Am J Physiol Lung Cell Mol Physiol
279: L408–L412, 2000.
special communication
Free radical production in hypoxic pulmonary artery
smooth muscle cells
Received 24 September 1999; accepted in final form 6 March 2000
Killilea, David W., Raymond Hester, Ronald Balczon,
Pavel Babal, and Mark N. Gillespie. Free radical production in hypoxic pulmonary artery smooth muscle cells. Am J
Physiol Lung Cell Mol Physiol 279: L408–L412, 2000.—This
study used an inexpensive and versatile environmental exposure system to test the hypothesis that hypoxia promoted
free radical production in primary cultures of rat main pulmonary artery smooth muscle cells (PASMCs). Production of
reactive species was detected by fluorescence microscopy
with the probe 2⬘,7⬘-dichlorodihydrofluorescein, which is converted to the fluorescent dichlorofluorescein (DCF) in the
presence of various oxidants. Flushing the airspace above the
PASMC cultures with normoxic gas (20% O2, 75% N2, and 5%
CO2) resulted in stable PO2 values of ⬃150 Torr, whereas
perfusion of the airspace with hypoxic gas (0% O2, 95% N2,
and 5% CO2 ) was associated with a reduction in PO2 values
to stable levels of ⬃25 Torr. Hypoxic PASMCs became increasingly fluorescent at ⬃500% above the normoxic baseline
after 60 min. Hypoxia-induced DCF fluorescence was attenuated by the addition of the antioxidants dimethylthiourea
and catalase. These findings show that PASMCs acutely
exposed to hypoxia exhibit a marked increase in intracellular
DCF fluorescence, suggestive of reactive oxygen or nitrogen
species production.
fluorescence microscopy; hypoxia; reactive oxygen species;
reactive nitrogen species; vascular smooth muscle cells
FOR SEVERAL PRACTICAL REASONS,
imaging cellular events
during hypoxia can be difficult. Removing cells from
hypoxia for immediate analysis is usually unacceptable because reoxygenation may induce additional
stress on cells and complicate interpretation of results
(11, 12). Mimicry of low oxygen conditions by adding
cyanide to cell cultures to cause “chemical hypoxia” has
been used as an alternative, but there has been persistent concern regarding a lack of correlation of effects of
Address for reprint requests and other correspondence: M. N.
Gillespie, Dept. of Pharmacology, College of Medicine, MSB 3130,
Univ. of South Alabama, Mobile, AL 36688 (E-mail: mgillesp
@jaguar1.usouthal.edu).
L408
chemical hypoxia to true hypoxia (1, 10). Cell cultures
can be made hypoxic by bubbling nitrogen gas through
the medium (3, 20), but this approach may compromise
sterility of the cultures and may interfere with imaging
devices. A different strategy has been the use of chambers that provide a closed environment but are still
optically permeable to imaging devices. However, the
closed environment itself presents certain problems,
including alteration of the environment by cell metabolism and the difficulty in rapidly switching experimental conditions. Therefore, chambers designed to
provide a continuous flow of a perfusing gas mixture,
such as modified Sykes-Moore chambers, are often
preferred, yet these can be expensive and/or technically difficult to fabricate in-house (18). This report
describes a simple and economic system for a continuous perfusion of atmosphere above living cell cultures
that enables simultaneous microscopic analysis during
exposure to the test environment.
To demonstrate the utility of our system, we tested
the specific hypothesis that hypoxia caused production
of reactive species in cultured rat pulmonary artery
smooth muscle cells (PASMCs) using fluorescence of
2⬘,7⬘-dichlorofluorescein (DCF) as a marker of reactive
species generation. Hypoxia-induced intracellular reactive species production has been demonstrated with
this approach in other cell types (4, 7), but it is not
known whether the PASMC, an important effector cell
in the acute and chronic response of the lung to hypoxia, responds similarly.
MATERIALS AND METHODS
Cell culture. PASMCs were isolated from the main pulmonary arteries of male 200- to 250-g Sprague-Dawley rats with
an explant technique previously described (2). The cells were
maintained in culture medium (1:1 Dulbecco’s modified EaThe 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.
1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society
http://www.ajplung.org
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DAVID W. KILLILEA,1 RAYMOND HESTER,2 RONALD BALCZON,3
PAVEL BABAL,1 AND MARK N. GILLESPIE1
Departments of 1Pharmacology and 3Structural and Cellular Biology and 2Biotechnical Services
Laboratory, College of Medicine, University of South Alabama, Mobile, Alabama 36688
HYPOXIA-INDUCED REACTIVE SPECIES PRODUCTION IN PASMCS
Fig. 1. Schematic of flow-through hypoxic chamber. Premixed gas
flows through a regulator (not shown) into the back of the thermal
conducting stage that is heated to 37°C. When the 60-mm2 culture
dish is placed inside, a partially closed chamber is formed because
the top of the plate rests flush on the inner ring of the stage. A port
allows gas to flow under the top of the plate and into the chamber; a
second port allows the gas to escape. Flow rate can be adjusted so
that backflow of air through the exit port is insignificant. The
opening in the bottom of the stage allows the oil objective to come
into direct contact with the glass coverslip glued into the bottom of
the plate (inset).
