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Biol. Bull. 209: 11-20. (August 2005)
© 2005 Marine Biological Laboratory

Hydrogen Sulfide Is Synthesized in the Gills of the Clam Mercenaria mercenaria and Acts Seasonally to Modulate Branchial Muscle Contraction

Louis F. Gainey, Jr.1,* and Michael J. Greenberg2

1 Department of Biological Sciences University of Southern Maine, Portland, Maine 04104
2 The Whitney Laboratory of the University of Florida, 9505 Ocean Shore Blvd., St. Augustine, Florida 32086

* To whom correspondence should be addressed. Department of Biological Sciences, University of Southern Maine, PO Box 9300, Portland, ME 04104-9300. E-mail: gainey{at}maine.edu


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Previously we showed that when the gill muscles of the venerid clam Mercenaria mercenaria are stimulated to contract by 5-hydroxytryptamine (5HT), the contraction is about doubled when another identical dose of 5HT is applied after washout. Furthermore, this "endogenous potentiation" is mimicked by nitric oxide (NO), which is synthesized in the gill. We now report that the isolated gills also synthesize H2S; the basal rate of synthesis was 0.70 µmol · g–1 · h–1 (se = 0.14; n = 24), but in the presence of 5HT (10–2 M), the rate increased markedly to 35.82 µmol · g–1 · h–1 (se = 4.93; n = 4). In addition, dithiothreitol (DTT; 2.2 mM) increased the rate of synthesis significantly to 4.9 µmol · g–1 · h–1 (se = 0.8; n = 8). Stimulation of H2S synthesis by 5HT (5 x 10–3 M) was seasonal; that is, the rates measured monthly from December through June are significantly lower than those measured from July through November. We also found that if isolated gills were pretreated with the H2S donor, sodium hydrosulfide (NaHS), their contractions in response to 5HT were potentiated. The threshold of the potentiation was 10–8 M NaHS, and the largest effect was at 10–6 M. During August, however, when endogenous and NO-induced potentiations are both absent, 10–6 M NaHS was also ineffective. Like the effect of NO, that of NaHS (10–6 M) was blocked by oxadiasoloquinoxalin (ODQ; 5 x 10–5 M), an inhibitor of soluble guanylate cyclase (sGC). Moreover, Rp-8-CPT-cGMPS (10–5 M), which inhibits protein kinase-G, also blocked the effect of NaHS (10–6 M). When isolated gills were treated with 2.2 mM DTT, the endogenous potentiation of a second 5HT-induced contraction more than doubled in comparison to untreated controls. In conclusion, H2S is synthesized in the gill and, along with NO, is a seasonal, endogenous modulator of branchial muscle contraction; its action may be mediated through a sGC/cGMP signaling cascade.

Abbreviations: AOAA, aminooxyacetic acid • ANOVA, analysis of variance • ASW, artificial seawater • CBS, cystathionine-ß-synthase • cGMP: 3'-5' cyclic quanosine monophosphate • CO, carbon monoxide • CSE, cystathionine-{gamma}-lyase • DMSO, dimethyl sulfoxide • DTT, dithiothreitol • 5HT, 5-hydroxytryptamine • H2S, hydrogen sulfide • NaHS, sodium hydrosulfide • NO, nitric oxide • NOS, nitric oxide synthase • ODQ, oxadiasoloquinoxalin • p, probability • PAG, propargylglycine • PK-G, protein kinase-G • Rp-8-CPT-cGMPS, (Rp-8-[(4-chlorophenyl)thio]-guanosine 3',5'-cyclic monophosphothioate triethylamine) • sGC, soluble guanylate cyclase • SNP, sodium nitroprusside • YC-1, 3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl indazole


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Literature Cited
 
Most bivalves are filter feeders, so the gills serve dual functions: feeding and respiration. The water current that supports both of these functions is driven by the lateral cilia, which constitute a simple on-off pump. Thus, the graded flow through the passages within the gill is determined by the diameter and shape of those passages, which are controlled by the tone of the branchial muscles. To learn how the actions of the lateral cilia and the muscles are integrated, we have been studying the neural control of these effectors in the gill of the quahog Mercenaria mercenaria (Linnaeus, 1758), a venerid clam (Gainey et al., 1999, Gainey et al., 2003). Recently, we discovered that a contraction of the gill muscles induced by 5HT potentiates subsequent contractions, and that this endogenous potentiation—which occurs seasonally—is mediated by a nitric oxide/cyclic GMP/protein kinase-G pathway (Gainey and Greenberg, 2003). Here we examine the possibility that another known gaseous modulator, hydrogen sulfide (H2S), is synthesized in Mercenaria gills and potentiates contractions induced by 5-hydroxytryptamine (5HT).

