Fatty acid translocase/CD36 mediates the uptake of palmitate
by type II pneumocytes
Florian
Guthmann1,
Renate
Haupt1,
A. Cornelis
Looman1,
Friedrich
Spener2, and
Bernd
Rüstow1
1 Abteilung Neonatologie,
Charité, Humboldt-Universität zu Berlin, D-10098 Berlin;
and 2 Institut für
Biochemie, Westfälische Wilhelms-Universität Münster,
D-48149 Münster, Germany
 |
ABSTRACT |
Type II pneumocytes, which synthesize, store,
and secrete pulmonary surfactant, require exogenous fatty acids, in
particular palmitic acid, for maximum surfactant synthesis. The uptake
of palmitate by type II pneumocytes is thought to be protein mediated, but the protein involved has not been characterized. Here we show by
RT-PCR and Northern blot analysis that rat type II pneumocytes express
the mRNA for fatty acid translocase (FAT/CD36), a membrane-associated protein that is known to facilitate the uptake of fatty acids into
adipocytes. The deduced amino acid sequence from rat type II
pneumocytes reveals 98% identity to the FAT/CD36 sequence obtained from rat adipocytes. The uptake of palmitate by type II pneumocytes follows Michaelis-Menten kinetics (Michaelis-Menten constant = 11.9 ± 1.8 nM; maximum velocity = 62.7 ± 5.8 pmol · min
1 · 5 × 105
pneumocytes
1)
and decreases reversibly under conditions of ATP depletion to 35% of
control uptake. Incubation of cells at 0°C inhibited the uptake of
palmitate almost completely, whereas depletion of potassium was without
effect. Preincubation of the cells with bromobimane or phloretin
decreases the uptake of palmitate significantly as does preincubation
with sulfo-N-succinimidyl oleate, the
specific inhibitor of FAT/CD36 (C. M. Harmon, P. Luce, A. H. Beth, and N. A. Abumrad. J. Membr. Biol. 121:
261-268, 1991). From these data, we conclude that FAT/CD36 is
expressed in type II pneumocytes and mediates the uptake of palmitate
in a saturable and energy-dependent manner. The data suggest that the
uptake process is independent of the formation of coated pits and
endocytotic vesicles.
fatty acid uptake; uptake kinetics; lung
 |
INTRODUCTION |
TYPE II PNEUMOCYTES synthesize, store, and secrete
pulmonary surfactant, a phospholipid-protein complex that prevents the collapse of the alveoli by reducing surface tension at the air-liquid interface. Surfactant is highly enriched in phospholipids, particularly dipalmitoylphosphatidylcholine (DPPC). The high rate of surfactant phospholipid synthesis requires sufficient sources of long-chain saturated fatty acids. On one hand, these fatty acids are synthesized de novo within the type II pneumocyte, in which lactate is the preferred substrate compared with glucose (5, 12, 21); on the other
hand, they are obtained from exogenous sources as free fatty acids or
in the form of triacylglycerols in lipoproteins (4, 8, 15, 22). The
importance of the various sources of fatty acids is not well understood
and may vary with nutritional status and development (for a review, see
Ref. 3). For instance, inhibitors of fatty acid biosynthesis decrease
the rate of DPPC synthesis in explants of fetal rat lung even when
palmitate is added (28), but in adult pneumocytes, maximum DPPC
synthesis is seen only in the presence of exogenous fatty acids (4, 6, 22). The latter emphasizes the necessity for these cells to transport
fatty acids across the plasma membrane.
How long-chain fatty acids move across the membrane has been a subject
of controversial discussion. Because of their lipophilic character,
they are thought to diffuse freely through the plasma membrane of cells
as has been shown for fatty acid methyl esters (19). This concept was
challenged by the finding of a rapid and saturable uptake that was
reduced by a preceding heat denaturation or protease treatment of cells
(36). Several plasma membrane-associated proteins capable of binding
fatty acids were isolated from various tissues and cells. Most proteins
identified as fatty acid transporters by diverse methods were
initially described in adipose tissue and liver (11, 14, 32, 35; for a review, see Ref. 31). A plasma membrane-associated fatty acid transporter of type II
pneumocytes has not been identified so far. However, from the results
of Maniscalco et al. (23), which show a saturable fatty acid uptake by
these cells, a protein-mediated transport across the membrane of type II pneumocytes might be hypothesized.
