ANALYTICAL
BIOCHEMISTRY
173-180
1%
(19%)
Measurement of Arachidonic Acid Release from Human Polymorphonuclear Neutrophils and Platelets: Comparison between Gas Chromatographic and Radiometric Assays Chakkodabylu
S. Ramesha’
and Leslie
Lipid Biochemistry Group, Department Syntex Research, Palo Alto, California
Received
June
A. Taylor
of Inflammation 94303
Biology, Institute
Academic
Press,
Inc.
Stimulated synthesis of eicosanoids has been implicated in the pathology of many of the inflammatory diseases (1,2). It is widely accepted that the release of ara-
1 To whom 7400. 0003-2697191
Copyright All
rights
Sciences,
20, 1990
A simple gas chromatographic method for the assay of phospholipase A, (PLA,) has been described in which arachidonic acid released from endogenous phospholipid pools is measured following its extraction and derivatization to pentafluorobenzyl esters. Using this asPLA, activities in control and calcium say, ionophore-stimulated human neutrophils, as well as in control, thrombin, and calcium ionophore stimulated human platelets, have been measured. These values are compared with those obtained by monitoring the release of radioactivity from [3H]- or [“Clarachidonic acid prelabeled cells. While the radiometric assay measures only the release of exogenously incorporated radioactive arachidonic acid, the gas chromatographic assay measures arachidonic acid released from all the endogenous pools. Thus, the apparent increase in PLA, activity in stimulated cells measured by the gas chromatographic assay is four- to fivefold higher than that by the radiometric assay. Inclusion of fatty acid free bovine serum albumin in the reaction buffer significantly increases the amount of arachidonic acid that is measured by gas chromatography. The gas chromatographic method has also been successfully utilized for measuring PLA, activity in cell-free preparations derived from physically disrupted human neutrophils. 0 1991
of Biological
correspondence
should
$3.00 0 1991 by Academic Press, of reproduction in any form
be addressed.
Fax:
(415)
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chidonic acid (20:4, n - 6)2 is the rate-limiting step in eicosanoid synthesis (3,4). Of the several pathways that have been documented for the release of arachidonic acid, which is almost exclusively esterified to the m-2 position of phospholipids, phospholipase A, (PLA,; EC 3.1.1.4) has been shown to play a major role (4,5). Therefore, inhibition of the PLA, activity that is responsible for the release of arachidonic acid may be therapeutically beneficial. Because of the direct involvement of neutrophils and other leucocytes in the inflammatory processes, there is increased interest in the characterization and pharmacological manipulation of PLA, activity in these cells (6,7). Radiometric assays have been extensively used to measure PLA, activity in intact cells and to monitor its inhibition by different drugs. In these assays, radiolabeled arachidonic acid is first incorporated into intact cells and then the release of label from the cells following stimulation is measured (8,9). These assays are not only very sensitive, but also less labor intensive. However, in such experiments it is usually assumed that the exogenously added 20:4 has equilibrated among different phospholipid pools and molecular species. The validity of such assumptions has been questioned because of the existence of heterogenous pools of 20:4-containing phospholipids and the complex kinetics involved in the relative rates of esterification and de-esterification of the exogenously added 20:4 with respect to these pools (10,111. It has been clearly demonstrated in recent stud-
* Abbreviations used: 20:4, arachidonic acid; BSA, bovine serum albumin; PMN, neutrophilic polymorphonuclear leukocyte; HBSS, Hanks’ balanced salt solution; DMSO, dimethyl sulfoxide; PLA,, phospholipase AZ; PFB, pentafiuorobenzyl bromide; GC, gas chromatography. 173
Inc. reserved.
174
RAMESHA
AND TAYLOR
ies that following radiolabeled 20:4 incorporation, the specific radioactivities of all phosphoglyceride molecular species varied with respect to each other at all time points studied. Even 2 h after incorporation, most of the radioactivity remained esterified to diacyl phosphoglycerides while the bulk of the endogenous 20:4 was associated with alkyl and alkenyl pools. Because of this discrepancy in the equilibration of the exogenous labeled 20:4 into endogenous pools, the radiometric assay measures the release of 20:4 mostly from the diacyl phospholipid pools. Thus, the amount of endogenous 20:4 released could be much higher than that measured by the radiometric assay, which would thus represent an underestimation of the total PLA, activity. In cell-free preparations, the PLA, activity is also often measured by monitoring the release of labeled fatty acids from synthetic phospholipid substrates containing radiolabeled fatty acids in sn-2 position. Such assays often give variable PLA, activities depending upon the nature and the amount of the substrate used, as well as the composition of the assay systems (12). In the present study we have taken a more direct approach to determining PLA, activity in stimulated neutrophils and platelets by measuring the amount of 20:4 released from endogenous pools. This assay has also been used for measuring the PLA, activity in cell-free preparations. The values thus obtained for PLA, activity have been compared with those obtained by radiometric methods. These findings not only pointed out major discrepancies between measuring mass and radiolabeled products to determine PLA, activity but also showed that the gas chromatographic assay is equal or more sensitive than the radiometric assay. MATERIALS
AND
METHODS
