2Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas and 3Laboratório de Tecido Conjuntivo, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ, 21941590, Brazil
Received on November 13, 1999; revised on February 18, 2000; accepted on February 28, 2000.
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Abstract |
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Key words: sulfated polysaccharides/heparin/smooth muscle/sarcoplasmic reticulum/Ca2+-ATPase/sea cucumber
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Introduction |
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The sea cucumber (Echinodermata, Holoturoidea) has five bundles of smooth muscles attached to its body wall. These muscles are related with physiological features of the sea cucumber, which can rapidly and reversibly alter its body length by more than 200% (Trotter and Koob, 1989). The contraction of the sea cucumber muscles like all others is promoted by an increase of the cytosolic Ca2+ concentration. In contrast with the mammalian skeletal muscles, the sarcoplasmic reticulum of the echinoderm muscles is scarce and difficult to identify (Hill et al., 1982
; Chen, 1986
). The contractionrelaxation cycle of the longitudinal smooth muscle involves not only the movement of Ca2+ between the extracellular medium and the cytosol of the muscle cells but also the release and accumulation of Ca2+ from intracellular stores (Suzuki, 1982
).
The study of these particular tissues of invertebrates may help to elucidate more general molecular mechanisms of muscle contraction, with possible implications in vertebrate tissues. This approach has already been employed in the case of connective tissues of some invertebrates (Vieira and Mourão, 1988; Vieira et al., 1991
, 1993; Pavã et al., 1994
).
We have previously described that heparin can, in vitro, inhibit and modulate the activity of different ATPases of the P-type family (de Meis and Suzano, 1994; Landeira-Fernandez et al., 1996
; Rocha et al., 1996
; Rocha et al., 1998
). Thus, several SERCA isoforms derived from rabbit muscle, human platelets and rat brain are not dependent on KCl but, in the presence of heparin, they are inhibited and become highly sensitive to K+. The inhibition can be antagonized by K+>Na+>Li+ (de Meis and Suzano, 1994
; de Meis, 1998
). A unique fucosylated chondroitin sulfate isolated from the sea cucumber connective tissue can mimic the effect of heparin in these SERCA isoforms (de Meis and Suzano, 1994
). However, the possible physiological implication of these results can be questioned since these highly sulfated polysaccharides (such as heparin and fucosylated chondroitin sulfate) are not normally found in mammalian tissues where these ATPases are located.
In this paper we concentrated our study on the muscles found on the body wall of the sea cucumber Ludwigothurea grisea. In contrast with the SERCA isoforms from mammalian tissues, the activity of the Ca2+-dependent ATPase from sea cucumber is dependent on monovalent cations. The echinoderm smooth muscle was found to have a high concentration of fucosylated chondroitin sulfate but as we purified vesicles derived from this muscle we found a new and highly sulfated polysaccharide. When added to vesicles obtained from rabbit muscle this unique compound inhibits the enzyme and mimics the dependence for monovalent cations found in the echinoderm ATPase. Thus, our results describe the isolation of a sulfated polysaccharide that modulates ATPase activity and coexists in the same cellular compartment as the enzyme.
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Results |
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When similar methodologies were applied to vesicles obtained from rabbit muscle we did not detect sulfated polysaccharides, as indicated by the absence of metachromasia on the Mono Q-FPLC (open circles in Figure 2C) and of metachromatic bands on polyacrylamide gel electrophoresis (lane 5, Figure 3).
Overall, these results indicate that sea cucumber muscles have a high content of fucosylated chondroitin sulfate, but vesicles derived from these muscles have, instead, a different sulfated polysaccharide, eluted from an anion exchange column at higher NaCl concentration and with a unique migration pattern on polyacrylamide gel electrophoresis. In contrast, vesicles derived from rabbit sarcoplasmic reticulum muscles have no detectable amounts of sulfated polysaccharides.
Sulfated polysaccharides from sea cucumber muscle modify Ca2+ uptake of rabbit vesicles
Both, the fucosylated chondroitin sulfate purified from the entire sea cucumber muscle and the unusual sulfated polysaccharide extracted from the vesicles inhibit Ca2+ transport by vesicles derived from rabbit sarcoplasmic reticulum (Figure 4A). The concentration required for half maximal inhibition of Ca2+ uptake was 10-fold lower for the sulfated polysaccharide extracted from sea cucumber vesicles than that for fucosylated chondroitin sulfate or heparin. In addition, the unusual polysaccharide from sea cucumber induces a monovalent cation dependence for Ca2+ uptake by rabbit vesicles (solid symbols in Figure 4B), as that already observed in the presence of heparin (solid symbols in Figure 1B).
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Discussion |
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When the muscle was stained with toluidine blue we observed strong metachromasia surrounding the fibers (not shown). However, when the sea cucumber muscle was homogenized and the vesicles derived from its sarcoplasmic reticulum purified, the fucosylated chondroitin sulfate was no longer detected. Instead, we observed a sulfated polysaccharide which eluted from the anion-exchange column at higher salt concentration (Figure 2B,C). The observed pattern of molecular mass distribution is totally unusual for sulfated polysaccharides. Besides vesicles obtained from the sarcoplasmic reticulum of rabbit muscles did not contain any detectable sulfated polysaccharide (open symbols in Figure 2C).
