(Received for publication, December 17, 1996)
From the Institute of Pharmaceutical Biology, Activated neutrophils release a variety of
eicosanoids into the extracellular medium including arachidonic acid,
5-hydroxyicosatetraenoic acid, and leukotriene A4 and
B4. In this study, the mechanism of arachidonic acid export
has been examined using inside-out plasma membrane vesicles from pig
polymorphonuclear leukocytes. Tritiated arachidonic acid associated
rapidly with the membrane vesicles and crossed the membrane into the
intravesicular space in a time-dependent and saturable
manner. Half the maximal influx rate was measured at an arachidonate
concentration of 5.7 µM, and a maximal influx velocity of
3.0 nmol/mg × min was determined at pH 6.8. Influx into vesicles
was sensitive to a number of common anion transport inhibitors
including pentachlorophenol, phloretin, diiodosalicylic acid, and
quercetin as well as to the proteases trypsin and Pronase, suggesting a
protein-dependent process. Furthermore, influx was
temperature-sensitive with an energy of activation of 11.6 kcal/mol.
Varying extravesicular concentration of ATP, Na+, or
K+ had no impact on arachidonate influx, whereas changes in
pH had a profound effect; optimum transport activity was observed at an
extravesicular pH of 6, whereas raising the pH to 9.5 essentially abolished uptake. These results indicate and initially characterize a
novel protein-facilitated arachidonate export mechanism in pig neutrophils.
Polymorphonuclear leukocytes (PMN)1
are the predominant cell type present in areas of acute inflammation.
Activated neutrophils release a number of eicosanoids such as
arachidonic acid (1-3), 5-hydroxyicosatetraenoic acid, and leukotriene
A4 (4) and B4 (5) into the plasma where they
may amplify or perpetuate the acute inflammatory response. Recent
progress in leukotriene research has led to a more detailed
understanding of the molecular biology and enzymology of the oxidative
metabolism of arachidonic acid and to the development of potent
inhibitors of biosynthesis as well as receptor antagonists interfering
with signal transduction (6, 7). The mechanism of how eicosanoids, once
biosynthesized, leave the producer cell to reach their target cell is,
however, still poorly understood. Recently, an ATP-driven export pump
for cysteinyl leukotrienes has been identified in hepatocytes (8), erythrocytes (9), and heart cells (9, 10). In addition, in intact PMN a
LTB4 export carrier has been identified and characterized (11). To study the cellular export mechanism, whole cell systems are
too complex to allow the accurate measurement of transport kinetics and
of the effects of inhibitors. Furthermore, the situation for PMN is
particularly complex since these cells do not only biosynthesize and
release eicosanoids but also avidly take up and metabolize arachidonic
acid, LTA4 (1, 4), and LTB4 (12).
To overcome those restrictions imposed by whole cell systems to the
investigation of transport mechanisms, we prepared inside-out plasma
membrane vesicles from PMN and investigated the usefulness of this
system to study eicosanoid transport using arachidonic acid as a
ligand. Inside-out plasma membrane vesicles without contamination with
right side-out vesicles were prepared by isolating phagocytic vesicles
from PMN. When a PMN engulfs particles, the ingested material is
encased within a vesicle derived from the plasma membrane with an
inside-out orientation (13). After phagocytosis of albumin-coated
mineral oil droplets, those phagocytic vesicles can readily be isolated
and have been used for Ca2+ export studies (14).
The mechanism of the translocation process of fatty acids through the
plasma membrane in general is controversial (15). On one hand, it is
argued that fatty acid movement through the phospholipid bilayer is an
entirely unregulated process in which diffusion of fatty acids in
either direction of the membrane is driven by concentration gradients
(16). Juxtaposed to the diffusion hypotheses, there is increasing
evidence for a facilitated protein-dependent transport
pathway for long chain fatty acids. Five putative fatty acid transport
proteins have been identified in the plasma membrane of several tissues
including adipocytes, liver, endothelium, brain, intestine, kidney,
lung, skeletal, and cardiac muscle. For three of these cDNA clones
have been isolated (reviewed in Ref. 15). Schaffer and Lodish (17)
first succeeded not only in expression cloning and purification of one
putative carrier protein but could also reconstitute transport
function, thus directly demonstrating its physiological role.
The results reported herein meet several criteria for a
protein-facilitated export mechanism for arachidonic acid in PMN plasma membrane vesicles, such as substrate saturation, inhibition by a
variety of anion transport inhibitors (18), and sensitivity toward
proteases. The ionic mechanism seems to be that of an H+ + arachidonate cotransport system operating with an energy of activation
of 11.6 kcal/mol. With the transport assay presented here, it may be
possible to separate the transport processes out of the complex cascade
of events starting after activation of PMN by various stimuli to study
the mechanism(s) of putative eicosanoid transporters and to investigate
the impact of leukotriene inhibitors on transport.
