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PubMed1991

Interaction of short chain aliphatic alcohols with muscarinic receptor-stimulated phosphoinositide metabolism in cerebral cortex from neonatal and adult rats.

Muscarinic receptor-stimulated phosphoinositide metabolism has been recently suggested as a possible target for the neurotoxic effects of ethanol during brain development. Since two other alcohols, tertiary butanol and n-propanol, have been shown to cause microencephaly in the rat when administered during the brain growth spurt, in the present study we investigated the in vitro effects of five short chain aliphatic alcohols on muscarinic receptor-stimulated phosphoinositide metabolism in cerebral cortical slices from 7 day-old rats. In neonatal animals all alcohols tested inhibited carbachol (1 mM)-stimulated [3H]inositol phosphates accumulation in a dose- and time-dependent manner. The order of potency was t-butanol greater than n-propanol greater than or equal to iso-propanol greater than ethanol greater than methanol. After 90 min of incubation, ethanol, n-propanol and t-butanol caused a significant inhibition of muscarinic receptor-stimulated inositol metabolism at doses as low as 15 - 50 mM, comparable to the blood concentrations reached after in vivo administration of doses able to induce developmental neurotoxicity. The inhibitory effect of ethanol was additive to that of iso-propanol or t-butanol. Differently from these effects in 7 day-old rats, in cortical slices from adult animals methanol and ethanol had no effect on carbachol-stimulated phosphoinositide metabolism, while the two propanol isomers and t-butanol were less effective than in neonatal animals. These results suggest that muscarinic receptor-coupled phosphoinositide metabolism might be a common neurochemical target for the developmental neurotoxicity of short chain aliphatic alcohols.

Alcohols↗

PubMed1990

Regulation by bradykinin of phosphoinositide metabolism in the endothelial cells of the pulmonary artery.

The effect of the vasodilatory peptide bradykinin on the regulation of phosphoinositide metabolism in endothelial cells was investigated. Activation of phosphoinositide metabolism by bradykinin in the endothelium of the bovine pulmonary artery was not blocked by pertussis toxin, which ADP-ribosylates a membrane protein of molecular mass 40 kDa, but botulinum toxin, which ADP-ribosylates a membrane protein of molecular mass 24 kDa, fully blocked bradykinin-stimulated phosphoinositide metabolism. The effect of bradykinin was potentiated by guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), an activator of GTP-binding proteins, and inhibited by guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S), an inhibitor of GTP-binding proteins. Activation of phosphoinositide metabolism by bradykinin was fully blocked by a B2-receptor antagonist, whereas a B1-receptor antagonist did not affect bradykinin action. It is concluded that the B2-receptor in endothelial cells is coupled to phospholipase C via a GTP-binding protein, which is a substrate for botulinum toxin.

Animals↗

PubMed1990

Desensitization of muscarinic stimulated hippocampal cell firing is related to phosphoinositide hydrolysis and inhibited by lithium.

The potency and efficacy of a series of muscarinic agonists for stimulation of neuronal firing rate was compared with stimulation of phosphoinositide (PPI) hydrolysis in similar hippocampal slice preparations. Carbachol, muscarine, pilocarpine, arecoline, bethanechol and oxotremorine varied in potency, but stimulated neuronal firing to a similar extent. At higher concentrations, all of the drugs except oxotremorine caused a decrease in firing rate (desensitization). A comparison of the concentration-response curves for PPI hydrolysis and neuronal firing rates showed that desensitization occurred at a threshold level of PPI hydrolysis. Although the concentration of drug that caused the decrease in firing rate was different for each agonist, the level of PPI hydrolysis at the desensitizing concentration was similar, with the exception of oxotremorine. Oxotremorine, the weakest agonist for stimulation of PPI hydrolysis, did not reach this PPI hydrolysis threshold and did not exhibit desensitization. Oxotremorine was also capable of both blocking and reversing the desensitization caused by carbachol. Low concentrations of pirenzepine, an M1 selective muscarinic antagonist, reversed carbachol desensitization. Concentrations of lithium that disrupt the phosphoinositide cycle by preventing recycling of free inositol (Allison, J. H., et al.: Biochem. Biophys. Res. Commun. 71: 664-670, 1976; Hallcher, L. M. and Sherman, W. R.: J. Biol. Chem. 225: 10896-10901, 1980) slowly reversed desensitization. Furthermore, inositol added to the buffer could re-establish desensitization in lithium-treated preparations. These studies suggest that muscarinic desensitization of hippocampal cell firing is related to large increases in phosphoinositide hydrolysis. Muscarinic receptor-stimulated increases in cell firing may be mediated by a subtype or state of muscarinic receptor different from that mediating phosphoinositide hydrolysis and desensitization.

