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Investigative Ophthalmology & Visual Science, Vol. 33, No. 9, August 1992
Copyright © Association for Research in Vision and Ophthalmology
Erhacrynic Acid Induces Reversible Shape and
Cyroskeleral Changes in Cultured Cells
Krisrine Erickson-Lamy, Alison Schroeder, and David L. Epstein
Cell cultures derived from trabecular meshworks of human and bovine eyes and from bovine vascular
endothelia were incubated at 37°C for 1 hr with ethacrynic acid (ECA, 0.1-0.5 mmol/1) dissolved in
culture medium. At 2 hr after the initial exposure, ECA at concentrations up to 0.4 mmol/1 induced a
reversible alteration in cell shape in all three cell types that was coincident with a change in the staining
pattern of major cytoskeletal components including actin, a-actinin, vinculin, and vimentin. Distinct
progressive alterations in /9-tubulin also occurred, with initial changes observed 10 min after ECA
exposure. The ECA-induced changes in tubulin were blocked in part by preincubation with taxol (which
stabilizes the microtubule structure), but they appeared to differ from those occurring with nocodazole
(which interferes with tubulin assembly). These results suggest the possibility that ECA-induced
increases in outflow facility may be mediated by alterations in the cytoskeletons of outflow pathway
cells. Invest Ophthalmol Vis Sci 33:2631-2640,1992
Previous reports from our laboratory document
substantial facility-increasing effects of various sulfhydryl (SH) agents in the monkey eye in vivo and in
vitro and in bovine and human eyes in vitro.1"5 Ethacrynic acid (ECA) is a SH-reactive drug that was developed originally as a systemic diuretic.6 Morphologic
studies of ECA-treated eyes show a correlation between changes in cell-cell and/or cell-substratum attachments and increased outflow facility.3"5 Collectively, these studies suggest the possibility that the induced changes in the cytoskeleton of cell populations
in the trabecular meshwork might have led to the increased outflow facility. Therefore, we undertook cell
culture studies to assess the effect of pharmacologically active doses of ECA on cell shape and components of the cytoskeleton more directly.
Our results indicate that ECA causes a dramatic
reversible change in cell shape in cultured calf and
human trabecular meshwork-derived cells and in bovine pulmonary artery endothelial cells. In addition,
the shape change is preceded by disruption, and subFrom the Department of Ophthalmology, Harvard Medical
School, Howe Laboratory of Ophthalmology, Boston, Massachusetts.
Supported by National Eye Institute (Bethesda, MD) grants
EYO7321 and EYO1894; National Glaucoma Research, a program of the American Health Assistance Foundation, Rockville,
Maryland; and the Massachusetts Lions Eye Research Foundation,
Stoneham, Massachusetts.
Submitted for publication: November 22, 1991; accepted February 26, 1992.
Reprint requests: Kristine Erickson-Lamy, PhD, Howe Laboratory of Ophthalmology, Massachusetts Eye and Ear Infirmary, 243
Charles Street, Boston, MA 02114.
sequent loss, of/3-tubulin staining. The loss in tubulin
staining precedes changes in actin, a-actinin, vimentin, and vinculin, which are coincident with the
changes in cell shape.
Materials and Methods
Cell Culture
Bovine trabecular meshwork: Enucleated eyes from
calves (age range, 3-14 days) were obtained from a
local abattoir. Immediately after enucleation, the eyes
were stored in a moist saline environment at 4°C and
were transported to the laboratory within 4 hr of
death. The eyes were soaked for 15 min in Dulbecco's
modified Eagle's medium (DMEM) containing 100
units/ml of penicillin, 100 /ig/ml of streptomycin,
and 250 ixg/m\ of amphotericin B before dissection.
The trabecular meshwork was dissected according^
an earlier method.7 The eyes were bisected at the
equator and placed into a sterile Petri dish (corneaside down). The uveal layer was removed by blunt
dissection, leaving the outflow tissue along with ciliary body remnants attached to the sclera. The remaining ciliary body then was scraped away gently, and the
outflow tissue removed from the sclera and placed
into a 60-mm culture dish. After approximately 1 wk
when several cells had migrated off the explant and
onto the culture dish, the outflow tissue was removed.