Once internalized, long-chain fatty acids are intracellularly
solubilized and translocated bound to cytoplasmic fatty acid binding
proteins (FABPs). The FABP binding of fatty acids maintains a gradient
between extra- and intracellular compartments; thus FABPs can stimulate
the uptake of fatty acids (33, 37). Recently, Guthmann et
al. (13) have shown that type II pneumocytes express the
epidermal type of FABP.
Based on the rationale that fatty acid translocase (FAT), a member of
the CD36-receptor family (18), is coexpressed with FABP for fatty acid
import (25), we considered this protein to be a strong candidate for
the putative fatty acid receptor of rat type II pneumocytes. Thus, in
this study, we address the identity of the fatty acid receptor in these
cells and examine its role in the mediation of cellular fatty acid import.
 |
METHODS |
Materials.
Oligonucleotides used for amplification of FAT cDNA from alveolar type
II cells were purchased from MWG Biotech (Ebersberg, Germany). The cDNA
coding sequence was amplified in four overlapping fragments with the
following primer sets [nucleotide (nt) numbering according to
Abumrad et al. (1)]: fragment 1 (nt 50-633), 5'-TGATTCTGCTGCACGAGGAG-3' and
5'-AAGAATGGATCTTTGTAACCCCAC-3', annealing temperature
54°C; fragment 2 (nt
547-1055), 5'-CAACAGCCTTATCAAAAAGTCC-3' and
5'-GCACACCATACGACGTACAG-3', annealing temperature
54°C; fragment 3 (nt
982-1334), 5'-ACTCCAGAACCCAGACAACCAC-3' and
5'-TCCCAGTCTCATTTAGCCACAG-3', annealing temperature
62°C; and fragment 4 (nt
1173-1518), 5'-ATAGGACATACTTGGATGTGG-3' and
5'-GTGCATAATGTAGGCTCATC-3', annealing temperature 54°C.
Sulfo-N-succinimidyl oleate (SSO) was
prepared with the method of Harmon et al. (14); bromobimane
(p-sulfobenzoyloxybromobimane) was
obtained from Calbiochem. Nigericin, valinomycin, and all other
chemicals (analytic grade) were from Sigma (Deisenhofen, Germany)
unless otherwise stated.
Primary culture of type II
pneumocytes. Wistar rats (body weight 100-120 g)
were from a local animal facility. Alveolar type II cells were isolated
by elastase digestion and by "panning" cells on immunoglobulin
G-coated bacteriological plastic dishes as described by Dobbs et al.
(9). Viability was judged by trypan blue staining and purity by
Harris-type hematoxylin staining of the isolated pneumocytes and ranged
from 90 to 95 and 87 to 93%, respectively. All experiments were done
after 18 h of pneumocyte culture.
Detection of FAT mRNA by PCR. Total
RNA from alveolar type II cells was isolated as described by
Chomczynski and Sacchi (7), and cDNA was synthesized with Moloney
murine leukemia virus reverse transcriptase (GIBCO, Eggenstein,
Germany) and random priming. PCR was performed with
Taq DNA polymerase (Promega,
Heidelberg, Germany) and primers as specified in
Materials at melting,
annealing, and extension temperatures of 94, 54-62, and 72°C,
respectively. After 35-40 cycles, PCR products were visualized by
ethidium bromide staining in 1.5% agarose gels.
Sequence analysis. PCR fragments
amplified from type II pneumocyte cDNA were purified from 1% agarose
gels with the Qiaex protocol (Qiagen, Hilden, Germany). The fragments
were sequenced from both strands with the primers as indicated in
Materials in a cycle sequence reaction
with biotinylated dideoxynucleotides (GATC, Constance,
Germany) and Thermo Sequenase (Amersham, Braunschweig, Germany). Terminated fragments were separated on a direct-blotting sequencing apparatus (GATC) and visualized by developing the blot with
streptavidin-coupled alkaline phosphatase, nitro blue tetrazolium, and
5-bromo-4-chloro-3-indolyl phosphate toluidin.
