Characterization of a Polyphosphoinositide Phospholipase C from the Plasma Membrane of Avena sativa

A phosphoinositide-specific phospholipase C activity was identified in oat root (Avena sativa, cv Victory) plasma membranes purified by separation in an aqueous two-phase polymer system. The enzyme is highly active toward inositol phospholipids but only minimally active toward phosphatidylethanolamine and phosphatidylcholine. Activity approaches maximal levels at 200 micromolar phosphatidylinositol 4-phosphate (PIP) and is highly dependent on calcium; it is inhibited by 1 millimolar EGTA and is activated by calcium with an apparent activation constant of 2 micromolar. At 10 micromolar calcium and 200 micromolar inositol phospholipid, the enzyme is specific for phosphatidylinositol 4,5-bisphosphate (PIP₂) and PIP, which are hydrolyzed at 10 and 4 times, respectively, the rate of phosphatidylinositol (PI) hydrolysis. The principle water soluble products of hydrolysis, as determined by high performance liquid chromatography, are inositol 1,4,5-trisphosphate from PIP₂, inositol 1,4-bisphosphate from PIP, and inositol phosphate from PI.     

Rapid changes in polyphosphoinositide metabolism associated with the response of Dunaliella salina to hypoosmotic shock

The inositol phospholipids phosphatidylinositol, phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) comprise 14.8, 1.2, and 0.3 mol %, respectively, of Dunaliella salina phospholipids. In isolated plasma membrane fractions, PIP and PIP2 are highly concentrated, together comprising 9.5 mol % of plasmalemma phospholipids. The metabolism of these inositol phospholipids and phosphatidic acid (PA) is very rapid under normal growth conditions. Within 5 min after introduction of 32Pi into the growth medium, over 75% of lipid-bound label was found in these quantitatively minor phospholipids. Within 2 min after a sudden hypoosmotic shock, the levels of PIP2 and PIP dropped to 65 and 79%, respectively, of controls. Within the same time frame, PA rose to 141% of control values. These data suggest that a rapid breakdown of the polyphosphoinositides may mediate the profound morphological and physiological changes which allow this organism to survive drastic hypoosmotic stress. In contrast to hypoosmotic shock, hyperosmotic shock induced a rise in PIP2 levels to 131% of control values, whereas the level of PA dropped to 56% of controls after 4 min. These two different types of osmotic stress affect inositol phospholipid metabolism in a fundamentally opposite manner, with only hypoosmotic shock inducing a net decrease in polyphosphoinositides.     

Stimulation of phosphoinositides breakdown by the heat stable E. coli enterotoxin in rat intestinal epithelial cells

Rat intestinal epithelial cells were labelled with [32P]Pi and extracted, and the phospholipids were analysed by thin-layer chromatography. 32P-incorporation in phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-phosphate (PIP2) were measured in control and heat stable enterotoxin (ST)-treated cells. ST was found to induce rapid degradation of PIP and PIP2. The degradation of inositol lipids was accompanied by an increase of water soluble inositol phosphate (IP1, IP2, IP3) compounds. There was a two-fold increase of radioactivity in IP2 and IP3 but no significant change was observed in IP1. Phospholipase C activity was increased tenfold with substrate PIP2 in ST-pretreated cells. The present study indicates that ST triggers another second messenger system by increasing the PIP2 hydrolysis with the enzyme phospholipase C.     

Serotonin-induced alterations in inositol phospholipid metabolism in human platelets

When human platelets were incubated for 5 min with [32P]orthophosphate and then stimulated with serotonin, the 32P content of phosphatidylinositol (PI) increased within seconds, compared with the control. The 32P content of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) only slightly increased during the first minute after addition of serotonin and became more apparent on prolonged stimulation. These changes were not caused by serotonin-induced change in the specific activity of ATP. Using inorganic phosphate determination for the chemical quantification of different inositol phospholipid pools, we found that the platelet PI content remained nearly constant; the amount of PIP increased while that of PIP2 decreased. When the platelets were first prelabeled for 80 min with [32P]orthophosphate, the changes in 32P-labeled inositol phospholipids after addition of serotonin were similar to their changes in mass. When the platelet inositol phospholipids were labeled with myo-[2-3H]inositol, serotonin induced an increase in [3H]inositol phosphates. From these data, it is concluded in addition to the earlier-reported effects on phospholipid metabolism (de Chaffoy de Courcelles, D. et al. (1985) J. Biol. Chem. 260, 7603-7608) that serotonin induces: a very rapid formation of PI; and alterations in inositol phospholipid interconversion that cannot be explained solely as a resynthesis process of PIP2.     

