Am J Physiol Heart Circ Physiol 288: H660 –H669, 2005.
First published September 23, 2004; doi:10.1152/ajpheart.00608.2004.
Imaging remodeling of the actin cytoskeleton in vascular smooth muscle cells
after mechanosensitive arteriolar constriction
Nicholas A. Flavahan, Simon R. Bailey, William A. Flavahan, Srabani Mitra, and Sheila Flavahan
Davis Heart and Lung Research Institute, Ohio State University, Columbus, Ohio
Submitted 18 June 2004; accepted in final form 20 September 2004
contraction to acetylcholine was associated with a decrease in
monomeric G-actin and that inhibitors of actin polymerization
(cytochalasin D, latrunculin A) reduced contractile activity.
These results suggested that alterations in actin polymerization
can contribute to regulation of contractile tone (18). Furthermore, the inhibitory effect of latrunculin A was dependent on
muscle length, suggesting that actin polymerization may be of
particular importance during mechanical strain (10, 11, 18).
Vascular smooth muscle cells (VSMCs) of small arteries and
arterioles are mechanosensitive, constricting in response to
elevations in transmural pressure (PTM) (5, 29). This constriction, termed the “myogenic response,” contributes to blood
flow autoregulation and the generation of basal vascular tone
(5, 29). Previous reports have observed impaired constrictor
responses to elevations in PTM and to other constrictor stimuli
in small arteries after inhibition of actin polymerization (9, 17,
30). Furthermore, PTM elevation has been reported to decrease
the VSMC content of G-actin in small cerebral arteries (4).
Actin polymerization may therefore be important for VSMC
constriction in response to agonist and mechanical stimulation
(9, 17, 30). However, the role played by actin polymerization
has not yet been clearly defined. Indeed, no previous studies
have assessed potential changes in the organization of the actin
cytoskeleton during arteriolar constriction. The aim of the
present study was, therefore, to determine the role of actin
polymerization and changes in the architecture of the VSMC
actin cytoskeleton in arteriolar constriction.
METHODS
myogenic response; laser scanning microscopy; cytochalasin D; latrunculin A; phenylephrine
Blood Vessel Chamber
THERE ARE TWO distinct actin-based filamentous systems in
contractile, differentiated smooth muscle cells: the cytoskeleton, which comprises predominantly nonmuscle actin (and
intermediate filaments), and the actin filaments of the smooth
muscle contractile apparatus that interact with smooth muscle
myosin (32, 33). One model for the structural organization of
these distinct systems proposes that the cytoskeletal elements
pervade the cytoplasm extending along the axial plane of the
cell, whereas smooth muscle contractile elements are oriented
at an angle to the cell axis and likely couple to the actin
cytoskeleton at dense bodies and dense plaques (32, 33).
Although the actin cytoskeleton has generally been considered
a passive structure in contractile smooth muscle cells, Mehta
and Gunst (18) demonstrated in tracheal smooth muscle that
Animal procedures were approved by the Ohio State University
Institutional Animal Care and Use Committee. Male mice (C57BL6)
were euthanized by CO2 asphyxiation. Distal tail arterioles were then
rapidly removed and placed in cold Krebs-Ringer bicarbonate solution
containing (in mmol/l) 118.3 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2
KH2PO4, 2.5 CaCl2, 25.0 NaHCO3, and 11.1 glucose (control solution) (22). Arterioles were cannulated at both ends with glass micropipettes, secured using 12-0 nylon monofilament sutures, and placed in
a microvascular chamber (Living Systems; Burlington, VT). The
chamber was superfused with control solution, maintained at 37°C,
pH 7.4 (gassed with 16% O2-5% CO2-balance N2), and placed on the
stage of an inverted microscope (⫻20, Nikon TMS-F) connected to a
video camera (Panasonic, CCTV camera). The vessel image was
projected onto a video monitor, and the internal diameter was continuously determined by a video dimension analyzer (Living Systems)
and monitored using a BIOPAC (Santa Barbara, CA) data-acquisition
system (22).
Address for reprint requests and other correspondence: N. A. Flavahan,
Heart and Lung Research Institute, 473 W. 12th Ave., Rm. 110E, Columbus,
OH 43210 (E-mail: [email protected]).
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.
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0363-6135/05 $8.00 Copyright © 2005 the American Physiological Society
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Flavahan, Nicholas A., Simon R. Bailey, William A. Flavahan,
Srabani Mitra, and Sheila Flavahan. Imaging remodeling of the
actin cytoskeleton in vascular smooth muscle cells after mechanosensitive arteriolar constriction. Am J Physiol Heart Circ Physiol 288:
H660 –H669, 2005. First published September 23, 2004; doi:10.1152/
ajpheart.00608.2004.—Experiments were performed to determine
whether remodeling of the actin cytoskeleton contributes to arteriolar
constriction. Mouse tail arterioles were mounted on cannulae in a
myograph and superfused with buffer solution. The ␣1-adrenergic
agonist phenylephrine (0.1–1 ␮mol/l) caused constriction that was
unaffected by cytochalasin D (300 nmol/l) or latrunculin A (100
nmol/l), inhibitors of actin polymerization. In contrast, each compound abolished the mechanosensitive constriction (myogenic response) evoked by elevation in transmural pressure (PTM; 10 – 60 or
90 mmHg). Arterioles were fixed, permeabilized, and stained with
Alexa-568 phalloidin and Alexa-488 DNAse I to visualize F-actin and
G-actin, respectively, using a Zeiss 510 laser scanning microscope.
