1951
The Journal of Experimental Biology 198, 1951–1961 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
EVIDENCE THAT CALCIUM-DEPENDENT CELLULAR PROCESSES ARE
INVOLVED IN THE STIFFENING RESPONSE OF HOLOTHURIAN DERMIS AND
THAT DERMAL CELLS CONTAIN AN ORGANIC STIFFENING FACTOR
JOHN A. TROTTER1 AND THOMAS J. KOOB2
1Department of Anatomy, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA and
2Skeletal Biology, Shriners Hospital for Crippled Children, Tampa, FL 33612, USA
Accepted 9 May 1995
Summary
Although previous investigations have shown that
experimental increases and decreases of the concentration
of extracellular Ca2+ produce correlated changes in the
stiffness of holothurian dermis, they have failed to
determine whether the Ca2+-correlated changes were due
to Ca2+-dependent cellular events or to direct effects of
Ca2+ on the viscosity of the extracellular matrix. We have
addressed this question by testing two explicit predictions
of the latter hypothesis: that dermal stiffness should be
correlated with the Ca2+ concentration in the absence of
viable cells; and that, in the presence of a normal
extracellular Ca2+ concentration, drugs that inhibit cellular
pathways dependent on Ca2+ should not affect dermal
stiffness. Our results are inconsistent with the hypothesis
and support the alternative hypothesis that Ca2+ is
important only in the cellular regulation of dermal
stiffness. In addition, we have extracted from dermal cells
an organic factor that stiffens the extracellular matrix.
Key words: echinoderm, sea cucumber, Cucumaria frondosa,
holothurian, mutable connective tissue, biomechanics, calcium
regulation, stiffening factor.
Introduction
Echinoderms have neurally regulated collagenous tissues
(Takahashi, 1967; Maeda, 1978; Wilkie, 1978a,b, 1984, 1988;
Motokawa, 1984, 1988; Morales et al. 1989). Resident
neurosecretory cells are thought to control the stiffness and
strength of their connective tissues through secretions that alter
the stress-transfer properties of the matrix between the spindleshaped collagen fibrils (Matsumura, 1973, 1974; Smith et al.
1981; Hidaka and Takahashi, 1983; Motokawa, 1984, 1988;
Wilkie, 1984, 1987, 1988, 1992; Trotter and Koob, 1989;
Trotter et al. 1994). The identities of the secreted molecules
and the mechanisms by which they affect the interfibrillar
matrix are unknown.
A number of experiments have demonstrated that fresh
echinoderm tissues containing viable cells are plasticized by
lowering the concentration of calcium ions in the bathing
medium (Hidaka, 1983; Diab and Gilly, 1984; Motokawa,
1984, 1987, 1988, 1994; Byrne, 1985; Wilkie, 1984, 1988,
1992; Hayashi and Motokawa, 1986; Shadwick and Pollock,
1988). They are stiffened by subsequent restoration of normal
calcium concentrations. These experimental results are
consistent with two distinct hypotheses. In the ‘extracellular
calcium regulation’ hypothesis, the neurosecretory cells
control the mechanical properties of the tissue by regulating
the free Ca2+ concentration in the extracellular matrix; the
strengths of the binding interactions of some macromolecular
constituents of the interfibrillar matrix are postulated to be
Ca2+-dependent. In the contrasting ‘cellular calcium
regulation’ hypothesis, the role of Ca2+ is postulated to be in
the regulation of one or more Ca2+-dependent cellular
processes, such as secretion (Motokawa, 1988). The two
hypotheses are not mutually exclusive, since the secretion of
Ca2+ could be Ca2+-dependent.
Recent experimental results have been interpreted to provide
support for both hypotheses. Szulgit and Shadwick (1994)
found that the spine ligament (‘catch apparatus’) of the sea
urchin Eucidaris tribuloides was plasticized by the chelation
of extracellular Ca2+ but was stiffened by the addition of the
non-ionic detergent Triton X-100. Their results were offered
in support of the idea that Ca2+ chelation had changed one or
more cellular activities, which secondarily resulted in a
decrease in tissue stiffness. The addition of the non-ionic
detergent was thought to have increased tissue stiffness by
releasing one or more stiffening factors from lysed cells. The
continual presence of a calcium chelator was thought to have
eliminated the possibility that the stiffening factor could be
Ca2+. Motokawa (1994) treated specimens of the dermis of a
sea cucumber, Holothuria leucospilata, with Triton X-100 in
combination with freezing and thawing, and subsequently
estimated their viscosities from the results of creep (tensile)
tests in the presence of widely varying cation concentrations.
1952 J. A. TROTTER AND T. J. KOOB
He also concluded that Ca2+ acted at the cellular level, but
argued in addition that Ca2+ acts directly as a stiffener of the
interfibrillar matrix. His results were thus taken to support the
hypothesis the dermis stiffening is caused by extracellular
Ca2+-dependent Ca2+-secretion.
The experiments reported here were undertaken to
discriminate between these hypotheses by testing the following
specific predictions of the ‘extracellular calcium regulation’
hypothesis. (1) The stiffness and strength of the extracellular
matrix should be Ca2+-dependent even in the absence of viable
cells. (2) The stiffness and strength of the extracellular matrix
should remain high in the presence of drugs that interfere with
calcium-dependent cellular events when the extracellular
concentration of Ca2+ is maintained at a normal level.
The results contradict both predictions and therefore fail to
support the ‘extracellular calcium regulation’ hypothesis.
However, the results are consistent with the ‘cellular calcium
regulation’ hypothesis. In addition, evidence has been obtained
for the existence of a cell-sequestered organic ‘stiffening
factor’.