When primary cultures became confluent, the cells
were detached from the culture dish by incubation
with 0.05% trypsin and 0.02% ethylenediaminetetraacetic acid in CA++ and Mg++ free phosphate-buffered saline and subcultured onto new culture dishes.
Cell cultures were maintained in DMEM containing
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / August 1992
1-glutamine and 10% newborn calf serum at 37°C in a
5% CO2 environment. Cell shape and cytoskeletal
studies were conducted on trabecular meshwork cells
from passages 1-5.
Human trabecular meshwork: The H-l cell line we
used was derived from a 16-year-old girl. The intact
enucleated eyes were shipped by overnight mail on ice
by the National Disease Research Interchange (Philadelphia, PA). Dissection and placement into cell culture occurred approximately 24 hr postmortem. Isolation of the anterior scleral shell (with attached trabecular meshwork) was done as described. After
removal of the scleral spur, the trabecular meshwork
was dissected along the anterior and posterior margins. It then was lifted away from the underlying
sclera with the aid offinejeweler's forceps. The meshwork was placed in a 30-mm culture dish and incubated at 37°C in a 5% CO2 environment in DMEM
with 1-glutamine and 20% fetal bovine serum (FBS).
Serial passages were made as described for the calf
meshwork cells. Passages 1-5 were used in this study.
Calfpulmonary artery endothelial cells: The established calf pulmonary artery endothelial cell line
(CPAE) was obtained from the American Type Culture Collection (CCL 209; Rockville, MD). The cells
were maintained in minimal essential medium containing 20% FBS. This cell line was used as a rough
model for the outflow cells lining Schlemm's canal,
the collector channels, and aqueous veins. We purchased FBS from Sigma (St. Louis, MO) or Hyclone
(Logan, UT). Growth supplements, antibiotics, and
media were obtained from Sigma. The newborn calf
serum was purchased from Gibco (Grand Island,
NY). Tissue culture dishes were obtained from Falcon (Lincoln Park, NJ).
Drugs and Concentrations
We dissolved ECA and cysteine (CYS) in culture
medium without serum to concentrations ranging
from 0.1-0.5 mmol/1 and 1.5-2.5 mmol/1, respectively. Cytochalasin B (CYTO B) was dissolved in dimethyl sulfoxide (DMSO) and diluted in culture medium without serum to give final concentrations of
0.05-0.2 mmol/1. Nocodazole (NOC) and taxol
(TAX) were dissolved in the same solvent and diluted
with culture medium without serum to givefinalconcentrations of 10~4 mol/1 and 10"5 mol/1, respectively.
All drugs were obtained from Sigma except for TAX,
which was provided by the National Cancer Institute
(Bethesda, MD).
For all procedures, the cultures were washed twice
with serum-free medium before drug administration.
In the case of ECA and CYS, the control incubations
consisted of culture medium without serum. In the
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case of CYTO B, NOC, and TAX, the control incubations consisted of 0.5%, 0.03%, and 0.03% DMSO in
culture medium, respectively. In all procedures, the
dishes were washed twice with serum-free medium.
The drug incubations were done for 1 hr, after which
the drug-containing medium was removed. The
dishes were washed twice with serum-free medium,
and the incubation was continued in drug-free culture
medium containing serum.
Effects of ECA and Cytoskeleton-Altering
Chemicals on Cell Shape
In experiments designed to examine changes in cell
shape, postconfluent cultures were incubated with
various concentrations of ECA (0.01-0.5 mmol/1),
ECA (0.3-0.5 mmol/1) and CYS (1.5-2.5 mmol/1),
CYTO B (0.05, 0.1, and 0.2 mmol/1), NOC (0.1 and
0.01 mmol/1), TAX (0.01 mmol/1), or ECA (0.1
mmol/1) and TAX (0.01 mmol/1 10"5 mol/1). With
ECA and ECA and CYS, the cultures were observed
by phase-contrast microscopy and photographed immediately after application, immediately after removal, and every 0.5 hr thereafter for 6 hr. In experiments with CYTO B, drug incubation was done for 1
hr, at which time the cultures were photographed in
the presence of CYTO B because cell shape changes
began to reverse immediately when the drug was removed. We incubated the cultures with NOC for 7 hr,
after which changes in cell shape were recorded. In
blocking experiments with ECA and TAX, TAX was
administered for 1 hr, after which ECA was added to
some cultures, and incubation continued in the presence of ECA and TAX or TAX alone for another
hour. Changes in cell shape were assessed as described
for ECA and ECA and CYS. In all cases, recovery of
normal morphology was documented 24 hr after drug
incubation.