Elevation in PTM, but not phenylephrine (1 ␮mol/l), significantly
increased the intensity of F-actin and significantly decreased the
intensity of G-actin staining in arteriolar vascular smooth muscle cells
(VSMCs). The increase in F-actin staining caused by an elevation in
PTM was inhibited by cytochalasin D. In VSMCs at 10 mmHg,
prominent F-actin staining was restricted to the cell periphery,
whereas after elevation in PTM, transcytoplasmic F-actin fibers were
localized through the cell interior, running parallel to the long axis of
the cells. Phenylephrine (1 ␮mol/l) did not alter the architecture of the
actin cytoskeleton. In contrast to VSMCs, the actin cytoskeleton of
endothelial or adventitial cells was not altered by an elevation in PTM.
Therefore, the actin cytoskeleton of VSMCs undergoes dramatic
alteration after elevation in PTM of arterioles and plays a selective and
essential role in mechanosensitive myogenic constriction.
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Vasomotor Responses
Arterioles were initially maintained at a PTM of 60 mmHg for 60
min, during which time they developed phasic and tonic vasomotor
activity (3, 22). Arterioles analyzed in the present study had basal
internal diameters of 50 –90 ␮m. To assess myogenic responses, PTM
was decreased to 10 mmHg for 30 min and then abruptly increased to
60 or 90 mmHg (22). When the effects of drugs on the myogenic
response were assessed, they were administered at a PTM of 10
mmHg, 15 min before elevations in PTM. Unless stated otherwise,
constrictor responses to the ␣1-adrenoceptor (␣1-AR) agonist phenylephrine were also evaluated at a PTM of 10 mmHg, because this PTM
is below the threshold for mechanosensitive signaling in arteriolar
VSMCs (22).
Laser Scanning Microscopy
Data Analysis
Unless stated otherwise, data are expressed as means ⫾ SE; n is the
number of animals from which blood vessels were taken. Alterations
Fig. 1. Effect of inhibitors of actin polymerization, cytochalasin D (Cyto D; 300 nmol/l),
or latrunculin A (Lat A; 100 nmol/l), on
constrictor responses evoked by increasing
transmural pressure (PTM; from 10 to 60 or
90 mmHg; A and B) or by the ␣1-adrenoceptor (␣1-AR) agonist phenylephrine (0.01–1
␮mol/l; C) in mouse tail arterioles. Responses to elevations in PTM are presented as
representative traces (A) or as means ⫾ SE
for n ⫽ 3– 8 experiments (B). In A, PTM was
increased when indicated (F, from 10 to 90
mmHg), and an upward deflection represents
an increase in diameter. The traces are offset
for clarity; neither Lat A nor Cyto D had an
effect on baseline diameter of the arterioles
at a PTM of 10 mmHg. In B and C, responses
are expressed as the percent change in the
baseline diameter at 10 mmHg in response to
increases in PTM from 10 to 60 or 90 mmHg
(B) or in response to phenylephrine (C).
Inhibition of actin polymerization by Cyto D
(300 nM) or Lat A (100 nM) abolished the
myogenic response (A and B) but did not
affect the constrictor response to phenylephrine (C; presented as means ⫾ SE for 3 or 4
experiments).
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Arterioles were mounted in specialized “flipper” chambers (Living
Systems) that enabled the blood vessel assembly to be rapidly (⬃ 1 s)
transferred from control solution to paraformaldehyde (3%, 4°C, 30
min). Unless stated otherwise, arterioles were fixed under control
conditions at a PTM of 10 mmHg or once the constrictor response to
an elevation of PTM or phenylephrine was stable (i.e., after ⬃8 min).
After fixation, arterioles were rinsed in PBS (3 ⫻ 10 min, 4°C),
permeabilized in TTX-100 (0.5%, 4°C) for 15 min, and then rinsed
again in PBS (3 ⫻ 10 min, 4°C). The arterioles were incubated
overnight (4°C) with Alexa 568-phalloidin (to label F-actin, 5 U/ml),
Alexa 488-DNAse I (to label G-actin, 9 ␮g/ml), or Sytox (to label
nuclei, 300 nM) (Molecular Probes). After being washed in PBS (3 ⫻
10 min, 4°C), the arterioles were cannulated in a specialized chamber
designed for use with an inverted fluorescent microscope (Living
Systems). The arterioles were then observed using a Zeiss 510 laser
scanning microscope equipped with a ⫻63 water immersion objective
(1.2 numerical aperture). Alexa 488-DNAseI or Sytox was excited
using the Argon/2 488-nm laser line, and the emission was processed
through a DM545 and a 500 –530BP filter; Alexa 568-phalloidin was
imaged using the HeNe 543-nm line and a DM 545 and LP 560 filter.
