Alkalinization stimulates the purified plasma-membrane Ca2+ pump by increasing its Ca2+ affinity.

The finding that negatively charged phospholipids activate the plasma-membrane (Ca2+ + Mg2+)-ATPase and that polycations counteract this stimulation suggest that negative charges in the environment of the ATPase protein could be important for its function. The aim of the present work was to investigate whether changing the charges on the ATPase protein itself by modifying the pH within the physiological range affects the activity of the purified plasma-membrane Ca2+ pump from stomach smooth muscle. Increasing the pH from 6.9 to 7.4 and using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA) as a Ca2+ buffer, doubled the ATPase activity at 0.3 microM-Ca2+ in the presence of 100% phosphatidylcholine (PC) or after substituting 20% of the PC by negatively charged phospholipids PtdIns, PtdIns4P, phosphatidylserine and phosphatidic acid. This stimulatory effect was due to an increased affinity of the enzyme for Ca2+, while the Vmax. remained unaffected. In the case of PtdIns(4,5)P2, a stimulatory effect upon alkalinization was only observed at a PtdIns(4,5)P2 concentration of 10%. When a concentration of 20% was used, alkalinization decreased the Vmax. and no stimulatory effect on the ATPase at 0.3 microM-Ca2+ could be observed. Alkalinization not only stimulated the purified Ca2+ pump, but it also increased the activity of the enzyme in a plasma-membrane-enriched fraction from stomach smooth muscle by a factor of 2.06. The ionophore A23187-induced Ca2+ uptake in closed inside-out vesicles also increased by a factor of 2.54 if the pH was changed from 6.9 to 7.4. This finding indicates that the effect of pH is most likely to be exerted at the cytoplasmic site of the Ca2+ pump protein.     

Breakdown of phosphatidylinositol 4,5-bisphosphate in a T-cell leukaemia line stimulated by phytohaemagglutinin is not dependent on Ca2+ mobilization.

Addition of phytohaemagglutinin (PHA) to the [32P]Pi-prelabelled JURKAT cells, a human T-cell leukaemia line, resulted in a decrease of [32P]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to about 35% of the control value. The decrease was almost complete within 30s after the PHA addition. This decrease was followed by an increase in the 32P-labelling of phosphatidic acid (maximally 2.8-fold at 2 min). The stimulation of myo-[2-3H]inositol-prelabelled JURKAT cells by PHA induced an accumulation of [2-3H]inositol trisphosphate in the presence of 5 mM-LiCl. The result indicates hydrolysis of PtdIns (4,5)P2 by a phospholipase C. The PHA stimulation of JURKAT cells induced about 6-fold increase in the cytosolic free Ca2+ concentration, [Ca2+]i, which was reported by Quin-2, a fluorescent Ca2+ indicator. Studies with partially Ca2+-depleted JURKAT cells, with the Ca2+ ionophore A23187, and with 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate indicate that the breakdown of PtdIns(4,5)P2 is not mediated through changes of [Ca2+]i. These results therefore indicate that the PHA-induced breakdown of PtdIns(4,5)P2 in JURKAT cells is not dependent on the Ca2+ mobilization.     

Receptor-mediated metabolism of the phosphoinositides and phosphatidic acid in rat lacrimal acinar cells.