The de novo phospholipid effect of insulin is associated with increases in diacylglycerol, but not inositol phosphates or cytosolic Ca2+.

We have previously reported that insulin increases the synthesis de novo of phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG) in BC3H-1 myocytes and/or rat adipose tissue. Here we have further characterized these effects of insulin and examined whether there are concomitant changes in inositol phosphate generation and Ca2+ mobilization. We found that insulin provoked very rapid increases in PI content (20% within 15 s in myocytes) and, after a slight lag, PIP and PIP2 content in both BC3H-1 myocytes and rat fat pads (measured by increases in 32P or 3H content after prelabelling phospholipids to constant specific radioactivity by prior incubation with 32Pi or [3H]inositol). Insulin also increased 32Pi incorporation into these phospholipids when 32Pi was added either simultaneously with insulin or 1 h after insulin. Thus, the insulin-induced increase in phospholipid content appeared to be due to an increase in phospholipid synthesis, which was maintained for at least 2 h. Insulin increased DAG content in BC3H-1 myocytes and adipose tissue, but failed to increase the levels of inositol monophosphate (IP), inositol bisphosphate (IP2) or inositol trisphosphate (IP3). The failure to observe an increase in IP3 (a postulated 'second messenger' which mobilizes intracellular Ca2+) was paralleled by a failure to observe an insulin-induced increase in the cytosolic concentration of Ca2+ in BC3H-1 myocytes as measured by Quin 2 fluorescence. Like insulin, the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the transport of 2-deoxyglucose and aminoisobutyric acid in BC3H-1 myocytes. These effects of insulin and TPA appeared to be independent of extracellular Ca2+. We conclude that the phospholipid synthesis de novo effect of insulin is provoked very rapidly, and is attended by increases in DAG but not IP3 or Ca2+ mobilization. The insulin-induced increase in DAG does not appear to be a consequence of phospholipase C acting upon the expanded PI + PIP + PIP2 pool, but may be derived directly from PA. Our findings suggest the possibility that DAG (through protein kinase C activation) may function as an important intracellular 'messenger' for controlling metabolic processes during insulin action.     

Transient increase in phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol trisphosphate during activation of human neutrophils

We recently showed that phosphatidylinositol trisphosphate (PIP3) was present in a unique lipid fraction generated in neutrophils during activation. Here, we demonstrate that the band containing this fraction isolated from thin layer chromatography consists primarily of PIP3 and that only small amounts of radiolabeled PIP3 exist prior to activation. In addition, high performance liquid chromatography of deacylated phospholipids from stimulated cells reveals an increase in a fraction eluting ahead of glycerophosphoinositol 4,5-P2. After removal of the glycerol we found that it coeluted with inositol 1,3,4-P3 when resubjected to high performance liquid chromatography. Thus, we have detected a second, novel form of phosphatidylinositol bisphosphate in activated neutrophils, PI-(3,4)P2. The elevation of PIP3 through the formyl peptide receptor is blocked by pretreatment with pertussis toxin, implicating mediation of the increase in PIP3 by a guanosine triphosphate-binding (G) protein. The rise in PIP3 is not secondary to calcium elevation. Buffering the rise in intracellular calcium did not diminish the increase in PIP3. The elevation of PIP3 appears to occur during activation with physiological agonists, its level varying with the degree of activation. Leukotriene B4, which elicits many of the same responses as stimulation of the formyl peptide receptor but with minimal oxidant production, stimulates a much attenuated rise in PIP3. Isoproterenol, which inhibits oxidant production also reduces the rise in PIP3. Hence formation of PI(3,4)P2 and PIP3 (presumed to be PI(3,4,5)P3) correlates closely with the early events of neutrophil activation.     