Northern blotting. For the generation
of radiolabeled single-strand DNA probes, the amplified PCR
fragment 2 was used as a template in
asymmetric 33-cycle PCR (16) with 0.26 µM
[32P]dCTP (3,000 Ci/mmol; ICN Biomedicals, Eschwege, Germany). The labeled probe was
separated from the deoxynucleotide triphosphates by chromatography on a
Sephadex G-50 column (Pharmacia). RNA was isolated (7) from freshly
prepared type II pneumocytes, and 10 µg of RNA were separated on a
1% formaldehyde-agarose gel and transferred overnight onto Hybond-N
nylon membranes (Amersham) by capillary blotting (30). The blots were
incubated (2 h) with prehybridization buffer [350 mM
Na2HPO4,
7% (wt /vol) SDS, 30% deionized formamide, and 1.0% (wt /vol)
BSA fraction V] and hybridized (overnight at 50°C) with the
DNA probe. The membranes were washed twice with 150 mM
Na2HPO4
containing 0.5% (wt /vol) SDS (10 min at 25°C) and once with 30 mM Na2HPO4 containing 0.1% (wt /vol) SDS
(10 min at 55°C) and were finally exposed to Kodak Biomax MS X-ray
film (Integra Biosciences, Fernwald, Germany) at
80°C.
Measurement of palmitate uptake.
Freshly isolated type II pneumocytes were plated at a density of 5 × 105 pneumocytes/well onto
24-mm tissue culture dishes for 18 h at 37°C in Dulbecco's
modified Eagle's medium (DMEM) containing 10% (wt/vol) fetal calf
serum. Pneumocytes were washed twice with serum-free DMEM and then
incubated with DMEM containing BSA and [3H]palmitic acid at
37°C. To this end, 460 µl of palmitic acid (1 mg/ml) with trace
[3H]palmitic acid (60 µl, 1 mCi/ml; Amersham), each dissolved in ethanol, were added to
Celite (Supelco), the ethanol was evaporated under nitrogen, and 8 ml
of DMEM and 880 µl of BSA (500 µM in phosphate-buffered saline,
fraction V; Boehringer Mannheim) were added to the dried palmitic
acid-Celite mixture. After incubation for 1 h at 37°C, Celite was
removed by centrifugation, the supernatant was recentrifuged (1,500 g for 10 min), and its volume was
adjusted to 100 ml by adding DMEM. The concentration of unbound
palmitate was calculated according to Richieri et al. (29). The
concentration of BSA remained unchanged at 4.4 µM in all experiments,
whereas the ratio of palmitic acid to BSA varied from 1 to 5.
Unless otherwise stated, the concentration of unbound palmitate was
40.6 nM in the incubation medium, and pneumocytes were incubated for 30 s at 37°C. Thereupon, the incubation medium was removed, and stop
solution was added for ~30 s [2 ml/well of ice-cold DMEM
containing 0.1% (wt /vol) BSA and 0.2 mM phloretin] and
subsequently replaced with ice-cold DMEM containing 0.1% (wt /vol)
BSA only. After 30 s, the medium was removed, and viability of the
pneumocytes was judged with trypan blue. Cells were lysed by adding 1 ml of 0.2% (wt /vol) SDS. This sample was finally dissolved in 8 ml of scintillation cocktail (Optiphase HI-Safe 2, Wallac), and
radioactivity was counted.
ATP depletion. Cells were ATP depleted
as previously described (24). Briefly, adherent type II pneumocytes
were washed twice with buffer [140 mM NaCl, 20 mM HEPES (pH 7.4),
1 mM CaCl2, and 1 mM
MgCl2] containing 5 mM
NaN3 and 50 mM 2-deoxyglucose (ATP depleted) or without NaN3 but
containing 50 mM glucose (control). Subsequently, the pneumocytes were
incubated for 30 min at 37°C either with
NaN3-containing buffer or with
buffer without NaN3 as described above.
In a second approach, type II cells were allowed to recover after ATP
depletion. The cells were once washed and incubated for 60 min with
NaCl-Pi containing 5 mM sodium
cyanide and 50 mM 2-deoxyglucose (ATP depleted) or 50 mM glucose
(control). The cells were allowed to recover during incubation in the
presence of DMEM containing 50 mM glucose for 90 min. Initial uptake of palmitate was measured after ATP depletion and after recovery of the
cells as described in Measurement of palmitate
uptake.