Materials Pentafluorobenzyl bromide and molecular sieves (size 5A) were purchased from Aldrich (Milwaukee, WI). Diisopropylethylamine, fatty acid free bovine serum albumin (BSA), sodium heparin, aprotinin, and calcium ionophore (A23187) were from Sigma (St. Louis, MO). Heneicosanoic acid was purchased from Nu Check (Elysian, MN). [5,6,8,9,11,12,14,15-3H]Arachidonic acid (specific activity, 94 to 240 Ci/mmol) and [1-“Clarachidonic acid (specific activity 55 mCi/mmol) were purchased from DuPont New England Nuclear (Boston, MA). All solvents were of analytical grade and were purchased from Burdick and Jackson (Muskegon, MI). and were kept anhydrous by storing over molecular sieve beads (size 5A). Methods Isolation of human neutrophils. Venous blood from healthy donors was collected in siliconized vacutainers
containing either EC.TA (5 mM) or sodium heparin (10 U/ml blood). The ne utrophilic polymorphonuclear leukocytes (PMN) were isolated by using Mono Poly Resolving Media (Flow Labs, McLean, VA) (13). Contaminating red blood cell 3 were removed by hypotonic lysis. The PMNs were suspended in either Tris (50 mM, pH 7.4) buffer containirtg NaCl (134 mM), KC1 (4.6 mM), MgCl, (1 mM), and &rcose (5 mM) or Hanks’ balanced salt solution (HBSS Gibco) containing Hepes (25 mM, pH 7.4) and glucose (9.6 mM). Isolation of humc:n platelets. Venous blood from healthy donors was collected as described above and centrifuged at 200g f )r 15 min. The platelet-rich plasma was removed and ct ntrifuged at 800g for 10 min. The supernatant was discarded and the platelet pellet was suspended in Tris bl offer (15 mM Tris) containing NaCl (134 mM), glucose (E mM), and EGTA (1 mM) (pH 7.4), and the platelets wtre washed once by centrifuging at 800g for 10 min. The! platelet pellet was resuspended in the above buffer to a final density of 2-3 x 10’ platelets/ml. Incorporation of rcldioactive fatty acid into neutrophils and platelets. Since low specific activity [i’C]arachidonic acid caused s gnificant clumping of PMNs, high specific activity [3H] arachidonic acid was used for labeling PMNs. Twenty microcuries of [3H]arachidonic acid was added with defttted serum albumin to 10 ml of the PMN suspensions. I’he PMNs were incubated at 37°C for 15-60 min. Lonl;er incubation time (30 and 60 min) did not enhance eitl ler the amount of the label released or the fold changes lipon stimulation. Therefore, 15-min incubation was choc en. The reaction was terminated by adding ice-cold bufler. The cells were removed by centrifugation (4OOg,l( min) and washed once with 10 ml of buffer at room temperature. The washed PMNs were resuspended to the: r original density in fresh buffer at room temperature. I’he platelets were similarly labeled, except that 2 &i o E[“Clarachidonic acid was used instead of [3H]arachi ionic acid. Preparation of Pil IiV homogenates. A 5- to lo-ml suspension of PMNs ill the Tris buffer (2-3 X 10’ cells/ml) was incubated witlt or without addition of CaCl, (2.5 mM, final concentrrltion) and A23187 (2.5 pM, final concentration) for 5 nin at 37°C. The incubation was stopped by adding 198% of the cells. The homoge-
ARACHIDONIC
ACID
MEASUREMENT
BY
GAS
nate was centrifuged at 400g for 10 min at 4“C to remove intact cells. The supernatant was used as the enzyme source for PLA, and as the source of membrane phospholipid substrate. Phospholipase A, assay on intact PMN. PMN (4-5 X lo6 cells) were preincubated in calcium-free buffer and in the absence or the presence of BSA (0.1 to 0.25%) at 37°C for 3 min and then were stimulated with Ca2+ (2.5 mM) alone or with Ca2+ and A23187 (2.5 PM), and incubated at 37°C for the desired lengths of time. The reaction was stopped by adding 1 ml of methanol to each tube. A known amount (l-l.5 pg) of heneicosanoic acid (21:0) was added as the internal standard and the lipids were extracted by the procedure of Bligh and Dyer (14). The lipids were dried under a stream of nitrogen. The free fatty acids were derivatized to pentafluorobenzyl (PFB) esters and were separated and quantitated by gas liquid chromatography as described below. The PLA, activity was expressed as picomoles of arachidonic acid released/lo’ cells. The PLA, activity in [3H]arachidonic acid prelabeled cells was measured by fi-scintillation counting after the reaction was stopped by adding EDTA (20 mM final concentration) and the cells were removed by centrifugation at 14,000g for 2 min; an aliquot of the supernatant was counted for radioactivity. The PLA, activity was expressed as counts per minute released/lo’ cells. Phospholipase A, assay in intact platelets. A 0.25- to 0.5-ml suspension of platelets (2-3.5 X 106platelets/ml) was preincubated in the absence or the presence of BSA (0.1 to 0.25%) at 37°C for 3 min and then the incubation was continued with buffer alone or with the addition of thrombin (5 U/ml) or A23187 (10 PM). After the desired incubation time, the reaction was stopped by adding 1 ml of methanol, and the samples were processed as described above for intact PMNs. PLA, activity was expressed as picomoles of arachidonic acid released/lO’ platelets. The PLA, activity in [14C]-20:4 labeled platelets was measured by P-scintillation counting after the reaction was stopped by adding EDTA (20 mM final concentration) and the platelets were removed by centrifugation at 14,000g for 2 min; an aliquot of the supernatant was counted for radioactivity. The PLA, activity was expressed as counts per minute released/lo8 platelets. Phospholipase A, assay in PMN homogenates. A 0.25 to 0.5 ml sample of PMN homogenate, representing l-2 X lo6 cells and containing 3 to 6 mg protein, was preincubated at 37°C for 3 min, and the reaction was started by adding 50 ~1 of buffer with or without 5 mM CaCl, (final concentration). The incubation was continued for selected time periods, the reaction was stopped by adding 1 ml of methanol, and the samples were processed as described for intact PMN. PLA, activity was expressed as picomoles of arachidonic acid released/ milligram of protein.