Images were obtained at an optical zoom of 2.0 or 2.5 (x-y pixel
dimensions of 0.14 ⫻ 0.14 ␮m and 0.11 ⫻ 0.11 ␮m, respectively),
and all images were obtained using a 512 ⫻ 512 frame and line
averaging of 4 scans. The resident pixel time was 12.8 ␮s (scan speed
5). When more than one fluorescent probe was imaged, channels were
switched after each frame. Laser power was generally 10% for the
Argon/2 488-nm and HeNe 543-nm lasers. Pinhole size (0.39 airy
units for F-actin staining) was set to generate optical slices (or “z”
resolution) of 0.6 ␮m, and z stacks were obtained at z steps of 0.6 ␮m.
Detector gain and amplifier offset were set to optimize the dynamic
range of the instrument.
When images were to be processed for quantitation of fluorescence,
two arterioles (control and test) were studied in parallel. These paired
arterioles were processed and imaged at the same time, under identical
conditions. Quantitation of fluorescence and analyses of nuclear
dimensions were performed using Zeiss laser scanning microscope
software and Volocity (Improvision; Lexington, MA), respectively.
All images are presented in their original state, and no processing,
thresholding, or photoediting has been performed.
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in arteriolar diameter were expressed as the percent change in the
basal prestimulation level. When global changes in fluorescence
intensity of optical slices were analyzed, images were processed at a
threshold cutoff of 22 (out of an 8-bit maximal intensity of 255) or
297 (out of a 12-bit maximal intensity of 4,095). This was performed
to mask the background and to prevent differences in background area
from influencing the results. When the profile of F-actin intensity
through individual VSMCs was analyzed, a central transverse line was
drawn through the widest part of the cells. Arterioles were first
visualized in orthogonal mode, and VSMCs that had the complete
transverse profile and the majority (⬎70%) of their longitudinal
profile contained within the z stack were selected for single-cell
analysis. The axial lengths of the cells were scanned in orthogonal
mode to determine the widest transverse profile of each cell. The x, y,
and z coordinates describing a central transverse line (single pixel
width) through that profile were then used to localize the line in the
x-y (two-dimensional) image for that z slice. The fluorescent intensity
of the line in the x-y image was then determined using Zeiss laser
scanning microscope “Profile” software. The edges of the cell were
defined as the first and final peaks in F-actin intensity. The width of
the cell was calculated as the distance between the two peaks. The
area under the curve (AUC; fluorescent intensity ⫻ distance, in ␮m)
was determined using a Riemann sum/trapezoidal rule calculation. For
this calculation, curves were restricted to first peak to final peak with
an additional 0.28 ␮m on either side. Statistical evaluation of the data
was performed by Student’s t-test for either paired or unpaired
observations. When more than two means were compared, ANOVA
was used. If a significant F-value was found, Scheffè’s test for
multiple comparisons was employed to identify differences among
groups. Values were considered to be statistically different when P
values were ⬍0.05.
RESULTS
Effect of Inhibitors of Actin Polymerization on
Arteriolar Constriction
Elevation in PTM from 10 to 60 or 90 mmHg caused tail
arterioles to initially dilate, reflecting passive dilation, and then
constrict, reflecting activation of the mechanosensitive “myogenic response” (Fig. 1) (22). Two inhibitors of actin polymerization were used: cytochalasin D, which acts by binding to the
rapidly growing barbed end of actin filaments, and latrunculin
A, which binds and sequesters actin monomers (34, 38).
Cytochalasin D (300 nmol/l) or latrunculin A (100 nmol/l)
abolished the myogenic constriction evoked by increasing PTM
from 10 to 60 or 90 mmHg (Fig. 1, A and B). In contrast,
cytochalasin D (300 nmol/l) or latrunculin A (100 nmol/l) had
no effect on the diameter of arterioles at a PTM of 10 mmHg
and did not affect constriction evoked by the ␣1-AR agonist
phenylephrine (0.01–1 ␮mol/l), assessed at a PTM of 10 mmHg
(Fig. 1C).
Imaging Arterioles
Analysis of VSMC actin polymerization. Laser scanning
microscopy generated optical sections through the arterial wall,
providing excellent visualization of vascular cells (Fig. 2).
