- Email: [email protected]
Activation of phospholipase A2 by low levels of fluoride in THP1 macrophages via altered Ca2+ and cAMP concentration
Prostaglandins, Leukotrienes and Essential Fatty Acids 86 (2012) 99–105
Contents lists available at SciVerse ScienceDirect
Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
Activation of phospholipase A2 by low levels of fluoride in THP1 macrophages via altered Ca2 þ and cAMP concentration I. Gutowska a,n, I. Baranowska-Bosiacka b, A. Siennicka c, A. Telesin´ski d, M. Stan´czyk-Dunaj e, T. Weso"owska c, M. Ga˛ssowska f, P. K"os g, H. Zakrzewska d, B. Machalin´ski g, D. Chlubek b, E. Stachowska a a
Department of Biochemistry and Human Nutrition, Pomeranian Medical University, ul. Z˙o!nierska 48, Szczecin, Poland ´ co ´w Wlkp 72, Szczecin, Poland Department of Biochemistry, ul. Powstan c ´ co ´w Wlkp 72, Szczecin, Poland Department of Clinical Biochemistry and Laboratory Diagnostics, ul. Powstan d Department of Biochemistry, West Pomeranian University of Technology, S!owackiego 17, 71-434 Szczecin, Poland e ´ co ´w Wlkp 72, Szczecin, Poland Department of Medical Chemistry, ul. Powstan f Department of Cellular Signaling, Mossakowski Medical Research Centre Polish Academy of Sciences, Pawinskiego 5, 02-106 Warsaw, Poland g ´ co ´w Wlkp 72, Szczecin, Poland Department of General Pathology, Pomeranian Medical University, Powstan b
a r t i c l e i n f o
abstract
Article history: Received 18 August 2011 Received in revised form 4 February 2012 Accepted 6 February 2012
Phospholipases (PLA’s) participate in the regulation of physiological and pathological processes in the cell, including the release of pro-inflammatory mediators and stimulation of inflammatory processes. It is also well known that fluoride can increase the inflammatory reactions. Therefore we decided to examine the effect of fluorides in concentrations determined in human serum on cPLA2 and sPLA2 activity. The incubation of macrophages in fluoride solutions significantly increased the amount of synthesized cellular cAMP, intracellular calcium and sPLA2 activity in a dose-dependent pattern. The cPLA2 activity, estimated by the amount of released arachidonic acid, increased significantly when 10 mM NaF was used. The results of our study suggest that fluoride may change the activity of phospholipases in macrophage cells. Probably, increased cAMP concentration activates protein kinase C (PKC) and thus stimulates PLA2. cAMP also regulates the passage of Ca2 þ through ion channels, which additionally influence PLA2 throughout Ca2 þ -calmodulin dependent protein kinase. & 2012 Elsevier Ltd. All rights reserved.
Keywords: Arachidonic acid Calcium cAMP Cytosolic PLA2 Fluoride Macrophage Secretory PLA2
1. Introduction Phospholipases participate in the regulation of physiological and pathological processes in the cell, including the release of proinflammatory mediators and stimulation of inflammatory processes [1]. Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 position of membrane phospholipids to release unsaturated fatty acids. The family of mammalian PLA2 enzymes includes cytosolic and secretory forms [1]. cPLA2 preferentially cleave arachidonic acid (AA) from the sn-2 position of phospholipids [2]. This enzyme is implicated in various pathological mechanisms, especially the release of pro-inflammatory mediators [3]. Secretory phospholipases are associated with the development of the atherosclerotic process, and expression of sPLA2 isozymes (for example IIA) increases dramatically during inflammation [4,5]. High levels of isozymes are found in human atherosclerotic, macrophage-rich arterial walls [6,7]. Macrophages play a key role in the inflammatory
n
Corresponding author. Tel.: þ48 91 466 15 15; fax: þ 48 91 466 15 16. E-mail address: [email protected] (I. Gutowska).
0952-3278/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2012.02.002
process [8], which may lead to development of the atherosclerotic process. Inflammatory reactions underlie pathogenesis of the atherosclerotic process, and oxygen free radicals formed during inflammatory reactions contribute to aggravation of atherosclerotic lesions [9]. A long-term exposure to fluorides leads to changes in the amount and catalytic properties of many enzymes [10–12]. Sources of fluoride include natural fluoride in foodstuffs and water, i.e., fluoridated water (usually at 1.0 mg/l), fluoride dentifrices (containing on average 1000 mg/kg), fluoride supplements (such as fluoride tablets), and professionally applied fluoride gel (containing on average 5000 mg/kg) [9,13]. Even at a low concentration but with long duration exposure, fluorides accumulate in the body and lead to numerous metabolic disorders [14]. It is well known that the toxicity of fluoride is associated with ROS induction [9,12,15], and that fluoride can increase the inflammatory reactions [16]. Inflammation and lipid peroxidation are hallmarks of atherogenesis. Inflammatory and lipid peroxidation products contribute to the initialization, progression and rupture of atherosclerotic plaque [17]. Epidemiological evidence demonstrates positive correlation between environmental and occupational fluoride exposure and
100
I. Gutowska et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 86 (2012) 99–105
risk to various cardio-respiratory disorders [18,19] in workers exposed to dust of various potentially toxic substances including fluoride [20]. Therefore we decided to examine the effect of fluorides on secretory and cytosolic phospholipase A2 activity in differentiated human THP1 monocytic cells at concentrations determined in the blood of persons environmentally exposed to fluorides.
2. Material and methods 2.1. Reagents and sources THP-1 was obtained from American Type Culture Collection (ATCC, Rockville, USA). Fetal bovine serum was from Gibco (Gibco, Paisley, UK). RPMI medium, glutamine, and antibiotics (penicillin and streptomycin) were from Sigma-Aldrich (Poznan, Poland). Phosphate buffered saline (PBS) was from PAP Laboratories (Vienna, Austria). The Bradford-based protein concentration measured kit, NaF and deionized water were from Sigma-Aldrich (Poznan, Poland). N,N,N0 ,N0 -tetramethyl-p-phenylenediamine, phorbol 12-myristate 13-acetate (PMA), Fura-2 pentakis(acetoxymethyl) ester (Fura 2-AM), Ethylene glycol-bis(2-aminoethylether)-N,N,N0 ,N0 -tetraacetic acid (EGTA) and digitonin were obtained from Sigma-Aldrich (Poznan, Poland). Ionophore A23187, solvents for thin-layer chromatography (TLC) (petroleum ether, diethylether, acetic acid) and trypan blue were purchased from Sigma-Aldrich (Poznan, Poland). High-purity standards, solvents, and reagents for gas chromatography (GC) were obtained from Sigma-Aldrich or Fluka (Poznan, Poland). Silica gel plates were from Merck (Germany). Kits for measurement of sPLA2 activity and cAMP concentration were from Cayman (Cayman Chemical Company, Michigan, USA). 2.2. Cell culture THP-1 cells were seeded at a density of 2 106 cells/well in 6 well plates, differentiated into macrophages by administering 100 nM PMA [21], and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 mg/mL) for 24 h at 37 1C in 5% CO2. The adherent macrophages were then washed three times with PBS and incubated with NaF solution for 48 h at 37 1C [21]. NaF concentration was selected on the basis of results of fluoride concentration in human serum determined by other authors (0.75–1.75 mM for healthy person and 6.5 mM for person with fluorosis) [14,22,23] and was used at final concentrations of 1, 3, 6 and 10 mM. Next the cells were harvested by scraping and a pellet was obtained by centrifugation (250g, 5 min). Cell viability was examined using a trypan blue dye exclusion method. The cell count was determined with a Bright Line Hemacytometer (SigmaAldrich, Poznan, Poland). Cell cultures with viability more than 97% were used for experiments [24]. Protein concentration was measured by Bradford method [25]. 2.3. Phospholipases A2 activity measurement Macrophages were incubated with 1, 3, 6 and 10 mM NaF for 48 h and then 5 uM ionophore (A23187) was added to the cells (1 h, 37 1C) with gentle agitation [26]. After incubation the cells were harvested by scraping, collected (with the medium), and extraction of total lipids in probe was done using Folch mixture (2:1 v/v chloroform/ methanol) containing 0.01% (w/v) butylated hydroxytoluene as antioxidant [27]. The obtained extract was evaporated until dry under nitrogen flow and then suspended in 100 ml of n-hexan. Thin layer chromatography plates, with applied extract, were developed with
petroleum ether/diethylether/acetic acid mixture 90/10/1 (v/v/v) [28]. The fraction of free fatty acids was scraped off the plate, extracted with Folch mixture, methylated with 20% (w/v) boron trifluoridemethanol, and extracted using hexane. The obtained extract was evaporated until dry under nitrogen flow and then suspended in 100 ml of hexane. Care was taken to minimize exposure of samples to air. The influence of fluoride on PLA2 activity was determined by gas chromatography. Methyl ester arachidonic acid analysis was performed with a Perkin Elmer AutoSystem XL chromatograph equipped with a flame-ionization detector (FID) and Elite 5 (60 m 0.25 mm 0.25 mm) column. Split injection (split ratio 1:10) was performed, with nitrogen as carrier gas at a flow of 1.1 cm3/min. The oven temperature was maintained at 170 1C for 16 min after injection then programmed at 1 1C/min to 210 1C, which was held for 30 min. The injection port temperature was 250 1C and the detector temperature was 280 1C. The hydrogen and air flows were 30 and 300 cm3/min, respectively. The retention time for methyl ester arachidonic acid was 60.1 min. AA was identified by comparison of its retention time with a pure standard (Sigma-Aldrich, Poznan, Poland). AA content in cells was divided by the number of cells in the well and expressed as mg per mg of protein. 2.4. sPLA2 measurement in macrophages After incubation with NaF, cells were harvested by scraping, and the concentration of sPLA2 was measured spectrophotometrically in the culture supernatants using a sPLA2 enzyme immunoassay kit (Cayman Chemical), according to the manufacturer’s protocol. This immunometric assay is based on a double-antibody technique. Monoclonal antibody used in this kit is specific for sPLA2 type IIA. The content of sPLA2 in the sample was compared to the amount of sPLA2 in the standards by comparison of the yellow color generated. The results obtained were expressed as enzyme concentration per mg of protein. 2.5. Intracellular cAMP measurements Cells were incubated with different concentrations of NaF as previously described. Then the medium from wells was aspirated and 0.1 M HCl was added for 20 min at room temperature. After incubation, cells were harvested by scraping, centrifugated (1000g, 10 min) and the concentration of cAMP was measured spectrophotometrically (ELISA) in the supernatant solutions using the enzyme immunoassay kit (Cayman Chemical), according to the manufacturer’s protocol. The results obtained were normalized to protein levels, as measured by Bradford assay. 2.6. Intracellular calcium [Ca2 þ ]i measurements Fura 2-fluorescence was measured with a Victor2V (1420 multilabel HTS Counter) fluorescence microplate reader with a 340/380 nm excitation wavelength, and emission wavelength of 510 nm. Briefly, the cells grown on microplates (in medium containing different NaF concentrations in conditions described in Section 2.2) for fluorescent studies (Eppendorf) were washed with Tyrod buffer (10 mM Hepes, 140 mM NaCl, 3 mM KCl, 0.5 mM MgCl2, 5 mM NaHCO3, 10 mM glucose; pH 7.4) and were loaded with 1 mM Fura 2 acetoxymethyl ester (FURA 2AM) in a humidified 95% air/CO2 atmosphere at 37 1C for 30 min. After incubation, cells were washed with Tyrod buffer and were kept at 37 1C with constant stirring throughout measurement. [Ca2 þ ]i values were determined from the ratio of Fura-2 fluorescence intensities at 340/380 nm excitation, according to Grynkiewicz et al. [29].