The metabolism of the inositol lipids and phosphatidic acid in rat lacrimal acinar cells was investigated. The muscarinic cholinergic agonist methacholine caused a rapid loss of 15% of [32P]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and a rapid increase in [32P]phosphatidic acid (PtdA). Chemical measurements indicated that the changes in 32P labelling of these lipids closely resembled changes in their total cellular content. Chelation of extracellular Ca2+ with excess EGTA caused a significant decrease in the PtdA labelling and an apparent loss of PtdIns(4,5)P2 breakdown. The calcium ionophores A23187 and ionomycin provoked a substantial breakdown of [32P]PtdIns(4,5)P2 and phosphatidylinositol 4-phosphate (PtdIns4P); however, a decrease in [32P]PtdA was also observed. Increases in inositol phosphate, inositol bisphosphate and inositol trisphosphate were observed in methacholine-stimulated cells, and this increase was greatly amplified in the presence of 10 mM-LiCl; alpha-adrenergic stimulation also caused a substantial increase in inositol phosphates. A23187 provoked a much smaller increase in the formation of inositol phosphates than did either methacholine or adrenaline. Experiments with excess extracellular EGTA and with a protocol that eliminates intracellular Ca2+ release indicated that the labelling of inositol phosphates was partially dependent on the presence of extracellular Ca2+ and independent of intracellular Ca2+ mobilization. Thus, in the rat lacrimal gland, there appears to be a rapid phospholipase C-mediated breakdown of PtdIns(4,5)P2 and a synthesis of PtdA, in response to activation of receptors that bring about an increase in intracellular Ca2+. The results are consistent with a role for these lipids early in the stimulus-response pathway of the lacrimal acinar cell.     

Ca2+ regulation of 1-(3-sn-phosphatidyl)-1D-myo-inositol 4-phosphate formation and hydrolysis on sarcoplasmic-reticular Ca2+-transport ATPase. A new principle of phospholipid turnover regulation.

Lipid phosphorylation was shown to occur on the isolated sarcoplasmic-reticulum (SR) Ca2+-transport ATPase. More than 95% of the radioactivity incorporated on incubation of the SR ATPase with [gamma-32P]ATPMg can be extracted with acidic organic solvents and was identified as 1-(3-sn-phosphatidyl)-1D-myo-inositol 4-phosphate (PtdIns4P) [Varsányi, Toelle, Heilmeyer, Dawson & Irvine (1983) EMBO J. 2, 1543-1548]. This lipid phosphorylation is only observed at nanomolar concentrations of free Ca2+; in the presence of micromolar free Ca2+ PtdIns4P disintegrates rapidly. Also, upon blockade of the kinase reaction PtdIns4P decomposes, indicating a PtdIns/PtdIns4P turnover. The PtdIns4P concentration is dependent on the free Ca2+ concentration, being half-maximal at 35 nM-Ca2+. PtdIns4P hydrolysis is catalysed by a PtdIns4P phosphomonoesterase; accordingly no diacylglycerol is formed, which would be a product of a phosphodiesteratic cleavage. Fluoride inhibits this phosphomonoesterase. Ca2+ does not influence directly either the PtdIns kinase or the PtdIns4P phosphomonoesterase. PtdIns4P forms a tight complex with the transport ATPase, from which it can be removed only by chromatography on heparin-agarose in the presence of Triton X-100. It is concluded that Ca2+ regulates the PtdIns/PtdIns4P turnover by availability of substrate, depending on the Ca2+-transport-ATPase conformation, which traps or exposes the respective lipid head groups.     

Effects of carbachol and pancreozymin (cholecystokinin-octapeptide) on polyphosphoinositide metabolism in the rat pancreas in vitro.