Portions of the data included in this report have been
published previously in abstract form (Trotter and Koob, 1992,
1994, 1995).
Materials and methods
Adult sea cucumbers, Cucumaria frondosa Hyman, were
obtained by dredging Frenchman Bay, an inlet of the Gulf of
Maine, and were maintained in mesh cages submerged beneath
the floating dock of Mount Desert Island Biological
Laboratory, Salsbury Cove, ME, USA. All experiments on
fresh specimens were carried out at Mount Desert Island
Biological Laboratory.
Test specimens were prepared from the two ventral
interambulacra, which lack tube feet. The body wall
musculature was stripped away from the dermis. To prepare
uniform specimens, the safety shields of single-edged razor
blades were glued together, either directly or with an
intervening metal shim, using commercially available
cyanoacrylate adhesive. This cutting apparatus was used to
produce equivalent specimens from the white inner dermis that
were approximately 3 cm long, 0.9 mm thick and either 1.7 or
1.8 mm wide. The long axis of each specimen was parallel to
the longitudinal axis of the animal. The 0.9 mm thick side was
in its radial dimension, and the wider side was in its
circumferential dimension.
Creep tests were carried out in a chamber constructed from
acrylic containing an epoxy-coated copper tube (Fig. 1).
Natural sea water from the pumped seawater system of Mount
Desert Island Biological Laboratory was passed through the
tube in order to maintain the temperature of the test solutions
at that of the water in which the animals lived, approximately
12–15 ˚C. The epoxy coating prevented the test solutions from
being contaminated by copper salts. The ends of the test
specimen were secured in acrylic clamps by the use of stainlesssteel screws and cyanoacrylate adhesive. A 3 mm wide by 2 mm
LVDT
SC
WT
S
C
RD
C
Fig. 1. Schematic drawing of the creep apparatus. See text for
explanation. The cooling device is not shown. SC, signal conditioner;
RD, recording device; S, specimen; WT, weight; C, clamp; LVDT,
linear variable differential transducer.
deep groove was milled in one of the two clamp members, and
a complementary ridge was milled into the other. The specimen
was retained in the groove by the glue and the pressure created
by tightening the screws. Both the glue and the screws were
found to be necessary to hold specimens firmly enough to be
tested without either slipping or breaking at the clamps. After
a specimen had been secured in both clamps, a digital caliper
was used to determine the distance between the two clamps at
which the specimen began to resist further extension. This was
taken as the undeformed length (L0). The bottom clamp was
retained by shelves in the test chamber. A threaded steel rod
extended from the top clamp. This rod was gripped by a collet
clamp attached to a braided stainless-steel wire, 0.6 mm in
diameter. The wire passed over two stainless-steel bearingmounted nylon pulleys at either end of the horizontal member
of a T-shaped aluminum stand. The other end of the wire was
attached to a metal container which was weighted with lead
shot. The container rested on a small platform. The specimen
chamber was bolted to the top of a small platform jack. Between
the two pulleys, the stainless-steel wire was glued to the core
of a linear variable differential transformer (LVDT: Schaevitz
model 100MHR, Lucas Schaevitz, Pennsauken, NJ, USA). The
hollow cylinder of the LVDT was held in place by a V-shaped
acrylic piece that could be positioned anywhere between the
two pulleys. The holder and LVDT were held in place by tape.
The output of the LVDT was passed from a digital transducer
readout (Lucas-Schaevitz model DTR451) to a chart recorder.
The system was calibrated by recording the deflection produced
on the pen of the chart recorder by specific length changes in a
micrometer. Once calibrated, all the settings of the signal
conditioner and the chart recorder were left unchanged. Prior to
The stiffening response of holothurian dermis 1953
each test, the weight was lowered until the bottom clamp made
contact with the retaining shelf, and the specimen became
straight. The LVDT was then positioned and taped. The creep
test was begun by lowering the platform jack, leaving the
weight hanging free, suspended only by the specimen. The
lengthening of the specimen was recorded for a period of
30 min, or until the specimen broke or the limit of the LVDT
was reached. When the test solutions contained only inorganic
salts, buffer and chelating agent, the specimens were fully
submerged during the tests. When drugs were included in the
test solutions, they were dripped onto the specimens during the
tests. The weight used in these tests was 200 g (producing a
force of 1.96 N) above the weight required to balance that of
the specimen clamps and specimen. The specimens were
0.9 mm31.8 mm in cross section, and the initial load was
therefore 1.21 MPa. The extension values for each time point
were divided by the L0 value for the specimen to obtain creep
plots, in which strain («=DL/L) is expressed as a function of
time.
Bending tests were carried out on 0.9 mm31.7 mm330 mm
specimens. The specimens were glued at their midpoints to
0.5 mm tungsten wire which had been bent into a Z shape with
two 90 ˚ angles. The wire containing the specimen was mounted
in a device consisting of a vertical plate of acrylic mounted in an
aluminum stand (Fig. 2). A groove had been cut from the top of
the plate. The top of the groove was closed by a slip of stiff foam
in which a slit was cut to accommodate the wire and to hold it
firmly in place. The bottom of the groove had a shallow slit for
the wire containing a small amount of Plasticine. A piece of
millimeter-interval graph paper was glued to the front of the
acrylic plate. A small ledge of acrylic was glued to the front of
the plate, to one side of the groove, so that its upper surface would
be at the same vertical position as the bottom of the specimen.