I. Gutowska et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 86 (2012) 99–105
101
2.7. Calculation of [Ca2 þ ]i The relationship between the fluorescence ratio 340/380 and [Ca2 þ ]i was obtained using the equation [28]: [Ca2 þ ]i ¼Kd(R Rmin)/(Rmax R) Fmin/Fmax where Rmax is the fluorescence ratio at saturating Ca2 þ ; Rmin is the ratio at zero Ca2 þ ; and Fmin and Fmax are fluorescence readings at 380 nm at zero, and saturating Ca2 þ , respectively. Kd is the effective dissociation constant of Fura-2 in appropriate conditions and was taken to be 224 nmol/l, according to Grynkiewicz et al. [29]. Calibration was performed by the following procedure: after each single experiment, cells were lysed with digitonin (5 mM) to obtain Rmax and Fmax. Thereafter to obtain Rmin and Fmin, 50 mM EGTA was added.
2.8. Imagine of Ca2 þ fluorescence assay The cells were plated at 1 104/well on a 96 well BD Falcon Imaging Plate (BD Biosciences, Bedford, MA, USA) and incubated in the same conditions we applied in quantification measurements. FURA-2AM was used in analytical procedure. Calcium response to different concentrations of fluorides was measured by a BD Pathway 855 (BD Biosciences, Rockville, MD, USA). 2.9. Statistical analysis The obtained results were analyzed statistically using the software package Statistica 6.1. Arithmetical mean and standard deviation (SD) were found for each of the studied parameters. As the distribution of variables in most cases deviated from normal (Shapiro–Wilk test), non-parametric tests were used for further analyses. For related samples, significance was first checked with ANOVA Friedmann’s analysis of variance, and significant results were subjected to the Wilcoxon matched-pair test. The level of significance was p r0.05.
Fig. 1. Effect of fluoride on sPLA2 concentration in macrophages cultured with NaF H2O, NaCl—positive control. Monocytes/macrophages were cultured with NaF for 48 h. After incubation cells were harvested by scraping and the concentration of sPLA2 was measured spectrophotometrically using the sPLA2 enzyme immunoassay kit. The results obtained from 4 separate experiments were normalized to protein levels. np o 0.02, significant difference vs. positive control H2O; # p o 0.04, ##po 0.02, significant difference vs. positive control NaCl.
3.4. [Ca2 þ ]i values increased in monocyte/macrophage after incubation with NaF Incubation of macrophages with increasing concentrations of fluorides caused a significant increase in the intracellular [Ca2 þ ] value in a dose dependent manner. NaF at a concentration of 3 uM increased [Ca2 þ ]i value by about 10% (p ¼0.032), at 6 mM by 29% (p ¼0.012) and NaF at 10 mM by about 20% (p ¼0.012). A 1 mM concentration of fluoride added to the culture did not result in any significant increase in [Ca2 þ ]i value in macrophages. Images from the fluorescent BD Pathway (Fig. 5A–C) show an increase in the Ca2 þ mobilization in cells (intensity of light) induced by the increase in NaF concentration and confirm obtained results of quantification measurements.
4. Discussion 3. Results 3.1. NaF at low concentrations can increase concentration of the sPLA2 enzyme in macrophages The increase in NaF concentration added to the culture was followed by an increase in the sPLA2 concentration in macrophages: the significance was observed for 1, 6 and 10 mM NaF (respectively: p¼ 0.030, 20%; p ¼0.028, 61%; p¼ 0.028, 66%). 3.2. NaF increased intracellular AA concentration in macrophages by activation of cPLA2 enzyme. The addition of ionophor A23187 to the macrophage culture with NaF caused an increase of intracellular AA concentration. For 1 uM NaF we observed 7%, for 3 uM NaF 19% (p¼ 0.07), for 6 uM NaF 19% and for 10 mM NaF 58% (p ¼0.027) increase in AA concentration (Fig. 2). 3.3. NaF increased intracellular cAMP concentration In macrophages cultured with NaF, concentration of cAMP significantly increased in a dose dependent manner (Fig. 1); 19% for 1 uM NaF (p¼ 0.028), 71% for 3 uM NaF (p ¼0.027), 174% for 6 uM NaF (p ¼0.027) and 221% for 10 mM NaF (p¼ 0.015).
Atherosclerosis is a pathological process that occurs in major arteries [30], and despite many years of research on the various effect of fluorides on the human body (both positive and negative), there are few papers on their role in the inflammatory process that may contribute to the production of atherosclerotic plaque [12,31–33], one of the major health problems in highly developed societies. Some of the recent findings of atherosclerosis at a molecular level include the role of macrophages [8,30]. Eicosanoid synthesis in macrophages is controlled by the availability of free arachidonic acid, and activation of the phospholipase A2 (PLA2) is an important mechanism leading to increased eicosanoid synthesis. Therefore, pathophysiological states can be controlled through modulation of activity of this enzyme [34]. To the best of our knowledge, the current investigation is the first such study to have described the influence low levels of fluorides on the activity of phospholipases, enzymes which play a key role in the inflammatory process. In our study, incubation of monocytes/macrophages with NaF caused a significant increase in cAMP concentration in cells. To our knowledge no data is available on the effect of micromolar concentrations of NaF on this parameter. It is known that water fluoride is highly significantly positively correlated with serum cAMP concentration. Shashi and Bhardwaj [35] noted that fluoride showed a positive dose dependent response effect on the serum concentration of cAMP in patients affected with fluorosis.
102
I. Gutowska et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 86 (2012) 99–105
Dieter and Fitzke [48], in their study on macrophages, observed no NaF-induced changes in the concentration of intracellular calcium. We suppose that increased intracellular calcium concentration noted in our study is associated with the fluoride-induced increase in inositol triphosphate (IP3) and diacyloglycerol through the stimulation of phospholipases C (probably by cAMP increase) [35]. It may results in the inflow of calcium ions to the cell through the dihydropyridine-sensitive calcium channels [46]. Another explanation may be that fluoride causes Ca2 þ -dependent ATP-ase activation [47]. There are only a few reports on the effect of fluorides on macrophages, and hence on the development of atherosclerosis through activation of PLA2 [31,32,48–51]. Phospholipase A2 catalyzes the sn-2 ester bonds of glycerophospholipids and appears to be the most influential subfamily in pro-inflammatory processes. This subfamily can be characterized as a group of calcium-dependent enzymes: cPLA2 and sPLA2, which have been involved in various physiological functions such as the release of pro-inflammatory mediators, inflammatory and lipid modification [7]. The production of the pro-inflammatory lipid mediators, the eicosanoids, is dependent on the availability of the precursor, the free arachidonic acid. The release of arachidonic acid promoted by sPLA2 is primarily based on increased enzyme activity. The results of our study suggest that fluoride
Cyclic AMP is a second messenger, used for intracellular signal transduction. In macrophages, cAMP plays an important role in regulating many activities such as phagocytosis and migration, but high intracellular levels of cAMP are negatively correlated with these activities [36]. Increased levels of cAMP in resident macrophages and inflammatory human peritoneal macrophages after NaF stimulation [36] were confirmed in our research. We suppose that it is associated with the influence of fluoride which causes an increase in cAMP production by stimulating adenylate cyclase [37], where the enzyme itself catalyzes the production of cAMP from ATP [38]. Probably, increased cAMP concentration activates protein kinase C (PKC) which phosphorylases serintreonin kinases and thus stimulates PLA2 [35]. cAMP also regulates the passage of Ca2 þ through ion channels [37], which additionally influence PLA2 throughout Ca2 þ -calmodulin dependent protein kinase [39]. Many studies have shown that PLA2 enzymes require Ca2 þ for activity and catalytically hydrolyze phospholipids in a Ca2 þ dependent manner [40–43]. They contain a conserved Ca2 þ binding loop, whereas the nearby catalytic site contains a his/asp pair conserved throughout [44]. Strokin et al. [43] showed evidence that arachidonic acid is released by Ca2 þ -dependent group IV cPLA2, and Ambs et al. [40] using rat liver macrophages suggested that an increase in [Ca2 þ ]i, but not cPLA2 phosphorylation, is necessary for arachidonic acid release. Less information concerning the effects of fluorides on [Ca2 þ ]i concentration [42] and phospholipases A2 activity is available. For the first time our study shows this phenomenon using micromolar concentrations of fluorides which can be determined in human blood serum. This aspect of the proatherogenic action of fluoride ions is more important as fluorides are commonly used in everyday life and could accumulate in the human body [9,13,14]. Researchers are not unanimous with regard to the potential participation of NaF in the regulation of the intracellular calcium concentration, a key ion in the stimulation of binding cPLA2 to cellular membrane and release of arachidonic acid [45]. In this investigation we have demonstrated that incubation of monocytes/macrophages with NaF caused a significant increase in calcium concentration in cells. It seems interesting that calcium concentration in macrophages distinctly increased along with the elevation of the added NaF concentration (Fig. 2). Similar results were noted by Murao et al. [46] and also Xu et al. [47], who observed an increase in intracellular calcium concentration in nephric epithelial cells after stimulation with NaF. However,
Fig. 3. Effect of fluoride on cAMP concentration in macrophages cultured with NaF H2O—positive control. Monocytes/macrophages were cultured with NaF for 48 h. Cells were then incubated with 0.1 M HCl for 20 min in room temperature. After incubation cells were harvested by scraping, centrifugated (1000g/10 min) and the concentration of cAMP was measured spectrophotometrically (ELISA) in the supernatant solutions. The results obtained from 6 separate experiments were normalized to protein levels. npo0.02, nnpo0.03—significant difference vs. positive control.