Mineral oil (M-5904), lipopolysaccharide
O26:B6 (L-3755), phenylmethylsulfonyl fluoride, Triton X-100, and PIPES
dipotassium salt were from Sigma. Ficoll-Paque, dextran T-500, and
ConA-Sepharose were obtained from Pharmacia Biotech Inc. Arachidonic
acid was from Fluka, and
[5,6,8,9,11,12,14,15-3H]arachidonic acid (230.5 Ci/mmol)
was purchased from DuPont NEN. Fatty acid-free bovine serum albumin was
from Boehringer Mannheim.
Arterial blood was
collected from 3 to 4-month old pigs using sterile Na2EDTA
(2.14 g/liter) as an anticoagulant. The whole blood was centrifuged at
200 × g for 15 min, and the platelet-rich supernatant
was discarded. PMN were prepared by dextran sedimentation, hypotonic
lysis of remaining erythrocytes, and centrifugation on Ficoll-Paque, as
described (19).
Purified PMN (50 × 106/ml) were resuspended in modified Hank's buffer (124 mM NaCl, 4.9 mM KCl, 0.66 mM
Na2HPO4, 0.64 mM
KH2PO4, 15 mM NaHCO3,
10 mM Tris-HCl, pH 7.4) and incubated with a mineral oil
emulsion, essentially as described (13). Briefly, 1 volume of mineral
oil and 4 volumes of modified Hank's buffer containing 20 mg/ml fatty
acid-free albumin were mixed and sonicated in a Branson Sonicator (Cell
Disrupter B15), with a micro tip for 2 × 1 min, setting 5. The
oil emulsion was centrifuged for 5 min at 1600 × g,
and the thin layer of coacervated droplets at the surface was removed.
The remaining stable emulsion was mixed with 1 volume of fresh pig
serum. One volume of this oil emulsion was added to 10 volumes of PMN
suspension at 4 °C. Phagocytosis was initiated by the addition of
0.9 mM CaCl2, 1.27 mM
MgCl2, and 10 ng/ml lipopolysaccharide and warmed to
37 °C. After 30 min incubation at 37 °C under gentle stirring,
cells were separated from residual emulsion by centrifugation at
250 × g for 5 min at 4 °C and washed twice with
Hank's buffer. Finally, the cells were suspended in modified
relaxation buffer (20) (100 mM KCl, 3 mM NaCl,
3.5 mM MgCl2, 10 mM PIPES-HCl, pH
6.8), treated with 5 mM MgCl2, 1 mM
1,4-dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and
0.25 mg/ml soybean trypsin inhibitor and subjected to nitrogen
cavitation (200 p.s.i. for 20 min). After addition of 1.25 mM EGTA, the suspension was centrifuged at 48,000 × g for 30 min. Liberated phagocytic vesicles floating on the
top of the tubes were collected with a spatula and resuspended in 250 mM sucrose, 10 mM Tris-HCl, pH 7.4. Inside-out
plasma membrane vesicles were frozen in liquid nitrogen and stored at
ConA affinity chromatography was performed at
4 °C as described by Del Buono et al. (22).
ConA-Sepharose was added to polypropylene columns to give a packed bed
volume of 3 ml, and columns were washed with degassed relaxation buffer
with and without addition of 0.1% BSA. Samples of membrane vesicles or
intact PMN in modified relaxation buffer were added to the columns in a
volume of 1-3 ml and incubated for 10 min. Nonadherent vesicles were
eluted with ice-cold modified relaxation buffer, and protein in the
fractions was quantified by the bicinchoninic acid assay (21). About
95-98% of the phagocytic vesicles were found in the first 6 ml of the eluate, whereas intact PMN almost completely (>95%) adhered to the
columns. Based on the assumption that ConA receptors are found exclusively on the extracellular surface of the plasma membrane of PMN
(22), from these experiments a low contamination of the inside-out
membrane vesicles by right side-out vesicles or plasma membrane
fragments was concluded.
