- Email: [email protected]
Coexpression of phospholipase A2 isoforms in rat striatal astrocytes
Neuroscience Letters 247 (1998) 83–86
Coexpression of phospholipase A2 isoforms in rat striatal astrocytes Patrizia Zanassi a, Mayra Paolillo a , b, Sergio Schinelli a ,* a
Istituto di Farmacologia della Facolta` di Farmacia, Universita` di Pavia, Viale Taramelli 14, 27100 Pavia, Italy b Casa di cura ‘Villa Chiarugi’, 84014 Nocera Inferiore, Italy Received 3 November 1997; received in revised form 6 March 1998; accepted 13 March 1998
Abstract The expression and activity of phospholipase A2 (PLA2) isoforms were investigated in primary cultures of striatal astrocytes. The calcium ionophore A23187 together with the protein kinase C activator phorbol ester was the most potent stimulus in eliciting [3H]arachidonic acid release in the extracellular medium. Reverse transcription coupled to polymerase chain reaction (RT-PCR) showed the presence of the 85 kDa cytosolic PLA2 mRNA and the 14 kDa secretory PLA2 mRNA in untreated astrocytes. Immunoblot experiments with isoform-specific antibodies showed the presence of the cytosolic PLA2 in untreated astrocytes, while the secretory PLA2 was detected only in lipopolysaccharide-treated astrocytes. These data suggest that the two PLA2 isoforms expressed in striatal astrocytes might play different roles in cellular processes mediated by astrocytes. 1998 Elsevier Science Ireland Ltd.
Keywords: Striatal astrocytes; PLA2 isoforms; Arachidonic acid
Phospholipase A2 (PLA2), which catalyzes the hydrolysis of membrane phospholipids releasing free fatty acids and lysophospholipids, is widespread distributed in the brain where it plays a crucial role in several cellular processes. Two main isoforms of mammalian Ca2+-dependent PLA2 have been cloned and sequenced: a 14 kDa secretory PLA (sPLA2) and a 85 kDa cytosolic PLA2 (cPLA2) [5,6]. These two isoforms have different subcellular distribution and Ca2+ requirement for activation: sPLA2 is secreted and activated by millimolar Ca2+ concentrations; cPLA2 selectively hydrolyzes arachidonic acid (AA) containing phospholipids, it is mainly cytosolic and it is activated by micromolar Ca2+ concentrations. In the brain, arachidonic acid (AA) and its metabolites are formed upon PLA2 activation and are involved in signal transduction pathways, modulation of ion channels and neurotransmitter release [10]. Previous works have shown that astrocytes are involved in the regulation of inflammatory processes [2] and in AA release [12], but the PLA2 isoforms that mediate these responses have not been characterized. In this study, we show, by * Corresponding author. Tel.: +39 382 507395; fax: +39 382 507405; e-mail: [email protected]
molecular and biochemical approaches, that unstimulated striatal astrocytes in culture express both the Ca2+ dependent 85 kDa cPLA2 and the 14 kDa sPLA2 mRNA together with the cytosolic Ca2+-dependent cPLA2 protein. In contrast, the 14 kDa sPLA2 protein and a PLA2 activity in membranes and extracellular medium are detectable only when striatal astrocytes are stimulated by an inflammatory agent such as lipopolysaccharide. Striata were removed from 14–15-dayold rat embryos (Morini, Italy) and astrocytes were prepared as reported [7]. When indicated, confluent striatal astrocytes were washed twice with Dulbecco’s modified Eagle’s medium and incubated for 10 h with 100 ng/ml bacterial lipopolysaccharide (LPS) in fresh growing medium. At the end of the incubation, the proteins in the extracellular medium were concentrated by microcon cartridge (Amicon) and the cells were gently scraped with a rubber policeman in 1 ml of ice-cold lysis buffer (10 mM Tris pH 7.4, 1 mM EGTA, 5 mg/ml aprotinin and 0.1 mM phenylmethylsulfonyl fluoride). The homogenate was sonicated for 5 s at 4°C and the suspension was centrifuged for 15 min at 40 000 × g to separate cytosolic fraction (surnatant) from membrane fraction (pellet). For AA release, untreated striatal astrocyte were labeled for 20 h with [3H]AA (200 Ci/mmol; Amer-
