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First published online February 15, 2008
Journal of Experimental Biology 211, 766-772 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.007658
Elasticity, unexpected contractility and the identification of actin and myosin in lobster arteries
1 Department of Biological Sciences, Faculty of Science, University of Calgary,
2500 University Drive N.W., Calgary, Alberta, T2N 1N4, Canada
2 Department of Physiology and Biophysics, Faculty of Medicine, University of
Calgary, 4440 Hospital Drive N.W., Calgary, Alberta, T2N 4N1, Canada
* Author for correspondence (e-mail: wilkens{at}ucalgary.ca)
Accepted 11 December 2007
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: actin, artery, cardiovascular system, lobster, microfilament, myosin, vascular resistance, Homarus americanus
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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).
Muscular cardioarterial valves are present at the origins of six of the
arteries (anterior median artery, AMA; anterior lateral arteries, ALAs;
hepatic arteries, HAs; and sternal artery, SA), but not at the origin of the
dorsal abdominal artery (DAA). At the back of the heart, a muscular flap is
situated at the entry into the bulbus arteriosus
(Kuramoto et al., 1992;
McMahon et al., 1997). The DAA
and the SA originate from the bulbus. Valves are also located at the origins
of the segmentally paired lateral arteries that arise from the DAA
(Davidson et al., 1998), but
they have not been found at the branch points along any of the other vessels.
Arteries divide until the finest arterioles terminate in capillary-sized
tissue spaces (McGaw and Reiber,
2002) (J.L.W., unpublished observations on lobsters). Haemolymph
collects in and returns to the heart through sinuses.
It seems imperative that a large crustacean be able to control the
distribution of haemolymph to different regions of the body to accommodate
changing physiological demands for oxygen, nutrients, and waste removal. Using
pulsed-Doppler techniques on intact animals, such distributional control has
indeed been observed in response to hypoxia
(Airriess and McMahon, 1992)
and after feeding (McGaw and Reiber,
2000; McGaw,
2005).
There are several possible mechanisms for controlling flow patterns among
the arteries. The cardioarterial valves are innervated
(Fujiwara-Tsukamoto et al.,
1992), and they are responsive to a variety of cardioactive drugs
(Kuramoto et al., 1995;
Wilkens and Kuramoto, 1998).
In addition, the arteries, if perfused with cardioactive drugs at a point
distal to the cardioarterial valve, show increased resistance to flow
(Wilkens, 1997;
Wilkens and Taylor, 2003). For
the DAA, the sites of drug action could be the striated muscle blocks in its
lateral walls (Martin et al.,
1989; Wilkens et al.,
1997b) as well as the valves at the branch points to the lateral
arteries (Davidson et al.,
1998). The other arteries do not have either striated muscle in
their walls (Wilkens et al.,
1997b; Chan et al.,
2006) or valves along their length. To date, all other contractile
(skeletal, cardiac and intestinal) tissues in crustaceans have been shown to
be striated muscles. It has been assumed that crustacean arteries, with the
possible exception of the DAA, are passive capacitance vessels. The goal of
the present study was to determine if lobster arteries are purely passive
vessels or whether they possess active properties.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Arteries and OOM were stored in ice-chilled saline and
tested over 2 days. The lobster saline used for dissection and perfusion
contained (in mmol l–1) 479.6 NaCl, 15.7 KCl, 25.9
CaCl2, 9.3 MgSO4 and 5.0 Tris buffer; the saline was
equilibrated with air and adjusted to pH 7.6.
Morphology and morphometry
Histochemistry
SAs were excised, cut longitudinally with iridectomy scissors, and opened
onto cover glasses. The spreads were ringed with petroleum jelly and flushed
with saline. A solution of 3.7% formaldehyde in saline was added to the
reservoirs for 10 min. The spreads were then washed with saline (2 min),
permeabilized with 0.1% Triton X-100 (5 min), washed with saline (2 min),
incubated in 1% bovine serum albumin in saline (30 min), stained with a
double-strength stock of Rhodamine–phalloidin (catalogue no. R-415;
Molecular Probes, Eugene, OR, USA) for 30 min, and washed with saline (2 min).
Rhodamine–phalloidin is a fluorescent phallotoxin probe for fibrous (F)
actin (Wieland, 1986). The
cover glasses were inverted and mounted on microscope slides with a solution
of equal parts of glycerol and saline. As a control, the
Rhodamine–phalloidin was omitted from the protocol.