Fig. 2. Effect of fluoride on intracellular AA concentration in macrophages cultured with NaF H2O—positive control. Monocytes/macrophages were cultured with NaF for 48 h and then with ionophor A23187 for 1 h. After incubation cells were harvested by scraping, and AA was isolated using Folch mixture and TLC method and measured using the GC method. The results obtained from 6 separate experiments were normalized to protein levels. np o0.03—significant difference vs. positive control.
Fig. 4. Effect of fluoride on [Ca2 þ ]i value in macrophages cultured with NaF H2O—positive control. Monocytes/macrophages were cultured with NaF for 48 h. Cells were then incubated with 1 mM Fura 2AM in humidified 95% air/CO2 atmosphere at 37 1C for 30 min. [Ca2 þ ]i values were measured by Victor2V (1420 multilabel HTS Counter) fluorescence microplate reader. The results obtained from 4 separate experiments were normalized to protein levels. npo0.02, nnpo0.04—significant difference vs. positive control.
I. Gutowska et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 86 (2012) 99–105
103
Fig. 5. Imaging of Ca2 þ influx in monocyte/macrophage cultured with (A) the control H2O, (B) 1 uM NaF, (C) 3 uM NaF and (D) 10 uM NaF H2O—positive control. Cells were incubated with 1 mM Fura 2AM in humidified 95% air/CO2 atmosphere at 37 1C for 30 min. Calcium mobilization in answer to different concentrations of fluorides (intensity of light) was measured by a BD Pathway 855 (BD Biosciences, Rockville, MD, USA).
may change activity of phospholipases in macrophage cells even in micromolar concentrations. We observed that sPLA2 activity increased significantly in cells incubated with NaF and that changes were in a dose-dependent manner (Fig. 1). Similar results were obtained by Bonney et al. [50] and Goldman et al. [32]. Probably the activation of PLA2 by fluoride is mediated by direct activation of PLA2 by the fluoride-induced generation of diacylglycerol [31]. The results also suggest that at least some of the pleiotropic effects of NaF in intact cells may not be mediated by G-protein activation but rather by depletion of ATP [12], which is essential for protein phosphorylation reactions. Macrophages are cells which can secrete sPLA2 [7], an acutephase protein expressed in response to a variety of pro-inflammatory cytokines. sPLA2 enzymes exhibit no preference for AA in the sn-2 position of phospholipids [7,52]. A calcium ion is an essential cofactor for catalysis, and is held in place by the calcium-binding loop and the Asp99 residue present in the consensus sequence of the catalytic site [53]. Therefore the cAMP increase (Fig. 3) followed by intracellular calcium concentration (Fig. 4) noted in this research may cause an activation of sPLA2. Another enzyme releasing arachidonic acid is Ca2 þ -dependent cPLA2, selective for arachidonate containing phospholipids [43]. For cPLA2, there are well-described mechanisms of activation involving an increase in concentration of intracellular Ca2 þ and/or phosphorylation of this enzyme by various mitogen-activated protein (MAP) kinases [54–57], which cause cPLA2-alfa translocation from the cytosol to the nuclear envelope triggered by increased intracellular calcium [56,58]. Recent reports show that p38 is the MAPK responsible for cPLA2 phosphorylation, and the NaF induced increase in intracellular Ca2 þ concentration ([Ca2 þ ]i) which led to p38 MAPK activation [42,59]. Arachidonic acid, released by PLA2, plays an important role as a second messenger and as a precursor of inflammatory lipid mediators; consequently its levels in cells are tightly regulated. Our results demonstrate an important role for calcium and cAMP
in arachidonic acid release induced by NaF through the activation of cPLA2. Probably, the fluoride-induced increase in intracellular Ca2 þ concentration was an upstream regulator in causing activation of p38 MAPK [42] leading to activation of cPLA2 [57,60]. Additionaly, an increase cAMP concentration may led to a translocation of protein kinase C from the cytosol to membranes [32,61] and phosphorylation of cPLA2 on Ser-505 which caused enzyme activation [57,62]. It has been proven in this study that application of NaF to macrophages at a very low dosage significantly increased the amount of released AA (Fig. 2). The same results were obtained by Dodam and Olson [33], but they used 30 mM NaF, about 103 fold higher NaF concentrations than in our experiment. The results obtained by Schulze-Specking et al. [61] suggest that the more sustained increase in calcium is contributing to the ability of NaF to induce a low level of arachidonic acid release. In several models, arachidonic acid release has been linked to the influx of extracellular calcium [46,47,61]. In the work of Bonney et al. [50], fluoride at a concentration of 10 mM stimulated a release of arachidonic acid from stromal cells and endometrial glands, and Goldman et al. [32] showed activation of PLA2 in several cell free systems. In future study it is worth checking out if inhibitors of cPLA2 and sPLA2 activity may blocks the effects of fluoride or they may influenced this effect in a competitive or synergistic manner. Although the results obtained in this study were not as spectacular as in other reports which used mM concentrations of NaF [33,36,42,50,61], they indicated that even in small concentrations fluorides may cause changes in the activity of enzymes taking part in the development of atherosclerosis.
Acknowledgment This study was supported by Grant no. N N404 228935 from the State Committee for Scientific Research, Poland.
104
I. Gutowska et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 86 (2012) 99–105
References [1] B.L. Diaza, J.P. Arm, Phospholipase A2, Prostaglandins Leukot. Essent. Fatty Acids 69 (2003) 87–97. [2] A. Dessen, J. Tang, H. Schmidt, et al., Crystal structure of human cytosolic phospholipase A2 reveals a novel topology and catalytic mechanism, Cell 97 (1999) 349–360. [3] A. Sapirstein, J.V. Bonventre, Specific physiological roles of cytosolic phospholipase A2 as defined by gene knockouts, Biochim. Biophys. Acta 1488 (2000) 139–148. [4] M. Alaoui-El-Azher, N. Havet, M. Singer, C. Dumarey, L. Touqui, Inhibition by unsaturated fatty acids of type II secretory phospholipase A2 synthesis in guinea-pig alveolar macrophages. Evidence for the eicosanoid-independent pathway, Eur. J. Biochem. 267 (2000) 3633–3639. [5] H. Kuwata, S. Yamamoto, Y. Miyazaki, et al., Studies on a mechanism by which cytosolic phospholipase A2 regulates the expression and function of type IIA secretory phospholipase A2, J. Immunol. 165 (2000) 4024–4031. [6] M. Hernandez, L. Fuentez, F.J. Fernandez-Aviles, M.S. Crespo, M.L. Nieto, Secretory phospholipase A(2) elicits proinflammatory changes and upregulates the surface expression of fas ligand in monocytic cells: potential relevance for atherogenesis, Circ. Res. 90 (2002) 38–45. [7] D. Divchev, B. Schieffer, The secretory phospholipases A2 group IIA: a missing link between inflammation, activated rennin-angiotensin system, and atherogenesis? Vasc. Health Risk Manage. 4 (2008) 597–604. [8] S.M. Lessner, H.L. Prado, E.K. Waller, Z.S. Galis, Atherosclerotic lesions grow through recruitment and proliferation of circulating monocytes in a murine model, Am. J. Pathol. 160 (2002) 2145–2155. [9] O. Barbier, L. Arreola-Mendoza, L.M. Del Razo, Molecular mechanisms of fluoride toxicity, Chem.-Biol. Interact. 188 (2010) 319–333. [10] S. Park, K. Ajtai, P. Burghardt, Inhibition of myosin ATPase by metal fluoride complexes, Biochem. Biophys. Acta 1430 (1999) 127–140. [11] M.L. Vani, K.P Reddy, Effects of fluoride accumulation on some enzymes of brain and gastrocnemius muscle of mice, Fluoride 33 (2000) 17–26. [12] I. Gutowska, I. Baranowska-Bosiacka, M. Bas´kiewicz, et al., Fluoride as a proinflammatory factor and inhibitor of ATP bioavailability in differentiated human THP1 monocytic cells, Toxicol. Lett. 196 (2010) 74–79. [13] H.M. Poureslami, P. Khazaeli, G.R. Noori, Fluoride in food and water consumed in Koohbanan (Kuh-E-Banan), Iran, Fluoride 41 (2008) 216–219. [14] P. Kalyanalakshmi, M. Vijayabhaskar, M. Dhananjaya Naidu, Lipid peroxidation and antioxidant enzyme status of adult males with skeletal fluorosis in Andhra Pradesh, India, Fluoride 40 (2007) 42–45. [15] J.A. Izquierdo-Vega, M. Sa´nchez-Gutie´rrez, L.M. Del Razo, Decreased in vitro fertility in male rats exposed to fluoride-induced oxidative stress damage and mitochondrial transmembrane potential loss, Toxicol. Appl. Pharmacol. 230 (2008) 352–357. [16] A.E. Vendrov, Z.S. Hakim, N.R. Madamanchi, M. Rojas, C. Madamanchi, M.S. Runge, Atherosclerosis is attenuated by limiting superoxide generation in both macrophages and vessel wall cells, Arterioscler. Thromb. Vasc. Biol. (2007) 2714–2721. [17] H.W. Niessen, P.A. Krijnen, C.A. Visser, C.J. Meijer, C.E. Hack, Type II secretory phospholipase A2 in cardiovascular disease: a mediator in atherosclerosis and ischemic damage to cardiomyocytes? Cardiovasc. Res. 60 (2003) 68–77. [18] S.J. Flora, V. Pachauri, M. Mittal, D. Kumar, Interactive effect of arsenic and fluoride on cardio-respiratory disorders in male rats: possible role of reactive oxygen species, Biometals 24 (2011) 615–628. [19] A. Rønneberg, Mortality and cancer morbidity in workers from an aluminium smelter with prebaked carbon anodes—part III: mortality from circulatory and respiratory diseases, Occup. Environ. Med. 52 (1995) 255–261. [20] S.V. Dhatrak, S.S. Nandi, Risk assessment of chronic poisoning among Indian metallic miners, Indian J. Occup. Environ. Med. 13 (2009) 60–64. [21] Y. Fu, N. Luo, M.F. Lopes-Virella, W.T. Garvey, The adipocyte lipid binding protein (ALBP/aP2) gene facilitates foam cell formation in human THP-1 macrophages, Atherosclerosis 165 (2002) 259–269. [22] J. Zawierta, E. Da˛bkowska, K. Jakubowska, M. Olszewska, I. Nocen´, The fluorine, iron, copper, zinc and selenium contents in plasma of healthy people living in Szczecin and its vicinity, Met. Fluorine (1998) 200–205. [23] J. Opydo-Szymaczek, M. Borysewicz-Lewicka, Variations in concentration of fluoride in blood plasma of pregnant women and their possible consequences for amelogenesis in a fetus, Homo 57 (2006) 295–307. [24] A. Nandi, D. Chandi, R. Lechesa, et al., Bifenthrin causes neurite retraction in the absence of cell death: a model for pesticide associated neurodegeneration, Med. Sci. Monit. 12 (2006) BR169–BR173. [25] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [26] J.D. Winkler, B.J. Bolognese, A.K. Roshak, A.K. Sung, L.A. Marshall, Evidence that 85 kDa phospholipase A2 is not linked to CoA-independent transacylasemediated production of platelet activating factor in human monocytes, Biochim. Biophys. Acta 1346 (1997) 173–184. [27] J. Folch, M. Lees, G.H.Sloane Stanley, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem. 226 (1957) 497–499. [28] R. Holman (Ed.), Progress in the chemistry of fats and other lipids, Chromatography, vol. VIII (Part 3), Pergamon Press, New York, 1966. [29] G. Grynkiewicz, M. Poenie, R.Y. Tsien, A new generation of Ca2 þ indicators with greatly improved fluorescence properties, J. Biol. Chem. 260 (1985) 3440–3450.