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sham, UK) and then washed three times (0.5 ml/well) with HEPES buffer HEPES buffer (composition in mM: NaCl 154, KCl 5.6, NaHCO3 3.6, CaCl2 1.3, MgCl2 0.5, HEPES 10, glucose 10, pH 7.4 containing 0.2% fatty-acid-free BSA, saturated with 5% CO2 –air mixture). The astrocytes were stimulated for 10 min at 37°C with indicated agents and [3H]AA release in the HEPES buffer was measured as previously described [11]. For RT-PCR experiments, total RNA was extracted from untreated astrocytes by a standard method [3] and then treated with 10 U ribonuclease-free deoxyribonuclease (DNase) I (Promega) for 15 min at 37°C to destroy possible contaminating genomic DNA. The DNase was then digested with 15 mg proteinase K (Promega) at 70°C for 20 min and the RNA was then phenol/chloroform extracted and ethanol precipitated. An aliquot of total RNA (2 mg) was added to 20 ml of RT buffer containing 1 mM random hexamer, 0.5 mM of each dNTP, 15 unit RNasin and 12.5 units of avian mieloblastoma virus (AMV) reverse transcriptase (all reagents obtained from Promega). After incubation for 45 min at 42°C, the reaction was stopped by heating the mixture to 95°C for 5 min and chilled on ice. The RT mixture was diluted to 100 ml with water and 5 ml of this solution was used as template for the PCR. An ‘hot start’ PCR protocol was carried out using AmpliWax PCR Gems (Perkin Elmer). A PCR mixture (total volume 30 ml) containing 50 pmol each of forward and reverse primer, 50 mM dNTPs and PCR buffer was dispensed into reaction tube and then an AmpliWax gem was added. The mixture was first heated to 80°C for 5 min to melt the wax gems and then cooled to 30°C; 20 ml of solution containing the template cDNA and two units of Taqpolymerase (Promega) were then layered over the solid wax. The PCR was carried out for 36 cycles (1 min at 92°C, 1 min at 60°C, 1 min at 72°C) with a final extension step of 10 min at 72°C. The primers were: 5′-ATTACGTTAATGGATGCCAATTAT-3′ forward and 5′-TTTCTCTGGAAA-ATCAGGGTGAGA-3′ reverse for cPLA2; 5′AGCCTTCTGGAGTTTGGGCA-AATG-3′ forward and 5′-TTGTTGGGGTAGAACTGGTACTTT-3′ reverse for sPLA2. An aliquot (10 ml) of PCR amplified mixture was separated by 1.7% agarose gel electrophoresis and bands visualized by ethidium bromide staining. For immunodetection experiments, proteins in the extracellular medium, cytosol and membranes were resolved by 7.5% polyacrylamide, 1% SDS gel electrophoresis for cPLA2 (30 mg proteins/lane) or 12.5% polyacrylamide, 1% SDS gel electrophoresis for sPLA2 (150 mg proteins/lane). The proteins in the gels were transferred onto nitrocellulose membranes by semidry blotting and, after blocking for 2 h at room temperature in Tris buffered saline (TBS; 50 mM Tris and 150 mM NaCl, pH 7.4) containing 5% low-fat dry milk, the blots were incubated for 6 h with the cPLA2 or sPLA2 rabbit polyclonal antibodies (dilution 1:1000, gift of Dr. Kramer; Eli Lilly, USA) in TBS-2.5% low-fat dry milk. After washing, the blots were incubated with alkaline phosphatase-conjugated (AP) goat anti-rabbit IgG (1:5000
dilution) and the signal was detected by adding the AP substrate 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium (BCIP/NBT) for 15 min. PLA2 activity in cytosol, membranes and extracellular medium obtained from untreated or LPS-treated astrocytes was measured as reported [13]. The PLA2 activity is expressed as nmol of pyrenedecanoate (PD) formed/mg protein/s. All data, unless otherwise indicated, are expressed as the mean ± SEM values of three independent determinations each performed in triplicate. Values of P were calculated by Student’s t-test or ANOVA followed by post hoc Student-Newman–Keul’s test. When different agents were tested for their ability in inducing [3H]AA release, the protein kinase C activator phorbol-12-myristate-13-acetate (PMA; 1 mM) weakly stimulated [3H]AA release (120% of basal); both 5 mM A23187 alone (165% of basal) and 5 mM A23187 plus 1 mM PMA (250% of basal) significantly increased [3H]AA release compared to the basal value (Fig. 1). The effect of A23187 plus PMA was synergistic, since the amount of released [3H]AA induced by these agents was more than additive compared to that induced by A23187 alone or PMA alone. The A23187 plus PMA-induced AA release was suppressed by 95.1 ± 7.5% in striatal astrocytes pretreated for 30 min with the aspecific PLA2 inhibitor mepacrine (50 mM), suggesting that AA results mainly from activation of PLA2. Moreover, preincubation of striatal astrocytes for 30 min with the sPLA2 inhibitor manoalide (1 mM) did not modify (99.7 ± 6.3% of control) the stimulated [3H]AA release and in the same conditions the compound p-bromophenacylbromide (BPB) augmented (122.3 ± 9.8% of control), rather than inhibited, stimulated [3H]AA release, probably by interfering with reuptake of [3H]AA into membrane phospholipids.