CHROMATOGRAPHY
AND
175
RADIOMETRY
21:o
21:o
Ah A
.
I
.
12
8
Retention
14 Time
16
16
20
(min)
FIG. 1. Gas chromatographic profiles of PFB esters of fatty acids released from (A) control PMNs and (B) 2.5 pM A23187-stimulated PMNs. Fatty acid 21:0 was added as internal standard.
Inhibition studies. Intact PMNs (4-5 X lo6 cells) were incubated for 15 min at 37°C with desired concentrations of inhibitors either in DMSO (l%), in ethanol (l%), or with the carrier vehicle alone. Cell activation was initiated by adding CaCl, (2.5 mM) and A23187 (2.5 PM), and the incubation was continued for 10 min at 37°C. The reaction was stopped by adding 1 ml methanol and the samples were processed for the PLA, assay. The effect of different compounds on PMN PLA, activity was expressed as percentage of the control value obtained in the presence of carrier vehicle alone. Protein estimation. Protein content in the PMN homogenate and in membrane preparations were estimated by the method of Bradford (16), using BSA as the standard. Derivatization of free fatty acids to PFB esters. The free fatty acids were derivatized to PFB esters as described by Strife and Murphy (15). To the dried total lipid extract from the PLA, reaction was added 200 ~1 of anhydrous methylene chloride, 10 ~1 of diisopropylethylamine, and 20 ~1 of 20% pentafluorobenzyl bromide in anhydrous acetonitrile. The contents were mixed well and allowed to stand at room temperature for 20 min. Solvents and excess reagents were dried under a stream of N, and the derivatives were redissolved in 200 to 400 ~1 of gas chromatographic grade ethyl acetate and taken for gas chromatographic analysis. Although large amounts of phospholipids and other neutral lipids were present in the total lipid extract, there was no need to separate fatty acids from these lipids either before the derivatization or before gas chromatographic analysis. A typical chromatogram is shown in Fig. 1. The fatty acids 21:0 and 20:4 were identified by GC-MS (15).
176
RAMESHA
AND
10’ 6.
0
15
30
Minutes
45
60
0
15
30
45
60
Minutes
FIG. 2. Time course of release of arachidonic acid: (A) Unlabeled PMN incubated in control buffer (0) or A23187 (2.5 pM) (0) and arachidonyl PFB esters measured by electron capture detection after separation by gas chromatography and (B) [3H]-20:4-labeled PMN incubated in control buffer (0) or A23187 (2.5 PM) (0) and the 3H-labe1 released was measured by radiometry. Each value represents mean + SE of three to six experiments performed in duplicate or triplicate.
Gas chromatography. Analysis of the fatty acid PFB esters was performed on a Shimadzu Model 15A gas chromatograph equipped with an electron capture detector, AOC-6 autoinjector, and C-R4A data processor. The PFB esters were separated on a fused silica capillary column (30 m X 0.25 mm i.d.) coated with SP2330 phase (Supelco, Ballafonte, PA). The injector temperature was 260°C and the detector temperature was 290°C. The initial column temperature was 6O”C, and was increased to 160°C at a rate of 30”Clmin and then to 240°C at a rate of lO”C/min. Helium was used as the carrier gas (0.8 ml/min) and nitrogen was used as the makeup gas. Two-microliter samples in ethyl acetate were injected in splitless mode and the arachidonic acid peak was quantitated using the internal standard heneicosanoic acid (C value, 21). Statistical analysis. The data were analyzed by using Student’s t test. RESULTS
Figure 2A shows the time course of unlabeled 20:4 release by A23187-stimulated human PMN. Unstimulated cells did not show any significant level of free arachidonic acid during the same period of time. Release of 3H-label from [3H]-20:4-labeled PMN (Fig. 2B) also followed the same pattern upon stimulation with A23187. However, [3H]arachidonic acid labeled cells also released a significant amount of radioactivity under basal conditions. When the stimulated increase in the PLA, activity was expressed as a multiple of the unstimulated value, significant differences were observed between the as-
TAYLOR
says for release of unlabeled and labeled 20:4 (Table 1). The apparent increase in the PLA, activity was significantly greater when measured by assessing total 20:4 release with the GC assay as compared to assessment by the radiometric assay. The same was true in the case of platelets, irrespective of the stimulus used, the platelets showed a greater apparent increase in the PLA, activity when assayed by GC than when assayed radiometrically (Table 2). When the unlabeled PMN or platelets were stimulated with A23187 in the presence of 0.1 to 0.25% fatty acid-free bovine serum albumin (BSA), there was a significant increase in the amount of 20:4 released (Table 3). Since the presence of BSA did not have any effect on the amount of 20:4 released by unstimulated cells, the apparent increase in PLA, activity in the A23187-stimulated cells was greater in the presence than in the absence of BSA. Since significant PLA, activity was observed in intact neutrophils, we assessedwhether this activity could be detected in disrupted cell preparations using the GC method. In this case, the reaction was initiated by adding calcium (5 mM, final concentration) to the cell-free preparation containing both the enzyme and membrane phospholipid substrate. Significant PLA, activity was found in homogenates prepared from both control and A23187-stimulated cells (Fig. 3). The amount of arachidonic acid released by the homogenate from A23187treated PMNs was higher than that released from control PMN homogenate at each time assessedup to 60 min, even though the Ca*+ concentration was identical in both preparations. Furthermore, the cell-free preparations from PMNs which were preincubated in the presence of CaCl, (2.5 mM) without ionophore for 5 min had a greater apparent PLA, activity than those from PMN which were preincubated in the absence of calcium. Most of the 20:4 release observed in the homogenate could be inhibited by 1 mM EGTA, indicating that the 20:4 release in this assay proceeded via a Ca2+-dependent process. When the PLA, activities in intact PMN and homogenates were compared, the homogenates from unstimulated cells showed 250 to 300% more PLA, activity than did the unstimulated intact cells, whereas the homogenate from A23187-stimulated PMN showed only 15 to 30% of the PLA, activity detected in corresponding intact cells (Table 4). Using intact PMN and both GC and radiometric assay methods, two known PLA, inhibitors were tested for their ability to inhibit the release of arachidonic acid from endogenous sources (Table 5). The concentrations of each inhibitor needed to achieve 50% inhibition of PLA, was essentially equivalent as assessed by each method. DISCUSSION
