Phospholipase C activation induced by noradrenaline in rat hippocampal slices is potentiated by GABA-receptor stimulation.

We have studied the effect of gamma-aminobutyric acid (GABA) and other GABA-receptor agonists (3-aminopropanesulphonic acid and muscimol) on the noradrenaline-induced stimulation of polyphosphoinositide metabolism in rat hippocampal slices. Formation of water-soluble inositol phosphates, and polyphosphoinositide metabolism were studied in hippocampal slices prelabelled with [3H]myoinositol. Noradrenaline induced formation of inositol mono-, bis- and trisphosphate during 10 min incubation in the presence of lithium; activation of phospholipase C by noradrenaline was also reflected by the hydrolysis of polyphosphoinositides and by the increased metabolism of phosphatidylinositol. GABA-receptor agonists were unable to activate per se phospholipase C; however, when added together with a low concentration of noradrenaline, they greatly potentiated the noradrenaline-stimulated polyphosphoinositide metabolism. We conclude that GABA-receptor agonists potentiate the effect of noradrenaline on polyphosphoinositide turnover and we discuss the role of this neurotransmitter interaction in the physiology of the hippocampus.     

Evidence for a GTP-binding protein coupling thrombin receptor to PIP2-phospholipase C in membranes of hamster fibroblasts

Two different methods were used to study directly alpha-thrombin modulation of polyphosphoinositide breakdown in membranes prepared from Chinese hamster lung (CHL) fibroblasts. In the first one we labelled the lipid pool by incubating the intact cells with myo-[3H]inositol prior to membrane isolation; in the other we used exogenous [3H]PIP2 with phosphatidylethanolamine (1:10) added as liposomes to freshly isolated membranes. A Ca2+-dependent PIP2 and PIP phospholipase C activity was characterized by measuring the rate of formation of inositol tris- and bisphosphate. Basal phospholipase C activity was stimulated up to 3-fold by GTP or GTP-gamma-S. Of the two mitogens, alpha-thrombin and EGF, known to stimulate DNA synthesis in Chinese hamster fibroblasts, only alpha-thrombin is a potent activator of PIP2 breakdown in intact cells. Consistent with this observation, alpha-thrombin but not EGF potentiated GTP-gamma-S-dependent phospholipase C activity in membrane preparations. These results strongly support the hypothesis that a GTP-binding protein couples alpha-thrombin receptor to PIP2 hydrolysis. Because both methods used to assay phospholipase C gave identical results, we conclude that the coupling is at the level of PIP2-phosphodiesterase activity.     

G-protein linked polyphosphoinositide phospholipase C activity in cell membranes from clonally derived and ras-transformed Madin-Darby canine kidney cells

Membranes prepared from clone D1 of Madin-Darby canine kidney (MDCK) cells contain activity that can be attributed to Gp, a guanine nucleotide binding protein linked to phosphatidylinositol 4,5-bisphosphate dependent phospholipase C. Polyphosphoinositides are produced by addition of GTP, nonhydrolyzable GTP analogs, or fluoroaluminate. This production is inhibited by guanosine 5'-(beta-thiodiphosphate). While Ca2+ at 1 microM or more can generate high yields of inositol phosphates, guanine nucleotide activation of Gp can potentiate this Ca2(+)-dependent yield at resting levels of the cation. Membranes from cells expressing large amounts of ras-p21 exhibit small differences in guanine nucleotide induced polyphosphoinositide quantities. The greatest difference between normal and ras membranes was seen with AlF4- incubation. Of the three inositol phosphates measured, only the inositol bisphosphate yield was greatly increased in ras membranes compared with membranes from both parental and the D-1 clone of MDCK cells. From these data, we conclude that the presence of ras-p21 may affect production of polyphosphoinositides in MDCK cell membranes by some means other than direct participation in phospholipase C activation.     

