Supplement 1 Detailed Material and Methods Platelet-activating factor reduces endothelial NO

Supplement 1
Detailed Material and Methods
Platelet-activating factor reduces endothelial NO
production - Role of acid sphingomyelinase
Yang Yang*1, Jun Yin*2, Werner Baumgartner3, Rudi Samapati2, Eike
Reppien4, Wolfgang M. Kuebler**2,5, Stefan Uhlig**1
*, these authors contributed equally to this study.
**, these authors share the last authorship.
of Pharmacology and Toxicology, Medical Faculty, RWTH Aachen
University, 52074 Aachen, Germany
for Physiology, Charité – Universitätsmedizin Berlin, 14195 Berlin, Germany
of Cellular Neurobionics, Institute of Biology 2, RWTH-Aachen, 52056
Aachen, Germany
Center Borstel, Division of Pulmonary Pharmacology, 23845 Borstel,
The Keenan Research Centre at the Li Ka Shing Knowledge Institute of St.
Michael´s Hospital, Toronto M5B 1W8, ON, Canada
Corresponding author: Stefan Uhlig, Institute of Pharmacology and Toxicology, Medical
Faculty, RWTH Aachen University, 52074 Aachen, Wendlingweg 2, Germany. Email:, Tel. +49 241 8089120, FAX 49 241 8082433
Materials and Methods
Male Sprague-Dawley rats (weight 350 to 450g; for in situ fluorescence imaging) and female
Wistar rats (weight 220 to 250g; for all other experiments) were obtained from Charles River
Laboratories (Germany, Sulzfeld) and kept under controlled conditions (22°C, 55% humidity,
12h day/night rhythm) on a standard laboratory chow and water ad libitum. All animals
received care in accordance with the Guide for the Care and Use of Laboratory Animals (NIH
Publication No. 86-23, revised 1985). The study was approved by the local animal care and
use committee of the local government authorities. Pentobarbital sodium (Nacoren, 400µl/kg)
was purchased from the Wirtschaftsgenossenschaft Deutscher Tierärzte (Hannover,
Substances and chemicals
Platelet-activating factor (PAF) was obtained from Sigma (Deisenhofen, Germany); acetyl
salicylic acid (ASA) from Grünthal (Aachen, Germany); imipramine from ICN Biomedicals
(Eschwege, Germany); L-NMMA from Cayman Chemical Company (MI, USA).
Silica-beads with a diameter of 5 µm were from Bangs Laboratories, Inc. (Fishers, IN, USA)
and chemicals for MBS-buffer and HBS-buffer were from Sigma Aldrich (Taufkirchen,
Germany). Aluminium chlorohydroxide, used to coat the silica beads was from PFATZ &
BAUER Inc. (Waterbury, CT, USA); Nycodenz from Axis-Shield PoC AS (Oslo, Norway)
and Triton-X-100 from Boehringer-Mannheim GmbH (Mannheim, Germany). Sucrose and all
other used chemicals were obtained from Sigma Aldrich.
Mouse monoclonal antibody to caveolin-1, eNOS, peNOS were from BD Biosciences
Pharmingen (Heidelberg, Germany). The secondary antibodies, Alexa-Fluor 680-anti-mouse
IgG and Alexa-Fluor700-anti-rabbit IgG as well as the NO-sensitive fluorescence dye 4amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM) and the NO donor Snitroso-N-acetylpenicillamine (SNAP) were obtained from Molecular Probes (Eugene, OR,
Preparation of isolated, ventilated and perfused rat lungs (IPL)
Rat lungs were prepared and perfused essentially as described [1,2]. Briefly, lungs were
perfused through the pulmonary artery at a constant hydrostatic pressure (12 cm H2O) with
Krebs-Henseleit-buffer. Perfusate buffer contained 2% albumin, 0.1% glucose and 0.3%
HEPES. Edema formation was assessed by continuously measuring the weight gain of the
lung. All equipment was obtained from Hugo Sachs Electronics (March-Hugstetten,
Germany). Imipramine (final concentration 10µM), D609 (300 µM), L-NAME (100 µM),
dexamethasone (10 µM) and PAPAnonoate (100 µM) were prepared in perfusate buffer and
were added in to the buffer reservoir 10 min prior to PAF administration. ASM (1 U/ml in
perfusate buffer) was continuously infused for 30 min into venular capillaries of isolated
lungs via a venous microcatheter (Ref. 800/110/100; SIMS Portex Ltd., Kent, UK). Lung
filtration coefficient was measured by the weight transient method as described in detail
before [3].
