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

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

Inositol 1,4,5-trisphosphate as fertilization signal in plants: testcase Chlamydomonas eugametos

Within the first 2 h of sexual reproduction, gametes of the green alga Chlamydomonas eugametos agglutinate, fuse via their mating structures, de-agglutinate and swim off as vis-à-vis pairs. During this period, increases in intracellular inositol 1,4,5-trisphosphate levels and changes in polyphosphoinositide synthesis were associated with cell fusion. The protein-kinase-C inhibitor staurosporine (0.1–0.2 M) inhibited the de-agglutination of pairs and therefore prevented them swimming away, while earlier stages of mating such as agglutination or cell fusion were unaffected. The results suggest that inositol 1,4,5-trisphosphate and diacylglycerol are fertilization signals in C. eugametos. The idea that they could also be fertilization signals in higher plants is discussed in relation to in vitro embryogenesis.Abbreviations DAG diacylglycerol - InsP₃ inositol 1,4,5-trisphosphate - mt⁺/mt^– mating-type plus or minus - PKC protein kinase C - PtdOH phosphatidic acid - PtdInsP phosphatidylinositol - 4-phosphate PtdInsP₂ phosphatidylinositol 4,5-bisphosphate     

Protein kinase C does not regulate diacylglycerol metabolism in aortic smooth muscle cells

Summary The effect of a reduction in protein kinase C activity on the metabolism of exogenous [³H]diC8 by freshly isolated smooth muscle cells from rabbit aorta and cultured A10 smooth muscle cells was determined. The metabolism of [³H]diC8 by both smooth muscle cell preparations was predominantly by hydrolysis to yield monoC8 and glycerol (lipase pathway); very little radioactivity was incorporated into phospholipids. Diacylglycerol lipase activity measured in vitro with A10 cell homogenates was much greater than diacylglycerol kinase activity. The addition of the protein kinase C inhibitor H-7 to incubations of isolated aortic smooth muscle cells and cultured A10 cells had no significant effect on the metabolism of [³H]diC8. Protein kinase C activity in cultured A10 cells preincubated for 20 h with a phorbol ester was reduced to 14% of control as a consequence of down-regulation, but diC8 metabolism was not changed. Therefore, protein kinase C does not regulate the metabolism of diacylglycerols in aortic smooth muscle cells.Abbreviations IP₃ inositol 1,4,5-trisphosphate - DG diacylglycerol - MG monoacylglycerol - PL phospholipid(s) - diC8 dioctanoylglycerol - H-7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride - monoC8 monooctanoylglycerol - PS phosphatidylserine - PDBu phorbol 12,13-dibutyrate     

Phosphoinositide fatty acids regulate phosphatidylinositol 5-kinase, phospholipase C and protein kinase C activities.

PtdIns(4,5)P(2) generally results from phosphorylation of PtdIns(4)P by the phosphatidylinositol 5-kinase (PtdIns5-K). Its hydrolysis by phospholipase C (PLC) yields inositol 1,4,5-trisphosphate and diacylglycerol, which stimulates protein kinase C (PKC). We show that epithelial cells of the cockroach rectum contain three different inositol lipids: PtdIns(4,5)P(2), PtdIns(4)P, and PtdIns. They are composed of six major fatty acids: palmitic (16:0) stearic (18:0), oleic (18:1n--9), linoleic (18:2n--6), linolenic (18:3n--3), and arachidonic (20:4n-6) acids. The fatty acid preference of each of the above enzymes was evaluated by incorporating different fatty acids in pairs into membrane lipids. Incorporation of 16:0 plus 18:1n--9 provoked an increase in PtdIns(4,5)P2-PLC activity and a decrease in PtdIns5-K activity. In contrast, incorporation of 16:0 plus 18:3n--3 led to a potentiation of PtdIns5-K activity and a decrease in PtdIns(4,5)P(2)-PLC activity. Furthermore, PLC and PtdIns5-K acted preferentially on substrates containing 18:3n--3, and 18:3n--3-containing diacylglycerol specifically potentiated PKC activity. Thus, we propose that the fatty acids that make up the phosphoinositides function as intracellular modulators of the activity of certain enzymes.     