Inositol Phospholipid Hydrolysis by Rat Sciatic Nerve Phospholipase C

Rat sciatic nerve cytosol contains a phosphodiesterase of the phospholipase C type that catalyzes the hydrolysis of inositol phospholipids, with preferences of phosphatidylinositol 4'-phosphate (PIP) greater than phosphatidylinositol (PI) much greater than phosphatidylinositol 4',5'-bisphosphate (PIP2), at a pH optimum of 5.5-6.0 and at maximum rates of 55, 13, and 0.7 nmol/min/mg protein, respectively. Analysis of reaction products by TLC and formate exchange chromatography shows that inositol 1,2-cyclic phosphate (83%) and diacylglycerol are the major products of PI hydrolysis. [32P]-PIP hydrolysis yields inositol bisphosphate, inositol phosphate, and inorganic phosphate, indicating the presence of phosphodiesterase, phosphomonoesterase, and/or inositol phosphate phosphatase activities in nerve cytosol. Phosphodiesterase activity is Ca2+-dependent and completely inhibited by EGTA, but phosphomonoesterase activity is independent of divalent cations or chelating agents. Phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) inhibit PI hydrolysis. They stimulate PIP and PIP2 hydrolysis up to equimolar concentrations, but are inhibitory at higher concentrations. Both diacylglycerols and free fatty acids stimulate PI hydrolysis and counteract its inhibition by PC and lysoPC. PIP2 is a poor substrate for the cytosolic phospholipase C and strongly inhibits hydrolysis of PI. However, it enhances PIP hydrolysis up to an equimolar concentration.     

Bradykinin-induced activation of phospholipase A2 is independent of the activation of polyphosphoinositide-hydrolyzing phospholipase C

This study evaluates the role of phosphatidylinositol 4,5-bisphosphate (PIP2) and its metabolites as possible mediators in the activation of phospholipases A2 in porcine aortic endothelial cells. We compared the time courses of bradykinin-induced turnover of phosphoinositides and the appearance of unesterified arachidonic acid (uAA) and eicosanoids. The metabolism of phosphoinositides was examined in cells prelabeled with [3H]inositol, which has a similar distribution as the endogenous inositol lipids. At 37 degrees C, bradykinin induced a rapid rise in lysophosphatidylinositol (lyso-PI) and inositol 1,4,5-trisphosphate (IP3) as well as a decrease in PIP2. Lyso-PI formation was detected at 10 s, as early as PIP2 degradation and IP3 formation. This suggests that the activation of PIP2-hydrolyzing phospholipase C and PI-hydrolyzing phospholipase A2 are simultaneous. However, at 30 degrees C, lyso-PI formation was detected in the absence of an increase in IP3 indicating that the activation of phospholipase A2 does not require the accumulation of IP3. The time course of formation of uAA and eicosanoids were examined in [3H]arachidonic acid-prelabeled cells. The 3H radioactivity was distributed among the phospholipid classes and subclasses the same as the endogenous phospholipids. Bradykinin stimulated the intracellular accumulation of uAA, detectable at 5 s, earlier than that of 1,2-diacylglycerol and phosphatidic acid. Such immediate formation of uAA further supports the notion that activation of phospholipase A2 is a very early event during the interaction of bradykinin with porcine endothelial cells, and that PIP2 hydrolysis is not prerequisite for the initial activation of phospholipase A2.     

Inositol phospholipid-induced suppression of F-actin-gelating activity of smooth muscle filamin.