We studied the possibility that hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] may be the initiating event for the increase in [32P]Pi incorporation into phosphatidic acid (PtdA) and phosphatidylinositol (PtdIns) during carbachol and pancreozymin (cholecystokinin-octapeptide) action in the rat pancreas. After prelabelling acini for 2h, [32P]Pi incorporation into PtdA, PtdIns(4,5)P2 and phosphatidylinositol 4-phosphate (PtdIns4P) had reached equilibrium. Subsequent addition of carbachol or pancreozymin caused 32P in PtdIns(4,5)P2 to decrease by 30-50% within 10-15 s, and this was followed by sequential increases in [32P]Pi incorporation into PtdA and PtdIns. Similar changes in 32P-labelling of PtdIns4P were not consistently observed. Confirmation that the decrease in 32P in chromatographically-purified PtdIns(4,5)P2 reflected an actual decrease in this substance was provided by the fact that similar results were obtained (a) when PtdIns(4,5)P2 was prelabelled with [2-3H]inositol, and (b) when PtdIns(4,5)P2 was measured as its specific product (glycerophosphoinositol bisphosphate) after methanolic alkaline hydrolysis and ion-exchange chromatography. The secretogogue-induced breakdown of PtdIns(4,5)P2 was not inhibited by Ca2+ deficiency (severe enough to inhibit amylase secretion and Ca2+-dependent hydrolysis of PtdIns), and ionophore A23187 treatment did not provoke PtdIns(4,5)P2 hydrolysis. The increase in the hydrolysis of PtdIns(4,5)P2 and the increase in [32P]Pi incorporation into PtdA commenced at the same concentration of carbachol in dose-response studies. Our findings suggest that the hydrolysis of PtdIns(4,5)P2 is an early event in the action of pancreatic secretogogues that mobilize Ca2+, and it is possible that this hydrolysis may initiate the Ca2+-independent labelling of PtdA and PtdIns. Ca2+ mobilization may follow these responses, and subsequently cause Ca2+-dependent hydrolysis of PtdIns and exocytosis.     

Multiple metabolic pools of phosphoinositides and phosphatidate in human erythrocytes incubated in a medium that permits rapid transmembrane exchange of phosphate.

1. A Hepes-based medium has been devised which allows rapid Pi exchange across the plasma membrane of the human erythrocyte. This allows the metabolically labile phosphate pools of human erythrocytes to come to equilibrium with [32P]Pi in the medium after only 5 h in vitro. 2. After 5-7 h incubation with [32P]Pi in this medium, only three phospholipids, phosphatidic acid (PtdOH), phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) are radioactively labelled. The concentrations of PtdIns4P and PtdIns4,5P2 remain constant throughout the incubation, so this labelling process is a reflection of the steady-state turnover of their monoester phosphate groups. 3. During such incubations, the specific radioactivities of the monoesterified phosphates of PtdIns4, PtdIns4,5P2 and PtdOH come to a steady value after 5 h that is only 25-30% of the specific radioactivity of the gamma-phosphate of ATP at that time. We suggest that this is a consequence of metabolic heterogeneity. This heterogeneity is not a result of the heterogeneous age distribution of the erythrocytes in human blood. Thus it appears that there is metabolic compartmentation of these lipids within cells, such that within a time-scale of a few hours only 25-30% of these three lipids are actively metabolized. 4. The phosphoinositidase C of intact human erythrocytes, when activated by Ca2+-ionophore treatment, only hydrolyses 50% of the total PtdIns4,5P2 and 50% of 32P-labelled PtdIns4,5P2 present in the cells: this enzyme does not discriminate between the metabolically active and inactive compartments of lipids in the erythrocyte membrane. Hence at least four metabolic pools of PtdIns4P and PtdIns4,5P2 are distinguishable in the human erythrocyte plasma membrane. 5. The mechanisms by which multiple non-mixing metabolic pools of PtdOH, PtdIns4P and PtdIns4,5P2 are sustained over many hours in the plasma membranes of intact erythrocytes are unknown, although some possible explanations are considered.     

Secretagogue-induced phosphoinositide metabolism in human leucocytes.