We have not been able yet to determine the chemical structure of this polysaccharide due to scarce material and its very complex nature. Thus, 1H NMR spectrum at 600 MHz of the polysaccharide recorded at 60°C showed broader and poorly resolved signals (not shown), indicating a clearly heterogeneous chemical structure. It was not possible to assign the peaks even with the two dimension techniques. Partial acid hydrolysis (150 mM H2SO4 100°C for 30 min) removes most of the sulfated fucose from the polymer but not galactose and hexosamines. Thus, the sulfated fucose residues are apparently branched units. Other attempts to prepare small fragments using partial hydrolysis procedures were unfruitful. This polysaccharide differs from mammalian glycosaminoglycans since it resists to chondroitin lyase digestion and to nitrous acid deamination.
Thus, the sea cucumber muscles contain two different pools of sulfated polysaccharides. Fucosylated chondroitin sulfate is present at very high concentration (almost similar to those found in the connective tissue of the invertebrate) and is located surrounding the muscle fibers. Another unique sulfated polysaccharide, almost undetectable when extraction was performed in the entire muscle, is found in the vesicles derived from the sarcoplasmic reticulum. The co-location of this polysaccharide along with the Ca2+-ATPase led us to speculate on a possible physiological relationship between the two.
We suggested that the sulfated polysaccharide present in the sea cucumber vesicles could act as an "endogenous" Ca2+-ATPase inhibitor. In fact, the sulfated polysaccharide obtained from vesicles of the sarcoplasmic reticulum of the sea cucumber muscle inhibits Ca2+ uptake in vesicles obtained from rabbit muscles even at lower doses than heparin (Figure 4A) and restores monovalent cation dependence (Figure 4B).
The effect of sulfated polysaccharide on Ca2+-ATPase from sarcoplasmic reticulum
The different proteins found in sarcoplasmic reticulum vesicles isolated from rabbit and sea cucumber muscles can be identified readily by SDS gel electrophoresis (MacLennan, 1970; MacLennan et al., 1978
) and both revealed a major band of 110 kDa. The major component is the Ca2+-ATPase, this is a single polypeptide chain with a molecular weight of 110 kDa and accounts for 7080% of the vesicles total protein content. The Ca2+-ATPase from sarcoplasmic reticulum is responsible for the active transport of Ca2+ from the cytosolic to the lumen of the reticulum controlling the muscle excitation-contraction cycle (de Meis, 1981
).
Sulfated polysaccharides have been shown to modify the activity of different E1-E2 transport ATPases and the inhibition is antagonized by monovalent cations in a manner similar to that observed with the sea cucumber Ca2+-ATPase. Kinetic evidence presented in previous reports indicate that the different sulfated polysaccharides including heparin stabilize the E2 conformation of the different E1-E2 ATPases (de Meis and Suzano, 1994; Landeira-Fernandez et al., 1996
; Rocha et al., 1998
). The antagonism of the inhibition does not appear to be due to neutralization of the negative charges of the sulfate residues. Lithium is the most electropositive ion among the alkali ions tested. Potassium, but not lithium, strongly antagonizes the effect of the different sulfated glycosaminoglycans in different preparations of sarcoplasmic reticulum Ca2+-ATPases. Thus, if the effect of the alkali ions were to neutralize the negative charges of heparin, then Li+ should be as effective as K+ or Na+.
It is unlikely that the calcium binding to the sulfated polysaccharides plays an important role for the effects of these compounds on the Ca2+-ATPase. The calcium affinity constant for fucosylated chondroitin sulfate (Ruggiero et al., 1994) and heparin (Mattai and Kwak, 1981
) is ~50 µM while the affinity of calcium for the ATPase is in the range of 0.1 µM (de Meis, 1981
). In addition, the concentration of sulfated polysaccharide used in the Ca2+ uptake experiments is very low (~10 µg/ml) to decrease the amount of free calcium in the medium.
Is there any physiological role for the sulfated polysaccharides in sea cucumber muscles?
The body wall of the sea cucumber is composed by connective tissue and smooth muscle layers. It can rapidly and reversibly alter its mechanical properties. These alterations, which are thought to be neuronally controlled, allow the tissue to change its length by more than 200% (Trotter and Koob, 1989). The connective tissue of this echinoderm contains parallel collagen fibrils which are able to slide past one another during length changes but are inhibited from sliding when the tissue is in "catch." It has been suggested that sulfated polysaccharides (specifically the fucosylated chondroitin sulfate) is an important component of the stress-transfer matrix in echinoderms (Trotter and Koob, 1989
).
In this paper we show that fucosylated chondroitin sulfate is not an exclusive component of this connective tissue but is also found in high concentrations surrounding the muscle fibers of the sea cucumber. The function of this polysaccharide in the echinoderm muscle is still not known. Perhaps its high negative charge density (Mourão et al., 1996) and consequent capacity to retain water in the extracellular matrix produces a "space" among muscle fibers allowing the intense change in length that may occur in this invertebrate (such as 200%) and are not observed in mammalian muscles.