The role of H2S as a gaseous modulator has been studied almost exclusively in mammals, and evidence from a variety of mammalian tissues (e.g., blood vessels, airways, gut, and excretory system) indicates that—like nitric oxide (NO)—H2S reduces smooth muscle tone (Abe and Kimura, 1996; Hosoki et al., 1997; Zhao et al., 2001; Kimura, 2002; Teague et al., 2002; Wang, 2002, Wang, 2003; Zhao and Wang, 2002). Although H2S and NO both relax mammalian smooth muscle, their mechanisms of action differ: whereas NO exerts its effects predominantly by stimulating soluble guanylate cyclase (sGC) and increasing the concentration of cGMP (reviewed by Buchwalow et al., 2002), H2S does not seem to stimulate sGC (Abe and Kimura, 1996; Zhao and Wang 2002). Experiments on rat vascular smooth muscle indicated that H2S binds directly to, and opens, a KATP channel (Zhao et al., 2001); yet the KATP channel blocker glibenclamide had no effect upon the H2S-induced relaxation of guinea-pig ileum (Teague et al., 2002). In summary, although H2S has come to be accepted as a gaseous modulator in mammalian nerve and muscle, its varied mechanisms of action are not well understood.

If we look beyond mammals, information is sparser, and the general picture becomes murkier. Recently, Dombkowski et al. (2004, Dombkowski et al. 2005) surveyed the effects of H2S on the vascular smooth muscles of the aortas and branchial or pulmonary arteries from representatives of virtually all the vertebrate classes. The effects were varied and often complex. First, the aortic muscles of the two marine osmoconformers surveyed—the hagfish Eptatretus stouti and the sandbar shark Carcharinus milberti— were relaxed by H2S, whereas those of the sea lamprey (Petromyzon marinus, captured in fresh water) and the air-breathers (toad, alligator, and duck) were contracted. Second, the effects of H2S on the branchial or pulmonary arteries were often triphasic; that is, relaxation, contraction, relaxation in steelhead and rainbow trout, but the opposite (contraction, relaxation, contraction) in toad and duck. Where these responses were monophasic, they were the same as those of the aorta (relaxation in the shark; contraction in the alligator). In summary, the effect of H2S on a restricted set of non-mammalian vertebrate vascular muscles depends on the species, the vascular bed, and possibly the environmental adaptation of the animal studied. As for the invertebrates, Julian et al. (2005) report that strips of circular muscle isolated from the body wall of the fat innkeeper worm Urechis caupo (Echiura), are contracted by H2S. Moreover, the contraction was greatly potentiated when the muscle was treated with both H2S and NO.

Many bivalves live in and are adapted to sediments with high concentrations of H2S, and its effects on bivalves are more complex than in other animals. Bivalves from several families that live in these sediments are known to harbor, within their gills, symbiotic bacteria that rely on H2S as a source of chemical energy, and these animals rely on an array of biochemical, behavioral, and anatomical adaptations to maintain their symbionts (reviewed in Fisher, 1990; Anderson, 1995; Kraus, 1995; Le Pennec et al., 1995; Dufour and Felbeck, 2003; Joyner et al., 2003). For example, various lucinid and thyasirid bivalves "mine" sulfide from the sediments with their feet (Dufour and Felbeck, 2003). In addition, Doeller and co-workers (Doeller, 1995; Doeller et al., 1999, 2001; Parrino et al., 2000) have shown that the gills of the ribbed mussel Geukensia demissa directly utilize H2S as a mitochondrial energy source for electron transport, and that the lateral cilia are stimulated by H2S. But there has been no evidence, until recently, that H2S is actually synthesized by molluscan tissues.

