special communication
A method for isolating adult and neonatal airway smooth
muscle cells and measuring shortening velocity
S. P. DRISKA, R. E. LAUDADIO, M. R. WOLFSON, AND T. H. SHAFFER
Physiology Department, Temple University School of Medicine, Philadelphia, Pennsylvania 19140
single-cell isolation; ovine airway smooth muscle; smooth
muscle contraction; enzymatic dissociation; papain; preterm;
trachea
THE INITIAL PREPARATION of isolated smooth muscle cells
from the stomach of the toad Bufo marinus in 1971 (2,
13) made possible a number of major advances in our
understanding of vertebrate smooth muscle. Studying
isolated cells allows muscle responses to be measured
without the influence of substances released from endothelial cells, epithelial cells (27), and nerve endings.
Fay and collaborators used this preparation to make
numerous important contributions in several areas,
such as ultrastructure (22), contractile mechanisms
(11, 37), Ca21 movements (3, 40), membrane transport
(24), and biochemical mechanisms regulating contraction (18). Later, several investigators isolated cells from
mammalian smooth muscle tissues including ferret
aorta (8), swine (10) and bovine (33, 39) carotid and
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coronary artery, rabbit intestine (4), and guinea pig
stomach (6).
Isolated airway smooth muscle (ASM) cells have
been prepared from several species (dog, rat, guinea
pig, human), and the contractile responses of the
canine cells have been studied (19). Whereas some
investigators have succeeded in measuring force development by the single cells isolated from other smooth
muscles (7, 38), force measurement of single cells is
extremely difficult and requires expensive specialized
equipment. In contrast, measuring the unloaded shortening velocity of a single smooth muscle cell is relatively easy and does not require the elaborate instrumentation required for single-cell force measurements.
In this study, we first describe a method for isolating
viable, elongated, and Ca21-tolerant muscle cells from
both adult and preterm sheep airways. Then we describe an apparatus and method that allow unloaded
shortening of the cells to be recorded in a perfusion
chamber where the agonist is delivered by the constant
flow of perfusion solution. Finally, we present a method
for analyzing the shortening response of the isolated,
unloaded cell that allows shortening velocities to be
calculated from measurements of the cell length at
various times on playback of a videotape.
Over the last several decades, considerable progress
has been made in characterizing developmental (preterm, newborn, and adult) differences in tracheal physiology and mechanics (pressure-volume and pressureflow relationships and the effects of mechanical
ventilation; Refs. 5, 14, 17, 26, 28–31). These studies,
with the use of the sheep and other species, lend
valuable insight into how the very premature infant
may differ from the later-term and full-term neonate
with respect to airway function. We wish to build on
these studies by learning about the mechanical properties of the ASM at the single-cell level.
METHODS
Tissue
Tracheae from adult (between 3 and 6 yr old) and neonatal
[110–135 days gestation; full-term gestation 5 147 6 3 (SE)
days] sheep were harvested. Ewes were given ketamine and
bupivacaine. Lambs received lidocaine, ketamine, pancuronium bromide, and sodium bicarbonate. All animals were
killed with a barbiturate and potassium chloride overdose
(27). The freshly gathered tissue was placed in cold collecting
solution. The animals were managed according to National
8750-7587/99 $5.00 Copyright r 1999 the American Physiological Society
427
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Driska, S. P., R. E. Laudadio, M. R. Wolfson, and T. H.
Shaffer. A method for isolating adult and neonatal airway
smooth muscle cells and measuring shortening velocity. J.
Appl. Physiol. 86(1): 427–435, 1999.—Methods are described
for isolating smooth muscle cells from the tracheae of adult
and neonatal sheep and measuring the single-cell shortening
velocity. Isolated cells were elongated, Ca21 tolerant, and
contracted rapidly and substantially when exposed to cholinergic agonists, KCl, serotonin, or caffeine. Adult cells were
longer and wider than preterm cells. Mean cell length in 1.6
mM CaCl2 was 194 6 57 (SD) µm (n 5 66) for adult cells and
93 6 32 µm (n 5 20) for preterm cells (P , 0.05). Mean cell
width at the widest point of the adult cells was 8.2 6 1.8 µm (n
5 66) and 5.2 6 1.5 µm (n 5 20) for preterm cells (P , 0.05).