Filamin, a high molecular weight actin-binding protein, cross-links actin filaments and produces a gel composed of F-actin. The effects of polyphosphoinositides on the gelating activity of smooth muscle filamin were examined by measuring the low shear viscosity of the F-actin solutions containing filamin incubated with phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), or phosphatidylinositol 4,5-bisphosphate (PIP2). Micelles of these inositol phospholipids bound to filamin inhibited the ability to form a gel of F-actin. The inhibiting activity of each phospholipid was in the following order, PIP2 greater than PIP greater than PI. The F-actin binding assay of filamin revealed that the inhibition of F-actin-gelation resulted in the loss of the F-actin-binding activity of filamin. Thus, polyphosphoinositides may play important roles in regulating the gelating activity of filamin.     

Herpes simplex virus-1-specific proteins are involved in alteration of polyphosphoinositide metabolism in baby-hamster kidney cells.

Herpes simplex virus type 1 (HSV-1) induces altered phosphoinositide metabolism in baby hamster kidney (BHK) cells, measured as incorporation of [3H]inositol or [32P]Pi [Langeland, Haarr & Holmsen (1986) Biochem. J. 237, 707-712]. We now report that this response in the inositol phospholipids is dependent on virus-specific proteins synthesized in the beta (early) stage of virus protein synthesis. This was demonstrated both by resistance to the inhibitory effect of cycloheximide after this stage of infection, and by the use of temperature-sensitive (ts) mutants of HSV-1; ts mutants in which protein synthesis was blocked so that only the alpha proteins were expressed showed a PIP2/PIP (phosphatidylinositol 4,5-bisphosphate/phosphatidylinositol 4-monophosphate) ratio similar to uninfected cells, while ts mutants which were defective in protein synthesis at a late beta stage or later showed increased PIP2/PIP ratios similar to cells infected by wild type HSV-1.     

Changes in phosphatidylinositol metabolism in response to hyperosmotic stress in Daucus carota L. cells grown in suspension culture.

Carrot (Daucus carota L.) cells plasmolyzed within 30 s after adding sorbitol to increase the osmotic strength of the medium from 0.2 to 0.4 or 0.6 osmolal. However, there was no significant change in the polyphosphorylated inositol phospholipids or inositol phosphates or in inositol phospholipid metabolism within 30 s of imposing the hyperosmotic stress. Maximum changes in phosphatidylinositol 4-monophosphate (PIP) metabolism were detected at 5 min, at which time the cells appeared to adjust to the change in osmoticum. There was a 30% decrease in [3H]inositol-labeled PIP. The specific activity of enzymes involved in the metabolism of the inositol phospholipids also changed. The plasma membrane phosphatidylinositol (PI) kinase decreased 50% and PIP-phospholipase C (PIP-PLC) increased 60% compared with the control values after 5 min of hyperosmotic stress. The PIP-PLC activity recovered to control levels by 10 min; however, the PI kinase activity remained below the control value, suggesting that the cells had reached a new steady state with regard to PIP biosynthesis. If cells were pretreated with okadaic acid, the protein phosphatase 1 and 2A inhibitor, the differences in enzyme activity resulting from the hyperosmotic stress were no longer evident, suggesting that an okadaic acid-sensitive phosphatase was activated in response to hyperosmotic stress. Our work suggests that, in this system, PIP is not involved in the initial response to hyperosmotic stress but may be involved in the recovery phase.     

Neomycin inhibits the phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate stimulation of plasma membrane ATPase activity

The inositol phospholipids, phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP₂), have been shown to increase the vanadate-sensitive ATPase activity of plant plasma membranes (AR Memon, Q Chen, WF Boss [1989] Biochem Biophys Res Commun 162: 1295-1301). In this paper, we show the effect of various concentrations of phosphatidyinositol, PIP, and PIP₂ on the plasma membrane vanadate-sensitive ATPase activity. PIP and PIP₂ at concentrations of 10 nanomoles per 30 microgram membrane protein per milliliter of reaction mixture caused a twofold and 1.8-fold increase in the ATPase activity, respectively. The effect of these negatively charged phospholipids on the ATPase activity was inhibited by adding the positively charged aminoglycoside, neomycin. Neomycin did not affect the endogenous plasma membrane ATPase activity in the absence of exogenous lipids.     

Evidence for a new inositol phospholipid in rat brain mitochondria.