Vesicles in
modified relaxation buffer were fixed in 3% glutaraldehyde at 4 °C
overnight. Material was rinsed with relaxation buffer, followed by a
1.5-h fixation with osmium tetroxide in the same buffer. For en
bloc staining, vesicles were incubated with 1% uranyl acetate in
20% acetone for 1.5 h, and dehydration was performed with a
graded acetone series. Samples were then infiltrated and embedded in
Spurr's low viscosity resin. After polymerization, sections were cut,
mounted on colloidin-coated copper grids, and post-stained with aqueous
lead citrate (3%, pH 13.0). All micrographs were taken on an EM 109 electron microscope (Zeiss, Oberkochen, Germany)
The standard incubation medium contained plasma membrane
vesicles (20 µg of protein) in modified relaxation buffer (100 mM KCl, 3 mM NaCl, 3.5 mM
MgCl2, 10 mM PIPES-HCl, pH 6.8) in a final volume of 200 µl. Tritiated arachidonic acid was added as a 50% ethanolic solution. The final ethanol concentrations never exceeded 0.5% and did not influence transport activity. Since a substantial portion of the added arachidonate bound to the walls of the container, the actual arachidonate concentration in each experiment was determined by scintillation counting of an aliquot of the incubations. All concentrations of arachidonate were corrected for adsorption losses to
container walls. The incubations were carried out at 37 °C and
terminated by the addition of 800 µl of ice-cold stop solution containing modified relaxation buffer, pH 6.8, 0.5 mM
pentachlorophenol, and 0.5% albumin and then further incubated for 5 min on ice. The sample was pipetted onto a Schleicher & Schuell filter
GF 52 (24 mm) and filtered using a filtration manifold (DN 025/1, Schleicher & Schuell, Dassel, Germany). Subsequently, vesicles were
washed with 1 ml of stop solution and 3 ml of relaxation buffer. The
filters were placed in scintillation vials and left overnight in 5 ml
of Rotizint Eco Plus (Roth, Karlsruhe, Germany) before radioactivity
was counted and corrected for adsorption of unbound arachidonate to the
filters.
Vesicles prepared for electron microscopy
consisted primarily of membrane-bound vesicles (0.1-1.2 µm) and some
associated granules. These isolated vesicles were identical to the
phagocytic vesicles seen in intact cells after phagocytosis of
albumin-coated mineral oil droplets. The vesicle membrane was clearly
trilaminar and identical in appearance and dimensions to the plasma
membrane. The membrane encloses the lipid droplet with an amorphous
electron-dense periphery representing the albumin coat of the lipid
droplet. There was essentially no contamination of the vesicles by
mitochondria, glycogen particles, nuclei, or other cytoplasmic matter
except for some granules associated with the vesicles, Fig.
1. Application of phagosomes to a ConA affinity column
revealed that only a small fraction (<2-5%) of the vesicles was
bound to the column, whereas intact PMN adhered to the affinity
material (>95%), demonstrating that the vesicles had indeed the
inside-out orientation with little contamination of plasma membranes
with exposed ConA receptors.
Various
concentrations of [3H]arachidonic acid were added as an
ethanolic solution to a vesicle suspension (20 µg of protein in
modified 200 µl of modified relaxation buffer) and incubated at
37 °C. A significant and constant portion of the added
[3H]arachidonate was bound to the walls of the incubation
container. About 50% of the added [3H]arachidonate was
recovered when the incubation mixture from the Eppendorf tube was
transferred to a scintillation vial and counted for radioactivity. From
the residual arachidonate in the buffer (50%), a constant arachidonate
portion (70.1%) associated with the vesicles in the concentration
range tested (0.24-9772 pmol/sample) and was recovered with the
vesicle membranes after rapid filtration (Fig. 2).
During a 30-min incubation, the vesicle-associated arachidonate
concentrations did not change.
Adsorption of arachidonate to container walls is a common observation
(23), which makes it necessary to correct arachidonate concentrations
for adsorption losses in every experiment. Vesicle association of
unbound arachidonate is thought to represent absorption to the plasma
membrane (24) and uptake into the intravesicular space. To measure
uptake accurately and to distinguish internalized ligand from that
merely bound to the external surface of the vesicles, it is essential
to employ a wash solution that blocks influx and efflux after certain
periods of incubation and removes ligand bound to the external membrane
of the vesicles (25).
In previous studies, washes with a 0.1-0.5% albumin solution with or
without addition of 200 µM of the transport inhibitor phloretin were generally used to remove fatty acids, which are exchangeably bound to the outer membrane leaflet, while preventing efflux (25-29). We employed a 0.4% albumin solution containing 0.4 mM pentachlorophenol (PCP) for this purpose since PCP is a stronger influx/efflux inhibitor in our system.