In the present study a new and more direct approach has been applied to measuring 20:4 releasing phospholi-
ARACHIDONIC
ACID
MEASUREMENT
BY
GAS
CHROMATOGRAPHY
TABLE Comparison
of PLA,
Activity
in Human
PMN
AND
1
Measured
by Gas
Chromatography
pmol
None (control) A23187 (2.5
Fold increase
of 20~4 released/ 10’ cells/l0 min 1.8 * 1.0 34.1 f 8.3*,*
PM)
and
Radiometry
Radiometry
GC
Treatment
177
RADIOMETRY
cpm released/lo7 cells/l0 min”
Fold increase
2511 -1- 900 10286 + 2381**
1 18.6
1 4.1
Note. Unlabeled PMN or PMN labeled with [3H]-20:4 were incubated at 37°C in the absence (control) or the presence of 2.5 pM A23187 for 10 min. The released arachidonic acid from unlabeled PMN was measured by gas chromatography. The released 3H-radioactivity from [3H]-20:4labeled PMN were measured by p-scintillation counting. Values are the mean + SE (n = 5-12). Each experiment was done in duplicate or triplicate. ’ Control and A23187-stimulated cells released 3-8 and 12-30% of the incorporated radioactivity, respectively. b P values: *97%) observed in the cell-free preparations was Cazf dependent (Fig. 3). However, it is interesting to note that the homogenate prepared from cells preincubated in Ca2+ (2.5 mM) containing buffer had significantly higher PLA, activity than that prepared from cells which were not exposed to Ca2+. Furthermore, the homogenate prepared from cells which were preincubated in Ca2+ (2.5 mM) and A23187 (2.5 pM) containing buffer had even more PLA, activity than that prepared from the cells which were preincubated with Ca2+ alone (Fig. 3); this occurred despite the fact that the final Ca2+ concentration in the assay mixture was the same (5 mM) in each case. In preliminary studies using Ca2+ -sensitive indo-l dye, when PMNs preincubated in Ca2+-free medium were subsequently exposed to Ca2+ (2.5 mM), there was an immediate rise in intracellular calcium which reached a peak between 2-3 min; the rise in the intracellular Ca2+ was even greater in the presence of both Ca2+ (2.5 mM) and ionomycin (2.5 PM), a nonfluorescent analogue of A23187 (Ramesha, C., unpublished data). Although the mechanism by which this initial increase in intracellular Ca2+
GAS
CHROMATOGRAPHY
AND
179
RADIOMETRY
might enhance the PLA, activity subsequently measured in a cell-free preparation has not been defined, it is possible that the Ca2+ might be involved in transloeating a soluble cytosolic PLA, into a membrane-associated compartment, wherein its catalytic activity was enhanced. Such Ca2+-dependent translocation of a cytosolic PLA, to a membrane-associated compartment form has been recently demonstrated for renal medullary cells (32). The results presented here demonstrate that in terms of sensitivity the gas chromatographic assay is equal or more sensitive than the radiometric assay. However, the advantage of the gas chromatographic assay over the radiometric assay for identifying inhibitors of cellular PLA, in intact cells and cell-free systems could not be rigorously validated because of paucity of potent inhibitors of cellular PLA,. The two compounds tested showed very similar PLA, inhibition in both gas chromatographic and radiometric assays (Table 4). This is not unexpected since these compounds have been shown to inhibit a wide variety of PLA, enzymes without specificity (33-35). However, the gas chromatographic assay may prove to be advantageous for selecting novel PLA, inhibitors because: (i) the PLA, activity measured is more physiological and less subjected to variation in substrate equilibrium, substrate composition and substrate presentation; (ii) the signal-to-noise ratio for stimulated vs basal PLA, activities are improved greatly; and (iii) the same assay can be used for measuring PLA, activities in intact cells and cell-free preparations. It should be noted that the gas chromatographic measurements of PLA, activities in these studies were based solely on the amount of arachidonic acid released from the endogenous pools, and that small quantities of certain other fatty acids were also released under the assay conditions. Since our main purpose was to measure that PLA, activity responsible for releasing 20:4 in these cells and cell-free preparations and to use these systems to select compounds that would inhibit the arachidonic acid releasing activity of PLA,, we restricted ourselves to the quantitation of 20:4. However, by modifying the gas chromatographic conditions, the present method could also be used for quantitating the extent of release of other fatty acids from endogenous membrane bound substrates. ACKNOWLEDGMENTS We thank Dr. D. V. K. Murthy reading of the manuscript.
and Dr. Robert
Lewis
for the critical
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BIOCHEMISTRY
173-180
1%
(19%)
Measurement of Arachidonic Acid Release from Human Polymorphonuclear Neutrophils and Platelets: Comparison between Gas Chromatographic and Radiometric Assays Chakkodabylu
S. Ramesha’
and Leslie
Lipid Biochemistry Group, Department Syntex Research, Palo Alto, California
Received
June
A. Taylor
of Inflammation 94303
Biology, Institute
Academic
Press,
Inc.