Phosphorylation of phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP) and diacylglycerol (DAG) was studied in rat brain cortex myelin, synaptosomal and mitochondrial fractions, with ATP as phosphate donor and endogenous phospholipids as substrate. All fractions had PI, PIP and DAG phosphorylating activity with their own characteristic subcellular distribution. However, in the mitochondrial fraction an unidentified lipid was phosphorylated, which had a slower Rf value than PIP2 on TLC. After hydrolysis of the polar head group of the lipid and separation on anion exchange columns, it appeared to be a phosphoinositide. The elution profile showed that it was not phosphatidylinositol trisphosphate, or a lyso-compound. The available evidence suggests that the unknown inositol phospholipid in rat brain mitochondria is a phosphatidylinositol 4,5-bisphosphate isomer, although the possibility of it being a glycosyl-phosphoinositide cannot be excluded.     

Alkylacetylglycerophosphocholine effects on the metabolism of phospholipids in rabbit platelets: effects of extracellular Ca2+ and prostacyclin

Alkylacetylglycerophosphocholine (AGEPC) stimulation of 32P-labeled lysophosphatidic acid formation in washed rabbit platelets was dependent on extracellular Ca2+. Its accumulation was slower and required a higher concentration of AGEPC in comparison to the degradation of inositol phospholipids and production of phosphatidic acid induced by the same agonist. These results suggest that the formation of lysophosphatidic acid is not directly related to the primary activation of rabbit platelets by AGEPC. AGEPC elicited a preferential degradation of inositol phospholipids in the following order: phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. The degradation of inositol phospholipids and subsequent production of phosphatidic acid were affected by pretreatment of platelets with prostacyclin or ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Synergistic inhibitions of these metabolic changes were observed in the platelets pretreated with both prostacyclin and EGTA. These results were compared with effects of prostacyclin and EGTA on serotonin release induced by AGEPC, and the possible roles of metabolic changes in phospholipids induced by AGEPC are discussed with respect to the mechanism of platelet activation.     

Regulation of high molecular weight bovine brain neutral protease by phospholipids in vitro

The activity of the heat stable, glycosylated high molecular weight bovine brain neutral protease (HMW protease) is differentially regulated by phospholipids. While phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidic acid (PA) had only marginal stimulatory effect (40–75%) on the activity of HMW protease, lysophoshatidylcholine (lysoPC) and lysophosphatidic acid (lysoPA) activated the enzyme by more than two-fold. Both lysoPC and lysoPA exhibited concentration-dependent saturation kinetics for the activation of HMW protease. Surprisingly, phosphoinositides (phosphatidylinositol, PI; phosphatidylinositol 4-phosphate, PIP; and phosphatidylinositol 4,5-bisphosphate, PIP₂) modulated the activity of protease differently: activation of the enzyme was higher with PIP (90%) as compared to PI (21%), whereas PIP₂ inhibited the enzyme (16%). The inhibition of the protease by PIP₂ was concentration-dependent. During receptor-coupled cell activation, phospholipase A₂ (PLA₂) converts PC and PA to lysoPC and lysoPA, respectively; PI is converted to PIP₂ by successive enzymatic phosphorylation by PI 4-kinase and PIP 5-kinase; and phospholipase C (PLC) degrades PIP₂ to diacylglycerol and inositol 1,4,5-trisphosphate. Therefore, the data suggest that HMW protease may be coupled to cell signal transduction where PLA₂, PI 4-kinase, PIP 5-kinase and PLC are involved.     

Changes in the concentration and fatty acid composition of phosphoinositides induced by hormones in hepatocytes