When the wire containing the specimen was in position in the
device, 15 mm of specimen extended beyond the wire as a
cantilever beam, while the other half of the specimen was
supported by the acrylic shelf. The testing device was mounted
in a flowing seawater tray, with the specimen positioned
approximately 4 cm above the surface of the water. This was to
maintain the temperature and humidity at relatively constant
values throughout all of the tests. During the mounting process,
the specimen was held in a slightly upwardly bent position using
the handle of a pair of tweezers to support it gently. To begin a
test, the free end of the specimen was released, and a stopwatch
was used to measure the time required for the specimen end to
move a vertical distance of 4 mm, beginning at a position no
lower than 1 mm above the horizontal position of the specimen,
and no higher than 3 mm above the horizontal. The time was
rounded up to the nearest second. Very plastic specimens
therefore had a uniform deflection time of 1 s. Because most tests
were completed in less than 2 min, and no tests took longer than
5 min, the potential problems arising from specimen desiccation
were not thought to be serious. Specimens from the dermis of C.
frondosa were suitable for bending tests because they retained
their shape even after lengthy incubation in test solutions.
The gravity-bending of a viscoelastic cantilever beam is
W
A
F
S
L
B
W
S
Fig. 2. Schematic drawing of the devices used for bending tests. In A
the test device is viewed from the front; in B the wire on which the
specimen is mounted is viewed from the side. The tungsten wire bent
into two 90 ˚ turns is indicated by W, and the specimen by S. The wire
is held in place by a foam clamp (F), and half of the specimen rests
on a Lucite shelf (L). The arrow indicates the bending of a specimen
during the test. Grid lines are 1 mm apart.
associated with complex tensile, compressive and shear
stresses. The magnitude and distribution of the stresses are
determined by the geometry and mass of the specimens. In
these tests, all the specimens had the same dimensions. All
specimens were blotted twice on absorbent paper prior to being
mounted. No noticeably consistent changes in specimen size,
due to absorption or loss of water during pre-test incubations,
were noted. Therefore, the stresses within all of the specimens
should have been nearly the same. Comparisons of the times
required for similar stresses to produce similar deformations
are therefore directly related to the viscous component(s) of
the tissue. This is because, for a Newtonion fluid,
h = s/«˙ ,
where h is viscosity, s is stress and «˙ is strain rate. Hence, for
specimens loaded with identical stresses, h~1/«˙. Because
1/«˙ = t/«, where t is time and « is strain, and the final strain
was the same for every specimen, therefore h~t. A similar
argument applies to the creep tests: the time required for the
specimen to reach a standard strain is directly proportional to
the viscosity of the tissue. These tests ignore the elasticity of
the collagen components, since it is assumed to remain
unchanged during the experiments. They also ignore the nonlinearities of the complex viscoelastic characteristics of this
tissue and would be inadequate to obtain measurements that
could be used to construct viscoelastic models. However, they
are adequate to obtain comparative information that is accurate
1954 J. A. TROTTER AND T. J. KOOB
enough to evaluate the effects of experimental manipulation on
tissue viscosities.
Initial experiments were carried out using creep tests. Some
difficulties inherent in this test modality, however, led to the
introduction of bending tests, which have several distinct
advantages. One major problem with the creep test was that it
was difficult to find clamping conditions that prevented
specimens from either slipping or breaking at a clamp. This
was especially true with plasticized specimens, which yielded
readily as the clamps were being tightened. A second problem
was that the time required for each creep test made it difficult,
with a single test apparatus, to conduct many tests within the
same time frame. This was a significant limitation, because the
animal-to-animal variability in viscosity measured on
specimens tested in artificial sea water (ASW) made it
necessary to conduct each set of experiments on specimens
from a single animal. For example, it can be seen in Fig. 4 that
the bending times of specimens from animals 7 and 9 averaged
more than 70 s, whereas those of specimens from animal 3
averaged less than 30 s. These difficulties partially offset the
inherently greater information content of creep tests when
compared with bending tests. One advantage of the bending
test was that the tissue was not distorted or damaged by being
clamped. A second advantage was that the rapidity of the tests
allowed many tests to be conducted within a short time, and
hence the number of repeated tests of each experimental
variation was sufficient to obtain statistical information. These
advantages partially compensated for the limitation that each
test produced only a single time point and thus lacked the more
complex information content of a creep test. Both test
modalities were applied to all of the experiments described in
the present paper, with the exception that the evaluations of the
stiffening effects of tissue extracts were made exclusively with
bending tests. In all cases where both test modalities were used,
comparable results were obtained.
The principal test solutions used in these studies were as
follows: (1) Mops-buffered artificial sea water (ASW), which
consisted of 0.5 mol l21 NaCl, 0.05 mol l21 MgCl2,
0.01 mol l21 CaCl2, 0.01 mol l21 KCl and 0.01 mol l21 3-(Nmorpholino)propane sulfonic acid (Mops), pH 7.8–8.0; and (2)
EGTA–ASW in which the CaCl2 was replaced by
0.0072 mol l21 EGTA. Standard specimens were cut from the
dermis and incubated in the test solutions at ambient seawater
temperature, with intermittent gentle agitation. Each
experiment comparing the effects of different treatments used
specimens from a single animal, and each experiment was
repeated using specimens from at least three different animals.
Approximately 30 specimens could be obtained from a single
animal. Each specimen was tested only once. Unless otherwise
noted, all incubations took place at seawater temperature
(12–15 ˚C), and the pH was maintained at 7.8–8.0.
