide response in synaptoneurosomes from immature rats. NaF mimicked the action of GTP(S) in stimulating PtdIns hydrolysis. Its effect was not affected by carbachol and was highly synergistic with that of AlCl₃, according to the concept that fluoroaluminate (AlF₄ ^–) is the active stimulatory species. No quantitative differences were found in the response to these salts between immature and adult animals. These results provide evidence that, in both the immature and adult rat brain, neuroreceptor activation is coupled to PtdIns hydrolysis through modulatory G-proteins.     

雷公藤对哮喘大鼠气道重构及磷脂酰肌醇3激酶表达的影响

目的:探讨雷公藤内酯醇对哮喘气道重构及磷脂酰肌醇3激酶(PI3K)表达的影响。方法:将40只SD大鼠随机分为5组(n=8):A组(正常对照组);B组(哮喘4周组);C组(哮喘6周组);D组(给药4周组);E组(给药6周组)。测定气道反应性并观察气道壁嗜酸性粒细胞浸润;图像分析软件测定支气管壁厚度、支气管平滑肌厚度及支气管平滑肌细胞核数量;免疫组织化学染色、逆转录聚合酶链式反应(RT-PCR)检测PI3K蛋白及mRNA表达。结果:①B组、C组PI3Kp85α的蛋白及mRNA表达水平显著高于A组(P均<0.01),E组上述指标较B组、C组、D组均显著降低(P<0.01、P<0.01、P<0.05);②B组及C组支气管壁厚度、支气管壁平滑肌厚度、支气管壁平滑肌细胞核数量均较A组明显增加(P均<0.01),而E组上述指标较B组、C组、D组均显著降低(P均<0.01);③B组、C组的气道反应性均高于A组(P均<0.01),E组较B组、C组、D组均显著降低(P<0.01、P<0.01、P<0.05)。结论:气道平滑肌增生是气道重构的一个显著特征,PI3K可能在此起促进作用。雷公藤内酯醇可能通过下调PI3K的表达而减轻哮喘气道高反应性及抑制气道平滑肌增生,对哮喘气道重构有一定治疗作用。     

Characterization and functional studies of a FYVE domain-containing phosphatase in C. elegans

The myotubularin (MTM) enzymes are phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 3,5-bisphosphate phosphatases. Mutation of MTM1, the founder member of this family, is responsible for X-linked myotubular myopathy in humans. Here, we have isolated and characterized a Caenorhabditis elegans homology of the enzymes designated ceMTM3. ceMTM3 preferably dephosphorylates PI3P and contains a FYVE lipid-binding domain at its C-terminus which binds PI3P. Immunoblotting analyses revealed that the enzyme is expressed during the early development and adulthood of the animal. Immunofluorescent staining revealed predominant expression of the enzyme in eggs and muscles. Knockdown of the enzyme by using feeding-based RNA interference resulted in an increased level of PI3P and caused severe impairment of body movement of the worms at their post-reproductive ages and significantly shortened their lifespan. This study thus reveals an important role of the MTM phosphatases in maintaining muscle function, which may have clinical implications in prevention and treatment of sarcopenia.     

Time-, concentration-, and age-dependent inhibition of muscarinic receptor-stimulated phosphoinositide metabolism by ethanol in the developing rat brain

We have previously reported that administration of ethanol (EtOH; 4 g/Kg/day) to rats from postnatal day 4 to day 10 causes microencephaly and decreases muscarinic receptor-stimulated inositol metabolism on days 7 and 10 (1). An identical exposure to EtOH of adult rats, which resulted in similar blood EtOH concentrations, did not have any effect on the same system. Initial in vitro studies have shown the presence of a differential sensitivity to EtOH of the phosphoinositide system coupled to muscarinic receptors during development (2). In the present study we have expanded these findings by investigating the concentration-, time-, and age-dependent effects of EtOH on accumulation of [³H]inositol phosphates ([³H]InsPs) in brain slices. EtOH caused a dose-dependent inhibition of carbachol-stimulated phosphoinositide metabolism in cerebral cortex slices from 7 day-old rats. When the time of incubation with EtOH was increased to 90 minutes, concentrations as low as 50 mM, which are reached following in vivo administration of EtOH, significantly inhibited the muscarinic response. The effect of EtOH was rather specific for the muscarinic receptors, since, even with longer incubation times, the accumulation of [³H]InsPs induced by norepinephrine or serotonin was inhibited only at concentrations of 150–500 mM. The effect of EtOH was more pronounced in cerebral cortex, hippocampus and cerebellum, and less in the brainstem. The potency of EtOH in inhibiting carbachol-stimulated phosphoinositide metabolism was also dependent on the age of the animals. Its effect was maximal in the 7 day-old rat and less pronounced in younger and older animals. These results confirm that the phosphoinositide system coupled to muscarinic receptors might represent a relevant target for the developmental neurotoxicity of EtOH.Presented in part at the 29th Annual Meeting of the Society of Toxicology (Toxicologist 1990; 10: 273).     