Cells were loaded into a perfusion dish maintained at 35°C
and exposed to agonists, and contractions were videotaped.
Cell lengths were measured from 30 video frames and plotted
as a function of time. Nonlinear fitting of cell length to an
exponential model gave shortening velocities faster than
most of those reported for airway smooth muscle tissues. For
a sample of 10 adult and 10 preterm cells stimulated with 100
µM carbachol, mean (6 SD) shortening velocity of the preterm cells was not different from that of the adult cells (0.64 6
0.30 vs. 0.54 6 0.27 s21, respectively), but preterm cells
shortened more than adult cells (68 6 12 vs. 55 6 11% of
starting length, respectively; P , 0.05). The preparative and
analytic methods described here are widely applicable to
other smooth muscles and will allow contraction to be studied
quantitatively at the single-cell level.
428
MEASURING SHORTENING VELOCITY OF SMOOTH MUSCLE CELLS
Institutes of Health regulations and the Guiding Principles in
the Care and Use of Animals of the American Physiological
Society. In addition, all procedures in this protocol were
approved by the Institutional Review Board.
Solutions
Enzymes and Reagents
Papain (type IV, P-4762) and DNase II (D-8764) were
obtained from Sigma Chemical (St. Louis, MO). DNase I (type
DP) was obtained from Worthington (Freehold, NJ), and
dithioerythritol (DTE) was obtained from Research Organics
(Cleveland, OH). Two lots of papain were used, with each
producing the same results.
Dissection and Tissue Digestion Conditions
The tissue was cut into sections approximately three to five
cartilage rings wide. The muscle layers were dissected from
the epithelium and the overlying connective tissue at room
temperature.
Papain (20 U/mg) was used at a concentration of 2 mg
protein/ml in MOPS-PSS. DNase II (1 mg/ml) was also added.
DTE (2 mM) was added to the digestion solution to activate
the papain by reducing the sulfhydryl groups. The DTE must
be fresh, or the papain will not be active or may not dissolve.
DTE was added from a 0.2 M stock solution kept under a
100% N2 atmosphere and sparged with N2 after each use. The
enzyme solution was warmed in a 37°C bath for 4–15 min
until the papain dissolved and was then immediately used.
Our laboratory (9) found previously that bubbling papain
solutions with oxygen during digestion is unadvisable, probably because it interferes with the reduction of the sulfhydryl
groups of papain.
The clean muscle was cut from the cartilage, added to the
digestion solution, and placed in the 37°C bath for 20–60 min,
depending on the amount of tissue, with an average of 30 min.
Once the tissue appeared swollen and frayed, it was removed
from the bath, rinsed gently with MOPS-PSS, and placed in a
90-mm-diameter plastic petri dish containing 20 ml MOPSPSS and 2 mg DNase I. DNase I was added to the petri dish
for better separation, settling, and collection of cells. Digestion with other enzymes (collagenase, dispase, elastase, and
chymopapain) did not produce satisfactory results.
Individual Cells and Microscopy
With the use of forceps, under a dissecting microscope
(Bausch and Lomb Stereozoom 37–30 magnification), individual cells were released into the MOPS-PSS when small
strips were gently teased from the digested tissue. These cells
could be seen falling off the strip if the mirror of the
microscope was set for off-axis illumination by transmitted
Optical System
The microscope image was passed through an Olympus
MTU adapter with a 32.5 lens attached to the monochrome
video camera (Dage-MTI NC70 Newvicon). The signal from
the camera was mixed with the signal from a time-date
generator and passed to a VHS videotape recorder. A microcomputer then digitized the monochrome signal into an 8-bit
image, 512 pixels wide 3 400 pixels high, and displayed it on
a monitor at video rates. With this optical configuration (340
objective), the full width of the image on the monitor screen
(,250 mm) corresponded to ,360 µm. Thus the final magnification was about 3700, and the width of each pixel represents
,0.7 µm. Cell dimensions were measured with imageanalysis software (BioQuant, R&M Biometrics, Nashville,
TN).