The relationship between receptor binding of the formylated peptide chemoattractant formylmethionylleucylphenylalanine (fMet-Leu-Phe), lysosomal enzyme secretion and metabolism of membrane phospholipids was evaluated in both human polymorphonuclear leucocytes (PMN) and the dimethyl sulphoxide (Me2SO)-stimulated human myelomonocytic HL-60 leukaemic cell line. In both cell types, exposure to fMet-Leu-Phe (100 nM) induced rapid lysosomal enzyme secretion (maximal release less than 30 s) and marked changes in the 32P-labelling of the inositol lipids phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] as well as phosphatidic acid (PtdA). Specifically, levels of [32P]PtdIns and [32P]PtdIns(4,5)P2 decreased rapidly (peak decrease at 10-15s), with a subsequent increase at 30 s and later. PtdIns4P and PtdA showed only an increase. In Me2SO-differentiated HL-60 cells prelabelled with [3H]inositol for 20 h, fMet-Leu-Phe caused a net increase in the cellular content of [3H]inositol phosphates, including a rapid increase in [3H]inositol 1,4,5-trisphosphate, suggesting that PtdIns(4,5)P2 breakdown occurs by a phospholipase C mechanism. Both lysosomal enzyme secretion and changes in phospholipid metabolism occur over the same agonist concentration range with a similar time course. Binding of [3H]fMet-Leu-Phe, although occurring over the same concentration range, exhibited markedly slower kinetics. Although depletion of extracellular Ca2+ had no effect on ligand-induced polyphosphoinositide turnover, PtdIns turnover, PtdA labelling and lysosomal enzyme secretion were severely curtailed. These studies demonstrate a receptor-mediated enhancement of phospholipid turnover that correlates with a specific biological response to fMet-Leu-Phe. Further, the results are consistent with the idea that phospholipase C-mediated degradation of PtdIns(4,5)P2, which results in the formation of inositol trisphosphate, is an early step in the stimulus-secretion coupling pathway of the neutrophil. The lack of correlation between these two responses and the equilibrium-binding condition suggests that either these parameters are responsive to the rate of ligand-receptor interaction or only fractional occupation is required for a full biological response.     

Acidic phospholipids, unsaturated fatty acids, and limited proteolysis mimic the effect of calmodulin on the purified erythrocyte Ca2+ - ATPase

The purified Ca2+-pumping ATPase of human erythrocyte membranes (Niggli, V., Adunyah, E. S., Penniston, J. T., and Carafoli, E. (1981) J. Biol. Chem. 256, 395-401) can be stimulated, in the absence of calmodulin, by other treatments. 1. A variety of acidic phospholipids (phosphatidylserine, cardiolipin, phosphatidylinositol, and phosphatidic acid) stimulate the Vmax and decrease the Km (Ca2+) of the isolated enzyme to the same extent as calmodulin. Unsaturated fatty acids (oleic and linoleic acid) have the same effect as phospholipids but at lower concentrations. Neutral phospholipids (phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine) have no effect on the enzyme. The minimal proportion of acidic phospholipids in the environment of the enzyme necessary for full stimulation is about 40%. 2. The isolated enzyme, after reconstitution in phosphatidylcholine liposomes in the absence of calmodulin, can be activated by limited proteolysis. The trypsinized enzyme has the same high Vmax and high affinity for Ca2+ of the enzyme in the presence of calmodulin.     

Inositol lipids, phosphatidate and diacylglycerol share stearoylarachidonoylglycerol as a common backbone in thrombin-stimulated human platelets.

Gel-filtered human platelets were stimulated with 5i.u. of thrombin/ml for times up to 1 min. The fatty acid composition of inositol-containing phospholipids, phosphatidic acid and diacylglycerol was determined by g.l.c. in control and thrombin-stimulated platelet suspensions. Inositol phospholipids were found to have similar proportions of stearic and arachidonic acids, the sum of these representing 86.6% of the total fatty acids in phosphatidylinositol (PtdIns), 76.9% in phosphatidylinositol 4-phosphate (PtdIns4P) and 85.4% in phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. However, arachidonic and stearic acids were less abundant in phosphatidic acid (PtdA) and diacylglycerols in non-stimulated platelets. A transient decrease in the mass of PtdIns(4,5)P2 was observed after 5-10s of thrombin stimulation, followed by an increase after 30s. The amounts of PtdIns4P and PtdIns decreased throughout the experiment. A transient accumulation of stearoylarachidonoylglycerol was observed at 5s, whereas stearoylarachidonoylglycerol 3-phosphate (PtdA) was produced in increasing amounts throughout the experiment. The decrease in inositol-containing phospholipids was not fully compensated for by the production of diacylglycerol or PtdA [or PtdIns(4,5)P2] at 1 min. All the changes in inositol phospholipids, as well as those observed in diacylglycerols and PtdA, were due to a parallel reduction or increase in the contents of stearic and arachidonic acids, with a stoichiometry equal to 1. Taken together, this suggests an interconversion of all these lipids with the utilization of a common backbone, stearoylarachidonoylglycerol. The deacylation of this diacylglycerol could account for up to 4-5nmol of arachidonate/10(9) platelets after 1 min stimulation by thrombin.     