[30] C. Lester, K. Nikogosian, R. Shrestha, C. Tucker, Atherosclerotic assault, Eukaryon 2 (2006) 40–46. [31] K. Wessel, K. Resch, V. Kaever, Aluminium fluoride enhances phospholipase A2 activity and eicosanoid synthesis in macrophages, Eicosanoids 2 (1989) 223–227. [32] R. Goldman, Y. Granot, U. Zor, A pleiotropic effect of fluoride on signal transduction in macrophages: is it mediated by GPT-binding proteins? J. Basic Clin. Physiol. Pharmacol. 6 (1995) 79–94. [33] J.R. Dodam, N.C. Olson, Effect of fluoride on cardiopulmonary function and release of eicosanoids in pigs, J. Appl. Physiol. 78 (1995) 569–577. [34] Z. Mallat, J. Benessiano, T. Simon, et al., Circulating secretory phospholipase A2 activity and risk of incident coronary events in healthy men and women: the EPIC-Norfolk study, Arterioscler. Thromb. Vasc. Biol. 27 (2007) 1177–1183. [35] A. Shashi, M. Bhardwaj, Study on blood biochemical diagnostic indices for hepatic function biomarkers in endemic skeletal fluorosis, Biol. Trace Elem. Res., 2011 (online first). [36] A.P. Houdijk, P.A. Van Leeuwen, M.J. Adolfs, I.L. Bonta, GTP-related difference in cyclic AMP production between resident and inflammatory human peritoneal macrophages, Int. J. Tissue React. 13 (1991) 279–285. [37] A. Shahed, D.W. Allman, Stimulation of adentlate cyclase activity by Na2 PO3F and NaF in intact rat hepatocytes and plasma membrane fractions, Fluoride 17 (1984) 210–217. [38] D. Kornegay, S. Pennigton, A review of the effect of fluoride ion on adenyl cyclase, Fluoride 6 (1973) 19–32. [39] S.G. Miller, M.B. Kennedy, Regulation of brain type II Ca2 þ /calmodulindependent protein kinase by autophosphorylation: a Ca2 þ -triggered molecular switch, Cell 44 (1986) 861–870. [40] P. Ambs, M. Baccarini, E. Fitzke, P. Dieter, Role of cytosolic phospholipase A2 in arachidonic acid release of rat-liver macrophages: regulation by Ca2 þ and phosphorylation, Biochem. J. 311 (1995) 189–195. [41] K. Hanasaki, K. Yamada, S. Yamamoto, et al., Potent modification of low density lipoprotein by group X secretory phospholipase A2 is linked to macrophage foam cell formation, J. Biol. Chem. 277 (2002) 29116–29124. [42] W.H. Liu, P.H. Kao, Y.L. Chiou, S. Lin, M.J. Wu, S.J. Chang, Catalytic activityindependent pathway is involved in phospholipases A2-induced apoptotic death of human leukemia U937 cells via Ca2 þ -mediated p38 MAPK activation and mitochondrial depolarization, Toxicol. Lett. 185 (2009) 102–109. [43] M. Strokin, M. Sergeeva, G. Reiser, Docosahexaenoic acid and arachidonic acid release in rat brain astrocytes is mediated by two separate isoforms of phospholipases A2 and is differently regulated by cyclic AMP and Ca2 þ , Br. J. Pharmacol. 139 (2003) 1014–1022. [44] E.A. Dennis, S.G. Rhee, M.M. Billah, Y.A. Hannuns, Role of phospholipases in generating lipid second messengers in signal transduction, FASEB J. 5 (1991) 2068–2077. [45] Z.H. Qui, M.A. Gijon, M.S. de Carvalho, D.M. Spencer, C.C. Leslie, The role of calcium and phosphorylation of cytosolic phospholipase A2 in regulating arachidonic acid release in macrophage, J. Biol. Chem. 273 (1998) 8203–8211. [46] H. Murao, N. Sakagami, T. Iguchi, T. Murakami, Y. Suketa, Sodium fluoride increases intracellular calcium in rat renal epithelial cell line NRK-52E, Biol. Pharm. Bull. 23 (2000) 581–584. [47] H. Xu, Y.L. Zhou, J.M. Zhang, H. Liu, L. Jing, G.S. Li, Effects of fluoride on the intracellular free Ca2 þ and Ca2 þ -ATPase of kidney, Biol. Trace Elem. Res. 116 (2007) 279–288. [48] P. Dieter, E. Fitzke, Formation of diacylglycerol, inositol phosphates, arachidonic acid and its metabolites in macrophages, Eur. J. Biochem. 218 (1993) 753–758. [49] N.V. Bogatcheva, P. Wang, A.A. Birukova, A.D. Verin, J.G.N. Garcia, Mechanism of fluoride-induced MAP kinase activation in pulmonary artery endothelial cells, Am. J. Physiol. Lung Cell. Mol. Physiol. 290 (2006) L1139–L1145. [50] R.C. Bonney, J.S. Beesley, S. Franks, Release of arachidonic acid from human endometrial cells in culture mediated by calcium ionophore (A23187) or fluoride, J. Reprod. Fertil. 93 (1991) 449–460. [51] P. Kova´cs, G. Csaba, PLA2 activity in Tetrahymena pyriformis. Effects of inhibitors and stimulators, J. Lipid Mediat. Cell Signal. 15 (1997) 233–247. [52] E. Valentin, G. Lambeau, Increasing molecular diversity of secreted phospholipases A2 and their receptors and binding proteins, Biochim. Biophys. Acta 1488 (2000) 59–70. [53] O.G. Berg, M.H. Gelb, M.D. Tsai, M.K. Jain, Interfacial enzymology: the secreted phospholipase A2-paradigm, Chem. Rev. 101 (2001) 2613–2654. [54] E.A. Nalefski, L.A. Sultzman, D.M. Martin, et al., Delineation of two functionally distinct domains of cytosolic phospholipase A2, a regulatory Ca(2 þ )dependent lipid-binding domain and a Ca(2 þ )-independent catalytic domain, J. Biol. Chem. 269 (1994) 18239–18249. [55] C.C. Leslie, Properties and regulation of cytosolic phospholipase A2, J. Biol. Chem. 272 (1997) 16709–16712. [56] T. Hirabayashi, K. Kume, K. Hirose, et al., Critical duration of intracellular Ca2 þ response required for continuous translocation and activation of cytosolic phospholipase A2, J. Biol. Chem. 274 (1999) 5163–5169. [57] W.K. Han, A. Sapirstein, C.C. Hung, A. Alessandrini, J.V. Bonventre, Cross-talk between cytosolic phospholipase A2 alpha (cPLA2 alpha) and secretory phospholipase A2 (sPLA2) in hydrogen peroxide-induced arachidonic acid release in murine mesangial cells: sPLA2 regulates cPLA2 alpha activity that is responsible for arachidonic acid release, J. Biol. Chem. 278 (2003) 24153–24163.
I. Gutowska et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 86 (2012) 99–105
[58] R.W. McNish, M. Peters-Golden, Redistribution of 5-lipoxygenase and cytosolic phospholipase A2 to the nuclear fraction upon macrophage activation, Biochem. Biophys. Res. Commun. 196 (1993) 147–153. [59] S.I. Syrbu, W.H. Waterman, T.F.P. Molski, D. Nagarkatti, J.J. Hajjar, R.I. Sha’afi, Phosphorylation of cytosolic phospholipase A2 and the release of arachidonic acid in human neutrophils, J. Immunol. 162 (1999) 2334–2340. [60] Z.H. Qiu, C.C. Leslie, Protein kinase C-dependent and -independent pathways of mitogen-activated protein kinase activation in macrophages by stimuli that activate phospholipase A2, J. Biol. Chem. 269 (1994) 19480–19487.
105
[61] A. Schulze-Specking, J. Duyster, P.J. Gebicke-Haerter, S. Wurster, P. Dieter, Effect of fluoride, pertussis and cholera toxin on the release of arachidonic acid and the formation of prostaglandin E2, D2, superoxide and inositol phosphates in rat liver macrophages, Cell Signal. 3 (1991) 599–606. [62] M.A. Gijo n, D.M. Spencer, A.R. Siddiqi, J.V. Bonventre, C.C. Leslie, Cytosolic phospholipase A2 is required for macrophage arachidonic acid release by agonists that do and do not mobilize calcium, J. Biol. Chem. 275 (2000) 20146–20156.