Stimulated synthesis of eicosanoids has been implicated in the pathology of many of the inflammatory diseases (1,2). It is widely accepted that the release of ara-
1 To whom 7400. 0003-2697191
Copyright All
rights
Sciences,
20, 1990
A simple gas chromatographic method for the assay of phospholipase A, (PLA,) has been described in which arachidonic acid released from endogenous phospholipid pools is measured following its extraction and derivatization to pentafluorobenzyl esters. Using this asPLA, activities in control and calcium say, ionophore-stimulated human neutrophils, as well as in control, thrombin, and calcium ionophore stimulated human platelets, have been measured. These values are compared with those obtained by monitoring the release of radioactivity from [3H]- or [“Clarachidonic acid prelabeled cells. While the radiometric assay measures only the release of exogenously incorporated radioactive arachidonic acid, the gas chromatographic assay measures arachidonic acid released from all the endogenous pools. Thus, the apparent increase in PLA, activity in stimulated cells measured by the gas chromatographic assay is four- to fivefold higher than that by the radiometric assay. Inclusion of fatty acid free bovine serum albumin in the reaction buffer significantly increases the amount of arachidonic acid that is measured by gas chromatography. The gas chromatographic method has also been successfully utilized for measuring PLA, activity in cell-free preparations derived from physically disrupted human neutrophils. 0 1991
of Biological
correspondence
should
$3.00 0 1991 by Academic Press, of reproduction in any form
be addressed.
Fax:
(415)
354-
chidonic acid (20:4, n - 6)2 is the rate-limiting step in eicosanoid synthesis (3,4). Of the several pathways that have been documented for the release of arachidonic acid, which is almost exclusively esterified to the m-2 position of phospholipids, phospholipase A, (PLA,; EC 3.1.1.4) has been shown to play a major role (4,5). Therefore, inhibition of the PLA, activity that is responsible for the release of arachidonic acid may be therapeutically beneficial. Because of the direct involvement of neutrophils and other leucocytes in the inflammatory processes, there is increased interest in the characterization and pharmacological manipulation of PLA, activity in these cells (6,7). Radiometric assays have been extensively used to measure PLA, activity in intact cells and to monitor its inhibition by different drugs. In these assays, radiolabeled arachidonic acid is first incorporated into intact cells and then the release of label from the cells following stimulation is measured (8,9). These assays are not only very sensitive, but also less labor intensive. However, in such experiments it is usually assumed that the exogenously added 20:4 has equilibrated among different phospholipid pools and molecular species. The validity of such assumptions has been questioned because of the existence of heterogenous pools of 20:4-containing phospholipids and the complex kinetics involved in the relative rates of esterification and de-esterification of the exogenously added 20:4 with respect to these pools (10,111. It has been clearly demonstrated in recent stud-
* Abbreviations used: 20:4, arachidonic acid; BSA, bovine serum albumin; PMN, neutrophilic polymorphonuclear leukocyte; HBSS, Hanks’ balanced salt solution; DMSO, dimethyl sulfoxide; PLA,, phospholipase AZ; PFB, pentafiuorobenzyl bromide; GC, gas chromatography. 173
Inc. reserved.
174
RAMESHA
AND TAYLOR
ies that following radiolabeled 20:4 incorporation, the specific radioactivities of all phosphoglyceride molecular species varied with respect to each other at all time points studied. Even 2 h after incorporation, most of the radioactivity remained esterified to diacyl phosphoglycerides while the bulk of the endogenous 20:4 was associated with alkyl and alkenyl pools. Because of this discrepancy in the equilibration of the exogenous labeled 20:4 into endogenous pools, the radiometric assay measures the release of 20:4 mostly from the diacyl phospholipid pools. Thus, the amount of endogenous 20:4 released could be much higher than that measured by the radiometric assay, which would thus represent an underestimation of the total PLA, activity. In cell-free preparations, the PLA, activity is also often measured by monitoring the release of labeled fatty acids from synthetic phospholipid substrates containing radiolabeled fatty acids in sn-2 position. Such assays often give variable PLA, activities depending upon the nature and the amount of the substrate used, as well as the composition of the assay systems (12). In the present study we have taken a more direct approach to determining PLA, activity in stimulated neutrophils and platelets by measuring the amount of 20:4 released from endogenous pools. This assay has also been used for measuring the PLA, activity in cell-free preparations. The values thus obtained for PLA, activity have been compared with those obtained by radiometric methods. These findings not only pointed out major discrepancies between measuring mass and radiolabeled products to determine PLA, activity but also showed that the gas chromatographic assay is equal or more sensitive than the radiometric assay. MATERIALS
AND
METHODS