Calcium promotes the accumulation of polyphosphoinositides in intact and permeabilized bovine adrenal chromaffin cells

1. Because cellular pools of phosphatidylinositol phosphate and phosphatidylinositol bisphosphate turn over rapidly during phospholipase C stimulation, the continuing production of inositol phosphates requires continuing synthesis from phosphatidylinositol of the polyphosphoinositides. In the present study in adrenal chromaffin cells, we examined the effects of nicotinic stimulation and depolarization in intact cells and micromolar Ca2+ in permeabilized cells on the levels of labeled polyphosphoinositides. We compared the effects to muscarinic stimulation in intact cells and GTP gamma S in permeabilized cells. 2. Nicotinic stimulation, elevated K+, and muscarinic stimulation cause similar production of inositol phosphates (D. A. Eberhard and R. W. Holz, J. Neurochem. 49:1634-1643, 1987). Nicotinic stimulation and elevated K+ but not muscarinic stimulation increased the levels of [3H]inositol-labeled phosphatidylinositol phosphate by 30-60% and [3H]phosphatidylinositol bisphosphate by 25-30%. The increase required Ca2+ in the medium, was maximal by 1-2 min, and was not preceded by an initial decrease in phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. 3. In digitonin-permeabilized cells, Ca2+ caused as much as a twofold increase in [3H]phosphatidylinositol phosphate and [3H]phosphatidylinositol bisphosphate. Similarly, Ca2+ enhanced the production of [32P]phosphatidylinositol phosphate and [32P]phosphatidylinositol bisphosphate in the presence of [gamma-32P]ATP. In contrast, GTP gamma S in permeabilized cells decreased polyphosphoinositides in the presence or absence of Ca2+. 4. The ability of Ca2+ to increase the levels of the polyphosphoinositides decayed with time after permeabilization. The effect of Ca2+ was increased when phosphoesterase and phospholipase C activities were inhibited by neomycin. 5. These observations suggest that Ca2+ specifically enhances polyphosphoinositide synthesis at the same time that it activates phospholipase C.     

The role of polyphosphoinositides and their breakdown products in A23187-induced release of arachidonic acid from rabbit polymorphonuclear leucocytes.

Stimulation of rabbit polymorphonuclear leucocytes with A23187 causes phospholipase C mediated breakdown of polyphosphoinositides, as evidenced by accumulation of [3H]inositol-labelled inositol bisphosphate and inositol trisphosphate. At the same time the polyphosphoinositides and the products of their breakdown, diacylglycerol and phosphatidic acid, label rapidly with radioactive arachidonic acid. Enhancement of polyphosphoinositide labelling is not as great as enhancement of diacylglycerol or phosphatidic acid labelling, suggesting additional early activation of a second independent synthetic pathway to the last named lipids. Experiments using double (3H/14C) labelling, to distinguish pools with different rates of turnover, suggest the major pool of arachidonic acid used for synthesis of lipoxygenase metabolites turns over more slowly than arachidonic acid in diacylglycerol, but at about the same rate as arachidonic acid esterified in phosphatidylcholine or phosphatidylinositol. Further, when cells are prelabelled with [14C]arachidonic acid, then stimulated for 5 min, it is only from phosphatidylcholine, and to a lesser extent phosphatidylinositol, that radiolabel is lost. Release of arachidonic acid is probably via phospholipase A2, since it is blocked by the phospholipase A2 inhibitor manoalide. The absence of accumulated lysophosphatides can be explained by reacylation and, in the case of lysophosphatidylinositol, deacylation. The importance of phospholipase A2 in phosphatidylinositol breakdown contrasts with the major role of phospholipase C in polyphosphoinositide hydrolysis. Measurements of absolute free fatty acid levels, as well as studies showing a correlation between production of radiolabelled hydroxyeicosatetraenoic acids and release of radiolabel from the phospholipid pool, both suggest that hydrolysis of arachidonic acid esterified into phospholipids is the limiting factor regulating formation of lipoxygenase metabolites. By contrast with A23187, fMet-Leu-Phe (a widely used polymorphonuclear leucocyte activator) is a poor stimulant for arachidonic acid release unless a 'second signal' (e.g. cytochalasin B, or a product of A23187-stimulated cells) is also present. In the presence of cytochalasin B, fMet-Leu-Phe, like A23187, stimulates release of radiolabelled arachidonic acid principally from phosphatidylcholine.     

Effect of glucose on polyphosphoinositide metabolism in isolated rat islets of Langerhans.

