PtdIns P2 Levels Research Articles

The Drosophila insulin/IGF receptor controls growth and size by modulating PtdInsP(3) levels.

Understanding the control of size is of fundamental biological and clinical importance. Insulin/IGF signaling during development controls growth and size, possibly by coordinating the activities of the Ras and PI 3-kinase signaling pathways. We show that in Drosophila mutating the consensus binding site for the Ras pathway adaptor Drk/Grb2 in Chico/IRS does not interfere with growth whereas mutating the binding sites of the PI 3-kinase adaptor p60 completely abrogates Chico function. Furthermore, we present biochemical and genetic evidence that loss of the homolog of the tumor suppressor gene, Pten, results in increased PtdInsP(3) levels and that these increased levels are sufficient to compensate for the complete loss of the Insulin/insulin-like growth factor receptor function. This reduction of Pten activity is also sufficient to vastly increase organism size. These results suggest that PtdInsP(3) is a second messenger for growth and that levels of PtdInsP(3) during development regulate organismal size.

Regulation and recruitment of phosphatidylinositol 4-kinase on immature secretory granules is independent of ADP-ribosylation factor 1.

Heterotrimeric G-proteins, as well as small GTPases of the Rho and ADP-ribosylation factor (ARF) family, are implicated in the regulation of lipid kinases, including PtdIns 4-kinases and PtdIns(4)P 5-kinases. Here, we describe a PtdIns 4-kinase activity on immature secretory granules (ISGs), regulated secretory organelles formed from the trans-Golgi network (TGN), and investigate the regulation of PtdIns4P levels on these membranes. Over 50% of the PtdIns 4-kinase activity on ISGs is inhibited by both a low concentration of adenosine and the monoclonal antibody 4C5G, a specific inhibitor of the type II PtdIns 4-kinase. Treatment of ISGs with mastoparan 7 (M7) stimulates the type II PtdIns 4-kinase via pertussis-toxin-sensitive G(i)/G(0) proteins, which, in contrast with previous results obtained with chromaffin granules [Gasman, Chasserot-Golaz, Hubert, Aunis and Bader (1998) J. Biol. Chem. 273, 16913-16920], does not require Rho A, B or C. M7 treatment also leads to an inhibition in the recruitment of ARF to ISG membranes: this inhibition is not dependent on G(i)/G(0) activation, and is not linked to the stimulation of PtdIns 4-kinase observed with M7. PtdIns 4-kinase activity on ISGs is not regulated by myristoylated ARF1-GTP, in contrast with results obtained with Golgi membranes [Godi, Pertile, Meyers, Marra, Di Tullio, Iurisci, Luini, Corda and De Matteis (1999) Nat. Cell Biol. 1, 280-287; Jones, Morris, Morgan, Kondo, Irvine and Cockcroft (2000) J. Biol. Chem. 275, 13962-13170], whereas ARF1-GTP does regulate the production of PtdIns(4,5)P(2). Our results suggest that the regulation of PtdIns 4-kinase on the ISGs differs in comparison with that on the TGN, and might be related to a specific requirement of ISG maturation.

Regulation and recruitment of phosphatidylinositol 4-kinase on immature secretory granules is independent of ADP-ribosylation factor 1

Heterotrimeric G-proteins, as well as small GTPases of the Rho and ADP-ribosylation factor (ARF) family, are implicated in the regulation of lipid kinases, including PtdIns 4-kinases and PtdIns(4)P 5-kinases. Here, we describe a PtdIns 4-kinase activity on immature secretory granules (ISGs), regulated secretory organelles formed from the trans-Golgi network (TGN), and investigate the regulation of PtdIns4P levels on these membranes. Over 50% of the PtdIns 4-kinase activity on ISGs is inhibited by both a low concentration of adenosine and the monoclonal antibody 4C5G, a specific inhibitor of the type II PtdIns 4-kinase. Treatment of ISGs with mastoparan 7 (M7) stimulates the type II PtdIns 4-kinase via pertussis-toxin-sensitive Gi/G0 proteins, which, in contrast with previous results obtained with chromaffin granules [Gasman, Chasserot-Golaz, Hubert, Aunis and Bader (1998) J. Biol. Chem. 273, 16913–16920], does not require Rho A, B or C. M7 treatment also leads to an inhibition in the recruitment of ARF to ISG membranes: this inhibition is not dependent on Gi/G0 activation, and is not linked to the stimulation of PtdIns 4-kinase observed with M7. PtdIns 4-kinase activity on ISGs is not regulated by myristoylated ARF1—GTP, in contrast with results obtained with Golgi membranes [Godi, Pertile, Meyers, Marra, Di Tullio, Iurisci, Luini, Corda and De Matteis (1999) Nat. Cell Biol. 1, 280–287; Jones, Morris, Morgan, Kondo, Irvine and Cockcroft (2000) J. Biol. Chem. 275, 13962–13170], whereas ARF1—GTP does regulate the production of PtdIns(4,5)P2. Our results suggest that the regulation of PtdIns 4-kinase on the ISGs differs in comparison with that on the TGN, and might be related to a specific requirement of ISG maturation.

