A HPLC-fluorescence detection method for determination of phosphatidic acid phosphohydrolase activity: Application in human myocardium

A HPLC-fluorescence detection method for determination of phosphatidic acid phosphohydrolase activity: Application in human myocardium

Available online at www.sciencedirect.com ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 374 (2008) 291–297 www.elsevier.com/locate/yabio A HPLC-flu...

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Available online at www.sciencedirect.com

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 374 (2008) 291–297 www.elsevier.com/locate/yabio

A HPLC-fluorescence detection method for determination of phosphatidic acid phosphohydrolase activity: Application in human myocardium Christof Burgdorf a,*, Antje Prey a, Gert Richardt b, Thomas Kurz a a

Medizinische Klinik II, Universita¨tsklinikum Schleswig-Holstein, 23538 Lu¨beck, Germany b Herzzentrum Segeberger Kliniken, 23795 Bad Segeberg, Germany Received 28 August 2007 Available online 4 November 2007

Abstract Phosphatidic acid phosphohydrolase (PAP) catalyzes the dephosphorylation of phosphatidic acid (PA) to diacylglycerol, the second messenger responsible for activation of protein kinase C. Despite the crucial role of PAP lipid signaling, there are no data on PAP signaling function in the human heart. Here we present a nonradioactive assay for the investigation of PAP activity in human myocardium using a fluorescent derivative of PA, 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphate (BODIPY-PA), as substrate in an in vitro PAP-catalyzed reaction. Unreacted BODIPY-PA was resolved from the PAP products by a binary gradient HPLC system and BODIPY-diacylglycerol was detected by fluorimetry. The reaction proceeded at a linear rate for up to 60 min and increased linearly with increasing amounts of cardiac protein in a range of 0.25 to 8.0 lg. This assay proved to be sensitive for accurate quantitation of total PAP activity, PAP-1 activity, and PAP-2 activity in human atrial tissue and right ventricular endomyocardial biopsies. Total PAP activity was approximately fourfold higher in ventricular myocardium than in atrial tissue. There was negligible PAP-1 activity in atrial myocardium compared with ventricular myocardium, indicating regional differences in activities and distribution pattern of PAP-1 and PAP-2 in the human heart. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Phosphatidic acid phosphohydrolase; Diacylglycerol; Phospholipase D; Phospholipase A2; Protein kinase C; Signal transduction; Human myocardium

Phosphatidic acid phosphohydrolase (PAP)1 functions in phospholipid metabolism by catalyzing the conversion of phosphatidic acid (PA) to diacylglycerol (DAG) [1]. PA has been implicated as a lipid second messenger in activation of mTOR, recruitment of Raf-1 to the membrane, vesicle trafficking, cytoskeletal rearrangements, and regula*

Corresponding author. Fax: +49 451 500 2363. E-mail address: [email protected] (C. Burgdorf). 1 Abbreviations used: PAP, phosphatidic acid phosphohydrolase; PA, phosphatidic acid; DAG, diacylglycerol; NEM, N-ethylmaleimide; BODIPY-PA, 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphate; BODIPY-fatty acid, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid; MAFP, methyl-arachidonyl-fluorophosphonate; DETAPAC, diethylenetriaminepentaacetic acid. 0003-2697/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.10.039

tion of cardiac function by its ability to stimulate sarcolemmal calcium-related transport systems [2–4]. Thus, the action of PAP is thought to attenuate the signaling function of PA [5]. On the other hand, by generating DAG, PAP represents an alternative and quantitatively more important pathway for DAG formation in comparison with the hydrolysis of phosphatidylinositol bisphosphate by phospholipase C [6–8]. PAP-catalyzed DAG formation has been shown to activate protein kinase C isozymes [9,10] that phosphorylate several myocardial proteins implicated in myocardial hypertrophy [11]. Two classes of mammalian PAP enzymes have been identified based on subcellular localization and biochemical properties [12]. PAP-1 is regulated by translocation from cytosol to endoplasmic reticulum. Its catalytic activity is Mg2+ dependent and sensitive