Recording Cell Contractions
Cell contractions were recorded in a perfusion chamber
made by the Biomedical Machine Shop at Temple Medical
School from a stainless steel disk. The outside edges of this
disk were slightly tapered to fit snugly into a water-heated
brass heat exchanger. The well in which cells were studied is
rhombus shaped. The acute angles of the rhombus are 45°,
and the resulting shape is ,20 mm long and 9 mm wide at its
widest point. Perfusion solution entered at one apex of the
rhombus and exited at the other through ports drilled into
the wall of the chamber ,1.5 mm above the floor. The floor of
the chamber is a 25-mm-diameter glass coverslip glued to the
base of the stainless steel disk. When the chamber is filled to
the top of the 3-mm-high walls, the chamber volume is ,270
µl. Water from a 37°C circulator warmed the apparatus to
34–35°C. Perfusion solutions entered the apparatus by gravity flow and passed through the heat exchanger, a switching
valve, and then through the port at the apex of the chamber.
Solutions were pumped out through a port in the opposite end
of the chamber by a peristaltic pump set to match the inflow
rate (,0.5 ml/min) so that the liquid level could be kept
approximately constant. With perfusion stopped, 100-µl aliquots of cells were loaded into the perfusion chamber and
allowed to settle. After the cells settled, MOPS-PSS containing 1.6 mM CaCl2 was allowed to flow in, removing debris and
dead cells. Many healthy cells remained, lightly adhering to
the floor of the chamber. Then the timer was started and the
switching valve was activated, allowing the agonist solution
to flow into the chamber and stimulate the cells. The Trypan
blue in the agonist solution permitted the leading edge of the
agonist solution to be visualized. The sucrose increased the
density of the solution so it flowed along the bottom of the
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MOPS-physiological salt solution (PSS). The composition
of MOPS-PSS was (in mM) 117.8 NaCl, 0.027 Na2EDTA, 6.0
KCl, 1.2 MgSO4, 24.3 MOPS (pH 7.5), and 5.6 glucose. It was
filtered through a 0.2-µm filter. This solution was nominally
Ca21 free.
Collecting solution. The collecting solution was identical to
MOPS-PSS except it lacked glucose and contained both
calcium (1.6 mM CaCl2 ) and phosphate (1.2 mM NaH2PO4 ).
Perfusion solution for the flow chamber studies. The standard solution for flow chamber studies was MOPS-PSS with
CaCl2 added to a final concentration of 1.6 mM.
Agonist perfusion solution for the flow chamber studies.
This solution was MOPS-PSS to which was added 100 µM
agonist, 1% sucrose, 1 mg/ml Trypan blue, and 1.6 mM CaCl2.
These solutions were filtered (0.2 µm) before use.
light. Surface tension pulled more cells off the strips when
they were lifted in and out of the solution. Cells then sank
toward the bottom of the dish. Viable adult cells settled on the
bottom of the dish, but viable neonatal cells did not always
reach the bottom. Sample aliquots (10 µl) of cells were
examined with bright-field, phase-contrast, or Nomarski optics by using an Olympus IMT-2 inverted microscope with
310, 20, or 40 objectives. All measurements were made by
using the 340 (0.60 numerical aperture) objective. When
viewed with phase-contrast optics, live, healthy cells appeared bright, without a visible nucleus. The perfusion
chamber used for cell contraction experiments (described in
Recording Cell Contractions) was open to the atmosphere,
and there was a meniscus at the top of the fluid level. The
meniscus made it impossible to use phase-contrast optics, so
bright-field optics were used when cell contractions were
videotaped.