To evaluate the effectiveness of this wash solution in incubations with
vesicle preparations, vesicle aliquots (20 µg of membrane protein)
were incubated at 37 °C with various amounts of
[3H]arachidonic acid at different time points, and
vesicle association of radioactivity was measured after treatment with
ice-cold BSA/PCP (stop/chase solution) as described under "Materials
and Methods." The vesicle-associated arachidonate resistance toward
BSA/PCP extraction increased linearly within the first 120 s of
incubation at 37 °C. The linear regression line of this time course
was extrapolated to zero time to give the amount of arachidonate that
could not be extracted by the BSA/PCP wash (determined for every
arachidonate concentration tested). As much as 98.3% of the merely
bound arachidonate at zero time could be extracted from the vesicles
with BSA/PCP stop solution. A similar result was obtained when vesicles
were incubated at 0 °C instead of 37 °C, even after prolonged
incubation times.
The non-extractable arachidonate increased in a linear fashion with
increasing amounts of arachidonate (0.24-9772 pmol), Fig. 2. Thus,
under the conditions employed, no saturable binding to putative
receptors became apparent in the concentration range (1.22 nM to 48.9 µM) tested.
Vesicles were incubated with 20 nM
[3H]arachidonate (4 pmol/200 µl) in modified relaxation
buffer, pH 6.8 at 37 °C, and reactions were terminated after various
time points by treatment with ice-cold BSA/PCP stop solution as
described under "Materials and Methods." During incubation, the
non-extractable portion of vesicle-associated arachidonate increased
from 68 fmol (1.7%) at zero time to approximately 1 pmol (58.8%)
after 60 min (total arachidonate corrected for losses to the test tube
represents 1.7 pmol = 100%), Fig. 3. Increase of
non-extractable arachidonate associated with the membrane vesicles could either reflect a time-dependent tight binding to high
affinity binding sites at or in the membrane vesicles or influx to an
as yet undefined compartment within the vesicles that is not accessible to BSA/PCP extraction. To distinguish between these possibilities, incubation of vesicles at 37 °C was terminated after 150 min by addition of ice-cold BSA/PCP stop solution and subjected to a 3 × 10-s sonication treatment on ice and filtered. Almost complete loss of
vesicle-associated arachidonate that had been accumulated during
incubation at 37 °C was observed after physical destruction of the
vesicles by sonication, Fig. 3. To check whether the loss of
radioactivity was due to the loss of sonicated membranes, aliquots of
the same incubation mixture were subjected to sonication and filtration
in the same way as before, except that the BSA/PCP solution was
replaced by albumin-free modified relaxation buffer. These control
experiments revealed that the recovery of vesicle membranes was not
influenced by the sonication procedure, suggesting that the loss of
radioactivity was due to liberation of arachidonic acid from the
intravesicular space. Thus, the time-dependent vesicle association of non-extractable arachidonate is consistent with influx
but not with binding to high affinity binding sites on the vesicle
membranes.
Vesicles (20 µg of protein/200 µl) were
incubated with 4 pmol of arachidonic acid at 37 °C, and internalized
[3H]arachidonic acid was quantitated after BSA/PCP
treatment after various time points by rapid filtration and
scintillation counting. Uptake was linear over a period of 120 s.
To measure influx inhibition by PCP, PCP was added (0.4 mM
final concentration) to aliquots of the suspension after 0 or 75 s. Addition of 0.4 mM PCP prevented influx of arachidonate.
Furthermore, internalized arachidonate concentrations at the time of
PCP addition remained almost at the same level over the entire
incubation period of 1000 s, demonstrating an efficient and long
lasting influx inhibition effect of PCP, Fig. 4.
Since PCP did not only block influx into vesicles over prolonged
incubation times at 37 °C, but also prevented losses of internalized arachidonic acid during the incubation, we tested the hypothesis whether PCP could also prevent vesicle efflux. Incubation of vesicles (20 µg of protein/200 µl) with 4 pmol of arachidonic acid resulted in a time-dependent increase of internalized arachidonate.
Addition of BSA (0.4% final concentration) stopped the accumulation of arachidonate in the vesicles, and a time-dependent loss of
intravesicular arachidonate was observed, reaching close to zero time
levels after 50 min. In contrast, addition of BSA/PCP (0.4 mM PCP, 0.4% BSA) only resulted in a very little loss of
internalized arachidonate.
Inhibition of vesicle association as well as vesicle dissociation of
the non-BSA/PCP extractable arachidonate cannot be explained on the
basis of high affinity binding to putative receptors. Furthermore, inhibition of influx and efflux of arachidonate by the anion transport inhibitor PCP suggested a non-diffusional uptake and release of arachidonate.