Free Ca2+ concentrations were estimated using the B&S
stability constants (Brooks, 1992) in the computer program
Maxchelator v6.5, obtained from Dr Chris Patton, Stanford
University, USA. The total calcium contents of specimens
were determined using atomic absorption spectroscopy (model
460 AA spectrophotometer, Perkin-Elmer Corp., Norwalk, CT,
USA) on tissue extracts made in 1 mol l21 HCl. Preliminary
experiments showed that 1 mol l21 HCl extracted 100 % of the
calcium, as judged by the inability of sequential treatment with
5.25 % sodium hypochlorite (household bleach) and 70 %
HNO3 to extract more. Specimens from the dermis of C.
frondosa were suitable for direct measurements of the calcium
associated with soft tissue elements because the adults of this
species lack calcareous ossicles (Hyman, 1955). This fact was
verified by microscopic examination of the contents of tissues
that had been dissolved in sodium hypochlorite.
Tissue extracts were prepared from the same region of the
dermis as that used for mechanical tests. The dermis was
minced into pieces of about 1 mm3, and the mince was then
extracted at seawater temperature in 5 volumes of
EGTA–ASW for 90–180 min. A direct freeze–thaw extract
was prepared by freezing the mixture of tissue and
EGTA–ASW at 260 ˚C for at least 2 h, followed by incubation
at seawater temperature until the liquid was completely
thawed. These steps were repeated for a total of five
freeze–thaw cycles. An EGTA–ASW extract (without
freezing) was made by separating the tissue from the liquid
after 180 min of incubation at seawater temperature. The tissue
from this procedure was then resuspended in the same volume
of fresh EGTA–ASW, and was frozen and thawed five times
as described above. A direct freeze–thaw extract was also made
from the longitudinal muscles of the body wall. In all cases,
the extracts were clarified by centrifugation at 27 000 g for
30 min and were stored frozen.
In preparation for transmission electron microscopy, fresh
and treated tissues were fixed for 36 h at 20 ˚C in 2.5 %
glutaraldehyde, 0.1 mol l21 Mops, 0.41 mol l21 NaCl,
0.05 mol l21 MgCl2, 0.01 mol l21 CaCl2, 0.01 mol l21 KCl,
pH 7.9. They were then rinsed for 24 h in several changes of
the same solution lacking glutaraldehyde, post-fixed for 2 h in
1 % OsO4, 0.1 mol l21 sodium cacodylate, pH 7.3, stained for
2 h in the dark in 0.5 % uranyl acetate in water, dehydrated in
increasing concentrations of ethanol, and embedded in Spurr’s
resin. Ultrathin sections were stained with uranyl acetate and
lead citrate and examined in Hitachi H-600 electron
microscopes.
All chemicals were reagent grade or better. Verapamil,
3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester
(TMB-8), Mops and Triton X-100 were from Sigma Chemical
Company, St Louis, MO, USA.
Results
Fresh specimens incubated in 7.2 mmol l21 EGTA–ASW
were markedly plastic in comparison with those incubated in
ASW. The plasticization caused by EGTA was fully reversed
by incubation in complete ASW (Figs 3, 4). This tissue thus
shows the same mechanical responses to calcium removal and
replacement that have previously been demonstrated in other
echinoderm collagenous tissues. In the presence of
7.2 mmol l21 EGTA, the maximal free Ca2+ concentration in
The stiffening response of holothurian dermis 1955
50
50
A
B
E
40
E
40
E
E
E
Length change (%)
E
30
30
C
ET
EC
EC
ET
ET
20
10
10
0
0
0
10
20
Time (min)
30
EW
C
EW
EC
EF
20
40
0
10
20
Time (min)
30
40
Fig. 3. Creep tests. In A the specimens were pre-incubated and tested in artificial sea water (ASW) containing the normal concentration of
calcium (C); pre-incubated and tested in ASW in which the Ca2+ had been replaced by EGTA–ASW (E); pre-incubated in EGTA–ASW followed
by ASW and tested in ASW (EC); or pre-incubated in EGTA–ASW and followed by EGTA–ASW containing Triton X-100, and tested in the
latter solution (ET). In B the specimens labelled E, C and EC were treated as described for A. Those labelled EW were pre-incubated in
EGTA–ASW, then in deionized water, and then in EGTA–ASW. They were tested in EGTA–ASW. The specimen labelled EF was pre-incubated
in EGTA–ASW, after which it was frozen and thawed five times and subsequently tested in EGTA–ASW. All the specimens in A came from
a single animal. Those in B also came from a single animal, which was different from that used for the experiment shown in A.
1 ml of solution containing a standard specimen
0.09 cm30.18 cm33 cm, assuming that the specimen
originally contained 10 mmol kg21 total calcium, was
calculated to be 52 mmol l21.
To determine whether the calcium-dependent stiffening
effect would occur in tissues which did not contain viable cells,
test specimens that had been incubated in EGTA–ASW for at
least 90 min were subjected to three separate treatments that
were predicted to lyse cellular membranes by different
mechanisms. Some specimens were exposed to 1 % Triton X100 in EGTA–ASW, others were exposed to deionized water
for 30 min, and others were frozen (260 ˚C) and thawed
(seawater temperature) five times in EGTA–ASW. They were
all subsequently evaluated in EGTA–ASW, using both creep
and bending tests. All three treatments resulted in viscosity
increases comparable to or greater than those seen when the
tissues with living cells were returned to ASW. Qualitatively
similar results were obtained using the two test modalities
(Figs 3, 4). In bending tests, the water and freeze–thaw
treatments consistently produced greater stiffening responses
than did detergent treatment. The cause of these differences is
not clear. It suggests that osmotic shock and freeze–thaw
treatments might be more effective at lysing cell membranes.