SA spreads were viewed and photographed with a Nikon Eclipse TE300 inverted fluorescence microscope. The microscope was calibrated with a stage micrometer (100 lines mm–1), and photomicrographs were made with Kodak Ektachrome 100 film. The transparencies were digitized with a Polaroid SprintScan 45i film scanner at a resolution of 600 dpi, and the images were stored as uncompressed TIFF files. Image files were converted to grayscale, adjusted, and printed with `Photoshop CS2 for Windows' (9.0) software (Adobe Systems, San Jose, CA, USA).
Ultrastructure
SAs destined for histology and ultrastructure were processed at slack
length. The isolated vessels were cannulated and perfused and bathed with
phosphate-buffered glutaraldehyde (Cloney
and Florey, 1968) for 5 min. The arteries were cut into short
cylindrical segments, and these rings were transferred to a container of fresh
fixative, where they remained for 60 min at room temperature.
The aldehyde-containing fixative was decanted, and bicarbonate-buffered
osmium tetroxide (Wood and Luft,
1965) was added to the container. The container was placed in an
ice bath for 60 min. At the end of osmication, the arterial rings were rinsed
with demineralized water, dehydrated with graded solutions of ethanol, and
treated with propylene oxide. The rings were infiltrated and embedded in
LX-112 epoxy resin (Ladd Research Industries, Williston, VT, USA).
Ultrathin sections (70–80 nm in thickness), cut with a diamond knife
on a Sorvall MT-6000 ultramicrotome, were collected on unsupported copper
grids and stained with aqueous solutions of uranyl acetate (saturated) and
lead citrate (Reynolds, 1963).
The sections were viewed with an Hitachi H-7000 transmission electron
microscope operated at 75 kV and equipped with an SIA-8A digital camera
(Scientific Instruments and Applications, Duluth, GA, USA). The microscope was
calibrated with a carbon replica of a diffraction grating (2158 lines
mm–1), and digital images were captured at a resolution of
2048x2048 pixels and saved as uncompressed TIFF files. Image files were
adjusted and printed with `Photoshop CS2 for Windows' (9.0) software.
Morphometry
Some aldehyde-fixed arterial rings were transferred to saline for
examination with a Nikon SMZ800 stereomicroscope. The microscope was
calibrated with a stage micrometer. The rings, ranging in length from 0.5 to 1
mm, were oriented on end and photographed with a Nikon Coolpix 5000 digital
camera using both reflected and transmitted light. The images were captured at
a resolution of 2560x1920 pixels and saved as uncompressed TIFF files.
Image files were imported into `SigmaScan Pro for Windows' (5.0) software
(Systat Software, Point Richmond, CA, USA) for morphometric analysis of wall
thickness and luminal area. Ten measurements of wall thickness and two
measurements of luminal area were made on each ring. Luminal (inside) diameter
was calculated by treating the luminal area as the area of a circle.
Alternatively, rings were cut open longitudinally and spread onto microscope
slides; cover glasses were mounted with saline. The slack circumference of a
vessel was measured as the width of the specimen, measured with an ocular
micrometer (10 lines mm–1) in a Wild Heerbrugg M5
stereomicroscope.
Biochemistry
Arterial rings (6 mm long), OOM, slow skeletal muscles from the crusher
claw and fast skeletal muscles from the cutter claw were homogenized in 40
µl sodium dodecyl sulfate (SDS) sample buffer. The sample buffer was kept
in an ice bath. Protein electrophoresis of the homogenates was carried out on
12% SDS-polyacrylamide discontinuous Laemmli gels (16x20 cm) for use in
a Bio-Rad Mini-Protean electrophoresis system (SDS-PAGE).
Physiology
Arterial wall and OOM force was measured using a strain gauge (resonant
frequency 286 Hz, SensoNor AE801; Sensor One, Sausalito, CA) connected to a
Gould Bridge amplifier. Rings, 1–2 mm in length, and longitudinal strips
were cut from the arteries. Insect pins glued to the force transducer and an
ergometer were placed inside a ring or inserted into the ends of a strip. The
manipulator-mounted transducer was moved to apply a small stretch to the
arterial wall. The linear compliance component of the transducer wafer and
insect pin (0.167 mm mN–1) was measured from the force while
the manipulator moved the pin against a rigid stop. After a stretched ring had
relaxed to equilibrium (relaxation time constant, 
=1/e, 7–16 s,
depending on the magnitude of the stretch), the active force generated in
response to drugs or electrical stimulation was determined. Tissues were
stimulated electrically with a pair of laterally placed platinum ribbon
electrodes. Arteries were stimulated with repeated 2 ms pulses delivered at 20
Hz. OOM received trains of stimuli (2 ms pulses delivered at 20 Hz, 100 ms
train duration, 2 trains min–1). Data were recorded using a
PowerLab/4SP A-D converter (sampling rate 100 Hz to 2 kHz) and analyzed with
`Chart for Windows' (4.2) software (ADInstruments, Colorado Springs, CO).