Action Potentials↗

PubMed1990

Phosphoinositide metabolism in adipocytes from fed and fasted rats.

Phosphoinositide turnover was investigated in adipocytes from fed and 48 hour fasted rats. Insulin stimulated phosphoinositide synthesis both in adipocytes from fed and fasted rats. Fasting enhanced this effect of insulin 2-fold. Hydrolysis of phosphoinositides to inositol phosphates was not activated by insulin, neither transient after 2 minutes nor after 60 minutes stimulation. Under similar conditions, alpha 1-adrenergic receptor stimulation induced a pronounced inositol phosphate production. Thus, it is suggested that phosphoinositide hydrolysis is not involved in insulin action. The alpha 1-adrenergic effect was similar in adipocytes from fed and fasting rats.

Adipose Tissue↗

PubMed1990

Stereoselectivity and mode of inhibition of phosphoinositide-coupled excitatory amino acid receptors by 2-amino-3-phosphonopropionic acid.

DL-2-Amino-3-phosphonopropionic acid, a phosphonate-substituted derivative of aspartic acid, has been shown to be an inhibitor of excitatory amino acid-stimulated phosphoinositide hydrolysis in rat brain slices. In this study, the enantiomers of 2-amino-3-phosphonopropionic acid were synthesized and used to further characterize the stereoselectivity and mechanism of interaction of this compound for inhibiting phosphoinositide-coupled (metabotropic) excitatory amino acid receptors. L-2-Amino-3-phosphonopropionic acid was 3-5 times more potent than D-2-amino-3-phosphonopropionic acid as an inhibitor of ibotenate-stimulated [3H]inositol monophosphate formation in slices of the rat hippocampus or quisqualate-stimulated [3H]inositol monophosphate formation in neonatal rat cerebral cortical slices. Carbachol-stimulated phosphoinositide hydrolysis was not inhibited by L-2-amino-3-phosphonopropionic acid, and L-2-amino-3-phosphonopropionic acid had no appreciable affinity for ionotropic excitatory amino acid receptors at concentrations required to inhibit metabotropic excitatory amino acid responses. The inhibitory effects of L-2-amino-3-phosphonopropionic acid or L-2-amino-4-phosphonobutyric acid on phosphoinositide hydrolysis were not competitive, because they could not be surmounted by increasing concentrations of ibotenate or quisqualate. L-2-Amino-3-phosphonopropionic acid inhibition also could not be prevented by washing the tissue before incubation with ibotenate. Thus, L-2-amino-3-phosphonopropionic acid is a stereoselective inhibitor of metabotropic excitatory amino acid receptors with little affinity for ionotropic receptors. However, the inhibitory effects of L-2-amino-3-phosphonopropionic acid or L-2-amino-4-phosphonobutyric acid were not readily reversed, and the site at which they act to inhibit metabotropic excitatory amino acid receptors remains to be determined.

Alanine↗

PubMed1989

Protein kinase C-mediated gonadotropin-releasing hormone receptor sequestration is associated with uncoupling of phosphoinositide hydrolysis.