Preparation of endothelial membrane fractions
Membrane fractions from endothelial cells were isolated from perfused rat lungs by use of
colloidal silica beads essentially as described [4,5]. After 40 min of perfusion, 5 nmol PAF
(final concentration 50 nM) was injected as a bolus directly into the perfusate. Ten minutes
later, the flow rate was reduced to 2-3 mL/min and perfusion with 1% cationic colloidal silica
beads in MES buffer saline (MBS, 25 mM MES-NaOH, pH 6.5, and 150 mM NaCl) was
Step 1: Purification of endothelial cell membranes. After perfusion with the silica beads,
lungs were immersed in cold HEPES buffered saline (HBS), minced, homogenized and
filtrated through a 0.65 µm and 0.45 µm Nytex net (GE Osmononics Inc, Minnetonka, USA).
The homogenate was mixed with an equal volume of 1.02 g/ml Nycodenz (containing 20 mM
KCl) and then layered over 0.5-0.7 g/ml Nycodenz containing 60 mM sucrose in a centrifuge
tube. After centrifugation in a Beckman SW 28 rotor at 20.000 rpm for 30 min at 4°C the
floating tissue debris was removed and the pellet containing the silica-coated endothelial
membranes fragments was resuspended with 1ml MBS.
Step 2: Isolation of membrane fractions from silica-coated endothelial cell membranes. 10%
cold Triton-X-100 (final concentration of 1%) was added to the membranes for 60 min at 4°C.
After incubation the suspension was homogenized and the homogenate mixed with 80%
sucrose to achieve a 40% membrane-sucrose-solution. A 30-5% sucrose gradient was layered
on top. Samples were centrifuged in a Beckman-centrifuge (SW55Ti rotor) at 4°C and at
30.000 rpm for 16-18h. Volumes of 3 x 150 µl were sampled from the top to the bottom and
collected as five membrane fractions. The pellet was solubilized in 150 µl MBS (pelletfraction) and 5 µg of protein were analyzed on the gels.
The fractions in Fig. 1 were prepared slightly different. After perfusion with the silica beads,
the lungs were minced in HBS and smoothly homogenized with a polytron mixer for
3x10min, subsequently incubated with collagenase (0.25%)+trypsin (0.25%), for 40min at
room temperature and again smoothly homogenized with polytron for another 3x10min. This
was followed by procedures as described in Step 1 and the resulting pellet was analyzed by
immunoblotting. This fraction is termed ‘total’ in Fig. 1d. In another set of experiments the
pellets were subjected to Step 1 and Step 2, before we analyzed the pooled fractions B/C
representing the caveolae and the fractions A/D/E plus the pellet representing the noncaveolar fractions. In this case, only 2.5 µg of protein were loaded on the gels.
Gel electrophoresis and immunoblotting
The protein content of each fraction was determined by a BCA-Assay kit (Pierce, Illinios,
USA). Equal amounts of protein (5µg) were separated by SDS-poly acrylamide gel
electrophoresis (12% for caveolin-1, 8% for eNOS) and transferred to nitrocellulose. After
transfer, nitrocellulose sheets were blotted with respective antibodies. For visualization, we
used Alexa-Fluor 680 anti-mouse IgG or Alexa-Fluor700 anti-rabbit IgG as secondary
antibodies. Western blot analyses were visualized in Odyssey® Imaging System (LI-COR
Biosciences GmbH, Bad Homburg, Germany). Protein bands (intensities) were quantified
with the Odyssey® Imaging System software at exactly the same settings for all parameters
such as background correction, contrast or channel settings.
Electron microscopy
Lungs were fixed by perfusion with 250 ml Caco-buffer (100 mM Na-cacodylate, 100 mM
NaCl, 2% formaldehyde, 2% glutaraldehyde and 2 mM CaCl2). Lungs were removed and cut
into pieces of about 5 x 5 x 5 mm and immersed in Caco-buffer over night. After washing
4x15 min in PBS, the tissue pieces were immersed in PBS containing 1% OsO4 followed by
washing 4 x 15 min in aqua bidest. Dehydration was performed by a graded EtOH-series and
embedded in Epon. Thin sections were cut and stained with uranyl acetate and lead citrate.