Fig. 1. [3H]AA release in untreated striatal astrocytes. Astrocytes were stimulated by 5 mM A23187 alone, 1 mM PMA alone or by 5 mM A23187 plus 1 mM PMA. Results are expressed as percentage of basal [3H]AA release (1.09 ± 0.11% of [3H]adenine converted to [3H]cAMP). *P , 0.05 compared to basal [3H]AA release.
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The expression of PLA2 isoforms mRNAs in untreated striatal astrocytes was studied by RT-PCR using specific primers for the two PLA2 isoforms. The PCR amplified products (Fig. 2), detected on ethidium bromide stained agarose gel, matched the expected size for cPLA2 (473 bp, lane 1) and sPLA2 (347 bp, lane 2). No PCR products were found when the RT step was omitted (not shown). In cytosolic extracts, a polyclonal antibody raised against cPLA2 recognized a protein of about 100 kDa; the amount of cPLA2 was not modified by the LPS treatment (Fig. 3a, lanes 1 and 2). It should be noted that although the cPLA2 cDNA sequence predicts a molecular weight of 85 kDa, the protein migrates with an apparent molecular weight of 100– 110 kDa on SDS-PAGE [5]. The negative control, carried out by preadsorbing the primary antibody with recombinant cPLA2, confirmed the identity of the observed signal (Fig. 3a, lane 3, PR). In parallel experiments, using 150 mg of membranes (Fig. 3b, lanes 1 and 2) or proteins found in the extracellular medium (Fig. 3b, lanes 3 and 4), the 14 kDa sPLA2 band was detected in astrocytes only following a 10 h treatment with bacterial LPS. Cytosol obtained from untreated striatal astrocytes showed either a basal Ca2+-independent (3.1 ± 0.6 nmol PD released/mg protein per s) and a Ca2+-dependent PLA2 activity (about 7.6-fold increase compared to basal value, P , 0.05); in membranes of untreated striatal astrocytes, the basal Ca2+-independent PLA2 activity (0.8 ± 0.1 nmol PD released/mg protein per s) was not significantly enhanced by the addition of Ca2+. In contrast, the LPS treatment evoked an increase of the Ca2+-dependent PLA2 activity either in membranes (about 2.1-fold, P , 0.05) and in the extracellular medium (about 5.4-fold, P , 0.05) without modifying the cytosolic Ca2+-dependent PLA2 activity (not shown). In this study we demonstrate for the first time that rat striatal astrocytes grown in primary culture coexpress the Ca2+-dependent 85 kDa cPLA2 mRNA and the 14 kDa sPLA2 mRNA, assessed by RT-PCR. Our results suggest that, although both PLA2 isoforms trancripts are present,
Fig. 2. Ethidium bromide staining of cPLA2 and sPLA2 RT-PCR products in striatal astrocytes. Total RNA (2 mg) was retrotranscribed and the cDNA was submitted to 36 PCR cycles. PCR products sizes are 473 bp for cPLA2 (lane 1) and 347 bp for sPLA2 (lane 2). The experiment was repeated twice with similar results.
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Fig. 3. cPLA2 and sPLA2 immunoblots of proteins extracts from LPS treated (+) and untreated (−) striatal astrocytes. (a) cPLA2 immunoblots of protein extracts obtained from the cytosol; PR indicates that the anti-cPLA2 antibody was incubated in the presence of recombinant cPLA2; (b) sPLA2 immunoblots of protein extracts obtained from membranes or extracellular medium. The experiment was repeated twice with similar results.