Gonadotropin-releasing hormone (GnRH) regulates pituitary gonadotropin release by a Ca2+-dependent mechanism involving receptor-mediated phosphoinositide hydrolysis. Previous studies indicate that activation of pituitary protein kinase C (PKC), while not required for acute gonadotropin release in response to GnRH, is likely involved in the chronic regulation of gonadotrope responsiveness. Studies from our laboratory have shown that activation of PKC by phorbol esters produces both the uncoupling of GnRH-stimulated phosphoinositide hydrolysis and the selective enhancement of GnRH agonist binding in pituitary cell cultures. In the present work, we have examined the possibility that these processes are related in mechanism. Dissociation of bound agonist radioligand at 23 degrees C was found to be reduced in the presence of phorbol esters, and ligand bound in the presence of phorbol ester was resistant to displacement by competing ligands at 4 degrees C. However, agonist bound in the presence of phorbol ester was dissociable by subsequently washing cells at pH 3. Receptor photoaffinity labeling studies confirmed that agonist association with membrane component(s) identified as the GnRH receptor was increased in the presence of phorbol ester. These results suggest that, in the presence of a phorbol ester PKC activator, agonist-occupied GnRH receptors remain at the cell surface, but are sequestered in some manner. In other experiments, cell preloaded with [3H]inositol were treated with GnRH agonist ligand and phorbol ester at 4 degrees C to form a pool of sequestered, agonist-occupied receptors, and then displaceable (nonsequestered) agonist was removed by incubation with antagonist ligand. After addition of LiCl and warming to 37 degrees C, [3H]inositol phosphate production (an index of phosphoinositide hydrolysis) in phorbol ester-treated cells was reduced to 67% of vehicle control, although residual specific agonist binding had been increased to greater than 300% of control. The appearance of sequestered receptors and inhibition of [3H]inositol phosphate production had similar phorbol ester concentration dependencies. These results suggest that the same agonist-occupied GnRH receptors sequestered as a result of PKC activation also are preferentially uncoupled from phosphoinositide hydrolysis.

Animals↗

PubMed1989

Thrombin and trypsin act at the same site to stimulate phosphoinositide hydrolysis and calcium mobilization.

Thrombin stimulates polyphosphoinositide hydrolysis in embryonic chick heart cells and in 1321N1 astrocytoma cells and increases intracellular Ca2+ in the 1321N1 cells. The serine protease trypsin mimics these actions in a dose-dependent fashion, whereas the proteolytically inactive thrombin derivatives diisopropyl fluorophosphate-thrombin (DIP-thrombin) and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-thrombin (PPACK-thrombin) are ineffective in this regard. The phosphoinositide responses to thrombin or trypsin and the muscarinic agonist carbachol are additive, but no additivity is observed between the responses to thrombin and trypsin. Unlike the response to carbachol, the phosphoinositide and Ca2+ responses to thrombin and trypsin desensitize, with no recovery of the calcium response even when Ca2+ stores are replenished. Cross-desensitization of phospholipase C activation and calcium mobilization between these proteases is also observed. In addition, PPACK-thrombin, which elicits no response itself, effectively inhibits trypsin-stimulated phosphoinositide hydrolysis. It is proposed that thrombin and trypsin act through the same receptor. Proteolysis appears to be important in the mechanism by which these agonists elicit phosphoinositide hydrolysis, calcium mobilization, and, perhaps, subsequent receptor desensitization.

Amino Acid Chloromethyl Ketones↗

PubMed1985

Agonist-induced desensitization of muscarinic receptor-mediated calcium efflux without concomitant desensitization of phosphoinositide hydrolysis.

Phosphoinositide hydrolysis does not appear to desensitize in 1321N1 astrocytoma cells. The evidence for this is that 1) the rate of accumulation of [3H]inositol 1-phosphate is linear for up to 90 min in the presence of carbachol, 2) pretreatment of cells with 100 microM carbachol for 75 min does not diminish the subsequent ability of carbachol to increase [3H]inositol 1-phosphate accumulation, and 3) the production of all of the [3H]inositol phosphates including the polyphosphoinositide metabolites [3H]inositol bis- and trisphosphate continues for up to 75 min in the presence of carbachol and declines rapidly when the muscarinic receptor antagonist atropine is added. Only when cells are treated with carbachol for 2.5 hr or longer is there a reduction in carbachol-stimulated phosphoinositide hydrolysis, and this is associated with a decrease in muscarinic receptor number. There does appear to be desensitization of hormone-stimulated Ca2+ mobilization in 1321N1 cells, because treatment of these cells with carbachol for 75 min leads to loss of the subsequent ability of carbachol to stimulate unidirectional 45Ca2+ efflux. Histamine-stimulated 45Ca2+ efflux also is lost in cells pretreated with carbachol, indicating that the desensitization is heterologous. We conclude that desensitization of hormone-stimulated, unidirectional 45Ca2+ efflux cannot be accounted for by a loss of receptor-mediated phosphoinositide hydrolysis. If phosphoinositide hydrolysis or inositol triphosphate formation are signals for calcium mobilization, the site at which the calcium response desensitizes must be distal to the initial receptor-mediated activation of phospholipase C.

