Skip Navigation

Cardiovascular Research 1998 40(1):182-190; doi:10.1016/S0008-6363(98)00113-8
© 1998 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Eskildsen-Helmond, Y. E.G.
Right arrow Articles by Lamers, J. M.J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Eskildsen-Helmond, Y. E.G.
Right arrow Articles by Lamers, J. M.J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1998, European Society of Cardiology

Phospholipid source and molecular species composition of 1,2-diacylglycerol in agonist-stimulated rat cardiomyocytes

Yvonne E.G. Eskildsen-Helmonda, Daniela Hahnelb, Ulrike Reinhardtb, Dick H.W. Dekkersa, Bernd Engelmannb and Jos M.J. Lamersa,*

aDepartment of Biochemistry, Cardiovascular Research Institute COEUR, Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, P.O.Box 1738, 3000 DR Rotterdam, The Netherlands
bPhysiologisches Institut der Universität München, Pettenkoferstrasse 12, 80336 München, Germany

* Corresponding author. Tel: +31 (10) 408 7335; Fax: +31 (10) 436 0615; E-mail: lamers@bc1.fgg.eur.nl

Received 2 October 1997; accepted 19 March 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The aim was to investigate the consequences of simultaneous stimulation of phospholipase C and D by agonists for the molecular species composition of 1,2-diacylglycerol and phospholipids in cardiomyocytes. Methods: Serum-free cultured neonatal rat cardiomyocytes were stimulated by endothelin-1, phenylephrine or phorbolester. The molecular species of 1,2-diacylglycerol (in mol%) and those derived from phosphatidylcholine and phosphatidylinositol were analyzed by high-performance liquid chromatography and their absolute total concentration (nmol per dish) by gas–liquid chromatography. Phospholipids were labelled with [14C]glycerol or double-labelled with [14C]16:0 and [3H]20:4n6 for measurements of respectively, the amount of or relative rate of label incorporation into 1,2-diacylglycerol. Results: The major molecular species of 1,2-diacylglycerol in unstimulated cells was found to be 18:0/20:4 (57 mol%). The same species was observed predominantly in phosphatidylinositol (73 mol% compared to 11 mol% in phosphatidylcholine). A significant decrease (about 10 mol%) was found for the 18:0/20:4 species of 1,2-diacylglycerol during stimulation (10–40 min) with endothelin-1 or phorbolester, but not phenylephrine. The results of the double-labelling experiments were consistent with the latter finding: the ratio [3H]20:4 over [14C]16:0 in 1,2-diacylglycerol decreased from 1.70 in the control to 1.40 during 10-min endothelin-1 or phorbolester stimulation, but not during phenylephrine stimulation. The [14C]glycerol incorporation into 1,2-diacylglycerol remained relatively constant under agonist-stimulated conditions as did the total concentration of 1,2-diacylglycerol. Conclusions: 1,2-Diacylglycerol present in unstimulated cardiomyocytes is likely derived from phosphatidylinositol. During stimulation with endothelin-1 and phorbolester, but not phenylephrine, phosphatidylcholine becomes an increasingly important source for 1,2-diacylglycerol due to sustained activation of phospholipase D. The 1,2-diacylglycerol level remains relatively constant during agonist stimulation which strongly indicates that particular molecular species of 1,2-diacylglycerol more than its total concentration determine the activation of protein kinase C isoenzymes.

KEYWORDS ET-1, endothelin-1; PMA, phorbol 12-myristate 13-acetate; PHE, phenylephrine; AngII, angiotensin II; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; Ins(1,4,5)P3, inositol-1,4,5-trisphosphate; 1,2-DAG, 1,2-diacylglycerol; TAG, triacylglycerol; PKC, protein kinase C; PLC, PLD and PLA2, respectively phospholipase C, D and A2; PtdChol, PtdIns, PtdEtn, PtdSer, respectively phosphatidylcholine, -inositol, -ethanolamine, -serine; (HP)TLC, (High-performance) thin-layer chromatography; HPLC and GLC, respectively, high-performance and gas–liquid chromatography; DPH, diphenyl-1,3,5 hexatrien; DNBC, 3,5-dinitrobenzoylchloride; stearic acid, 18:0; arachidonic acid, 20:4n6; palmitic acid, 16:0.


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Many stimuli such as the {alpha}1-adrenergic agonist phenylephrine (PHE) and endothelin-1 (ET-1) have been shown to be positive inotropic and to induce hypertrophy and changes in phenotype in myocardium. [1–3]. Studies have revealed that these agonists stimulate GTP-binding protein-coupled receptors linked to phospholipase C-β (PLC-β) leading to hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) into inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) and the lipid second messenger 1,2-diacylglycerol (1,2-DAG). More recently, PLC-{gamma} interacting with tyrosine-phosphorylated receptors has been proposed, for angiotensin II, to be an important downstream effector as well [4, 5]. At present, it is generally assumed that activation by 1,2-DAG (and Ca2+) of distinct protein kinase C (PKC)-isozymes results in activation of various sets of downstream targets such as raf-kinase, MAP-kinase and myofibrillar component proteins such as troponin I, C and C-protein which lead to the (patho)physiological responses [2, 6, 7].

Previously, we reported that PtdIns and PtdIns(4,5)P2 in cultured neonatal rat cardiomyocytes have a fatty acid composition that is very different from phosphatidylserine (PtdSer) and phosphatidylcholine (PtdChol) [8, 9]. Thus the molecular species of 1,2-DAG formed by PLC-β, PLC-{gamma} and/or PLD from these phospholipids will not be the same. 1,2-DAG, which is considered to be the major lipid activator of PKC might exert this action by its increased total level or by altered competition between particular molecular species [10]. However, at present there is no information on the molecular species composition of 1,2-DAG in cardiomyocytes before and after agonist stimulation.