Changes in the platelet phosphoinositides during the first minute after stimulation of washed rabbit platelets with thrombin.

Experiments with washed platelets from rabbits demonstrate that stimulation with a low concentration of thrombin (0.1 unit/ml) that causes maximal aggregation and partial release of granule contents does not significantly decrease the amount of phosphatidylinositol 4,5-bisphosphate [ PtdIns (4,5)P2] at 10s; this contrasts with ADP stimulation. The amount of PtdIns (4,5)P2 was significantly decreased by a higher concentration of thrombin (0.3 unit/ml). Increased turnover of the PtdIns (4,5)P2 at 60s was indicated by changes in labelling with [3H]glycerol in platelets stimulated with both concentrations of thrombin. An unexpected observation with the lower thrombin concentration was a significant increase in the amount of phosphatidylinositol ( PtdIns ) at 10s. This contrasts with data from other laboratories, which indicate that thrombin causes a significant decrease in PtdIns . At 60s, with the lower concentration of thrombin, PtdIns was significantly decreased. With the higher concentration of thrombin there was a significant decrease in the amount of PtdIns at 10s, in keeping with the data from other laboratories. The initial increase in PtdIns may not have been observed by other investigators because higher concentrations of thrombin were used. The reaction involved in this initial increase in the amount of PtdIns does not appear to be increased degradation of PtdIns4P or PtdIns (4,5)P2, since their total amount was unchanged at 10s. The magnitude of the increase in PtdIns is such that more than the existing pool of phosphatidic acid would have to be converted into PtdIns to account for the increase. It is suggested that synthesis of phosphatidic acid de novo from dihydroxyacetone phosphate and glycerol 3-phosphate might be the source of phosphatidic acid, which leads to increased PtdIns at 10s with the lower concentration of thrombin. Thus it appears that the initial response of platelets to thrombin does not require an early change in PtdIns (4,5)P2 and may involve stimulation of synthesis de novo of PtdIns via phosphatidic acid.     

Characterization of partially purified phospholipase C from human platelet membranes.

A phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-hydrolytic activity was found to be present in the human platelet membrane fraction, with 20% of the total activity of the homogenate. The membrane-associated phospholipase C activity was extracted with 1% deoxycholate (DOC). The DOC-extractable phospholipase C was partially purified approx. 126-fold to a specific activity of 0.58 mumol of PtdIns-(4,5)P2 cleaved/min per mg of protein, by Q-Sepharose, heparin-Sepharose and Ultrogel AcA-44 column chromatographies. This purified DOC-extractable phospholipase C had an Mr of approx. 110,000, as determined by Ultrogel AcA-44 gel filtration. The enzyme exhibits a maximal hydrolysis for PtdIns-(4,5)P2 at pH 6.5 in the presence of 0.1% DOC. The addition of 0.1% DOC caused a marked activation of both PtdIns(4,5)P2 and phosphatidylinositol (PtdIns) hydrolyses by the enzyme. The enzyme hydrolysed PtdIns(4,5)P2 and PtdIns in a different Ca2+-dependent manner; the maximal hydrolyses for PtdIns(4,5)P2 and PtdIns were obtained at 4 microM- and 0.5 mM-Ca2+ respectively. In the presence of 1 mM-Mg2+, PtdIns(4,5)P2-hydrolytic activity was decreased at all Ca2+ concentrations examined, but PtdIns-hydrolytic activity was not affected.     

Partial purification and reconstitution of inositol 1,4,5-trisphosphate receptor/Ca2+ channel of bovine liver microsomes.