Materials Pentafluorobenzyl bromide and molecular sieves (size 5A) were purchased from Aldrich (Milwaukee, WI). Diisopropylethylamine, fatty acid free bovine serum albumin (BSA), sodium heparin, aprotinin, and calcium ionophore (A23187) were from Sigma (St. Louis, MO). Heneicosanoic acid was purchased from Nu Check (Elysian, MN). [5,6,8,9,11,12,14,15-3H]Arachidonic acid (specific activity, 94 to 240 Ci/mmol) and [1-“Clarachidonic acid (specific activity 55 mCi/mmol) were purchased from DuPont New England Nuclear (Boston, MA). All solvents were of analytical grade and were purchased from Burdick and Jackson (Muskegon, MI). and were kept anhydrous by storing over molecular sieve beads (size 5A). Methods Isolation of human neutrophils. Venous blood from healthy donors was collected in siliconized vacutainers
containing either EC.TA (5 mM) or sodium heparin (10 U/ml blood). The ne utrophilic polymorphonuclear leukocytes (PMN) were isolated by using Mono Poly Resolving Media (Flow Labs, McLean, VA) (13). Contaminating red blood cell 3 were removed by hypotonic lysis. The PMNs were suspended in either Tris (50 mM, pH 7.4) buffer containirtg NaCl (134 mM), KC1 (4.6 mM), MgCl, (1 mM), and &rcose (5 mM) or Hanks’ balanced salt solution (HBSS Gibco) containing Hepes (25 mM, pH 7.4) and glucose (9.6 mM). Isolation of humc:n platelets. Venous blood from healthy donors was collected as described above and centrifuged at 200g f )r 15 min. The platelet-rich plasma was removed and ct ntrifuged at 800g for 10 min. The supernatant was discarded and the platelet pellet was suspended in Tris bl offer (15 mM Tris) containing NaCl (134 mM), glucose (E mM), and EGTA (1 mM) (pH 7.4), and the platelets wtre washed once by centrifuging at 800g for 10 min. The! platelet pellet was resuspended in the above buffer to a final density of 2-3 x 10’ platelets/ml. Incorporation of rcldioactive fatty acid into neutrophils and platelets. Since low specific activity [i’C]arachidonic acid caused s gnificant clumping of PMNs, high specific activity [3H] arachidonic acid was used for labeling PMNs. Twenty microcuries of [3H]arachidonic acid was added with defttted serum albumin to 10 ml of the PMN suspensions. I’he PMNs were incubated at 37°C for 15-60 min. Lonl;er incubation time (30 and 60 min) did not enhance eitl ler the amount of the label released or the fold changes lipon stimulation. Therefore, 15-min incubation was choc en. The reaction was terminated by adding ice-cold bufler. The cells were removed by centrifugation (4OOg,l( min) and washed once with 10 ml of buffer at room temperature. The washed PMNs were resuspended to the: r original density in fresh buffer at room temperature. I’he platelets were similarly labeled, except that 2 &i o E[“Clarachidonic acid was used instead of [3H]arachi ionic acid. Preparation of Pil IiV homogenates. A 5- to lo-ml suspension of PMNs ill the Tris buffer (2-3 X 10’ cells/ml) was incubated witlt or without addition of CaCl, (2.5 mM, final concentrrltion) and A23187 (2.5 pM, final concentration) for 5 nin at 37°C. The incubation was stopped by adding 198% of the cells. The homoge-
ARACHIDONIC
ACID
MEASUREMENT
BY
GAS
nate was centrifuged at 400g for 10 min at 4“C to remove intact cells. The supernatant was used as the enzyme source for PLA, and as the source of membrane phospholipid substrate. Phospholipase A, assay on intact PMN. PMN (4-5 X lo6 cells) were preincubated in calcium-free buffer and in the absence or the presence of BSA (0.1 to 0.25%) at 37°C for 3 min and then were stimulated with Ca2+ (2.5 mM) alone or with Ca2+ and A23187 (2.5 PM), and incubated at 37°C for the desired lengths of time. The reaction was stopped by adding 1 ml of methanol to each tube. A known amount (l-l.5 pg) of heneicosanoic acid (21:0) was added as the internal standard and the lipids were extracted by the procedure of Bligh and Dyer (14). The lipids were dried under a stream of nitrogen. The free fatty acids were derivatized to pentafluorobenzyl (PFB) esters and were separated and quantitated by gas liquid chromatography as described below. The PLA, activity was expressed as picomoles of arachidonic acid released/lo’ cells. The PLA, activity in [3H]arachidonic acid prelabeled cells was measured by fi-scintillation counting after the reaction was stopped by adding EDTA (20 mM final concentration) and the cells were removed by centrifugation at 14,000g for 2 min; an aliquot of the supernatant was counted for radioactivity. The PLA, activity was expressed as counts per minute released/lo’ cells. Phospholipase A, assay in intact platelets. A 0.25- to 0.5-ml suspension of platelets (2-3.5 X 106platelets/ml) was preincubated in the absence or the presence of BSA (0.1 to 0.25%) at 37°C for 3 min and then the incubation was continued with buffer alone or with the addition of thrombin (5 U/ml) or A23187 (10 PM). After the desired incubation time, the reaction was stopped by adding 1 ml of methanol, and the samples were processed as described above for intact PMNs. PLA, activity was expressed as picomoles of arachidonic acid released/lO’ platelets. The PLA, activity in [14C]-20:4 labeled platelets was measured by P-scintillation counting after the reaction was stopped by adding EDTA (20 mM final concentration) and the platelets were removed by centrifugation at 14,000g for 2 min; an aliquot of the supernatant was counted for radioactivity. The PLA, activity was expressed as counts per minute released/lo8 platelets. Phospholipase A, assay in PMN homogenates. A 0.25 to 0.5 ml sample of PMN homogenate, representing l-2 X lo6 cells and containing 3 to 6 mg protein, was preincubated at 37°C for 3 min, and the reaction was started by adding 50 ~1 of buffer with or without 5 mM CaCl, (final concentration). The incubation was continued for selected time periods, the reaction was stopped by adding 1 ml of methanol, and the samples were processed as described for intact PMN. PLA, activity was expressed as picomoles of arachidonic acid released/ milligram of protein.