MEASURING SHORTENING VELOCITY OF SMOOTH MUSCLE CELLS
chamber where the cells were located. Sucrose, by itself, did
not cause contractions. The blue agonist solution moved
across the center of the chamber with a linear velocity of ,0.6
mm/s. Using this flow velocity, the volume flow rate (0.5
ml/min) of the solution, and the width of the chamber (9 mm),
one can calculate the depth of the agonist solution layer to be
1.5 mm. These parameters ensure that all parts of a 300-µmlong cell would be exposed to agonist within 500 ms. Rapid
delivery of agonist is necessary for synchronous contraction of
a cell and high shortening velocities.
A number of agonists were used, including caffeine, KCl,
and cholinergic agonists (acetylcholine, bethanachol, carbachol, and methacholine). Carbachol was used in most experiments because it is more resistant to choline esterases.
Calculating Shortening Velocity
Accuracy of Cell Length and Velocity Measurements
Any factor that causes an error in the measurement of time
or cell length can lead to errors in the shortening velocity. The
effective temporal resolution of the measurements (33 ms)
was set by the use of a standard NTSC video signal (30
frames/s).
The accuracy of the cell-length measurements depends on a
number of factors, including the microscope and camera
optics, the resolution of the digital image, the accuracy and
resolution of the digitizing tablet used to trace the image of
the cell, and the ability of a human operator to trace the
image. The 512 3 400 pixel image allowed 0.25% resolution,
and the digitizing tablet used to trace the cell images was
capable of 0.15% resolution. After analyzing these factors, we
concluded that the precision of the human operator tracing
the image is likely to be the limiting factor in most measurements. Repeated measurements of the same cell are usually
within 2% of each other. Measurement errors occurring if one
end of the cell is out of the plane of focus are discussed in the
DISCUSSION, but, taking these errors into account, we estimate
that cell-length measurements are accurate to within 5% of
the true cell length. It is important to use high optical
magnification to maintain this accuracy, and we used a 340
objective for the data reported here.
RESULTS
Characteristics of the Cells
Examples of elongated adult and preterm cells are
shown in Fig. 1. The adult cell is 249 µm long and
appears to be completely relaxed. The preterm cell is
also fairly elongated but may have partially contracted;
it is 121 µm long. When cells have contracted slightly,
they sometimes have a serpentine appearance, but
other cells contract evenly without any apparent twisting or writhing. With extreme contraction, cells become
very thick and show ‘‘blisters’’ or membrane ‘‘blebs,’’
behavior that is common for isolated smooth muscle
cells. Both cells have relatively smooth margins, indicating an extended cell. The cells shown in Fig. 1 look quite
similar to cells isolated from other smooth muscles, but
some elongated airway cells (both neonatal and adult)
have a slightly different appearance, with angular
protrusions looking almost like thorns. It is unclear
whether this morphology is due only to adhering connective tissue or whether this represents the underlying
cellular structure.
Nuclei are not visible in Nomarski, bright-field, or
phase-contrast images of healthy adult cells. Nuclei in
damaged or lysed adult and preterm cells are readily
Fig. 1. Tracheal smooth muscle cells
isolated from adult and preterm sheep.
Both images were obtained with 340
differential interference contrast (Nomarski) optics by using an Olympus
IMT-2 inverted microscope. Gray-scale
images (8 bits) were captured directly
to a computer file with a Dage-MTI
Newvicon video camera. Top: smooth
muscle cell from a preterm trachea; cell
length is 121 µm. Bottom: smooth
muscle cell from an adult trachea; cell
length is 249 µm.
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Estimation of the cell shortening velocity was determined
by nonlinear fitting by using the SAS statistical program. Cell
length was measured in 30 video frames covering the range
before, during, and after contraction. The time (to the nearest
0.01 s) associated with each frame was also noted. A plot of
cell length vs. time was made.
Cell lengths were fit to the following exponential model:
length 5 Lmin 1 (Lmax 2 Lmin ) exp[2v (time 2 lag)], where
Lmax is the longest length measured, Lmin is the shortest
length reached, time is the time elapsed since the switching
valve started delivering agonist solution, lag is the time at
which rapid contraction starts, and v is the exponential rate
constant. These parameters are explained below.