To study the effect of common anion transport inhibitors
on arachidonate transport, a variety of structurally different
inhibitors were preincubated with vesicles in modified relaxation
buffer for 15 min before 4 pmol/200 µl of
[3H]arachidonic acid were added. To determine
internalized arachidonate after various incubation periods, 200-µl
aliquots of the suspension (20 µg protein) were pipetted at desired
time points into 0.8 ml of cold 0.5% BSA, 0.5 mM PCP and
filtered after 5 min extraction time. Incubations were performed in
triplicate, and initial influx rates were computed from the slope of
the linear regression line over the first 120 s after arachidonate
addition. Strong inhibition of the initial influx was observed by 1 mM quercetin (11.5 ± 1.5% of control, mean ± S.D.), 0.5 mM phloretin (9 ± 5.6%), 0.5 mM diiodosalicylic acid (10.3 ± 6.8%), and 0.5 mM pentachlorophenol (1.3 ± 0%), respectively (Fig.
5). N-Ethylmaleimide had little, if any,
effect on arachidonate influx. However, three other compounds that
inhibit monocarboxylate (18) or prostaglandin transport (30),
4-acetamido-4
Proteolytic digestion of vesicles is often employed
as a key experiment to decide whether or not proteins are involved in transport processes. Since proteins facilitating transmembrane movement
of ligands are often partially exposed on the membrane surface, they
can be digested by proteases without damaging the membrane barrier
(31). Vesicles were preincubated in modified relaxation buffer with or
without addition of Pronase (1 mg/ml) or trypsin (2.5 mg/ml) overnight.
Preincubation with either Pronase or trypsin at 37 °C strongly
inhibited the initial arachidonate influx into the vesicles. Pronase
and trypsin digestion reduced initial influx rates to 12.7 ± 4.6 and 5.6 ± 2.1% of control (mean ± S.D., n = 3), respectively. Preincubation at 0 °C had no effect on the
influx rates, suggesting that influx inhibition is indeed mediated by
the enzymatic properties of the proteases and not by unspecific protein
binding as seen with albumin.
Inhibition of arachidonate
influx by proteases suggested that at least one step in the mechanism
of arachidonate influx is protein-dependent. Thus,
arachidonic acid influx should be saturable by arachidonate. Initial
arachidonic acid influx rates were determined as described above after
addition of various amounts of arachidonate (final concentration 1.2 nM to 86.5 µM, corrected for adsorption losses to the container). At low ligand concentrations, the initial influx rates increased in a linear fashion with increasing arachidonic acid concentrations and appeared to saturate at higher arachidonate concentration (Fig. 6). The data were fitted against the
Michaelis-Menten equation by nonlinear regression analysis. At a
vesicle protein concentration of 100 µg/ml, a half-maximal influx
rate was calculated for an arachidonate concentration of 5.7 ± 1.9 µM, and a Vmax of 3.0 ± 0.2 nmol/min × mg was obtained. The data were replotted according
to Lineweaver-Burk giving a linear regression line (Fig. 6,
inset).
To determine
whether arachidonate influx is a temperature-dependent
process, vesicles were incubated at temperatures between 0 and
71 °C, and initial influx rates were determined over the first
120 s as described above. Arachidonate influx demonstrated a
marked temperature dependence, Fig. 7. At 0 °C influx
was almost completely abrogated (0.22 pmol/min × mg), and an
optimum transport rate was measured at 51 °C (7.2 pmol/min × mg), Fig. 7, inset. The temperature dependence of the
initial influx rates (0-47 °C) was tested according to Arrhenius
where ln Vmax is plotted against 1/T
as shown in Fig. 7, inset. A linear relationship was found (r =
Essentially no influx could be observed at 71 °C (0 pmol/min × mg) and transport competence could not be restored by lowering the
temperature back to 37 °C. Inactivation of transport function might
be due to denaturation of a putative carrier protein or physical damage
of the vesicles. Electron micrographic pictures taken from the vesicles
before and after a 10-min incubation at 70 °C showed that the
vesicles were indeed heavily damaged by heat treatment. In fact, it was
not possible to find any intact vesicle in representative pictures of
more than 100 vesicles (data not shown).
To study the effect
of pH on initial arachidonic acid influx, the extravesicular pH was
varied from pH 9.8 to 3. At an alkaline pH higher than 8 almost no
influx could be measured (0.1 pmol/mg × min at pH 9.8) and was
indistinguishable from similar incubations where PCP had been added.
Lowering the pH to 6.0 increased the initial influx (12.7 pmol/mg × min) which could completely be inhibited by PCP, Fig.