Phosphorylation of myocardial sarcolemma proteins during induction of the phosphatidylinositol cycle in isolated perfused rabbit heart]

Phosphorylation of cardiac sarcolemma proteins under stimulation of M-receptors by agonist carbacholine used to stimulate phosphatidylinositide cycle, was investigated in the isolated, rabbit heart perfused with 32Pi. Carbacholine (10(-7) stimulates the polyphosphoinositide metabolism which is expressed in the activated incorporation of 32P from [gamma-32P]ATP in polyphosphoinositide as well as in the increased content of the labelled inositol trisphosphate released through phosphatidylinositol-4,5-bisphosphate break-down by phospholipase C. The diacylglycerol produced simultaneously with inositol triphosphate as a second messenger activates the protein kinase C. This was confirmed by considerable activation of phosphorylation sarcolemma proteins-substrates of protein kinase C, with Mr 94, 87, 78, 51 and 46 kDa.     

Rapid dephosphorylation of protein kinase C substrates by protein kinase A activators results from inhibition of diacylglycerol release

The biochemical events encompassing the dephosphorylation of protein kinase C substrates by protein kinase A activators have been investigated in a neurotumor cell line, NCB-20. Treatment of [32P]orthophosphate-labeled cells with protein kinase A activators (e.g. forskolin, dibutyryl cAMP, prostaglandin E1) resulted in an inhibition of protein kinase C activity due to a failure of the protein kinase C complex to translocate into the membrane. Phospholipase C activity, as measured by the synchronous release of diacylglycerol and inositol phosphates (inositol 1,4,5-trisphosphate, inositol 1,4-bisphosphate, and inositol 1-phosphate) in response to bradykinin, was inhibited up to 50% following exposure to protein kinase A activators. At the same time, phospholipase C-specific inositol phospholipid substrates (phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate) were found to accumulate in NCB-20 cells following treatment with protein kinase A activators. This suggests that phospholipase C may be altered through protein kinase A-mediated protein phosphorylation. Second messenger generation (inositol phosphates, diacylglycerol, and Ca2+) is therefore inhibited through cyclic AMP-mediated shutdown of the inositol lipid cycle at the level of phospholipase C.     

Transmembrane Signaling via Phosphatidylinositol 4,5-Bisphosphate Hydrolysis in Plants

Recent investigations have confirmed the presence of the polyphosphoinositides, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PIP₂), as well as inositol phospholipid-specific phospholipase C in higher plant and microalgal cells. In addition, it has been shown that stimulation of some photosynthetic cell types by environmental or hormonal challenge is accompanied by degradation of the polyphosphoinositides. The products of phospholipase C-catalyzed PIP₂ hydrolysis, inositol 1,4,5-trisphosphate and diacylglycerol, appear to be capable of releasing organelle-bound Ca²⁺ and stimulating protein kinase C-like activity in vitro. However, a direct cause and effect relationship between stimulated PIP₂ breakdown and changes in intracellular calcium, protein phosphorylation, or cell function has not yet been unequivocally established. Despite a number of technical difficulties slowing progress in this field, it is likely that photosynthetic organisms will soon be shown to transmit physiologically significant extracellular signals across their plasma membranes by a PIP₂-mediated transduction mechanism.     

Polyphosphoinositol lipids inChlamydomonas eugametos gametes

InChlamydomonas eugametos gametes, phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) comprised 0.4 and 0.3% of the whole-cell phospholipids. They were concentrated in the plasma membrane around the cell body and were present in low concentrations in the flagellar membrane. When gametes were fed³²PO ₄ ^- , the label was rapidly incorporated into PtdInsP and PtdInsP₂ and only slowly incorporated into structural lipids such as phosphatidylethanolamine and phosphatidylglycerol. Similarly, when a pulse of³²PO ₄ ^- was chased with PO ₄ ^- , the label was rapidly lost from the polyphosphoinositol lipids but not from the structural lipids. The major fatty acids in the polyphosphoinositides were C-22 carbon polyenoic acids (70%). The significance of these results in relationship to intracellular signalling via inositol phosphates and Ca²⁺ is discussed.Abbreviations InsP₃ inositol 1,4,5-trisphosphate - mt^–/mt⁺ mating-type plus or minus - PtdA phosphatidic acid - PtdEtn phosphatidylethanolamine - PtdGro phosphatidylglycerol - PtdIns phosphatidylinositol - PtdInsP phosphatidylinositol 4-phosphate; - PtdInsP₂ phosphatidylinositol 4,5-bisphosphate - TCA trichloroacetic acid We thank Frank Schuring for Fig. 5A and Susan Kenter, Hans Kruisselbrink, Saskia Bijvank and Nelleke Corbett for their enthousiastic assistance.     

