Calcium modulation of phosphoinositide kinases in transverse tubule vesicles from frog skeletal muscle

Highly purified transverse tubule membranes isolated from frog skeletal muscle phosphorylate phosphatidylinositol to phosphatidylinositol 4-phosphate and phosphatidylinositol (4,5)-bisphosphate. The two phosphorylation reactions have different calcium requirements. Phosphorylation of phosphatidylinositol to phosphatidylinositol 4-phosphate, which takes place in both isolated transverse tubules and sarcoplasmic reticulum membrane, is independent of calcium in a range of concentrations from 10(-9) to 10(-6) M, and is progressively inhibited to 10% of the maximal values by increasing calcium to 10(-4) M or higher (K0.5 = 5 X 10(-6) M). In contrast, phosphorylation of phosphatidylinositol 4-phosphate to phosphatidylinositol (4,5)-bisphosphate, a reaction exclusively present in transverse tubule membranes, is maximal at calcium concentrations higher than 2 X 10(-6) M and decreases to 30% of maximal values at calcium concentrations of 2 X 10(-7) M or lower (K0.5 = 10(-6) M). Unlike frog membranes, transverse tubules from rabbit muscle need exogenous phosphatidylinositol 4-phosphate in order to produce the bisphosphate derivative in the same range of calcium concentrations. Inositol (1,4,5)-trisphosphate has been proposed recently as a chemical messenger in excitation-contraction coupling in skeletal muscle. Calcium regulation of the synthesis of phosphatidylinositol (4,5)-bisphosphate, the membrane-bound precursor of inositol (1,4,5)-trisphosphate, might have physiological implications regarding modulation of excitation-contraction coupling by intracellular calcium levels.     

Phosphatidylinositol 4,5-bisphosphate formation in rabbit skeletal and heart muscle membranes

Incubation of rabbit skeletal muscle microsomes or isolated triads with gamma 32P-ATP/Mg2+ in the absence and in the presence of added phosphatidylinositol resulted in the formation of phosphatidylinositol 4-phosphate catalyzed by phosphatidylinositol kinase. When phosphatidylinositol 4-phosphate was added as exogenous substrate, phosphatidylinositol 4,5-bisphosphate was also formed demonstrating the presence of a membrane bound phosphatidylinositol 4-phosphate kinase. Triads were broken mechanically in a French press and separated on a continuous sucrose gradient. Incubation of these fractions with gamma 32P-ATP/Mg2+ resulted in a rapid labeling of phospholipid in a membrane fraction banding between transverse tubules and the terminal cisternae. Partial triad breakage and triad reformation experiments indicated that this phosphatidylinositol kinase was associated with T-tubules. When exogenous phosphatidylinositol 4-phosphate was employed as substrate phosphatidylinositol 4,5-bisphosphate and phosphatidic acid were formed, indicating the presence of all the enzymes of the polyphosphoinositide signaling system in this special membrane fraction. In contrast, heart muscle microsomes or plasma membranes can catalyze this reaction sequence from endogenous formed phosphatidylinositol 4-phosphate.     

Synthesis of polyphosphoinositides in vertebrate photoreceptor membranes

Rod outer segments isolated from bovine retinas incorporated 32P into phospholipids after incubation with [gamma-32P]ATP in a Mg2+-containing medium. Only phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidate were labelled. The incorporation of label into lipids was detected as early as 20 s after the start of incubation and the products were stable for at least 10 min. The reactions were time, protein and ATP-concentration dependent. Entire rod outer segments showed higher diacylglycerol kinase and lower phosphatidylinositol and phosphatidylinositol 4-phosphate kinase activities than the disc membranes obtained from them. Exogenously added phosphatidylinositol (up to 1 mM) in the presence of Triton X-100 increased phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate labelling in rod outer segments (8- and 6-fold, respectively). Triton X-100 at a concentration of 0.4% stimulated phosphorylation of endogenous phosphoinositides. Diacylglycerol kinase activity was largely suppressed by the detergent, but this effect was partially reversed by addition of phosphatidylinositol. It is suggested that the rod outer segments contain phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase bound to disc membranes, as well as an active diacylglycerol kinase occurring either as a soluble or a peripherally bound protein in disc membranes.     