Endothelial cells were aligned with blood flow and were
characterized by F-actin staining that delineated cell borders
(Fig. 2). Endothelial cells had intense staining for monomeric
G-actin throughout the cell interior (Fig. 2). Adventitial cells,
likely representing adventitial fibroblasts, were identified by
nuclear staining and by characteristic high-intensity staining
for G-actin but minimal staining for F-actin (Fig. 2). The most
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Fig. 2. Representative laser scanning microsopy (LSM) images of mouse tail
arterioles demonstrating the presence of three cell types within the blood vessel
wall. LSM images were obtained as described in the text. Orientation of the
blood vessel is presented in the inset drawing, with the red square representing
the optical slices. A: oblique section through an arteriole wall that had been
stained with Alexa 568-phalloidin (light blue) to label F-actin and Sytox (red)
to label nuclei. The endothelial cells line the lumen and are oriented in the
direction of flow (most prominent in the bottom part of the image). Vascular
smooth muscle cells (VSMCs), rich in F-actin fibers, are oriented in a circular
fashion around the arteriole, perpendicular to the endothelial cells (VSMCs
most prominent in the top part of the image). Adventitial cells are also present
and are easily distinguished from VSMCs by their lack of F-actin staining. B:
oblique section through an arteriole wall that had been stained with Alexa
568-phalloidin (red) to label F-actin and Alexa 488-DNAse I (green) to label
G-actin. Images are presented for individual channels (top two images) and for
the composite image (bottom image). All three cell types stained strongly for
G-actin. Arterioles were fixed at a PTM of 60 mmHg. Bar ⫽ 5 ␮m.
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Fig. 3. Representative LSM images of mouse tail arterioles that were fixed at
a PTM of 10 (A), 60 (B), or 90 mmHg (C) and stained with Alexa 568phalloidin to label F-actin (light blue). At 60 and 90 mmHg, arterioles were
fixed after stabilization of the myogenic constrictor response (⬃8 min after
PTM was increased). The LSM images represent stacks containing multiple
sequential LSM sections. The series of sections was obtained through the front
surface of the arterioles (drawing) and are presented as an orthogonal view. In
this mode, the sections are stacked on top of one another, and only one of those
sections is visible (taken approximately from the middle of the stack). The
narrow picture at the right of each image represents an orthogonal view
through the stack of sections (red arrow in cartoon) and was obtained at the
position of the red vertical line on each main image. The alternate orthogonal
view (green arrow in drawing), which was obtained at the position of the green
horizontal line on each main image, is presented at the top of each picture. The
position of each main image, relative to the orthogonal side views, is indicated
by the blue lines in the side views. Bar ⫽ 5 ␮m.
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prominent cells were VSMCs, which comprised one to two cell
layers and were oriented in a circular fashion around the blood
vessel, perpendicular to the luminal endothelial cells. VSMs
stained intensely for F-actin and G-actin (Fig. 2).
Global changes in VSMC actin dynamics were assessed by
quantifying fluorescence from optical slices through the media
of the arterioles, excluding the endothelium. When present,
adventitial cells were excluded from the analyses by defining
regions of interest (ROI). Elevation of PTM from 10 to 60 or 90
mmHg was associated with increased intensity of F-actin
staining (23.6 ⫾ 6.1%, n ⫽ 8, P ⬍ 0.01) and decreased
intensity of G-actin staining of VSMCs (⫺22.8 ⫾ 8.3%, n ⫽
8, P ⫽ 0.01), indicating actin polymerization. The increase in
F-actin staining was similar when PTM was elevated from 10 to
60 mmHg (23.5 ⫾ 5.8% increase, n ⫽ 4, P ⬍ 0.05) or from 10
to 90 mmHg (30.4 ⫾ 12.7% increase, n ⫽ 7, P ⬍ 0.05). In
contrast to elevations in PTM, constriction of arterioles with the
␣1-AR agonist phenylephrine (to ⬃ 70% of baseline diameter,
1 ␮mol/l) at a constant PTM of 10 mmHg was not associated
with significant changes in the intensity of F-actin [0.5 ⫾
10.9%, n ⫽ 3, P ⫽ not significant (NS)] or G-actin staining in
VSMCs (⫺7.9 ⫾ 12.6%, n ⫽ 3, P ⫽ NS).
Analyzing VSMC cytoskeleton architecture. At a PTM of 10
mmHg, F-actin staining of arteriolar VSMCs resembled a
cortical actin structure: intense staining for F-actin was localized at the cell periphery, with greatly reduced staining in the
cell interior (Fig. 3A). After an elevation of PTM (to 60 or 90
mmHg), there was a dramatic reorganization of the actin
cytoskeleton, with fibers dispersed through the interior of the
VSMCs, running parallel to the long axis of the cells (Fig. 3, B
and C). To quantify remodeling of the VSMC actin cytoskeleton, the profile of F-actin staining through a central transverse
axis of VSMCs was determined from orthogonal z sections
(Fig. 4). At a PTM of 10 mmHg, cytoskeleton organization was
characterized by two prominent peaks of F-actin staining,
delineating the cell periphery, with a dramatic reduction in
staining between the two peaks, representing the interior of the
cells (Fig. 4). The intensity of F-actin staining in the cell
interior was significantly increased at 60 mmHg and further
significantly increased at 90 mmHg: PTM elevation from 10 to
60 mmHg increased the AUC (fluorescence intensity ⫻ distance, in ␮m) from 351.3 ⫾ 39.8 to 771.2 ⫾ 81.2 (n ⫽ 16 cells,
P ⫽ 0.0001), and an elevation of PTM from 10 to 90 mmHg
increased the AUC from 440.5 ⫾ 52.8 to 1,008.7 ⫾ 51.8 (n ⫽
18 cells, P ⬍ 0.0001; P ⬍ 0.02 for comparison between 60 and
90 mmHg; Fig. 4). In contrast, constriction of arterioles with
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ACTIN REMODELING AND ARTERIOLAR CONSTRICTION
the ␣1-AR agonist phenylephrine (to ⬃ 70% baseline diameter)
at a PTM of 10 mmHg was not associated with changes in the
architecture of F-actin staining in arteriolar VSMCs: AUC of
446.7 ⫾ 32.3 and 480.1 ⫾ 30.2 for control and phenylephrinetreated arterioles, respectively (n ⫽ 26 cells, P ⫽ NS; Figs. 4
and 5).