The passive force–length and stress–strain relationships were
determined for three arteries to evaluate their relative compliance and to
determine the magnitude of diameter change that a contraction would produce.
Strain was defined as stretch (
l) divided by slack
circumferential length (Lo), and stress was defined as
force divided by wall area (ring length multiplied by wall thickness).
The preparation was continuously superfused with saline at 12°C (4 ml
min–1 in a 1.5 ml bath). The source to the superfusion pump
could be switched between normal saline and saline containing the
neurotransmitter glutamic acid (GLU, 10 mmol l–1; Sigma
Chemical Co., St Louis, MO) or the neuropeptide hormone proctolin (PR, 1.0
µmol l–1; Sigma Chemical Co.). These concentrations were
effective in causing increased flow resistance in perfused arteries
(Wilkens, 1997;
Wilkens and Taylor, 2003) and
contraction in OOM (Wilkens et al.,
2005). Although the GLU level appears high, it may reflect the
effective concentration in glutaminergic synapses. Drug exposure was
continuous until a response reached a stable maximum, often requiring more
than 5 min.
The actin inhibitor cytochalasin D (CD; Sigma Chemical Co.) was tested on arterial rings and OOM pretreated for 5–10 min at concentrations from 0.5 to 10.0 µg ml–1. Since rings recovered extremely slowly (>90 min) from drug-induced contractions, successive rings were used for each test (e.g. rings one and three would be exposed to PR and ring two would be pretreated with CD and then exposed to PR diluted in the same concentration of CD).
Statistical comparisons between data sets were performed using Student's t-test. Differences where P<0.05 were considered significant.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). The tunica
interna is an elastic connective tissue adjoining the lumen of the vessel, and
the tunica externa is a collagenous connective tissue anchoring the vessel to
the adventitia of the body. The highest concentration of cells is found in the
tunica intermedia. In the small vessels (ALAs, AMA and HAs) leaving the
anterior end of the heart, the cells of the tunica intermedia aggregate into
compact masses. In the larger vessels (SA and DAA) departing from the rear of
the heart, the cells of the tunica intermedia are less compacted, being
infiltrated by connective-tissue elements and, in the case of the DAA, by
blocks of striated muscle cells.
Cells in the tunica intermedia of the ALA
(Chan et al., 2006), the AMA
(Cavey et al., 2008) and the SA
(present study) stain intensely after exposure to Rhodamine–phalloidin
(Fig. 1A), correlating with the
presence of bundled microfilaments in the cells of this layer
(Fig. 1B). These microfilament
bundles traverse the cytoplasm and frequently associate with filamentous mats
on the inner surface of the plasmalemma.
|
) when
position of the molecular marker distribution had been scaled for P75 and
actin. The band labelled P1, 2 at 
105 kDa may represent paramyosin. The
myofibrillar protein P75 was present in fast, but not slow, skeletal muscle.
Numerous other bands are common to the different homogenates, but we did not
attempt to identify them.
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Arterial rings slowly contracted upon perfusion with PR or GLU (Figs 4, 5; Table 2). These contractions often required 5–8 min to reach a maximum; recovery was much slower and proportional to drug exposure time, requiring more than 90 min after a 5 min drug treatment. The magnitude of the contractions varied from 0 to as much as 50% of the original passive equilibrium force. In the DAA, drug-induced changes in force could have arisen from either the striated muscle cells or the muscle-free areas of the vessel wall. The amplitude of PR-induced contractions was similar to that during electrical stimulation, but the rate of rise of force was much lower, requiring 20 s to reach peak active force during electrical stimulation and more than 5 min during PR exposure (Fig. 5; Table 2). The rate and amplitude of GLU-induced contractions of a DAA ring were greater than for PR and could represent responses of both the muscle and the muscle-free regions. None of the other arteries contracted when stimulated electrically. Longitudinal strips of arteries did not exhibit force generation during exposure to PR or GLU or during tetanic electrical stimulation. Similar tests were performed on OOM; GLU (data not shown), PR, and electrical stimulation caused large contractions that reached maximum values in seconds, as opposed to minutes for the arterial rings (see Fig. 7A,C).