Plasma membrane phosphatidylinositol 4,5-bisphosphate levels decrease with time in culture.

During the stationary phase of growth, after 7 to 12 d in culture, the levels of phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)) decreased by 75% in plasma membranes of the red alga Galdieria sulphuraria. Concomitant with the decrease in PtdInsP(2) levels in plasma membranes, there was an increase in PtdInsP(2) in microsomes, suggesting that the levels of plasma membrane PtdInsP(2) are regulated differentially. The decline of PtdInsP(2) in plasma membranes was accompanied by a 70% decrease in the specific activity of PtdInsP kinase and by reduced levels of protein cross-reacting with antisera against a conserved PtdInsP kinase domain. Upon osmotic stimulation, the loss of PtdInsP(2)from the plasma membrane increased from 10% in 7-d-old cells to 60% in 12-d-old cells, although the levels of inositol 1,4,5-trisphosphate (InsP(3)) produced in whole cells were roughly equal at both times. When cells with low plasma membrane PtdInsP(2) levels were osmotically stimulated, a mild osmotic stress (12.5 mM KCl) activated PtdInsP kinase prior to InsP(3) production, whereas in cells with high plasma membrane PtdInsP(2), more severe stress (250 mM KCl) was required to induce an increase in PtdInsP kinase activity. The differential regulation of a plasma membrane signaling pool of PtdInsP(2) is discussed with regard to the implications for understanding the responsive state of cells.

Inositol lipids are regulated during cell cycle progression in the nuclei of murine erythroleukaemia cells.

Previous data suggest the existence of discrete pools of inositol lipids, which are components of a nuclear phosphoinositide (PI) cycle. However, it is not known whether the contents of these pools are regulated during cell proliferation. In the present study we demonstrate that the mass levels of three important constituents of the nuclear PI cycle are regulated during the cell cycle. Radioactive label incorporation into PtdIns(4,5)P(2) was seen to increase dramatically as synchronized cells entered S-phase. This did not coincide with any significant changes in the nuclear mass levels of this lipid, suggesting that the rate of turnover of this molecule was increased. Levels of PtdIns4P, the major substrate for PtdIns(4,5)P(2) production by Type I PtdInsP kinases (PIPkins), were regulated during the cell cycle and indicated a complex relationship between these two lipids. An alternative substrate for PtdIns(4,5)P(2), PtdIns5P, phosphorylated by Type II PIPkins, was present in nuclei at much smaller amounts than the PtdIns4P, and thus is unlikely to contribute significantly to PtdIns(4,5)P(2) turnover. However, a large increase in nuclear PtdIns5P mass was observed when murine erythroleukaemia cells are in G(1), and this could represent a potential pool of nuclear inositol lipid that has a specific signalling role. Analysis of extracted lipid fractions indicated the absence of any PtdIns3P in these nuclei.