Three principal types of electron-dense granules were seen
in the cells of the deep dermis (Fig. 5A). The first type was
round and small (approximately 200 nm in diameter) and very
electron dense. The second type was larger (approximately
500 nm in diameter), frequently ellipsoidal and less electron-
dense. The third type was larger still (greater than 1 mm in
diameter) and highly variable in electron density, even within
a single granule. No two granule types were observed in the
same cell, and they may therefore represent three separate cell
types. Cell processes containing granules were enclosed by a
continuous external lamina. The plasmalemmae of the granular
cells were lysed by all three treatments (Fig. 5B–D). Some
granules of all three types remained in the treated tissues, but
the number of granules was markedly decreased. These
treatments thus resulted in exposure of the extracellular matrix
to the contents of the cells, including the contents of the
granules, and coincidentally the tissue became stiff, even in the
continual presence of the calcium chelator EGTA. These
results suggested that the cells contain one or more substances
capable of stiffening the extracellular matrix and that Ca2+ is
not one of these substances.
To determine how effective the EGTA treatments were in
removing calcium from the specimens, the total calcium
content was determined by atomic absorption spectroscopy of
identically treated specimens from five different animals. The
results (mean ± S.D., N=5) showed that EGTA alone reduced
the total calcium concentration from 8.9±1.2 to
0.58±0.22 mmol kg21 wet mass. Water, detergent and
freeze–thaw treatments in the presence of EGTA reduced the
total calcium further to 0.22±0.18, 0.14±0.04 and
0.08±0.10 mmol kg21, respectively. Hence, more calcium was
chelated in those specimens in which the cells had been lysed.
Presumably this was cellular calcium that had been released
1956 J. A. TROTTER AND T. J. KOOB
Time (s)
Fig. 4. The results of bending
1000
tests on specimens from nine
animals. Each animal is
indicated by a number above the
x-axis. The y-axis shows the
time (in seconds) on a
logarithmic scale required for
100
the ends of the specimens to
deflect a vertical distance of
4 mm. Each bar shows the mean
and standard deviation of five
tests, each on a different
specimen. The animal–animal
10
variability is seen in the bending
times of specimens incubated
sequentially in EGTA–ASW
and ASW (EGTA→ASW). The
effect of Triton X-100 on
1 2 3 4 5 6 7 8 9
1 2 3 4 5 6
7 8 9
1 2 3
4 5 6
7 8 9
specimens
tested
in
1
EGTA–ASW (E→TX) was very
ErTX
ErWater
ErFT
EGTArASW
EGTA
similar to that of the normal
Ca2+ concentration in ASW. The effects of both water (E→Water) and freezing and thawing (E→FT) were generally greater than those of Ca2+.
and made available to the chelator by cell lysis. The
observation that the specimens which had been frozen and
thawed had both the highest viscosities and the lowest total
calcium contents made it very improbable that the release of
calcium from lysed cells participated in the stiffening response.
The results just described suggested that the mechanical effect
of Ca2+ replacement on live tissues is on one or more cellular
processes, such as secretion, rather than directly on the
extracellular matrix. To determine whether calcium-dependent
cellular processes are involved in the stiffening responses of live
tissues, specimens that had been plasticized by incubation in
EGTA–ASW were subsequently incubated in ASW or in ASW
containing verapamil or 3,4,5-trimethoxybenzoic acid 8(diethylamino)octyl ester (TMB-8). Verapamil is known to
block certain voltage-dependent Ca2+ channels in a number of
different cell types (Triggle, 1981), while TMB-8 is known to
have pleiotropic effects on calcium-dependent cellular processes
(Malagodi and Chiou, 1974; Gordon and Chang, 1989; Ishihara
and Karaki, 1991). At 1 mmol l21, both drugs blocked the
stiffening effect of Ca2+ replacement, and their effects were
reversed by returning the specimens to ASW without drugs.
Both drugs were equally effective at plasticizing tissues that had
not been incubated in EGTA–ASW, but had instead been placed
directly into ASW containing the drug (Figs 6, 7). They were
both maximally effective at doses approaching 1 mmol l21 and
were ineffective at doses lower than 0.1 mmol l21. Neither drug
affected the stiffness of tissues in which the cells had been lysed
by freezing and thawing (not shown).
These results indicated that experimental manipulation of the
extracellular Ca2+ concentration affects tissue viscosity
indirectly, possibly through an effect on cell secretion, and that
cell lysis causes tissue stiffening, even in the presence of a
calcium chelator. It might be expected, then, that cell lysis would
result in the release of a soluble stiffening factor that acts on the
extracellular matrix. Evidence for the presence of a cell-derived
stiffening factor was obtained from experiments in which fresh
tissues were incubated for 90 min in EGTA–ASW to plasticize
the extracellular matrix, followed by incubation in one of four
distinct tissue extracts, made in the same solution. The extracts
were obtained by freezing and thawing minced dermis (five
cycles) in EGTA–ASW (freeze–thaw extract: FTE in Fig. 8), by
extracting minced dermis in EGTA–ASW without freezing and
thawing (EGTA extract: EgE in Fig. 8), by freezing and thawing
the dermis residue from the previous step in fresh EGTA–ASW
[EGTA–ASW-extracted tissue subsequently extracted by
freezing and thawing in EGTA–ASW: (Eg)FTE in Fig. 8], and
by freezing and thawing body wall muscles in EGTA–ASW
(MuscE in Fig. 8). The results showed that the lysed cells
released a factor that caused matrix stiffening in the presence of
EGTA (Fig. 8). No stiffening effect was obtained in extracts of
dermis made without freeze–thaw cycles or in freeze–thaw
extracts of muscle. The stiffening factor was detected in four
different preparations. It was stable to repeated freezing and
thawing and was non-dialyzable (using dialysis tubing with a
molecular mass cut-off of 14 kDa), but was destroyed by boiling
for 15 min.