Contents lists available at SciVerse ScienceDirect
Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
Activation of phospholipase A2 by low levels of fluoride in THP1 macrophages via altered Ca2 þ and cAMP concentration I. Gutowska a,n, I. Baranowska-Bosiacka b, A. Siennicka c, A. Telesin´ski d, M. Stan´czyk-Dunaj e, T. Weso"owska c, M. Ga˛ssowska f, P. K"os g, H. Zakrzewska d, B. Machalin´ski g, D. Chlubek b, E. Stachowska a a
Department of Biochemistry and Human Nutrition, Pomeranian Medical University, ul. Z˙o!nierska 48, Szczecin, Poland ´ co ´w Wlkp 72, Szczecin, Poland Department of Biochemistry, ul. Powstan c ´ co ´w Wlkp 72, Szczecin, Poland Department of Clinical Biochemistry and Laboratory Diagnostics, ul. Powstan d Department of Biochemistry, West Pomeranian University of Technology, S!owackiego 17, 71-434 Szczecin, Poland e ´ co ´w Wlkp 72, Szczecin, Poland Department of Medical Chemistry, ul. Powstan f Department of Cellular Signaling, Mossakowski Medical Research Centre Polish Academy of Sciences, Pawinskiego 5, 02-106 Warsaw, Poland g ´ co ´w Wlkp 72, Szczecin, Poland Department of General Pathology, Pomeranian Medical University, Powstan b
a r t i c l e i n f o
abstract
Article history: Received 18 August 2011 Received in revised form 4 February 2012 Accepted 6 February 2012
Phospholipases (PLA’s) participate in the regulation of physiological and pathological processes in the cell, including the release of pro-inflammatory mediators and stimulation of inflammatory processes. It is also well known that fluoride can increase the inflammatory reactions. Therefore we decided to examine the effect of fluorides in concentrations determined in human serum on cPLA2 and sPLA2 activity. The incubation of macrophages in fluoride solutions significantly increased the amount of synthesized cellular cAMP, intracellular calcium and sPLA2 activity in a dose-dependent pattern. The cPLA2 activity, estimated by the amount of released arachidonic acid, increased significantly when 10 mM NaF was used. The results of our study suggest that fluoride may change the activity of phospholipases in macrophage cells. Probably, increased cAMP concentration activates protein kinase C (PKC) and thus stimulates PLA2. cAMP also regulates the passage of Ca2 þ through ion channels, which additionally influence PLA2 throughout Ca2 þ -calmodulin dependent protein kinase. & 2012 Elsevier Ltd. All rights reserved.
Keywords: Arachidonic acid Calcium cAMP Cytosolic PLA2 Fluoride Macrophage Secretory PLA2
1. Introduction Phospholipases participate in the regulation of physiological and pathological processes in the cell, including the release of proinflammatory mediators and stimulation of inflammatory processes [1]. Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 position of membrane phospholipids to release unsaturated fatty acids. The family of mammalian PLA2 enzymes includes cytosolic and secretory forms [1]. cPLA2 preferentially cleave arachidonic acid (AA) from the sn-2 position of phospholipids [2]. This enzyme is implicated in various pathological mechanisms, especially the release of pro-inflammatory mediators [3]. Secretory phospholipases are associated with the development of the atherosclerotic process, and expression of sPLA2 isozymes (for example IIA) increases dramatically during inflammation [4,5]. High levels of isozymes are found in human atherosclerotic, macrophage-rich arterial walls [6,7]. Macrophages play a key role in the inflammatory
n
Corresponding author. Tel.: þ48 91 466 15 15; fax: þ 48 91 466 15 16. E-mail address: [email protected] (I. Gutowska).
0952-3278/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2012.02.002
process [8], which may lead to development of the atherosclerotic process. Inflammatory reactions underlie pathogenesis of the atherosclerotic process, and oxygen free radicals formed during inflammatory reactions contribute to aggravation of atherosclerotic lesions [9]. A long-term exposure to fluorides leads to changes in the amount and catalytic properties of many enzymes [10–12]. Sources of fluoride include natural fluoride in foodstuffs and water, i.e., fluoridated water (usually at 1.0 mg/l), fluoride dentifrices (containing on average 1000 mg/kg), fluoride supplements (such as fluoride tablets), and professionally applied fluoride gel (containing on average 5000 mg/kg) [9,13]. Even at a low concentration but with long duration exposure, fluorides accumulate in the body and lead to numerous metabolic disorders [14]. It is well known that the toxicity of fluoride is associated with ROS induction [9,12,15], and that fluoride can increase the inflammatory reactions [16]. Inflammation and lipid peroxidation are hallmarks of atherogenesis. Inflammatory and lipid peroxidation products contribute to the initialization, progression and rupture of atherosclerotic plaque [17]. Epidemiological evidence demonstrates positive correlation between environmental and occupational fluoride exposure and
100
I. Gutowska et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 86 (2012) 99–105
risk to various cardio-respiratory disorders [18,19] in workers exposed to dust of various potentially toxic substances including fluoride [20]. Therefore we decided to examine the effect of fluorides on secretory and cytosolic phospholipase A2 activity in differentiated human THP1 monocytic cells at concentrations determined in the blood of persons environmentally exposed to fluorides.
2. Material and methods 2.1. Reagents and sources THP-1 was obtained from American Type Culture Collection (ATCC, Rockville, USA). Fetal bovine serum was from Gibco (Gibco, Paisley, UK). RPMI medium, glutamine, and antibiotics (penicillin and streptomycin) were from Sigma-Aldrich (Poznan, Poland). Phosphate buffered saline (PBS) was from PAP Laboratories (Vienna, Austria). The Bradford-based protein concentration measured kit, NaF and deionized water were from Sigma-Aldrich (Poznan, Poland). N,N,N0 ,N0 -tetramethyl-p-phenylenediamine, phorbol 12-myristate 13-acetate (PMA), Fura-2 pentakis(acetoxymethyl) ester (Fura 2-AM), Ethylene glycol-bis(2-aminoethylether)-N,N,N0 ,N0 -tetraacetic acid (EGTA) and digitonin were obtained from Sigma-Aldrich (Poznan, Poland). Ionophore A23187, solvents for thin-layer chromatography (TLC) (petroleum ether, diethylether, acetic acid) and trypan blue were purchased from Sigma-Aldrich (Poznan, Poland). High-purity standards, solvents, and reagents for gas chromatography (GC) were obtained from Sigma-Aldrich or Fluka (Poznan, Poland). Silica gel plates were from Merck (Germany). Kits for measurement of sPLA2 activity and cAMP concentration were from Cayman (Cayman Chemical Company, Michigan, USA). 2.2. Cell culture THP-1 cells were seeded at a density of 2 106 cells/well in 6 well plates, differentiated into macrophages by administering 100 nM PMA [21], and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 mg/mL) for 24 h at 37 1C in 5% CO2. The adherent macrophages were then washed three times with PBS and incubated with NaF solution for 48 h at 37 1C [21]. NaF concentration was selected on the basis of results of fluoride concentration in human serum determined by other authors (0.75–1.75 mM for healthy person and 6.5 mM for person with fluorosis) [14,22,23] and was used at final concentrations of 1, 3, 6 and 10 mM. Next the cells were harvested by scraping and a pellet was obtained by centrifugation (250g, 5 min). Cell viability was examined using a trypan blue dye exclusion method. The cell count was determined with a Bright Line Hemacytometer (SigmaAldrich, Poznan, Poland). Cell cultures with viability more than 97% were used for experiments [24]. Protein concentration was measured by Bradford method [25]. 2.3. Phospholipases A2 activity measurement Macrophages were incubated with 1, 3, 6 and 10 mM NaF for 48 h and then 5 uM ionophore (A23187) was added to the cells (1 h, 37 1C) with gentle agitation [26]. After incubation the cells were harvested by scraping, collected (with the medium), and extraction of total lipids in probe was done using Folch mixture (2:1 v/v chloroform/ methanol) containing 0.01% (w/v) butylated hydroxytoluene as antioxidant [27]. The obtained extract was evaporated until dry under nitrogen flow and then suspended in 100 ml of n-hexan. Thin layer chromatography plates, with applied extract, were developed with
petroleum ether/diethylether/acetic acid mixture 90/10/1 (v/v/v) [28]. The fraction of free fatty acids was scraped off the plate, extracted with Folch mixture, methylated with 20% (w/v) boron trifluoridemethanol, and extracted using hexane. The obtained extract was evaporated until dry under nitrogen flow and then suspended in 100 ml of hexane. Care was taken to minimize exposure of samples to air. The influence of fluoride on PLA2 activity was determined by gas chromatography. Methyl ester arachidonic acid analysis was performed with a Perkin Elmer AutoSystem XL chromatograph equipped with a flame-ionization detector (FID) and Elite 5 (60 m 0.25 mm 0.25 mm) column. Split injection (split ratio 1:10) was performed, with nitrogen as carrier gas at a flow of 1.1 cm3/min. The oven temperature was maintained at 170 1C for 16 min after injection then programmed at 1 1C/min to 210 1C, which was held for 30 min. The injection port temperature was 250 1C and the detector temperature was 280 1C. The hydrogen and air flows were 30 and 300 cm3/min, respectively. The retention time for methyl ester arachidonic acid was 60.1 min. AA was identified by comparison of its retention time with a pure standard (Sigma-Aldrich, Poznan, Poland). AA content in cells was divided by the number of cells in the well and expressed as mg per mg of protein. 2.4. sPLA2 measurement in macrophages After incubation with NaF, cells were harvested by scraping, and the concentration of sPLA2 was measured spectrophotometrically in the culture supernatants using a sPLA2 enzyme immunoassay kit (Cayman Chemical), according to the manufacturer’s protocol. This immunometric assay is based on a double-antibody technique. Monoclonal antibody used in this kit is specific for sPLA2 type IIA. The content of sPLA2 in the sample was compared to the amount of sPLA2 in the standards by comparison of the yellow color generated. The results obtained were expressed as enzyme concentration per mg of protein. 2.5. Intracellular cAMP measurements Cells were incubated with different concentrations of NaF as previously described. Then the medium from wells was aspirated and 0.1 M HCl was added for 20 min at room temperature. After incubation, cells were harvested by scraping, centrifugated (1000g, 10 min) and the concentration of cAMP was measured spectrophotometrically (ELISA) in the supernatant solutions using the enzyme immunoassay kit (Cayman Chemical), according to the manufacturer’s protocol. The results obtained were normalized to protein levels, as measured by Bradford assay. 2.6. Intracellular calcium [Ca2 þ ]i measurements Fura 2-fluorescence was measured with a Victor2V (1420 multilabel HTS Counter) fluorescence microplate reader with a 340/380 nm excitation wavelength, and emission wavelength of 510 nm. Briefly, the cells grown on microplates (in medium containing different NaF concentrations in conditions described in Section 2.2) for fluorescent studies (Eppendorf) were washed with Tyrod buffer (10 mM Hepes, 140 mM NaCl, 3 mM KCl, 0.5 mM MgCl2, 5 mM NaHCO3, 10 mM glucose; pH 7.4) and were loaded with 1 mM Fura 2 acetoxymethyl ester (FURA 2AM) in a humidified 95% air/CO2 atmosphere at 37 1C for 30 min. After incubation, cells were washed with Tyrod buffer and were kept at 37 1C with constant stirring throughout measurement. [Ca2 þ ]i values were determined from the ratio of Fura-2 fluorescence intensities at 340/380 nm excitation, according to Grynkiewicz et al. [29].