In 2002, however, Julian et al. reported that tissue homogenates of the Manila clam Tapes philippinarum synthesize H2S from L-cysteine. Further, when L-cysteine and the enzyme co-factor pyridoxal-5'-phosphate were added to the reaction mixture, the rate of synthesis by gill homogenate was the highest of the six tissues assayed. H2S synthesis was completely inhibited by aminooxyacetic acid (AOAA), a specific inhibitor of cystathionine-ß-synthase (CBS); moreover, addition of either 2.2 mM 2-mercaptoethanol or dithiothreitol (DTT) stimulated H2S synthesis by 80%. This latter effect was taken as evidence for the presence of an activated L-serine sulfhydrase pathway in which L-cysteine reacts with specific thiols to form H2S and a thioether.

Because the genera Mercenaria and Tapes are both in the family Veneridae, and because the highest rate of H2S synthesis was found in homogenates of Tapes gill, we came readily to the hypothesis that Mercenaria gills would also synthesize H2S. In previous experiments, we demonstrated that 5HT stimulates the Mercenaria gill to synthesize NO, and that the gas, in turn, potentiates 5HT-induced contractions of the branchial muscles (Gainey and Greenberg, 2003). We therefore speculated that H2S might bear similar relationships with 5HT and muscle tone.

To test these notions, we measured the synthesis of H2S by isolated gills of Mercenaria and the stimulation of this synthesis by 5HT. We also used isolated gills in a pharmacological study of the effects of H2S on branchial muscle. In addition, we tested the possibility that H2S might be acting—like NO—through a sGC/cGMP/PK-G signaling cascade. Finally, we asked whether these phenomena would exhibit the persistent seasonality observed with endogenous and NO-induced potentiation (Gainey and Greenberg, 2003). Some of these data were presented in preliminary form to the Society of Integrative and Comparative Biology (Gainey et al., 2000).


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Literature Cited
 
Animals
Specimens of Mercenaria mercenaria (L., 1758) that had been dug from various locations along the northeast Atlantic coast (mostly Cape Cod) were purchased from seafood retailers in Portland, Maine. For most of the year, the clams were held at 10 °C on a 12-h light/dark cycle, but during July, August, and September they were held at room temperature (about 20 °C) in ambient lighting. All animals were held a minimum of 3 days in 30{per thousand} natural seawater from Casco Bay, Maine.

Gill preparation and experimental conditions
Gills were dissected away from the body wall, separated into demibranchs, and the branchial nerves removed. All experiments, except those on endogenous potentiation with dithiothreitol (DTT), were carried out at 10 °C in degassed artificial seawater [ASW; made according to the Marine Biological Laboratory recipe (ASW #1; http://www.mbl.edu/BiologicalBulletin/COMPENDIUM/Comp-ASW.html)].

Measurement of H2S synthesis
The conversion of sulfide into methylene blue was used to determine the rate of H2S synthesis by isolated demibranchs. The reagents were those described by Abe and Kimura (1996), but we modified their procedure since we were measuring H2S synthesis by intact demibranchs, rather than by tissue homogenates. The four demibranchs from single clams were dissected out, and each one was placed into 5 ml of degassed ASW in a 15-ml test tube. With a demibranch in the tube, an air space of 5 to 8 ml remained above the ASW. 5HT was added to the ASW in the tubes containing three of the demibranchs, whereas the fourth demibranch served as an untreated control. Finally, the four test tubes were covered with Parafilm and incubated overnight at 10 °C.

The next morning, the Parafilm covering the tubes containing the highest concentration of 5HT (10–2 M), but not the controls, was bulging outward, presumably due to the evolution of H2S into the air space above the ASW. Moreover, when the Parafilm was removed from these tubes, but not the controls, the distinctive odor of H2S was evident. These observations also indicate that the Parafilm seal was quite tight. After removing the Parafilm, 500 µl of the ASW in each tube was added to 500 µl of 1% zinc acetate. The ASW–zinc acetate solutions were then added to tubes containing reaction mixture (see below), which were then vortexed and allowed to stand for 30 min at room temperature; the absorbance was then read at 670 nm.