Discussion
Previously published data have shown that the tensile
stiffness of sea urchin spine ligaments (Hidaka, 1983; Diab and
Gilly, 1984; Shadwick and Pollock, 1988), crinoid
intervertebral ligaments (Wilkie, 1983), ophiuroid
intervertebral ligaments (Wilkie, 1988, 1992), sea cucumber
dermis (Eylers, 1982, 1989; Motokawa, 1984, 1988, 1994;
Hayashi and Motokawa, 1986) and several other echinoderm
collagenous tissues (Byrne, 1985) can be experimentally
altered by manipulating the concentration of free Ca2+ in the
The stiffening response of holothurian dermis 1957
B
A
✽
✽
✽
C
Fig. 5. Transmission electron micrographs
of specimens that had been incubated in
artificial sea water (A), Triton X-100 (B) or
deionized water (C), or had been frozen and
thawed five times in EGTA–ASW (D).
(A) A folded external lamina surrounding
several cell processes (arrow) and cell
processes containing small dense granules
(small arrowhead), larger dense granules
(medium arrowhead) and large granules of
varied density (large arrowhead) can be
seen. (B,C,D) Regions of cellular debris
(asterisks), and some unlysed granules can
be seen. Magnification of all micrographs is
85003; the bar in D is 2 mm long.
D
✽
bathing medium. It has been pointed out that the plasticizing
effect caused by depleting the tissues of free Ca2+ could have
resulted either from an inhibition of one or more Ca2+dependent macromolecular associations in the extracellular
matrix or from an inhibition of one or more cellular events,
such as cell secretion (Motokawa, 1988; Wilkie, 1988).
Nevertheless, the former explanation has been favored, and
models have been published which hypothesize that calcium
plays an essential role in the self-association of the highly
✽
✽
✽
sulfated glycosaminoglycans (GAGs), which are known to be
present in the tissues (Wilkie, 1988, 1992). The increased selfassociation of these GAGs has been suggested to be the
molecular mechanism underlying the increased stiffness or
viscosity observed when the free calcium concentration is
increased (Wilkie, 1988, 1992; Kariya et al. 1990; Motokawa,
1994). However, most of the previously published experiments
showing a calcium-dependence of tissue stiffness have been
performed using fresh tissues, containing viable cells, and it
1958 J. A. TROTTER AND T. J. KOOB
60
140
50
Bending time (s)
Bending time (s)
120
100
80
60
40
EGTA
ASW
1
0.3
0.1
Verapamil
0.03
(mol l−1)
Fig. 6. The effect of verapamil on bending times. Freshly prepared
specimens were incubated for 90 min in ASW containing the indicated
concentrations of verapamil prior to testing. It is seen that 1 mmol l21
verapamil is as effective a plasticizer as 7.2 mmol l21 EGTA, whereas
0.1 mmol l21 verapamil has no effect. This is a representative
experiment conducted on specimens from a single animal. Each bar
represents the mean and standard deviation of five tests, each on a
different specimen.
Bending time (s)
30
20
10
20
0
40
100
90
80
70
60
0
E
E →A
E+FTE
E+EgE E+(Eg)FTE E+MuscE
Fig. 8. The effects of tissue extracts on bending times. With the
exception of those that were pre-incubated in EGTA–ASW followed
by ASW (E→A), the specimens were pre-incubated in EGTA–ASW
followed by EGTA–ASW (E) containing tissue extracts. E contained
no extract; E+FTE contained the extract made by five freeze–thaw
cycles in EGTA–ASW; E+EgE contained the extract made by
incubating tissue in EGTA–ASW without freezing and thawing;
E+(Eg)FTE contained the extract made by freezing and thawing, in
EGTA–ASW, the tissue that had been pre-extracted in EGTA–ASW;
E+MuscE contained a freeze–thaw extract of body wall longitudinal
muscle in EGTA–ASW. The freeze–thaw extracts of dermis were
almost as effective at stiffening specimens as was the normal Ca2+
content of ASW. In contrast, the EGTA extract made without
freeze/thaw cycles (E+EgE), and the freeze–thaw extract of muscle
(E+MuscE) had little or no stiffening activity. Each bar shows the
mean and standard deviation of five tests on different specimens. All
specimes came from a single animal.
50
40
30
20
10
0
EGTA
ASW
1
0.3
TMB-8 (mol l−1)
Fig. 7. The effect of TMB-8 on bending times. Freshly prepared
specimens were incubated for 90 min in ASW containing the indicated
concentrations of TMB-8 prior to testing. 1 mmol l21 TMB-8 was
almost as effective a plasticizer as 7.2 mmol l21 EGTA, whereas
0.3 mmol l21 TMB-8 had no effect. This is a representative
experiment conducted on specimens from a single animal. Each bar
represents the mean and standard deviation of five tests, each on a
different specimen.
has thus been impossible to decide between the two different
explanations of the results.
Motokawa (1994) used a combination of 1 % Triton X-100
and freeze–thaw treatments to lyse cells in the dermis of H.
leucospilata. He compared the creep rates of specimens in
which the cells had been lysed with those of intact specimens
from the same animals. If the intact specimens were incubated
in the presence of EGTA, they showed the same responses to
large changes in the concentrations of monovalent cations as
were shown by detergent-treated specimens. He interpreted
these results as evidence for a cellular effect of extracellular
Ca2+, which is in agreement with the present findings.