At a PTM of 10 mmHg, VSMC nuclei were present in areas
devoid of F-actin staining, whereas at PTMs of 60 or 90 mmHg,
Fig. 4. F-actin intensity profiles for individual VSMCs determined from paired
analyses of arterioles at PTMs of 10 and 60 mmHg (top), PTMs of 10 and 90
mmHg (middle), and a PTM of 10 mmHg in the absence (control) and presence
of phenylephrine (constricted to ⬃70% baseline diameter, bottom). The intensity profiles were generated from LSM z stacks of arterioles, which were fixed
(at a PTM of 10 mmHg or 8 min after elevation in PTM to 60 or 90 mmHg or
8 min after exposure to phenylephrine) and then stained with Alexa 568phalloidin to label F-actin. As explained in METHODS, paired arterioles were
processed and analyzed at the same time under identical conditions. All images
in this analysis were 8-bit, so the maximal intensity for F-actin fluorescence
was 255. With the use of the orthogonal mode for viewing the z stack, a central
transverse line was drawn through the widest part of the cells. The edges of the
cell were defined as the first and final peaks in F-actin intensity. To facilitate
graphical representation of these intensity profiles, the first peak was defined as
0 and the final peak was normalized to 1. The data are presented as means ⫾
SE for intensity and for relative distance; n ⫽ 16 –26 cells. The width of the
cells, calculated as the absolute distance between the two peaks, increased as
a result of arteriolar constriction: elevation in PTM from 10 to 60 mmHg was
associated with an increase in width from 4.8 ⫾ 0.4 to 6.6 ⫾ 0.5 ␮m (n ⫽ 16
cells, P ⬍ 0.005; top), elevation of PTM from 10 to 90 mmHg increased width
from 5.1 ⫾ 0.3 to 6.6 ⫾ 0.5 ␮m (n ⫽ 18 cells, P ⫽ 0.01; middle), and
phenylephrine-induced constriction increased width from 4.9 ⫾ 0.3 to 6.0 ⫾
0.3 ␮m (n ⫽ 26 cells, P ⬍ 0.01; bottom). Because of differences in absolute
distance, not all cells are represented at every relative distance. However,
because of careful clustering of adjacent points in the analyses, the SEs for
relative distance are smaller than the symbols and are therefore not visible.
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the transcytoplasmic F-actin fibers were observed compressing
the VSMC nuclei (Fig. 6). Indeed, an elevation of PTM from 10
to 60 mmHg was associated with a significant elongation of
VSMC nuclei. Although there was no change in the volume of
VSMC nuclei (170.0 ⫾ 9.1 and 178.1 ⫾ 10.4 ␮m3 at 10 and
60 mmHg, respectively, n ⫽ 11 cells, P ⫽ NS), an elevation of
PTM was associated with an increased length (20.1 ⫾ 1.5 and
28.6 ⫾ 1.7 ␮m at 10 and 60 mmHg, respectively, n ⫽ 11 cells,
P ⬍ 0.002) but reduced diameter (3.3 ⫾ 0.1 and 2.8 ⫾ 0.1 ␮m
at 10 and 60 mmHg, respectively, n ⫽ 11 cells, P ⬍ 0.05) of
VSMC nuclei. This nuclear compression occurred despite an
increase in the transverse width of VSMCs associated with
pressure-induced myogenic constriction: an elevation in PTM
from 10 to 60 mmHg was associated with an increase in the
diameter of VSMCs from 4.8 ⫾ 0.4 to 6.6 ⫾ 0.5 ␮m (n ⫽ 16
cells, P ⬍ 0.005). For comparison, an elevation of PTM from 10
to 90 mmHg increased VSMC diameter from 5.1 ⫾ 0.3 to
6.6 ⫾ 0.5 ␮m (n ⫽ 18 cells, P ⫽ 0.01), whereas phenylephrine-induced constriction increased VSMC diameter from
4.9 ⫾ 0.3 to 6.0 ⫾ 0.3 ␮m (n ⫽ 26 cells, P ⬍ 0.01). These
changes in VSMC width are similar to those previously reported during constriction of cremaster arterioles to norepinephrine (16).