Recently, we and others [11, 12]obtained evidence that during ET-1 stimulation of serum-free cultured rat cardiomyocytes, the PKC activator 1,2-DAG might not only be derived from receptor-dependent hydrolysis of (PtdIns(4,5)P2) through immediate action of PLC-β detectable by measurement of [3H]inositolphosphates ([3H]InsPn) but after a lag-phase of about 5–10 min from PtdChol by the action of phospholipase D (PLD), the latter detectable by [3H]choline formation [11]. We could confirm this action of PLD by showing in parallel occurrence, [14C]phosphatidylgroup transfer from endogenous [14C]16:0-labelled PtdChol to exogenous ethanol. [3H]Choline formation appeared not to be due to the action of PtdChol-directed PLC, because phospho-[3H]choline was not found to be increased. However, the action of PC-PLC cannot be totally excluded on the basis of the unaffected phosphocholine concentration, because phosphocholine is likely degraded to choline. No phosphocholine phosphatase inhibitors are presently available to block this pathway. The previous results indicated that 1,2-DAG production from PtdChol-specific PLD is slow in onset and sustained, in contrast to the rapid and more transient production of 1,2-DAG from PtdIns(4,5)P2-specific PLC-β [11]. On the other hand, phosphatidic acid (PtdOH), the first product of PLD action is implicated as an intracellular mediator of several cellular processes, such as mitogenesis and actin assembly. This product can be rapidly converted to 1,2-DAG by PtdOH phosphohydrolyse or to lyso-PtdOH by phospholipase A2. We also obtained evidence that during ET-1 stimulation cross-talk between PLC-β and PLD occurs through PKC-{varepsilon} activation. We proved activation of both PLC and PLD by ET-1, but not by PHE or phorbol 12-myristate 13-acetate (PMA). PHE only stimulates PLC-β and PMA, as expected only PLD, PMA, but not PHE, induced a cellular translocation/activation of PKC-{varepsilon}, -{delta} as well as -{alpha} [12], confirming the results of Bogoyevitch et al. [13]and Clerk et al. [14].

Studies in cell-types other than cardiomyocytes (e.g. fibroblasts) indicate that 1,2-DAG formed in the early (15 s–1 min) transient phase of receptor-stimulation predominantly shows fatty acids present in the PtdIns pool (stearic acid 18:0, arachidonic acid 20:4n6) whereas the later phase shows more saturated fatty acids such as palmitic acid 16:0 typically found in PtdChol ([15, 16]and reviews [17, 18]). However, Hermans et al. [19]showed in pancreatic acini that in the later phase, 1,2-DAG remains derived from both PtdIns and from PtdChol.

Therefore, the aim of the present study was to investigate the changes in the molecular species of 1,2-DAG and PtdChol and PtdIns of neonatal rat cardiomyocytes that are stimulated by ET-1, PHE or PMA, possibly more relevant for activation of PKC than the absolute amount of 1,2-DAG. High-performance liquid chromatography (HPLC) was used for the separation of the different molecular species in free 1,2-DAG and 1,2-DAG obtained from PtdChol and PtdIns exogenously treated by PLC purified from respectively, C. Welchii and B. Cereus. Moreover, the relative incorporation of [14C]16:0 (a 10-fold higher percentage of 16:0 is present in PtdChol compared to PtdIns) (compare also [8]) and [3H]20:4n6 (a 2-fold higher percentage of 20:4 is present in PtdIns compared to PtdChol) (compare also [8]) into 1,2-DAG was followed during agonist stimulation.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1985).

2.1 Materials
Integrid culture dishes (154 cm2) and 20-cm2 dishes were from Falcon (Becton-Dickinson, Oxnard, CA, USA) and culture dishes (4-well Multidish, 1.8 cm2 per well) were from Nunc (Roskilde, DK), while the culture media DMEM and M199 were both obtained from Gibco (UK); fetal calf serum, horse serum, penicillin/streptomycin, trypsin and ET-1 were obtained from Boehringer Mannheim (Germany). [1-14C]Palmitic acid (16:0) (57 mCi/mmol) and [3H]arachidonic acid (20:4n6) (210 Ci/mmol) were from Dupont NEN products (Boston MA, USA). [U-14C]Glycerol (156 mCi/mmol) was from Amersham International PLC (Amersham, UK). PMA, PHE, DPH (diphenyl-1,3,5 hexatrien), difluorescein, phospholipase C (Perfingens, C. Welchii), phospholipase C (B. Cereus, PtdIns-specific), DNBC (3,5-dinitrobenzoylchloride), BF3 and the internal standard 21:0 were all obtained from Sigma (St. Louis, MO, USA). High-performance thin-layer chromatography (HPTLC) plates and thin-layer chromatography (TLC) plates (Kieselgel 60) were obtained from Merck (Darmstadt, Germany). Instagel-plus was from Canberra Packard Benelux N.V./S.A. (Groningen, The Netherlands).

2.2 Culture of neonatal rat ventricular myocytes
Primary cultures of neonatal ventricular myocytes were prepared from hearts from 1–2 day old Wistar rats as described before [20]using a single preplating step (surface area of approximately 10 cm2 per rat heart) to further increase cardiomyocyte to noncardiomyocyte ratio. The cultures contain 90–95% myocytes as routinely assessed by microscopical observations of cells and by usage of the periodic-acid Schiff (PAS) staining method to distinguish myocytes from nonmyocytes. Cardiomyocytes were seeded in 1.8, 20 or 154-cm2 wells/dishes (for respectively labelling experiments, GLC and HPLC analysis) at 150 to 175x103 cells per cm2 giving a confluent monolayer of spontaneously contracting cells after 24 h. Cardiomyocytes were kept in 5% CO2 at 37°C in complete growth medium consisting of DMEM/M199 (4:1), 25 mM HEPES supplemented with 5% fetal calf serum and 5% horse serum, 100 U penicillin per ml and 100 µg streptomycin per ml for the first 24 h. Growth medium was renewed 24 h after seeding with serum-free growth medium and every 48 h thereafter. Experiments were routinely performed at 5 to 6 days after plating of the cells.