Astrocytoma↗

PubMed1985

Insulin-stimulated phosphoinositide metabolism in isolated fat cells.

Treatment of isolated fat cells with insulin produced increases of up to 4.8-fold in the incorporation of [3H]inositol into phosphatidylinositol. This effect of insulin was both time- and dose-dependent with half-maximal stimulation at 30 microunits/ml of insulin. Insulin increased the labeling of phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate but not phosphatidylinositol 4-monophosphate in cells which had been preincubated with [3H]inositol for 90 min. Incubation of the cells in a Ca2+-free buffer increased the basal level of phosphatidylinositol labeling and enhanced the effect of insulin. Glucagon and isoprenaline, both of which stimulate lipolysis, had no effect on phosphatidylinositol labeling but did potentiate insulin-stimulated incorporation of [3H]inositol into phosphatidylinositol. Phosphoinositide breakdown was measured by the accumulation of inositol phosphates. Insulin did not increase the level of the inositol phosphates at all concentrations of the hormone tested. By comparison, phenylephrine and vasopressin were able to stimulate phosphoinositide breakdown. Pretreatment of the cells with insulin enhanced the effect of phenylephrine on inositol phosphates' accumulation, suggesting that insulin may potentiate phenylephrine-mediated phosphoinositide turnover. From these data we conclude that insulin stimulates the de novo synthesis of phosphatidylinositol and phosphatidylinositol 4,5-biphosphate, but has no effect on phosphoinositide breakdown.

Adipose Tissue↗

PubMed1984

Relationships between phosphoinositide and calcium responses to muscarinic agonists in astrocytoma cells.

Activation of muscarinic receptors in human astrocytoma (1321N1) cells stimulates phosphoinositide metabolism and calcium mobilization. The muscarinic effect on phosphoinositide turnover is evidenced by increased formation of [3H]inositol 1-phosphate (Ins1P) and by increased [3H]inositol incorporation into PtdIns. The muscarinic effect on calcium mobilization is seen as a large increase in undirectional 45Ca2+ efflux from cells equilibrated with 45Ca2+ and a small increase in unidirectional 45Ca2+ influx. A series of muscarinic agonists was used to explore the relationship between phosphoinositide metabolism and unidirectional 45Ca2+ efflux. The maximal increases in [3H]Ins1P formation produced by carbachol and acetylcholine are similar and are much larger than those caused by oxotremorine and pilocarpine. The effects of these agonists on 45Ca2+ efflux are similar: carbachol and acetylcholine cause equivalent maximal increases in the rate of 45Ca2+ efflux whereas oxotremorine and pilocarpine cause submaximal 45Ca2+ efflux responses. The Kact values of carbachol and acetylcholine for stimulation of [3H]Ins1P formation are 40 microM and 1.5 microM, respectively. These values are only 2- to 3-fold higher than the respective Kact values for stimulating 45Ca2+ efflux. The finding that each of the muscarinic agonists tested has nearly identical efficacy and similar potency for stimulating [3H]Ins1P formation and 45Ca2+ efflux supports the idea that hormonal stimulation of phosphoinositide hydrolysis leads to calcium mobilization.

Astrocytoma↗

PubMed1983

Impairment of membrane phosphoinositide metabolism by aminoglycoside antibiotics: streptomycin, amikacin, kanamycin, dibekacin, gentamicin and neomycin.