Analysis of the metabolic turnover of the individual phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Validation of novel analytical techniques by using 32P-labelled lipids from erythrocytes.

We have developed methods that yield estimates of the 32P content of each of the individual phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, thus extending the information available from studies of the labelling of these lipids in intact cells or membrane preparations. The analyses are undertaken with the deacylated lipids. Assay of the 5-phosphate of phosphatidylinositol 4,5-bisphosphate is achieved by the use, under conditions of first-order kinetics, of a 5-phosphate-specific phosphomonoesterase present in isolated erythrocyte membranes [Downes, Mussat & Michell (1982) Biochem. J. 203, 169-177]. Assay of the 4-phosphate of phosphatidylinositol 4-phosphate and of the total monoester phosphate content (4-phosphate plus 5-phosphate) of phosphatidylinositol 4,5-bisphosphate employs alkaline phosphatase from bovine intestine. The radioactivity of the 1-phosphate is that remaining as organic phosphate after exhaustive alkaline phosphatase treatment. The methodology has been validated by using lipids from human erythrocytes: these contain no 32P in their 1-phosphate. These methods should be of substantial value in studies of the many cells that show rapid hormonal perturbations of phosphatidylinositol 4,5-bisphosphate metabolism.     

Generation of phosphatidylinositol 4,5-bisphosphate proceeds through an intracellular route in rat hepatocytes

The subcellular distribution in rat hepatocytes of enzymes participating in the entire generation cycle of phosphatidylinositol 4,5-bisphosphate, and phosphorylated intermediates of this pathway, has been examined by Nycodenz gradient centrifugation. Our results indicate that the synthesis of phosphatidylinositol takes place in the endoplasmic reticulum, and that its phosphorylation to phosphatidylinositol 4-phosphate occurs intracellularly in low-density membranes before translocation to the plasma membrane, where it is further phosphorylated to phosphatidylinositol 4,5-bisphosphate. The intracellular formation of PIP implies a vesicular transport to the plasma membrane.     

Intracellular localization of inositol-phospholipid-metabolizing enzymes in rabbit fast-twitch skeletal muscle. Can D-myo-inositol 1,4,5-trisphosphate play a role in excitation-contraction coupling?

Rabbit fast-twitch skeletal muscle microsomes have been separated by isopycnic centrifugation on a linear sucrose gradient into triads and light sarcoplasmic reticulum. In both fractions phosphatidylinositol-kinase activity is found [Varsányi et al. (1986) Biochem. Biophys. Res. Commun. 138, 1395]. In contrast, phosphatidylinositol-4-phosphate kinase is nearly exclusively associated with triads. The phosphatidylinositol-4,5-bisphosphate-phosphodiesterase activity shows a biphasic distribution: approximately 50% of the activity is associated with triads and 50% appears in the overlay. Triads have been broken mechanically into transverse tubules and terminal cisternae, then separated by isopycnic sucrose-gradient centrifugation. Both fractions exhibit phosphatidylinositol-kinase activity; the activities of phosphatidylinositol-4-phosphate kinase and phosphatidylinositol-4,5-bisphosphate phosphodiesterase are associated mainly with the transverse tubules. Consequently, in rabbit fast-twitch skeletal muscle all necessary enzymes for production of D-myo-inositol 1,4,5-trisphosphate are associated with transverse tubules. Phosphatidylinositol-4,5-bisphosphate phosphodiesterase associated with triads shows a pH optimum at 6.8. The enzyme is maximally active between pCa 5 and pCa 4. Mg2+ inhibits the enzyme activity half-maximally at about 1 mM. Guanine-nucleotide-binding proteins seem not to be involved in the regulation of enzyme activity; guanosine 5'-[gamma-thio]triphosphate does not influence phosphatidylinositol-4,5-bisphosphate phosphodiesterase activity. It correlates well with the observation that neither alpha 1-adrenergic nor muscarinic receptors have been found in fast-twitch rabbit skeletal muscle. On basis of the respective enzyme activities estimations on maximal phosphatidylinositol turnover were made and a possible involvement of this signal pathway in excitation-contraction coupling has been discussed. Furthermore, calculations show that during a single twitch D-myo-inositol 1,4,5-trisphosphate concentration does not reach more than 2 nM. However, during a 4-s tetanus D-myo-inositol 1,4,5-trisphosphate can accumulate to a level which could effect force generation [Thieleczek and Heilmeyer (1986) Biochem. Biophys. Res. Commun. 135, 662] and aldolase distribution (Thieleczek et al., unpublished results).     