in normal conditions in striatal astrocytes the Ca2+-dependent cPLA2 isoform mainly mediates the receptor-dependent stimulation of AA release. A synergistic effect in eliciting [3H]AA release is induced by the PKC activator PMA and the calcium ionophore A23187, in agreement with the observation that the Ca2+-dependent cPLA2 activity is synergistically stimulated by calcium and phorbol esters via a PKC-dependent phosphorylation of cPLA2 [8]. Moreover, the [3H]AA release was prevented by the non-selective PLA2 inhibitor mepacrine, but not by BPB or manoalide, two compounds that inhibit sPLA2 selectively over cPLA2 [2]. Finally, the cytosol obtained from untreated astrocytes showed a Ca2+-dependent PLA2 activity and contains an immunoreactive 100 kDa band (cPLA2) whose molecular weight corresponds to that of the cloned Ca2+-dependent 85 kDa cPLA2. Interestingly, the finding that striatal astrocytes possess also a cytosolic Ca2+-independent PLA2 activity seems to suggest that these cells may express Ca2+independent PLA2 isoforms recently described in other cell systems [5]. Several papers have described the presence and regulation of cPLA2 expression in astrocytes. Stephenson et al. [15] reported the colocalization of glial fibrillary acidic protein together with cPLA2 immunoreactivity in human cerebral cortex sections; these authors also found that hippocampal astrocytes and microglia obtained from rats subjected to ischemia showed a 3-fold increase of cPLA2 immunoreactivity [4]. A new role of cPLA2 in modulating the antiinflammatory response in astrocytes has been emphasized in a recent paper by Stella et al. [14]; these authors found that in murine striatal astrocytes a prolonged
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treatment with interleukin-1b induces an increase in Ca2+dependent cPLA2 protein expression and Ca2+-dependent cPLA2-induced AA release. Since we report here that the Ca2+-dependent cPLA2 expression is not changed by LPS treatment, further studies are necessary to elucidate the exact mechanisms by which different proinflammatory stimuli regulate PLA2 isoforms expression in astrocytes. Our results indicate that either sPLA2 levels and PLA2 activity associated with the membranes or released in the extracellular medium are extremely low in unstimulated astrocytes; the sPLA2 protein and a PLA2 activity became detectable only after LPS treatment. Our data, together with the finding that an increase of PLA2 activity in the incubation medium and of sPLA2 mRNA synthesis were found in LPS-treated rat astrocytes [9], strongly suggest that the increased PLA2 activity and sPLA2 levels in membranes or extracellular medium are due to new synthesis of sPLA2 protein and not to a stimulus-dependent secretion of preexisting cellular sPLA2 protein. Two explanations are possible for our results; a fraction of the newly formed sPLA2 is first delivered to the membranes before being released in the extracellular medium or alternatively sPLA2 is released in the extracellular medium and then an aliquot reassociates with the membranes. The coexpression of at least two PLA2 isoforms raises the intriguing possibility that in astrocytes different PLA2 isoforms could regulate the spatiotemporal pattern of AA release in response to extracellular stimuli. This mechanism has been described in LPS-pretreated murine P388D1 macrophages stimulated by platelet activating factor, in which cPLA2 mediates the cell-associated AA accumulation whereas sPLA2 mediates AA release into the incubation medium [1]. Given the multitude of cellular physiological and pathological processes activated by PLA2 activation in astrocytes, further studies aimed to elucidate the specific contribution of PLA2 isoforms in these processes are necessary to targeting of new therapeutic interventions. Part of this work was performed when S.S. was a recipient of an EMBO short term fellowship at INSERM U.109, Paris, France. The authors thank Dr. Piomelli for constructive criticism, Dr. R.M. Kramer (Eli Lilly, USA) and Dr. J. Ishizaki (Shionogi Research Laboratories, Japan) for providing PLA2 antibodies and sPLA2 cDNA.