Inositol lipids are regulated during cell cycle progression in the nuclei of murine erythroleukaemia cells

Previous data suggest the existence of discrete pools of inositol lipids, which are components of a nuclear phosphoinositide (PI) cycle. However, it is not known whether the contents of these pools are regulated during cell proliferation. In the present study we demonstrate that the mass levels of three important constituents of the nuclear PI cycle are regulated during the cell cycle. Radioactive label incorporation into PtdIns(4,5)P2 was seen to increase dramatically as synchronized cells entered S-phase. This did not coincide with any significant changes in the nuclear mass levels of this lipid, suggesting that the rate of turnover of this molecule was increased. Levels of PtdIns4P, the major substrate for PtdIns(4,5)P2 production by Type I PtdInsP kinases (PIPkins), were regulated during the cell cycle and indicated a complex relationship between these two lipids. An alternative substrate for PtdIns(4,5)P2, PtdIns5P, phosphorylated by Type II PIPkins, was present in nuclei at much smaller amounts than the PtdIns4P, and thus is unlikely to contribute significantly to PtdIns(4,5)P2 turnover. However, a large increase in nuclear PtdIns5P mass was observed when murine erythroleukaemia cells are in G1, and this could represent a potential pool of nuclear inositol lipid that has a specific signalling role. Analysis of extracted lipid fractions indicated the absence of any PtdIns3P in these nuclei.

Cellular regulation of cytosolic group IV phospholipase A2 by phosphatidylinositol bisphosphate levels.

Cytosolic group IV phospholipase A2 (cPLA2) is a ubiquitously expressed enzyme with key roles in intracellular signaling. The current paradigm for activation of cPLA2 by stimuli proposes that both an increase in intracellular calcium and mitogen-activated protein kinase-mediated phosphorylation occur together to fully activate the enzyme. Calcium is currently thought to be needed for translocation of the cPLA2 to the membrane via a C2 domain, whereas the role of cPLA2 phosphorylation is less clearly defined. Herein, we report that brief exposure of P388D1 macrophages to UV radiation results in a rapid, cPLA2-mediated arachidonic acid mobilization, without increases in intracellular calcium. Thus, increased Ca2+ availability is a dispensable signal for cPLA2 activation, which suggests the existence of alternative mechanisms for the enzyme to efficiently interact with membranes. Our previous in vitro data suggested the importance of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in the association of cPLA2 to model membranes and hence in the regulation of cPLA2 activity. Experiments described herein show that PtdInsP2 also serves a similar role in vivo. Moreover, inhibition of PtdInsP2 formation during activation conditions leads to inhibition of the cPLA2-mediated arachidonic acid mobilization. These results suggest that cellular PtdInsP2 levels are involved in the regulation of group IV cPLA2 activation.

Inositol phospholipid 3-kinase is activated by cellular stress but is not required for the stress-induced activation of glucose transport in L6 rat skeletal muscle cells.

A characteristic response of cells subjected to a stress stimulus is a rapid activation of cellular glucose transport. The mechanisms governing this increase in glucose transport are poorly understood, but it has been suggested that the response may involve the intracellular-signaling components that also participate in the hormonal activation of glucose transport. In skeletal muscle and fat tissue, inositol phospholipid 3-kinase plays an integral role in the regulation of both basal and insulin-stimulated glucose transport. In this study, we have investigated whether inositol phospholipid 3-kinase is activated by chemical stress and, if so, whether it has a role to play in the stress-induced increase in glucose transport in L6 muscle cells. Furthermore, we have attempted to assess the basis by which inositol phospholipid 3-kinase may participate in the regulation of basal glucose transport. Acute exposure (30 min) of L6 muscle cells to 0.5 mM arsenite induced an 80% stimulation in glucose transport. This activation was due to a rise in the number of cell-surface glucose transporters, based on an increase in the Vmax of glucose transport and the observation that arsenite increases the plasma membrane content of GLUT1 and GLUT4 glucose transporters by 95% and 60%, respectively, from an intracellular compartment. Arsenite induced rapid activation (< 2 min) of inositol phospholipid 3-kinase with an approximately fourfold increase in phosphatidylinositol 3,4,5-trisphosphate (PtdIns3,4,5P3). In contrast, phosphatidylinositol 3-phosphate (PtdIns3P) levels were unaffected. Prior treatment of L6 cells with 100 nM wortmannin suppressed the arsenite-induced increase in PtdIns3,4,5P3 and reduced the cellular content of PtdIns3P by 50%. Under these conditions however, wortmannin failed to prevent the stress-induced activation of glucose transport, but suppressed basal glucose transport by 60% with an IC50 of about 10 nM. In the absence of arsenite, wortmannin caused a dose-dependent inhibition in the cellular levels of PtdIns3P and PtdIns3,4,5P3 with IC50 values of about 10 nM and 100 nM, respectively. In summary, the present results demonstrate that chemical stress activates inositol phospholipid 3-kinase and glucose transport in L6 muscle cells, but unlike the hormonal responses of these cells the activation of inositol phospholipid 3-kinase is not responsible for the stress-induced increase in glucose transport. This implies that stress-induced and hormonal stimulated increases in PtdIns3,4,5P3 levels are functionally distinct. By contrast, the maintenance of PtdIns3P levels, presumably involving a PtdIns-specific, wortmannin-sensitive inositol phospholipid 3-kinase may be required to support basal glucose transport.