The exponential rate constant v, with units of reciprocal
seconds (s21 ), is used as the shortening velocity. It represents
velocity in cell lengths per second and allows shortening
velocities of different sized cells to be compared. Lag is the
earliest time point that is fit by the curve. The fitting program
omits a few points at the beginning of the shortening phase, if
necessary, to get the best fit. Because times are measured by
the video stopwatch from the moment the switching valve is
activated, ‘‘lag’’ has no physiological significance because it
mostly represents the time required for agonist solution to
flow from the switching valve into the dish and reach the cell
and, therefore, is quite variable depending on flow rates and
cell location. It is not critical to determine the exact instant
when a cell starts to contract.
Usually the arrival of the agonist solution containing the
Trypan blue dye was plainly visible, and it would have been
possible to start the timer at the instant that the blue solution
reached the cell. This would have given smaller values of lag
that would represent only the time required for a cell to
respond to a stimulus. Sometimes, though, the boundary
between the clear and blue solutions was not sharp enough.
Therefore, we always started the timer the moment the
switching valve was activated, and the recorded videotape
had the timer information on each video frame.
429
430
MEASURING SHORTENING VELOCITY OF SMOOTH MUSCLE CELLS
Table 1. Dimensions of isolated airway
smooth muscle cells
Cells
n
Length, µm
Width, µm
Volumecyl , pl
Volumecone , pl
Adult
Preterm
66
20
194 6 57
93 6 32*
8.2 6 1.8
5.2 6 1.5*
10.2
2.0
3.4
0.7
Results for length and width are means 6 SD; n 5 no. of cells. Cells
were measured in the presence of 1.6 mM CaCl2 at 35°C. Volumecyl ,
cell volume assuming cylindrical geometry; Volumecone , estimated
volume treating cell as two cones joined at the base. Volumes were
calculated by using mean lengths and widths as given, for each cell
type. * Significantly different from adult (P , 0.05, Tukey-Kramer
honestly significant difference test).
Contraction of Cells
Observations of these smooth muscle cells in the
flow-through chamber revealed that the cells were Ca21
tolerant and that they contracted rapidly and substantially in response to agonists. Cells were loaded into the
perfusion chamber on the microscope stage, allowed to
settle with no solution flowing for 0.5–4 min, and then
perfused with 1.6 mM CaCl2 MOPS-PSS. After 1–10
min of perfusion with the CaCl2 solution, agonists were
introduced. Because Ca21-tolerant cells (cells that remained elongated in 1.6 mM CaCl2 ) were selected, the
contractions we videotaped were caused by the agonist,
not merely the exposure to CaCl2.
Figure 2 depicts the contraction of an adult ASM cell.
Figure 2A shows a plot of cell length (measured from
still frames of videotaped contractions) as a function of
time. Time was measured from the instant when the
switching valve was activated, not the instant when the
agonist reached the cell. Trypan blue in the agonist
solution allowed the arrival of the agonist at the cell to
be seen. Usually the cells contracted within 2–3 s of the
DISCUSSION
We believe that a smooth muscle cell preparation can
produce the highest quality results only if it can meet
the following criteria: 1) the cells should be relaxed and
elongated so that substantial contraction can be demonstrated; 2) they should be Ca21 tolerant at physiological temperatures, meaning that they should maintain
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seen with bright-field or phase-contrast optics. However, in healthy, Ca21-tolerant, preterm cells that
contract in response to agonists, nuclei can sometimes
be seen. Presumably, the smaller amount of contractile
material in the preterm cells makes the nuclei easier to
locate.
Average cell lengths, in 1.6 mM CaCl2, for a sample of
adult and preterm cells are given in Table 1. Adult cells
were much longer than neonatal cells [194 6 57 (SD)
µm for adults and 93 6 32 µm for neonates]. Preterm
cells were also narrower, averaging 5 µm, compared
with 8 µm wide for adult cells. These differences were
statistically significant (P , 0.05). Despite their shorter
length, preterm cells are long enough to contract substantially. However, it was more difficult to isolate cells
from neonatal trachea, probably because the trauma is
greater in the smaller, more fragile tissue. The cell
volumes estimated from these mean length and width
measurements are also shown, assuming two simple
geometries, a cylinder and two cones joined at the base.