The polyphosphoinositide cycle exists in the nuclei of Swiss 3T3 cells under the control of a receptor (for IGF-I) in the plasma membrane, and stimulation of the cycle increases nuclear diacylglycerol and apparently induces translocation of protein kinase C to the nucleus.

When Swiss 3T3 cells are treated with Insulin-like Growth Factor I, a rapid decrease in the mass of polyphosphoinositol lipids (phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate) occurs within the nuclei, with a concomitant increase in nuclear diacylglycerol and translocation of protein kinase C to the nuclear region. This is in contrast to the effects of the regulatory peptide, bombesin, which causes similar inositol lipid changes in the plasma membrane, has no effect on nuclear inositide levels and causes a translocation of protein kinase C to post-nuclear membranes. These results suggest the existence of a discrete nuclear polyphosphoinositide signalling system entirely distinct from the well-known plasma membrane-located system, which is under regulatory control by cell surface-located receptors.     

BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation

Deciphering many roles played by inositol lipids in signal transduction and membrane function demands experimental approaches that can detect their dynamic accumulation with subcellular accuracy and exquisite sensitivity. The former criterion is met by imaging of fluorescence biosensors in living cells, whereas the latter is facilitated by biochemical measurements from populations. Here, we introduce BRET-based biosensors able to detect rapid changes in inositol lipids in cell populations with both high sensitivity and subcellular resolution in a single, convenient assay. We demonstrate robust and sensitive measurements of PtdIns4P, PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃ dynamics, as well as changes in cytoplasmic Ins(1,4,5)P₃ levels. Measurements were made during either experimental activation of lipid degradation, or PI 3-kinase and phospholipase C mediated signal transduction. Our results reveal a previously unappreciated synthesis of PtdIns4P that accompanies moderate activation of phospholipase C signaling downstream of both EGF and muscarinic M3 receptor activation. This signaling-induced PtdIns4P synthesis relies on protein kinase C, and implicates a feedback mechanism in the control of inositol lipid metabolism during signal transduction.     

Phorbol ester and the actions of phosphatidylinositol 4,5-bisphosphate specific phospholipase C and protein kinase C in microsomes prepared from cultured cardiomyocytes

Microsomes were prepared from cultured neonatal rat cardiomyocytes. Incubation of microsomes in buffer containing 5µM CaCl₂, 5 mM cholate and 100 nM [3H-]Phosphatidylinosito14,5-bisphosphate (PtdIns(4,5) P₂) resulted in the formation of [³H-]InsP ₃. GTP-gamma-S (125 µM) stimulated the production of [³H-]InsP ₃. Microsomes prepared from phorbol ester-treated (100 nM phorbol 12-myristate 13-acetate, PMA) cardiomyocytes showed decreased activities of basal as well as GTP-gamma-S-stimulated [³H-]Ptdlns(4,5)P ₂ hydrolysis. In the microsomes a 15 kD protein was demonstrated to be the major substrate phosphorylated by intrinsic protein kinase C, which was activated by 0.5 mM Ca²⁺. Addition of phorbol ester (100 nM PMA) enhanced the ³²P-incorporation into the 15 kD protein. Protein kinase C, purified from rat brain, in the presence of Ca²⁺, diglyceride, and phosphatidylserine did not change the phosphorylation pattern any further. In conclusion, it was shown that phorbol ester pretreatment of neonatal rat cardiomyocytes reduces microsomel GTP-gamma-S-stimulated Ptdlns(4,5)P ₂-specific phospholipase C activity, as estimated with exogenous substrate, and that in cardiomyocyte microsomes phorbol ester activates protein kinase C-induced 15 kD protein phosphorylation. The results indicate that phorbol ester may down-regulate -adrenoceptor mediated Ptdlns(4,5)P ₂ hydrolysis by activation of protein kinase C-induced 15 kD protein phosphorylation.List of abbreviations ATP Adenosine 5-Trphosphate - CSU Catalytic Subunit of cyclic AMP-dependent protein kinase - DG Diacylglycerol - DMSO Dimethylsulfoxide - DTT DL-dithiothreitol - EDTA Ethylenedinitrilotetraacetic Acid - EGTA Ethyleneglycol-0,0-bis(aminoethyl)-N,N,N,N,-tetraacetic acid - GTP-gamma-S Guanosine 5-O-(3-thiotriphosphate) - HPTLC High Performance Thin Layer Chromatography - InsP ₃ Inositol monophosphate - InsP ₂ Inositol bisphosphate - InsP ₃ Inositol trisphosphate - MES 2-Morpholinoethanesulfonic acid - MOPS 3-[N-Morpholino]Propanesulfonic acid - PAGE Polyacrylamide-gel Electrophoresis - PKC Protein Kinase C - PLase C Phospholipase C - PMA Phorbol 12-Myristate 13-Acetate - PMSF Phenylmethylsulfonyl Fluoride - PtdSer Phosphatidylserine - PtdIns Phosphatidyl inositol - PT Pertussis Toxin - Ptdlns(4)P Phosphatidylinositol 4-monophosphate - Ptdlns (4,5)PZ-Phosphatidylinositol4,5-bisphosphate - SDS-Sodium Dodecyl Sulfate Tris-Tris(hydroxymethyl) aminomethane     