Phosphatidylinositol 3,5-bisphosphate defines a novel PI 3-kinase pathway in resting mouse fibroblasts.

PtdIns(3,5)P2 is identified as the product of an agonist-independent, wortmannin-sensitive pathway in resting mouse cells. Results are presented here to indicate that PtdIns(3,5)P2 is formed by phosphorylation of PtdIns3P at the D-5 position, and they suggest that relatively constant cellular levels of PtdIns3P and PtdIns(3, 5)P2 are maintained by the concerted action of PtdIns3P 5-kinase and PtdIns(3,5)P2 5-phosphatase. These studies imply a novel mechanism for the action of PtdIns-specific phosphoinositide 3-hydroxykinases in mammalian cells.

Phorbol esters stimulate phosphatidylinositol 3,4,5-trisphosphate production in 3T3-L1 adipocytes: implications for stimulation of glucose transport.

The effects of insulin and phorbol 12-myristate 13-acetate (PMA) on the levels of cellular phosphoinositides were investigated in 3T3-L1 adipocytes. Stimulation for 4 min with PMA (1 microM) or insulin (10 nM) increased levels of PtdIns(3,4,5)P3 approx. 2-fold and 6-fold respectively. PMA also had a small effect on the cellular levels of PtdIns4P, whereas insulin had no effect on PtdIns4P levels; levels of PtdIns(4,5)P2 and PtdIns3P were not significantly affected by either agent. Insulin increased the levels of the p85 alpha subunit of phosphoinositide (PI) 3-kinase associated with membranes, whereas PMA decreased levels of membrane-associated p85 alpha. PMA did not increase PI 3-kinase activity in anti-phosphotyrosine or anti-p85 immunoprecipitates. The stimulation of glucose transport by insulin or PMA was blocked by 100 nM wortmannin or 10 ng/ml LY294002, indicating that PI 3-kinase is essential for stimulation by both agents. In summary, these results demonstrate: (1) that PMA and insulin stimulate PtdIns(3,4,5)P3 production by distinct mechanisms in 3T3-L1 adipocytes, and (2) that stimulation of PtdIns(3,4,5)P3 production by PMA is likely to be important in signalling pathways leading from PMA stimulation to end-point responses such as glucose transport.

Possible involvement of a tyrosine kinase-dependent pathway in the regulation of phosphoinositide metabolism by vanadate in normal mouse islets

The potential roles of protein tyrosine kinases (TKs) and of phosphotyrosine phosphatases (PTPs) in pancreatic islet function are not known. In this study, we investigated whether vanadate, a potent PTP inhibitor, affects phosphoinositide (PI) metabolism by a TK-dependent pathway in isolated mouse islets. To avoid the confounding effects of changes in Ca2+ influx, all experiments were performed in the absence of Ca2+. In the presence of 15mM glucose, vanadate, acetylcholine (ACh) or [Arg]vasopressin (AVP) strongly stimulated InsP production. Vanadate also increased PtdInsP levels in membranes. The TK inhibitor genistein (not its inactive analogues genistin and daidzein) significantly reduced vanadate effects, but was without effect in the absence of stimulation or in the presence of ACh or AVP. Islet proteins resolved by SDS/PAGE were analysed by immunobloting with anti-phosphotyrosine antibody. Under control conditions, several phosphotyrosyl-proteins (PYPs) were present. Vanadate increased phosphotyrosine residues on several PYPs, notably two proteins of 145 and 85 kDa. This effect was prevented by genistein, p145 and p85 could correspond to phospholipate Cgamma(PLCgamma) and the regulatory subunit of PtdIns-3-kinase (PtdIns-3K) respectively. Both proteins are expressed in islets, as revealed by immunoblots with specific antibodies. Tungstate, another PTP inhibitor, reproduced vanadate effects, but inhibition of PtdIns-3K by wortmannin failed to affect vanadate-increased PtdInsP levels. Incubation of the islets in the presence of 10% (v/v) fetal calf serum instead of BSA increased InsP production and this effect was prevented by genistein. These results suggest that inhibition of PTP increases InsP production in mouse islets by a TK-dependent pathway. They also provide evidence for a potential role of TK and PTP in pancreatic B-cell function.