Viability Studies
The effect of increasing concentrations of ECA on
cell viability was assessed by an earlier method.8 After
incubation with ECA, the cells that were grown on
glass cover slips were rinsed gently with phosphatebuffered saline and stained with 2 ng of fluorescein
diacetate and 0.6 /u.g of propidium iodide (Sigma) for 3
min at room temperature. The cells then were kept at
4°C until the photographs were taken.
Cytoskeletal Studies
For identification of cytoskeletal proteins, the cells
were grown on glass cover slips. For all procedures,
control samples were done to verify the specificity of
the antibody staining. These control experiments in-
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eluded substitution of the primary antibody with nonimmune serum, exclusion of the primary antibody
incubation step, and exclusion of both primary and
secondary antibodies. After the antibody-staining
procedures, the cover slips were mounted onto slides
using a fluorescence-stabilizing mounting medium
containing n-propyl gallate in glycerol (Sigma) with
polyvinyl alcohol (Air Products & Chemicals, Allentown, PA). Fluorescence was visualized using a Zeiss
(Thornwood, NY) IM 35 fluorescence photomicroscope system.
Filamentous Actin
Filamentous actin (F-actin) was labeled using rhodamine phalloidin (Molecular Probes, Eugene, OR).
Briefly, glass cover slips containing cultured cells were
rinsed gently in buffered salt solution (BS, containing
137 mmol/1 NaCl, 5 mmol/1 KC1, 1.1 mmol/1
Na2HPO4 • 2H2O, 0.4 mmol/1 KH2PO4, 5.5 mmol/1
glucose, 4 mmol/1 NaHCO3, 2 mmol/1 MgCl2, and 2
mmol/1 EGTA, pH 6), fixed with a mixture of 3%
paraformaldehyde and 0.5% Triton X-100 for 30 min
and washed with BS for 10 min. They were incubated
with rhodamine-phalloidin for 1 hr at room temperature. The cells then were washed for 15 min in BS and
mounted.
0-Tubulin
/3-tubulin was labeled using monoclonal anti-rat
brain j8-tubulin (Clone TUB 2.1; Sigma) and rhodamine-conjugated anti-mouse immunoglobulin G
(Cappel, Durham, NC). The cells grown on glass
cover slips were rinsed gently in BS and permeabilized
in 0.5% Triton X-100 and 0.25% glutaraldehyde for 2
min at room temperature. The cells were rinsed in BS,
fixed in 1 % glutaraldehyde for 20 min at room temperature, and rinsed again in BS. The cover slips were
transferred to ice-cold NaBH4 for 15 min and washed
for 20 min in BS. The cells were incubated with monoclonal anti-^-tubulin for 1 hr at room temperature,
rinsed several times with BS, and incubated with the
secondary antibody for 30 min. Then the cells were
washed for 15 min in BS and mounted.
Vimentin
Vimentin was labeled using monoclonal antivimentin (Sigma) and rhodamine-conjugated antimouse immunoglobulin G. Glass cover slips with cultured cells were gently rinsed twice in BS and fixed in
cold (-20°C) methanol for 5 min. The cover slips
were transferred to cold (-20°C) acetone for 2 min,
dried briefly, and washed in BS for 10 min. The cells
were incubated with monoclonal antivimentin for 1
hr at room temperature, rinsed several times with BS,
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and incubated with the secondary antibody for 30
min. The cover slips then were washed and mounted
as before.