Finally, the sulfated polysaccharide extracted from the sarcoplasmic reticulum vesicles has probably a totally different physiological role. The effect of this polysaccharide as an inhibitor of Ca2+ uptake by rabbit skeletal vesicles, producing its monovalent cation dependence, suggests that this compound may act as an "endogenous" Ca2+-ATPase regulator in sea cucumber smooth muscles. This possibility, however, requires future investigation.
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Materials and methods |
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Ca2+ uptake by microsomes prepared from muscles of sea cucumber and rabbit
Ca2+ uptake was measured by the filtration method using 45Ca (Chiesi and Inesi, 1979). Briefly, microsomes (20 µg/ml, as protein) in 50 mM MOPS/TRIS (pH 7.0), 1 mM MgCl2, 50 µM 45Ca-CaCl2 (from Dupont, Wilmington, DE), 5 mM oxalate, 1mM ATP and increasing concentrations of either KCl, NaCl, LiCl, in the presence or absence of sulfated polysaccharides were incubated at 25°C for sea cucumber vesicles and at 35°C for rabbit vesicles. Ca2+ uptake was measured by filtration on Millipore filter. The filters were washed five times with 5 ml of 3 mM La(NO3)3 and the radioactivity remaining on the filters was counted on a liquid scintillation counter.
Extraction of sulfated polysaccharides from intact muscles and from microsome preparations at different stages of vesicle purification
Sea cucumber muscles were cut into small pieces, immersed immediately in acetone, and kept for 24 h at 4°C. The sulfated polysaccharides were extracted from the dried tissue (~2 g) by papain digestion and partially purified by cetylpyridinium chloride and ethanol precipitation (Vieira et al., 1991). In the case of microsome preparations we employed a more simplified procedure and the cetylpyridinium precipitation step was omitted (Vieira and Mourão, 1988
). Approximately 4.0 ml of the microsome preparation was mixed with 4 ml of 0.1 M sodium acetate buffer (pH 6.0) containing 5 mg of papain, 5 mM EDTA and 5 mM cysteine. The incubation mixture was then centrifuged (2000 x g for 15 min at 10°C), and the sulfated polysaccharides in the supernatant were precipitated with 5 volumes of ethanol. After 24 h at 4°C, the precipitate formed was collected by centrifugation (2000 x g for 15 min at 10°C), washed with 10 ml of 80% ethanol and once with 95% ethanol. The final precipitate was dried at 60°C for 24 h (~3 mg, dry weight), dissolved in 5 ml of distilled water, and lyophilized.
Purification of the sulfated polysaccharides
Anion exchange chromatography. Sulfated polysaccharides extracted from the muscles or from the microsome preparations (~10 mg of each) were applied to a DEAE-cellulose column (7 x 2 cm) equilibrated with 0.05 M sodium acetate buffer (pH 5.0) and washed with 100 ml of the same buffer. The sulfated polysaccharides were eluted stepwise with 20 ml of 1.0 M NaCl in the same buffer. Eluates were dialyzed against distilled water and lyophilized. The DEAE-cellulose-purified polysaccharides were applied to a Mono Q-FPLC column (HR 5/5) from Amersham Pharmacia Biotech, equilibrated with 20 mM Tris:HCl (pH 8.0). The column was developed by a linear gradient of 03 M NaCl in the same buffer. The flow rate of the column was 0.5 ml/min, and fractions of 0.5 ml were collected. The fractions were assayed for metachromatic property using 1,9-dimethylmethylene blue (Farndale et al., 1986). The NaCl concentration in the fractions was estimated by conductivity. The fractions containing the sulfated polysaccharides were pooled, dialyzed against distilled water, and lyophilized.
Gel filtration chromatography. The Mono Q-purified polysaccharides were applied on a Superose-12 (HR 10/30) column, linked to a FPLC system from Amersham Pharmacia Biotech, equilibrated with 0.1 M Tris:HCl and 1 mM EDTA (pH 8.0). The column was eluted with the same solution, at a flow rate of 0.5 ml/min and fractions of 0.5 ml were collected and assayed by metachromasia using 1,9-dimethylmethylene blue (Farndale et al., 1986). The various subfractions were pooled, dialyzed against distilled water, and lyophilized.
Polyacrylamide gel electrophoresis. Sulfated polysaccharides (~10 µg) were applied to a 1-mmthick 6% polyacrylamide slab gel and after electrophoresis at 100 V for ~1 h in 0.06 M sodium barbital (pH 8.6), the gel was stained with 0.1% toluidine blue in 1% acetic acid. After staining, the gel was washed for ~6 h in 1% acetic acid.
Chemical analyses.
After acid hydrolysis of the polysaccharide (5.0 M trifluoroacetic acid for 5 h at 100°C), sulfate was measured by the BaCl2/gelatin method (Saito et al., 1968). The percentages of hexoses, 6-deoxyhexoses and hexosamines were estimated by gas-liquid chromatography of derived alditol acetates (Kircher, 1960
). Hexuronic acid was estimated by the carbazole reaction (Dische, 1947
).
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Acknowledgments |
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Footnotes |
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References |
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