The tubes of reaction mixture were made immediately before use. Each contained—in addition to 1.75 ml degassed distilled water—200 µl of 20 mM dimethyl-p-phenylenediamine oxalate in 7.2 M HCl and 200 µl of 30 mM FeCl3 in 1.2 M HCl; both solutions were made in degassed distilled water. The NaHS standards were, however, made in degassed ASW and ranged from 0 to 50 mM. When the assay was finished, the demibranchs were dried overnight at 70 °C, and weighed.

The rate of H2S synthesis, expressed as micromoles per hour per gram of tissue (µmol · h–1 · g–1), was calculated by dividing the amount of sulfide in the tube by the duration of the reaction (about 15 h) and the dry weight of the demibranch. In the experiments with 5HT, rates of H2S synthesis are expressed as the treatment rate minus the control rate. The total rate determined in our experiments was underestimated because of two factors: diffusion of H2S into the air space, and oxidation.

To determine whether sulfide production might have been due to bacteria on the gills, we repeated an experiment at 5 x 10–3 M 5HT, and added chloramphenicol to the overnight incubation at a concentration of 5 mg · l–1—a dose sufficient to kill sulfide-producing bacteria on clam gills (de Zwaan et al., 2001).

Muscle contraction
Four isolated demibranchs were suspended in separate organ baths and attached with thread to isometric force transducers (Grass FT03 and UFI 1030) equipped with light springs. The transducers were connected to Biopac DA 100 amplifiers and a Biopac MP100 analog-to-digital converter. The magnitude of the contractions was measured with AcqKnowledge vers. 3.5 (Biopac Systems).

Preparation of H2S
Before each experiment, a 10–2 M stock solution of NaHS was prepared in degassed ASW. In water, the HS– participates in the following equilibria: H2S {leftrightarrow} H+ + HS– {leftrightarrow} 2H+ + S–2. The value of pK1 is 6.76, at 10 °C and 30{per thousand} seawater (Millero et al., 1988), and the range of values for pK2 is 12–17 (Savenko, 1972). Thus, at the pH of our degassed ASW (7.86), the second dissociation is negligible, while the ratio of HS– to H2S is 1/0.08 at 10 °C and 30{per thousand}. Therefore, although H2S, HS–, and S–2 are all present in solution, we refer to their sum as H2S. In none of the experiments using NaHS were the organ baths aerated, for this would have both degassed and oxidized the H2S. After the addition of any of the compounds tested, we used a Pasteur pipette and bulb, with three cycles of filling and emptying, to mix both treatment and control organ baths.

Experimental designs
Because each clam has two inner and two outer demibranchs, we used a paired comparisons design, with one inner and one outer demibranch serving as untreated controls, to test the effects of various treatments (Gainey and Greenberg, 2003).

External contraction ratio design (Fig. 1a).
Each of the four demibranchs from one clam was suspended in an organ bath and attached to a force transducer. After 15 min of relaxation, one inner and one outer demibranch were exposed, for an appropriate time, to a treatment [i.e., NaHS, ODQ (oxadiasoloquinoxalin), Rp-8-CPT-cGMPS (Rp-8-[(4-chlorophenyl)thio]-guanosine 3',5'-cyclic monophosphothioate triethylamine). ODQ was prepared as a 10–2 M stock solution in dimethyl sulfoxide (DMSO) and kept frozen at –20 °C between experiments; the untreated demibranchs were exposed to an equal volume of DMSO. Rp-8-CPT-cGMPS and 5HT were made as 10–2 M stock in distilled water and kept frozen at –20 °C between experiments; all other solutions were made up fresh in distilled water unless specified. All four demibranchs were then exposed to 2 x 10–5 M 5HT, the concentration we have used for all of our previously published potentiation experiments (Gainey and Greenberg, 2003). For each demibranch, the magnitude of the 5HT-induced contraction was expressed as a percentage of its initial length. External contraction ratios were constructed by dividing the response of each treated inner and outer demibranch by that of its corresponding control demibranch.