In contrast to VSMCs, the actin cytoskeleton of endothelial
or adventitial cells was not altered by elevations in PTM (data
not shown)
Effect of cytochalasin D. Cytochalasin D (300 nM), administered at a PTM of 10 mmHg, significantly decreased the
intensity of F-actin staining observed in VSMCs after an
elevation in PTM to 60 mmHg (19.8 ⫾ 1.5% decrease compared with paired, untreated control arteries at a PTM of 60
mmHg, n ⫽ 3, P ⬍ 0.01), which was equivalent to an 84%
reduction in the response to PTM elevation. This was associated
with marked deterioration in the architecture of F-actin staining, with fragmentation and disordering of F-actin fibers (Fig.
7). This deterioration in structural organization of the cytoskeleton was associated with irreversible impairment of arteriolar
function. As noted in Fig. 1, cytochalasin D (300 nM) abol-
ACTIN REMODELING AND ARTERIOLAR CONSTRICTION
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Cytochalasin D (300 nM) did not inhibit constriction to
phenylephrine at a PTM of 10 mmHg (Fig. 1). However, after
the arterioles were exposed to an elevated PTM (60 mmHg, 30
min) in the presence of cytochalasin D, subsequent constriction
to the ␣1-AR agonist at a PTM of 10 mmHg (and absence of
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Fig. 5. Representative LSM image of a mouse tail arteriole that was fixed
during constriction with the ␣1-AR agonist phenylephrine (to ⬃70% of
baseline diameter) at a PTM of 10 mmHg. The arteriole was fixed after
stabilization of the phenylephrine-induced constriction (after ⬃8 min) and
subsequently stained with Alexa 568-phalloidin to label F-actin (light blue).
The LSM image represents a stack containing multiple sequential LSM
sections. The series of sections was obtained through the front surface of the
arteriole (drawing) and is presented as an orthogonal view. In this mode, the
sections are stacked on top of one another, and only one of those sections is
visible (taken approximately from the middle of the stack). The narrow picture
at the right of the image represents an orthogonal view through the stack of
sections (red arrow in the drawing) and was obtained at the position of the red
vertical line on the main image. The alternate orthogonal view (green arrow in
image), which was obtained at the position of the green horizontal line on the
main image, is presented at the top of each picture. The position of the main
image, relative to the orthogonal side views, is indicated by the blue lines in the
side views. Bar ⫽ 5 ␮m.
ished the myogenic constriction evoked by an elevation in PTM
from 10 to 60 mmHg (Fig. 8A). Subsequent removal of
cytochalasin D from the perfusate failed to completely restore
the normal constriction to PTM elevation (Fig. 8A): the response to PTM elevation (from 10 to 60 mmHg) was a slight
decrease in diameter of 2.2 ⫾ 12.8% before cytochalasin D, an
increase in diameter of 87.9 ⫾ 12.1% during exposure to
cytochalasin D, and an increase in diameter of 40.4 ⫾ 15.6%
after subsequent removal of cytochalasin D (n ⫽ 3, P ⬍ 0.01
for responses before cytochalasin D and after removal of
cytochalasin D; Fig. 8A). In contrast, when arteriolar PTM was
maintained at 10 mmHg, an equivalent transient exposure to
cytochalasin D (300 nM) did not impair the myogenic constrictor response (Fig. 8B). Likewise, cytochalasin D (300 nM)
did not affect the pattern of F-actin staining in VSMCs when
arteriolar PTM was maintained at 10 mmHg (data not shown).
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Fig. 6. Representative LSM images of mouse tail arterioles that were fixed at
a PTM of 60 (A) or 10 mmHg (B) and stained with Alexa 568-phalloidin to label
F-actin (light blue) and Sytox (red) to label nuclei. The arteriole at 60 mmHg
was fixed at the peak of the passive increase in diameter, ⬃15 s after the
increase in PTM. The images were obtained through the front surface of the
arterioles (inset drawing). Images are presented for individual channels (top
two images in each collage) and for the composite image (bottom image in
each collage). Bar represents 5 ␮m.
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ACTIN REMODELING AND ARTERIOLAR CONSTRICTION
cytochalasin D) was dramatically reduced (Fig. 8C). Therefore,
the irreversible functional impairment associated with pressure-induced disruption of the actin cytoskeleton was evident
for both myogenic and ␣1-AR constriction. When constriction
to phenylephrine was assessed at a PTM of 60 mmHg, instead
of 10 mmHg, cytochalasin D also dramatically reduced constriction to the agonist (Fig. 8D).