the plate was equipped with a 1-ml syringe attached to the
Luer hub of the stopcock. If a drug was to be added during an
experiment, the syringe containing the drug would be attached to the stopcock, and the stopcock was placed in the
open position before gas perfusion, allowing equilibration of
the drug with the desired atmosphere. If no drug was to be
added, the stopcock was closed. An additional stopcock added
in-line preceding the apparatus allowed rapid switching of
environmental conditions if multiple gas mixtures were attached. To verify hypoxia in the medium, PO2 was measured
with a blood-gas analyzer (model ABL 30, Radiometer, Cleveland, OH).
Fluorescence microscopy. The production of reactive species as detected by DCF fluorescence during hypoxia was
measured with an ACAS 570 confocal laser scanning microscope (Meridian Instruments, Okemos, MI). Nonfluorescent
2⬘,7⬘-dichlorodihydrofluorescein diacetate (DCFH-DA; Molecular Probes, Eugene, OR) was loaded into cell cultures at a
final concentration of 5 ␮M for 20 min. Reactive oxygen
species (ROS) can oxidize DCFH-DA to the fluorescent DCF
(6, 13, 16, 22). The complete apparatus was mounted on the
stage of an Olympus IMT-2 inverted microscope (Olympus,
Lake Success, NY). The 488-nm line of an argon laser was
used at 100 mW to excite DCF with fluorescence emission
collected through a ⫻100 oil-immersion objective (numerical
aperture ⫽ 1.3) and a 530/30 band-pass filter in front of the
photomultiplier tube.
Other instrument settings included a 3% scan strength
and a 1% neutral density filter. Fluorescence intensity was
obtained in arbitrary units after background subtraction
with Meridian Software version 3.29.
RESULTS
Initial studies determined whether the apparatus
described above afforded maintenance of stable normoxic or hypoxic environments. Premixed normoxic
(20% O2, 5% CO2, and 75% N2) or hypoxic (5% CO2 and
95% N2) gas was perfused at 25 ml/min through the
flow-through chamber, with a headspace of ⬃20 cm3
over 5 ml of culture medium in a 60-mm-diameter
culture dish. Direct sampling of medium from these
experiments indicated that although medium PO2 during exposure to normoxic gas remained stable at ⬃150
Torr, medium PO2 during hypoxia decreased exponentially to ⬃20 Torr after 30 min of exposure (Fig. 2A).
This PO2 level remained stable over the duration of
hypoxic exposure. The pH of the medium was also
stable during both normoxic and hypoxic exposures,
indicating that the changes in fluorescence were not
due to pH gradients established by hypoxia (Fig. 2B).
Sampling from plates with fluorescent dye had no
effect on observed PO2 or pH (data not shown).
To demonstrate the utility of the above-described
perfusion system, we determined whether hypoxia promoted generation of ROS or reactive nitrogen species.
PASMC cultures were placed in the apparatus, and 5
␮M DCFH-DA was added. The cells were allowed to
take up dye for 30 min in a normoxic atmosphere
before the initiation of hypoxia. On exposure to the
hypoxic gas mixture, the cells became increasingly
fluorescent as medium PO2 decreased (Fig. 3). After 60
min, DCF fluorescence was ⬃500% over normoxic baseline. When the chamber was flushed with normoxic
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gle’s medium-Ham’s F-12 medium, 10% fetal bovine serum,
100 U/ml of penicillin, and 100 ␮g/ml of streptomycin) and
passaged with trypsin-EDTA treatment. All cell culture reagents were obtained from GIBCO BRL (Life Technologies,
Grand Island, NY). The cells were used for experiments
within 20 passages and studied when they were at ⬃80%
confluence.
Flow-through chamber design. Standard 20 ⫻ 20-mm glass
coverslips were washed sequentially with water and 70%
ethanol to remove any factory residue. Holes 15 mm in
diameter were punched into standard 60-mm-diameter tissue culture dishes with a chassis punch. The edges of the
holes were smoothed with a fine-grain file, and the plates
were washed with water to remove debris. A ring of waterresistant glue was applied on the inside of the plates ⬃2 mm
from the edge of the hole. Dry coverslips covering the glue
were immediately placed in the plates, taking care not to
spread the glue into the chamber of the plate or under the
coverslip, and were allowed to dry upside-down. A 4-mm hole
was punched off-center into the top of the tissue culture dish
lid, which allowed one arm of a stopcock to fit inside (Fig. 1).