Both phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase products are increased by antigen receptor signaling in B cells.

The effects of Ag binding on B cell development and activation are mediated by intracellular signals initiated by the B cell AgR. In this report, we show that the B cell AgR regulates the production of inositol phospholipids involved in two different signal transduction pathways, the phosphatidylinositol 3-kinase (PtdIns 3-kinase) pathway and the phospholipase C (PLC) pathway. Phosphatidylinositol 3-phosphate (PtdIns3P), phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2], and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] are produced by PtdIns 3-kinase, an enzyme that appears to be an essential component of tyrosine kinase-mediated signaling. Both PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are likely to function as second messengers in vivo because they can activate the zeta isoform of protein kinase C (PKC) in vitro. We show that cross-linking of the B cell AgR with anti-Ig antibodies caused a five- to sixfold increase in the levels of PtdIns(3,4)P2 in both the mature B cell line BAL 17 and the immature B cell line WEHI-231. PtdIns(3,4)P2 levels increased within 15 s of anti-Ig addition and remained elevated for at least 5 min. AgR cross-linking also caused a slower increase in PtdIns3P levels (approximately 50% over control) and a small, transient increase in PtdIns(3,4,5)P3 levels. Thus, the B cell AgR activates the PtdIns 3-kinase pathway. The other inositol phospholipid signaling pathway involves PLC, which cleaves phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], yielding second messengers that increase intracellular calcium and activate other isoforms of PKC. We analyzed the effects of AgR signaling on PtdIns(4,5)P2 and its precursor, phosphatidylinositol 4-phosphate (PtdIns4P). Consistent with its ability to activate PLC, AgR ligation decreased the levels of PtdIns(4,5)P2. In contrast, AgR cross-linking increased the levels of PtdIns4P. Increased synthesis of PtdIns4P followed by phosphorylation at the D-5 position may prevent depletion of PtdIns(4,5)P2. Thus, signaling by the B cell AgR increases the levels of PtdIns 4-kinase products and PtdIns 3-kinase products. The simplest interpretation of our results is that the B cell AgR activates both PtdIns 3-kinase and PtdIns 4-kinase.

Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses.

Phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) is rapidly produced upon exposure of neutrophils to the chemoattractant N-formylmethionyl-leucylphenylalanine (fMLP), and has been proposed to act as a second messenger mediating actin polymerization and respiratory-burst activity. Here we present evidence that wortmannin, a known inhibitor of respiratory-burst activity, acts on PtdIns 3-kinase, the enzyme producing PtdInsP3 from PtdIns(4,5)P2. Pretreatment of 32P-labelled human neutrophils with 100 nM wortmannin totally abolished fMLP-mediated PtdInsP3 production, raised PtdInsP2 levels, and did not affect cellular PtdInsP and PtdIns contents. The inhibitory effect on PtdInsP3 formation in intact cells was dose-dependent, with an IC50 of approximately 5 nM. Similar results were obtained with PtdIns 3-kinase immunoprecipitated by antibodies against the p85 regulatory subunit: wortmannin totally inhibited PtdIns3P production in immunoprecipitates at concentrations of 10-100 nM (IC50 approximately 1 nM). These results illustrate the direct and specific inhibition of PtdIns 3-kinase by wortmannin. Since agonist-mediated respiratory-burst activation is most sensitive to wortmannin (IC50 = 12 nM), this suggests that agonist-mediated PtdInsP3 formation is indispensable for this cell response. Neutrophils pretreated with wortmannin develop oscillatory changes in F-actin content, but actin polymerization in response to fMLP is not inhibited. This, and the absence of PtdInsP3 under these conditions, are in agreement with a modulatory role for PtdInsP3 in cytoskeletal rearrangements, but imply that PtdInsP3 production is not a primary event triggering elongation of actin filaments in neutrophils.