However, he also found that Ca2+ chelation by EGTA
significantly reduced the viscosity of both intact and detergenttreated specimens, and interpreted this to be evidence that Ca2+
plays a specific role as a stiffener of the extracellular matrix.
This finding conflicts with the present results and with those
of Szulgit and Shadwick (1994). The latter authors found that
sea urchin spine ligaments responded to Triton X-100 by
stiffening in the continual presence of EGTA. Moreover, the
spine ligaments have also been observed to stiffen after
freezing and thawing, using a protocol similar to that described
in this report (G. K. Szulgit, personal communication). The
causes of these different findings are not clear. Species
variation might be important since Motokawa found that water
(osmotic lysis) and freeze–thaw treatments of H. leucospilata
were less effective in cell lysis than was Triton X-100.
The three methods employed in the present study to destroy
cellular functions were all predicted to produce membrane
disruption and cell lysis, leading to a release of those
cytoplasmic, granular and nuclear contents that are soluble in
artificial sea water containing EGTA. Triton X-100 produces
chemical lysis by dissolving membrane lipids (Helenius and
Simons, 1975). Freezing and thawing lyses membranes,
probably through the physical damage caused by ice crystals
(Mazur, 1966). Water produces osmotic lysis because of the
high solute concentrations within cells and cell organelles.
Electron microscopy confirmed that cell and organelle lysis
The stiffening response of holothurian dermis 1959
resulted from all three treatments. That all three methods of
lysis caused the stiffness of the dermis to increase even in the
presence of EGTA strongly suggested that the cells contain one
or more stiffening factors that affect the extracellular matrix
independently of the extracellular Ca2+ concentration. Because
the stiffening effect is observed in the presence of EGTA, it is
clear that released intracellular Ca2+ alone could not be the
stiffening factor. This conclusion is supported by the
observation that the tissues with the highest viscosities were
those that had the lowest total calcium concentration.
The plasticizing effect of EGTA and the stiffening effect of
Triton X-100, water and freeze–thaw treatments were
qualitatively similar in creep and bend tests. Creep tests of
intact specimens in EGTA exhibited a rapid initial rate of
elongation which, in some animals, became slower after
5–10 min. Fig. 3B shows an example of this phenomenon,
which was not seen in specimens in which the cells had been
lysed. This loading response could indicate that the effector
cells in the dermis possess one or more mechanotransduction
mechanisms that operate – independently of the concentration
of extracellular Ca2+ – to stiffen the tissue in response to
internal shearing strains. These effects could not be observed
in bend tests, because these only measured what would have
been equivalent to the initial deformation rates seen in the
creep tests. Because of this difference, the results of the creep
and bend tests were quantitatively different, although they
showed the same qualitative effect of the different treatments.
It might be thought that the different ionic treatments used
(EGTA–ASW and ASW) could have produced their effects by
artifactually causing either swelling or shrinking of the
specimens. Such effects could potentially be large, because the
tissue is a discontinuous fiber-reinforced composite material
(Smith et al. 1981; Trotter and Koob, 1989; Trotter et al. 1994),
in which the viscosity (or shear strength) of the interfibrillar
matrix as well as the distance between fibrils both strongly
affect the resistance to tensile loads. The effects on bending
could be greater than those on creep rates because the rate of
bending would change with the mass of the beam, which would
change with the amount of water in the tissue. Although these
considerations would carry considerable weight in any effort to
deduce from the data the absolute material properties of the
specimens, they are of much less concern in the comparative
studies reported here. This is because the comparisons from
which the main conclusions have been drawn were made
between treatments in which the ionic conditions were identical.
Thus, verapamil- and TMB-8-treated specimens were much
more plastic in ASW than were untreated specimens in ASW;
and Triton X-100-, water- and freeze–thaw-treated specimens
were much stiffer in EGTA–ASW than were untreated
specimens in EGTA–ASW. Finally, and perhaps most
significantly, specimens in EGTA–ASW containing a
freeze–thaw extract of dermis were much stiffer than were
specimens in EGTA–ASW containing either an EGTA–ASW
extract of dermis or a freeze–thaw extract of muscle. These
results cannot have been caused by differential water contents
in tissues exposed either to ASW or to EGTA–ASW.
A major prediction of the extracellular matrix calcium
hypothesis is that the tissue ought to remain stiff in the presence
of pharmacological agents which inhibit or disrupt cellular
calcium-dependent events, provided that the calcium
concentration in the extracellular matrix is kept within a normal
range. Although the tissues remained stiff for many hours after
they had been dissected from the animals and stored in ASW,
they became plastic after 90 min in ASW containing EGTA and
they became stiff again when the normal calcium concentration
was restored. Significantly, the stiffening observed when
calcium was restored was inhibited by the presence of verapamil
or TMB-8. Neither of these drugs would be predicted to interact
directly with components of the extracellular matrix, and neither
had a plasticizing effect on tissues that had been frozen and
thawed. The plasticizing effects of both drugs were reversed by
washing them out of the medium. They are, therefore, unlikely
to have affected cell viability. Importantly, both drugs also
caused a reversible plasticization of tissues continuously
maintained in ASW. This result showed that interference with
cellular signalling pathways not only prevented the stiffening
response to the restoration of normal Ca2+ concentrations, but
also caused a reversible plasticization of tissues in which the
Ca2+ concentration was unchanged. These results are
inconsistent with the prediction of the extracellular calcium
regulation hypothesis and strongly suggest that the effects on
tissue stiffness caused by experimental modulation of the
calcium concentration are cellular effects.