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to sulfhydryl-reactive reagents such as N-ethylmaleimide (NEM). Recently, Han and coworkers demonstrated that the single lipin gene ortholog in Saccharomyces cerevisiae, PAH1, exhibits PAP-1 activity that has a key role in de novo lipid synthesis [13]. Furthermore, recombinant human lipin-1 expressed in Escherichia coli also shows PAP-1 activity [13]. A second PAP activity, PAP-2, was characterized in mammalian cells based on a lack of requirement for bivalent cations and insensitivity to inhibition of NEM. PAP-2 is primarily localized to the plasma membrane and plays an important role in modulating the signaling functions of PA generated by phosphatidylcholine-specific phospholipase D [7,12]. Alterations of myocardial PAP activity have been shown in diabetic rats and in acute ischemic and chronic infarcted rat hearts [14–18]. There are, however, no data on PAP signaling function in the human heart. This prompted us to develop a nonradioactive assay for the investigation of PAP activity in human myocardium. Previous in vitro assays for PAP activity have used either 14C-labeled or 32 P-radiolabeled PA as substrate and have assessed radioactive DAG by thin-layer chromatography [19–21]. These methods, however, require time-consuming and potentially hazardous steps such as scraping of visualized lipid spots from the thin-layer chromatography plates prior to radioactivity counting. As an alternative, here we report the development of a highly sensitive nonradioactive assay for quantitation of myocardial PAP activity, its validation in rat myocardium, and its application in human myocardial tissue samples.

rinsed in ice-cold saline, and frozen in liquid nitrogen. Frozen myocardial tissue was pulverized and homogenized in ice-cold lysis buffer (5 mM Tris and 2 mM EDTA, pH 7.5). The homogenate was centrifuged at 1000g for 10 min to remove cell debris and nuclei, and the supernatant was centrifuged at 100,000g for 30 min. The resulting pellet (membrane fraction) was resuspended in incubation buffer (20 mM Tris, 2 mM EDTA, and 1 mM EGTA, pH 7.5) and stored at 80 °C prior to assay. Human atrial tissue was obtained during routine cardiac surgery from patients undergoing coronary artery bypass grafting or valve replacement after informed consent had been obtained. A specimen of 100 to 200 mg was harvested from the tip of the right atrial appendage during venous cannulation for extracorporal circulation. Frozen tissue from three or four specimens was pooled, pulverized in liquid nitrogen, and homogenized in ice-cold buffer in a ratio of 2 ml buffer per 100 mg tissue. The homogenate was centrifuged at 1000g and 4 °C for 10 min, and the supernatant was frozen in liquid nitrogen and stored at –80 °C in small aliquots. Right ventricular endomyocardial biopsies were taken from patients undergoing cardiac catheterization for suspected myocarditis, who turned out to have normal left ventricular function at cardiac catheterization and no signs of inflammatory disease in the biopsies. Biopsies (2–5 mg) were homogenized as described above. Protein concentration in the myocardial preparations was determined according to Bradford [22].

Materials and methods

An aliquot of BODIPY-PA was dissolved in 10 mM Triton X-100, giving a final concentration of 100 lM BODIPY-PA (starting reagent). The reaction was initiated by the addition of 5 ll starting reagent to 45 ll incubation buffer (20 mM Tris, 5 mM MgCl2, 2 mM EDTA, and 1 mM EGTA, pH 7.5). PAP-2 activity was assayed in the absence of MgCl2 in incubation buffer containing 5 mM NEM. Reactions were terminated by the addition of 300 ll icecold methanol and 150 ll chloroform containing 0.005% (w/v) butylated hydroxytoluene. After the addition of 150 ll chloroform and 220 ll of 150 mM KCl, samples were centrifuged for 3 min at 13,000g. The lower phase was evaporated to dryness under an N2 stream. The residue was dissolved in 100 ll hexane–isopropanol–acetic acid (50:50:1, v/v/v) plus triethylamine (0.08%, v/v) prior to chromatography. The final concentration of the phospholipase A2 inhibitor MAFP in the enzyme assay was 1 lM.

This study was carried out in accordance with the ethical standards as formulated in the Helsinki Declaration of 1975 (revised 1983). The experiments on animals were in accordance with the EU (86/609/EEC) and National Institutes of Health guidelines, and the study protocol was approved by an institutional ethics committee. Reagents 2-(4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphate (BODIPY-PA) and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-pentanoic acid (BODIPY-fatty acid) were obtained from Molecular Probes. Methyl-arachidonyl-fluorophosphonate (MAFP) was obtained from Calbiochem. DAG kinase from Escherichia coli was obtained from Sigma–Aldrich. All other chemicals were of analytical grade. Preparation of myocardial tissue Seven-week-old male Wistar rats (200–250 g body weight, Charles River) were anesthetized with 150 mg/kg thiopental sodium (Byk Gulden) intraperitoneally. The thorax was opened, and the heart was immediately excised,