I. Gutowska et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 86 (2012) 99–105
101
2.7. Calculation of [Ca2 þ ]i The relationship between the fluorescence ratio 340/380 and [Ca2 þ ]i was obtained using the equation [28]: [Ca2 þ ]i ¼Kd(R Rmin)/(Rmax R) Fmin/Fmax where Rmax is the fluorescence ratio at saturating Ca2 þ ; Rmin is the ratio at zero Ca2 þ ; and Fmin and Fmax are fluorescence readings at 380 nm at zero, and saturating Ca2 þ , respectively. Kd is the effective dissociation constant of Fura-2 in appropriate conditions and was taken to be 224 nmol/l, according to Grynkiewicz et al. [29]. Calibration was performed by the following procedure: after each single experiment, cells were lysed with digitonin (5 mM) to obtain Rmax and Fmax. Thereafter to obtain Rmin and Fmin, 50 mM EGTA was added.
2.8. Imagine of Ca2 þ fluorescence assay The cells were plated at 1 104/well on a 96 well BD Falcon Imaging Plate (BD Biosciences, Bedford, MA, USA) and incubated in the same conditions we applied in quantification measurements. FURA-2AM was used in analytical procedure. Calcium response to different concentrations of fluorides was measured by a BD Pathway 855 (BD Biosciences, Rockville, MD, USA). 2.9. Statistical analysis The obtained results were analyzed statistically using the software package Statistica 6.1. Arithmetical mean and standard deviation (SD) were found for each of the studied parameters. As the distribution of variables in most cases deviated from normal (Shapiro–Wilk test), non-parametric tests were used for further analyses. For related samples, significance was first checked with ANOVA Friedmann’s analysis of variance, and significant results were subjected to the Wilcoxon matched-pair test. The level of significance was p r0.05.
Fig. 1. Effect of fluoride on sPLA2 concentration in macrophages cultured with NaF H2O, NaCl—positive control. Monocytes/macrophages were cultured with NaF for 48 h. After incubation cells were harvested by scraping and the concentration of sPLA2 was measured spectrophotometrically using the sPLA2 enzyme immunoassay kit. The results obtained from 4 separate experiments were normalized to protein levels. np o 0.02, significant difference vs. positive control H2O; # p o 0.04, ##po 0.02, significant difference vs. positive control NaCl.
3.4. [Ca2 þ ]i values increased in monocyte/macrophage after incubation with NaF Incubation of macrophages with increasing concentrations of fluorides caused a significant increase in the intracellular [Ca2 þ ] value in a dose dependent manner. NaF at a concentration of 3 uM increased [Ca2 þ ]i value by about 10% (p ¼0.032), at 6 mM by 29% (p ¼0.012) and NaF at 10 mM by about 20% (p ¼0.012). A 1 mM concentration of fluoride added to the culture did not result in any significant increase in [Ca2 þ ]i value in macrophages. Images from the fluorescent BD Pathway (Fig. 5A–C) show an increase in the Ca2 þ mobilization in cells (intensity of light) induced by the increase in NaF concentration and confirm obtained results of quantification measurements.
4. Discussion 3. Results 3.1. NaF at low concentrations can increase concentration of the sPLA2 enzyme in macrophages The increase in NaF concentration added to the culture was followed by an increase in the sPLA2 concentration in macrophages: the significance was observed for 1, 6 and 10 mM NaF (respectively: p¼ 0.030, 20%; p ¼0.028, 61%; p¼ 0.028, 66%). 3.2. NaF increased intracellular AA concentration in macrophages by activation of cPLA2 enzyme. The addition of ionophor A23187 to the macrophage culture with NaF caused an increase of intracellular AA concentration. For 1 uM NaF we observed 7%, for 3 uM NaF 19% (p¼ 0.07), for 6 uM NaF 19% and for 10 mM NaF 58% (p ¼0.027) increase in AA concentration (Fig. 2). 3.3. NaF increased intracellular cAMP concentration In macrophages cultured with NaF, concentration of cAMP significantly increased in a dose dependent manner (Fig. 1); 19% for 1 uM NaF (p¼ 0.028), 71% for 3 uM NaF (p ¼0.027), 174% for 6 uM NaF (p ¼0.027) and 221% for 10 mM NaF (p¼ 0.015).
Atherosclerosis is a pathological process that occurs in major arteries [30], and despite many years of research on the various effect of fluorides on the human body (both positive and negative), there are few papers on their role in the inflammatory process that may contribute to the production of atherosclerotic plaque [12,31–33], one of the major health problems in highly developed societies. Some of the recent findings of atherosclerosis at a molecular level include the role of macrophages [8,30]. Eicosanoid synthesis in macrophages is controlled by the availability of free arachidonic acid, and activation of the phospholipase A2 (PLA2) is an important mechanism leading to increased eicosanoid synthesis. Therefore, pathophysiological states can be controlled through modulation of activity of this enzyme [34]. To the best of our knowledge, the current investigation is the first such study to have described the influence low levels of fluorides on the activity of phospholipases, enzymes which play a key role in the inflammatory process. In our study, incubation of monocytes/macrophages with NaF caused a significant increase in cAMP concentration in cells. To our knowledge no data is available on the effect of micromolar concentrations of NaF on this parameter. It is known that water fluoride is highly significantly positively correlated with serum cAMP concentration. Shashi and Bhardwaj [35] noted that fluoride showed a positive dose dependent response effect on the serum concentration of cAMP in patients affected with fluorosis.
102
I. Gutowska et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 86 (2012) 99–105
Dieter and Fitzke [48], in their study on macrophages, observed no NaF-induced changes in the concentration of intracellular calcium. We suppose that increased intracellular calcium concentration noted in our study is associated with the fluoride-induced increase in inositol triphosphate (IP3) and diacyloglycerol through the stimulation of phospholipases C (probably by cAMP increase) [35]. It may results in the inflow of calcium ions to the cell through the dihydropyridine-sensitive calcium channels [46]. Another explanation may be that fluoride causes Ca2 þ -dependent ATP-ase activation [47]. There are only a few reports on the effect of fluorides on macrophages, and hence on the development of atherosclerosis through activation of PLA2 [31,32,48–51]. Phospholipase A2 catalyzes the sn-2 ester bonds of glycerophospholipids and appears to be the most influential subfamily in pro-inflammatory processes. This subfamily can be characterized as a group of calcium-dependent enzymes: cPLA2 and sPLA2, which have been involved in various physiological functions such as the release of pro-inflammatory mediators, inflammatory and lipid modification [7]. The production of the pro-inflammatory lipid mediators, the eicosanoids, is dependent on the availability of the precursor, the free arachidonic acid. The release of arachidonic acid promoted by sPLA2 is primarily based on increased enzyme activity. The results of our study suggest that fluoride
Cyclic AMP is a second messenger, used for intracellular signal transduction. In macrophages, cAMP plays an important role in regulating many activities such as phagocytosis and migration, but high intracellular levels of cAMP are negatively correlated with these activities [36]. Increased levels of cAMP in resident macrophages and inflammatory human peritoneal macrophages after NaF stimulation [36] were confirmed in our research. We suppose that it is associated with the influence of fluoride which causes an increase in cAMP production by stimulating adenylate cyclase [37], where the enzyme itself catalyzes the production of cAMP from ATP [38]. Probably, increased cAMP concentration activates protein kinase C (PKC) which phosphorylases serintreonin kinases and thus stimulates PLA2 [35]. cAMP also regulates the passage of Ca2 þ through ion channels [37], which additionally influence PLA2 throughout Ca2 þ -calmodulin dependent protein kinase [39]. Many studies have shown that PLA2 enzymes require Ca2 þ for activity and catalytically hydrolyze phospholipids in a Ca2 þ dependent manner [40–43]. They contain a conserved Ca2 þ binding loop, whereas the nearby catalytic site contains a his/asp pair conserved throughout [44]. Strokin et al. [43] showed evidence that arachidonic acid is released by Ca2 þ -dependent group IV cPLA2, and Ambs et al. [40] using rat liver macrophages suggested that an increase in [Ca2 þ ]i, but not cPLA2 phosphorylation, is necessary for arachidonic acid release. Less information concerning the effects of fluorides on [Ca2 þ ]i concentration [42] and phospholipases A2 activity is available. For the first time our study shows this phenomenon using micromolar concentrations of fluorides which can be determined in human blood serum. This aspect of the proatherogenic action of fluoride ions is more important as fluorides are commonly used in everyday life and could accumulate in the human body [9,13,14]. Researchers are not unanimous with regard to the potential participation of NaF in the regulation of the intracellular calcium concentration, a key ion in the stimulation of binding cPLA2 to cellular membrane and release of arachidonic acid [45]. In this investigation we have demonstrated that incubation of monocytes/macrophages with NaF caused a significant increase in calcium concentration in cells. It seems interesting that calcium concentration in macrophages distinctly increased along with the elevation of the added NaF concentration (Fig. 2). Similar results were noted by Murao et al. [46] and also Xu et al. [47], who observed an increase in intracellular calcium concentration in nephric epithelial cells after stimulation with NaF. However,
Fig. 3. Effect of fluoride on cAMP concentration in macrophages cultured with NaF H2O—positive control. Monocytes/macrophages were cultured with NaF for 48 h. Cells were then incubated with 0.1 M HCl for 20 min in room temperature. After incubation cells were harvested by scraping, centrifugated (1000g/10 min) and the concentration of cAMP was measured spectrophotometrically (ELISA) in the supernatant solutions. The results obtained from 6 separate experiments were normalized to protein levels. npo0.02, nnpo0.03—significant difference vs. positive control.