Neither of these geometries is an exact description of
the shape of the cell, but the true volume would be
between these two estimates. The mean volume of the
preterm cell is only about one-fifth the mean volume of
the adult cell.
arrival of agonist. In the example shown, the carbacholTrypan blue solution reached the cell at 31 s, the cell
was contracting at 33 s, and the contraction was
complete by 41 s. Figure 2B shows images of this cell as
it contracted from 322 to 85 µm.
The shortening response was treated as an exponential process, as described in previous work (9). Obviously, the cells do not contract to zero length, but rather
their length asymptotically approaches some minimum
length, representing the maximum contraction of which
they are capable under the chosen conditions [physiological Ca21 concentrations and high (100 µM) agonist
concentrations]. Usually the cells contract to one-half
or less of their starting length for both adult and
preterm cells.
The shortening velocity v can be compared with
estimates of unloaded shortening velocity of multicellular preparations expressed in lengths per second. The
value of 0.503 s21 for the cell shown in Fig. 2 is greater
than most estimates from multicellular ASM preparations. These results demonstrate that the contractile
system in the single cells is well preserved after the
isolation procedure.
Figure 3 shows the contraction of a preterm ASM cell
in response to carbachol. As shown, Figure 3A is a plot
of cell length vs. time. The carbachol-Trypan blue
solution reached the preterm cell at 41.5 s, the cell was
contracting at 44 s, and the contraction was essentially
complete by 50 s. As with the adult cell, the contraction
was substantial (final length ,22% of initial length).
The shortening velocity of this cell was 0.788 s21, which
is more than the value for the adult cell shown. Figure
3B shows images of the preterm cell as it contracted
from a length of 179 µm to a final length of 40 µm.
Table 2 summarizes the shortening velocities and the
amounts shortened by adult and preterm cells. The
shortening velocities of the preterm cells were not
significantly different from the adult cells. However,
there was a significant difference in the amount shortened: the preterm cells shortened a greater fraction of
their cell length (68%) than the adult cells did (55%).
Other agonists besides carbachol cause rapid contractions of these cells. In a preliminary study, a sample of
10 adult cells stimulated with 10 mM caffeine in 1.6
mM CaCl2 exhibited a mean shortening velocity of
0.59 s21 (range 0.42–0.74 s21 ). Furthermore, the shortening velocity with 100 µM serotonin was similar
(mean 0.56 s21; range 0.46–0.65 s21 ). Thus the response of the cells and the suitability of the measurement method are not limited to carbachol stimulation.
Further experiments need to be done to fully characterize the response of ASM cells to these and other
agonists.
MEASURING SHORTENING VELOCITY OF SMOOTH MUSCLE CELLS
431
their elongated state when transferred from Ca21-free
solutions to ones with physiological (mM) Ca21 concentrations; 3) they should exclude Trypan blue; 4) they
should contract within seconds of being exposed to
contractile agonists; and 5) the contractions should be
substantial, meaning one-half of the cell’s starting
length. One of the reasons that studies on the Bufo cells
have produced so many important results is that the
cells used were of very high quality and met these
standards. Using the method described herein, we have
been able to isolate elongated, relaxed, single smooth
muscle cells from both adult and preterm sheep tra-
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Fig. 2. Contraction of an adult ASM
cell. A: plot of cell length as a function of
time during shortening. Agonist solution was 100 µM carbachol, containing
1% sucrose to increase density (see METHODS ) and 1 mg/ml Trypan blue so flow
could be monitored. Flow rate was ,0.5
ml/min, and apparent linear velocity of
agonist past cell was 0.6 mm/s. Agonist
reached cell at a time corresponding to
31 s, denoted by downward arrow.