Like many amphiphilic cationic drugs, aminoglycosides are able to produce phospholipidosis, mainly by inhibiting enzymes involved in phospholipid metabolism. Phosphoinositides have been suggested to function as receptors for aminoglycosides. Therefore, we investigated the influence of these drugs upon phosphoinositide metabolism by measuring the 32P-incorporation into the polyphosphoinositides, using the rat erythrocyte membrane as a model. Depending upon the experimental conditions, neomycin induced a decrease and/or an increase in the 32P-labeling of triphosphoinositides (TPI) and of diphosphoinositides (DPI), respectively. These variations were rapid and depended upon the drug concentration. At 0.3 mM, neomycin reversed the distribution of radioactivities associated with DPI and TPI without modifying the total radioactivity incorporated. This drug concentration altered neither the Mg++-activated TPI-specific phosphomonoesterase activity nor the Ca++-activated polyphosphoinositide phosphodiesterase activity. It appears likely that the drug inhibits the DPI-kinase activity, by interacting with DPI and thereby lowering the substrate availability. Over the range of concentrations studied (up to 1-2 mM), gentamicin, kanamycin and dibekacin behave as neomycin. However, their effects could be observed only at drug concentrations higher than those of neomycin. By contrast, streptomycin and amikacin did not alter the 32P-labeling of TPI and of DPI. The order of potency of aminoglycosides for the impairment of the phosphoinositide interconversion was neomycin, gentamicin, dibekacin, kanamycin. A possible relationship between the toxicity of aminoglycosides and their capacity to impair the phosphoinositide metabolism is discussed.

Amikacin↗

PubMed1994

Dual effects of nordidemnin on WRK1 cells: inhibition of phosphoinositide metabolism and cell proliferation.

Nordidemnin (NorD), a cyclodepsipeptide isolated from marine invertebrates, exhibits antiproliferative and antitumoral properties identical to didemnin B on many cell lines. On WRK1 cells, a rat mammary tumor cell line, NorD considerably reduced the vasopressin-stimulated accumulation of inositol phosphates. This effect was more pronounced on dividing cells and of weak amplitude on quiescent ones. It was observed with nanomolar concentrations of NorD and became significative after 3 hr of incubation at 37 degrees C. The maximal effect was observed after a 14-hr incubation period. In contrast, the inactive analog epinordidemnin, as well as the structurally related immunosuppressive cyclosporin A, had no significant effect on phosphoinositide metabolism. More detailed analysis demonstrated that NorD reduced the amounts of all intracellular inositol phosphate isomers, including inositol pentakisphosphate and inositol hexakisphosphate. Vasopressin-stimulated inositol (1,4,5)-trisphosphate accumulation was reduced by 80% and, as a consequence, the intracellular calcium mobilization was strongly affected. Similarly, NorD reduced both the level of inositol (1,4,5)-trisphosphate and the intracellular free calcium concentration of unstimulated cells. NorD blocked phosphoinositide metabolism by reducing the myoinositol transporter and, by a consequence, the pool of inositol lipids. NorD also strongly inhibited WRK1 cell proliferation with the same EC50 as that observed for the effect on phosphoinositide metabolism. Epinordidemnin, which was unable to inhibit inositol phosphate accumulation, had no effect on cell growth. Cyclosporin A, which slightly inhibited WRK1 cell growth, did not significantly affect the calcium-phosphatidylinositol cascade. Taken together, these results suggest that NorD might interfere with WRK1 cell growth by inhibiting phosphoinositide turnover.

Animals↗

PubMed1994

Thyrotropin-releasing hormone (TRH) receptor number determines the size of the TRH-responsive phosphoinositide pool. Demonstration using controlled expression of TRH receptors by adenovirus mediated gene transfer.

We use an adenovirus vector, AdCMVmTRHR, to express thyrotropin-releasing hormone (TRH) receptors (TRH-Rs) to determine whether the size of the hormone-responsive phosphoinositide pool in mammalian cells is directly related to receptor number. Infection of HeLa cells with increasing numbers of AdCMVmTRHR caused time-dependent graded expression of TRH-Rs. Measurement of cytoplasmic free Ca2+ in individual cells permitted quantitation of the fraction of cells responsive to TRH. Infection with 100 AdCMVmTRHR particles/cell or more led to TRH responsiveness in > or = 90% of HeLa cells. Measurement of prelabeled phosphoinositides hydrolyzed during prolonged TRH stimulation assesses the size of the TRH-responsive pool. In cells infected with AdCMVmTRHR for 24 h, the size of the TRH-responsive phosphoinositide pool increased with increasing TRH-R expression. The TRH-responsive pool also increased with time after infection as the number of TRH-Rs increased. Similar observations were made in GHY and KB cells. These data confirm our previous suggestion (Cubitt, A. B., Geras-Raaka, E., and Gershengorn, M. C. (1990) Biochem. J. 271, 331-336) that there are hormone-responsive and -unresponsive pools of cellular phosphoinositides and that the maximal size of the TRH-responsive pool is directly related to the number of TRH-Rs.