Phosphatidylinositol availability and polyphosphoinositide synthesis in pancreatic islet cell membranes

Polyphosphoinositide synthesis in isolated islets of the rat was determined by the phosphorylation of endogenous phosphatidylinositol (PtdIns) by PtdIns kinase and [γ- 32P]ATP to form [ 32P]-phosphatidylinositol 4-phosphate (PtdInsP) in cell homogenates. Glucose stimulation of intact islets resulted in a time- and concentration-dependent reduction in PtdInsP synthesis. Similarly, the stimulation of intact islets with carbachol (CCh), cholecystokinin (CCK-8S), or tolbutamide for 15 min reduced PtdInsP production in a concentration-dependent manner. The effects of glucose, tolbutamide and CCh were reversible. PtdInsP hydrolysis did not account for the reduction in PtdInsP recovery. The addition of exogenous PtdIns to the PtdIns kinase assay significantly increased basal PtdInsP levels. In addition, exogenous PtdIns completely reversed the inhibitory effects of glucose and increased PtdIns kinase activity in homogenates of glucose-stimulated islets to levels found in control homogenate with PtdIns. Exogenous PtdIns also increased PtdIns kinase activity in CCK-8S-treated islets, although exogenous PtdIns did not overcome the tolbutamide-induced inhibition of PtdIns kinase. The V max of PtdIns kinase in homogenates of islets treated with tolbutamide was reduced significantly, although glucose did not affect the V max. In addition, the K m values for ATP and PtdIns were not altered by exposure of the islets to cell stimuli. The results suggest that the level of PtdIns in islet cell membranes is rate limiting for PtdInsP synthesis, and that tolbutamide is a noncompetitive inhibitor of PtdIns kinase.

Phosphoinositide metabolism in intestinal smooth muscle: preferential production of Ins(1,4,5)P3 in circular muscle cells.

The pattern of inositol phospholipid metabolism in response to stimulation by contractile agonists was examined in muscle cells isolated separately from circular and longitudinal muscle layers of guinea pig intestine. Addition of cholecystokinin octapeptide (CCK-8) to circular muscle cells caused a prompt decrease in phosphatidylinositol 4,5-bisphosphate (PtdInsP2) (50 +/- 6%) and PtdInsP (38 +/- 9%), which reached a nadir in 15 s. Addition of CCK-8 to longitudinal muscle cells caused a decrease in PtdInsP only (42 +/- 6%); PtdInsP2 levels, which were three times lower in this cell type, did not change throughout the period of stimulation. Hydrolysis of PtdInsP2 in circular muscle cells was accompanied by a concentration-dependent increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]; the maximal increase was 10 to 16 times greater in circular muscle cells as measured by radioreceptor assay and by ion-exchange chromatography. The small amounts of InsP3 produced in longitudinal muscle cells did not result from more rapid degradation, since the rates of disappearance of exogenous Ins(1,4,5)P3 in both cell types were similar. Within 5 s after addition of CCK-8, InsP3 comprised 73 +/- 5% of total inositol phosphates produced in circular muscle cells and 2.0 +/- 0.2% in longitudinal muscle cells. These divergent patterns indicate that inositol phospholipid metabolism is channeled toward generation of InsP3 in circular muscle cells only; the metabolic pattern in these cells parallels the selective presence of high-affinity InsP3 receptors and the pattern of intracellular Ca2+ mobilization.