The metabolism of inositol-containing phospholipids during insulin secretion was studied in rat islets of Langerhans preincubated with [3H]inositol to label their phospholipids. Glucose (20 mM) caused a rapid breakdown of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate and an accumulation of inositol trisphosphate and inositol bisphosphate. This effect was maximal at 60s, did not require the presence of extracellular Ca2+, and was abolished by mannoheptulose (15 mM), but not by noradrenaline (1 microM). Mannose (20 mM) and DL-glyceraldehyde (10 mM) produced similar effects to those of glucose, but galactose (20 mM) and KCl (30 mM) were without effect. These results are compatible with the hypothesis that an early event in the stimulus-secretion coupling mechanism in the pancreatic B-cell is the rapid breakdown of polyphosphoinositides catalysed by phospholipase C. Moreover, they suggest that the breakdown of polyphosphoinositides is linked to sugar metabolism in the B-cell. This observation is important, since it demonstrates that events in a cell other than plasma-membrane receptor occupancy can promote polyphosphoinositide hydrolysis.     

Polyphosphoinositide hydrolysis by phospholipase C is accelerated by thyrotropin releasing hormone (TRH) in clonal rat pituitary cells (GH3 cells)

Thyrotropin releasing hormone (TRH) accelerates the turnover of phosphatidylinositol in GH3 cells ('phospholipid response'). From the analysis of inositol phosphates in the presence of Li+ which inhibits their dephosphorylation, it can be concluded that the hydrolysis of phosphatidylinositol 4,5-biphosphate, and possibly of phosphatidylinositol 4-phosphate by phospholipase C is markedly accelerated by TRH. It appears that this reaction initiates the acceleration of phosphatidylinositol turnover. The specificity of hormonally regulated phospholipase C reaction for polyphosphoinositides has important implications for the potential role of the phospholipid response as a mechanism of membrane signal transduction.     

Thyrotropin-releasing hormone rapidly activates the phosphodiester hydrolysis of polyphosphoinositides in GH3 pituitary cells. Evidence for the role of a polyphosphoinositide-specific phospholipase C in hormone action

The early actions of thyrotropin-releasing hormone (TRH) have been studied in hormone-responsive clonal GH3 rat pituitary cells. Previous studies had demonstrated that TRH promotes a "phosphatidylinositol response" in which increased incorporation of [32P]orthophosphate into phosphatidylinositol and phosphatidic acid was observed within minutes of hormone addition. The studies described here were designed to establish whether increased labeling of phosphatidylinositol and phosphatidic acid resulted from prior hormone-induced breakdown of an inositol phosphatide. GH3 cells were prelabeled with [32P]orthophosphate or myo-[3H]inositol. Addition of TRH resulted in the rapid disappearance of labeled polyphosphoinositides, whereas levels of phosphatidylinositol and other phospholipids remained unchanged. TRH-promoted polyphosphoinositide breakdown was evident by 5 S and maximal by 15 s of hormone treatment. Concomitant appearance of inositol polyphosphates in [3H]inositol-labeled cells was observed. In addition, TRH rapidly stimulated diacylglycerol accumulation in either [3H]arachidonic- or [3H]oleic acid-labeled cultures. These results indicate that TRH rapidly causes activation of a polyphosphoinositide-hydrolyzing phospholipase C-type enzyme. The short latency of this hormone effect suggests a proximal role for polyphosphoinositide breakdown in the sequence of events by which TRH alters pituitary cell function.     

Inhibition of plant plasma membrane phosphoinositide phospholipase C by the actin-binding protein, profilin

A key event in signal transduction in many eukaryotes is activation of polyphosphoinositide-specific phospholipase C (PIC). This enzyme hydrolyses the plasma membrane-associated lipid, phosphatidylinositol(4,5)bisphosphate (Ptdlns(4,5)P₂) which leads to the production of the two second messenger molecules: inositol(1,4,5)trisphosphate (Ins(1,4,5)P₃) and 1,2-diacylglycerol (DG). In plants, an enzyme which functionally resembles mammalian PIC is known to exist in the plasma membrane, but little is understood about how its activity is regulated. The recent discovery of several plant proteins with 30–40% homology to the mammalian actin- and phosphoinositide-binding protein, profilin, has prompted an investigation as to whether these proteins (plant profilins) are able to interact with polyphosphoinositides and, if so, whether such interactions have physiological relevance for signal transduction via the plant phosphoinositide system.
In this study it is demonstrated that a direct and highly specific interaction does exist between plant profilin and polyphosphoinositides and that these interactions dramatically affect the ability of plant plasma membrane phosphoinositide phospholipase C to utilize phosphoinositides for second messenger production. These data are the first to demonstrate a functional role of plant profilin in controlling polyphosphoinositide turnover and also provide the first evidence for a direct effect of an actin-binding protein on a membrane-associated signalling enzyme. These findings indicate a novel mechanism for control of plant phosphoinositide turnover, and suggest a possible link between plant cell activation, second messenger production and modulation of cytoskeletal dynamics.     