The binding of inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] to bovine liver microsomes was characterized. The Ins(1,4,5)P3 receptor of the microsomes was solubilized by 1% Triton X-100 and purified by sucrose density gradient, Heparin-Sepharose, DEAE-Toyopearl, ATP-Agarose, and Ins(1,4,5)P3-Sepharose column chromatographies. More than 1,000-fold enrichment of the Ins(1,4,5)P3-binding activity was achieved. Kd values of the binding activity were 2.8 nM in microsomes and 3.0 nM in the partially purified receptor, respectively, and the binding activity was optimal in the medium containing 100 mM KCl and at pH between 7.5 and 8.5. The presence of Ca2+ failed to inhibit the binding. Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PtdIns), and phosphatidylinositol-4-monophosphate [PtdIns(4)P] showed no effect on the Ins(1,4,5)P3 binding. However, soybean phospholipids asolectin and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] strongly inhibited the binding activity. PtdIns(4,5)P2 inhibited the activity competitively with a half-maximal inhibitory concentration of 30 micrograms/ml. The partially purified Ins(1,4,5)P3 receptor was reconstituted into proteoliposomes. Fluorescence measurements using Quin 2 indicated that Ins(1,4,5)P3 stimulated Ca2+ influx into the proteoliposomes. The EC50 of Ins(1,4,5)P3 on Ca2+ influx was 50 nM. This result strongly suggest that Ins(1,4,5)P3 binding protein of liver microsomes acts as a physiological Ins(1,4,5)P3 receptor/Ca2+ channel.     

Inositol lipid turnover and adenosine 3,5 cyclic monophosphate in the salt-secreting rectal gland of the dogfish (Scyliorhinus canicula)

The rectal gland of the dogfish is rich in inositol lipids. Total phospholipids from the gland contained 9.1 mol% of phosphatidylinositol (PtdIns), 1.0 mol% of phosphatidylinositol 4-phosphate (PtdIns4P) and 0.9 mol% of phosphatidylinositol 4,5-biphosphate (PtdIns4,5P2). [32P]Orthophosphate was readily incorporated into PtdIns, phosphatidic acid (PtdA) and especially into PtdIns4P and PtdIns4,5P2 in salt gland slices incubated in elasmobranch Ringer with glucose and no other additions over a 2 hr period. The calcium ionophore A23187 stimulated incorporation into PtdIns and PtdA, but not into PtdIns4P or PtdIns4,5P2. Oxygen uptake by rectal gland slices was maximally stimulated by 0.08mM forskolin, 2.5mM 8-chlorophenylthio cyclic AMP, 2.0mM dibutyryl cyclic AMP and 0.25mM theophylline. Stimulated oxygen uptake was inhibited by 0.1mM ouabain in all cases. Incorporation of [32P]orthophosphate into PtdIns, PtdA, PtdIns4P and PtdIns4,5P2 was inhibited by 0.08mM forskolin and 2.0mM dibutyryl cyclic AMP over a 2 hr period. The results are discussed in relation to the control of salt secretion by the rectal gland.     

Increased oxidation of uroporphyrinogen by an inducible liver microsomal system. Possible relevance to drug-induced uroporphyria.

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.     

Schwann Cell Proliferation Is Accompanied by Enhanced Inositol Phospholipid Metabolism

Inositol phospholipid metabolism during mitogen-induced Schwann cell proliferation has been examined. Addition of axolemma- and myelin-enriched membrane fractions (AXL and MYE, respectively) to cultured Schwann cells stimulated 32P incorporation into phosphatidylinositol 4-monophosphate [PtdIns(4)P] and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. During the first 5 min of incubation with the mitogens, the amount of 32P incorporated into PtdIns(4)P and PtdIns(4,5)P2 was four- to fivefold above control values. The phosphorylation of the inositol phospholipids was dependent on the concentration of membrane mitogens and was maximal within 1 h. Schwann cells that were prelabeled with [3H]glycerol and then stimulated with AXL and MYE displayed a 30-70% increase in the amounts of [3H]PtdIns(4)P and [3H]PtdIns(4,5)P2 and a 60-80% increase in the amount of [3H]phosphatidic acid. A concomitant 20% decrease in the content of [3H]PtdIns was observed after stimulation. These results suggest that the increased metabolism of PtdIns, PtdIns(4)P, and PtdIns(4,5)P2 may be one of the initial molecular events in the transduction of the mitogenic signal across the Schwann cell plasma membrane.     