2.3 Molecular species of free 1,2-DAG and 1,2-DAG derived from PtdIns and PtdChol
Cardiomyocytes were either used as control or stimulated with ET-1 (10–8 M), PHE (10–5 M) or PMA (10–6 M). Lipids (from 2 to 3 154 cm2 culture dishes) were extracted by a modified method of Bligh and Dyer [21](using chloroform containing 50 mg/l butylated hydroxytoluene). Throughout the procedure, samples were kept under N2 and if required overnight, at –20°C. 1,2-DAG was separated from other lipids by thin-layer chromatography (TLC) using hexane–diethylether–formic acid (30:70:2 v/v/v) as solvent. The phospholipids remaining at the origin were scraped off again and three times reextracted in chloroform–methanol (2:1) and then separated by two-dimensional TLC according to Bütikofer et al. [22]with running solvents, for the first dimension, chloroform–methanol–NH4OH–H2O (90:74:12:8 v/v/v/v) and for the second dimension, chloroform–methanol–acetone–acetic acid–H2O (80:30:30:24:16 v/v/v/v/v). Plates were sprayed with 1% K-oxalate in MeOH/H2O (2:3 v/v), dried and activated at 90°C for 1 h before separation. Spots were then visualized by spraying with DPH (0.03% in chloroform), scraped off and lipids were extracted from the silica by the method of Arvidson [23]. The phospholipids were subsequently dispersed by sonication in 2 ml of either 30 mM K2HPO4, 30 mM boric acid (pH 7.0; for PtdOH), or 30 mM Tris, 30 mM boric acid (pH 7.4; for PtdIns), or 50 mM Tris, 5mM CaCl2, 30 mM boric acid (pH 7.4; for PtdChol). PLC from B. cereus [P7147, for PtdOH (12 U) and P8804, PtdIns-specific (0.2 U)] and phospholipase C from C. Welchii [P4039, for PtdChol (5 U)] was added followed by 5 ml of diethylether. The mixtures were incubated under argon overnight in a shaking water bath (37°C). After completion of the PLC-catalyzed formation of 1,2-DAG, which was routinely checked by subjecting an aliquot of the ether phase to one-dimensional TLC using diethylether/hexane (3:2, v/v) as a running solvent, the ether phase was collected. The lower phase was extracted again with diethylether and combined ether phases were blown to dryness with nitrogen and subsequently dried under vacuum overnight. After addition of 25 mg of DNBC, the mixture was again dried overnight under vacuum. Derivatization of the 1,2-DAGs was achieved by addition of 1 ml of dry pyridine and subsequent incubation for 15 min at 64°C. The extract was chilled on ice for 15 s and 3 ml of ice-cold water was added together with 2 ml of hexane. The water phase was extracted twice with 2 ml of hexane and the combined hexane phases were dried under nitrogen. The extract was redissolved in 2 ml hexane and successively washed with 2 ml of NaCl (1 M) and 2 ml of water. The 1,2-DAG subclass was separated by HPTLC using hexane–diethylether (7:3, v/v) as a running solvent. The spots were visualized by dichlorofluorescein (0.001% in 2 mM NaOH) and extracted with diethylether. The extract was then washed with distilled water in order to remove the water-soluble degradation products. After evaporation of the ether phase the extract was dissolved in 100 µl of acetonitrile–isopropanol (8:2, v/v) and subjected to reversed-phase HPLC using an ODS Hypersil column (5 µm C18, 200x2.1 mm, Hewlett Packard, Böblingen, Germany). The column was eluted with acetonitrile–isopropanol (8:2, v/v) at a flow-rate of 0.25 ml/min. The elution profile of the single DNBC-diacylglycerols was monitored at 254 nm by means of a Hewlett Packard 1050 UV detector. Molecular species analysis was performed essentially as described. [24]. The molecular species of the derivatized 1,2-DAGs were identified on the basis of the retention times obtained with standard dinitrobenzoyl–diacylglycerols and/or the relative retention times reported by Takamura et al. [25]. An example of an HPLC separation of PtdChol is shown in Fig. 1.


Figure 1
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 HPLC separation pattern of DNBC-1,2-DAGs representative for PLC-treated PtdChol that were extracted from cardiomyocytes stimulated for 40 min by ET-1. The identity of numbered peaks is: (1) 18:1/22:6n3; (2) 16:0/22:6n3; (3) 16:0/20:4n6; (4) 16:0/18:2n6; (5) 18:0/20:4n6; (6) 18:0/22:5n6; (7) 18:1/18:1; (8) 16:0/18:1+18:0/18:2n6; (9) 16:0/16:0; (10) 18:0/18:1; (11) 18:0/16:0. The DNBC-1,2-DAGs were measured at 254 nm.

 
2.4 Fatty acid composition of PtdIns and PtdChol
For gas–liquid chromatography (GLC) analysis, phospholipids were transmethyl esterified using BF3 as originally described by Morrison and Smith [26]and as used by us in other studies [8, 27]. For the gas chromatographic separation of the fatty acid methylesters, a CP9000 capillary column chromatograph (Chrompack, Middelburg, The Netherlands), equipped with a CP-Sil 88-coated fused-silica capillary column (Chrompack, WCOT 50 mx0.25 mm, 0.2-µm film), was used. The separated peaks were identified and quantified on the basis of the retention times as compared to those of known standard amounts [27]. The absolute total concentrations (nmol per dish) of 1,2-DAG, PtdChol and PtdIns were also measured by GLC.