PAP activity assay

Conversion of DAG to PA by exogenous DAG kinase Fractions collected after separation of myocardial lipids by HPLC were evaporated under N2. Dried lipids were solubilized in 100 ll reaction buffer (1 mM cardiolipin, 0.2 mM diethylenetriaminepentaacetic acid [DETAPAC], 50 mM octyl-ß-glucopyranoside, 50 mM NaCl, 50 mM imidazole–HCl [pH 6.6], 12.5 mM MgCl2, 1 mM EDTA, 2 mM

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dithiothreitol, 1 mM ATP, and 5 lg DAG kinase [activity 6.6 U/mg protein]). After mixing, the reaction was allowed to proceed at 25 °C for 30 min. Lipids were extracted as described above. HPLC separation and quantification Lipid separation was accomplished by normal phase HPLC on a LiChrospher 100 Diol (5 lm) 250  4-mm column (Merck). The analyses were carried out by binary gradient elution with mobile phase solvents of hexane– isopropanol–acetic acid (82:17:1, v/v/v, solvent A) and isopropanol–water–acetic acid (85:14:1, v/v/v, solvent B) at a flow rate of 1 ml/min. Triethylamine (0.08%, v/v) was added to the solvents. The gradient profile started at 5% for solvent B and was increased to 40% B in 25 min, after which it was increased to 100% B in 5 min. Aliquots (50 ll) of lipid samples were injected onto the column, which was kept at 45 °C at all runs. Reequilibration time of the HPLC column prior to injection of a new sample was 30 min. A MIDAS automatic sample injector (Spark) was used to enable continuous sample processing. The HPLC system was interfaced with a fluorescence detector (Kontron SFM 25) set at excitation and emission wavelengths of 475 and 515 nm, respectively. The detector signal was recorded and integrated by a personal computer and a software program (Andromeda, Techlab). BODIPY-PA was routinely used as standard for calculation of the fluorescent products because the fluorescent properties of the different BODIPY lipid products are very similar [23]. When the standard BODIPY-PA was subjected to HPLC and fluorimetric detection, a linear standard curve exhibiting a correlation coefficient of 0.999 was obtained. Results Identification of fluorescent hydrolysis products in cardiac membranes Fig. 1 shows the HPLC separation of lipids extracted from rat cardiac membranes that were incubated using BODIPY-PA as fluorescent substrate. Four major fluorescent peaks could be detected (Fig. 1A). The peak at 20 min corresponds to BODIPY-PA, the substrate for the enzymatic reaction. The peak at 12 min could not be further identified and presents a contaminant fluorescent substance present in the BODIPY-PA standard that was not reacted in the presence of myocardial membranes. Peaks at 3 and 6 min were identified as DAG and free fatty acid, respectively. When cardiac membranes were incubated with BODIPY-PA in the presence of MAFP (1 lM), a selective inhibitor of both calcium-dependent and calcium-independent phospholipase A2 [24], the peak at 6 min was nearly completely suppressed (Fig. 1A). This peak could be further evidenced as fluorescent free fatty acid by its identical retention time to chromatographically resolved BODIPYfatty acid standard (Fig. 1B). Thus, in cardiac membranes,

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BODIPY-PA is used as substrate for fatty acid formation by endogenous phospholipase A2 activity. MAFP was used routinely in the assay buffer to prevent interference of DAG formation by concomitant phospholipase A2 activity. The peak at 3 min could be clearly identified as DAG by back-conversion to PA in a reaction using exogenous DAG kinase. Collected fractions of chromatographically separated lipids of myocardial samples after PAP assay reaction were subjected to enzymatic conversion by DAG kinase. As shown in Fig. 1C, BODIPY-PA was formed by DAG kinase from fluorescent lipids in the collected fraction containing the chromatographic peak at 3 min. This back-conversion was complete when the enzymatic reaction was allowed to proceed for 120 min (data not shown). In contrast, no formation of fluorescent PA was observed in the fraction containing the chromatographic peak at 6 min (Fig. 1D). Fig. 2 represents a typical chromatogram of the incubation products of rat myocardial membranes in the presence of the phospholipase A2 inhibitor MAFP (1 lM). Effect of incubation time and membrane protein concentration on cardiac PAP activity The generation of fluorescent DAG proceeded at a linear rate (correlation coefficient of 0.985) for up to 30 min (Fig. 3). The formation of product was linear (correlation coefficient of 0.994) for concentrations of membrane protein between 0.25 and 8 lg. Total PAP, PAP-1, and PAP-2 activities in rat myocardium Total PAP activity (specific activity relative to protein) was approximately 14-fold enriched in rat myocardial membranes as compared with myocardial homogenate (Fig. 4). Rat myocardial homogenate contained approximately 70% NEM-insensitive PAP-2 activity and 30% NEM-sensitive and Mg2+-dependent PAP-1 activity. This distribution contrasts with that in crude myocardial membranes that displayed predominantly PAP-2 activity (>90%), whereas there was relatively little PAP-1 activity (Fig. 4). The apparent Km value for total PAP in myocardial homogenate was 24 lM, which is in close agreement with previously reported values of 5 and 11 lM for the rat microsomal and mitochondrial enzymes, respectively [19]. Total PAP, PAP-1, and PAP-2 activities in human atrial and ventricular myocardium Total PAP activity (specific activity relative to protein) was approximately fourfold higher in human ventricular myocardium as compared with atrial tissue (Fig. 5). There was negligible PAP-1 activity in human atrial myocardium (< 1% of total PAP activity). Likewise, in human ventricular myocardium, PAP-2 activity was still the predominant class of PAP activity. PAP-1 activity, however, amounted