Adenoviridae↗

PubMed1993

Phosphoinositide second messenger system is enriched in striosomes: immunohistochemical demonstration of inositol 1,4,5-trisphosphate receptors and phospholipase C beta and gamma in primate basal ganglia.

The neurochemical organization of the basal ganglia has been studied extensively with respect to neurotransmitters, neuropeptides, and their receptors. The chemoarchitecture of the striatum has been found particularly striking, because it distinguishes many substances by their relative distributions within the striosome and matrix compartments of the striatum. Very little is yet known about the differential distribution of second messenger systems in the basal ganglia, however, and no information is available about whether the distribution of second messenger systems is related to the prominent neurochemical compartmentalization of the striatum. We have examined the distribution of the phosphoinositide second messenger system in the primate basal ganglia and substantia nigra, as detected with polyclonal antisera against the inositol 1,4,5-trisphosphate receptor (IP3R), and monoclonal antisera against phospholipase C beta (PLC beta) and phospholipase C gamma (PLC gamma). In the striatum, immunostaining for each of the three proteins was present predominantly in medium-sized neuronal perikarya and in the neuropil. Circumscribed zones of enhanced IP3R, PLC beta, and PLC gamma immunoreactivity appeared in a background of generally weaker staining, and these zones corresponded to striosomes as identified by calbinidin D28k and substance P immunostaining in adjacent sections. Thus, the richest representation of the phosphoinositide system in the primate striatum appears to be in striosomes. In the substantia nigra pars compacta, neurons and neuropil were immunopositive, but in the substantia nigra pars reticulata and in each segment of the globus pallidus, immunostaining was mainly confined to the neuropil. Perikaryal PCL gamma immunoreactivity in the absence of detectable PLC beta or IP3R immunolabeling was found in the magnocellular neurons embedded in the medullary layer between the putamen and the globus pallidus. These observations demonstrate that the phosphoinositide second messenger system is selectively enhanced in neuronal subsystems of the basal ganglia, including striosomes, and suggest that signaling by phosphoinositide pathways elicits discrete effects on input-output processing by the basal ganglia.

Animals↗

PubMed1996

Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase.

Pleckstrin homology (PH) domains may act as membrane localization modules through specific interactions with phosphoinositide phospholipids. These interactions could represent responses to second messengers, with scope for regulation by soluble inositol polyphosphates. A biosensor-based assay was used here to probe interactions between PH domains and unilamellar liposomes containing different phospholipids and to demonstrate specificity for distinct phosphoinositides. The dynamin PH domain specifically interacted with liposomes containing phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and, more weakly, with liposomes containing phosphatidylinositol-4-phosphate [PI(4)P]. This correlates with phosphoinositide activation of the dynamin GTPase. The functional GTPase of a dynamin mutant lacking the PH domain, however, cannot be activated by PI(4,5)P2. The phosphoinositide-PH domain interaction can be abolished selectively by point mutations in the putative binding pocket predicted by molecular modelling and NMR spectroscopy. In contrast, the Bruton's tyrosine kinase (Btk)PH domain specifically bound liposomes containing phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3]: an interaction requiring Arg28, a residue found to be mutated in some X-linked agammaglobulinaemia patients. A rational explanation for these different specificities is proposed through modelling of candidate binding pockets and is supported by NMR spectroscopy.

Amino Acid Sequence↗

PubMed1996

[Catabolism of membrane phosphatidylcholines during early stages of mitogenic initiation of phosphoinositide cycle in lymphocytes].