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Will the contractile force generated by an arterial ring be sufficient to alter vessel radius and, thus, the resistance to haemolymph flow? One estimate was taken from force-length relationships of rings. After the rings were incrementally stretched, the stretch was reduced to 2–2.3xLo where the passive force was in the range of transmural force and drugs were applied (Fig. 6). As shown for the SA in the middle panel of Fig. 6, the magnitude of contractile force was used to evaluate how much this force would have decreased the circumference of the ring. From the responses of three animals so tested, PR would have reduced ALA, SA and DAA radii by 13.6±6.0, 36.8±27.0 and 6.3±3.8% (mean ± s.e.m.), respectively, and GLU would have caused a 24.8±19.2% reduction in DAA radius. These values are similar to or greater than those shown in Table 2, where drugs were tested over greater length ranges. Alternatively, contractile force will shorten a ring due to the transducer compliance. The PR-induced shortening of arteries shown in Fig. 6 would have reduced the radius for the ALA, SA and DAA by 5.1%, 5.2% and 2.7%, respectively. GLU reduced the radius of the DAA by 4.1%. This calculated amount of circumferential shortening may underestimate the shortening in vivo, because shortening in these experiments occurred against the compliant transducer.
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We explored the effect of cytochalasin D in order to study the involvement of microfilaments in generation and maintenance of the PR-induced force by these arteries. Pretreating with CD prevented PR-induced contraction in two ALAs, whereas five untreated rings contracted. For the SA, CD abolished or reduced PR-induced contraction in three of six rings taken from two animals (Fig. 7A,B). One of the SAs that failed to respond to PR during CD treatment contracted – but only weakly – when perfused with PR following 80 min of washing. CD (0.5 or 1.0 µg ml–1, 5–10 min) treatment caused an irreversible decrease in tonus and a decrease in tetanic force in each of five OOMs from two animals. CD effects on OOM were reversible when exposure was of shorter duration (Fig. 7C).
|
DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
To et al., 2004), including
those in the lateral walls of the dorsal abdominal artery (DAA)
(Martin et al., 1989;
Wilkens et al., 1997b), have
proved to consist of striated cells. It has been assumed that other crustacean
arteries only serve as haemolymph conduction vessels since electron
micrographs reveal no evidence of muscle cells
(Chan et al., 2006;
Wilkens et al., 1997b).
However, we know that several cardioactive compounds do increase the
resistance to flow in in situ perfused arteries
(Wilkens, 1997;
Wilkens and Taylor, 2003).
Although seemingly unlikely, it was important to determine whether these
cardioactive agents could cause arteries to contract.
Rings of the anterior lateral artery (ALA), sternal artery (SA) and DAA
exhibit non-linear elasticity, characteristic of the interplay of elastic and
collagenous connective tissues, as are found in the tunica interna and the
tunica externa, respectively (Shadwick et
al., 1990; Wilkens et al.,
1997a; Chan et al.,
2006) (present study). Although vessel diameters are similar, the
wall thicknesses reported here are 2–3.7 times greater than those cited
previously for the crab and the lobster
(Shadwick et al., 1990).
ALAs, HAs and SA do develop force when stimulated by proctolin (PR) and
glutamic acid (GLU), observations suggesting that the cells with actin-based
microfilaments also contained myosin. Protein analysis by SDS-PAGE
demonstrated that actin and myosin are present in all three arteries as well
as the cardiac ostia (OOM), and the slow and fast skeletal muscle. In
addition, there are bands in all of our homogenates that correspond to
paramyosin (P1, 2, 105 kDa), tropomyosin (38 kDa), and troponins T and I
(Fig. 2). The bands correspond
closely with those found in skeletal muscle by Medler and Mykles
(Medler and Mykles, 2003). For
slow and fast skeletal muscles in lobsters, there are differences in the
isoforms of myosin heavy chains (S1, S2, and fast) and tropomyosin (S1, S2,
and fast) and the protein P75 which is found in fast but not in slow skeletal
muscle (Meiss et al., 1981;
Mykles, 1985;
Medler and Mykles, 2003);
however, these isoform differences are not distinguishable in our gels.
A clear distinction must be made between the DAA, with striated muscle
cells in its architecture (Wilkens et al.,
1997b), and the other six arteries. Consistent with the presence
of muscle, DAA rings (but not longitudinal strips) contract during electrical
stimulation; none of the other arteries respond to electrical stimulation. The
striated muscle cells of the OOM contract rapidly when stimulated
electrically.