Evidence that the inositol phospholipids are necessary for exocytosis. Loss of inositol phospholipids and inhibition of secretion in permeabilized cells caused by a bacterial phospholipase C and removal of ATP

We directly manipulated the levels of PtdIns, PtdInsP and PtdInsP2 in digitonin-treated adrenal chromaffin cells with a bacterial phospholipase C (PLC) from Bacillus thuringiensis and by removal of ATP. The PtdIns-PLC acted intracellularly to cause a large decrease in [3H]inositol- or [32P]phosphate-labelled PtdIns, but did not directly hydrolyse PtdInsP or PtdInsP2. [3H]PtdInsP and [3H]PtdInsP2 levels declined markedly, probably because of the action of phosphatases in the absence of synthesis. Removal of ATP also caused marked decreases in [3H]PtdInsP and [3H]PtdInsP2. The decrease in polyphosphoinositide levels by PtdIns-PLC treatment or ATP removal was reflected by the inhibition of the production of inositol phosphates upon subsequent activation of the endogenous PLC by Ca2(+)-dependent catecholamine secretion from permeabilized cells was strongly inhibited by PtdIns-PLC treatment and by ATP removal. Ca2(+)-dependent secretion was similarly correlated with the sum of PtdInsP and PtdInsP2 when the level of these lipids was changed by either manipulation. PtdIns-PLC inhibited only the ATP-dependent component of secretion and did not affect ATP-dependent secretion. Both PtdIns-PLC and ATP removal inhibited the late slow phase of secretion, but had little effect on the initial rapid phase. Although we found a tight correlation between polyphosphoinositide levels and secretion, endogenous phospholipase C activity (stimulated by Ca2+, guanine nucleotides and related agents) was not correlated with secretion. Additional experiments indicated that neither the products of the PtdIns-PLC reaction (diacylglycerol and InsP1) nor the inability to generate products by subsequent activation of the endogenous PLC is likely to account for the inhibition of secretion. Incubation of permeabilized cells with neomycin in the absence of ATP maintained the level of polyphosphoinositides and more than doubled subsequent Ca2(+)-dependent secretion. The data suggest that: (1) Ca2(+)-dependent secretion has a requirement for the presence of inositol phospholipids; (2) the enhancement of secretion by ATP results in part from increased polyphosphoinositide levels; and (3) the role for inositol phospholipids in secretion revealed in these experiments is independent of their being substrates for the generation of diacylglycerol and InsP3.

Evidence for the calcium-dependent activation of phospholipase D in thrombin-stimulated human erythroleukaemia cells

Human erythroleukaemia (HEL) cells were exposed to thrombin and other platelet-activating stimuli, and changes in radiolabelled phospholipid metabolism were measured. Thrombin caused a transient fall in PtdInsP and PtdInsP2 levels, accompanied by a rise in diacylglycerol and phosphatidic acid, indicative of a classical phospholipase C/diacylglycerol kinase pathway. However, the rise in phosphatidic acid preceded that of diacylglycerol, which is inconsistent with phospholipase C/diacylglycerol kinase being the sole source of phosphatidic acid. In the presence of ethanol, thrombin and other agonists (platelet-activating factor, adrenaline and ADP, as well as fetal-calf serum) stimulated the appearance of phosphatidylethanol, an indicator of phospholipase D activity. The Ca2+ ionophore A23187 and the protein kinase C activator phorbol myristate acetate (PMA) also elicited phosphatidylethanol formation, although A23187 was at least 5-fold more effective than PMA. Phosphatidylethanol production stimulated by agonists or A23187 was Ca2(+)-dependent, whereas that with PMA was not. These result suggest that phosphatidic acid is generated in agonist-stimulated HEL cells by two routes: phospholipase C/diacylglycerol kinase and phospholipase D. Activation of the HEL-cell phospholipase D in response to agonists may be mediated by a rise in intracellular Ca2+.

Metabolic and structural evidence for the existence of a third species of polyphosphoinositide in cells: d-phosphatidyl-myo-inositol 3-phosphate