Measurement of Hippocampal Levels of Cellular Second Messengers Following In Situ Freezing

Abstract: The in situ freezing technique has been widely used to fix labile metabolites and cellular second messengers in cerebral cortex. In this study, we isolated specific brain regions at 0°C from coronal sections of frozen heads following in situ brain freezing and measured regional concentrations of labile metabolites and cellular messengers. These levels in the cortex were compared with those in cortical punches obtained at freezing temperature (less than −40°C) from the same in situ frozen brains and those of cortex dissected from decapitated animals. In both isoflurane- and pentobarbital-anesthetized animals, we observed that the levels of lactate, free fatty acids, inositol 1,4,5-trisphosphate, and diacylglycerol, as well as the proportion of protein kinase C associated with the membrane fraction, were similar in cortical punches taken at freezing temperature and those dissected at 0°C. However, with animals decapitated at room temperature, cortical and hippocampal levels of lactate, free fatty acids, and inositol 1,4,5-trisphosphate and the proportion of membrane protein kinase C were significantly higher than those of corresponding brain regions isolated at 0°C from in situ frozen brains ( p < 0.05). These results indicate that dissection of cortex and hippocampus at 0°C following in situ freezing will eliminate decapitation-induced production of artifacts and changes in the levels of cellular second messengers such as inositol 1,4,5-trisphosphate, diacylglycerol, and protein kinase C. The present technique, used in conjunction with in situ freezing, will fix cellular second messengers and labile metabolites in several regions of brain and may facilitate accurate characterization of molecular and cellular mechanisms underlying CNS function.     

Epidermal growth factor stimulates the production of phosphatidylinositol monophosphate and the breakdown of polyphosphoinositides in A431 cells

The effects of epidermal growth factor (EGF) on the metabolism of phosphatidylinositol were examined using A431 cells labeled with either 32PO3(4)- or myo-[3H] inositol. EGF was found to increase the incorporation of phosphate into phosphatidic acid, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-diphosphate as early as 15 s after addition of hormone. These changes were found to be due to two effects of EGF on the phosphatidylinositol cycle. First, EGF stimulated the breakdown of phosphatidylinositol 4,5-diphosphate to diacylglycerol and an inositol triphosphate. In addition, EGF induced a rise in the levels of phosphatidylinositol 4-monophosphate. The EGF-dependent increases in both inositol triphosphate production and phosphatidylinositol 4-monophosphate levels were inhibited by pretreatment of the cells with 12-O-tetradecanoylphorbol-13-acetate. Treatment of the cells with pertussis toxin did not inhibit either of these responses. However, treatment of the cells with cholera toxin selectively abolished the ability of EGF to stimulate the rise in phosphatidylinositol monophosphate levels but did not alter the ability of the hormone to induce the breakdown of phosphatidylinositol diphosphate. The effects of cholera toxin were not mimicked by forskolin, cAMP analogs, or isobutyl-methylxanthine. These data demonstrate that EGF stimulates the production of inositol triphosphate. In addition, the findings are consistent with the hypothesis that EGF independently stimulates a phosphatidylinositol kinase. Based on the effects of cholera toxin and the inability of cyclic nucleotides to mimic this response, the effect of EGF on the phosphatidylinositol kinase may be mediated via a guanine nucleotide-binding protein that is not involved in cAMP production.     