Subcellular localisation of inositol lipid kinases in rat liver

The subcellular distribution of the enzymes which phosphorylate phosphatidylinositol sequentially to form phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was investigated in rat liver. We demonstrate that whilst phosphatidylinositol kinase is present in Golgi, lysosomes and plasma membranes, the kinase that forms phosphatidylinositol 4,5-bisphosphate is localised predominantly at the plasma membrane. The role of the inositol lipid kinases in cell function is discussed.     

Annexin VI is attached to transverse-tubule membranes in isolated skeletal muscle triads

Annexin VI is a 68-kDa protein of the Annexin family, a group of Ca2+-dependent phospholipid-binding proteins widely distributed in mammalian tissues including skeletal muscle. We investigated a) which membrane system contributes Annexin VI to skeletal muscle triads, and b) whether Annexin VI removal affects triad integrity or function. Annexin VI was present in isolated triads and transverse tubules but not in heavy sarcoplasmic reticulum vesicles, indicating that Annexin VI binds to either free or triad-attached transverse tubules. Extraction with EGTA of Annexin VI from triads did not alter their migration as a single band in sucrose density gradients or their ouabain binding-site density, indicating that triad integrity does not require Annexin VI. Caffeine-induced Ca2+ release kinetics and Ca2+ uptake rates were likewise not affected by Annexin VI removal from triads, suggesting that Annexin VI is not involved in these functions. Annexin VI purified from rabbit skeletal muscle displayed Ca2+-dependent binding to liposomes containing phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine. Binding saturated at 1/20 molar ratio phosphatidylinositol 4,5-bisphosphate/phosphatidylcholine and was optimal at free [Ca2+] > or = 20 mM. Extraction of Annexin VI from triads did not affect the generation of phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, or phosphatidic acid by endogenous lipid kinases, suggesting that despite its capacity to bind to negatively charged phospholipids, Annexin VI does not affect the kinase activities responsible for their generation.     

Polyamines stimulate the phosphorylation of phosphatidylinositol in membranes from A431 cells

The phosphorylation of phosphatidylinositol in plasma membranes from A431 cells was investigated using [gamma-32P]ATP as the substrate. Phosphatidylinositol 4-phosphate was found to be the major product after an incubation time of 5-10 min. Little, if any, phosphatidylinositol 4,5-bisphosphate was found under these conditions. Epidermal growth factor (EGF) had no effect on the formation of phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate. On the other hand, the polyamines spermidine and spermine stimulated the phosphatidylinositol kinase activity about eightfold yielding almost exclusively phosphatidylinositol 4-phosphate as the reaction product. Half-maximum stimulation by spermidine occurred under near physiological conditions (1.5 mM). Furthermore various proteins and amino acid polymers containing clustered basic amino acid residues (e.g. histones and polylysine) stimulated the formation of phosphatidylinositol 4-phosphate to a similar extent. Half-maximal concentrations for the activation were considerably lower ranging from 1.5 microM to 80 microM. The ATP specificity of the phosphatidylinositol kinase(s) was investigated with a small set of selected ATP derivatives. In the presence of spermidine the specificity changed significantly indicating that (a) spermidine acts on a kinase and not on a phosphatase, (b) this activity is distinct from the EGF-receptor protein kinase activity. The results do not suggest an involvement of the EGF receptor in the growth-factor-dependent formation of phosphatidylinositol phosphates. It is proposed that the phosphorylation of phosphatidylinositol by polyamines might be a mechanism to replenish the pool of inositolphospholipids.     