Vinculin and a-Actinin
Vinculin was labeled using monoclonal antivinculin and rhodamine-conjugated anti-mouse immunoglobulin G. a-Actinin was labeled using monoclonal
anti-a-actinin and rhodamine-conjugated anti-mouse
immunoglobulin G. Both vinculin and a-actinin
cover slips werefixedwith a mixture of 3% formaldehyde and 0.5% Triton X-100 for 30 min and washed
with BS for 10 min. Incubation with the primary antibody was done for 1 hr at room temperature. The
cover slips were rinsed several times with BS and incubated with the secondary antibody for 30 min at
room temperature. Then the cells were washed for 15
min in BS and mounted.
Results
Incubation with ECA resulted in a dramatic and
reversible retraction of the cells and apparent attenuation of cell-cell attachments in calf trabecular meshwork (CTM), H-1, and CPAE cells at concentrations
ranging from 0.1-0.3 mmol/1 (Figs. 1A-F). Concentrations of ECA up to 0.4 mmol/1 did not result in any
apparent toxicity, as shown by the lack of uptake of
propidium iodide (Figs. 2A-C). By contrast, with concentrations of 0.5 mmol/1 and higher, the shape
changes were not reversible, and cell toxicity was evident (Figs. 2D-F).
To determine whether the shape changes were related to the SH reactivity of ECA, the cell cultures
were incubated with ECA and CYS (which binds to
ECA's SH reactive site). Incubation with 2.5 mmol/1
CYS alone caused no change in cell shape. In combination with a usually lethal concentration of ECA (0.5
mmol/1), CYS completely prevented the expected
ECA-induced shape change (Figs. 3A-D).
The changes in cell shape (Figs. 1-3) suggested the
likelihood of a disruption of actin filaments. Therefore, cell cultures were incubated with CYTO B
(which is known to disrupt actin filaments selectively). These results were compared with those obtained with ECA. Incubation with CYTO B resulted
in a change in cell shape similar to that occurring with
ECA at concentrations ranging from 0.02-0.2 mmol/
1 (Figs. 4A-B). However, the kinetics of the shape
change with CYTO B differed from those occurring
with ECA. The former's effect was virtually immediate and recovery occurred as soon as the drug was
removed. By contrast, the shape changes after the latter were seen first approximately 60 min after removal of the drug's incubation medium (120 min
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / Augusr 1992
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Fig. 1. Effect of ethacrynic acid (ECA) on the morphology of cultured cells. The appearance of post-confluent cell cultures was documented
by phase microscopy after incubation with control media (the appropriate culture medium without serum; a, c, e) or in culture medium with
added 0.2 mmol/l ECA (b, d, f). After a 1 hr incubation, the control and ECA containing media were replaced with the appropriate culture
media plus serum. In all cases, cell shape changes were documented in normal media 2 hr after the initial exposure to ECA or control media (ie,
1 hr after ECA/control media removal), (a) Calf trabecular meshwork cells (CTM), control culture. (Original magnification X 25.6.) (b) CTM
cells after exposure to 0.2 mmol/l ECA. Incubation with ECA resulted in a cell retraction and apparent attenuation of cell-to-cell contacts. This
change in cell shape was fully reversible by 24 hr after ECA exposure. (Original magnification X 25.6.) (c) Human trabecular meshwork-derived (HTM) cells, control culture, (Original magnification X161.3.)(d) HTM cells after exposure to 0.2 mmol/l ECA. ECA induced cell shape
changes similar to those in CTM cells (b). (Original magnification x 161.3.) (e) Calf pulmonary artery endothelial (CPAE) cells, control culture.
(Original magnification X25.6.) (f) CPAE cells after exposure to 0.2 mmol/l ECA. Cell shape changes are similar to those occurring in CTM (b)
and HTM (d) cells. (Original magnification X25.6.)
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Fig. 2. The dose-related effect of ethacrynic acid (ECA) on cell viability. Post-confluent cultures of calf pulmonary artery endothelial cells
(CPAE) were exposed for 1 hr to ECA (0.1 -0.5 mmol/l). One hour later (after replacement of normal culture media), cultures were stained with
fluoresceindiacetate(FDA)and propidium iodide (PI), as described in Materials and Methods. Positive staining with FDA in conjunction with
negative staining with PI is presumptive evidence for cell viability.8 Data show that CPAE cells are viable after exposure to 0.1 mmol/l ECA
(a-c) but are nonviable after exposure to 0.5 mmol/l ECA (d-f). (Original magnification X25.6.) (a) Phase contrast micrograph of CPAE cells
after 0.1 mmol/l ECA. (b) Fluorescence micrograph of the CPAE field shown in (a), indicating that the majority of cells stain positive for FDA.