The hormonal regulation of phosphoinositide levels in isolated hepatocytes was studied using chemical means. Extracted inositol phospholipids were adsorbed to neomycin-coated glass beads and then eluted and quantitated by charring after separation by thin layer chromatography on silica gel. The amounts (in nanograms/mg wet weight) of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol (PI) were 20 +/- 1, 16 +/- 1, and 1790 +/- 140, respectively). Incubation of the cells with 100 nM vasopressin decreased the value for PIP2 to 10 +/- 0.2 at 15 s, 12 +/- 1.5 at 1 min, and 14 +/- 2.1 at 5 and 30 min. In contrast, the hormone increased 1,2-diacylglycerol plus phosphatidate by over 200 ng/mg wet weight at 5 min under similar conditions (Bocckino, S. B., Blackmore, P. F., Wilson, P. B., and Exton, J. H. (1987) J. Biol. Chem. 262, 15309-15315). PIP2 was also significantly decreased at 15 s by angiotensin II (100 nM), ATP (100 microM), and epinephrine (1 microM). In contrast, PIP was not significantly changed, and PI was significantly decreased (by approximately 15%) at later times (15 and 30 min). The changes in phosphoinositide mass were well correlated with changes in labeled phosphoinositides in hepatocytes previously incubated with [3H]inositol for 90 min. The amounts of inositol phospholipids in liver plasma membranes (in micrograms/mg protein) were 2.1 +/- 0.2 for PIP2, 0.24 +/- 0.03 for PIP, and 23 +/- 4 for PI. Comparison of these values with those for whole cells suggests that PIP2 is enriched in the plasma membrane, whereas PIP is present elsewhere in the cell. The fatty acid composition of whole cell PIP2 showed significant differences from that of PI. The percentages of palmitic, stearic, linoleic, and arachidonic acids were, respectively, 14, 41, 10, and 25 for PIP2 and 10, 34, 7, and 37 for PI. Vasopressin treatment for 15 s did not alter the fatty acid composition of PIP2. The corresponding fatty acid percentages for liver plasma membranes were 13, 41, 11, and 21 for PIP2 and 8, 34, 0, and 40 for PI. The fatty acid composition of PIP in whole cells and plasma membranes resembled that of PIP2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Involvement of phosphoinositide metabolism in potentiation by adrenaline of ADP-induced aggregation of rabbit platelets.

Changes in phosphoinositide metabolism were examined in washed rabbit platelets stimulated with 0.5 microM-ADP, 50 microM-adrenaline, or ADP and adrenaline in combination. Adrenaline does not stimulate platelet aggregation when used alone, but does potentiate aggregation stimulated by ADP. In platelets prelabelled with [32P]Pi and [3H]glycerol, adrenaline was found to potentiate the ADP-induced changes in platelet phospholipids, causing larger increases in the amount and labelling of phosphatidylinositol 4-phosphate (PIP) and phosphatidic acid than was observed with ADP alone. The combination of ADP and adrenaline did not produce a greater decrease in phosphatidylinositol 4,5-bisphosphate (PIP2) than was produced by ADP alone. In platelets prelabelled with [3H]inositol, adrenaline potentiated the increases in labelling of inositol phosphate and inositol bisphosphate stimulated by ADP; no increase in inositol trisphosphate labelling was detected with ADP alone or with the combination of ADP and adrenaline. Phentolamine, an alpha-adrenergic-receptor antagonist, blocked potentiation by adrenaline of ADP-induced changes in phosphoinositide metabolism. Propranolol and sotalol, beta-adrenergic-receptor antagonists, augmented the potentiation; this is consistent with the concept that the effect of adrenaline is mediated by beta-adrenergic receptors. The effect of adrenaline on phosphoinositide metabolism appears to be to potentiate the mechanisms by which ADP causes turnover of PIP and possibly degradation of PI, rather than the mechanism by which PIP2 is decreased.     

Purification of a phospholipase C from rat liver cytosol that acts on phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate

