Mammalian cell PtdIns (phosphatidylinositol) in vivo is enriched in the sn-1-stearoyl 2-arachidonoyl species, the physiological precursor of phosphatidylinositol 4,5-bisphosphate. Mechanisms regulating this specificity are unclear but are typically lost for cells in culture. We used ESI-MS (tandem electrospray ionization-mass spectrometry) to determine the molecular species of PtdIns synthesized by mouse tissues in vivo compared with cultured cells in vitro. After incorporation of deuteriated myo-d6-inositol over 3 h, endogenous and newly synthesized PtdIns and lysoPtdIns species were quantified from precursor scans of m/z 241 and m/z 247 respectively. PtdIns was synthesized as a wide range of species irrespective of the final membrane composition. Analyses of isotope enrichments argued against acyl remodelling as the major regulatory mechanism: composition of the lysoPtdIns pool under all conditions reflected that of either endogenous or newly synthesized PtdIns and was always at equilibrium. The kinetics of PtdIns synthesis, together with the prolonged time scale required for achieving final equilibrium compositions suggest that selective transport between membranes and/or hydrolysis of selected molecular species are the most probable mechanisms regulating compositions of PtdIns and, ultimately, phosphatidylinositol 4,5-bisphosphate.

Introduction

Mammalian cells in vivo maintain a remarkably consistent and simple molecular species composition of membrane PtdIns (phosphatidylinositol), dominated by a single species containing stearate (18:0) at the sn-1 and arachidonate (20:4n-6) at the sn-2 position [1]. In contrast, the dominant membrane phospholipids PtdCho (phosphatidylcholine) and phosphatidylethanolamine are much more complex, comprising in excess of 30–50 molecular species [2]. Evidence suggests that the specificity of PtdIns composition is functionally associated with the intracellular signalling functions of polyphosphoinositides. Therefore phosphatidylinositol 4,5-bisphosphate is synthesized from PtdIns and hydrolysed by PtdIns-specific phospholipase C to generate the second messengers DAG (diacylglycerol) and inositol trisphosphate [3] and the precise molecular species composition of DAG mobilized appears to be critical for activation of DAG-sensitive isoforms of protein kinase C [4].

The mechanisms whereby cells acquire and maintain this composition of PtdIns are not well understood and cells in culture tend to lose this specificity. Acyl remodelling reactions are integral to PtdCho synthesis de novo in many tissues [5] where selective lipase actions generate lyso-PtdCho intermediates that are then reacylated to generate equilibrium PtdCho compositions. Similar mechanisms at sn-1 and sn-2 positions of liver PtdIns have been proposed [6,7] but their molecular specificity in vivo has not been systematically investigated. Moreover, it is uncertain whether synthetic mechanisms identified in liver are generally applicable to other cell types.

Here we used ESI-MS (electrospray ionization-mass spectrometry), previously shown to be effective for analysing choline-d9 incorporation into newly synthesized PtdCho in vitro and in vivo [8,9], to define incorporation patterns of deuteriated myo-d6-inositol into molecular species of PtdIns and lysoPtdIns. Comparison of newly synthesized and endogenous PtdIns and lysoPtdIns molecular species compositions permitted calculation of relative incorporation rates of deuteriated label. Relative enrichments in turn allowed assessment of whether obligatory remodelling of all newly synthesized PtdIns via lysoPtdIns determined the pattern of PtdIns synthesis in mouse tissues in vivo and in a range of cultured mammalian cells in vitro.

Materials and methods

myo-d6-Inositol was obtained from C/D/N Isotopes Inc (Pointe-Claire, Quebec, Canada), organic solvents were from Fisher Scientific (Loughborough, Leics., U.K.) and aminopropyl BondElut sample preparation cartridges (100 mg) were obtained from Phenomenex (Macclesfield, Cheshire, U.K.). PtdIns internal standard was from Avanti Polar Lipids (Alabaster, AL, U.S.A.). Human IMR-32 neuroblastoma cells, Jurkat T-lymphoma cells and, CHO-K1 (where CHO stands for Chinese-hamster ovary) cells were cultured in 75 cm2 flasks in Dulbecco's modified Eagle's medium containing 25 mM Hepes and fetal bovine serum at 10% (5% for CHO-K1 cells). Subconfluent cells were labelled with myo-d6-inositol for up to 3 h as described elsewhere [10]. Black CB6 mice were injected intraperitoneally with 1 mg of myo-d6-inositol in 200 μl saline for 3 h before killing and tissue removal and storage at −20°C. After subsequent cell/tissue lipid extraction [11] and phospholipid class purification by solid-phase extraction [8], ESI-MS/MS of endogenous and newly synthesized PtdIns in the acidic phospholipid fraction was undertaken on a Micromass Ultima Quatro electrospray ionization tandem mass spectrometer by direct injection employing precursor scans of m/z 241 and m/z 247 as described previously [8] and lysoPtdIns species were incorporated into the analyses by extending the scans to encompass the range of masses from m/z 500 to 1000.