Rings of all arteries contract during exposure to cardioactive drugs. Since
the responses are clear and reproducible, they represent a reactive property
of the vessels. PR induces stronger contractions in both ALA and SA than does
GLU, whereas the reverse is true for the DAA, presumably reflecting different
mechanisms of action of the different drugs on the muscle. Contraction
probably arises from the formation of cross-bridges and will affect the
resistance to flow in these arteries. The time course of the contractions is
slow but similar to the changes in resistance to flow (approx. two- to
threefold) seen in perfused arteries in situ
(Wilkens, 1997;
Wilkens and Taylor, 2003).
It is noteworthy that arteries taken from at least half of the animals were
more responsive to PR and GLU 1 or 2 days after removal from the animal than
on the day of dissection. The vessels in vivo may exist in a
tonically `contracted' state, a state that relaxes slowly once the tissues
have been removed from the milieu interieur of the animal. This and
the reduced responses to repeated application of a drug may be related to
tissue receptor desensitization (contrasting denervation hypersensitivity).
Thus, on the day of dissection, the tissues may not have been fully relaxed,
but after 2 or 3 days in cold saline, the baseline tonus of the walls may have
decreased so they become more responsive to drugs. This suggests that the
animal can both actively contract and relax vessel walls to control arterial
resistance. Since the arteries do not appear to be innervated (M.J.C., K. S.
Chan and J.L.W., unpublished), cardioactive drugs must be acting directly on
the vessel walls to cause contraction while an unknown relaxing factor,
possibly nitric oxide (Mahadevan et al.,
2004), circulating in the haemolymph or released by surrounding
tissues, could cause vessel dilation. Such local control of arterial radius,
plus the participation of cardioarterial valves and DAA-lateral artery valves,
will be important in regional haemolymph distribution patterns among the seven
arteries leaving the heart (Airriess and
McMahon, 1992; Davidson et al.,
1998; McGaw and Reiber,
2002; Guadagnoli and Reiber,
2005).
Poiseuille's law states that the drug-induced decreases in vessel radius, measured here, will increase the resistance to flow by the inverse fourth power of that change. Further, since serial resistances are additive, it is obvious that even a very small decrease in radius repeated along the length of an artery and its branches can have a profound effect on flow through that vessel.
The motor that drives circumferential contraction in lobster arteries is
probably based on actomyosin interactions. We show above that myosin is
clearly present, and actin-containing microfilaments in the cells of these
arteries may complete the contractile apparatus
(Fig. 2). Three morphological
observations on lobster arteries – the presence of prominent bundles of
actin-based microfilaments in cells of the tunica intermedia, the association
of microfilament bundles with the plasmalemma, and the tendency of
microfilament bundles to orient circularly with respect to the vessel lumen
– prompted us to investigate the role of microfilaments with
cytochalasin D (CD). Pretreatment with CD, a drug that inhibits actin
polymerization and disrupts filament organization, reduces or eliminates the
slow contractions of both ALA and SA, and it reduces the force generated by
OOM. CD inhibits contraction of unstriated smooth muscle
(Adler et al., 1983;
Saito et al., 1996;
Mehta and Gunst, 1999) and
cardiac striated muscle (Wu et al.,
1998) in vertebrates. Polymerization of globular (G) actin into
F-actin may be necessary to force generation in some smooth muscle cells
(Adler et al., 1983;
Mehta and Gunst, 1999). In the
arterial cells and the OOM of the lobster, the filament bundles are obviously
pre-existing. However, the density of F-actin depends on the rates of
polymerization and depolymerization. CD will shift the balance toward
depolymerization and, hence, the density of force-bearing actin filaments may
decrease after CD treatment. In addition, CD might also disrupt the normally
close spacing of the microfilaments, reducing the probability of cross-bridge
formation between myosin and actin molecules.
Quantitative study of transmural arterial pressure and diameter of lobster
arteries is still needed, but it is clear from our data that PR and GLU may
increase wall stress substantially. To a first approximation, the magnitude of
the increased wall stress is sufficient to constrict the arteries in
vivo (at arterial pressure=1.0 kPa) by 20% and hence explain the
observed increase in the resistance to flow by a factor of two to three
(Wilkens, 1997;
Wilkens and Taylor, 2003). If
these predictions can indeed be corroborated in vivo, it will
emphasize that PR and GLU play important active roles in the haemodynamics of
crustaceans.
LIST OF ABBREVIATIONS
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Acknowledgments |
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