Role of Phospholipase A2 Isozymes in Agonist-Mediated Signaling in Proximal Tubular Epithelium
Abstract—Angiotensin II in proximal tubule epithelium is known to stimulate the release of arachidonic acid after stimulation of phospholipase A2 (PLA2) independent of phospholipase C–mediated signaling. Furthermore, an angiotensin II type 2 receptor subtype has been linked to this signaling cascade. We investigated the regulation and differential stimulation of PLA2s by comparing the PLA2 activities associated with the membranes and cytosol of rabbit renal proximal tubular epithelial cells after stimulation with angiotensin II, epidermal growth factor, and bradykinin. Both fractions demonstrated PLA2 activity that was dithiothreitol insensitive, required micromolar concentrations of Ca2+ for optimal activity, and was inhibited in a dose-dependent manner by an antiserum to a cytosolic PLA2 with a molecular mass of 85 kD. However, membrane-associated PLA2 did not demonstrate significant substrate specificity, whereas 1-steroyl-2-[14C]arachidonylphosphatidyl choline was the preferred substrate for cPLA2. An antiserum generated against mastoparan, a known PLA2 activator, inhibited membrane- but not cytosol-associated PLA2 activity. Membrane fractions showed a broad pH range (7.5 to 8.5) for optimal PLA2 activity, whereas cytosol was maximum at pH 9.5. Angiotensin II stimulated membrane-associated PLA2 activity by 88%, whereas bradykinin and epidermal growth factor inhibited activity by 54% and 41%, respectively. The three agonists stimulated cPLA2. Moreover, angiotensin II–induced activation of membrane-associated PLA2 preceded the activation of cPLA2. These results demonstrate differential localization and regulation of proximal tubular epithelial PLA2 isozymes, which may determine the pattern of subsequent arachidonic acid metabolism by the cytochrome P450 system.
- phospholipase A2
- angiotensin II
- mitogen-activated protein kinase
PLA2 isozymes as a group hydrolyze the sn-2 fatty acyl ester bond of membrane phospholipids, generating free fatty acids and lysophospholipids.1 2 This mechanism of AA release is believed to be a rate-limiting step in the synthesis of a variety of eicosanoids that are critical modulators of transport, vasoreactivity, and inflammation.1 PLA2 isozymes have been broadly classified into sPLA2 and cPLA2 isozymes.3 The sPLA2 isozymes have a molecular mass of ≃14 to 18 kD and are regulated by Ca2+.3 The cPLA2 isozymes have been classified as either Ca2+ regulated or Ca2+ independent.3 The Ca2+-regulated isoforms have a molecular mass of ≃85 to 110 kD,4 5 6 whereas the Ca2+-independent isozyme has a molecular mass of ≃40 kD. There is negligible sequence homology between the secreted and cytosolic PLA2 isoforms.7 Membrane-associated PLA2 isozymes (20 to 45 kD) also have been described; however, they are less well characterized.6 8
A variety of PLA2 isozymes have been described in renal tissues ranging in molecular mass from 14 to 110 kD.4 5 6 9 Despite the fact that cPLA2 has been linked to a variety of agonists in mesangial cells, including vasopressin, PLA2 isoforms and their regulation have been poorly described in kidney epithelial cells. During anoxic renal injury, both membrane-associated and cytosolic PLA2 activations occur.9 10 An angiotensin II type 2 receptor subtype has been linked to Ca2+-independent apically oriented PLA2 activation in kidney epithelial cells.11 Furthermore, Ang II has been demonstrated to release AA from BBMVs independent of PLC,12 13 providing more support for the involvement of membrane-associated PLA2. We reasoned that the initiation of this signaling cascade would involve activation of a membrane-associated PLA2 followed by the release of AA. We have shown that Ang II and/or AA activates a series of kinases, including MAPK,14 which are then known to phosphorylate and activate PLA2 activity in the cytosol.
The aim of the present study was to test the hypothesis that activation of a membrane-associated PLA2 by Ang II is independent of cPLA2. By using membrane and cytosolic fractions isolated from the rabbit renal epithelial cells, we demonstrate that the membrane-associated PLA2 activity may be regulated differently than the activity present in the cytosol. Ang II stimulates membrane-associated PLA2, whereas other agonists that bind to a kinase receptor (EGF) and a PLC-coupled receptor (BK) inhibit this isozyme. All three agonists stimulate PLA2 activity associated with the cytosol. This, coupled with their differences in temporal stimulation and their reactivity toward various antisera, further emphasizes that the membrane-associated PLA2 activity may be regulated differently than that present in the cytosol.