2.5 Double-label experiments
Cardiomyocytes were labelled with both [14C])16:0 (0.5 µCi/ml) and [3H]20:4n6 (0.1 µCi/ml) for 24 h in complete serum-free growth medium supplemented with 0.04% fat-free Bovine Serum Albumin (BSA). Cells were extensively washed (three times) with radioactive-free medium (containing 0.2% fat-free BSA), twice the amount of volume as during labelling. Cells were then preincubated (15 min, 37°C) in medium containing 130 mM NaCl; 4.7 mM KCl; 1.3 mM CaCl2; 0.44 mM NaH2PO4; 1.1 mM MgSO4; 20 mM NaHCO3; 0.2% glucose; 10 mM HEPES; pH 7.4, 37°C and stimulated with either ET-1, PHE or PMA. After termination, lipids were extracted as described before [28]. TLC plates were pretreated by impregnation with 1% boric acid in CH3OH and activated. CHCl3:CH3COCH3 (94:4 v/v) was used as the solvent system. The labelled lipids were visualized by means of autoradiography and the 1,2-DAG spots were scraped off and counted by liquid-scintillation counting.

2.6 1,2-DAG level by [3H]glycerol incorporation
Cardiomyocytes were labelled with 2 µCi (U-[14C])glycerol per ml for 24 h in complete serum-free growth medium. Cells were extensively washed (three times) with radioactive-free medium, twice the amount of volume as during labelling. Cells were then preincubated (15 min, 37°C) with incubation buffer (130 mM NaCl; 4.7 mM KCl; 1.3 mM CaCl2; 0.44 mM NaH2PO4; 1.1 mM MgSO4; 20 mM NaHCO3; 0.2% glucose; 10 mM HEPES; pH 7.4, 37°C) and stimulated with either ET-1, PHE or PMA. After incubation the [14C]glycerol-containing lipids were extracted, TLC separated and the 1,2-DAG were counted as described before [28].

2.7 Statistics
The statistical significance of any of the observed effects was evaluated by the Students t-test and significance was set at probability less than 0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Molecular species of free 1,2-DAG and 1,2-DAG derived from PtdIns and PtdChol in control and agonist-stimulated cardiomyocytes
PtdIns(4,5)P2, of which small amounts are endogenously formed from PtdIns by the action of PtdIns kinases, and the bulk of phospholipids PtdChol, are the endogenous substrates of PLC-β and PLD, respectively. Therefore, we treated PtdIns and PtdChol extracted from cardiomyocytes in vitro with PLC purified from C. Welchii (for PtdChol) and from B. cereus (specific for PtdIns) to obtain their constitutive 1,2-DAG molecular species. Although the PtdIns(4,5)P2 content in cardiomyocytes is too low for accurate determination of its molecular species, it is the direct substrate for PLC-β [9]. Table 1 shows the mol% of the major molecular species of PtdIns and PtdChol of cells unstimulated and stimulated by ET-1, PHE and PMA during 40 min. No significant agonist-dependent differences were found over this time period. The 18:1/18:1 (26 mol%) appeared to be a major molecular species in PtdChol, whereas PtdIns was mainly composed of 18:0/20:4n6 (73 mol%).


View this table:
[in this window]
[in a new window]

  		Table 1 Molecular species composition (mol%) of DNBC-1,2-DAG derived from PtdChol and PtdIns of control and agonist stimulated myocytes

 
In order to obtain an estimate for the absolute amounts, we measured the total concentration of fatty acid methylesters obtained from PtdChol, PtdIns and 1,2-DAG in control and ET-1-stimulated cardiomyocytes by GLC. No significant changes due to ET-1 stimulation in the absolute concentration (nmol per dish) of phospholipid and 1,2-DAG occurred (Table 2).


View this table:
[in this window]
[in a new window]

  		Table 2 Absolute concentration (nmol/dish) of PtdChol, PtdIns and 1,2-DAG of control and ET-1 stimulated cardiomyocytes

 
The concentration of 1,2-DAG in the cardiomyocytes is very low compared to that of the phospholipids independent of whether the cells were stimulated by agonists or not (Table 2). Therefore, in order to measure the molecular species composition, it was necessary to combine at least two to three 154-cm2 culture dishes for accurate HPLC analysis. The results are shown in Table 3. Firstly, it can be noted that 18:0/20:4n6 is the major 1,2-DAG molecular species (about 57 mol%) present in unstimulated control cardiomyocytes. This indicates that under control conditions, basal turnover of PtdIns and PLC-β activity contributes mostly to the 1,2-DAG pool in cardiomyocytes. When the cardiomyocytes were stimulated with ET-1 and PMA for 10 min, a decrease of about 10 mol% was found in the relative content of 18:0/20:4n6. In ET-1-stimulated cells, this decrease was only accompanied by a significant increase in relative content of 18:0/18:1, and in PMA-stimulated cells in the relative content of 18:1/18:1. Previously, we reported that PHE is a strong stimulator of PLC-β, however in comparison to ET-1 and PMA, it had only small effects on PLD [12]. In agreement with the latter finding is that 10-min PHE stimulation did not alter the molecular species composition of 1,2-DAG (Table 3). When the cardiomyocytes were stimulated with ET-1, PHE or PMA for 40 min, the mol% of 18:0/20:4 did not decrease further. Comparison of Tables 1 and 3Go may raise the suggestion that the tendency to the nonsignificant decrease of 18:0/20:4 in PtdChol accounts for the reduction of 18:0/20:4 in 1,2-DAG. This can, however, almost be excluded on the basis of the relative and absolute 1,2-DAG content (Table 2). The total concentration of PtdChol and PtdIns is respectively 100- and 10-fold higher than that of free 1,2-DAG (Table 2). We have not measured the molecular species composition of 1,2-DAG earlier than 10 min in order to trace the early action of PLC-β as shown in other cell types [15–18]. On the other hand, the unusual high initial 18:0/20:4 content (Table 3) of 1,2-DAG in unstimulated cardiomyocytes complicates detection of the relative increase of this molecular species.