Temperature. Adherent type II
pneumocytes were preincubated at 37 (control) or 0°C for 20 min,
and uptake of palmitate was measured as described in
Measurement of palmitate uptake but at the respective temperature.
Potassium depletion. Adherent type II
pneumocytes were depleted of potassium. Cells were washed three times
with buffer containing 140 mM NaCl, 20 mM HEPES (pH 7.4), 1 mM
CaCl2, 1 mM
MgCl2, and 5.5 mM
D-glucose with 10 mM KCl
(control) or without KCl (potassium depleted). Subsequently, the cells
were incubated for 5 min at 37°C with or without
potassium-containing buffer, respectively, now diluted 1:1 with
distilled water in either case. Before measurement of palmitate uptake,
the cells were again washed three times with the above isotonic buffer
with or without potassium, respectively.
Potassium ionophores. Type II cells
were incubated with DMEM containing 3 µM nigericin for 60 min or with
DMEM containing 3 µM valinomycin for 20 min. Subsequently, initial
uptake of palmitate was measured as described in
Measurement of palmitate uptake.
 |
RESULTS |
Identification and expression of FAT/CD36 in type II
pneumocytes. Using primers specific for FAT/CD36, we
obtained DNA fragments migrating as single bands of the
expected sizes on an agarose gel (data not shown). We investigated the
expression of FAT/CD36 thus identified in type II pneumocyte RNA with
Northern blotting. Total RNA obtained from type II pneumocytes was
blotted and hybridized with the FAT/CD36 probe. As shown in Fig.
1, a single band was detected at ~2.9 kb,
the size reported for FAT/CD36 from rat adipocytes (1).

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Fig. 1.
Expression of fatty acid translocase (FAT) in type II pneumocytes. RNA
(10 µg/lane) was analyzed by Northern blotting and probed with
32P-labeled
fragment 2 of FAT cDNA. 18S and 28S,
18S and 28S rRNA, respectively.
|
|
Sequence analysis. Analysis of four
overlapping PCR fragments obtained by respective cDNA amplification
from Wistar rat type II pneumocytes resulted in a sequence of 1,468 bp,
which covers the entire protein coding sequence and 23 bases upstream
as well as 29 bases downstream (GenBank accession number AF072411). It
showed 99% identity with the nucleotide sequence obtained from Sprague-Dawley rat adipocytes (1). The deduced amino acid sequence showed 98.1% identity with the adipocyte FAT protein and differed at
positions 66 (Val
Ile), 85 (Ile
Lys), 214 (Ser
Phe), 256 (Leu
Phe), 257 (Gly
Val), 261 (Arg
Gln), 340 (Asn
Ile), 362 (Thr
Asn), and 383 (Ser
Ala) [numbering according to Abumrad et al.
(1)]. All amino acid exchanges, with the exception of position
261, are identical with the deduced protein sequence from mouse CD36
platelet glycoprotein (10). Position 261 corresponds with the sequence
from human platelet CD36 (26).
Palmitate uptake. After the
demonstration of FAT/CD36 expression at the mRNA level, we then probed
the cells for protein dependence of fatty acid uptake by administering
[3H]palmitate under
various physiological conditions of the cells. We incubated type II
pneumocytes for 0-300 s with DMEM containing [3H]palmitate (40.6 nM
unbound palmitate) and determined the amount of palmitate that was
taken up by the cells (Fig. 2). The uptake was fast and revealed linearity during the first minute of incubation.

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Fig. 2.
Time course of palmitate uptake in adherent type II pneumocytes. Before
assay, pneumocytes were maintained at 37°C for 18 h in
FCS-containing DMEM and then washed with serum-free DMEM twice. Uptake
of [3H]palmitate was
assayed at 37°C with a concentration of unbound palmitate of 40.6 nM. After 15-300 s, uptake was stopped by removing medium and
adding stop solution. Pneumocytes were then washed with ice-cold
DMEM-albumin and lysed in 1 ml of 0.2% SDS before radioactivity was
measured.
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|
Uptake kinetics. Uptake of palmitate
by adherent type II pneumocytes was saturable and followed
Michaelis-Menten kinetics (Fig. 3), with a
Michaelis-Menten constant of 11.9 ± 1.8 nM. This value is ~10-
and 5-fold lower than values found for the uptake of oleate into
hepatocytes (34) and of palmitate into rabbit type II pneumocytes (23),
respectively. Maximum velocity was 62.7 ± 5.8 pmol
palmitate · min
1 · 5 × 105 type II
pneumocytes
1, again lower
than the maximum velocity for oleate uptake by hepatocytes (30) but
almost the same velocity as that shown by rabbit type II pneumocytes
(23).