(c) Fluorescence micrograph of the CPAE field shown in (a) and (b), indicating that the majority of celts stain negative for PI. (d) Phase
micrograph of CPAE cells after exposure to 0.5 mmol/t ECA. (e) Fluorescence micrograph of the same field shown in (d), showing that the
majority of cells stain negative for FDA. (f) Fluorescence micrograph of the same field shown in (d and e), indicating that most of the cells stain
positive for PI.
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INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / Augusr 1992
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Fig. 3. Blockage of ethacrynic acid (ECA)-induced cell shape changes in cultured cells by cysteine (CYS). Changes in cell shape were noted
(as described in Materials and Methods and in Fig. I) in calf pulmonary artery endothelial (CPAE) cells 2 hr after the initial exposure to a I hr
incubation with CYS, ECA, or ECA plus CYS. (Original magnification X25.6.) (a) CPAE cells incubated in control medium, (b) CPAE cells
after exposure to 2.5 mmol/1 CYS. The appearance of the cells is indistinguishable from the appearance of the control culture shown in (a), (c)
CPAE cells after exposure to (2.5 mmol/1) plus ECA (0.5 mmol/1). There is no obvious change from the appearance of the control culture (a) or
the CYS culture (b). (d) CPAE cells after exposure to ECA (0.5 mmol/1). Cell retraction is obvious with this toxic concentration of ECA.
after the drug was added). Recovery was not complete
until approximately 24 hr later.
Incubation with NOC (0.01 mmol/1, which is
known to disrupt microtubules by preventing tubulin
assembly) also resulted in a reversible change in cell
shape (Fig. 4C). Similar to the finding with ECA and
CYTO B, the cells were no longer in close juxtaposition. However, the shape change appeared to be different than that occurring with ECA or CYTO B. The
characteristic spider-like appearance of a round cell
body with long tenuous attachments assumed by cells
exposed to either ECA or CYTO B was not apparent.
In addition, the shape change was not observed until
approximately 7 hr after exposure to NOC. It required continuous drug exposure. Similar to the finding with ECA, recovery occurred approximately 24 hr
after NOC.
The changes in cell shape that occurred suggested
that, like CYTO B and NOC, ECA might affect the
cytoskeleton. Therefore, the influence of ECA on cytoskeletal components was studied using immunocytochemical analysis at various times after ECA administration.
The administration of this drug resulted in various
alterations of cytoskeletal protein staining in CTM,
H-l, and CPAE cells. The staining of F-actin was altered dramatically by 0.1 mmol/1 ECA. Instead of discrete longfilamentousbeams, the staining in the cytoplasm after ECA took on a punctate appearance. In
general, the cell borders and processes were outlined
distinctly. This change appeared to coincide with the
rounding of the cell shape (Figs. 5A-B). In contrast,
tubulin disruption was seen first after only 10 min of
incubation with 0.1 mmol/1 ECA (Fig. 5D). Progres-
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CHANGES IN CELL SHAPE AND CYTOSKELETON WITH ECA / Erickson-Lamy er al
sive alteration of /3-tubulin staining occurred during
the 1 hr of ECA incubation, with seemingly early loss
of peripheral tubulin staining and the observation of
coiled filaments around the cell nucleus (Fig. 5E).
Distinct curled fragments of tubulin staining were observed in the peripheral cytoplasm. The ECA-induced
alteration of tubulin staining appeared distinct from
that after NOC (Fig. 5F) or TAX (Fig. 5G) treatment.
Pretreatment with TAX both attenuated the ECA-induced disruption of/3-tubulin staining (Fig. 5H) and
delayed changes in cell shape. The loss of tubulin
staining after incubation with ECA was completely
reversible. The tubulin pattern recovered within 4 hr
of drug withdrawal.