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  		Figure 1. Experimental designs used to construct external and internal contraction ratios. External contraction ratios were used to determine if H2S potentiates the effects of 5HT on muscle contraction, and to determine if H2S works via stimulation of sGC and PK-G. One inner or one outer demibranch was exposed to a treatment followed by 5HT, while the other demibranch was exposed only to 5HT. Internal contraction ratios were used to determine if H2S is synthesized fast enough, and in sufficient quantities, to participate in endogenous potentiation. A single demibranch was exposed to 5HT, followed by DTT, and then a second exposure to 5HT. Traces represent contraction of the gill muscles in response to 5-hydroxytryptamine (5HT; up arrows) followed by flushing of the organ bath and relaxation of the muscle (down arrows). The blocks represent the enzymes inferred for the signaling pathway. Enzyme inhibitors = –, stimulators = +. Abbreviations: hc = height of control contraction; ht = height of treatment contraction. sGC = soluble guanylate cyclase; ODQ = a sGC inhibitor; cGMP = cyclic GMP; PK-G = protein kinase G; Rp-8-CPT-cGMPS = a PK-G inhibitor; DTT = a stimulator of H2S synthesis via the activated L-serine sulfhydrase pathway.

 
Internal contraction ratio design (Fig. 1b).
This experimental design was used to test whether the gills synthesize H2S fast enough, and in a sufficient concentration, to potentiate 5HT-induced muscle contraction; these experiments were carried out in aerated ASW, again at 10 °C. After 15 min of relaxation, each of the four demibranchs was exposed to 2 x 10–5 M 5HT. When the resulting contractions had stabilized, typically after 5 min, the baths were flushed, and the demibranchs allowed to relax. One inner and one outer demibranch were exposed to 2.2 mM DTT, while the remaining demibranch of each pair was untreated and served as a control. Thirty minutes after the addition of DTT, an identical dose of 5HT was applied to all of the demibranchs. The results were expressed as the ratio between the second and first 5HT-induced contractions from the same demibranch and are thus internal contraction ratios. In this experimental design, the first exposure to 5HT served as an internal control, while the second exposure to 5HT served to test for potentiation by the gas. Because the contraction ratios of untreated gills are potentiated, that is, greater than one (Gainey and Greenberg, 2003), we compared the contraction ratios of the demibranchs exposed to DTT to those of the untreated controls with paired t tests.

Because neither external nor internal contraction ratios are normally distributed, we used a logarithmic transformation to achieve normality (Gainey et al., 2003). For external contraction ratios, the ln-transformed ratios were tested against a mean of 0 (since ln 1 = 0) with a one-sample t test. This is mathematically equivalent to a paired t test because the contractions used to construct the ratios were from the same clam. Although the statistical tests were performed on the ln-transformed data, tabular and graphical data are presented untransformed in the Results section for the sake of clarity. Depending upon the particular experiment, the P-values reported for these tests are either two- or one-tailed probabilities, and are noted in the results; P values less than 0.05 were considered significant.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Literature Cited
 
H2S synthesis by the gill: dose-dependent stimulation by 5HT
The mean rate of H2S synthesis in untreated demibranchs was 0.70 µmol · g–1 · h–1 (se = 0.14; n = 24), and this basal rate is significantly greater than 0 (one-tailed P < 0.001). When isolated demibranchs were exposed to increasing concentrations of 5HT, there was a dose-dependent increase in the rate of H2S synthesis. The threshold of a detectable response that is significantly greater than that of the untreated controls was at 10–4 M 5HT; the mean rate was 1.33 µmol · g–1 · h–1 (se = 0.25, n = 8). The highest rate of synthesis [35.82 µmol · g–1 · h–1 (se = 4.93, n = 4; Fig. 2)] was at 10–2 M 5HT. This rate is more than 50 times the basal level of H2S synthesis. These assays were done during October and November.



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  		Figure 2. Mean rates of H2S synthesis in response to increasing concentrations of 5HT. Error bars (where visible) are ± 1 se; n = 4 or 8 at each concentration of 5HT; c: rate of H2S synthesis in gills not exposed to 5HT.

 
To determine if bacteria were synthesizing H2S, we exposed the gills from two clams to the antibiotic chloramphenicol and compared the rates of H2S synthesis to those of two other untreated clams. Hydrogen sulfide synthesis in all of the clams was measured in response to 5 x 10–3 M 5HT. The mean rates of H2S synthesis are equal in the two groups (mean chloramphenicol = 5.50 µmol · g–1 · h–1, se = 0.86; mean untreated = 6.87 µmol · g–1 · h–1, se = 0.98; two-tailed P = 0.32; n = 6 for each group).