A soluble phospholipase C from rat liver was purified to homogeneity using phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate. After ammonium sulfate fractionation, the purification involved chromatography on phosphocellulose, DEAE-Sepharose CL-6B, hydroxylapatite, Reactive Blue 2 dye-linked agarose, and Mono S cation exchanger. Under the conditions of the assay, the pure enzyme had a specific activity of 407 mumol/mg protein/min. It migrated as a single band with a molecular mass of 87 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The water-soluble product formed during the hydrolysis of PIP2 by the purified enzyme was inositol 1,4,5-trisphosphate. The enzyme shows one-half of maximum velocity at 2 microM Ca2+ with PIP2 as substrate. Between 0 and 100 microM Ca2+, the enzyme shows approximately the same activity with phosphatidylinositol 4-phosphate (PIP) as it does with PIP2, and very low activity with phosphatidylinositol. The enzyme is activated by low concentrations of basic proteins; for example, with PIP2 as substrate, 1 microgram/ml histone activates the enzyme 3.6-fold. The enzyme shows an almost absolute requirement for monovalent salts which can be met by different alkali metal halides. A second, minor peak of PIP2-hydrolyzing phospholipase C activity was resolved during chromatography of the enzyme on hydroxylapatite. The substrate specificity suggests that PIP and PIP2 are normal substrates of this enzyme. Under physiological conditions of activation, the enzyme may therefore generate inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate in amounts determined by the ratio of PIP and PIP2 present in the cellular membranes.     

Inhibition of inositol phospholipids metabolism and calcium mobilization by cyclic AMP-increasing agents and phorbol ester in neutrophils

Cyclic AMP-increasing agents such as PGE2 and dibutyryl cAMP inhibited the fMLP-induced inositol phospholipids metabolism mainly through the suppression of the conversion of phosphatidylinositol(PI) to phosphatidylinositol 4,5-bisphosphate(PIP2). A part of this inhibition was found to be caused by the inhibitory effect of cAMP on PI kinase using isolated plasma membranes. On the other hand, 12-O-tetradecanoyl phorbol acetate(TPA) mainly inhibited the conversion of phosphatidylinositol 4-phosphate(PIP) to PIP2 without a significant effect on the fMLP-induced breakdown of PIP2, though direct effect of TPA on PI and PIP kinases was not demonstrated in isolated plasma membranes. Concerning Ca2+ mobilization, both cAMP-increasing agents and TPA inhibited the fMLP-induced second phase of Ca2+ elevation, while they did not affect the first phase of Ca2+ rapid increase. However, Ca2+ ionophore ionomycin-induced Ca2+ elevation was only inhibitable by TPA but not PGE2. These results suggest that cAMP inhibits the fMLP-induced Ca2+ influx, while TPA stimulates Ca2+ removal from cytosol.     

Early effects of luteinizing hormone on mitochondrial phosphoinositides in ovarian follicles

It is not clear if luteinizing hormone (LH) stimulates breakdown as well as synthesis of phosphoinositides in ovarian tissue. Possibly, LH stimulation results in hydrolysis of ovarian phosphoinositides in discrete subcellular compartments while increasing their synthesis at other sites. To investigate this hypothesis, we determined the effects of LH on phosphoinositide metabolism in whole homogenates and mitochondria of ovarian follicles. Medium (3-7 mm) follicles from porcine ovaries were preincubated for 2 h in phosphate (PO4)-free medium with 32PO4, and incubated without or with LH (1 microgram/ml). Phosphatidylinositol (PI) and related compounds, phosphatidic acid (PA), phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP2), accounted for 40% of the radiolabeled phospholipids in whole homogenates and over 60% in mitochondria from preincubated follicles. After 5 min, LH caused a significant decrease in radiolabeling of PIP2 and PIP in mitochondria, but not in whole homogenates. Luteinizing hormone increased radiolabeling of PIP2, PIP, PI and PA within 10 min in whole homogenates, and within 20 to 30 min in mitochondria. This delayed increase in radiolabeling of mitochondrial phosphoinositides after LH treatment was accompanied by decreases in PIP2, PIP and PI radiolabeling in whole homogenates. Follicles also were preincubated for 4 h with [3H]inositol, then for 15 min with 10 mM LiCl (an inhibitor of inositol phosphate hydrolysis). Inositol phosphate accumulation in 30 min was 2.7 times higher in homogenates of LH-treated follicles then in untreated follicles. Also, LH significantly decreased inositol bisphosphate, but did not change inositol trisphosphate accumulation. Accumulation of inositol phosphates in mitochondria was not measurable.(ABSTRACT TRUNCATED AT 250 WORDS)