Thrombin- and nucleotide-activated phosphatidylinositol 4,5-bisphosphate phospholipase C in human platelet membranes

Thrombin, nucleotides, and chelators elicited a phosphatidylinositol 4,5-bisphosphate (PtdIns-P2) phospholipase C activity that was associated with human platelet membranes. Both alpha- and gamma-thrombin enhanced phospholipase C activity, whereas active site-inhibited alpha-thrombin did not stimulate PtdIns-P2 hydrolysis. PtdIns-P2 phospholipase C was also activated by nucleoside triphosphates, citrate, EDTA, and NaF. Magnesium was an inhibitor of PtdIns-P2 hydrolysis stimulated by nucleotides and chelators. Only PtdIns-P2 was degraded by the phospholipase C activated by alpha-thrombin, nucleotides, and chelators. The soluble fraction phospholipase C activity was also stimulated at low protein concentrations by nucleotides; however, soluble fraction phospholipase C activity cleaved both PtdIns-P2 and phosphatidylinositol 4-phosphate and was inhibited by chelators, suggesting the presence of a different enzyme in this compartment. The pH optimum for the membrane-associated phospholipase C in the presence of alpha-thrombin or nucleotides was 6.0, and the PtdIns-P2 phospholipase C was inhibited by neomycin and high detergent concentrations. Guanine nucleotides did not synergistically activate phospholipase C in the presence of alpha-thrombin. The characteristics of the membrane-associated PtdIns-P2 phospholipase C suggest that this enzyme is involved in platelet activation by the low-affinity alpha- or gamma-thrombin-dependent pathway.     

Phospholipid turnover during cell-cycle traverse in synchronous Chinese-hamster ovary cells. Mitogenesis without phosphoinositide breakdown.

The turnover of phospholipids was investigated in quiescent serum-starved Chinese-hamster ovary (CHO-K1) cells stimulated to progress through the cell cycle by the addition of dialysed bovine serum. A variety of radiolabelling techniques were employed to study the rapid effects of serum on phospholipids and later events during G1 and S phases of the cell cycle. Pulse-labelling studies using [32P]Pi revealed that there was a stimulation of the synthesis rate of all phospholipids investigated during the initial few hours after serum addition. The greatest stimulation (20-fold) was observed in phosphatidylcholine, and the smallest in the polyphosphoinositides (PPIs). Mock stimulation with serum-free medium caused a similar increase in PPI turnover, but little or no effect on turnover of other phospholipids. This effect could be accounted for by a stimulation of the turnover of cellular ATP pools increasing [32P]ATP specific radioactivity. Late G1 and S phases were associated with a decrease in the rate of synthesis of all phospholipids. Phosphatidic acid was the only phospholipid whose labelling fell below that in mock-stimulated cells during the period of the cell cycle. Stimulation of serum-starved cells that had been prelabelled with myo-[2-3H]inositol caused no change in the amounts of inositol trisphosphate, but both serum-stimulated and mock-stimulated cells exhibited similar small decreases in both inositol bisphosphate and inositol monophosphate, of approx. 30% after 30 s. When cells were serum-stimulated in the presence of 10 mM-Li+, there was no increase in the size of the total inositol phosphate pool. We conclude that mitogenic stimulation and cell-cycle traverse cause profound and complex effects on phospholipid turnover in CHO-K1 cells, but there is no evidence for a role of inositol lipid turnover in the proliferative response to serum in this cell line.     