Fig. 2. Effect of fluoride on intracellular AA concentration in macrophages cultured with NaF H2O—positive control. Monocytes/macrophages were cultured with NaF for 48 h and then with ionophor A23187 for 1 h. After incubation cells were harvested by scraping, and AA was isolated using Folch mixture and TLC method and measured using the GC method. The results obtained from 6 separate experiments were normalized to protein levels. np o0.03—significant difference vs. positive control.
Fig. 4. Effect of fluoride on [Ca2 þ ]i value in macrophages cultured with NaF H2O—positive control. Monocytes/macrophages were cultured with NaF for 48 h. Cells were then incubated with 1 mM Fura 2AM in humidified 95% air/CO2 atmosphere at 37 1C for 30 min. [Ca2 þ ]i values were measured by Victor2V (1420 multilabel HTS Counter) fluorescence microplate reader. The results obtained from 4 separate experiments were normalized to protein levels. npo0.02, nnpo0.04—significant difference vs. positive control.
I. Gutowska et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 86 (2012) 99–105
103
Fig. 5. Imaging of Ca2 þ influx in monocyte/macrophage cultured with (A) the control H2O, (B) 1 uM NaF, (C) 3 uM NaF and (D) 10 uM NaF H2O—positive control. Cells were incubated with 1 mM Fura 2AM in humidified 95% air/CO2 atmosphere at 37 1C for 30 min. Calcium mobilization in answer to different concentrations of fluorides (intensity of light) was measured by a BD Pathway 855 (BD Biosciences, Rockville, MD, USA).
may change activity of phospholipases in macrophage cells even in micromolar concentrations. We observed that sPLA2 activity increased significantly in cells incubated with NaF and that changes were in a dose-dependent manner (Fig. 1). Similar results were obtained by Bonney et al. [50] and Goldman et al. [32]. Probably the activation of PLA2 by fluoride is mediated by direct activation of PLA2 by the fluoride-induced generation of diacylglycerol [31]. The results also suggest that at least some of the pleiotropic effects of NaF in intact cells may not be mediated by G-protein activation but rather by depletion of ATP [12], which is essential for protein phosphorylation reactions. Macrophages are cells which can secrete sPLA2 [7], an acutephase protein expressed in response to a variety of pro-inflammatory cytokines. sPLA2 enzymes exhibit no preference for AA in the sn-2 position of phospholipids [7,52]. A calcium ion is an essential cofactor for catalysis, and is held in place by the calcium-binding loop and the Asp99 residue present in the consensus sequence of the catalytic site [53]. Therefore the cAMP increase (Fig. 3) followed by intracellular calcium concentration (Fig. 4) noted in this research may cause an activation of sPLA2. Another enzyme releasing arachidonic acid is Ca2 þ -dependent cPLA2, selective for arachidonate containing phospholipids [43]. For cPLA2, there are well-described mechanisms of activation involving an increase in concentration of intracellular Ca2 þ and/or phosphorylation of this enzyme by various mitogen-activated protein (MAP) kinases [54–57], which cause cPLA2-alfa translocation from the cytosol to the nuclear envelope triggered by increased intracellular calcium [56,58]. Recent reports show that p38 is the MAPK responsible for cPLA2 phosphorylation, and the NaF induced increase in intracellular Ca2 þ concentration ([Ca2 þ ]i) which led to p38 MAPK activation [42,59]. Arachidonic acid, released by PLA2, plays an important role as a second messenger and as a precursor of inflammatory lipid mediators; consequently its levels in cells are tightly regulated. Our results demonstrate an important role for calcium and cAMP
in arachidonic acid release induced by NaF through the activation of cPLA2. Probably, the fluoride-induced increase in intracellular Ca2 þ concentration was an upstream regulator in causing activation of p38 MAPK [42] leading to activation of cPLA2 [57,60]. Additionaly, an increase cAMP concentration may led to a translocation of protein kinase C from the cytosol to membranes [32,61] and phosphorylation of cPLA2 on Ser-505 which caused enzyme activation [57,62]. It has been proven in this study that application of NaF to macrophages at a very low dosage significantly increased the amount of released AA (Fig. 2). The same results were obtained by Dodam and Olson [33], but they used 30 mM NaF, about 103 fold higher NaF concentrations than in our experiment. The results obtained by Schulze-Specking et al. [61] suggest that the more sustained increase in calcium is contributing to the ability of NaF to induce a low level of arachidonic acid release. In several models, arachidonic acid release has been linked to the influx of extracellular calcium [46,47,61]. In the work of Bonney et al. [50], fluoride at a concentration of 10 mM stimulated a release of arachidonic acid from stromal cells and endometrial glands, and Goldman et al. [32] showed activation of PLA2 in several cell free systems. In future study it is worth checking out if inhibitors of cPLA2 and sPLA2 activity may blocks the effects of fluoride or they may influenced this effect in a competitive or synergistic manner. Although the results obtained in this study were not as spectacular as in other reports which used mM concentrations of NaF [33,36,42,50,61], they indicated that even in small concentrations fluorides may cause changes in the activity of enzymes taking part in the development of atherosclerosis.
Acknowledgment This study was supported by Grant no. N N404 228935 from the State Committee for Scientific Research, Poland.
104
I. Gutowska et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 86 (2012) 99–105
References [1] B.L. Diaza, J.P. Arm, Phospholipase A2, Prostaglandins Leukot. Essent. Fatty Acids 69 (2003) 87–97. [2] A. Dessen, J. Tang, H. Schmidt, et al., Crystal structure of human cytosolic phospholipase A2 reveals a novel topology and catalytic mechanism, Cell 97 (1999) 349–360. [3] A. Sapirstein, J.V. Bonventre, Specific physiological roles of cytosolic phospholipase A2 as defined by gene knockouts, Biochim. Biophys. Acta 1488 (2000) 139–148. [4] M. Alaoui-El-Azher, N. Havet, M. Singer, C. Dumarey, L. Touqui, Inhibition by unsaturated fatty acids of type II secretory phospholipase A2 synthesis in guinea-pig alveolar macrophages. Evidence for the eicosanoid-independent pathway, Eur. J. Biochem. 267 (2000) 3633–3639. [5] H. Kuwata, S. Yamamoto, Y. Miyazaki, et al., Studies on a mechanism by which cytosolic phospholipase A2 regulates the expression and function of type IIA secretory phospholipase A2, J. Immunol. 165 (2000) 4024–4031. [6] M. Hernandez, L. Fuentez, F.J. Fernandez-Aviles, M.S. Crespo, M.L. Nieto, Secretory phospholipase A(2) elicits proinflammatory changes and upregulates the surface expression of fas ligand in monocytic cells: potential relevance for atherogenesis, Circ. Res. 90 (2002) 38–45. [7] D. Divchev, B. Schieffer, The secretory phospholipases A2 group IIA: a missing link between inflammation, activated rennin-angiotensin system, and atherogenesis? Vasc. Health Risk Manage. 4 (2008) 597–604. [8] S.M. Lessner, H.L. Prado, E.K. Waller, Z.S. Galis, Atherosclerotic lesions grow through recruitment and proliferation of circulating monocytes in a murine model, Am. J. Pathol. 160 (2002) 2145–2155. [9] O. Barbier, L. Arreola-Mendoza, L.M. Del Razo, Molecular mechanisms of fluoride toxicity, Chem.-Biol. Interact. 188 (2010) 319–333. [10] S. Park, K. Ajtai, P. Burghardt, Inhibition of myosin ATPase by metal fluoride complexes, Biochem. Biophys. Acta 1430 (1999) 127–140. [11] M.L. Vani, K.P Reddy, Effects of fluoride accumulation on some enzymes of brain and gastrocnemius muscle of mice, Fluoride 33 (2000) 17–26. [12] I. Gutowska, I. Baranowska-Bosiacka, M. Bas´kiewicz, et al., Fluoride as a proinflammatory factor and inhibitor of ATP bioavailability in differentiated human THP1 monocytic cells, Toxicol. Lett. 196 (2010) 74–79. [13] H.M. Poureslami, P. Khazaeli, G.R. Noori, Fluoride in food and water consumed in Koohbanan (Kuh-E-Banan), Iran, Fluoride 41 (2008) 216–219. [14] P. Kalyanalakshmi, M. Vijayabhaskar, M. Dhananjaya Naidu, Lipid peroxidation and antioxidant enzyme status of adult males with skeletal fluorosis in Andhra Pradesh, India, Fluoride 40 (2007) 42–45. [15] J.A. Izquierdo-Vega, M. Sa´nchez-Gutie´rrez, L.M. Del Razo, Decreased in vitro fertility in male rats exposed to fluoride-induced oxidative stress damage and mitochondrial transmembrane potential loss, Toxicol. Appl. Pharmacol. 230 (2008) 352–357. [16] A.E. Vendrov, Z.S. Hakim, N.R. Madamanchi, M. Rojas, C. Madamanchi, M.S. Runge, Atherosclerosis is attenuated by limiting superoxide generation in both macrophages and vessel wall cells, Arterioscler. Thromb. Vasc. Biol. (2007) 2714–2721. [17] H.W. Niessen, P.A. Krijnen, C.A. Visser, C.J. Meijer, C.E. Hack, Type II secretory phospholipase A2 in cardiovascular disease: a mediator in atherosclerosis and ischemic damage to cardiomyocytes? Cardiovasc. Res. 60 (2003) 68–77. [18] S.J. Flora, V. Pachauri, M. Mittal, D. Kumar, Interactive effect of arsenic and fluoride on cardio-respiratory disorders in male rats: possible role of reactive oxygen species, Biometals 24 (2011) 615–628. [19] A. Rønneberg, Mortality and cancer morbidity in workers from an aluminium smelter with prebaked carbon anodes—part III: mortality from circulatory and respiratory diseases, Occup. Environ. Med. 52 (1995) 255–261. [20] S.V. Dhatrak, S.S. Nandi, Risk assessment of chronic poisoning among Indian metallic miners, Indian J. Occup. Environ. Med. 13 (2009) 60–64. [21] Y. Fu, N. Luo, M.F. Lopes-Virella, W.T. Garvey, The adipocyte lipid binding protein (ALBP/aP2) gene facilitates foam cell formation in human THP-1 macrophages, Atherosclerosis 165 (2002) 259–269. [22] J. Zawierta, E. Da˛bkowska, K. Jakubowska, M. Olszewska, I. Nocen´, The fluorine, iron, copper, zinc and selenium contents in plasma of healthy people living in Szczecin and its vicinity, Met. Fluorine (1998) 200–205. [23] J. Opydo-Szymaczek, M. Borysewicz-Lewicka, Variations in concentration of fluoride in blood plasma of pregnant women and their possible consequences for amelogenesis in a fetus, Homo 57 (2006) 295–307. [24] A. Nandi, D. Chandi, R. Lechesa, et al., Bifenthrin causes neurite retraction in the absence of cell death: a model for pesticide associated neurodegeneration, Med. Sci. Monit. 12 (2006) BR169–BR173. [25] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [26] J.D. Winkler, B.J. Bolognese, A.K. Roshak, A.K. Sung, L.A. Marshall, Evidence that 85 kDa phospholipase A2 is not linked to CoA-independent transacylasemediated production of platelet activating factor in human monocytes, Biochim. Biophys. Acta 1346 (1997) 173–184. [27] J. Folch, M. Lees, G.H.Sloane Stanley, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem. 226 (1957) 497–499. [28] R. Holman (Ed.), Progress in the chemistry of fats and other lipids, Chromatography, vol. VIII (Part 3), Pergamon Press, New York, 1966. [29] G. Grynkiewicz, M. Poenie, R.Y. Tsien, A new generation of Ca2 þ indicators with greatly improved fluorescence properties, J. Biol. Chem. 260 (1985) 3440–3450.