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Fig. 1. (A) Chromatogram of the incubation products of rat myocardial membranes. Membranes (10 lg protein) were incubated for 30 min at 37 °C, pH 7.5, using BODIPY-PA as substrate as described in Materials and Methods. Incubations were carried out either in the absence or in the presence of the phospholipase A2 inhibitor MAFP (1 lM). (B) Chromatogram of BODIPY-fatty acid directly subjected to HPLC. (C,D) Collected fractions of chromatographically separated lipids corresponding to the peaks at 3 min (C) and 6 min (D) were subjected to enzymatic conversion by exogenous DAG kinase as described in Materials and Methods. BODIPY-PA was formed by DAG kinase from fluorescent lipids in the collected fraction containing the chromatographic peak at 3 min, whereas no formation was observed in the fraction containing the chromatographic peak at 6 min.

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Fig. 2. Representative chromatogram of the incubation products of rat myocardial membranes. Myocardial membranes (2 lg protein) were incubated for 30 min at 37 °C, pH 7.5, using BODIPY-PA as substrate as described in Materials and Methods. Incubations were carried out in the presence of the phospholipase A2 inhibitor MAFP (1 lM).

to approximately 20% of total PAP activity, suggesting differences in the regional distribution pattern of PAP-1 and PAP-2 activities in the human heart.

Discussion We have developed a nonradioactive assay for cardiac PAP activity in vitro. A fluorescent PA derivative was used as substrate for PAP-catalyzed reaction in myocardial sam-

ples. Unreacted PA was resolved from the PAP products by HPLC, and DAG was detected by fluorimetry. This assay proved to be sensitive enough for accurate detection and quantitation of myocardial PAP activity. The detection limit of this assay was found to be approximately 1 pmol BODIPY-DAG in the HPLC system. The reaction proceeded at a linear rate for up to 30 min and increased linearly with increasing amounts of membrane protein in a range of 0.25 to 8 lg. Thus, this PAP assay can be used over a fairly broad range of incubation times and myocardial protein concentrations depending on the experimental needs and the availability of myocardial tissue. The initial assay conditions revealed the profound formation of fluorescent fatty acid in myocardial samples. From this finding, we assumed the presence of myocardial phospholipase A2 activity to be responsible for fatty acid formation because the fluorescent derivative of PA used as substrate in the assay was labeled at the fatty acid residue located in the sn-2 position of the glycerol backbone. This assumption was confirmed by nearly complete suppression of fluorescent fatty acid formation when the assay was carried out in the presence of MAFP, a selective inhibitor of both calcium-dependent and calcium-independent phospholipase A2 [24]. Therefore, MAFP was included in the PAP assay system to inhibit phospholipase A2 activity that competes with PAP for the fluorescent substrate by conversion of PA to lyso-PA. This is of particular importance in disease states of up-regulated phospholipase A2

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Fig. 3. Effect of incubation time (left panel) and membrane protein concentration (right panel) on PAP activity of rat cardiac membranes. The reaction was carried out for the indicated times with 2 lg membrane protein (left panel) or for the indicated membrane protein concentrations at an incubation time of 15 min (right panel). The formation of BODIPY-DAG in the membranes was quantitated by fluorimetric detection after separation of total lipids by HPLC as described in Materials and Methods. Product formation was calculated using BODIPY-PA as standard. Values shown are means ± SD obtained from three separate experiments.