8. At pH values below 6, initial influx is again
suppressed, and influx at pH 3 is indistinguishable from that observed
in the presence of PCP (0.45 pmol/mg × min). The enhancement of
arachidonate influx by acidification and suppression by alkalinization
in the pH range from 6 to 9 is compatible with an H+ + arachidonate cotransport system but may also be due to allosteric effects of pH on the hypothetical carrier protein. Electron microscopic examination of vesicle integrity at low pH values revealed that vesicles preincubated at pH 4.5 were almost indistinguishable from
those incubated at neutral pH, whereas at pH 3.5 about 20% of the
vesicles appeared to be damaged. Thus, decrease of transport activity
at low pH values appears not to be predominantly due to vesicle
destruction. Notably, PCP inhibited influx almost completely over the
whole pH range tested, further supporting the hypotheses that
arachidonic acid influx is non-diffusive in nature even at pH values
below 6 where arachidonic acid should be in a non-ionic state. A
pH-induced change in the binding of arachidonate to putative receptors
appears unlikely since sonication of vesicles just prior to filtration
liberates almost all of the arachidonate (Fig. 2) without affecting
membrane recovery.
We next investigated the
influence of extravesicular Na+ and K+
concentrations. The relaxation buffer was replaced in different experiments by buffers (10 mM HEPES-KOH, pH 6.8) containing
0-200 mM NaCl or buffers (10 mM HEPES-NaOH, pH
6.8) containing 0-200 mM KCl. In a series of experiments,
modified relaxation buffer containing various ATP concentrations
(0.5-10 mM) or an ATP-regenerating system was used as
external medium. All these replacements had no impact on the initial
arachidonate influx or efflux rates (data not shown).
Fatty acid trafficking through membranes is very often thought to
be purely by passive diffusion driven by fatty acid gradients (16). In
contrast, our findings on arachidonate export support the view (15, 28,
31) that transmembrane fatty acid movement is a
protein-dependent and saturable process that cannot be
explained by free diffusion. Although a protein that functions as long
chain fatty acid transporter has recently been cloned, the
translocation mechanism still remains to be elucidated. Three models
for the protein-facilitated transport mechanism of hydrophobic fatty
acids across the phospholipid bilayer that are compatible with the data collected so far have been proposed recently (32, 33). First, the
transport protein may form a pore through which fatty acids pass,
similar to the function of classical facilitated diffusion transporters, or the transport protein may bind specifically fatty acids from the aqueous phase to its external surface. The fatty acid
moves along the surface of the protein until it reaches the interface
of the protein and the phospholipid bilayer. The transport protein
catalyzes a "flip" of the fatty acid from the outer to the inner
leaflet of the bilayer, and the fatty acid is then released into the
aqueous phase; or the transport protein may bind the fatty acid already
associated with one leaflet of the plasma membrane and catalyze a
"flip flop" of the fatty acid in the membrane.
The studies presented herein indicate that pig neutrophils possess a
transport mechanism for arachidonic acid that facilitates the efflux of
arachidonic acid into the extracellular medium. In the experimental
model used, arachidonic acid associated rapidly with the outer leaflet
of the inverted plasma membrane vesicles from where it was translocated
by a protease-sensitive, time-dependent, and saturable
process to the vesicle interior. Arachidonate associated with the
external membrane leaflet is extracted by extravesicular albumin,
whereas internalized arachidonate is inaccessible. Thus, albumin
extraction in the presence of an appropriate efflux inhibitor can be
used to distinguish internalized arachidonate from that associated with
the external membrane leaflet. In PMN, arachidonic acid once
translocated to the outer leaflet of the plasma membrane may easily
partition into the plasma and bind to plasma albumin. Because of its
high binding affinity for fatty acids with a Kd of
about 15 nM (34), albumin has been found to extract fatty acids from membranes (24).
In activated neutrophils, activation of various phospholipases (PLA)
(35) leads to increased intracellular levels of free arachidonate, some
of which is used for leukotriene biosynthesis and some of which is
released into the extracellular medium (3, 36, 37). However, the
sources of arachidonate released into the medium as well as the
lipase(s) involved in this process are still a matter of controversy.
Studies using activated mast cells and macrophages support evidence for
the possibility of different pools of arachidonate generated by
different lipases (38, 39). Thus in macrophages, most of the
arachidonate released into the medium is derived from a secreted type
II PLA2 (sPLA2) acting on the outer surface of
the cell, while a group IV PLA2 (cPLA2) acting
intracellularly is coupled to eicosanoid biosynthesis and is only
responsible for a portion of the arachidonate released into the medium.
In PMN, the situation may be similar, although direct evidence for
secretion of sPLA2 activity has been elusive (40).
Alternatively, activated neutrophils may also generate arachidonate by
the concerted action of phospholipase C or D (41) and a lipase located
at the inner leaflet of the plasma membrane.
Regardless of the source, unesterified arachidonate dissolved in plasma
membranes has been shown to influence a variety of membrane properties
in different tissues including K+ channels (42, 43),
Cl Differential sensitivity toward anion transport inhibitors (18, 30, 49)
makes it possible to discriminate between different transport pathways.