Renal proximal tubule epithelial cells were isolated from male New Zealand White rabbits as described previously.15 16 Briefly, the method involves homogenization of the renal cortex and separation of fully dissociated cells on a discontinuous 30% to 60% Percoll gradient. Cells with a density of ≈1.026 g/mL were removed and cultured in Costar tissue culture flasks according to the method of Chung et al.17 These cells have been described as possessing the usual characteristics of proximal tubular epithelium.15 The standard growth medium was a 50:50 mixture of Dulbecco’s modified Eagle’s and Ham’s F12 media supplemented with 15 mmol/L HEPES buffer, pH 7.35, 1.2 mg/mL sodium bicarbonate, 192 IU/mL penicillin, 200 μg/mL streptomycin, 5 μg/mL bovine insulin, 5 μg/mL human transferrin, 5×10−8 mol/L hydrocortisone, and 5% FBS. Cells from the 1.026 g/mL Percoll fraction had been shown previously to be derived mainly from the proximal tubule.13 15 16 18 Cells were passaged after 2 weeks after dissociation with trypsin and EDTA. These first-passage cells were deprived of serum for 16 hours before the following experiments were performed.
Agonists or a diluent was added to the medium with 0.5% fatty acid–free BSA. The cells were stimulated with the agonists for 10 minutes (unless otherwise stated), and the incubation was terminated by discarding the medium, rinsing three times, and scraping the cells into 1 mL of homogenization buffer consisting of 50 mmol/L HEPES, pH 7.4, 0.25 mol/L sucrose, 1 mmol/L EDTA, 1 mmol/L EGTA, 60 μmol/L leupeptin, 60 μmol/L pepstatin, 1 mmol/L phenylmethylsulfonyl fluoride, and 1000 U/mL Trasylol. Cells were homogenized with 30 strokes in a Dounce homogenizer. The homogenate was spun at 280 000g for 1 hour in a Beckman ultracentrifuge with a Ti 65 rotor. The resulting high-speed supernatant was designated as cytosol fraction, and the pellet was designated as the membrane fraction.
Assay of PLA2 Activity
PLA2 activity was assayed according to a variation of the method of Ballou et al19 using APC as a substrate unless stated otherwise. Cytosol and membrane fractions from diluent or agonist exposed cells were diluted to achieve identical protein concentrations. Substrates were dried under nitrogen and resuspended in dimethylsulfoxide. Two microliters of APC or another substrate (15 μmol/L) was pipetted into an Eppendorf microcentrifuge tube. Reactions were initiated by the addition of 34 μL of 5 to 25 μg of protein and 3 mmol/L free Ca2+; the reaction mixture was incubated for 30 minutes at 37°C. The reaction was terminated by the addition of 40 μL of ethanol with 2% (v/v) acetic acid and 10% of 5 mg/mL free AA. Released AA was analyzed using silica gel thin-layer chromatography plates (LK6D; Whatman). Fifty microliters of the reaction mixture was spotted, and the plates were developed in the organic phase of ethyl acetate/isooctane/water/acetic acid (55:75:100:10), respectively. Lipids were visualized by iodine staining. With this system, free AA migrates near the front (Rf of ≈0.5), whereas unhydrolyzed phospholipid remains at the origin. The free AA bands were scraped and counted in 5 mL of Bio-safe scintillation fluid (Research Products International Corp). Blank samples were run routinely containing no cell extract. PC was quantified in membranes in agreement with reported values for rat kidney cortical tubules.20 PE was undetectable with this method in the membrane fraction. Both were below the limit of detectability in cytosol. Hence, corrections are made for total (85.6 nmol/mg of protein) based on these estimated and reported values for PE.20
Immunochemical Inactivation of Enzyme Using Antiserum Raised Against Mastoparan
The membranes were solubilized after incubation with 0.4% octylglucopyranoside (final concentration) on ice for 90 minutes. Cytosol was incubated with the detergent as a control. Membranes and cytosol were preincubated with a 1:200 dilution of mastoparan antiserum at 37°C for 15 minutes before the assay for the PLA2 activities.