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Fig. 3.
Kinetics of [3H]palmitate
uptake. Initial uptake of
[3H]palmitate was
assayed at 37°C for 30 s with concentrations of unbound palmitate
from 5.1 to 97.9 nM, determined according to Richieri et al. (29).
Albumin concentration remained constant.
Inset: Lineweaver-Burk plot of the
same data. Linear regression revealed a Michaelis-Menten constant of
11.9 ± 1.8 nM, and maximum velocity was 62.7 ± 5.8 pmol · min 1 · 5 × 105
pneumocytes 1
(r2 = 0.98).
Values are means ± SD of
n = 4 experiments.
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Lowering the temperature to 0°C diminished the initial palmitate
uptake to 7% of the control value (Fig.
4), and ATP depletion of the
cells by sodium azide significantly lowered palmitate uptake to 28% of
that for nondepleted cells (Fig. 4). To substantiate the lack of
nonrelated toxic or irreversible changes as the cause of decreased
palmitate uptake, we let cells recover as described in
METHODS. The initial palmitate uptake
of cyanide-treated type II pneumocytes decreased to 59% of that of the
control uptake. Recovery of the cells for 90 min restored the palmitate
uptake rate nearly to that of the control uptake (Fig.
5). These data clearly show that movement
of palmitate across the membrane of type II pneumocytes is an
energy-dependent process. The cellular uptake via the endocytotic
pathway depends on the intracellular concentration of potassium. We
tested the effect of potassium depletion on palmitate uptake by type II
pneumocytes and found that there was no significant difference between
control and depleted cells (Fig. 4). In addition, incubation of the
cells with nigericin or valinomycin, both potent potassium ionophores,
was without significant effect on initial palmitate uptake (Fig. 4).
This indicates that palmitate uptake is independent of the formation of
coated pits and endocytotic vesicles.

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Fig. 4.
Influence of temperature, ATP depletion ( ATP), and potassium
depletion ( K+) on initial
palmitate uptake rates. Uptake of
[3H]palmitate was
assayed for 1 min at a concentration of unbound palmitate of 40.6 nM as
described in METHODS. Values are means ± SD of n = 3 or 2(*)
experiments.
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Fig. 5.
Recovery of ATP-depleted type II pneumocytes. Type II cells were ATP
depleted with cyanide and 2-deoxyglucose for 60 min, and subsequently,
initial uptake of palmitate was determined (open bar) as described in
METHODS. Cells were allowed to recover
in presence of DMEM containing 50 mM glucose for 90 min before
palmitate uptake was measured (solid bar). Values are means ± SD of
n = 2 experiments.
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Inhibition of palmitate uptake by
bromobimane. In a more direct experimental approach to
prove the involvement of membrane-bound proteins in fatty acid uptake,
we incubated type II pneumocytes with bromobimane (Table
1). Bromobimane reacts with thiol groups of
membrane-bound proteins (17) and might inhibit in this way receptor-mediated transport processes. In contrast to other thiol group-reacting compounds, bromobimane is not able to penetrate cells;
therefore its inhibition of palmitate uptake is mediated by
membrane-bound proteins only. The inhibition of palmitate uptake was
~50%.
Inhibition of FAT by SSO. Finally, to
attack FAT/CD36 specifically in the process of fatty acid import into
the pneumocyte, we applied SSO, which was described as a specific,
nontoxic, membrane-impermeable inhibitor of FAT when preincubated with
adipocytes for 25 min (14). When a similar procedure was applied here,
palmitate uptake by type II pneumocytes was drastically inhibited by
SSO in a concentration-dependent manner (Fig.
6). The half-maximal effect was seen at a
concentration < 0.5 mM SSO, and the maximal inhibition of palmitate
uptake was ~80%, similar to that affected by ATP depletion.

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Fig. 6.
Inhibition of initial palmitate uptake by
sulfo-N-succinimidyl oleate (SSO).