Changes in polyphosphoinositide levels in rat liver nuclei in response to prolactin, a known hepatic mitogen.

The effect of prolactin action on nuclear polyphosphoinositide synthesis was investigated in isolated rat liver nuclei. An increased uptake of phosphate from [gamma 32P] adenosinetriphosphate was observed in both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate with a maximum response at 10(-12) M concentration of hormone. Pulse-chase experiments in isolated nuclei following prolactin treatment indicate that the observed increase in accumulation of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate is mainly due to a decrease in their rate of turnover possibly induced by a change in activity of polyphosphoinositide-specific monoesterases. In vitro prolactin also reduces the activity of nuclear phospholipase C specific for phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Moreover, this feature is strongly supported by the concomitant decrease in nuclear diacylglycerol mass. Thus these data suggest that once prolactin reaches the nucleus an intranuclear signalling is evoked through inositol lipid metabolism.     

Polyphosphoinositides undergo charge neutralization in the physiological pH range: a 31P-NMR study

The charge state of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was determined as a function of pH by way of 31P-NMR spectroscopy. The pK values for the first protonation of the phosphomonoester residues in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were found to be 6.2 and 6.6, respectively, for the 4-phosphate moiety, and 7.7 for the 5-phosphate moiety.     

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.     

Coated vesicles contain a phosphatidylinositol kinase

When coated vesicles (CVs) are incubated with [gamma-32P]ATP, radioactivity is rapidly incorporated into a compound identified by thin layer chromatography as phosphatidylinositol 4-phosphate. This activity has been identified in CVs isolated from bovine brain as well as from rat liver and chick embryo skeletal muscle. Phosphatidylinositol (PI) kinase is not separated from CVs during agarose electrophoresis, which produces CVs of greater than 95% purity, indicating that the activity present does not derive from contamination. The specific activity of these highly purified CVs was demonstrated to be approximately twice that of synaptic plasma membranes, further ruling out contamination from this source. The PI kinase remains associated with the vesicle upon removal of clathrin and its associated proteins and is solubilized by nonionic detergents, suggesting it is an integral membrane protein. We have been unable to demonstrate the formation of significant amounts of phosphatidylinositol 4,5-bisphosphate in any of our CV preparations. In the presence of exogenous PI, activity is stimulated, with maximal phosphorylation occurring at 0.1 mM. The enzyme appears to be maximally stimulated by 200 mM MgCl2 and 1 mM ATP and is most active at pH 7.25. Calculations indicate that, under optimal conditions, approximately 25 molecules of PIP are produced per CV within 60 s, suggesting that these structures may play an important role in cellular PI metabolism.     

Dopamine D2 Receptors Attenuate Phosphatidylisitol 4,5-Bisphosphate in Synaptosomal Membranes from Rat Neostriatum

The effects of dopamine on [32P]ATP-labelled phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidic acid were analyzed by TLC in synaptosomal membranes of the rat neostriatum. The incorporation of 32P into these compounds was found to be stable within 1 min and was maintained during the 30 min of incubation. Dopamine (0.1-10 microM) was found to attenuate the levels of phosphatidylinositol 4,5-bisphosphate without affecting the levels of phosphatidylinositol 4-phosphate or phosphatidic acid. The maximal decrease (-35 +/- 4%) was reached at 10 microM of dopamine after 30 min of incubation. The dopamine (0.1 microM)-induced decrease was blocked by the D2 selective antagonist raclopride (1 microM), but not by the D1 selective antagonist SCH 23390 (1 microM). These findings indicate the existence of an intramembrane coupling of dopamine D2 receptors to phosphoinositide turnover and may underlie some of the physiological effects of D2 receptor stimulation.     