CHROMATOGRAPHY
AND
175
RADIOMETRY
21:o
21:o
Ah A
.
I
.
12
8
Retention
14 Time
16
16
20
(min)
FIG. 1. Gas chromatographic profiles of PFB esters of fatty acids released from (A) control PMNs and (B) 2.5 pM A23187-stimulated PMNs. Fatty acid 21:0 was added as internal standard.
Inhibition studies. Intact PMNs (4-5 X lo6 cells) were incubated for 15 min at 37°C with desired concentrations of inhibitors either in DMSO (l%), in ethanol (l%), or with the carrier vehicle alone. Cell activation was initiated by adding CaCl, (2.5 mM) and A23187 (2.5 PM), and the incubation was continued for 10 min at 37°C. The reaction was stopped by adding 1 ml methanol and the samples were processed for the PLA, assay. The effect of different compounds on PMN PLA, activity was expressed as percentage of the control value obtained in the presence of carrier vehicle alone. Protein estimation. Protein content in the PMN homogenate and in membrane preparations were estimated by the method of Bradford (16), using BSA as the standard. Derivatization of free fatty acids to PFB esters. The free fatty acids were derivatized to PFB esters as described by Strife and Murphy (15). To the dried total lipid extract from the PLA, reaction was added 200 ~1 of anhydrous methylene chloride, 10 ~1 of diisopropylethylamine, and 20 ~1 of 20% pentafluorobenzyl bromide in anhydrous acetonitrile. The contents were mixed well and allowed to stand at room temperature for 20 min. Solvents and excess reagents were dried under a stream of N, and the derivatives were redissolved in 200 to 400 ~1 of gas chromatographic grade ethyl acetate and taken for gas chromatographic analysis. Although large amounts of phospholipids and other neutral lipids were present in the total lipid extract, there was no need to separate fatty acids from these lipids either before the derivatization or before gas chromatographic analysis. A typical chromatogram is shown in Fig. 1. The fatty acids 21:0 and 20:4 were identified by GC-MS (15).
176
RAMESHA
AND
10’ 6.
0
15
30
Minutes
45
60
0
15
30
45
60
Minutes
FIG. 2. Time course of release of arachidonic acid: (A) Unlabeled PMN incubated in control buffer (0) or A23187 (2.5 pM) (0) and arachidonyl PFB esters measured by electron capture detection after separation by gas chromatography and (B) [3H]-20:4-labeled PMN incubated in control buffer (0) or A23187 (2.5 PM) (0) and the 3H-labe1 released was measured by radiometry. Each value represents mean + SE of three to six experiments performed in duplicate or triplicate.
Gas chromatography. Analysis of the fatty acid PFB esters was performed on a Shimadzu Model 15A gas chromatograph equipped with an electron capture detector, AOC-6 autoinjector, and C-R4A data processor. The PFB esters were separated on a fused silica capillary column (30 m X 0.25 mm i.d.) coated with SP2330 phase (Supelco, Ballafonte, PA). The injector temperature was 260°C and the detector temperature was 290°C. The initial column temperature was 6O”C, and was increased to 160°C at a rate of 30”Clmin and then to 240°C at a rate of lO”C/min. Helium was used as the carrier gas (0.8 ml/min) and nitrogen was used as the makeup gas. Two-microliter samples in ethyl acetate were injected in splitless mode and the arachidonic acid peak was quantitated using the internal standard heneicosanoic acid (C value, 21). Statistical analysis. The data were analyzed by using Student’s t test. RESULTS
Figure 2A shows the time course of unlabeled 20:4 release by A23187-stimulated human PMN. Unstimulated cells did not show any significant level of free arachidonic acid during the same period of time. Release of 3H-label from [3H]-20:4-labeled PMN (Fig. 2B) also followed the same pattern upon stimulation with A23187. However, [3H]arachidonic acid labeled cells also released a significant amount of radioactivity under basal conditions. When the stimulated increase in the PLA, activity was expressed as a multiple of the unstimulated value, significant differences were observed between the as-
TAYLOR
says for release of unlabeled and labeled 20:4 (Table 1). The apparent increase in the PLA, activity was significantly greater when measured by assessing total 20:4 release with the GC assay as compared to assessment by the radiometric assay. The same was true in the case of platelets, irrespective of the stimulus used, the platelets showed a greater apparent increase in the PLA, activity when assayed by GC than when assayed radiometrically (Table 2). When the unlabeled PMN or platelets were stimulated with A23187 in the presence of 0.1 to 0.25% fatty acid-free bovine serum albumin (BSA), there was a significant increase in the amount of 20:4 released (Table 3). Since the presence of BSA did not have any effect on the amount of 20:4 released by unstimulated cells, the apparent increase in PLA, activity in the A23187-stimulated cells was greater in the presence than in the absence of BSA. Since significant PLA, activity was observed in intact neutrophils, we assessedwhether this activity could be detected in disrupted cell preparations using the GC method. In this case, the reaction was initiated by adding calcium (5 mM, final concentration) to the cell-free preparation containing both the enzyme and membrane phospholipid substrate. Significant PLA, activity was found in homogenates prepared from both control and A23187-stimulated cells (Fig. 3). The amount of arachidonic acid released by the homogenate from A23187treated PMNs was higher than that released from control PMN homogenate at each time assessedup to 60 min, even though the Ca*+ concentration was identical in both preparations. Furthermore, the cell-free preparations from PMNs which were preincubated in the presence