View this table:
[in this window]
[in a new window]

  		Table 3 Molecular species composition (mol%) of DNBC-1,2-DAG of control and agonist stimulated myocytes

 
PtdOH is the first product formed by PLD-catalyzed PtdChol degradation. Therefore, we also tried to determine its 1,2-DAG molecular species content. Unlike 1,2-DAG, the PtdOH content was too low for accurate analysis on HPLC. Its fatty acid composition could, however, be determined on GLC and was found to have closest resemblance to that of PtdChol (Table 4). On the other hand, 1,2-DAG and PtdOH are generally believed to be interconvertible through the action of PtdOH-hydrolase and 1,2-DAG kinase [12, 29, 30], but no evidence for this proposal was obtained.


View this table:
[in this window]
[in a new window]

  		Table 4 Fatty acid composition (mol%) of PtdChol, PtdIns and PtdOH and TAG of control and ET-1 stimulated cardiomyocytes

 
3.2 Fatty acid composition of PtdIns and PtdChol
Small, but nonsignificant, changes due to agonist stimulation in the 1,2-DAG molecular species composition of PtdChol and PtdIns were observed (Table 1). For this reason the fatty acid composition was also determined by GLC. On the basis of the HPLC results, on one hand a slightly different pattern of total fatty acid composition of PtdChol and PtdIns should have been expected on GLC, but more importantly, the results on GLC prove once again that no changes of fatty acid patterns of PtdChol and PtdIns occur during agonist stimulation. From the data on fatty acid composition, it can also be derived that 16:0 is a major component of PtdChol (25 mol%) in contrast to its minimal presence in PtdIns (4 mol%). On the other hand, PtdIns contains 2-fold more (mol%) of the major component 20:4n6 than PtdChol. This information was used for the design of the double-label experiments (see below).

3.3 Incorporation of [14C]16:0 and [3H]20:4 into 1,2-DAG
ET-1 and PMA stimulation of cardiomyocytes caused a small, but significant decrease in mol% of 18:0/20:4n6 in 1,2-DAG after 10-min stimulation, which was accompanied by an increase of mol% 18:0/18:1 and 18:1/18:1, respectively (Table 3). Experiments were designed based upon the analyzed fatty acid composition of PtdChol and PtdIns (Table 4), not showing significant changes between 40-min control and ET-1 stimulation, providing additional proof for the shift from PLC-β-induced hydrolysis of PtdIns to PLD-induced hydrolysis of PtdChol as became apparent by HPLC analysis of 1,2-DAG. For this purpose, cardiomyocytes were labelled with [14C]16:0 and [3H]20:4. Subsequently, the cardiomyocytes were stimulated by agonists and the relative incorporation of [14C] and [3H] into free 1,2-DAG was counted. After 10- and 40-min stimulation with either ET-1 or PMA, a significant decrease of the ratio [3H]20:4/[14C]16:0 was found (Fig. 2). The ratio changed from 1.70 to 1.40 for both ET-1 and PMA after 10 min and from 1.70 to 1.56 for PMA and from 1.70 to 1.39 for ET-1 after 40 min. The absolute incorporation of [3H]20:4 and [14C]16:0 into 1,2-DAG both increased 9% and 32% respectively, after 10-min ET-1 and by 16% and 40% after 10-min PMA stimulation, which is likely due to agonist-induced increase of 1,2-DAG content (see also Fig. 3). In agreement with the results on molecular species composition, there were no significant changes in labelling ratio of 1,2-DAG in control-incubated cardiomyocytes and those stimulated by PHE.


Figure 2
View larger version (39K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Incorporation of [3H]20:4 relative to [14C]16:0 in 1,2-DAG in control and agonist-stimulated cardiomyocytes. Data are presented as means±SEM of three separate experiments with triplicate measurements for each condition. Datapoints marked with * are significantly different from zero time and those with + are significantly different from the corresponding control (p<0.05).

 

Figure 3
View larger version (27K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 [14C]Glycerol content of 1,2-DAG in control and agonist-stimulated cardiomyocytes. The [14C]glycerol content was standardized on the basis of the total [14C]glycerol incorporation (in water-soluble and -insoluble fraction) to correct for variation in number of cells extracted as done before [28]. Results are presented as means±SEM of 4 experiments. Points marked with * are significantly different from the corresponding control (p<0.05).