[1] Balsinde, J. and Dennis, E.A., Distinct roles in signal transduction for each of the phospholipase A2 enzymes present in P388D1 macrophages, J. Biol. Chem., 271 (1996) 6758–6765. [2] Benveniste, E.N., Inflammatory cytokines within the central nervous system: sources, function, and mechanism of action, Am. J. Physiol., 263 (1992) C1–C16. [3] Chomczynski, P. and Sacchi, N., Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem., 162 (1987) 156–159. [4] Clemens, J.A., Stephenson, D.T., Smalstig, E.B., Roberts, E.F., Johnstone, E.M., Sharp, J.D., Little, S.P. and Kramer, R.M., Reactive glia express cytosolic phospholipase A2 after transient global forebrain ischemia in the rat, Stroke, 27 (1996) 527–535. [5] Dennis, E.A., The growing phospholipase A2 superfamily of signal transduction enzymes, Trends Biochem. Sci., 22 (1997) 1–2. [6] Glaser, K.B., Mobilio, D., Chang, J.Y. and Senko, N., Phospholipase A2 enzymes: regulation and inhibition, Trends Pharmacol. Sci., 14 (1993) 92–98. [7] Grimaldi, M., Pozzoli, G., Navarra, P., Preziosi, P. and Schettini, G., Vasoactive intestinal peptide and forskolin stimulate interleukin 6 production by rat cortical astrocytes in culture via a cAMP-dependent prostaglandins-independent mechanism, J. Neurochem., 63 (1994) 344–350. [8] Lin, L.L., Wartmann, M., Lin, A.Y., Knopf, J.L., Seth, A. and Davis, R.J., cPLA2 is phosphorylated and activated by MAP kinase, Cell, 72 (1993) 269–278. [9] Oka, S. and Arita, H., Inflammatory factors stimulate expression of group II phospholipase A2 in rat cultured astrocytes, J. Biol. Chem., 266 (1991) 9956–9960. [10] Piomelli, D., Eicosanoids in synaptic transmission, Crit. Rev. Neurobiol., 5 (1994) 274–280. [11] Schinelli, S., Paolillo, M. and Corona, G.L., Opposing actions of D1 and D2-dopamine receptors on arachidonic acid release and cAMP production in striatal neurons, J. Neurochem., 62 (1994) 944–949. [12] Stella, N., Tence´, M., Glowinski, J. and Pre´mont, J., Glutamateevoked release of arachidonic acid from mouse brain astrocytes, J. Neurosci., 14 (1994) 568–575. [13] Stella, N., Pellerin, L. and Magistretti, P.J., Modulation of the glutamate-evoked release of arachidonic acid from mouse cortical neurons: involvement of a pH-sensitive membrane phospholipase A2, J. Neurosci., 15 (1995) 3307–3317. [14] Stella, N., Estelles, A., Siciliano, J., Tence´, M., Desagher, S., Piomelli, D., Glowinski, J. and Pre´mont, L., Interleukin-1 enhances the ATP-evoked release of arachidonic acid from mouse astrocytes, J. Neurosci., 17 (9) (1997) 2939–2946. [15] Stephenson, D.T., Manetta, J.V., White, D.L., Chiou, X.G., Cox, L., Gitter, B., May, P.C., Sharp, J.D., Kramer, R.M. and Clemens, J.A., Calcium-sensitive cytosolic phospholipase A2 is expressed in human brain astrocytes, Brain Res., 637 (1994) 97–105.
Coexpression of phospholipase A2 isoforms in rat striatal astrocytes Patrizia Zanassi a, Mayra Paolillo a , b, Sergio Schinelli a ,* a
Istituto di Farmacologia della Facolta` di Farmacia, Universita` di Pavia, Viale Taramelli 14, 27100 Pavia, Italy b Casa di cura ‘Villa Chiarugi’, 84014 Nocera Inferiore, Italy Received 3 November 1997; received in revised form 6 March 1998; accepted 13 March 1998
Abstract The expression and activity of phospholipase A2 (PLA2) isoforms were investigated in primary cultures of striatal astrocytes. The calcium ionophore A23187 together with the protein kinase C activator phorbol ester was the most potent stimulus in eliciting [3H]arachidonic acid release in the extracellular medium. Reverse transcription coupled to polymerase chain reaction (RT-PCR) showed the presence of the 85 kDa cytosolic PLA2 mRNA and the 14 kDa secretory PLA2 mRNA in untreated astrocytes. Immunoblot experiments with isoform-specific antibodies showed the presence of the cytosolic PLA2 in untreated astrocytes, while the secretory PLA2 was detected only in lipopolysaccharide-treated astrocytes. These data suggest that the two PLA2 isoforms expressed in striatal astrocytes might play different roles in cellular processes mediated by astrocytes. 1998 Elsevier Science Ireland Ltd.
Keywords: Striatal astrocytes; PLA2 isoforms; Arachidonic acid
Phospholipase A2 (PLA2), which catalyzes the hydrolysis of membrane phospholipids releasing free fatty acids and lysophospholipids, is widespread distributed in the brain where it plays a crucial role in several cellular processes. Two main isoforms of mammalian Ca2+-dependent PLA2 have been cloned and sequenced: a 14 kDa secretory PLA (sPLA2) and a 85 kDa cytosolic PLA2 (cPLA2) [5,6]. These two isoforms have different subcellular distribution and Ca2+ requirement for activation: sPLA2 is secreted and activated by millimolar Ca2+ concentrations; cPLA2 selectively hydrolyzes arachidonic acid (AA) containing phospholipids, it is mainly cytosolic and it is activated by micromolar Ca2+ concentrations. In the brain, arachidonic acid (AA) and its metabolites are formed upon PLA2 activation and are involved in signal transduction pathways, modulation of ion channels and neurotransmitter release [10]. Previous works have shown that astrocytes are involved in the regulation of inflammatory processes [2] and in AA release [12], but the PLA2 isoforms that mediate these responses have not been characterized. In this study, we show, by * Corresponding author. Tel.: +39 382 507395; fax: +39 382 507405; e-mail: [email protected]