Silicone sealant (Silastic 732 RTV Adhesive/Sealant, Dow
Corning, Midland, MI) was applied around the opening on
the outside of the lid to secure the stopcock and prevent
airflow around it. The lids and plates were washed sequentially with water to remove residual curing agents and 70%
ethanol to sterilize. After they were dry, the plates were
ready to be seeded with cells.
The cultures were placed in a model 5000 thermal conducting stage from 20/20 Technology (Whitehouse Station, NJ) as
shown in Fig. 1. The apparatus maintained the cultures at
37°C and provided a partially closed environment when a
60-mm-diameter culture dish was fitted inside. Through one
opening, certified gas (Air Products, Jacksonville, FL) at a 25
ml/min flow rate was allowed to perfuse the airspace above
the medium. Gas exited through the second opening, preventing any pressure buildup or backflow of air. The top of
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HYPOXIA-INDUCED REACTIVE SPECIES PRODUCTION IN PASMCS
gas, DCF fluorescence returned to baseline values
within 20 min (data not shown).
To provide confirmatory evidence that hypoxiainduced DCF fluorescence was mediated by reactive
species, an antioxidant, dimethylthiourea (DMTU),
was used to suppress the production of DCF fluorescence during hypoxia. After 40 min of hypoxia, 100
␮M DMTU dissolved in 0.5 ml of culture medium was
added to the cells in the chamber through the syringe
Fig. 3. Representative experiment
showing increase in dichlorofluorescein (DCF) fluorescence during exposure to hypoxia. A: pulmonary artery
smooth muscle cells (PASMCs) were
exposed to hypoxia, and images of the
same 100-␮m2 field were captured at
intervals of 0, 5, 10, 15, 20, 30, and 60
min. Fluorescence scale in arbitrary
units ranges from low (purple) to high
(red) fluorescence. B: summary of hypoxic data. ■, Hypoxia; ‚, dimethylthiourea (100 ␮M) was added to the culture
medium after 40 min of hypoxic exposure; , PASMCs were pretreated for
6 h with 300 U/ml of catalase before
induction of hypoxia. Values are
means ⫾ SE normalized as a percentage of normoxic baseline control for 10
cells in each of at least 3 experiments.
* Addition of dimethylthiourea caused
significant reductions in DCF fluorescence in hypoxic PASMCs. Catalase
prevented hypoxia-induced increases
in DCF fluorescence.
port. Acute administration of DMTU in this manner
reduced DCF fluorescence intensity to ⬃50% of peak
levels within 20 min after introduction into the hypoxic chamber (Fig. 3B). Incubation of PASMCs with
catalase (300 U/ml) for 6 h, a condition known to
promote substantial elevations in systemic vascular
smooth muscle cell catalase activity (17), also prevented hypoxia-induced increases in DCF fluorescence (Fig. 3B).
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Fig. 2. PO2 (A) of culture medium decreases exponentially on exposure to hypoxia, whereas pH (B) does not change
in the hypoxic environment (n ⱖ 4 cultures/point).
HYPOXIA-INDUCED REACTIVE SPECIES PRODUCTION IN PASMCS
DISCUSSION
literature (22), there is some controversy regarding
exactly which ROS or reactive nitrogen species is responsible (6). DCFH-DA does not react efficiently with
superoxide radical or hydrogen peroxide; metals such
as those involved in Fenton-type chemistry are likely
necessary for oxidation to DCF. Nitric oxide can oxidize
DCFH-DA directly but not at physiological levels; however, peroxynitrite can readily oxidize DCFH-DA at
low concentrations. Hypochlorous acid can also oxidize
DCFH but with low efficiency. The present data show
that DMTU, a low molecular weight, thiol-based antioxidant, attenuates hypoxia-induced DCF formation at
micromolar concentrations. At such concentrations,
DMTU has a weak scavenging affinity for most ROS
(9), although it reacts at near diffusion-limited kinetics
with the hydroxyl radical (8, 21). Our data also show
that catalase inhibits hypoxia-induced DCF fluorescence, thus suggesting involvement of hydrogen peroxide at some point in the free radical reactions evoked by
hypoxic exposure. Based on the above considerations, it
seems likely that Fenton-active superoxide or hydrogen peroxide plays a significant role in the hypoxiainduced DCF fluorescence. The specific contribution of
nitric oxide as well as the potential involvement of
mitochondria as a source of reactive species in hypoxic
PASMCs should be facilitated with the apparatus described in this report.
In summary, this report describes a simple apparatus for exposing cells to selected gaseous environments
while simultaneously examining them on a standard
inverted microscope. Using this preparation, we found
that hypoxia causes rat PASMCs in culture to generate
reactive species as detected by increased DCF fluorescence. The PASMC thus joins a growing list of cells
that respond to hypoxia with intracellular free radical
generation and provides further support for the concept that ROS or reactive nitrogen species play important roles in hypoxic signal transduction.
This investigation was supported by National Heart, Lung, and
Blood Institute Grants HL-38495 and HL-58243.
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