Circles, measured length of smooth
muscle cell at times indicated; curved
line, best-fit line to these points (see
Calculating Shortening Velocity). B: photomicrographs of same cell at selected
points in contraction. Images were acquired with a 340 objective and digitized. Composite image was created with
the program NIH Image. Brightness
and contrast of images were adjusted to
compensate for dye mixed with agonist
solution. First panel: cell at a time (23 s)
before carbachol has reached it, cell
length is 322 µm; second panel: time 5
34 s (3 s after contact with carbachol),
cell length is 237 µm; third panel:
time 5 35.3 s (4.3 s after carbachol), cell
length is 162 µm; fourth panel: cell after
contraction was complete, time 5 41 s,
cell length is 85 µm.
432
MEASURING SHORTENING VELOCITY OF SMOOTH MUSCLE CELLS
cheae. These cells remain relaxed in millimolar concentrations of Ca21 and contract substantially and rapidly
when exposed to cholinergic agonists.
A common problem if smooth muscle cell preparations are to be used for contractile studies is that the
isolated cells are extremely short, possibly because
they have already contracted during the isolation procedure. The conclusion that cells are precontracted is only
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Fig. 3. Contraction of a preterm ASM cell. Same
conditions as in Fig. 2. A: plot of cell length as a
function of time during shortening. Agonist
reached cell at a time corresponding to 41.5 s,
denoted by downward arrow. Symbols are as
described in Fig. 2 legend. B: photomicrographs of
same cell at selected points in contraction. First
panel: cell at a time (42.5 s) after carbachol
reached it but before it started to contract, cell
length is 179 µm; second panel: time 5 44 s (2.5 s
after contact with carbachol), cell length is 140
µm; third panel: time 5 45 s (3.5 s after carbachol), cell length is 90 µm; fourth panel: cell after
contraction was complete, time 5 58 s, cell length
is 40 µm.
valid if it can be later demonstrated that elongated cells
can be prepared from the species and tissue in question.
Cells in other species and tissues might just be intrinsically shorter, or differences in the amount and nature of
cytoskeletal proteins expressed might be responsible.
Maximum shortening of 70% of the starting length can
be seen in healthy, elongated ASM cells. When our
preparations deteriorate, ,8 h postdigestion, they com-
MEASURING SHORTENING VELOCITY OF SMOOTH MUSCLE CELLS
Table 2. Contraction of isolated airway
smooth muscle cells
Cells
n
Velocity, s21
Amount Shortened,
%original length
Adult
Preterm
10
10
0.54 6 0.27
0.64 6 0.30
55 6 11
68 6 12*
Results are means 6 SD; n, no. of cells. Cells contracted in response
to 100 µM carbachol in presence of 1.6 mM CaCl2 at 35°C. * Significantly different from adult (P , 0.05, Tukey-Kramer honestly significant difference test).
variability in batches of collagenase. An advantage of
papain is that it is a crystalline enzyme, and we have
never purchased an unsatisfactory lot. Another advantage of papain is that it does not require Ca21 for
enzyme activation, so Ca21-free conditions can be used
if desired.
We routinely used high concentrations (100 µM) of
carbachol to cause maximal contractions, but lower
concentrations (10 and 1 µM) also cause contractions.
We have not yet determined the responsiveness to
threshold doses of cholinergic agonists, nor have we
systematically investigated other potential contractile
agonists. Many smooth muscle cell preparations are
more sensitive to cholinergic agonists than are the
intact tissue from which they were derived, but this is
not always the case. For example, toad stomach cells
are more sensitive to acetylcholine than the muscle
strips are, yet the isolated cells are less sensitive to
carbachol than are the muscle strips, needing nearly
1024 M carbachol for a maximum cell response (12).
Adult sheep tracheal strips require high concentrations
of acetylcholine (1023 M) to achieve maximum force
(25). Preliminary studies showed that KCl, caffeine,
and serotonin caused isolated adult cells to contract.
The preliminary results with caffeine are important
because they demonstrate that the rapid shortening
response is not restricted to cholinergic agonists. We
presume that carbachol contracts these cells through
inositol trisphosphate-mediated release of Ca21 from
the sarcoplasmic reticulum. Caffeine is believed to have
a simpler mode of action, directly causing Ca21 release
from the sarcoplasmic reticulum, although activation of
plasma membrane cation channels may also take place
(15). The similarity of shortening velocities with carbachol, caffeine, and serotonin is reassuring and suggests
that the contractions we observed may be the maximum of which these cells are capable under these
conditions. Further studies need to be done to determine whether there are differences between the adult
and neonatal cells with respect to functional receptors
expressed and their sensitivity.