Carbachol induces a rapid and sustained hydrolysis of polyphosphoinositide in bovine tracheal smooth muscle measurements of the mass of polyphosphoinositides, 1,2-diacylglycerol, and phosphatidic acid

The effects of carbachol on polyphosphoinositides and 1,2-diacylglycerol metabolism were investigated in bovine tracheal smooth muscle by measuring both lipid mass and the turnover of [3H]inositol-labeled phosphoinositides. Carbachol induces a rapid reduction in the mass of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate and a rapid increase in the mass of 1,2-diacylglycerol and phosphatidic acid. These changes in lipid mass are sustained for at least 60 min. The level of phosphatidylinositol shows a delayed and progressive decrease during a 60-min period of carbachol stimulation. The addition of atropine reverses these responses completely. Carbachol stimulates a rapid loss in [3H]inositol radioactivity from phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate associated with production of [3H]inositol trisphosphate. The carbachol-induced change in the mass of phosphoinositides and phosphatidic acid is not affected by removal of extracellular Ca2+ and does not appear to be secondary to an increase in intracellular Ca2+. These results indicate that carbachol causes phospholipase C-mediated polyphosphoinositide breakdown, resulting in the production of inositol trisphosphate and a sustained increase in the actual content of 1,2-diacylglycerol. These results strongly suggest that carbachol-induced contraction is mediated by the hydrolysis of polyphosphoinositides with the resulting generation of two messengers: inositol 1,4,5-trisphosphate and 1,2-diacylglycerol.     

Pertussis toxin inhibits thrombin-induced activation of phosphoinositide hydrolysis and Na+/H+ exchange in hamster fibroblasts.

Prior treatment with pertussis toxin of G0-arrested hamster fibroblasts (CCL39) results in a dose-dependent inhibition of two early events of the mitogenic response elicited by alpha-thrombin: accumulation of inositol phosphates in Li+-treated cells, and activation of the Na+/H+ antiport, measured either by the amiloride-sensitive 22Na+ influx or by the increase in intracellular pH. At 10(-1) U/ml of alpha-thrombin, the maximal inhibition was approximately 50% for these two early cellular responses, but the pertussis toxin effect was more pronounced at lower thrombin concentrations. In contrast, pertussis toxin does not affect the Na+/H+ antiport activation induced by phorbol esters or EGF, the action of which is not mediated by the phosphoinositide-metabolizing pathway in CCL39 cells. Therefore, our data suggest the following. A GTP-binding regulatory protein is probably involved in signal transduction between thrombin receptors and the phosphatidylinositol 4,5-bisphosphate-specific phospholipase C. This regulation does not seem to be exerted via modulations of cyclic AMP levels. The thrombin-induced activation of Na+/H+ antiport is, at least in part, mediated by the protein kinase C, as a consequence of stimulation of phosphatidylinositol turnover.     

Antigen-stimulated metabolism of inositol phospholipids in the cloned murine mast-cell line MC9.

Cells of the murine mast-cell clone MC9 grown in suspension culture were sensitized with an anti-DNP (dinitrophenol) IgE and subsequently prelabelled by incubating with [32P]Pi. Stimulation of these cells with DNP-BSA (bovine serum albumin) caused marked decreases in [32P]polyphosphoinositides (but not [32P]phosphatidylinositol) with concomitant appearance of [32P]phosphatidic acid. Whereas phosphatidylinositol monophosphate levels returned to baseline values after prolonged stimulation, phosphatidylinositol bisphosphate levels remained depressed. Stimulation of sensitized MC9 cells with DNP-BSA increased rates of incorporation of [32P]Pi into other phospholipids in the order: phosphatidylcholine greater than phosphatidylinositol greater than phosphatidylethanolamine. In sensitized cells prelabelled with [3H]inositol, release of inositol monophosphate, inositol bisphosphate and inositol trisphosphate, was observed after stimulation with DNP-BSA. When Li+ was added to inhibit the phosphatase activity that hydrolysed the phosphomonoester bonds in the sugar phosphates, greater increases were observed in all three inositol phosphates, particularly in inositol trisphosphate. The IgE-stimulated release of inositol trisphosphate was independent of the presence of extracellular Ca2+. In addition, the Ca2+ ionophore A23187 caused neither the decrease in [32P]polyphosphoinositides nor the stimulation of the release of inositol phosphates. These results demonstrate that stimulation of the MC9 cell via its receptor for IgE causes increased phospholipid turnover, with effects on polyphosphoinositides predominating. These data support the hypothesis that hapten cross-bridging of IgE receptors stimulates phospholipase C activity, which may be an early event in stimulus-secretion coupling of mast cells. The results with the Ca2+ ionophore A23187 indicate that an increase in intracellular Ca2+ alone is not sufficient for activation of this enzyme.     

