Chemolithoheterotrophy in a metazoan tissue: thiosulfate production matches ATP demand in ciliated mussel gills
1 Department of Biology, University of Alabama at Birmingham, 1300 University Boulevard, Birmingham, AL 35294-1170, USA and
2 Institut für Zoophysiologie, Heinrich-Heine-Universität, Universitätsstraße 1, 40225 Düsseldorf, Germany
*e-mail: doeller{at}uab.edu
Accepted August 10, 2001
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Summary |
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Key words: sulphide, gills, sulphide oxidation, bromobimane HPLC, succinate, mussel, Geukensia demissa, Mytilus edulis.
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Introduction |
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Although the biochemistry of sulfide oxidation in metazoan tissue is only partially understood, thiosulfate has been shown to be the major product of sulfide oxidation in the animals studied thus far [for a review, see Grieshaber and Völkel (Grieshaber and Völkel, 1998)]. In the present study, the products of sulfide oxidation in G. demissa gills were determined as a function both of ambient sulfide concentration and of tissue ATP demand. The rates of sulfide oxidation and oxygen consumption were measured in the same gill preparation to determine the relationship between sulfide oxidation and ATP demand. In addition, the gill contents of succinate were estimated as an indication of anaerobic metabolism: when cytochrome c oxidase can no longer transfer electrons to oxygen because of either limiting ambient oxygen tensions or sulfide inhibition, fumarate is reduced to succinate (Grieshaber et al., 1994) and steady-state levels of succinate increase. Gills from the blue mussel Mytilus edulis, from intertidal low-sulfide habitats, were examined for comparative purposes.
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Materials and methods |
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Experimental protocol
Identification of thiol compounds and determination of their production and release kinetics
Whole gills were excised from two living mussels and placed in Millipore-filtered (0.45 µm) ASW at maintenance salinity and room temperature (1820°C) for at least 30 min prior to experimentation to allow removal of excess mucus. Gills were then cut into small pieces; mean tissue wet mass was 13.6±3.7 mg (mean ± S.D., N=42). Individual pieces, randomly mixed from the two animals, were placed in incubation vials, one piece per vial, containing 5 ml of aerated sea water with either 0.5 µmol l1 (low) or 10 µmol l1 (high) 5-hydroxytryptamine (5-HT) to stabilize ciliary beating at 510 Hz or 2025 Hz, respectively (Doeller et al., 1999). 5-Hydroxytryptamine, also known as serotonin, is an endogenous neurotransmitter in the bivalve gill that stimulates beating of the lateral cilia (Clemmesen and Jørgensen, 1987) probably via cAMP signal-transduction pathways [for a review, see Satir and Sleigh (Satir and Sleigh, 1990)]. At time zero, 100 µmol l1 Na2S (25 µl of 20 mmol l1 Na2S stock) was added to the incubation vials. Stock solutions of 20 mmol l1 Na2S were prepared by dissolving washed crystals of Na2S.9H2O in N2-saturated filtered ASW, with pH adjusted to 8.0 with 0.1 mol l1 HCl (Wohlgemuth et al., 2000). Stock solutions of 0.01 mmol l1 and 1 mmol l1 5-HT were prepared by dissolving crystals in filtered ASW. At 0, 5, 10, 20, 30 and 50 min, six tissue pieces were removed; three pieces were prepared immediately and three were frozen in liquid nitrogen for high-performance liquid chromatography (HPLC) determination of thiol compounds (see below). All pieces were prepared individually. Ambient seawater samples were taken at the same times and prepared for thiol HPLC. Control gill pieces placed in incubation vials containing only either a low or a high 5-HT concentration in aerated seawater were removed after 50 min.
Determination of the ratio of oxygen consumed to sulfide oxidized and of anaerobic end-products
Gill pieces, weighing approximately 50 mg, were placed in respirometer chambers and exposed to either 0.5 µmol l1 or 10 µmol l1 5-HT and to 01000 µmol l1 Na2S at time zero. After 10 min, during which time a stable oxygen consumption rate was recorded (see below), a small piece of gill tissue, weighing 1020 mg, was excised from the experimental tissue and frozen in liquid nitrogen for thiol HPLC (see below). Another gill piece was treated in the same way for determination of succinate levels (see below). The remaining gill tissue was weighed, dried for at least 48 h at 70°C and reweighed. Ambient seawater samples were taken at the same time and frozen in liquid nitrogen for thiol HPLC. These experiments were repeated three times, each time with a different animal.