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Fig. 4. PAP activity of rat myocardial membranes (left panel) and rat myocardial homogenate (right panel). Rat myocardial membranes (2 lg membrane protein) or rat myocardial homogenate (3 lg myocardial protein) was incubated for 15 min either in reaction buffer containing 5 mM MgCl2 for determination of total PAP activity (open bars) or in reaction buffer without MgCl2 containing 5 mM NEM for determination of PAP-2 activity. PAP-1 activity was calculated as the difference of total PAP and PAP-2 activity. The formation of DAG was quantitated by fluorimetric detection after separation of total lipids by HPLC as described in Materials and Methods. Product formation was calculated using BODIPY-PA as standard. Bars represent means ± SEM obtained from six experiments.

activity (e.g., congestive heart failure [25]) displaying an exclusive or relative selectivity for PA as substrate [26–28] that otherwise might interfere with the PAP analysis. Furthermore, it implies that the fluorescent PA used in the assay is a preferred substrate for myocardial phospholipase A 2. Initial biochemical characterization of PAP activity in rat liver demonstrated that the enzyme activity could be separated into two components [12]. PAP-1 was defined as a cytosolic/endoplasmic reticulum-localized activity that was both dependent on Mg2+ and inactivated by NEM. Conversely, an Mg2+-independent NEM-insensitive membrane-associated activity was classified as PAP-2. Based on these biochemical properties, we assayed PAP-1 and

PAP-2 activities in rat myocardium. In agreement with the cellular distribution of PAP-1 and PAP-2 shown previously in rat cardiac myocytes [29], we found both activities in rat myocardial homogenate and more than 90% membrane-associated PAP-2 activity. Human myocardial PAP activity was fourfold (atrial myocardium) and 20-fold (ventricular myocardium) higher than in rat heart, indicating profound species differences in enzyme activity. Moreover, there are regional differences in cardiac PAP activity, as indicated by fourfold higher PAP activity in human ventricular myocardium as compared with atrial tissue. Notably, essentially no PAP-1 activity was observed in human atrial myocardium, whereas ventricular myocardium displayed PAP-1 activity. This may reflect the role of PAP-1

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Phosphatidic acid phosphohydrolase activity / C. Burgdorf et al. / Anal. Biochem. 374 (2008) 291–297 125 PAP activity (nmol DAG/h/mg protein)

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Fig. 5. PAP activity of human atrial tissue (left panel) and human right ventricular endomyocardial biopsies (right panel). Human atrial tissue (2 lg myocardial protein) or ventricular endomyocardial biopsies (2 lg myocardial protein) were incubated for 15 min either in reaction buffer containing 5 mM MgCl2 for determination of total PAP activity (open bars) or in reaction buffer without MgCl2 containing 5 mM NEM for determination of PAP-2 activity. PAP-1 activity was calculated as the difference of total PAP and PAP-2 activity. The formation of DAG was quantitated by fluorimetric detection after separation of total lipids by HPLC as described in Materials and Methods. Product formation was calculated using BODIPY-PA as standard. Bars represent means ± SEM obtained from six experiments.

in glycerolipid biosynthesis and, thus, may relate to the enhanced energy demand covered by b-oxidation of fatty acids in ventricular myocardium. The assay has been validated for quantifying the activity of PAP against PA as substrate. This is appropriate for selectively assaying PAP activity in investigations of the phospholipase D-PAP signal transduction pathway in which the interest lies on the metabolic fate of phospholipase D-derived PA. PAP-2, however, has been renamed lipid phosphate phosphatase because of its ability to hydrolyze lipids such as lyso-PA, DAG pyrophosphate, ceramide1-phosphate, and sphingosine-1-phosphate in addition to PA [30]. Because no such BODIPY-labeled lipids are available currently, this is a potential disadvantage of our assay in instances where assaying PAP activity against substrates other than PA is of importance. Furthermore, previous results from studies using the radioactive PAP assay are not directly comparable with our findings due to differences in myocardial sample processing. Swanton and Saggerson found total PAP activity of approximately 240 nmol DAG/h/mg protein in rat crude myocardial membranes dependent on the Mg2+ concentration of 5 mM in the reaction buffer [29]. Yu and coworkers and Asemu and coworkers described PAP-2 activity of approximately 330 to 450 nmol DAG/h/mg protein in left ventricular sarcolemmal membranes and of 550 nmol DAG/h/mg protein in right ventricular sarcolemmal membranes, respectively, obtained from Sprague–Dawley rats [17,18]. In conclusion, this nonradioactive assay proved to be sensitive for accurate quantitation of myocardial PAP activity. The assay was applied for quantitation of total PAP activity, PAP-1 activity, and PAP-2 activity in human myocardial samples, providing first evidence of regional differences in activities and distribution pattern of PAP-1 and PAP-2 in the human heart.

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