 
3.4 1,2-DAG concentration ([14C]glycerol incorporation)
Previously, we demonstrated by [3H]glycerol-labelling of serum-grown cardiomyocytes, that the [3H]1,2-DAG level raised not more than 1.2-fold after 15-min stimulation of cells by ET-1 and PHE [28]. We repeated these measurements for serum-free cultured cells, except that this time [14C]glycerol was used. Again, only a small rise in the [14C]1,2-DAG after ET-1 stimulation was observed, which disappeared after 40-min stimulation (Fig. 3). These results are consistent with the GLC analysis (Table 2). PMA stimulation also did not result in a clear increase of [14C]1,2-DAG. These results together with the previous [12]indicate that relative changes in concentration of particular molecular species of 1,2-DAG, have more importance in the activation of PKC isoenzymes than its overall concentration.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The results of this study provide for the first time, evidence that during ET-1- and PMA-, but not PHE-stimulation of cardiomyocytes, PtdChol slowly becomes a significantly contributing source for formation of 1,2-DAG through sustained activation of PLD. This finding is consistent with our previous data on InsPn-formation (PLC-β activity measurements) and choline-formation (PLD activity measurements) during agonist stimulation [12]. Although the rate of formation of labelled-InsPn and choline does not have to parallel that of 1,2-DAG when PLC-β and PLD are activated, the fact that PtdChol becomes a significant source of 1,2-DAG proves that a part of 1,2-DAG arises from the dephosphorylation of PLD-generated PtdOH by PtdOH phosphohydrolase [31]. Another, though unexpected finding in this study, is that the molecular species composition of 1,2-DAG in unstimulated cells closely resembles that of PtdIns. Furthermore, no significant elevation of the total concentration of 1,2-DAG was observed during prolonged ET-1 and PMA stimulation, for ET-1 measured by both [3H]glycerol incorporation and GLC of fatty acid methylesters. It was previously shown by Western blotting that under these conditions (ET-1 and PMA stimulation) several PKC isozymes (such as {epsilon}, {alpha} and {delta}) become translocated/activated [12–14]. Therefore, the question was becoming increasingly important whether elevation of the total 1,2-DAG content and/or change in particular molecular species of 1,2-DAG determines the activation of PKC-isozymes. On the other hand, it cannot be excluded that 1,2-DAG formed from TAG and phospholipids is distinctly compartmentalized, that elevated levels of second messenger molecular species of 1,2-DAG cannot be detected on top of the pool(s) of 1,2-DAG derived from basal TAG and phospholipid turnover. In one experiment, we also analyzed the fatty acid composition of TAG (Table 4), which showed a pattern different from what would be expected on the basis of the molecular species composition of 1,2-DAG in unstimulated cells.

In contrast to some other reports, neither amounts of 1-alk-1-enyl-2-acyl-glycerophospholipids nor of 1-alkyl-2-acyl-glycerophospholipids in PtdChol were found, although they can easily be separated from 1,2-DAG during the preparation of DNBC-derivatives [19]. Post et al. [32]could detect small amounts of alkyl-acyl-glycero-P-choline in serum-grown neonatal rat cardiomyocytes. In the present study, however, the neonatal cardiomyocytes were kept in serum-free medium for a period of 48 h prior to the experiments. The reason for choosing these culture conditions is that, in contrast to, in the presence of serum, PHE, ET-1 and PMA induce hypertrophy. Serum addition in itself produces hypertrophy of cardiomyocytes, because it contains growth factors and fatty acids which may also influence phospholipid turnover. Hermans et al. [19]found 1-alk-1-enyl-2-acyl in PtdChol of these species in pancreatic acini using the methodology for 1,2-DAG separation and identification as used in this study. Thus, it appears that the analysis used must have been sensitive enough to detect these species if they were present, and that other factors (such as long-term presence of serum) are likely the cause for the absence of alkyl-acyl or alkenyl-acyl species.

We are not the first to show subtle time-dependent changes in the endogenous 1,2-DAG molecular species with only certain polyunsaturated fatty acid containing species showing significant agonist-stimulated decrease [15, 17, 19]. Studies of {alpha}-thrombin-stimulated IIC9 chinese hamster embryo fibroblasts [15], bombesin-stimulated Swiss 3T3 mouse fibroblasts [17]and bradykinin-stimulated human fibroblasts [16]support the idea that PtdIns(4,5)P2 could be the source of 1,2-DAG at early stimulation times (15 s–1 min), but PtdChol at all times. The changes in 1,2-DAG species observed in the aforementioned cell types and in the present study demonstrate not only that there may be differences in phospholipase activity or phospholipid substrate with time of agonist stimulation, but also differences in cellular responses (e.g. activation/translocation of PKC isoenzymes). Although until now there is limited information concerning the specificity of molecular 1,2-DAG species in activating individual PKC isoforms, it was hypothesized by Pettitt and Wakelam [17]that distinct changes in 1,2-DAG molecular species patterns may relate to a distinct pattern of differential activation of PKC isoforms and protein phosphorylation. In this view it is important to note that it was shown by us and others [12–14]that during ET-1 stimulation of neonatal rat cardiomyocytes PKC-{varepsilon} becomes rapidly translocated/activated, whereas no translocation/activation was found during PHE stimulation. Thus, the present results which also show no effect of PHE on 1,2-DAG molecular species are indicating a critical role of the pattern of changes in 1,2-DAG molecular species for PKC isoenzyme activation.

The double-label experiments designed to find support for the conclusions from the data on molecular species composition of 1,2-DAG demonstrated indeed that less [14C]20:4n6 compared to [3H]16:0 is incorporated into 1,2-DAG during ET-1 and PMA, but not PHE stimulation. These results are consistent with the increased contribution of PtdChol to formation of 1,2-DAG after prolonged stimulation, because 6-fold more 16:0 is found to be present in PtdChol compared to PtdIns and 1.5-fold less of 20:4n6 is present in PtdChol compared to PtdIns (Table 4). Moreover, PMA was definitely proven to uniquely activate PLD [12]. On the other hand, PHE only stimulates PLC-β [12], which is in accordance with the finding of no changes in the [14C]20:4n6/[3H]16:0 incorporation ratio.

In the [14C]glycerol-labelling experiments, for the estimation of concentration changes in 1,2-DAG during agonist stimulation, only small elevations of 1,2-DAG in the cells during ET-1 and PMA stimulation were found. Also this finding provides evidence that the increase of cellular 1,2-DAG level is less important in the transduction of receptor-mediated signals to PKC. However, it should be noted that 1,2-DAG may have functions apart from stimulating PKC isoenzymes. For example 1,2-DAG can become a source for free 20:4n6 which is the main substrate for cyclo- and lipoxygenase to form prostaglandins and leukotrienes, respectively [33].