Factors that regulate the activity of the phosphatidylinositol kinase present in oocyte membranes of Xenopus laevis

1. Phosphatidylinositol kinase present in the membranes of Xenopus laevis oocytes was characterized. 2. The enzyme requires Mg2+ or Mn2+ at 10 mM and exogenous phosphatidylinositol (50 microM) increases the formation of phosphatidylinositol-4-phosphate. 3. The oocyte phosphatidylinositol kinase cannot use GTP as a phosphate donor but this compound inhibits competitively the utilization of ATP. 4. Addition of phosphatidylserine and phosphatidylinositol-4,5-bisphosphate stimulates the phosphorylation of phosphatidylinositol but 2,3-bisphosphoglycerate at 5 mM concentration is a strong inhibitor of the reaction.     

Purification and characterization of a phosphoinositide-specific phospholipase C from guinea pig uterus. Phosphorylation by protein kinase C in vivo

Two peaks of phosphoinositide-specific phospholipase C (PI-PLC) activity were resolved when guinea pig uterus cytosolic proteins were chromatographed on a DEAE-Sepharose column. The first peak of enzyme activity eluting from the DEAE-Sepharose column (PI-PLC I) was further purified to homogeneity, whereas the second peak of enzyme activity was enriched 300-fold. PI-PLC I migrated as a 62-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. Antibodies prepared against PI-PLC I failed to react with PI-PLC II. PI-PLC I hydrolyzed all three phosphoinositides, exhibiting a greater Vmax for phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. Hydrolysis of phosphatidylinositol was calcium-dependent, whereas significant hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate occurred in the presence of 2.5 mM EGTA. At physiological concentrations of calcium, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were the preferred substrates. Antibodies specific for PI-PLC I reacted with a 62-kDa protein in both the cytosol and membrane fractions from guinea pig uterus. Quantitation of the immunoblots revealed that 25% of the 62-kDa protein was membrane-associated, whereas only 5% of the total enzyme activity was membrane-associated. Approximately 20% of the membrane-bound phospholipase C activity and immunoreactive material were loosely bound, whereas the remainder required detergent extraction for complete solubilization. The 62-kDa protein associated with the membrane fractions did not bind lectin affinity columns, suggesting that it was not glycosylated. PI-PLC I was identified as a phosphoprotein in [32P]orthophosphate-labeled rat basophilic leukemia (RBL-1) cells by two-dimensional gel electrophoresis followed by immunoblotting. In untreated cells, 32P-labeled PI-PLC I was found in the cytosolic fraction. Treatment of RBL-1 cells with those phorbol esters which are known to activate the Ca2+/phospholipid-dependent enzyme protein kinase C, resulted in a time-dependent increase in the phosphorylation of both membrane-bound and cytosolic PI-PLC I. Thus, in RBL-1 cells, protein kinase C may play an important role in the regulation of phospholipase C through protein phosphorylation.     

12-O-Tetradecanoylphorbol 13-acetate stimulates inositol lipid phosphorylation in intact human platelets

The phorbol esters are among the most potent tumor promoters. On addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) to isolated human platelets prelabelled with [32P]orthophosphate we found a rapid increase in 32P incorporation into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. In view of similar findings with cells infected with the oncogene Rous sarcoma virus, it is suggested that inositol lipid phosphorylation might be a key event in the molecular action of phorbol esters.     

Metabolism of lysopolyphosphoinositides by rat brain and liver microsomes

Lysophosphatidylinositol 4,5-bisphosphate has been reported to form ion-conducting channels in artificial membranes. If formed in vivo, mechanisms for its removal from cellular membranes would be required. Thus, possible pathways were explored in rat brain and liver microsomes. Since neither lysophosphatidylinositol 4-phosphate nor lysophosphatidylinositol 4,5-bisphosphate were acylated in experiments with [3H]arachidonic acid or [14C]oleoyl CoA, polyphosphoinositides do not participate directly in a deacylation-reacylation cycle as proposed for the postsynthesis enrichment of phosphatidylinositol with arachidonic acid. Similar enrichment in polyphosphoinositides can occur only via the rapid phosphorylation-dephosphorylation cycle linking all three phosphoinositides. Lysophosphatidyl[2-3H]inositol 4,5-bisphosphate and lysophosphatidyl[2-3H]inositol 4-phosphate were rapidly dephosphorylated to 1-acyl-sn-glycero(3)phospho(1)-D-myo-inositol by microsomes from both tissues. Appearance of only trace quantities of radioactive lysophosphatidylinositol monophosphate during the catabolism of lysophosphatidyl[2-3H]inositol 4,5-bisphosphate indicated that the second dephosphorylation step, which was cation independent, was at least as fast as the first step which required Mg2+. In the presence of ATP, CoA, and arachidonic acid, the lysophosphatidylinositol was converted to phosphatidylinositol. This acylation reaction was rate limiting in brain microsomes. Dephosphorylation of lysophosphatidylinositol 4,5-bisphosphate was rate limiting in liver microsomes. Neither the lysopolyphosphoinositides nor the lysophosphatidylinositol produced from them in the reactions were degraded by acyl hydrolases or phosphodiesterases in microsomes from either tissue. Therefore, any lysopolyphosphoinositide formed in vivo would probably be removed by dephosphorylation and recycled to phosphatidylinositol.     