SDS-PAGE and Immunoblotting
The cytosolic and membrane fractions were suspended in SDS-sample buffer and electrophoresed in 8% gels (BioRad) at 90 mA for 18 to 20 hours at room temperature. Standard proteins (high molecular mass from BioRad), prestained standards (BioRad), and recombinant cPLA2 (human cPLA2) as positive control (kindly provided by Dr Ruth Kramer, Eli Lilly, Indianapolis, Ind) also were routinely electrophoresed. After electrophoresis, the proteins were transferred at 60 V for 3 hours (BioRad) onto a Millipore polyvinylidene difluoride membrane. The membranes were blocked overnight at 4°C in blocking buffer containing 5% milk. Membranes were then incubated at 4°C with the primary antibodies (rabbit anti-human cPLA2 antiserum [690, 13/B061493]; kindly provided by Dr Ruth Kramer) in the blocking solution for 2 hours. After being washed three times (15 minutes each), the membranes were incubated in the secondary antibodies (1:5000 dilution), conjugated with horseradish peroxidase, in the blocking solution at room temperature for 1 hour. They were washed five times (15 minutes each) and then developed using enhanced chemiluminescence (ECL Kit; Amersham).
Induction of Polyclonal Antiserum Against Mastoparan
Mastoparan (3 mg) was conjugated to keyhole limpet hemocyanin (10 mg) by the use of glutaraldehyde (21 mmol/L) in the presence of 0.1 mol/L phosphate buffer, pH 7.0. The conjugated peptide (65 μg) was dialyzed against the phosphate buffer to remove glutaraldehyde, mixed with an equal volume of complete Freund’s adjuvant, and subcutaneously injected into New Zealand White rabbits. Four weeks later, rabbits received a booster immunization with 32.5 mg of the conjugated peptide in incomplete Freund’s adjuvant. Rabbits then received a booster immunization every 4 weeks, and antisera were collected 2 weeks after the third booster immunization.
Total phospholipids were analyzed as described previously.21 Phospholipids were extracted according to the Bligh-Dyer method from the membrane and cytosol fractions. Samples (in duplicate), along with phospholipid standards, were separated on thin-layer chromatographic plates that were coated with silica gel in chloroform/methanol/20% methylamine (60:36:10, vol/vol/vol). The bands corresponding to PC and PE were cut and transferred to borosilicate glass culture tubes. To the separated PC and PE bands we added 40 μL of 10 N H2SO4 and 70% perchloric acid. The samples were heated to 190°C until dry. After cooling, to the samples, 75 mL of water along with 400 mL of freshly filtered ammonium molybdate/malachite green (1:3) dye was added, and absorbance was measured at 660 nm. The amount of PC and PE was calculated after the standard curve was plotted.
Radioactive phospholipid substrates, APC, and APE were obtained form Amersham. Silica gel LK6D plates were from Whatman. Protein was measured with the use of a protein kit from BioRad. Other reagents were obtained from Sigma Chemical.
Values are given as mean±SEM. Unpaired Student’s t test was used for comparison between two groups. All n values represent the number of experiments conducted with cells from different animals, with each experiment performed in triplicate. All Ca2+ concentrations represent free Ca2+ calculated using the computer program Free Ca (Fabiato). Two-way ANOVA was used for comparison of multiple groups. Statistical significance was considered to be at the level of P<.05.
Previous observations have documented that after the activation of cPLA2, there is translocation to membranes,3 whereas in our experiments, cellular fractions were prepared in the presence of EGTA and EDTA, which results in dissociation of the cPLA2 from the membranes. The PLA2 activity in our membrane fraction was designated as membrane-associated PLA2 activity; hence, it was important to determine the temporal relationship to identify the difference between the PLA2 activities in the membrane and cytosol after stimulation of intact cells with Ang II (Fig 1⇓). Membrane-associated PLA2 was significantly increased at 5 minutes, after exposure of intact cells to Ang II, whereas cPLA2 was increased at 10 minutes.
Ca2+ Requirement for Activity of PLA2 in Membrane and Cytosolic Fractions
We observed that Ca2+ was necessary to optimize the activity of both compartments, with a maximal increase occurring at 1 μmol/L (Fig 2⇓).
Optimal PLA2 activity in the cytosol was observed at pH 9.5 as described previously,3 whereas the membrane-associated PLA2 activity was observed over a broad range with a plateau between pH 7.5 and 8.5 (Fig 3⇓).