molecular and biochemical approaches, that unstimulated striatal astrocytes in culture express both the Ca2+ dependent 85 kDa cPLA2 and the 14 kDa sPLA2 mRNA together with the cytosolic Ca2+-dependent cPLA2 protein. In contrast, the 14 kDa sPLA2 protein and a PLA2 activity in membranes and extracellular medium are detectable only when striatal astrocytes are stimulated by an inflammatory agent such as lipopolysaccharide. Striata were removed from 14–15-dayold rat embryos (Morini, Italy) and astrocytes were prepared as reported [7]. When indicated, confluent striatal astrocytes were washed twice with Dulbecco’s modified Eagle’s medium and incubated for 10 h with 100 ng/ml bacterial lipopolysaccharide (LPS) in fresh growing medium. At the end of the incubation, the proteins in the extracellular medium were concentrated by microcon cartridge (Amicon) and the cells were gently scraped with a rubber policeman in 1 ml of ice-cold lysis buffer (10 mM Tris pH 7.4, 1 mM EGTA, 5 mg/ml aprotinin and 0.1 mM phenylmethylsulfonyl fluoride). The homogenate was sonicated for 5 s at 4°C and the suspension was centrifuged for 15 min at 40 000 × g to separate cytosolic fraction (surnatant) from membrane fraction (pellet). For AA release, untreated striatal astrocyte were labeled for 20 h with [3H]AA (200 Ci/mmol; Amer-
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sham, UK) and then washed three times (0.5 ml/well) with HEPES buffer HEPES buffer (composition in mM: NaCl 154, KCl 5.6, NaHCO3 3.6, CaCl2 1.3, MgCl2 0.5, HEPES 10, glucose 10, pH 7.4 containing 0.2% fatty-acid-free BSA, saturated with 5% CO2 –air mixture). The astrocytes were stimulated for 10 min at 37°C with indicated agents and [3H]AA release in the HEPES buffer was measured as previously described [11]. For RT-PCR experiments, total RNA was extracted from untreated astrocytes by a standard method [3] and then treated with 10 U ribonuclease-free deoxyribonuclease (DNase) I (Promega) for 15 min at 37°C to destroy possible contaminating genomic DNA. The DNase was then digested with 15 mg proteinase K (Promega) at 70°C for 20 min and the RNA was then phenol/chloroform extracted and ethanol precipitated. An aliquot of total RNA (2 mg) was added to 20 ml of RT buffer containing 1 mM random hexamer, 0.5 mM of each dNTP, 15 unit RNasin and 12.5 units of avian mieloblastoma virus (AMV) reverse transcriptase (all reagents obtained from Promega). After incubation for 45 min at 42°C, the reaction was stopped by heating the mixture to 95°C for 5 min and chilled on ice. The RT mixture was diluted to 100 ml with water and 5 ml of this solution was used as template for the PCR. An ‘hot start’ PCR protocol was carried out using AmpliWax PCR Gems (Perkin Elmer). A PCR mixture (total volume 30 ml) containing 50 pmol each of forward and reverse primer, 50 mM dNTPs and PCR buffer was dispensed into reaction tube and then an AmpliWax gem was added. The mixture was first heated to 80°C for 5 min to melt the wax gems and then cooled to 30°C; 20 ml of solution containing the template cDNA and two units of Taqpolymerase (Promega) were then layered over the solid wax. The PCR was carried out for 36 cycles (1 min at 92°C, 1 min at 60°C, 1 min at 72°C) with a final extension step of 10 min at 72°C. The primers were: 5′-ATTACGTTAATGGATGCCAATTAT-3′ forward and 5′-TTTCTCTGGAAA-ATCAGGGTGAGA-3′ reverse for cPLA2; 5′AGCCTTCTGGAGTTTGGGCA-AATG-3′ forward and 5′-TTGTTGGGGTAGAACTGGTACTTT-3′ reverse for sPLA2. An aliquot (10 ml) of PCR amplified mixture was separated by 1.7% agarose gel electrophoresis and bands visualized by ethidium bromide staining. For immunodetection experiments, proteins in the extracellular medium, cytosol and membranes were resolved by 7.5% polyacrylamide, 1% SDS gel electrophoresis for cPLA2 (30 mg proteins/lane) or 12.5% polyacrylamide, 1% SDS gel electrophoresis for sPLA2 (150 mg proteins/lane). The proteins in the gels were transferred onto nitrocellulose membranes by semidry blotting and, after blocking for 2 h at room temperature in Tris buffered saline (TBS; 50 mM Tris and 150 mM NaCl, pH 7.4) containing 5% low-fat dry milk, the blots were incubated for 6 h with the cPLA2 or sPLA2 rabbit polyclonal antibodies (dilution 1:1000, gift of Dr. Kramer; Eli Lilly, USA) in TBS-2.5% low-fat dry milk. After washing, the blots were incubated with alkaline phosphatase-conjugated (AP) goat anti-rabbit IgG (1:5000