Measurement of thiol compounds by HPLC
Levels of thiol compounds in gill tissue homogenates and ambient sea water were measured using the monobromobimane HPLC method (Fahey et al., 1981; Newton et al., 1981; Vetter et al., 1989) as described by Völkel and Grieshaber (Völkel and Grieshaber, 1992; Völkel and Grieshaber, 1994), with the following modification: tissues that had previously been frozen in liquid nitrogen were then homogenized while thawing in the bimane reaction mixture. Thiol levels determined in previously frozen tissue were not significantly different from thiol levels determined in tissue processed immediately for HPLC (t-test; data not shown). The compounds of interest were the inorganic thiols sulfide, thiosulfate and sulfite, as possible components of the sulfide oxidation reaction, and the organic thiols glutathione and cysteine, as possible components involved in cellular oxidation/reduction reactions and sulfur storage, respectively. This method produced linear results for all thiol standards within the concentration range 2100 µmol l1 in the assay volume (data not shown). Blank tissue thiol levels were measured in the absence of added sulfide, and blank ambient seawater thiol levels were measured in the absence of tissue.
Measurement of gill oxygen consumption rate
Gill oxygen consumption rate was measured in a dual closed-chambered respirometer (Oroboros Oxygraph, model 67097; Paar, Graz, Austria) as described by Lee et al. (Lee et al., 1996). Briefly, a section of excised gill was placed on a stainless-steel screen shelf inside a respirometer chamber containing 5 ml of stirred (500 revs min1) ASW at 20°C. The second chamber containing identical apparatus minus gill served as a control for blank oxygen consumption rates. During experiments, microliter additions of 5-HT and Na2S stock solutions were made through an injection port in the stopper of each chamber using a Hamilton syringe; rates of oxygen consumption were measured within 10 min following additions.
Measurement of succinate levels
Tissue was extracted according to the method of Beis and Newsholme (Beis and Newsholme, 1975), and succinate was measured spectrophotometrically according to the method of Beutler (Beutler, 1985).
Data presentation and statistical analyses
Data are presented as means ± S.D. (number of repetitions). Two-sample comparisons were made using paired or unpaired one-tailed t-tests assuming equal variance (Microsoft Excel). Multiple comparisons were made using analysis of variance (ANOVA) using the Bonferroni post-hoc test (SAS Institute Inc.; StatView). Significance was accepted at the 5 % level.
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Results |
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Thiol levels in G. demissa gills during a 50 min incubation in sea water injected with 100 µmol l1 Na2S at time zero are shown in Fig. 1 (see time course of ambient seawater Na2S concentration in Fig. 2). At both 0.5 µmol l1 and 10 µmol l1 5-HT, the main product of sulfide oxidation was thiosulfate; control tissue in the absence of sulfide did not produce thiosulfate during the 50 min incubation time. In low-5-HT gills, thiosulfate levels increased significantly at 10 and 20 min to near 1000 µmol kg1 wet mass, then declined (Fig. 1A) following the drop in ambient sulfide level. In high-5-HT gills, thiosulfate levels increased significantly within 5 min and reached a maximum of approximately 2000 µmol kg1 wet mass at 20 min, then declined (Fig. 1B) following the drop in ambient sulfide level. Sulfite showed patterns in both low- and high-5-HT gills that resembled those of thiosulfate, although at less than 5 % of the thiosulfate concentration (Fig. 1). At 10 min, levels of both thiosulfate and sulfite in high-5-HT gills, those with a higher ciliary beat frequency, were significantly higher than levels in low-5-HT gills. In contrast to thiosulfate and sulfite, levels of sulfide, glutathione and cysteine remained relatively constant throughout the incubation.
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Thiol levels in Geukensia demissa gills versus Mytilus edulis gills
A comparison of initial levels of thiol compounds in gills of mussels taken directly from maintenance aquaria (Table 1) showed that thiosulfate, glutathione and sulfide levels were significantly greater in G. demissa gills than in M. edulis gills, whereas cysteine and sulfite levels were not significantly different. A comparison of inorganic thiol levels measured 10 min after injection of 100 µmol l1 Na2S at time zero in respirometer chambers (Fig. 3) indicated that thiosulfate in both low- and high-5-HT gills and sulfite levels in high-5-HT gills were significantly greater in G. demissa than in M. edulis; sulfide levels were not significantly different between the species in either low- or high-5-HT gills. A comparison of organic thiol levels showed that glutathione levels were significantly greater in G. demissa gills than in M. edulis gills and that cysteine levels were not significantly different (Fig. 3).