With the use of 15 μmol/L APC or APE as exogenous substrates, cytosol-associated PLA2 activity was 25 times more active toward APC than toward APE, whereas membrane-associated activity was only 3.2 times more active toward APC than toward APE (Table⇓).
Because reducing agents have been shown to inhibit sPLA2 activity, DTT was used to determine the influence on PLA2 activity in the membrane versus the cytosolic fractions. DTT (1 mmol/L) did not inhibit enzyme activity associated with either membranes or cytosol, consistent with the absence of sulfhydryl bonds3 (data not shown).
Immunochemical Modulation of Enzyme Activity
We used cPLA2 antisera that recognizes an 85-kD cPLA2 to determine whether differential modulation of enzyme activity might occur (Fig 4⇓). At dilutions of 1:10 000, 1:1000, and 1:100, 19±9%, 83±3%, and 80±4% (n=2) inhibition below basal levels occurred in membrane fraction and 17±2.4%, 75±11%, and 71±9% (n=2) inhibition occurred in cytosolic fractions. Because these values did not differ significantly, there is a suggestion that these isozymes have a similar epitope, mastoparan, a polypeptide isolated from wasp venom has been demonstrated to stimulate AA release from membrane phospholipids by activating PLA2.22 Polyclonal antiserum generated against the polypeptide mastoparan was also used to determine whether membrane-associated versus cytosolic PLA2 activities would be affected. The antiserum, at dilutions of 1:200, significantly inhibited membrane-associated activity by 67±9% (n=3, P<.05) below the basal level; in contrast, cytosolic activity was inhibited by only 21±14% (n=5), which did not differ significantly from basal activity (Fig 5⇓). Preimmune serum was used as control and did not affect either the membrane-associated or the cytosolic PLA2 activity (data not shown). These observations are consistent with differential regulation of PLA2 activity in these cellular compartments.
Agonist Modulation of PLA2 Isozymes
Differential agonist-induced modulation was also observed in that Ang II significantly stimulated the membrane-associated PLA2 activity by 88±34% (n=9, P<.05), whereas bradykinin and EGF inhibited activity by 54±8% (n=7, P<.05) and 41±19% (n=2, P<.05), respectively (Fig 6⇓). However, all agonists significantly increased the cytosolic activity: Ang II by 81±13% (n=10, P<.05), bradykinin by 54±23% (n=5, P<.05), and EGF by 38±5% (n=2, P<.008) (Fig 6⇓). Among this group, Ang II is the only agonist that is not linked to inositol-specific PLC in this cell type, suggesting direct stimulation of membrane-associated PLA2 activity.
Immunoblot Analysis of PLA2
Phosphorylation of cPLA2 by MAPK and translocation from cytosol to membranes have been associated with enzyme activation.23 Phosphorylation, which shifts PLA2 activity in the cytosol (≃100 kD) to a slower migrating isoform,1 was observed with all agonists and was more pronounced with EGF and BK than with Ang II (Fig 7⇓) despite the fact that in our assay, Ang II–stimulated PLA2 activity in both fractions was higher than the activities that resulted from stimulation with either EGF or BK. Furthermore, EGF- and BK-induced phosphorylation of the cytosolic PLA2 activity was also observed in membrane fractions, consistent with translocation from cytosol to membrane (Fig 7⇓). It is of interest that antisera also identified a higher molecular mass band of ≃126 kD in the membrane fraction that was not observed in the cytosol.
Several PLA2 isoforms have been found in the kidney, including cPLA2s (≃40 to 110 kD),10 24 sPLA2 (≃14 to 18 kD),25 and mPLA2 (20 to 45 kD).9 12 26 Membrane-associated PLA2 has been described in proximal tubular epithelium, and activity is modulated after Ang II stimulation of BBMVs and after brief anoxic exposure.9 12 A Ca2+-independent plasmalogen-selective isoform has also been implicated in hypoxic injury to proximal tubular cells.10
The present results document that both membrane-associated and cytosolic PLA2 activation is associated with agonist-induced signaling in renal proximal tubular epithelium. Membrane-associated PLA2 activity appears to be regulated differently than that in the cytosol. Our assay conditions were designed to detect the “classic” cPLA2, as described by a number of groups, in that homogenizations were performed in Ca2+-free buffer with EGTA.4 5 6 We verified that all agonists activate cPLA2 in renal epithelial cells despite differing properties. EGF, which is linked to a kinase receptor, has been shown previously to activate cPLA2 in glomerular mesangium, and the same signaling occurs in epithelial cells.27 28 29 30 BK, which has been shown to be linked to inositol-specific PLC,31 also activates PLA2. Our observations document that Ang II also activates the cPLA2 but by a unique mechanism and may involve prior activation of a membrane-associated PLA2 and release of AA, which we have documented temporally. Moreover, both AA and Ang II have been shown to activate MAPK, which has been linked to phosphorylation of cPLA2.14 Thus, a novel mechanism for MAPK-mediated activation of the PLA2 activity in the cytosol is supported by these observations.