From the results it can be concluded that 1,2-DAG present in unstimulated neonatal rat cardiomyocytes is likely derived from PtdIns(4,5)P2. During ET-1 and PMA, but not PHE stimulation, PtdChol becomes an increasingly important source for 1,2-DAG due to only early PLC-β and sustained-PLD activation [12]. Unlike that of 1,2-DAG, the molecular species composition of PtdChol and PtdIns remained constant during agonist stimulation. The 1,2-DAG level only rises slightly during agonist stimulation, whereas PKC isoenzymes have been shown to become translocated/activated under these conditions. The latter result provides strong evidence that particular molecular species of 1,2-DAG, more than its overall concentration, determine the activation of PKC isoenzymes.

Time for primary review 35 days


   Acknowledgements
 
This study was supported by grant nr 900-516-146 from the Netherlands Organization for Scientific Research (NWO) and by the Wilhelm-Sander Stiftung to B.E.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Chien K.R., Knowlton K.U., Zhu H., et al. Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response. FASEB J (1991) 5:3037–3046.[Abstract]
  2. De Jonge H.W., Van Heugten H.A.A., Lamers J.M.J. Signal transduction by the phosphatidylinositol cycle in myocardium. J Mol Cell Cardiol (1995) 27:93–106.[Web of Science][Medline]
  3. Van Heugten H.A.A., Eskildsen-Helmond Y.E.G., De Jonge H.W., et al. Phosphoinositide-generated messengers in cardiac signal transduction. Mol Cell Biochem (1996) 157:5–14.[Web of Science][Medline]
  4. Pawson T., Gish G.D. SH2 and SH3 domains: from structure to function. Cell (1992) 71:359–362.[CrossRef][Web of Science][Medline]
  5. Marrero M.B., Schieffer B., Ma H., et al. Ang II-induced tyrosine phosphorylation stimulates phospholipase C-gamma1 and Cl-channels in mesangial cells. Am J Physiol (1996) 270:C1834–1842.[Web of Science][Medline]
  6. Bogoyevitch M.A., Marshall C.J., Sugden P.H. Hypertrophic agonists stimulate the activities of the protein kinase c-Raf and A-Raf in cultured ventricular myocytes. J Biol Chem (1995) 270:26303–26310.[Abstract/Free Full Text]
  7. Bezstarosti K., Soei L.K., Verdouw P.D., et al. Phosphorylation by protein kinase C and the responsiveness of Mg2+-ATPase to Ca2+ of myofibrils isolated from stunned and nonstunned porcine myocardium. Mol Cell Biochem (1997) 000:211–218.
  8. Lamers J.M.J., Dekkers D.H.W., De Jong N., et al. Modification of fatty acid composition of the phospholipids of cultured rat ventricular myocytes and the rate of phosphatidylinositol-4,5-bisphosphate hydrolysis. J Mol Cell Cardiol (1992) 24:605–618.[CrossRef][Web of Science][Medline]
  9. Lamers J.M.J., Dekkers D.H.W., Mesaeli N., et al. Myocardial phosphoinositides do not share the same fatty acid profile. Biochem Biophys Res Commun (1993) 191:487–494.[CrossRef][Web of Science][Medline]
  10. Nishizuka Y. Intracellular signalling by hydrolysis of phospholipids and activation of protein kinase C. Science (1992) 258:607–614.[Abstract/Free Full Text]
  11. Clerk A., Sugden P.H. Regulation of phospholipase C and D in rat ventricular myocytes: stimulation by endothelin-1, bradykinin and phenylephrine. J Mol Cell Cardiol (1997) 29:1593–1604.[CrossRef][Web of Science][Medline]
  12. Eskildsen-Helmond Y.E.G., Bezstarosti K., Dekkers D.H.W., et al. Cross-talk between receptor-mediated phospholipase C-β and D via protein kinase C as an intracellular signal possibly leading to hypertrophy in serum-free cultured cardiomyocytes. J Mol Cell Cardiol (1997) 29:2545–2559.[CrossRef][Web of Science][Medline]
  13. Bogoyevitch M.A., Parker P.J., Sugden P.H. Characterization of protein kinase C isotype expression in adult rat heart. Circ Res (1993) 72:757–767.[Abstract/Free Full Text]
  14. Clerk A., Bogoyevitch M.A., Anderson M.B., et al. Differential activation of protein kinase C isoforms by endothelin-1, and phenylephrine and subsequent stimulation of p42 and p44 mitogen-activated protein kinases in ventricular myocytes cultured from neonatal rat hearts. J Biol Chem (1994) 269:32848–32857.[Abstract/Free Full Text]
  15. Pessin M.S., Raben D.M. Molecular species analysis of 1,2-diglycerides stimulated by {alpha}-thrombin in cultured fibroblasts. J Biol Chem (1989) 264:8729–8738.[Abstract/Free Full Text]
  16. Van Blitterswijk W.J., Hilkman H., De Widt J., et al. Phospholipid metabolism in bradykinin-stimulated human fibroblasts. J Biol Chem (1991) 266:10344–10350.[Abstract/Free Full Text]
  17. Pettitt T.R., Wakelam M.J.O. Bombesin stimulates distinct time-dependent changes in the sn-1,2-diradylglycerol molecular species profile from Swiss 3T3 fibroblasts analyses by 3,5-dinitrobenzoyl derivatization and HPLC separation. Biochem J (1993) 289:487–495.[Web of Science][Medline]
  18. Exton J.H. Phosphatidylcholine breakdown and signal transduction. Biochim Biophys Acta (1994) 1212:26–42.[Medline]
  19. Hermans S.W.G., Engelmann B., Reinhardt U., et al. Diradylglycerol formation in cholecystokinin-stimulated rabbit pancreatic acini: assessment of precursor phospholipids by means of molecular species analysis. Eur J Biochem (1996) 235(1, 2):73–81.[Web of Science][Medline]