DISCUSSION
In the present study, cytochalasin D or latrunculin A, two
mechanistically distinct inhibitors of actin polymerization,
abolished constriction of tail arterioles evoked by elevations in
PTM but did not directly affect constriction evoked by ␣1-AR
stimulation with phenylephrine. Furthermore, elevations in
PTM, but not phenylephrine, increased the intensity of F-actin
staining and decreased the intensity of G-actin staining in
arteriolar VSMCs. These results suggest that actin polymerization is activated by elevation in PTM and plays an essential and
selective role in the pressure-induced myogenic constriction of
arterioles. Actin polymerization, however, does not appear to
play a general role in arteriole constriction, as judged by the
response to phenylephrine. In these experiments, responses to
phenylephrine and to myogenic stimulation were both initiated
at a low PTM (10 mmHg), which is below the threshold for
mechanosensitive signaling in arterioles and so minimizes any
indirect effects associated with prior mechanical strain (22).
Indeed, when assessed at a PTM of 60 mmHg, inhibition of
actin polymerization abolished the constriction evoked by
AJP-Heart Circ Physiol • VOL
phenylephrine. This likely resulted from indirect depression of
the ␣1-AR response. After inhibition of actin polymerization,
the arterioles did not tolerate elevations in PTM, which caused
disruption of the VSMC actin cytoskeleton and irreversible
impairment of arteriolar constriction. Cytochalasin D is a
reversible inhibitor of actin polymerization, and arterioles
could be transiently exposed to the agent at a PTM of 10 mmHg
without significant alteration in arteriole function. However,
combined exposure to elevated PTM and to cytochalasin D
impaired constriction to myogenic or ␣1-AR activation, even
after PTM was returned to 10 mmHg and cytochalasin D was
removed from the superfusate. Although the present study
analyzed the response to only 60 and 90 mmHg, the irreversible impairment in arteriolar function makes it unlikely that
inhibition of actin polymerization merely shifted myogenic
responsiveness to higher levels of PTM. Previous studies have
observed generalized depression in constriction of small arteries/arterioles after inhibition of actin polymerization, with
reduced responses to KCl, angiotensin II, or an elevation in
PTM (9, 17, 30). However, those previous studies analyzed
constrictor responses when the arteries were concurrently exposed to significant mechanical strain (PTMs of 50 –70 mmHg)
(9, 17, 30). Furthermore, the effects were observed using
agents (cytochalasin B) or concentrations of cytochalasin D
that cause cleavage of actin filaments rather than inhibition of
actin polymerization (38). The present study, by analyzing
arterial constriction in the absence of significant mechanical
strain, by directly analyzing actin polymerization and by uti-
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Fig. 7. Representative LSM images of mouse tail arterioles demonstrating the effects of Cyto D (300 nM) on VSMCs. The arterioles were fixed at a PTM of 90
mmHg and stained with Alexa 568-phalloidin to label F-actin (light blue). Arterioles were treated with Cyto D (300 nM) before the elevation in PTM and were
fixed ⬃8 min after the increase in PTM. The left image was obtained as a single optical section through the side wall of the arteriole (drawing). Disruption of
the actin cytoskeleton is indicated by the arrows. The right image represents a stack containing multiple sequential LSM sections. The series of sections was
obtained through the front surface of the arteriole (drawing) and is presented as an orthogonal view. In this mode, the sections are stacked on top of one another,
and only one of those sections is visible (taken approximately from the middle of the stack). The narrow picture at the right of the image represents an orthogonal
view through the stack of sections (red arrow in drawing) and was obtained at the position of the red vertical line on the main image. The alternate orthogonal
view (green arrow in drawing), which was obtained at the position of the green horizontal line on the main image, is presented at the top of the picture. The
position of the main image, relative to the orthogonal side views, is indicated by the blue lines in the side views. Bar ⫽ 5 ␮m.
ACTIN REMODELING AND ARTERIOLAR CONSTRICTION
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lizing agents that selectively inhibit actin polymerization, demonstrates that actin polymerization is not required for arteriolar
constriction but is essential for arterioles to function correctly
at an elevated PTM.
No previous studies have analyzed the three-dimensional
organization of the actin cytoskeleton of VSMCs or its potential modulation during arteriolar constriction. Indeed, the organization of the actin cytoskeleton has not been clearly
defined (32, 33). One model, based on histological analyses of
chicken gizzard smooth muscle cells, proposes that cytoskeletal elements pervade the cytoplasm extending along the axial
plane of the cell, whereas smooth muscle contractile elements
are oriented at an angle to the cell axis, coupling to the actin
cytoskeleton at dense bodies and dense plaques (32, 33). The
present study demonstrates that, at a PTM of 10 mmHg, F-actin
staining in arteriolar VSMCs resembled a cortical actin organization, with intense staining at the cell periphery and minimal staining in the cell interior. Indeed, when visualized in
orthogonal reconstruction, VSMCs appeared as hollow tubes
with intense peripheral bands of F-actin and minimal or lowintensity diffuse staining in the interior. This latter staining
may represent thin F-actin filaments that are beyond the resolution of the present images. At this low PTM, VSMC nuclei
AJP-Heart Circ Physiol • VOL
were localized in areas devoid of F-actin staining, although
these areas were not exclusively reserved for nuclei. After an
elevation of PTM to 60 or 90 mmHg, transcytoplasmic F-actin
fibers were localized running through the cell interior, parallel
to the long axis of the cells. The VSMC nuclei were observed
compressed between the F-actin fibers in areas that had a
reduced fiber density. Indeed, the dramatic change in the
organization of the actin cytoskeleton was matched by an
equally dramatic elongation of VSMC nuclei. Therefore, although the architecture of the actin cytoskeleton observed at
elevated pressure is consistent with the proposed model of
VSMCs and with previous histological analyses (19 –21, 32,
33), the structural organization observed at low pressure is
markedly different.