So far, three different Cl We thank Dr. T. M. Kutchan for linguistic
help in the preparation of the manuscript. We are grateful to Dr.
Jesper Haeggström (Karolinska Institute, Stockholm, Sweden) for
helpful discussion.
Botanical
Institute,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Biochemicals
20 °C until use. Protein was determined using the bicinchoninic
acid assay (21).
Vesicle Preparation
Fig. 1.
Electron micrographs of pig PMN-derived
phagosomes. The picture on the left shows isolated
vesicles with some adhering granules. On the right, a single
isolated vesicle shows the trilaminar membrane (arrows)
enclosing the albumin-coated mineral oil droplet from which the lipid
has been extracted during sample preparation.
[View Larger Version of this Image (93K GIF file)]
Fig. 2.
Binding of arachidonate to vesicle
membranes. Inside-out plasma membrane vesicles (20 µg of protein
in 200 µl of modified relaxation buffer) were incubated with
[3H]arachidonate (0.24-9772 pmol, corrected for
adsorption losses to the container) at 37 °C and treated with 800 µl of ice-cold relaxation buffer with () or without addition of
0.4% BSA, 0.4 mM PCP (
) for 5 min. Vesicle-associated
radioactivity was determined after rapid filtration and scintillation
counting of the filters.
[View Larger Version of this Image (16K GIF file)]
Fig. 3.
Time course of arachidonate uptake into
phagosomes. Vesicles (20 µg of protein) were incubated with 4 pmol of [3H]arachidonate in 200 µl of modified
relaxation buffer, pH 6.8 at 37 °C. Reactions were terminated by
transferring an aliquot into 4 volumes of ice-cold stop/chase solution
containing 0.5% BSA and 0.5 mM PCP. After 5 min on ice,
incubations were filtered, and vesicle-associated radioactivity was
determined by scintillation counting of the filters. An aliquot of the
incubation at 150 min (arrow) was treated with stop/chase
solution in a similar way but was sonicated three times for 10 s
during the extraction period before recovery of membrane
(vesicle)-associated radioactivity was determined (mean ± S.D.,
n = 6).
[View Larger Version of this Image (15K GIF file)]
Fig. 4.
Inhibition of arachidonate influx and efflux
by PCP. Vesicles, 20 µg of protein in 200 µl of modified
relaxation buffer, were incubated with 4 pmol of
[3H]arachidonic acid at 37 °C. Vesicle-associated
arachidonate after various time points was determined in the incubation
mixtures (with or without additions) as described under "Materials
and Methods." A, at time points indicated
(arrow), aliquots of the incubation mixtures received an
addition of 0.4 mM PCP, and incubation at 37 °C was
continued (,
). An aliquot was incubated with no addition of PCP
to monitor influx without inhibitor (
). B, at time point
indicated (arrow), half of the incubation mixture received either 0.4% BSA, 0.4 mM PCP (
) or 0.4% BSA (
), and
incubation was continued at 37 °C before internalized arachidonate
was determined.
[View Larger Version of this Image (24K GIF file)]
-isothiocyanostilbene-2,2
-disulfonic acid,
-cyano-4-hydroxycinnamate, and the dye bromcresol green, showed no
effect on arachidonate influx. The effectiveness of common anion
transport inhibitors is suggestive of non-diffusive uptake of
arachidonic acid into the vesicles.
Fig. 5.
Effect of anion transport inhibitors on
arachidonate influx. Vesicles (20 µg of protein) were
preincubated with various inhibitors in modified relaxation buffer (200 µl) for 15 min at 37 °C. Reactions were started by addition of 20 nM arachidonate, and internalized arachidonate was measured
as described in the legend for Fig. 3 over the first 120 s (five
time points). Initial influx rates were calculated from the slope of
the linear regression line from experiments performed in triplicate
(mean ± S.D.). AA, arachidonic acid; DISA,
3,5-diiodosalicylic acid; NEM,
N-ethylmaleimide.
[View Larger Version of this Image (22K GIF file)]
Fig. 6.
Saturable uptake of arachidonate into
phagosomes. Various concentrations of
[3H]arachidonate were incubated with vesicles at a
protein concentration of 100 µg/ml. Initial influx rates were
determined as described in the legend to Fig. 5 and plotted against the
arachidonate concentration (corrected for adsorption losses to the
container walls). For the calculation of the kinetic parameters, data
were fitted against the Michaelis-Menten equation by nonlinear
regression analysis based on the least-squares method
(line). A half-maximal influx rate was observed at an
arachidonate concentration of 5.7 ± 1.9 µM, and a
Vmax of 3.0 ± 0.2 nmol/min × mg was
obtained (mean ± S.D., n = 3-6). The data were
replotted according to Lineweaver-Burk giving a straight line with no
apparent breaks in the curve (inset). AA,
arachidonic acid.