  20. Van Heugten H.A.A., De Jonge H.W., Bezstarosti K., et al. Calcium and the endothelin-1 and {alpha}1-adrenergic stimulated phosphatidylinositol cycle in cultured rat cardiomyocytes. J Mol Cell Cardiol (1994) 26:1081–1093.[CrossRef][Web of Science][Medline]
  21. Bligh E.G., Dyer W.J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol (1959) 37:911–918.[Medline]
  22. Bütikofer P., Wen Lin Z., Kuypers F.A., et al. Chlorpromazine inhibits vesiculation, alters phosphoinositide turnover and changes deformability of ATP-depleted RBCs. Blood (1989) 73:1699–1704.[Abstract/Free Full Text]
  23. Arvidson G.A.E. Structural and metabolic heterogeneity of rat liver glycerophosphatides. Eur J Biochem (1968) 4:478–486.[Web of Science][Medline]
  24. Engelmann B., Bräutigam C., Kulschar R., et al. Reversible reduction of phospholipid bound arachidonic acid after low density lipoprotein apheresis. Evidence for rapid incorporation of plasmalogen phosphatidylethanolamine into the red blood cell membrane. Biochim Biophys Acta (1994) 1196:154–164.[Medline]
  25. Takamura H., Narita H., Park H.J., et al. The modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim Biophys Acta (1984) 779:89–137.[Medline]
  26. Morrison W.R., Smith L.M. Preparation of fatty methylesters and dimethylacetals from lipids with boron fluoride-methanol. J Lipid Res (1964) 5:600–608.[Abstract]
  27. Montfoort A., Van der Werf L., Hartog J.M., et al. The influence of fish oil diet and norepinephrine treatment on fatty acid composition of rat heart phospholipids and the positional fatty acid distribution in phosphatidylethanolamine. Basic Res Cardiol (1986) 81:289–302.[CrossRef][Web of Science][Medline]
  28. De Jonge H.W., Van Heugten H.A.A., Bezstarosti K., et al. Distinct {alpha}1-adrenergic agonist- and endothelin-1-evoked phosphoinositide cycle responses in cultured neonatal rat cardiomyocytes. Biochem Biophys Res Commun (1994) 203:422–429.[CrossRef][Web of Science][Medline]
  29. El Bawab S., Macovschi O., Thevenon C., et al. Contribution of phosphoinositides and phosphatidylcholines to the production of phosphatidic acid upon concanavalin A stimulation of rat thymocytes. J Lipid Res (1996) 37:2098–2108.[Abstract]
  30. Wakelam M.J.O., Briscoe C.P., Stewart A., et al. Phosphatidylcholine hydrolysis: a source of multiple lipid messenger molecules. Biochem Soc Trans (1993) 21:874–877.[Medline]
  31. Cook S.J., Wakelam M.J. Epidermal growth factor increases sn-1,2-diacylglycerol levels and activates phospholipase D-catalysed phosphatidylcholine breakdown in Swiss 3T3 cells in the absence of inositol–lipid hydrolysis. Biochem J (1992) 285:247–253.[Web of Science][Medline]
  32. Post J.A., Verkleij A.J., Roelofsen B., et al. Plasmalogen content and distribution in the sarcolemma of cultured neonatal rat myocytes. FEBS Lett (1988) 240:78–82.[CrossRef][Web of Science][Medline]
  33. Van Bilsen M., Van der Vusse G.J. Phospholipase-A2-dependent signalling in the heart. Cardiovasc Res (1995) 30:518–529.[Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Exp. Biol. Med.Home page
M. Hlavackova, J. Neckar, J. Jezkova, P. Balkova, B. Stankova, O. Novakova, F. Kolar, and F. Novak
Dietary Polyunsaturated Fatty Acids Alter Myocardial Protein Kinase C Expression and Affect Cardioprotection Induced by Chronic Hypoxia
Experimental Biology and Medicine, June 1, 2007; 232(6): 823 - 832.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
T. Asai, K. Okumura, R. Takahashi, H. Matsui, Y. Numaguchi, H. Murakami, R. Murakami, and T. Murohara
Combined therapy with PPAR{alpha} agonist and L-carnitine rescues lipotoxic cardiomyopathy due to systemic carnitine deficiency
Cardiovasc Res, June 1, 2006; 70(3): 566 - 577.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
R. Takahashi, K. Okumura, T. Asai, T. Hirai, H. Murakami, R. Murakami, Y. Numaguchi, H. Matsui, M. Ito, and T. Murohara
Dietary fish oil attenuates cardiac hypertrophy in lipotoxic cardiomyopathy due to systemic carnitine deficiency
Cardiovasc Res, November 1, 2005; 68(2): 213 - 223.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
R. Takahashi, K. Okumura, H. Matsui, Y. Saburi, H. Kamiya, K. Matsubara, T. Asai, M. Ito, and T. Murohara
Impact of {alpha}-tocopherol on cardiac hypertrophy due to energy metabolism disorder: the involvement of 1,2-diacylglycerol
Cardiovasc Res, June 1, 2003; 58(3): 565 - 574.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
Y. Saburi, K. Okumura, H. Matsui, K. Hayashi, H. Kamiya, R. Takahashi, K. Matsubara, and M. Ito
Changes in distinct species of 1,2-diacylglycerol in cardiac hypertrophy due to energy metabolic disorder
Cardiovasc Res, January 1, 2003; 57(1): 92 - 100.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
J. C. Lenz, H. P. Reusch, N. Albrecht, G. Schultz, and M. Schaefer
Ca2+-controlled competitive diacylglycerol binding of protein kinase C isoenzymes in living cells
J. Cell Biol., October 28, 2002; 159(2): 291 - 302.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Eskildsen-Helmond, Y. E.G.
Right arrow Articles by Lamers, J. M.J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Eskildsen-Helmond, Y. E.G.
Right arrow Articles by Lamers, J. M.J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?