Role of inositol trisphosphate as a second messenger in signal transduction processes: An essay

This essay attempts to summarize some of the best evidence for the role of inositol trisphosphate as a second messenger in signal transduction processes. The following aspects are addressed in the essay: (a) The synthesis of inositol trisphosphate and other inositol lipids, (b) Receptor-phosphatidylinositol bisphosphate phospholipase C coupling and the N-ras protooncogene, (c) Inositol trisphosphate and intracellular calcium, (d) Cell growth and oncogenes, (e) Receptors linked to the phosphatidylinositol cycle, (f) Phototransduction and (g) Interactions between inositol trisphosphate and other second messengers.Abbreviations Cyclic AMP Adenosine 3,5-cyclic monophosphate - Cyclic GMP Guanosine 3,5-cyclic monophosphate - DG sn, 1,2-Diacylglycerol - EGF Epidermal growth factor - GDP Guanosine diphosphate - GTP Guanosine triphosphate - IP Inositol 1-monophosphate - IP₂ Inositol 1,4-diphosphate - IP₃ Inositol 1,4,5-trisphosphate - PA Phosphatidic acid - PDGF Platelet-derived growth factor - PI Phosphatidylinositol - PIP Phosphatidylinositol 4-monophosphate - PIP₂ Phosphatidylinositol 4,5-bisphosphate - PIP₃ Phosphatidylinositol 3,4,5-trisphosphate - PLC Phospholipase C     

Combination and positional distribution of fatty acids in lipids from blue-green algae

The main glycerolipids (monogalactosyl-, digalactosyl-, sulphoquinovosyl diacylglycerol, phosphatidylglycerol) from five blue-green algae (Microcystis, Anabaena, Nostoc, Oscillatoria, Tolypothrix) were analyzed for fatty acid composition, occurrence of diglyceride species and positional distribution of fatty acids between thesn-1- andsn-2-position of glycerol. In contrast to eucaryotic plants biosynthetically closely related lipids (monogalactosyl-, digalactosyl-, trigalactosyl diacylglycerol) show nearly identical diglyceride moieties, whereas sulphoquinovosyl diacylglycerol and phosphatidylglycerol are separated from galactolipids by composition as well as occurrence of fatty acids. On the other hand the positional distribution of fatty acids in all lipids is controlled exclusively by chain length and not by degree of unsaturation with C₁₈-fatty acids at thesn-1- and C₁₆-fatty acids at thesn-2-position. These results show that in procaryotic organisms the diversity in diglyceride portions of lipids is reduced as compared to eucaryotic organisms, but nevertheless does exist.Abbreviations MGD, DGD, TGD, SQD monogalactosyl-, digalactosyl-, trigalactosyl-, sulphoquinovosyl diacylglycerol - PG phosphatidyl glycerol     

Mechanisms of action of calcium-mobilizing agonists: some variations on a young theme

It is now accepted that many hormones and neurotransmitters exert their effects through G protein-mediated activation of a phospholipase C, which breaks down phosphatidylinositol bisphosphate. This releases inositol trisphosphate, which mobilizes intracellular calcium, and diacylglycerol, which, in turn, activates protein kinase C. However, recent evidence indicates that other mechanisms are involved. In some cells, the increases in cytosolic calcium elicited within 1-2 s by high concentrations of agonists or at later times by low, physiological concentrations of agonists occur without any detectable changes in inositol phosphates and calcium mobilization, and result from the opening of plasma membrane channels that are permeable to Ca2+. This response appears to be mediated more directly by G proteins. These findings question the postulated roles of inositol phosphates and calcium mobilization in the stimulation of calcium influx. Measurements of the mass and fatty acid composition of the inositol phospholipids and of the diacylglycerol and phosphatidic acid generated by agonists in several cell types indicate that phosphatidylinositol bisphosphate is probably a minor source of these lipids. On the other hand, measurements of phosphatidylcholine, choline, and phosphocholine indicate that this phospholipid is a major source, and that its breakdown involves both phospholipase C and D. These findings indicate that phosphatidylcholine breakdown may be more important than phosphoinositide hydrolysis in the regulation of protein kinase C and perhaps other cell functions.     