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Discussion |
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Thiosulfate
Thiosulfate is the main sulfide oxidation product in a variety of macrofaunal sulfide inhabitants, accumulating in the tissues and body fluids [for a review, see Grieshaber and Völkel (Grieshaber and Völkel, 1998)]. Thiosulfate has also recently been shown to be the sulfide detoxification product in rat cecal mucosa (Levitt et al., 1999). The peak thiosulfate concentrations reported here for low-5-HT and high-5-HT gills of G. demissa exposed to 100 µmol l1 Na2S in aerated sea water, 1000 and 2200 µmol kg1 wet mass, respectively, are similar to values reported for body wall tissue of the lugworm Arenicola marina exposed to Na2S in aerated sea water [1424 µmol kg1 wet mass exposed to 200 µmol l1 Na2S (Hauschild and Grieshaber, 1997); near 1 mmol l1 exposed to 117 µmol l1 Na2S (Wohlgemuth et al., 2000)]. In A. marina, sulfide is oxidized in the mitochondria to thiosulfate (Völkel and Grieshaber, 1996; Völkel and Grieshaber, 1997), which accumulates in the body wall, coelomic fluid and blood (Hauschild and Grieshaber, 1997). In the echiuroid worm Urechis caupo exposed to sulfide, thiosulfate, the product of sulfide oxidation primarily by hematin in coelomocytes (Powell and Arp, 1989), accumulates in the coelomic fluid (Julian et al., 1999). In G. demissa gills, sulfide is oxidized in gill mitochondria (Parrino et al., 2000), and we show here that thiosulfate accumulated in the gill tissue for a brief period before detectable release into the ambient sea water.
Biological sulfide oxidation in prokaryotes is thought to occur mainly by enzymes linked to the respiratory chain, such as the sulfide quinone oxidoreductase (Arieli et al., 1994; Schütz et al., 1997; Schütz et al., 1999) and the sulfide cytochrome c oxidoreductase, also called flavocytochrome c or sulfide dehydrogenase (Schneider and Friedrich, 1994; Visser et al., 1997; Sorokin et al., 1998). The putative products of these enzymes are elemental sulfur or sulfate (Schütz et al., 1997; Schneider and Friedrich, 1994; Visser et al., 1997; Sorokin et al., 1998). The gene from a yeast mitochondrial sulfide-oxidizing enzyme has recently been shown to have homology with genes from other prokaryotes and eukaryotes, extending the family of sulfur chemistry enzymes to the eukaryotes (Vande Weghe and Ow, 1999). The sulfide-oxidizing enzyme(s) in G. demissa gills may also belong to this enzyme family, as was suggested for A. marina (Völkel and Grieshaber, 1996). Mitochondrial enzymes such as thiosulfate thiotransferase (rhodanese) and sulfite oxidase may be responsible for thiosulfate production (OBrien and Vetter, 1990).
The soluble, non-toxic thiosulfate may represent a favorable alternative to the other products of sulfide oxidation; in addition, its production requires less oxygen than sulfate production (OBrien and Vetter, 1990; Grieshaber and Völkel, 1998). Sulfite was shown to be an intermediate in the production of thiosulfate in mitochondria from the sulfide-tolerant clam Solemya reidi (OBrien and Vetter, 1990). In this reaction, a single sulfide molecule undergoes a six-electron oxidation to sulfite, and a second sulfide molecule is added with a further two-electron oxidation (OBrien and Vetter, 1990). On the basis of the similar kinetic pattern between thiosulfate and sulfite [compare Fig. 1 in this paper with Fig. 4 in OBrien and Vetter (OBrien and Vetter, 1990)], we suggest that sulfite is also an intermediate in G. demissa gills.