[30] C. Lester, K. Nikogosian, R. Shrestha, C. Tucker, Atherosclerotic assault, Eukaryon 2 (2006) 40–46. [31] K. Wessel, K. Resch, V. Kaever, Aluminium fluoride enhances phospholipase A2 activity and eicosanoid synthesis in macrophages, Eicosanoids 2 (1989) 223–227. [32] R. Goldman, Y. Granot, U. Zor, A pleiotropic effect of fluoride on signal transduction in macrophages: is it mediated by GPT-binding proteins? J. Basic Clin. Physiol. Pharmacol. 6 (1995) 79–94. [33] J.R. Dodam, N.C. Olson, Effect of fluoride on cardiopulmonary function and release of eicosanoids in pigs, J. Appl. Physiol. 78 (1995) 569–577. [34] Z. Mallat, J. Benessiano, T. Simon, et al., Circulating secretory phospholipase A2 activity and risk of incident coronary events in healthy men and women: the EPIC-Norfolk study, Arterioscler. Thromb. Vasc. Biol. 27 (2007) 1177–1183. [35] A. Shashi, M. Bhardwaj, Study on blood biochemical diagnostic indices for hepatic function biomarkers in endemic skeletal fluorosis, Biol. Trace Elem. Res., 2011 (online first). [36] A.P. Houdijk, P.A. Van Leeuwen, M.J. Adolfs, I.L. Bonta, GTP-related difference in cyclic AMP production between resident and inflammatory human peritoneal macrophages, Int. J. Tissue React. 13 (1991) 279–285. [37] A. Shahed, D.W. Allman, Stimulation of adentlate cyclase activity by Na2 PO3F and NaF in intact rat hepatocytes and plasma membrane fractions, Fluoride 17 (1984) 210–217. [38] D. Kornegay, S. Pennigton, A review of the effect of fluoride ion on adenyl cyclase, Fluoride 6 (1973) 19–32. [39] S.G. Miller, M.B. Kennedy, Regulation of brain type II Ca2 þ /calmodulindependent protein kinase by autophosphorylation: a Ca2 þ -triggered molecular switch, Cell 44 (1986) 861–870. [40] P. Ambs, M. Baccarini, E. Fitzke, P. Dieter, Role of cytosolic phospholipase A2 in arachidonic acid release of rat-liver macrophages: regulation by Ca2 þ and phosphorylation, Biochem. J. 311 (1995) 189–195. [41] K. Hanasaki, K. Yamada, S. Yamamoto, et al., Potent modification of low density lipoprotein by group X secretory phospholipase A2 is linked to macrophage foam cell formation, J. Biol. Chem. 277 (2002) 29116–29124. [42] W.H. Liu, P.H. Kao, Y.L. Chiou, S. Lin, M.J. Wu, S.J. Chang, Catalytic activityindependent pathway is involved in phospholipases A2-induced apoptotic death of human leukemia U937 cells via Ca2 þ -mediated p38 MAPK activation and mitochondrial depolarization, Toxicol. Lett. 185 (2009) 102–109. [43] M. Strokin, M. Sergeeva, G. Reiser, Docosahexaenoic acid and arachidonic acid release in rat brain astrocytes is mediated by two separate isoforms of phospholipases A2 and is differently regulated by cyclic AMP and Ca2 þ , Br. J. Pharmacol. 139 (2003) 1014–1022. [44] E.A. Dennis, S.G. Rhee, M.M. Billah, Y.A. Hannuns, Role of phospholipases in generating lipid second messengers in signal transduction, FASEB J. 5 (1991) 2068–2077. [45] Z.H. Qui, M.A. Gijon, M.S. de Carvalho, D.M. Spencer, C.C. Leslie, The role of calcium and phosphorylation of cytosolic phospholipase A2 in regulating arachidonic acid release in macrophage, J. Biol. Chem. 273 (1998) 8203–8211. [46] H. Murao, N. Sakagami, T. Iguchi, T. Murakami, Y. Suketa, Sodium fluoride increases intracellular calcium in rat renal epithelial cell line NRK-52E, Biol. Pharm. Bull. 23 (2000) 581–584. [47] H. Xu, Y.L. Zhou, J.M. Zhang, H. Liu, L. Jing, G.S. Li, Effects of fluoride on the intracellular free Ca2 þ and Ca2 þ -ATPase of kidney, Biol. Trace Elem. Res. 116 (2007) 279–288. [48] P. Dieter, E. Fitzke, Formation of diacylglycerol, inositol phosphates, arachidonic acid and its metabolites in macrophages, Eur. J. Biochem. 218 (1993) 753–758. [49] N.V. Bogatcheva, P. Wang, A.A. Birukova, A.D. Verin, J.G.N. Garcia, Mechanism of fluoride-induced MAP kinase activation in pulmonary artery endothelial cells, Am. J. Physiol. Lung Cell. Mol. Physiol. 290 (2006) L1139–L1145. [50] R.C. Bonney, J.S. Beesley, S. Franks, Release of arachidonic acid from human endometrial cells in culture mediated by calcium ionophore (A23187) or fluoride, J. Reprod. Fertil. 93 (1991) 449–460. [51] P. Kova´cs, G. Csaba, PLA2 activity in Tetrahymena pyriformis. Effects of inhibitors and stimulators, J. Lipid Mediat. Cell Signal. 15 (1997) 233–247. [52] E. Valentin, G. Lambeau, Increasing molecular diversity of secreted phospholipases A2 and their receptors and binding proteins, Biochim. Biophys. Acta 1488 (2000) 59–70. [53] O.G. Berg, M.H. Gelb, M.D. Tsai, M.K. Jain, Interfacial enzymology: the secreted phospholipase A2-paradigm, Chem. Rev. 101 (2001) 2613–2654. [54] E.A. Nalefski, L.A. Sultzman, D.M. Martin, et al., Delineation of two functionally distinct domains of cytosolic phospholipase A2, a regulatory Ca(2 þ )dependent lipid-binding domain and a Ca(2 þ )-independent catalytic domain, J. Biol. Chem. 269 (1994) 18239–18249. [55] C.C. Leslie, Properties and regulation of cytosolic phospholipase A2, J. Biol. Chem. 272 (1997) 16709–16712. [56] T. Hirabayashi, K. Kume, K. Hirose, et al., Critical duration of intracellular Ca2 þ response required for continuous translocation and activation of cytosolic phospholipase A2, J. Biol. Chem. 274 (1999) 5163–5169. [57] W.K. Han, A. Sapirstein, C.C. Hung, A. Alessandrini, J.V. Bonventre, Cross-talk between cytosolic phospholipase A2 alpha (cPLA2 alpha) and secretory phospholipase A2 (sPLA2) in hydrogen peroxide-induced arachidonic acid release in murine mesangial cells: sPLA2 regulates cPLA2 alpha activity that is responsible for arachidonic acid release, J. Biol. Chem. 278 (2003) 24153–24163.
I. Gutowska et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 86 (2012) 99–105
[58] R.W. McNish, M. Peters-Golden, Redistribution of 5-lipoxygenase and cytosolic phospholipase A2 to the nuclear fraction upon macrophage activation, Biochem. Biophys. Res. Commun. 196 (1993) 147–153. [59] S.I. Syrbu, W.H. Waterman, T.F.P. Molski, D. Nagarkatti, J.J. Hajjar, R.I. Sha’afi, Phosphorylation of cytosolic phospholipase A2 and the release of arachidonic acid in human neutrophils, J. Immunol. 162 (1999) 2334–2340. [60] Z.H. Qiu, C.C. Leslie, Protein kinase C-dependent and -independent pathways of mitogen-activated protein kinase activation in macrophages by stimuli that activate phospholipase A2, J. Biol. Chem. 269 (1994) 19480–19487.
105
[61] A. Schulze-Specking, J. Duyster, P.J. Gebicke-Haerter, S. Wurster, P. Dieter, Effect of fluoride, pertussis and cholera toxin on the release of arachidonic acid and the formation of prostaglandin E2, D2, superoxide and inositol phosphates in rat liver macrophages, Cell Signal. 3 (1991) 599–606. [62] M.A. Gijo n, D.M. Spencer, A.R. Siddiqi, J.V. Bonventre, C.C. Leslie, Cytosolic phospholipase A2 is required for macrophage arachidonic acid release by agonists that do and do not mobilize calcium, J. Biol. Chem. 275 (2000) 20146–20156.