Type II pneumocytes were preincubated for 25 min with different amounts
of SSO. Uptake of
[3H]palmitate was
assayed at 37°C for 1 min at a concentration of unbound palmitate
of 40.6 nM. Values are means ± SD expressed in arbitrary units (AU)
of n = 3 or 2(*) experiments.
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Additive effect of phloretin and SSO on palmitate
uptake. For myocardial fatty acid uptake, the
involvement of several membrane proteins has been proposed (31). The
uptake kinetics and the inhibition of the palmitate influx by
bromobimane and SSO strongly indicate that FAT facilitates the uptake
of palmitate by type II pneumocytes, but these results did not exclude
the participation of other membrane-bound proteins. To address this
question, we used phloretin, a potent nonspecific inhibitor of membrane
transport proteins (20), and SSO, a specific, membrane-impermeable
inhibitor of FAT. Table 2 shows the effect
of both compounds on palmitate uptake. The combined inhibitory action
of phloretin and SSO significantly exceeds the effect reached by each
substance alone. From these data and from the maximal inhibition seen
with SSO (Fig. 6), we conclude that FAT/CD36 is the principal protein
facilitating palmitate transport into type II pneumocytes. The
existence of another fatty acid carrier besides FAT/CD36 is likely.
 |
DISCUSSION |
We have shown here that the uptake of palmitate by type II pneumocytes
is mediated by a protein of the surface membrane in a saturable,
energy-dependent process. Type II pneumocytes express the mRNA of FAT,
and an FAT-specific inhibitor reduced their uptake of palmitate. FAT is
a CD36-related class B scavenger receptor (2) facilitating fatty acid
uptake by rat adipocytes (14). It was cloned in adipocytes and detected
in various tissues by Northern blot analysis (1). Most members of the
class B scavenger-receptor family are localized in raftlike membrane
domains (18). These microdomains are involved in numerous cellular
functions including endocytosis, for which the formation of coated pits
and clathrin-coated vesicles is not essential (27). Potassium depletion
inhibits the formation of coated pits and clathrin-coated vesicles but does not affect the uptake of exogenous palmitate by type II
pneumocytes. This reveals that the influx of palmitate is not dependent
on coated pits and clathrin-coated vesicles and is consistent with the
localization envisaged for the CD36-related receptor proteins.
Because five different plasma membrane-associated proteins have been
described for myocardial fatty acid uptake (for a review, see Ref. 31),
it might be supposed that type II pneumocytes might express additional
fatty acid transporters other than FAT. However, the maximum inhibition
of palmitate uptake by preincubation with the FAT-specific inhibitor
was ~75%, indicating that FAT seems to be the main protein involved
in fatty acid transfer across the membrane of type II pneumocytes. On
the other hand, the inhibitory effect of phloretin on the uptake of
palmitate was additive to the inhibition available with the
FAT-specific inhibitor. Thus a second, quantitatively less important
protein-mediated mechanism for type II pneumocyte fatty acid uptake may
also exist.
After protein-mediated movement of fatty acids across the membrane, the
water-insoluble long-chain fatty acids can leave the inner leaflet of
the surface membrane only when a cytosolic protein solubilizes the
fatty acids. Therefore, the intracellular binding of fatty acids to
cytosolic FABP might be an important element in the regulation of the
fatty acid influx. However, whether the concentration of epidermal
FABP, which was shown to be expressed in type II pneumocytes (13),
modulates the uptake of fatty acids is still unknown.
From our results, we conclude that the uptake of exogenous fatty acids
by type II pneumocytes of adult rats is predominantly mediated by FAT.
The regulation of this receptor-mediated uptake of fatty acids by type
II pneumocytes might be an important factor affecting the biosynthesis
of lung surfactant and could gain clinical significance. Further
investigations are now possible to understand how the FAT/CD36-mediated
uptake of fatty acids is regulated.
 |
ACKNOWLEDGEMENTS |
We thank Sandra Bebenroth for expert technical help.
 |
FOOTNOTES |
This work was supported by grants from the Deutsche
Forschungsgemeinschaft (Ru 517/2-1 and Sp 135/10).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: B. Rüstow,
Humboldt-Universität zu Berlin, Universitäts-Kinderklinik
der Charité, Neonatologie, D-10098 Berlin, Germany.
Received 15 September 1998; accepted in final form 11 March 1999.
 |
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