Occurrence and functions of the phosphatidylinositol cycle in the myocardium

In the last decade a great deal of attention was awarded to a signal transduction pathway which is utilized primarily by Ca²⁺ mobilizing signal molecules and which involves the hydrolysis of a quantitatively minor phospholipid, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) by a PtdIns-specific phospholipase C (PLC). The evidence for the existence of receptor-mediated GTP binding protein-coupled PLC in myocardium and its possible functions are briefly summarized. The minireview is concentrated on the following aspects: 1) cellular localization and synthesis of polyphospho-PtdIns from PtdIns, 2) desensitization of the ₁-adrenergic agonist and endothelin-1 mediated PtdIns responses, 3) oscillatory Ca²⁺ transients initiated by Ptdlns(4,5)P₂ hydrolysis, 4) polyunsaturated fatty acids as constituents of polyphospho-PtdIns and of the protein kinase C activator 1,2-diacylglycerol (DAG), 5) source other than Ptdlns(4,5)P₂ contributing to the stimulated DAG, 6) role of the PtdIns pathway in cardiomyocyte growth and gene expression during the hypertrophic response. (Mol Cell Biochem116: 59–67, 1992)Abbreviations Phosphatidylinositol 4,5-bisphosphate PtdIns(4,5)P₂ - Phosphatidylinositol 4-monophosphate PtdIns(4)P₄ - Phosphatidylinositol PtdIns - Inositol 1,4,5-triphosphate Ins(1,4,5)P₃ - Inositol 1,3,4,5-tetrakisphosphate Ins(1,3,4,5)P₄ - Inositol 1-monophosphate Ins(1)P - Inositol 1,4-bisphosphate Ins(1,4)P₂ - Inositol Ins - Inositolphosphates InsP_n - Guanine 5'-triphosphate GTP - GTP binding protein G-protein - Phosphatidylinositolspecific phospholipase C PLC - Protein kinase C PKC - 1,2-Diacylglycerol DAG - Monoacylglycerol MAG - cytidyldiphoshate-diacylglycerol CDP-DAG - Sarcolemma SL - Sarcoplasmic reticulum SR - Stearic acid 18:0 - Polyunsaturated fatty acids PUFA - Arachidonic acid 20:4n-6 - Linoleic acid 18:2n-6 - Eicosapentaenoic acid 20:5n-3 - Docosahexaenoic acid 22:6n-3 - Phosphatidic acid PtdOH - Phospholipase D PLD - Phosphatidylcholine PtdChol     

Protein kinase C activation by cis-fatty acid in the absence of Ca2+ and phospholipids

Protein kinase C has been shown to be a phospholipid/Ca2+-dependent enzyme activated by diacylglycerol (Nishizuka, Y. (1984) Nature 308, 693-697; Nishizuka, Y. (1984) Science 225, 1365-1370). We have reported that unsaturated fatty acids (oleic acid and arachidonic acid) can activate protein kinase C independently of Ca2+ and phospholipid (Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193). This study shows that other cis-fatty acids such as linoleic acid also fully activate protein kinase C in the same manner. None of the saturated fatty acids (C:4 to C:18) nor the detergents (sodium dodecyl sulfate and Triton X-100) tested here were as effective as oleic acid. Unlike oleic acid, these detergents strongly inhibited protein kinase C activity induced by Ca2+/phosphatidylserine (PS) and diacylglycerol. Lowering the critical micelle concentration of oleic acid by increasing ionic strength also strongly inhibited oleic acid activation of protein kinase C activity. Dioleoylphosphatidylserine activated protein kinase C effectively (Ka = 7.2 microM). On the other hand, dimyristoylphosphatidylserine, which contains saturated fatty acids at both acyl positions, failed to activate protein kinase C even in the presence of Ca2+. These observations suggest that: protein kinase C activation by free fatty acid is specific to the cis-form and is not due to their detergent-like action, cis-fatty acid activation is due to the direct interaction of protein kinase C with the monomeric form of cis-fatty acids and not with the micelles of fatty acids, and cis-fatty acids at acyl positions in PS are also important for Ca2+/PS activation of protein kinase C.     