Sodium ion and the neutrotransmitter-stimulated 32P labelling of phosphoinositides and other phospholipids in the iris muscle

The effects of Na+, other cations and the neurotransmitters, acetylcholine and norepinephrine on 32Pi incorporation into phospholipids of the rabbit iris smooth muscle were investigated [1]. The basal 32P-labelling of phospholipids including phosphatidic acid, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine and the polyphosphoinositides increased with Na+ concentration [2]. The neurotransmitter-stimulated 32P labelling of phosphatidic acid, phosphatidylinositol and phosphatidylcholine is dependent on the presence of extracellular Na+ [3]. The monovalent cation requirement for Na+ specific. Of the monovalent cations Li+, NH+4, K+, Choline+ and Tris, only Li+ partially substituted for Na+ [4]. A significant decrease in 32P labelling of phospholipids in response to acetylcholine was observed when Ca2+ and/or K+ were added to an isoosmotic medium deficient of Na+ [5]. Ouabain, which blocks the Na+-pump, inhibited the basal 32Pi incorporation into phosphatidylcholine and the acetylcholine-stimulated 32P labelling of phosphatidic acid, phosphatidylinositol and phosphatidylcholine [6]. It was suggested that phosphoinositide breakdown is associated with Ca2+ influx as we have previously reported (Akhtar, R.A. and Abdel-Latif, A.A. (1978) J. Pharmacol. Exp. Ther. 204, 655-668) and that the enhanced 32P-labelling of phosphoinositides could be associated with Na+ outflux, via the Na+-pump mechanism.     

Phosphatidylinositol 4,5-bisphosphate anchors cytosolic group IVA phospholipase A2 to perinuclear membranes and decreases its calcium requirement for translocation in live cells

The eicosanoids are centrally involved in the onset and resolution of inflammatory processes. A key enzyme in eicosanoid biosynthesis during inflammation is group IVA phospholipase A2 (also known as cytosolic phospholipase A2alpha, cPLA2alpha). This enzyme is responsible for generating free arachidonic acid from membrane phospholipids. cPLA2alpha translocates to perinuclear membranes shortly after cell activation, in a process that is governed by the increased availability of intracellular Ca2+. However, cPLA2alpha also catalyzes membrane phospholipid hydrolysis in response to agonists that do not mobilize intracellular Ca2+. How cPLA2alpha interacts with membranes under these conditions is a major, still unresolved issue. Here, we report that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] promotes translocation of cPLA2alpha to perinuclear membranes of intact cells in a manner that is independent of rises in the intracellular Ca2+ concentration. PtdIns(4,5)P2 anchors the enzyme to perinuclear membranes and allows for a proper interaction with its phospholipid substrate to release arachidonic acid.     

Receptor-mediated net breakdown of phosphatidylinositol 4,5-bisphosphate in parotid acinar cells.

The metabolism of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] in rat parotid acinar cells was investigated, particularly with regard to the effects of receptor-active agonists. Stimulation of cholinergic-muscarinic receptors with methacholine provoked a rapid disappearance of 40--50% of [32P]PtdIns(4,5)P2, but had no effect on PtdIns4P. Adrenaline, acting on alpha-adrenoceptors, and Substance P also stimulated net loss of PtdIns(4,5)P2. The beta-adrenoceptor agonist, isoprenaline, and the Ca2+ ionophore, ionomycin, failed to affect labelled PtdIns(4,5)P2 or PtdIns4P. By chelation of extracellular Ca2+ with excess EGTA, and by an experimental protocol that eliminates cellular Ca2+ release, it was demonstrated that the agonist-induced decrease in PtdIns(4,5)P2 is independent of both Ca2+ influx and Ca2+ release. These results may suggest that net PtdIns(4,5)P2 breakdown is an early event in the stimulus-response pathway of the parotid acinar cell and could be directly involved in the mechanism of agonist-induced Ca2+ release from the plasma membrane.     