The polyphosphoinositide phosphodiesterase of erythrocyte membranes

1. A new assay procedure has been devised for measurement of the Ca(2+)-activated polyphosphoinositide phosphodiesterase (phosphatidylinositol polyphosphate phosphodiesterase) activity of erythrocyte ghosts. The ghosts are prepared from cells previously incubated with [(32)P]P(i). They are incubated under appropriate conditions for activation of the phosphodiesterase and the released (32)P-labelled inositol bisphosphate and inositol trisphosphate are separated by anion-exchange chromatography on small columns of Dowex-1 (formate form). When necessary, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate can be deacylated and the released phosphodiesters separated on the same columns. 2. The release of both inositol bisphosphate and inositol trisphosphate was rapid in human ghosts, with half of the labelled membrane-bound phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate broken down in only a few minutes in the presence of 0.5mm-Ca(2+). For both esters, optimum rates of release were seen at pH6.8-6.9. Mg(2+) did not provoke release of either ester. 3. Ca(2+) provoked rapid polyphosphoinositide breakdown in rabbit erythrocyte ghosts and a slower breakdown in rat ghosts. Erythrocyte ghosts from pig or ox showed no release of inositol phosphates when exposed to Ca(2+). 4. In the presence of Mg(2+), the inositol trisphosphate released from phosphatidylinositol 4,5-bisphosphate was rapidly converted into inositol bisphosphate by phosphomonoesterase activity. 5. Neomycin, an aminoglycoside antibiotic that interacts with polyphosphoinositides, inhibited the breakdown of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, with the latter process being appreciably more sensitive to the drug. Phenylmethanesulphonyl fluoride, an inhibitor of serine esterases that is said to inhibit phosphatidylinositol phosphodiesterase, had no effect on the activity of the erythrocyte polyphosphoinositide phosphodiesterase. 6. These observations are consistent with the notion that human, and probably rabbit and rat, erythrocyte membranes possess a single polyphosphoinositide phosphodiesterase that is activated by Ca(2+) and that attacks phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate with equal facility. Inhibition of this activity by neomycin seems likely to be due to interactions between neomycin and the polyphosphoinositides, with the greater inhibition of phosphatidylinositol 4,5-bisphosphate breakdown consistent with the greater affinity of the drug for this lipid. In addition, erythrocyte membranes possess Mg(2+)-dependent phosphomonoesterase that converts inositol 1,4,5-triphosphate into inositol bisphosphate.     

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.     

Basic fibroblast growth factor does not initiate mitogenic signalling via phosphoinositide hydrolysis in PC12 cells

Basic fibroblast growth factor (b FGF) was found to be equally potent mitogen as compared to alpha-thrombin to reinitiate DNA synthesis in quiescent PC12 cells. Whereas thrombin was found to be an activator of phospholipase C as judged by a rapid increase in the formation of inositol triphosphate, inositol biphosphate and a massive accumulation of inositol phosphate when 20 mM LiCl was present as an inhibitor of inositol mono phosphatases, basic FGF failed to induce the breakdown of the polyphosphoinositides in quiescent PC12 cells to any appreciable levels, however, a simultaneous increase in the level of diacylglycerol was observed. b FGF also failed to stimulate protein kinase C which is believed to be activated by diacylglycerol. It is therefore concluded that bFGF receptor mediated 'signalling is not via phospholipase C activation and bFGF's early mitogenic responses and DNA synthesis are initiated independent of the inositol lipids and protein kinase C activation. Thus bFGF must have its own unique signal transducing mechanism independent of inositol pathways.     