dilution) and the signal was detected by adding the AP substrate 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium (BCIP/NBT) for 15 min. PLA2 activity in cytosol, membranes and extracellular medium obtained from untreated or LPS-treated astrocytes was measured as reported [13]. The PLA2 activity is expressed as nmol of pyrenedecanoate (PD) formed/mg protein/s. All data, unless otherwise indicated, are expressed as the mean ± SEM values of three independent determinations each performed in triplicate. Values of P were calculated by Student’s t-test or ANOVA followed by post hoc Student-Newman–Keul’s test. When different agents were tested for their ability in inducing [3H]AA release, the protein kinase C activator phorbol-12-myristate-13-acetate (PMA; 1 mM) weakly stimulated [3H]AA release (120% of basal); both 5 mM A23187 alone (165% of basal) and 5 mM A23187 plus 1 mM PMA (250% of basal) significantly increased [3H]AA release compared to the basal value (Fig. 1). The effect of A23187 plus PMA was synergistic, since the amount of released [3H]AA induced by these agents was more than additive compared to that induced by A23187 alone or PMA alone. The A23187 plus PMA-induced AA release was suppressed by 95.1 ± 7.5% in striatal astrocytes pretreated for 30 min with the aspecific PLA2 inhibitor mepacrine (50 mM), suggesting that AA results mainly from activation of PLA2. Moreover, preincubation of striatal astrocytes for 30 min with the sPLA2 inhibitor manoalide (1 mM) did not modify (99.7 ± 6.3% of control) the stimulated [3H]AA release and in the same conditions the compound p-bromophenacylbromide (BPB) augmented (122.3 ± 9.8% of control), rather than inhibited, stimulated [3H]AA release, probably by interfering with reuptake of [3H]AA into membrane phospholipids.
Fig. 1. [3H]AA release in untreated striatal astrocytes. Astrocytes were stimulated by 5 mM A23187 alone, 1 mM PMA alone or by 5 mM A23187 plus 1 mM PMA. Results are expressed as percentage of basal [3H]AA release (1.09 ± 0.11% of [3H]adenine converted to [3H]cAMP). *P , 0.05 compared to basal [3H]AA release.
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The expression of PLA2 isoforms mRNAs in untreated striatal astrocytes was studied by RT-PCR using specific primers for the two PLA2 isoforms. The PCR amplified products (Fig. 2), detected on ethidium bromide stained agarose gel, matched the expected size for cPLA2 (473 bp, lane 1) and sPLA2 (347 bp, lane 2). No PCR products were found when the RT step was omitted (not shown). In cytosolic extracts, a polyclonal antibody raised against cPLA2 recognized a protein of about 100 kDa; the amount of cPLA2 was not modified by the LPS treatment (Fig. 3a, lanes 1 and 2). It should be noted that although the cPLA2 cDNA sequence predicts a molecular weight of 85 kDa, the protein migrates with an apparent molecular weight of 100– 110 kDa on SDS-PAGE [5]. The negative control, carried out by preadsorbing the primary antibody with recombinant cPLA2, confirmed the identity of the observed signal (Fig. 3a, lane 3, PR). In parallel experiments, using 150 mg of membranes (Fig. 3b, lanes 1 and 2) or proteins found in the extracellular medium (Fig. 3b, lanes 3 and 4), the 14 kDa sPLA2 band was detected in astrocytes only following a 10 h treatment with bacterial LPS. Cytosol obtained from untreated striatal astrocytes showed either a basal Ca2+-independent (3.1 ± 0.6 nmol PD released/mg protein per s) and a Ca2+-dependent PLA2 activity (about 7.6-fold increase compared to basal value, P , 0.05); in membranes of untreated striatal astrocytes, the basal Ca2+-independent PLA2 activity (0.8 ± 0.1 nmol PD released/mg protein per s) was not significantly enhanced by the addition of Ca2+. In contrast, the LPS treatment evoked an increase of the Ca2+-dependent PLA2 activity either in membranes (about 2.1-fold, P , 0.05) and in the extracellular medium (about 5.4-fold, P , 0.05) without modifying the cytosolic Ca2+-dependent PLA2 activity (not shown). In this study we demonstrate for the first time that rat striatal astrocytes grown in primary culture coexpress the Ca2+-dependent 85 kDa cPLA2 mRNA and the 14 kDa sPLA2 mRNA, assessed by RT-PCR. Our results suggest that, although both PLA2 isoforms trancripts are present,
Fig. 2. Ethidium bromide staining of cPLA2 and sPLA2 RT-PCR products in striatal astrocytes. Total RNA (2 mg) was retrotranscribed and the cDNA was submitted to 36 PCR cycles. PCR products sizes are 473 bp for cPLA2 (lane 1) and 347 bp for sPLA2 (lane 2). The experiment was repeated twice with similar results.