Rapid breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in rat hepatocytes stimulated by vasopressin and other Ca2+-mobilizing hormones.

Rat hepatocytes rapidly incorporate [32P]Pi into phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]; their monoester phosphate groups approach isotopic equilibrium with the cellular precursor pools within 1 h. Upon stimulation of these prelabelled cells with Ca2+-mobilizing stimuli (V1-vasopressin, angiotensin, alpha 1-adrenergic, ATP) there is a rapid fall in the labelling of PtdIns4P and PtdIns(4,5)P2. Pharmacological studies suggest that each of the four stimuli acts at a different population of receptors. Insulin, glucagon and prolactin do not provoke disappearance of labelled PtdIns4P and PtdIns(4,5)P2. The labelling of PtdIns4P and PtdIns(4,5)P2 in cells stimulated with vasopressin or angiotensin initially declines at a rate of 0.5-1.0% per s, reaches a minimum after 1-2 min and then returns towards the initial value. The dose-response curves for the vasopressin- and angiotensin-stimulated responses lie close to the respective receptor occupation curves, rather than at the lower hormone concentrations needed to evoke activation of glycogen phosphorylase. Disappearance of labelled PtdIns4P and PtdIns(4,5)P2 is not observed when cells are incubated with the ionophore A23187. The hormone-stimulated polyphosphoinositide disappearance is reduced, but not abolished, in Ca2+-depleted cells. These hormonal effects are not modified by 8-bromo cyclic GMP, cycloheximide or delta-hexachlorocyclohexane. The absolute rate of polyphosphoinositide breakdown in stimulated cells is similar to the rate previously reported for the disappearance of phosphatidylinositol [Kirk, Michell & Hems (1981) Biochem. J. 194, 155-165]. It seems likely that these changes in polyphosphoinositide labelling are caused by hormonal activation of the breakdown of PtdIns(4,5)P2 (and may be also PtdIns4P) by the action of a polyphosphoinositide phosphodiesterase. We therefore suggest that the initial response to hormones is breakdown of PtdIns(4,5)P2 (and PtdIns4P?), and that the simultaneous disappearance of phosphatidylinositol might be a result of its consumption for the continuing synthesis of polyphosphoinositides.     

Coupled inositide phosphorylation and phospholipase D activation initiates clathrin-coat assembly on lysosomes.

Adaptors appear to control clathrin-coat assembly by determining the site of lattice polymerization but the nucleating events that target soluble adaptors to an appropriate membrane are poorly understood. Using an in vitro model system that allows AP-2-containing clathrin coats to assemble on lysosomes, we show that adaptor recruitment and coat initiation requires phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) synthesis. PtdIns(4,5)P2 is generated on lysosomes by the sequential action of a lysosome-associated type II phosphatidylinositol 4-kinase and a soluble type I phosphatidylinositol 4-phosphate 5-kinase. Phosphatidic acid, which potently stimulates type I phosphatidylinositol 4-phosphate 5-kinase activity, is generated on the bilayer by a phospholipase D1-like enzyme located on the lysosomal surface. Quenching phosphatidic acid function with primary alcohols prevents the synthesis of PtdIns(4, 5)P2 and blocks coat assembly. Generating phosphatidic acid directly on lysosomes with exogenous bacterial phospholipase D in the absence of ATP still drives adaptor recruitment and limited coat assembly, indicating that PtdIns(4,5)P2 functions, at least in part, to activate the PtdIns(4,5)P2-dependent phospholipase D1. These results provide the first direct evidence for the involvement of anionic phospholipids in clathrin-coat assembly on membranes and define the enzymes responsible for the production of these important lipid mediators.