Further studies on the mechanism of action of substance P in rat brain,involving selective phosphatidylinositol hydrolysis

We have suggested that substance P, in cerebral cortex, causes a phosphatidylinositol (PI) breakdown by a dual mechanism suggesting the involvement of either phospholipase A₂ or phospholipase C. We have presently characterized further these effects. Substance P (65 pM) provoked an increase in lysoPI concomitant with a decrease in PI level. This finding confirms the involvement of phospholipase A₂ activation. To study the involvement of phospholipase C in the action of higher doses (0.65 M) of the peptide, we used pulse-chase experiments (where phospholipid depletion was monitored) and short-term³²P-labeled slices (where phospholipid synthesis was studied). Substance P evoked an acceleration of both hydrolysis and resynthesis of PI as early as 15 s. A prolonged exposure (30 min) resulted in stimulation of PI hydrolysis without subsequent resynthesis. The peptide did not cause any effect on inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate. These alterations in PI metabolism take place simultaneously with a generation of diacylglycerol which showed two maxima at both indicated times.     

Phosphoinositide 3‐kinases in cell migration

Cell migration is essential for many biological processes in animals and is a complex highly co‐ordinated process that involves cell polarization, actin‐driven protrusion and formation and turnover of cell adhesions. The PI3K (phosphoinositide 3‐kinase) family of lipid kinases regulate cell migration in many different cell types, both through direct binding of proteins to their lipid products and indirectly through crosstalk with other pathways, such as Rho GTPase signalling. Emerging evidence suggests that the involvement of PI3Ks at different stages of migration varies even within one cell type, and is dependent on the combination of external stimuli, as well as on the signalling status of the cell. In addition, it appears that different PI3K isoforms have distinct roles in cell polarization and migration. This review describes how PI3K signalling is regulated by pro‐migratory stimuli, and the diverse ways in which PI3K‐mediated signal transduction contributes to different aspects of cell migration.     

Characterization of ouabain-induced phosphoinositide hydrolysis in brain slices of the neonatal rat

The effect of the Na/K-ATPase inhibitor ouabain on phosphoinositide (Ptdlns) hydrolysis was studied in rat brain cortical slices. Ouabain induced a dose-dependent accumulation of inositol phosphates (InsPs) which was much higher in neonatal rats (1570±40% of basal) than in adult animals (287±18% of basal). For this reason, all experiments were conducted with 7 day-old rats. Strophantidin caused a similar stimulation of Ptdlns hydrolysis, although it was less potent than ouabain. The order of potency for ouabain-stimulated InsPs accumulation in brain areas was hippocampus>cortex>brainstem>cerebellum. The effect of ouabain was not blocked by antagonists for the muscarinic, alpha₁-adrenergic and glutamate receptors. Also ineffective were the K⁺ channel blockers 4-aminopyridine and tetraethylammonium, the sodium channel blocker tetrodotoxin, and the calcium channel blocker verapamil, whereas the Na/Ca exchanger blocker amiloride partially antagonized the effect of ouabain. The accumulation of InsPs induced by ouabain was additive to that of carbachol and norepinephrine, as well as to that induced by high K⁺ and veratrine, but not to that of glutamate. Removal of Na⁺ ions from the incubation buffer completely prevented the accumulation of InsPs induced by ouabain. The effect of ouabain was also dependent upon extracellular calcium and was under negative feedback control of protein kinase C. Despite the higher effect of ouabain on Ptdlns hydrolysis of immature rats, the density of [³H]ouabain binding sites, as well as the activity of Na/K-ATPase were higher in adult animals. Furthermore, a poor correlation was found between ouabain-stimulated Ptdlns hydrolysis and [³H]ouabain binding in brain regions. These results suggest an involvement of Na⁺ pump in the hydrolysis of Ptdlns, possibly related to an effect on Na⁺ and Ca²⁺ homeostasis. The immature rat appear to be an useful model for studying the relationship between Na/K-ATPase and inositol metabolism.     

Crosstalk between the phosphatidylinositol cycle and MAP kinase signaling pathways in Xenopus mesoderm induction

Recent studies have established a role for the phosphoinositide (PI) cycle in the early patterning of Xenopus mesoderm. In explants, stimulation of this pathway in the absence of growth factors does not induce mesoderm, but when accompanied by growth factor treatment, simultaneous PI cycle stimulation results in profound morphological and molecular changes in the mesoderm induced by the growth factor. This suggests the possibility that the PI cycle exerts its influence via crosstalk, by modulating some primary mesoderm-inducing pathway. Given recent identification of mitogen-activated protein kinase (MAPK) as an intracellular mediator of some mesoderm-inducing signals, the present study explores MAPK as a potential site of PI cycle-mediated crosstalk .We report that MAPK activity, like PI cycle activity, increases in intact embryos during mesoderm induction. Phosphoinositide cycle stimulation during treatment of explants with basic fibroblast growth factor (bFGF) synergistically increases late-phase MAPK activity and potentiates bFGF-induced expression of Xbra , a MAPK-dependent mesodermal marker.     

Oxytocin and vasopressin stimulate inositol phosphate production in human gestational myometrium and decidua cells

The involvement of phosphoinositide hydrolysis in the action of oxytocin and vasopressin on the uterus was investigated in gestational myometrium and decidua cells by measuring the production of inositol phosphates. Both peptides stimulated a dose related increase in all three inositol phosphates in myometrium. This may be related to the control of sarcoplasmic Ca⁺⁺ levels in the myometrium. Oxytocin and vasopressin also stimulated inositol 1-phosphate (IP) production in decidua cells. The hydrolysis of phosphatidylinositol by decidua homogenates exhibited a precursor-product relationship for diacylglycerol and arachidonic acid accumulation. Hence both peptides may mobilise free arachidonic acid, for prostaglandin biosynthesis, from decidua cell phosphoinositides by the sequential action of phospholipase C and diacylglycerol lipase.