Although the concentrations of verapamil required to affect
the dermis of C. frondosa were much greater than those
required to inhibit voltage-sensitive Ca2+ channels in mammals
(Triggle, 1981), they were similar to the concentrations of
organic channel-blockers required to block the voltagesensitive Ca2+ channels in echinoderm nerves (Berrios et al.
1985). Thus, a Ca2+ channel might be involved in the stiffening
response. It should also be noted, however, that high
concentrations of verapamil may inhibit the activity of protein
kinase C by interfering with its interaction with
phosphatidylserine-containing membranes (Mori et al. 1980).
TMB-8 affects multiple cellular pathways, including Ca2+
movement across membranes and phosphatidylserinestimulated protein kinase C activity (Malagodi and Chiou,
1974; Kojima et al. 1985; Gordon and Chiang, 1989; Ishihara
and Karaki, 1991). Therefore, no specific site of action is
indicated by the experimental results. The results with both
drugs are consistent with an active stiffening mechanism that
involves the translocation of Ca2+ across the cell membrane
and/or with the activation of protein kinase C.
Taken together, the results suggest that, in this experimental
model, the dependence of extracellular matrix stiffness on
experimentally manipulated changes in the extracellular Ca2+
concentration is cell-mediated. Diab and Gilly (1984) and
Szulgit and Shadwick (1994) have come to the same
conclusion concerning the role of Ca2+ in the spine ligaments
(‘catch apparatus’) of sea urchins, and Motokawa (1994) has
found that Ca2+ affects cellular activity in sea cucumber
dermis. Diab and Gilly (1984) also found that the polyamines
1960 J. A. TROTTER AND T. J. KOOB
putrescine and cadaverine, at a concentration of 10 mmol l21,
inhibited the stiffening of sea urchin ligaments in response to
mechanical agitation. They suggested that transglutaminasemediated protein crosslinking might be part of the stiffening
response. More recent studies have shown, however, that
polyamines are potent inhibitors of cation – including Ca2+ and
K+ – channels in a number of cell membranes (Ficker et al.
1994; Scott et al. 1993) and also affect other membrane
functions (Schuber, 1989). Diab and Gilly’s results are thus
also consistent with the cell membrane being a site for
stiffening inhibition in the presence of normal extracellular
Ca2+ concentrations. In the sea urchin spine ligament (‘catch
apparatus’) as well as in the sea cucumber dermis, then, the
depletion of extracellular Ca2+ or interference with normal
cellular membrane functions causes the tissue to become
plastic, while restoration of the Ca2+ or reversing the inhibition
of membrane functions stiffens the tissue.
It is generally believed that the granules of the cells observed
in the dermis, and in other mutable collagenous tissues of
echinoderms, secrete the substances that regulate the stiffness
of the interfibrillar matrix (Wilkie, 1979, 1984, 1988; Holland
and Grimmer, 1981; Smith et al. 1981; Motokawa, 1982a,
1984, 1988; Hidaka and Takahashi, 1983). Since all of the cells
were lysed by the treatments used in the present studies, it is
impossible to attribute the stiffening effect to any specific
granule, nor was that an objective of the experiments. It is
clear, however, that the stiffening effect of cell lysis is not due
to the release of Ca2+, since the total calcium contents of tissues
in which the cells had been lysed were lower than those of
tissues that had not been exposed to lytic agents. Thus, the
calcium identified histochemically in certain granules of the
cells in brittlestar ligaments (Wilkie, 1979) and in the dermis
of the sea cucumber Stichopus chloronotus (Matsuno and
Motokawa, 1992) is likely to have a function other than as a
secreted stiffening agent. Because the quantity of calcium
identified by pyroantimonate precipitation in the vacuoles of
the ‘vacuole cells’ in the dermis of S. chloronotus seemed to
be reduced in tissues stimulated to become stiff, it was thought
possible that this cell regulates dermal stiffness by secreting
and sequestering calcium (Matsuno and Motokawa, 1992). If
S. chloronotus dermis is physiologically similar to C. frondosa
dermis, the loss of Ca2+ from the vacuoles may be related to a
secretory process, but the calcium per se is probably not the
stiffening agent. No ‘vacuole cells’ were observed in C.
frondosa dermis and they were not observed in an earlier study
of S. chloronotus dermis (Motokawa, 1982b).
The results reported here are consistent with the existence
of an organic stiffening agent that acts on the matrix in calcium
concentrations that are at least two orders of magnitude below
that of sea water. The need for cell lysis and the failure to
obtain stiffening activity in lytic extracts of muscle support the
idea that the stiffener is a specialized dermis cell product. The
observations that the activity of the stiffener is non-dialyzable
and is destroyed by boiling suggest that it is macromolecular,
possibly protein, and unlikely to be a neurotransmitter.
Although stiffening was assayed in tissues containing living
cells and the activity theoretically could have been a cellular
or an extracellular effect, it is more likely to have been
extracellular because it occurred in the presence of EGTA,
which consistently plasticizes tissues containing living cells. In
these respects it differs markedly from the ‘stiffening factor’
prepared from sea cucumber coelomic fluid (Motokawa,
1982a), which was thought to act via the nervous system.
Experiments are under way to purify and characterize the
macromolecule(s) responsible for the stiffening activity, to
determine the site or sites of action and to determine whether
a plasticizer can also be identified and characterized.
This research was supported by grants from the National
Science Foundation and the Office of Naval Research. The
authors thank Robert Shadwick, Greg Szulgit, Gillian LyonsLevy, Stephen Andrews and three anonymous reviewers for
their helpful comments on the manuscript.
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