Electron microscopy pictures were digitally recorded using a Zeiss EM10.
Acid sphingomyelinase activity
Acid sphingomyelinase (ASM) activity was determined by using 14C-labelled sphingomyelin.
For all samples, 10 μg protein diluted to 10 μl was incubated at 37°C for 2 h with 40 μl
substrate (73 nmol
C-labelled sphingomyelin + 400 nmol sphingomyelin). Lipids were
separated by chloroform/methanol extraction, 4 ml scintillation liquid was added and
radioactivity counted in a β-counter.
In situ fluorescence microscopy
In situ imaging of endothelial NO production was performed as previously described [6]. In
brief, lungs were excised and continuously perfused with 14 ml/min autologous blood at
37°C. Lungs were constantly inflated with a gas mixture of 21% O2, 5% CO2, balance N2 at a
positive airway pressure (PAW) of 5 cmH2O. Left atrial pressure (PLA) was set to 3 cmH2O,
yielding pulmonary artery pressure (PPA) of 10±1 cmH2O. PAW, PLA, and PPA were
continuously monitored and recorded (DASYlab 32; Datalog GmbH, Moenchengladbach,
Germany). Lungs were positioned on a custom-built vibration-free microscope stage and
superfused with normal saline at 37°C.
For in situ imaging of endothelial NO production, membrane-permeant DAF-FM diacetate (5
μM/L), which de-esterifies intracellularly to cell-impermeant, NO-sensitive DAF-FM was
infused for 20 min into pulmonary capillaries via a venous microcatheter. Intracellular DAFFM is converted by an NO-dependent, irreversible reaction to an intensely fluorescent
benzotriazole derivative with fluorescence intensity linearly reflecting NO concentration [7].
Single venular capillaries were viewed at a focal plane corresponding to maximum diameter
(17-28 μm). Endothelial DAF-FM fluorescence was excited at 480 nm by a near
monochromatic beam generated by a digitally controled galvanometric scanner (Polychrome
IV; TILL Photonics, Martinsried, Germany) from a 75-watt xenon light source. Fluorescence
emission was collected through an upright microscope (Axiotechvario 100HD; Zeiss, Jena,
Germany) equipped with an apochromat objective (UAPO 40x W2/340; Olympus, Hamburg,
Germany) and dichroic and emission filters (FT 510, LP 520; Zeiss, Jena, Germany) by a
CCD camera (Sensicam; PCO, Kelheim, Germany) and subjected to digital image analysis
(TILLvisION 4.0; TILL Photonics). Exposure time for each single image was limited to 5
milliseconds. Fluorescence images obtained in 10 s intervals were background-corrected and
fluorescence intensity (F) was expressed relative to its individual baseline (F0). Since the
conversion of DAF-FM to the benzotriazole derivative is irreversible, NO production is
reflected by changes of the ratio F/F0 (Δ F/F0) over time and was determined in 5 min
intervals. At the end of experiments, the exogenous NO donor SNAP (1 mmol/L) was added
to test whether endotheliale cells still contained unconverted DAF-FM.
Measurement of alveolar fluid influx and reabsorption.
Fluid fluxes into and out of the alveolar space were quantified by a double-indicator dilution
technique as previously reported [8]. Briefly, a high-molecular-weight fluorescence tracer,
texas red dextran (70 kDa; Molecular Probes, Eugene, OR), was instilled into the alveolar
space for determination of alveolar net fluid shift while a low-molecular-weight tracer, Na+
fluorescein (376 Da; Sigma-Aldrich, Taufkirchen, Germany), was added to the perfusate to
allow for differentiation between alveolar fluid influx and alveolar fluid reabsorption. At time
-10 min, 0 min, and 60 min, samples were drawn from both compartments, and alveolar fluid
reabsorption, alveolar fluid influx, and net fluid shift were calculated as previously described
assuming a two-compartmental distribution model [8].
Statistics. In case of heteroscedasticity data were transformed by the Box-Cox transformation
prior to analysis. Data were analyzed by two-sided t-tests or by the Dunnett test (JMP 7).
Fluorescence data were analyzed by the Kruskal-Wallis and Mann-Whitney U-test. The data
in Fig. 1d were analyzed by 2-way ANOVA (factors: treatment, fraction) with the
experimental ID-number as the blocking factor. If required, p-values were corrected for
multiple comparisons according to the false-discovery rate procedure using the “R” statistical
package [9].
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