[View Larger Version of this Image (20K GIF file)]
0.9890), and from the slope of the Arrhenius
plot an activation energy of 11.6 ± 0.3 kcal/mol (mean ± S.D., n = 3) could be calculated.
Fig. 7.
Temperature dependence of arachidonate
influx. Temperature dependence (0-71 °C) of arachidonate
influx into phagosomes. Initial influx rates were determined as
described in the legend for Fig. 5. The data (temperature range
0-47 °C) was replotted according to Arrhenius (inset).
Graph indicates strictly linear relationship with no apparent breaks in
the curve. Slope signifies activation energy of 11.6 ± 0.3 kcal/mol. Results are from three experiments. AA,
arachidonic acid.
[View Larger Version of this Image (22K GIF file)]
Fig. 8.
Effect of pH on arachidonate uptake into
vesicles. Initial influx rates were determined as described in
Fig. 5 in the absence or presence of 0.4 mM PCP in medium
wherein pH0 was varied between 3 and 9.8. Optimum transport
activity was observed at an extravesicular pH of 6.
[View Larger Version of this Image (13K GIF file)]
channels (44), proton pumping (45), Ca2+
permeability (46), and membrane ATPase (47). Therefore, it may be
important that unesterified arachidonate in the inner leaflet of the
plasma membrane is removed by an efficient export mechanism to clear
the membrane from arachidonic acid to prevent disturbance of normal
membrane function. Thus, export might be an additional mechanism apart
from the rapid reacylation in the cytosol to keep the arachidonate
level well below 3 pmol/million PMN (48) and may contribute to the
release of arachidonate from PMN during cell activation.
channels and four different
anion transport activities have been identified in neutrophils as
follows: a Cl
/HCO3
exchanger (50), a sulfate carrier (51), a H+ + lactate-carrier (52), and a H+ + lipoxin A4
cotransporter (49). Lactic acid fluxes are completely blocked by
N-ethylmaleimide, and the sulfate carrier is very sensitive to 4-acetamido-4
-isothiocyanostilbene-2,2
-disulfonic acid,
whereas arachidonate export is sensitive to neither of these inhibitors at roughly comparable concentrations. The
Cl
/HCO3
exchanger
displays a pH dependence opposite to that for arachidonate transport.
In contrast, many of the kinetic characteristics and the inhibitor
profile for arachidonate export are shared by the H+ + LXA4 cotransporter that imports the eicosanoid
LXA4 into PMN. Similar to LXA4 uptake into PMN,
arachidonate influx into phagosomes displays a striking pH dependence
with almost no transport activity at pH 8 and highest activity at pH 6. ATP, Na+, or K+ dependence was not noted in
either case. With respect to the inhibitors, both transporters are
sensitive toward PCP, 3,5-diiodosalicylic acid, but not to
N-ethylmaleimide, iodoacetate, and
4-acetamido-4
-isothiocyanostilbene-2,2
-disulfonic acid. The energy of
activation for both transporters was relatively low, 15.0 kcal/mol for
the trihydroxylated eicosanoid LXA4 and 11.6 kcal/mol for
arachidonic acid. Substrate saturation was not seen with the
LXA4 transporter but was evident in the case of arachidonate transport at relatively high concentrations. It has not
yet been clarified if arachidonate influx and efflux in PMN is mediated
by the same protein, facilitating the electroneutral diffusion of
arachidonate + H+ species through the membrane similar to
the lactate + H+ cotransporter in PMN (52) and erythrocytes
(18). Alternatively, two independent transport systems may exist. The
question of which other eicosanoids and fatty acids are accepted by the
arachidonate transporter has not been addressed in this investigation
and remains to be elucidated in the future.
*
This work was supported by the Deutsche
Forschungsgemeinschaft, Bonn (MU 1105/2-1).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence and reprint requests should be addressed:
Universität München, Institut für Pharmazeutische
Biologie, Karlstr. 29, D-80333 München, Germany. E-mail:
Martin.Mueller{at}lrz.uni-muenchen.de. Tel.: 89-5902-386; Fax:
89-5902-611.
1
The abbreviations used are: PMN,
polymorphonuclear leukocyte(s); LT, leukotriene; PIPES,
piperazine-N,N-bis(2-ethanesulfonic acid); BSA,
bovine serum albumin; PCP, pentachlorophenol; PLA2, phospholipase A2; LXA4, lipoxin
A4.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.