of CaCl, (2.5 mM) without ionophore for 5 min had a greater apparent PLA, activity than those from PMN which were preincubated in the absence of calcium. Most of the 20:4 release observed in the homogenate could be inhibited by 1 mM EGTA, indicating that the 20:4 release in this assay proceeded via a Ca2+-dependent process. When the PLA, activities in intact PMN and homogenates were compared, the homogenates from unstimulated cells showed 250 to 300% more PLA, activity than did the unstimulated intact cells, whereas the homogenate from A23187-stimulated PMN showed only 15 to 30% of the PLA, activity detected in corresponding intact cells (Table 4). Using intact PMN and both GC and radiometric assay methods, two known PLA, inhibitors were tested for their ability to inhibit the release of arachidonic acid from endogenous sources (Table 5). The concentrations of each inhibitor needed to achieve 50% inhibition of PLA, was essentially equivalent as assessed by each method. DISCUSSION
In the present study a new and more direct approach has been applied to measuring 20:4 releasing phospholi-
ARACHIDONIC
ACID
MEASUREMENT
BY
GAS
CHROMATOGRAPHY
TABLE Comparison
of PLA,
Activity
in Human
PMN
AND
1
Measured
by Gas
Chromatography
pmol
None (control) A23187 (2.5
Fold increase
of 20~4 released/ 10’ cells/l0 min 1.8 * 1.0 34.1 f 8.3*,*
PM)
and
Radiometry
Radiometry
GC
Treatment
177
RADIOMETRY
cpm released/lo7 cells/l0 min”
Fold increase
2511 -1- 900 10286 + 2381**
1 18.6
1 4.1
Note. Unlabeled PMN or PMN labeled with [3H]-20:4 were incubated at 37°C in the absence (control) or the presence of 2.5 pM A23187 for 10 min. The released arachidonic acid from unlabeled PMN was measured by gas chromatography. The released 3H-radioactivity from [3H]-20:4labeled PMN were measured by p-scintillation counting. Values are the mean + SE (n = 5-12). Each experiment was done in duplicate or triplicate. ’ Control and A23187-stimulated cells released 3-8 and 12-30% of the incorporated radioactivity, respectively. b P values: *97%) observed in the cell-free preparations was Cazf dependent (Fig. 3). However, it is interesting to note that the homogenate prepared from cells preincubated in Ca2+ (2.5 mM) containing buffer had significantly higher PLA, activity than that prepared from cells which were not exposed to Ca2+. Furthermore, the homogenate prepared from cells which were preincubated in Ca2+ (2.5 mM) and A23187 (2.5 pM) containing buffer had even more PLA, activity than that prepared from the cells which were preincubated with Ca2+ alone (Fig. 3); this occurred despite the fact that the final Ca2+ concentration in the assay mixture was the same (5 mM) in each case. In preliminary studies using Ca2+ -sensitive indo-l dye, when PMNs preincubated in Ca2+-free medium were subsequently exposed to Ca2+ (2.5 mM), there was an immediate rise in intracellular calcium which reached a peak between 2-3 min; the rise in the intracellular Ca2+ was even greater in the presence of both Ca2+ (2.5 mM) and ionomycin (2.5 PM), a nonfluorescent analogue of A23187 (Ramesha, C., unpublished data). Although the mechanism by which this initial increase in intracellular Ca2+
GAS
CHROMATOGRAPHY
AND
179
RADIOMETRY
might enhance the PLA, activity subsequently measured in a cell-free preparation has not been defined, it is possible that the Ca2+ might be involved in transloeating a soluble cytosolic PLA, into a membrane-associated compartment, wherein its catalytic activity was enhanced. Such Ca2+-dependent translocation of a cytosolic PLA, to a membrane-associated compartment form has been recently demonstrated for renal medullary cells (32). The results presented here demonstrate that in terms of sensitivity the gas chromatographic assay is equal or more sensitive than the radiometric assay. However, the advantage of the gas chromatographic assay over the radiometric assay for identifying inhibitors of cellular PLA, in intact cells and cell-free systems could not be rigorously validated because of paucity of potent inhibitors of cellular PLA,. The two compounds tested showed very similar PLA, inhibition in both gas chromatographic and radiometric assays (Table 4). This is not unexpected since these compounds have been shown to inhibit a wide variety of PLA, enzymes without specificity (33-35). However, the gas chromatographic assay may prove to be advantageous for selecting novel PLA, inhibitors because: (i) the PLA, activity measured is more physiological and less subjected to variation in substrate equilibrium, substrate composition and substrate presentation; (ii) the signal-to-noise ratio for stimulated vs basal PLA, activities are improved greatly; and (iii) the same assay can be used for measuring PLA, activities in intact cells and cell-free preparations. It should be noted that the gas chromatographic measurements of PLA, activities in these studies were based solely on the amount of arachidonic acid released from the endogenous pools, and that small quantities of certain other fatty acids were also released under the assay conditions. Since our main purpose was to measure that PLA, activity responsible for releasing 20:4 in these cells and cell-free preparations and to use these systems to select compounds that would inhibit the arachidonic acid releasing activity of PLA,, we restricted ourselves to the quantitation of 20:4. However, by modifying the gas chromatographic conditions, the present method could also be used for quantitating the extent of release of other fatty acids from endogenous membrane bound substrates. ACKNOWLEDGMENTS We thank Dr. D. V. K. Murthy reading of the manuscript.
and Dr. Robert
Lewis
for the critical
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