Deacylation of the phosphatidylcholine fraction in plasma membranes of rat thymocytes and human blood lymphocytes was studied as well as its relationship to the activation of phosphoinositide-specific phosphodiesterase at early stages of mitogen-initiated phosphoinositide cycle. The data indicate that in lymphocyte membranes, enzymatic system of cascade deacylation of the phosphatidylcholine fraction includes calcium-activated phospholipase A1 and lysophospholipase. The enzyme system contributes to the rapid and reversible modification of the lipid bilayer of lymphocyte plasma membrane and can cooperate with phosphoinositide-specific phosphodiesterase during its activation at early stages of the initiation of phosphoinositide pathway during translocation of the external mitogenic signal.

Animals↗

PubMed1996

Phosphoinositide-dependent in vitro phosphorylation of profilin by protein kinase C. Phospholipid specificity and localization of the phosphorylation site.

Phosphoinositides bind to profilin and regulate actin-based cytoskeletal protein assembly. We report here that profilin is phosphorylated in vitro by protein kinase C (PKC) in the presence of phosphoinositides and micromolar concentrations of calcium. PKC-mediated phosphorylation of profilin was observed only in the presence of phosphoinositides; phosphatidylserine and diacylglycerol (known activators of PKC) and other lipids, including phosphatidic acid and phosphatidylglycerol phosphate, did not activate the phosphorylation. The activation of PKC-mediated phosphorylation of profilin by phosphoinositides was as follows: phosphatidylinositol (PI) 4-phosphate (K(m) = 18 microM) > PI 4,5-bisphosphate (K(m) = 30 microM) > PI (no activation). About 0.5 mol phosphate was incorporated per mol of profilin. Phosphorylation of profilin by PKC was not affected by the presence of various concentrations of actin. Phospho-amino acid analysis showed serine to be the only amino acid phosphorylated. The amino acid sequence of a phosphopeptide from CNBr-digested profilin corresponded to the COOH-terminal peptide of profilin (Ala-Ser-His-Leu-Arg-Ser-Gln-Tyr). Further digestion of this phosphopeptide by trypsin generated two phosphopeptides (Arg-Ser-Gln-Tyr and Ser-Gln-Tyr), thereby confirming that the phosphorylation site was the antepenultimate Ser (Ala-Ser-His-Leu-Arg-Arg-Ser(P)-Gln-Tyr).

Actins↗

PubMed1990

Phosphoinositide metabolism in cultured glioma and neuroblastoma cells: subcellular distribution of enzymes indicate incomplete turnover at the plasma membrane.

The hypothesis that the small portion of cellular phosphoinositide participating in signal transduction might be preferentially recycled within the plasma membrane was tested in rat glioma (C6) and murine neuroblastoma (N1E-115) cells. Percoll density gradient centrifugation was used to isolate a purified plasma membrane fraction and the subcellular distribution of all enzymes mediating phosphoinositide turnover was assessed. A small but significant proportion of PtdInsP2-specific phosphodiesterase was located in the plasma membrane but only two of the five enzymes required to replace PtdInsP2 (diacylglycerol kinase and PtdInsP kinase) also were present. CTP:phosphatidate cytidylyltransferase and CMP-phosphatidate:inositol phosphatidyltransferase were located exclusively in a microsomal fraction containing enriched levels of endoplasmic reticulum markers. Thus, diacylglycerol from agonist-stimulated cleavage of PtdInsP2, or phosphatidic acid formed from it, must be transferred to the endoplasmic reticulum for conversion to PtdIns. Plasma membrane also lacked PtdIns kinase. If the soluble PtdIns kinase has access to membrane-bound substrate, PtdIns may be phosphorylated to PtdInsP before or during transport to the plasma membrane. Phosphorylation by the predominantly plasma membrane PtdInsP kinase to form PtdInsP2 completes the cycle. PtdInsP phosphatase was present in all membrane fractions suggesting that PtdInsP can be returned to the PtdIns pool in plasma membrane and elsewhere. PtdInsP2 phosphatase was almost exclusively in the cytosol suggesting that reversible interchange between PtdInsP and PtdInsP2 in the plasma membrane may be modulated by the ability of this phosphatase to act on PtdInsP2 in the membrane. Thus, PtdIns resynthesis in the plasma membrane of these cells does not occur and is not required for phosphoinositide-mediated signal transduction.

Animals↗