When human 1321 N1 astrocytoma cells were labelled to steady state with [3H]inositol and briefly with [32P]orthophosphate, a compound which contained both radiotracers and which co-migrated with phosphatidylinositol-myo-inositol 4-phosphate during t.l.c. could be extracted in acidic chloroform/methanol. Treatment with methylamine under conditions which lead to deacylation of conventional glycerophospholipids yielded a water-soluble moiety which was labelled with both radioisotopes and was eluted from an anion-exchange h.p.l.c. column with a retention time similar to, but distinct from, that of glycerophosphoinositol 4-phosphate. Experiments using sodium periodate and selective phosphatase enzymes to degrade this compound systematically generated a series of products which suggested the structure of the parent phospholipid was phosphatidyl-myo-inositol 3-phosphate (PtdIns3P). PtdIns3P is metabolically closely related to the pool(s) of inositol phospholipid(s) that serves as substrate(s) for an agonist-sensitive phosphoinositidase C, as the levels of PtdIns3P fell significantly when 1321 N1 cells were stimulated with carbachol. The relative rate of turnover of the inositol moiety of PtdIns3P is similar to that of both of the major polyphosphoinositides and significantly higher than that of total cellular phosphatidyl-myo-inositol. This suggests that all three polyphosphoinositides are synthesized from a common, rapidly metabolized, pool of phosphatidyl-myo-inositol.

Effects of pre-weaning undernutrition and post-weaning rehabilitation on polyphosphoinositide pools in rat brain regions.

In order to assess the effects of undernutrition during the pre-weaning period on polyphosphoinositide (PolyPI) pools in rat cerebral cortex, brain stem, and cerebellum, dams were fed 5% (L-) or 22% (L+) protein diets from birth to weaning and the pups were used at this age for analyses. To examine rehabilitation post-weaning, L- and L+ pups were fed 22% protein diets (P+) for an additional six week period. Rats were decapitated and the dissection begun either immediately ("0 min" samples) or 10 min later (10 min samples). Body and tissue weights, and cerebroside levels were determined in addition ot PolyPI concentrations. In brain the extent of disappearance of PolyPI during the 10 min post-mortem period paralleled the content of gray matter: cerebral cortex greater than cerebellum greater than brain stem in all groups regardless of diet. Levels of PtdIns4P and PtdIns4,5P2 were decreased by 40% and 70% respectively in cerebral cortex of L- "0 min" samples. Deficits of both lipids in brain stem and cerebellum were 40-50%. In the L- 10 min samples, deficits were 20-30% in all three regions as compared with L+ 10 min levels, indicating the presence of a portion of both lipids affected only moderately by nutritional insufficiency. The effects on this relatively inert pool, much of it localized in myelin, were reversed on nutritional rehabilitation. The PolyPI pool lost post-mortem in L+ brain regions was practically absent in L- brain regions and was not restored in L-P+ animals. Thus, this study indicates that a metabolically labile pool, primarily located in gray matter structures, is more sensitive to nutritional deprivation during the pre-weaning period than the more stable pool. The precise role and function of these pools remain to be determined.

The alleged kimberlite-carbonatite relationship; additional contrary mineralogical evidence

<h3>Abstract</h3> Lysosomes orchestrate degradation and recycling of exogenous and endogenous material, thus controlling cellular homeostasis. Little is known how this organelle changes during malignant transformation. We investigate the intracellular landscape of lysosomes in a cellular model of bladder cancer. Employing standardized cell culture on micropatterns we identify a phenotype of peripheral lysosome positioning prevailing in bladder cancer but not normal urothelium. We show that lysosome positioning is controlled by transcription factor EB (TFEB) and that lysosomal dispersion results from TFEB activation downstream of lysosomal Ca²⁺ release. Remarkably, we find that TFEB regulates phosphatidylinositol-3-phosphate (PtdIns3P) levels on endomembranes which recruit FYVE-domain containing proteins, such as the motor adaptor protrudin, for anterograde movement of lysosomes. Altogether, we uncover lysosome positioning as result of PtdIns3P activation downstream of TFEB as a potential biomarker for bladder cancer. Moreover, we reveal a novel role of TFEB in regulating cellular PtdIns levels, conceptually clarifying the dual role of TFEB as regulator of endosomal maturation and autophagy, two distinct processes controlled by PtdIns3P. <h3>Statement of significance</h3> Here we provide the first atlas for the landscape of the lysosomal compartment in bladder cancer and reveal the mechanistic role of TFEB in regulating endosomal PtdIns3P levels and subsequent lysosomal dispersion. We unveiled lysosomal positioning as a potential biomarker for malignant bladder cancer which might arise as an actionable target for cancer therapy.