Polylysine and polyamine stimulation of the phosphatidylinositol kinases of amphibian oocyte membranes

Phosphatidylinositol kinase present in Xenopus laevis oocyte membranes catalyzes the formation of phosphatidylinositol 4-phosphate using phosphatidylinositol and ATP as substrates while the activity of a second enzyme, phosphatidylinositol-4-phosphate kinase, results in the synthesis of phosphatidylinositol 4,5-bisphosphate. Large (Mr greater than 20,000) homopolymers of L-lysine or L-ornithine can stimulate the activity of both of these enzymes by at least 2-fold at 10-20 microM concentrations. Under similar conditions poly-L-arginine fails to stimulate the reaction causing a partial inhibition. Smaller polylysine (25 lysines) or lysine-rich oligopeptides such as one corresponding to the last 14 amino acids of the carboxyl end of c-Ki-ras 2 protein produce appreciable stimulation of phosphatidylinositol but at concentrations of 300-500 microM. Spermine and spermidine at millimolar concentrations also stimulate exogenous phosphatidylinositol phosphorylation. The amino-glycoside antibiotic neomycin has a biphasic effect, stimulating the phosphatidylinositol kinase at concentrations below 0.5 mM and strongly inhibiting at higher concentrations. Polylysine also moderately stimulates the loss of radioactivity of phosphatidylinositol-4-[32P] phosphate observed in oocyte membranes. Polylysine and polyornithine do not change the apparent Km for ATP of the phosphatidylinositol kinase but increase the Vmax of the reaction.     

Breakdown of polyphosphoinositides and not phosphatidylinositol accounts for muscarinic agonist-stimulated inositol phospholipid metabolism in rat parotid glands.

The molecular mechanisms underlying the ability of muscarinic agonists to enhance the metabolism of inositol phospholipids were studied using rat parotid gland slices prelabelled with tracer quantities of [3H]inositol and then washed with 10 mM unlabelled inositol. Carbachol treatment caused rapid and marked increases in the levels of radioactive inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate and an accumulation of label in the free inositol pool. There were much less marked changes in the levels of [3H]phosphatidylinositol, [3H]phosphatidylinositol 4-phosphate and [3H]phosphatidylinositol 4,5-bisphosphate. At 5 s after stimulation with carbachol there were large increases in [3H]inositol 1,4-bisphosphate and [3H]inositol 1,4,5-trisphosphate, but not in [3H]inositol 1-phosphate. After stimulation with carbachol for 10 min the levels of radioactive inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate greatly exceeded the starting level of radioactivity in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate respectively. When carbachol treatment was followed by addition of sufficient atropine to block all the muscarinic receptors the radioactive inositol phosphates rapidly returned towards control levels. The carbachol-evoked changes in radioactive inositol phosphate and phospholipid levels were blocked in the presence of 2,4-dinitrophenol (an uncoupler of oxidative phosphorylation). The results suggest that muscarinic agonists stimulate a polyphosphoinositide-specific phospholipase C and that these lipids are continuously replenished from the labelled phosphatidylinositol pool. [3H]Inositol 1-phosphate in the stimulated glands probably arises via hydrolysis of inositol 1,4-bisphosphate and not directly from phosphatidylinositol.