N-3 polyunsaturated fatty acid supplementation alters inositol phosphate metabolism and protein kinase C activity in adult porcine cardiac myocytes

Several mechanisms have been proposed to explain the anti-arrhythmic effects of n-3 polyunsaturated fatty acids. One mechanism is the effect of modifying cell membrane phospholipid and their subsequent effect on intracellular cell signaling via the second messengers, Ins(1,4,5)P(3) and diacylglycerol. Isolated cardiac myocytes from adult pig hearts were used to investigate the effect of n-3 polyunsaturated fatty acids, eicosapentaenoic acid and docosahexaenoic acid, on the inositol phosphate metabolism and protein kinase C activity. Adult porcine cardiac myocytes were grown in media supplemented with 400 μM arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid. After 24 hr, fatty acid analyses of total lipids by TLC in supplemented cells showed that eicosapentaenoic acid and docosahexaenoic acid were selectively incorporated into the phosphatidylinositol fraction. In the diacylglycerol fraction, there was a small incorporation of both eicosapentaenoic acid and docosahexaenoic acid but it was not significantly different from that of controls. To study the effect of membrane phospholipid modification on the phospholipase C mediated inositol lipid cycle, cardiac myocytes were labeled with 4μCi/ml myo-[2-(3)H]Ins for 48 hr. After stimulation with epinephrine and phenylephrine (alpha-receptor agonist) the water soluble [(3)H]Ins products were separated by chromatography on Dowex AG 1-X8 and measured by scintillation counting. After stimulation, the levels of [(3)H]Ins(1,4,5)P(3) and [(3)H]Ins(1,3,4,5)P(4) in eicosapentaenoic acid and docosahexaenoic acid supplemented myocytes were significantly reduced (P < 0.05) compared to arachidonic acid supplemented myocytes. Similarly, eicosapentaenoic acid and docosahexaenoic acid supplemented cells had reduced levels of protein kinase C activity after stimulation compared to arachidonic acid supplemented cells. From these experiments, it is evident that n-3 PUFA supplementation modulates intracellular cell signaling suggesting a possible anti-arrhythmic mechanism.     

U46619双向调节系膜细胞DNA合成的研究

Mene用血清或佛波醇肉豆寇乙酸(PMA)预失活化系膜细胞的PKC,可抑制U46619所致的IP合成及细胞内Ca~(2 )的升高,提示PKC预先活化后,可对再次活化PLC-PKC的物质起负反馈抑制作用。血清中含有血液凝固时血小板释放的血小板源性生长因子(PDGF),可活化PLC及PKC,可能对U46619的作用起负反馈抑制,使U46619促增殖作用丧失。第三,TXA_2可能直接或间接刺激前列腺素(PG)E_2或PGI_2的合成,而后两者有抑制细胞增殖的作用。不论何种机制参与,U46619这种抑制作用在病理上可能有积极意义:在正常状态下,体内肾小球系膜细胞为静息状态,肾小球合成的PG及TX极少,当肾小球产生炎症时,肾小球白细胞及血小板浸润增多,合成的PG、TX及PDGF增多,这时TX的升高可能有抑制PDGF所致细胞增殖的作用。 摘要 本实验用[~3H]TdR掺入法测定TXA_2类似物U46619对大鼠系膜细胞DNA合成的调节作用,测定系膜细胞合成的DAG及1,4,5-IP_3量,用PKC抑制剂Calphostin C预处理系膜细胞,观察其对U46619促增殖作用的影响。结果表明,U46619促进生长停滞的系膜细胞的DNA合成及IP_3的生成。本文首次证实U46619也促进DAG的生成并发现PKC抑制剂可抑制U46619的促增殖作用,提示U46619使生长停滞的系膜细胞的PLC活化,促进IP及DAG生成,进而激活PKC,促进系膜细胞DNA合成