It has been shown previously that EGF induces ω-hydroxylase metabolism of AA, whereas Ang II induces epoxygenase metabolism in proximal tubular epithelium.32 Currently, we observed that Ang II stimulates but BK and EGF inhibit the membrane-associated PLA2 activity, whereas all agonists stimulate the cytosolic activity. The mechanism or mechanisms by which EGF and BK inhibit the membrane activity have yet to be determined. It will be interesting to determine whether the topography of different epithelial PLA2 isozymes and cytochrome P450 isoforms influence the pattern of downstream metabolism of AA by various agonists.
We performed SDS-PAGE and immunoblotting using antiserum raised against cPLA2 to further characterize agonist interactions with the PLA2 activities associated with the membrane and cytosol. We observed that BK and EGF caused a complete phosphorylation-induced shift of PLA2 in both the membrane and cytosolic fractions. Similar phosphorylation shifts involving the activation of PLA2 have been observed in other cell types.1 23 33 The Ang II effect on phosphorylation of cytosolic PLA2 activity was less pronounced, consistent with an alternative mechanism of activation.
We attempted to characterize proximal tubular PLA2 isozymes with respect to physical properties for comparison with other PLA2 isozymes. Both the epithelial PLA2 activities were DTT insensitive, required micromolar concentrations of Ca2+ for optimal activity, and were inhibited in a dose-dependent manner by cPLA2 antiserum, characteristics shared with the majority of intracellular PLA2 isozymes.3 Several differences, in addition to agonist modulation, include the observation that antiserum raised against the peptide mastoparan significantly inhibited the membrane-associated PLA2 activity by 70% in contrast to a negligible effect on the cytosol. We were unsuccessful in determining the molecular mass of the protein or proteins interacting with this antiserum in kidney epithelium; however, in whole kidney, the protein migrates at ≃40 kD on SDS-PAGE (Chung-Ho Chang, unpublished observations). It is possible that the antiserum interacts directly with the activity associated with the membrane or, alternatively, with a regulatory protein.
The optimal pH for cPLA2 activity is known to be in the alkaline range,3 consistent with 9.5 in our cytosolic fraction. However, membrane fraction demonstrated a broad range, which plateaued between pH 7.5 and 8.5. Thus, the pH at which the membrane-associated PLA2 activity was measured failed to optimize cPLA2 activity, supporting the involvement of distinct isozymes. Other differences relate to substrate preferences in which the activity in the cytosolic fraction showed a 25-fold higher activity with APC compared with APE. With membranes, there was only 1.6-fold more activity with APC compared with APE as substrate, further reenforcing the hypothesis that two distinct isoenzymes are involved.
Although our results indicate a strong possibility of the existence of two distinct PLA2 activities, the possibility of the same cytosolic PLA2 activity existing at the membrane that is differentially regulated by different agonists cannot be completely ruled out without isolation, purification, and cloning of the proteins involved.
Thus, in summary, we documented differential regulation of membrane-associated and cytosol PLA2 activity by vasoactive hormones and EGF in proximal tubular epithelium. Ang II, a PLC-independent agonist, activates membrane-associated PLA2 activity and initiates AA metabolism. Activation of the cPLA2 activity follows this initial signaling. In contrast, BK and EGF activate the cytosol-associated PLA2 activity independent of this initial signaling. Some biochemical properties differ in these cellular compartments, suggesting that they represent distinct PLA2 isoforms.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|BBMV||=||brush border membrane vesicles|
|cPLA2||=||cytosolic phospholipase A2|
|EGF||=||epidermal growth factor|
|MAPK||=||mitogen-activated protein kinase|
|mPLA2||=||membrane-associated phospholipase A2|
|PAGE||=||polyacrylamide gel electrophoresis|
|sPLA2||=||secretory phospholipase A2|
- Received October 7, 1996.
- Revision received November 19, 1996.
- Accepted October 15, 1997.
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