Since thiosulfate is a charged molecule, thiosulfate elimination would require cell and possibly mitochondrial membrane transport mechanisms; however, the nature of these mechanisms is unknown at the present. Whole-animal thiosulfate elimination has been shown to occur by passive diffusion in A. marina (Hauschild et al., 1999) and U. caupo (Julian et al., 1999), crossing the body wall and the hindgut, respectively. The estimated thiosulfate gradient, based on volume average cellular and ambient concentrations, suggests that thiosulfate release does not require active transport. Thiosulfate probably moves through epithelia via paracellular spaces following a concentration gradient in the outward direction (Hauschild et al., 1999; Julian et al., 1999). In G. demissa gills, thiosulfate accumulated for approximately 510 min before release was detectable. Because mussel gill epithelia consist of two cell layers, cellular release of thiosulfate represents an important mechanism for thiosulfate elimination. Direct transport of thiosulfate into the ambient medium is proposed.
Glutathione
Glutathione is a major cellular thiol participating in cellular redox reactions and in the elimination of H2O2 and organic hydroperoxides [for a review, see Sies (Sies, 1999)]. Reduced glutathione is also essential for the activity of sulfur-oxidizing enzyme in several species of thiobacilli [for a review, see Kelly (Kelly, 1999)]. Our results indicate that glutathione levels in G. demissa gills are approximately three times greater than in M. edulis gills (Table 1; Fig. 3, Fig. 5, Fig. 6). Glutathione levels show initial, although non-significant, decreases upon sulfide exposure (Fig. 1), indicating a possible correspondence between glutathione and sulfide oxidation. Although Vismann (Vismann, 1991) states that glutathione does not play an important role in the detoxification of sulfide in the isopod Saduria entomon, we propose that the high glutathione level in G. demissa gills compared with M. edulis gills represents an adaptation to environmental sulfide exposure and, thus, that glutathione may function in sulfide detoxification.
Cysteine
Cysteine is a non-essential amino acid in mammals, synthesized from serine and homocysteine, a breakdown product of methionine; in plants and microorganisms, cysteine is synthesized from serine and sulfide (Cooper, 1983) and may therefore represent a storage form of sulfide. In the gills of both mussel species, cysteine levels show significant increases in response to changing ambient sulfide levels, indicating that cysteine production may be influenced by sulfide exposure.
Sulfide
The initial level of gill tissue sulfide for M. edulis taken from sulfide-free seawater aquaria was 18 µmol kg1 wet mass (Table 1). Values reported for body wall tissue of A. marina under control conditions in the absence of ambient sulfide were 2428 µmol l1, attributed to the presence of mercapto groups of body wall proteins not to free sulfide (Hauschild and Grieshaber, 1997; Wohlgemuth et al., 2000). This may also be the case for mussel gills. In M. edulis gills, tissue sulfide concentration rose to 6080 µmol kg1 wet mass in the presence of ambient Na2S levels up to 200 µmol l1 (Fig. 6). In contrast, the initial level of gill tissue sulfide for G. demissa taken from sulfide tanks was 83 µmol kg1 wet mass (Table 1), and the value remained at 40100 µmol kg1 wet mass at ambient Na2S concentrations up to 1000 µmol l1 (Fig. 5). In the gills of both mussel species, tissue sulfide concentration appears to remain relatively low at the ambient Na2S concentrations tested. However, even these relatively low values may be inhibitory in M. edulis gills, as demonstrated by significant succinate production under these conditions compared with G. demissa gills (see below).
Anaerobic metabolism
In the presence of toxic levels of sulfide, oxygen consumption could become limited as a result of cytochrome c oxidase poisoning (Nicholls, 1975), and animals may exhibit anaerobic metabolism to maintain ATP turnover [for a review, see Grieshaber and Völkel (Grieshaber and Völkel, 1998)]. Two animals, the symbiont-containing clam S. reidi and the symbiont-free lugworm A. marina, produce the anaerobic product succinate under aerated conditions in the presence of sulfide concentrations greater than 250 µmol l1 (Anderson et al., 1990; Völkel and Grieshaber, 1994). We have shown that, in the absence of oxygen, the gills of both mussel species exhibit anaerobic heat dissipation (Doeller et al., 1990; Doeller et al., 1993; Doeller and Kraus, 1992) and a build-up of succinate (Fig. 8). However, under aerated conditions in the presence of 100 µmol l1 Na2S, only the gills of M. edulis produced significant levels of succinate, nearly as high as those seen in anaerobic gills; this is probably evidence of sulfide poisoning of aerobic metabolism. The gills of G. demissa did not produce significant levels of succinate. These data indicate that, in contrast to the gills of M. edulis, the gills of G. demissa do not undergo anaerobic metabolism at 100 µmol l1 Na2S. In fact, succinate levels in G. demissa gill were not significantly greater than those of the control until the gills were exposed to 500 and 1000 µmol l1 Na2S (data not shown).