Shortening velocities of the isolated airway cells
were greater than most reported values of maximum
shortening velocity in multicellular ASM tissues (35,
36) and are about three times faster than the shortening velocities of isolated arterial smooth muscle cells
(9). The more rapid shortening of isolated cells compared with intact multicellular tissues may be due to
the elimination of tissue elements that reduce shortening velocity in the multicellular preparations.
The technique described here for delivering agonist
to cells by switching to a slightly denser agonist solution has advantages compared with other approaches.
It is less complicated than trying to rapidly move a
multiwell apparatus underneath a cell held by a microtool. Another approach is to use a device that sprays
picoliter amounts of agonist solution onto a cell in a
flowing solution. With the ‘‘spray’’ method, the agonist
concentrations are not uniform around the cell, nor is
the agonist concentration known. The presence of 1%
sucrose in the agonist solution increases osmolarity
Downloaded from http://jap.physiology.org/ by 10.220.32.247 on June 18, 2017
monly lose their Ca21 tolerance and shorten, and these
damaged cells are then only capable of 10–20% shortening when agonists are added.
ASM cells suitable for patch-clamp studies of ion
currents have been isolated and used successfully in a
number of studies (16, 19, 20, 32, 34). In one case, the
electrophysiological studies were combined with simultaneous measurement of contraction, a very powerful
approach (19). These canine tracheal cells had a mean
length of 119 µm and rapidly shortened 20–40% of their
initial length when exposed to acetylcholine. Additionally, muscarinic receptors were studied in isolated
bovine, canine, and porcine ASM cells (19, 21, 23, 34).
After contraction in response to 100 µM carbachol,
the ASM cells usually do not elongate, even though the
agonist-containing solution is washed out of the perfusion dish. This behavior is typical of vascular smooth
muscle cells as well. Alexander and Cheung (1) found
that, although their arterial cells did not elongate after
washout of the stimulus, the free Ca21 concentration
(estimated by using fura-2) did return to the initial
resting value. Thus the failure of a smooth muscle cell
to elongate after contraction is not necessarily an
indication of abnormal Ca21 handling. When our ASM
cells contract in response to lower concentrations of
carbachol, a variable amount of elongation can be
observed after the agonist is washed out (not shown). In
the best example, a 187-µm cell briefly stimulated with
10 µM carbachol shortened to a length of 83 µm and
then elongated to 123 µm after the carbachol was
washed out, a 48% increase in length.
The only real disadvantage of our isolation method is
the relatively low yield of cells. We estimate that it
might be possible to isolate ,5 3 105 cells from the
tracheal smooth muscle in a 4-cm segment of sheep
trachea, if it were completely teased into fine strips.
Our method is fundamentally different from most
cell-preparation methods in that papain is only used to
soften the tissue; the actual release of cells takes place
when fine strips are dissected from the digested tissue
in a Ca21-free solution. Most other methods use crude
collagenase mixtures, and many investigators now also
add papain to these collagenase mixtures. In those
preparative methods, relatively large numbers of cells
are liberated by the combined action of proteolytic
enzymes and agitation, and the teasing of fine strips
from the digested tissue is not required.
Researchers who use collagenase mixtures for cell
dissociation periodically have difficulties caused by the
433
434
MEASURING SHORTENING VELOCITY OF SMOOTH MUSCLE CELLS
The authors are grateful to Robert Roache for technical assistance.
This study was partially supported by a Temple University
Grant-in-Aid of Research (to S. P. Driska).
Address for reprint requests: S. P. Driska, Physiology Dept.,
Temple Univ. School of Medicine, Philadelphia, PA 19140 (E-mail:
[email protected]).
Received 16 June 1997; accepted in final form 22 September 1998.
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