Alterations in other cytoskeletal proteins including
vimentin, vinculin, and a-actinin also were observed
after ECA treatment. Incubation with ECA for 30
min caused a slight reduction of peripheral staining of
tf-actinin and vimentin and no apparent change in
vinculin. A complete disruption of tubulin staining
occurred after only 10 min of incubation with ECA.
Similar to the timing of changes in actin staining, a
dramatic loss of staining in a-actinin, vimentin, and
vinculin occurred at the time of the observed changes
in cell shape. Thus, ECA-induced changes in tubulin
staining preceded those of other studied cytoskeletal
proteins by 1-2 hr (Fig. 6).
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of the cytoskeleton including F-actin, a-actinin, vinculin, and vimentin. By contrast, distinct changes in
the staining pattern of jS-tubulin were observed as
soon as 10 min after incubation with ECA.
The kinetics of the altered /?-tubulin staining pattern correlate with changes in outflow facility thattypi-
Discussion
Our results show that, by a SH-reactive mechanism, ECA causes a reversible change in the shape of
cultured trabecular meshwork and vascular endothelial cells, first evident by phase-contrast microscopy
approximately 2 hr after exposure to the drug. The
shape changes were coincident with reversible alterations in the staining patterns of major components
Fig. 4. Effects of cytochalasin B (CYTO B) and nocodazole
(NOC) on the morphology of calf pulmonary artery endothelial
(CPAE) cells. Post-confluent cultures of CPAE cells were incubated
in: (a) control medium (minimal essential medium, without serum,
containing 0.5% dimethyl sulfoxide; (b) 0.2 mmol/1 CYTO B in
control medium; or (c) 10 ^mol/l NOC in control medium. (Original magnification X 51.2.) (a) CPAE cells after a 1 hr incubation in
control medium. The morphology retained a normal appearance
even with extended (ie, 12 hr) incubations in control medium, (b)
CPAE cells after a 1 hr incubation with CYTO B. Cells are retracted
and cell-to-cell attachments are attenuated. Unlike the changes
with ethacrynic acid (ECA; Fig. If), the change in cell shape occurred within 15 min, and recovery of normal morphology occurred within 30 min of the removal of CYTO B. (c) CPAE cells
after a 7 hr incubation with NOC. A disorganization of the triangular cell shape and the juxtaposition of cells has occurred, leaving
large intercellular spaces. Cells do not appear to be retracted as with
CYTO B (b) or ECA; Fig. If).
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / August 1992
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Fig. 5. Effect of ethacrynic acid (ECA), nocodazole (NOC), and taxol on
cytoskeletal elements. Immunofluorescent staining of
f-actin (a, b) and beta tubulin (c-h) was carried out in
human trabccular meshwork (HTM) or calf pulmonary artery endothelial
(CPAE) cells after exposure
to control media or cytoskeletal-active agents, as described in Materials and
Methods. (Original magnification X 161.3.) (a) Rhodamine phalloidin staining
of f-actin in HTM cells
incubated in Dulbecco's
modified Eagle's medium
(DMEM). (b) Rhodamine
phalloidin staining of filamentous actin in HTM cells
2 hr after the initial exposure to a 1 hr incubation
with 0.2 mmol/1 ECA in
DMEM.(c)Immunofluorcscent localization of beta tubulin in CPAE cells incubated in minimal essential
medium (MEM), (d) Immunofluorescent localization
of beta tubulin in CPAE
cells after a 10 min incubation with 0.2 mmol/1 ECA.
Note the disrupted staining
pattern at this early time
point, (c) Immunofluorescent localization of beta tubulin in CPAE cells after a I
hr incubation with 0.2
mmol/1 ECA. (f) Disruption
of microtubule staining in
CPAE cells after a 7 hr incubation with 10 Mmol/1 NOC
in 0.3% dimethyl sulfoxide
(DMSO)/MEM. (g) Condensation ofperinuclearimmunofluorescent beta tubulin staining in CPAE cells
after a I hr incubation with
10 Mmol/1 taxol in 0.03%
DMSO/MEM.(h)lmmunofluorescent localization of
beta tubulin in CPAE cells
after a I hr pretreatment
with 10 /xmol/1 taxol, in
0.03% DMSO/MEM, followed by a 1 hr incubation
with 0.2 mmol/1 ECA and
10 Mmol/l taxol. Note the
preservation, by taxol, of the
beta tubulin structure.