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Fig. 3. cPLA2 and sPLA2 immunoblots of proteins extracts from LPS treated (+) and untreated (−) striatal astrocytes. (a) cPLA2 immunoblots of protein extracts obtained from the cytosol; PR indicates that the anti-cPLA2 antibody was incubated in the presence of recombinant cPLA2; (b) sPLA2 immunoblots of protein extracts obtained from membranes or extracellular medium. The experiment was repeated twice with similar results.
in normal conditions in striatal astrocytes the Ca2+-dependent cPLA2 isoform mainly mediates the receptor-dependent stimulation of AA release. A synergistic effect in eliciting [3H]AA release is induced by the PKC activator PMA and the calcium ionophore A23187, in agreement with the observation that the Ca2+-dependent cPLA2 activity is synergistically stimulated by calcium and phorbol esters via a PKC-dependent phosphorylation of cPLA2 [8]. Moreover, the [3H]AA release was prevented by the non-selective PLA2 inhibitor mepacrine, but not by BPB or manoalide, two compounds that inhibit sPLA2 selectively over cPLA2 [2]. Finally, the cytosol obtained from untreated astrocytes showed a Ca2+-dependent PLA2 activity and contains an immunoreactive 100 kDa band (cPLA2) whose molecular weight corresponds to that of the cloned Ca2+-dependent 85 kDa cPLA2. Interestingly, the finding that striatal astrocytes possess also a cytosolic Ca2+-independent PLA2 activity seems to suggest that these cells may express Ca2+independent PLA2 isoforms recently described in other cell systems [5]. Several papers have described the presence and regulation of cPLA2 expression in astrocytes. Stephenson et al. [15] reported the colocalization of glial fibrillary acidic protein together with cPLA2 immunoreactivity in human cerebral cortex sections; these authors also found that hippocampal astrocytes and microglia obtained from rats subjected to ischemia showed a 3-fold increase of cPLA2 immunoreactivity [4]. A new role of cPLA2 in modulating the antiinflammatory response in astrocytes has been emphasized in a recent paper by Stella et al. [14]; these authors found that in murine striatal astrocytes a prolonged
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treatment with interleukin-1b induces an increase in Ca2+dependent cPLA2 protein expression and Ca2+-dependent cPLA2-induced AA release. Since we report here that the Ca2+-dependent cPLA2 expression is not changed by LPS treatment, further studies are necessary to elucidate the exact mechanisms by which different proinflammatory stimuli regulate PLA2 isoforms expression in astrocytes. Our results indicate that either sPLA2 levels and PLA2 activity associated with the membranes or released in the extracellular medium are extremely low in unstimulated astrocytes; the sPLA2 protein and a PLA2 activity became detectable only after LPS treatment. Our data, together with the finding that an increase of PLA2 activity in the incubation medium and of sPLA2 mRNA synthesis were found in LPS-treated rat astrocytes [9], strongly suggest that the increased PLA2 activity and sPLA2 levels in membranes or extracellular medium are due to new synthesis of sPLA2 protein and not to a stimulus-dependent secretion of preexisting cellular sPLA2 protein. Two explanations are possible for our results; a fraction of the newly formed sPLA2 is first delivered to the membranes before being released in the extracellular medium or alternatively sPLA2 is released in the extracellular medium and then an aliquot reassociates with the membranes. The coexpression of at least two PLA2 isoforms raises the intriguing possibility that in astrocytes different PLA2 isoforms could regulate the spatiotemporal pattern of AA release in response to extracellular stimuli. This mechanism has been described in LPS-pretreated murine P388D1 macrophages stimulated by platelet activating factor, in which cPLA2 mediates the cell-associated AA accumulation whereas sPLA2 mediates AA release into the incubation medium [1]. Given the multitude of cellular physiological and pathological processes activated by PLA2 activation in astrocytes, further studies aimed to elucidate the specific contribution of PLA2 isoforms in these processes are necessary to targeting of new therapeutic interventions. Part of this work was performed when S.S. was a recipient of an EMBO short term fellowship at INSERM U.109, Paris, France. The authors thank Dr. Piomelli for constructive criticism, Dr. R.M. Kramer (Eli Lilly, USA) and Dr. J. Ishizaki (Shionogi Research Laboratories, Japan) for providing PLA2 antibodies and sPLA2 cDNA.
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