Results and discussion

Incorporation of myo-d6-inositol into mouse liver PtdIns

As anticipated, the endogenous composition of mouse tissue PtdIns molecular species was dominated by 18:0/20:4. However, the pattern of myo-d6-inositol incorporation into liver PtdIns and lysoPtdIns (Figure 1) revealed that newly synthesized PtdIns 3 h after intraperitoneal injection of the label was relatively enriched in the PtdIns16:0/20:4 molecular species (Figure 1c) compared with the endogenous, equilibrium composition (Figure 1d). Similar observations were noted for other mouse tissues. Together, these imply lack of enzymic selectivity for 18:0/20:4 species into PtdIns at the level of CDPdiglyceride incorporation. Moreover, enhanced turnover of PtdIns16:0/20:4 is suggested during the transition to the equilibrium composition, from comparison of the lysoPtdIns species in the same liver sample (Figures 1a and 1b), where newly acquired lysoPtdIns16:0 is relatively enriched. Importantly, in none of the time-course analyses of tissue or cultured cells did the deuterium enrichment in lysoPtdIns molecular species ever exceed that of the corresponding PtdIns pool as would be expected if all newly synthesized PtdIns passed through a mandatory lysoPtdIns remodelling process.

ESI-MS analyses of mouse liver PtdIns and lysoPtdIns compositions and synthesis

Figure 1
ESI-MS analyses of mouse liver PtdIns and lysoPtdIns compositions and synthesis

The acidic phospholipid fraction of a chloroform–methanol extract of mouse liver 3 h after i.p. myo-d6-inositol with selected molecular species indicated. (a) Precursor scan of m/z 247 fragment over mass range 525–725 showing newly synthesized lysoPtdIns species. (b) Precursor scan of m/z 241 fragment over mass range 525–725 showing endogenous lysoPtdIns species. (c) Precursor scan of m/z 247 over mass range 750–930 showing newly synthesized PtdIns species. (d) Precursor scan of m/z 241 fragment over mass range 750–930 showing endogenous PtdIns species.

Figure 1
ESI-MS analyses of mouse liver PtdIns and lysoPtdIns compositions and synthesis

The acidic phospholipid fraction of a chloroform–methanol extract of mouse liver 3 h after i.p. myo-d6-inositol with selected molecular species indicated. (a) Precursor scan of m/z 247 fragment over mass range 525–725 showing newly synthesized lysoPtdIns species. (b) Precursor scan of m/z 241 fragment over mass range 525–725 showing endogenous lysoPtdIns species. (c) Precursor scan of m/z 247 over mass range 750–930 showing newly synthesized PtdIns species. (d) Precursor scan of m/z 241 fragment over mass range 750–930 showing endogenous PtdIns species.

Incorporation of myo-d6-inositol into PtdIns of cells in culture

In contrast with mouse tissue PtdIns, the most striking observation from analyses of cultured cell PtdIns, whether endogenous or newly synthesized from myo-d6-inositol, was their relatively lower abundance of the 18:0/20:4 molecular species. This was seen, for example, in the composition of PtdIns from CHO-K1 cells (Figure 2), where molecular species containing monounsaturated acyl chains predominate, but was also true of Jurkat cells and (to a lesser extent) IMR-32 cells (results not shown). Ability to enhance the abundance of endogenous 18:0/20:4 species was retained to a small degree but never reflected the capacity of whole mouse tissues in vivo.

ESI-MS of PtdIns composition and synthesis in CHO-K1 cells

Figure 2
ESI-MS of PtdIns composition and synthesis in CHO-K1 cells

The acidic phospholipids fraction of a chloroform–methanol extract of CHO-K1 cells 3 h after incubation with myo-d6-inositol. (a) Precursor scan of m/z 247 fragment over mass range 820–920 showing newly synthesized PtdIns. (b) Precursor scan of m/z 241 fragment over mass range 820–920 showing endogenous PtdIns species.

Figure 2
ESI-MS of PtdIns composition and synthesis in CHO-K1 cells

The acidic phospholipids fraction of a chloroform–methanol extract of CHO-K1 cells 3 h after incubation with myo-d6-inositol. (a) Precursor scan of m/z 247 fragment over mass range 820–920 showing newly synthesized PtdIns. (b) Precursor scan of m/z 241 fragment over mass range 820–920 showing endogenous PtdIns species.

Accordingly, from thorough analyses of incorporation patterns over time we conclude the following:

  1. Synthesis of PtdIns de novo at the ER does not involve rapid early transit of all molecular species through lyso-PtdIns as an obligatory step.

  2. Our results are consistent with a slower, selective, enhanced removal of molecular species which do not comprise 18:0/20:4 moieties perhaps linked to downstream intracellular transport phenomena.

  3. Proportionately lower PtdIns18:0/20:4 abundance in cultured cell membranes does not appear to preclude adequate growth, division and (presumably) signalling.

Research Colloquia: Research Colloquia at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by M. Bouvier (Montreal, Canada), G. Milligan (Glasgow, U.K.), V. O'Donnell (Cardiff, U.K.), M. Brand (MRC-Dunn Human Nutrition Unit, Cambridge, U.K.), M. Schweizer (Heriot-Watt University, Edinburgh, U.K.), R. Insall (Birmingham, U.K.), A. Ridley (Ludwig Institute for Cancer Research, London, U.K.) and M. Sutcliffe (Leicester, U.K.). The first eight papers featured in this Section were presented as a part of the GPCR Regulation and Signalling Research Colloquium, incorporating the GPCR–Ion Channel Interactions Pfizer-Sponsored Research Colloquium.

Abbreviations

     
  • DAG

    diacylglycerol

  •  
  • ESI-MS

    electrospray ionization-mass spectrometry

  •  
  • PtdCho

    phosphatidylcholine

  •  
  • PtdIns

    phosphatidylinositol

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