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Fig. 6. Effect of ethacrynic acid (ECA) on the
staining of alpha actinin, vimentin, and vinculin. Calf
pulmonary artery endothelial (CPAE) cells were incubated for 30 min in control
medium (minimal essential
medium) alone or with
added 0.2 mmol/l ECA. Immunofluorescent localization of alpha actinin (a, b),
vimentin (c, d), and vinculin
(c, f) in CPAE cells after incubation in control medium
(a, c, e) or 0.2 mmol/l ECA
(b, d, f). Note the slight reduction of peripheral staining in alpha actinin and vimentin and no apparent
change in vinculin staining
(b, d, f) after 30 min incubation with 0.2 mmol/l ECA.
Tubulin staining at this time
is completely disrupted (Fig.
5d). Dramatic absence of
staining in alpha actinin, vimentin, and vinculin occurs
90 min later, at the time of
observed changes in cell
shape. {Original magnification X161.3.)
cally occur within 20-30 min of ECA administration.4-5 These findings, in conjunction with the observation that outflow facility increases in the rabbit eye
after administration of colchicine,9 suggest that an alteration in tubulin metabolism may play a role in the
mechanism of ECA-induced changes in outflow facility.
Previous studies found that ECA binds to tubulin
isolated from trabecular cells.10 The observation that
preincubation with TAX (which stabilizes the microtubule structure) blocks the ECA-induced tubulin
changes suggests that ECA may affect tubulin metabolism directly. The staining pattern of /?-tubulin after
exposure to NOC was different from that observed
after ECA exposure. This suggests that ECA may be
affecting aspects of tubulin metabolism other than or
in addition to tubulin assembly. Additional study
should clarify the nature of ECA's effect on tubulin
and what, if any, alteration there is on outflow physiology.
We found the time course of discernible changes in
. ECA-induced cell shape and actin staining was much
longer than reported for increases in outflow facility
induced previously be this drug.4'5 However, a causative role for cell shape and/or actin changes cannot
be. excluded in the facility-increasing mechanism
of ECA.
The obvious cell shape change we observed may be
an end-stage phenomenon, preceded by more subtle
changes in cell adhesion and junctional permeability.
The results of morphologic studies in the monkey eye
in vivo4 and the human eye in vitro5 show a correlation between changes in tissue architecture and increased outflow facility after perfusion with ECA.
These studies showed that perfusion with this drug
resulted in breaks in the inner wall endothelial lining
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / August 1992
of Schlemm's canal and loss of cell-cell attachments
between the inner wall and the subendothelial trabecular cells and between trabecular cells themselves. Similar morphologic changes were seen in the monkey eye
in vivo after perfusion with CYTO B and with calcium chelators,""16 agents that also cause large increases in outflow facility. Furthermore, shape
changes in cultured cells were seen after CYTO B administration in our and previous studies.1718
Collectively, the results of these data suggest that
ECA belongs to the class of "trabecular-acting" drugs
that exert their physiologic effects on outflow facility
by induced changes in the cytoskeleton.4'5 Although
ECA-induced changes in the staining of filamentous
actin were dramatic, we observed distinct earlier
changes in the staining pattern of /3-tubulin. It is possible that this drug's facility-increasing action is mediated by changes in actin (and cell shape) and tubulin. Therefore, in addition to changes in F-actin or the
junctions themselves, assembly and disassembly processes involving tubulin1920 may mediate the observed changes in tissue architecture, which ultimately may result in increased outflow facility.
Our findings with ECA and other drugs that influence cell shape suggest a possible dynamic role for the
cytoskeleton of outflow pathway cells in mediating
outflow resistance. Additional research should clarify
whether changes in the cytoskeleton of outflow pathway cells and changes in outflow facility are related
causally or whether both are derived secondarily from
some other mechanism.
Key words: trabecular meshwork, ethacrynic acid, outflow
facility, glaucoma, cytoskeleton
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