A receptor and G-protein-regulated polyphosphoinositide-specific phospholipase C from turkey erythrocytes. I. Purification and properties

Eighty-three percent of polyphosphoinositide-specific phospholipase C activity was recovered in a cytosolic fraction after nitrogen cavitation of turkey erythrocytes. This activity has been purified approximately 50,000-fold when compared to the starting cytosol with a yield of 1.7-5.0%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phospholipase C preparation revealed a major polypeptide of 150 kDa. The specific activity of the purified enzyme was 6.7-14.0 mumol/min/mg of protein with phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 4-phosphate as substrate. Phospholipase C activity was markedly dependent on the presence of Ca2+. The phospholipase C showed an acidic pH optimum (pH 4.0). At neutral pH, noncyclic inositol phosphates were the major products formed by the phospholipase C, while at pH 4.0, substantial formation of inositol 1:2-cyclic phosphate derivatives occurred. Properties of the purified 150-kDa turkey erythrocyte phospholipase C were compared with the approximately 150-kDa phospholipase C-beta and -gamma isoenzymes previously purified from bovine brain (Ryu, S. H., Cho, K. S., Lee, K. Y., Suh, P. G., and Rhee, S. G. (1987) J. Biol. Chem. 262, 12511-12518). The turkey erythrocyte phospholipase C differed from the two mammalian phospholipases with respect to the effect of sodium cholate on the rate of polyphosphoinositide hydrolysis observed. Moreover, when presented with dispersions of pure inositol lipids, phospholipases C-beta and -gamma displayed comparable maximal rates of polyphosphoinositide and phosphatidylinositol hydrolysis. By contrast, the turkey erythrocyte phospholipase C displays a marked preference for polyphosphoinositide substrates.     

Characteristics of a phosphatidylinositol exchange activity of soybean microsomes

Microsome fractions from hypocotyls of dark-grown soybean (Glycine max [L.] Merrill) seedlings incorporated myo-inositol into phosphatidylinositol by an exchange reaction stimulated by Mn²⁺ (optimum at 10 mm) and cytidine nucleotides (CMP = CDP CTP) but not by Mg²⁺ or nucleotides other than cytidine nucleotides. The activity was membrane associated, with an optimum pH of 8, stimulated by auxin, and inhibited by certain thiol reagents or by heating above 40°C. With radioactive inositol, phosphatidylinositol was the only radioactive product. That turnover was by myo-inositol exchange was verified from experiments where unlabeled inositol replaced already incorporated inositol with approximately the same kinetics as for the incorporation of label. Both the incorporation and the displacement reactions were stimulated by Mn²⁺ and CMP and both were responsive to auxin with comparable dose dependency. Corresponding exchange activities with choline or ethanolamine were not observed. The phosphatidylinositol-myo-inositol exchange activity was low or absent from plasma membrane, tonoplast, and mitochondria enriched fractions. The activity co-localized on free-flow electrophoresis and aqueous two-phase partition with NADPH cytochrome c reductase and latent IDPase, markers for endoplasmic reticulum and Golgi apparatus, respectively. With microsomes incubated with both ATP and inositol, polyphosphoinositides were unlabeled demonstrating separate locations for the inositol exchange and phosphatidylinositol kinase reactions. Thus, the auxin-responsive inositol turnover activity of soybean membranes is distinct from the usual de novo biosynthetic pathway. It is not the result of a traditional D-type phospholipase and appears not to involve plasma membrane-associated polyphosphoinositide metabolism. It most closely resembles previously described phosphatidylinositol-myo-inositol exchange activities of plant and animal endoplasmic reticulum.     

The Ca2+-activated polyphosphoinositide phosphodiesterase of human and rabbit neutrophil membranes.

Addition of Ca2+ to a plasma-membrane fraction derived from human or rabbit neutrophils led to the specific breakdown of polyphosphoinositides. The degradation products were identified as diacylglycerol and inositol bis- and tris-phosphate, thus demonstrating the presence of a Ca2+-activated phospholipase C. The newly generated diacylglycerol resembled the polyphosphoinositides in its fatty acid composition, and in the presence of MgATP2- it was converted into phosphatidate. These results therefore demonstrate the presence in neutrophil plasma membranes not only of polyphosphoinositide phosphodiesterase but also of diacylglycerol kinase.