Sulfide oxidation and energy demand
We have previously proposed that sulfide oxidation in G. demissa gills functions in cellular ATP production (Doeller et al., 1999; Parrino et al., 2000). The evidence includes (i) a turning on and off of gill ciliary beating by sulfide exposure and removal, respectively, in the presence of antimycin A, (ii) a fall in the ratio of gill ciliary beat frequency to oxygen consumption rate to a level that is quantitatively consistent with electrons from sulfide oxidation entering the mitochondrial electron transport chain at the level of cytochrome c, (iii) gill cytochrome c reduction in the presence of sulfide and (iv) sulfide-supported coupled respiration and ATP production by isolated gill mitochondria, with an ADP/O ratio of 1.
A further piece of evidence consistent with the hypothesis that G. demissa gills function in sulfide-supported chemolithoheterotrophy, presented here, is the near unity ratio of oxygen consumed to sulfide oxidized at both low and high 5-HT concentration or ciliary beat frequency (Fig. 4), which demonstrates a coupling between sulfide oxidation and ATP demand in gills and ATP production in mitochondria. We interpret this ratio as follows: when sulfide is oxidized to thiosulfate, eight electrons are released in the oxidation of two sulfide molecules, or four electrons per sulfide (OBrien and Vetter, 1990). If these electrons were to enter the mitochondrial electron transport chain, four electrons would lead to the reduction of two atomic oxygens or one molecular diatomic oxygen to water; therefore, the oxidation of one sulfide molecule would lead to the reduction of one oxygen molecule. Our data show that this ratio is the same at low and high ciliary beat frequency or ATP demand. If sulfide oxidation in G. demissa gills at 100 µmol l1 Na2S functions mainly to detoxify sulfide, then the rate of sulfide oxidation should be the same whether the tissue has a low or high ATP demand. Instead, the sulfide oxidation rate follows ATP demand. Additional evidence suggests that, if terminal oxidases suspected of participation in the sulfide oxidation pathway are inhibited with cyanide or salicylhydroxamic acid (SHAM), the gill exhibits decreases in both oxygen consumption rate and thiosulfate production (D. Kraus and J. Doeller, unpublished observations), as has been shown for A. marina (Völkel and Grieshaber, 1997).
In contrast to the gills of G. demissa, the gills of M. edulis show roughly one-fifth the rates of sulfide oxidation and oxygen consumption in the presence of sulfide. In addition, the difference in oxygen/sulfide ratios between G. demissa gills and M. edulis gills remains over a range of sulfide concentrations (Fig. 7). One interpretation of the high oxygen/sulfide ratios in the gills of M. edulis is that sulfide oxidation results in the production of sulfate or polythiols, which also requires oxygen. However, in most macrofaunal sulfide inhabitants studied thus far, thiosulfate was shown to be the major product of sulfide oxidation (Grieshaber and Völkel, 1998). We interpret the elevated oxygen/sulfide ratio in M. edulis gills as indicating a possible uncoupling of sulfide oxidation from oxygen consumption and energy demand. In contrast, the near unity oxygen/sulfide ratio in G. demissa gills up to 1000 µmol l1 Na2S indicates that electrons from sulfide oxidation probably enter the mitochondrial electron transport chain.
In conclusion, we have shown (i) that thiosulfate is the main product of sulfide oxidation in mussel gills, (ii) that the gills of G. demissa have much higher rates of thiosulfate production and levels of glutathione than the gills of M. edulis and (iii) that sulfide oxidation and mitochondrial oxidative phosphorylation appear to be coupled in G. demissa gills, with no input from anaerobic metabolism, and uncoupled in M. edulis gills. This latter conclusion further supports our hypothesis that G. demissa gills function in sulfide-supported chemolithoheterotrophy, able to use either carbon compounds or sulfide as respiratory substrate. How the gills make the choice between substrates is the subject of ongoing research.
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Acknowledgments |
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