The emergence of transcytoplasmic F-actin fibers could be
observed as early as 15 s after the change in PTM (e.g., Fig. 6),
which is too rapid for the fibers to be generated by de novo
actin polymerization (6). Indeed, when assessed at the cellular
level (e.g., Fig. 4), the pressure-induced increase in F-actin
intensity was more striking than changes determined from
global quantitation of optical sections. Furthermore, at the
cellular level, elevations in PTM from 10 to 60 or 90 mmHg
caused a stepwise increase in F-actin intensity, whereas global
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Fig. 8. A and B: representative tracings
demonstrating the effects of Cyto D (300
nM) on arteriolar function. An upward deflection represents an increase in diameter.
Elevation in PTM (from 10 to 60 mmHg, F)
generated a robust myogenic constrictor response in mouse tail arterioles. A: if Cyto D
(300 nM) was introduced between the first
and second myogenic responses (15 min
before the second response), then the response was abolished. Removing Cyto D
from the superfusate immediately after the
second response and enabling a 30-min recovery period did not restore the myogenic
response on the third challenge. B: if the
arteriole was incubated with Cyto D for an
equivalent period of time, but PTM was
maintained at 10 mmHg during the exposure, then removal of Cyto D from the superfusate enabled a normal myogenic constrictor response. Similar results were obtained in two other experiments. Boxes
represent the presence of Cyto D in the
superfusate. C: constriction to phenylephrine, assessed at a PTM of 10 mmHg, was
analyzed under control conditions or after
the artery had been exposed to an elevation
in PTM (60 mmHg, 30 min) in the presence
of Cyto D (300 nM), followed by removal of
Cyto D from the superfusate and return of
PTM to 10 mmHg (30-min recovery period
as in A). D: constriction to phenylephrine,
assessed at a PTM of 60 mmHg, was analyzed under control conditions or in the presence of Cyto D (300 nM). In C and D,
responses are expressed as the percent
change in the baseline diameter at 10 (C) or
60 mmHg (D) and are presented as means ⫾
SE for n ⫽ 3.
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ACTIN REMODELING AND ARTERIOLAR CONSTRICTION
AJP-Heart Circ Physiol • VOL
adventitial cells were often in close apposition to VSMCs and
may represent adventitial fibroblasts or vascular “interstitial
cells of Cajal” (ICCs) (23, 24). ICCs are smooth muscle/
fibroblast-like cells that are present in the gastrointestinal tract,
urinary tract, mesenteric resistance arteries, and portal vein,
where they are thought to function as pacemakers, regulating
the phasic activity of smooth muscle cells (23, 24). Although
mouse tail arterioles display phasic constrictor activity, the
mechanism(s) underlying this activity has not been defined.
Elevations in PTM did not alter the actin cytoskeleton of
adventitial cells or of the endothelial cells, suggesting that they
are either not responsive to changes in PTM or that they respond
differently from VSMCs. Endothelial cells are mechanosensitive and respond to shear stress or to axial stretch of arteries
with reorganization of the actin cytoskeleton (2, 31).
Therefore, the present study, by analyzing arterial constriction in the absence of significant mechanical strain, by directly
analyzing actin polymerization and by utilizing agents that
selectively inhibit actin polymerization, demonstrates that actin
polymerization plays a selective and essential role in the
response of arterioles to elevations in PTM. The myogenic
constrictor response of mouse tail arterioles was selectively
attenuated by two mechanistically distinct inhibitors of actin
polymerization: cytochalasin D and latrunculin A. Moreover,
elevations in PTM stimulated actin polymerization in VSMCs,
which was associated with a dramatic reorganization of the
actin cytoskeleton. Actin polymerization and cytoskeletal remodeling did not occur during arteriolar constriction to the
␣1-AR agonist phenylephrine (at a low PTM), suggesting that it
is not an inherent part of the contractile process. However,
these results do not rule out an interaction between mechanosensitive and agonist-induced signaling in modulating actin
dynamics at higher PTMs. These results are consistent with
mediation of the myogenic response by two parallel signaling
pathways: remodeling of the actin cytoskeleton, which enables
the arteriole to withstand the increase in PTM, and activation of
the MLC-based signaling cascade to cause constriction of the
arteriole.
GRANTS
This study was supported by grants from the Scleroderma Research Foundation and National Heart, Lung, and Blood Institute Grant HL-67331 (to
N. A. Flavahan).
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