Modulators of H2S synthesis
Hydrogen sulfide production in response to 5 x 10–3 M 5HT was measured in gills exposed to either 1 mM propargylglycine (PAG), an inhibitor of cystathionine-{gamma}-lyase, or 1 mM aminooxyacetic acid (AOAA), an inhibitor of cystathionine-ß-synthase. The rates of H2S synthesis in gills exposed to either inhibitor are not significantly different from those of the controls (Fig. 3; these data were collected in Jan 2005, which is two years later than and a different month from the data presented in Fig. 2). In contrast, the basal rate of H2S synthesis was stimulated nearly 5-fold by 2.2 mM dithiothreitol (DTT), which stimulates synthesis via the activated L-serine sulfhydrase pathway. This stimulation is statistically significant (one-tailed P = 0.001; Fig. 3).



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  		Figure 3. The effects of putative modulators of H2S synthesis. AOAA [aminooxyacetic acid, an inhibitor of cystathionine-ß-synthase (CBS)]. The effect of 1 mM AOAA on the rate of H2S synthesis stimulated by 5 x 10–3 M 5HT is compared with controls, which were treated only with 5HT (n = 12 for both treatment and control). PAG (propargylglycine, an inhibitor of cystathionine-{gamma}-lyase). The effect of 1mM PAG on the rate of H2S synthesis stimulated by 5 x 10–3 M 5HT is compared with controls, which were treated only with 5HT (n = 6 for both treatment and control). DTT (dithiothreitol, a stimulator of CBS). The effect of 2.2 mM DTT on the basal rate of sulfide synthesis is compared with controls, which were untreated (n = 8 for both treatment and control). Values are means ± 1 se. (*): Statistically different values (treatment vs. control) with a paired t test between pairs of inner and outer demibranchs from the same clam; two-tailed P = 0.002.

 
Monthly H2S synthesis
To test the hypothesis that H2S synthesis in 5HT-treated gills would show a seasonal component, we exposed isolated demibranchs to 5 x 10–3 M 5HT in each month for a year, and measured the rates of H2S synthesis. These rates appear to fall into two groups: a "low" season from December through June, and a "high" season from July through November (Fig. 4). In the low season, the rates of H2S synthesis ranged from 6.18 µmol · g–1 · h–1 (se = 0.65; n = 12) in January to 8.77 µmol · g–1 · h–1 (se = 0.37; n = 6) in April. ANOVA revealed that the monthly rates are all equal during this low season (mean = 7.27 µmol · g–1 · h–1; se = 0.31; n = 46; F (6, 39) = 1.32; P = 0.27). In the high season, the rates of H2S synthesis ranged from 10.28 µmol · g–1 · h–1 (se = 1.25; n = 4) in November to 14.26 µmol · g–1 · h–1 (se = 2.02; n = 6) in August. Again, ANOVA revealed that the monthly rates are equal during this season (mean = 12.43 µmol · g–1 · h–1; se = 0.99; n = 27; F (4, 22) = 0.73; P = 0.58). Finally, comparing the mean seasonal rates of H2S synthesis revealed that the mean rate during the high season is significantly greater than that during the low season (one-tailed P < 0.001; n = 83).



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  		Figure 4. Monthly rates of H2S synthesis in response to 5 x 10–3 M 5HT. Data are means; error bars are ± 1 se; n = 6 except for November (4) and January (12). Closed circles are rates of synthesis in the "low" season; open circles are rates of synthesis in the "high" season.

 
Effect of H2S on muscle contraction
We used the external contraction ratio design (Fig. 1a) to test whether H2S potentiates gill muscle contraction. One inner and one outer demibranch were exposed to NaHS for 5 min; these two treatment demibranchs, together with two control demibranchs, were then exposed to 2 x 10–5 M 5HT. NaHS, by itself, had no effect upon the contractile state of the demibranchs (Fig. 5). At 10–9 M NaHS, the 5HT-induced contractions of the treated and control demibranchs are equal; that is, the mean contraction ratio was 0.95 (se = 0.2; n = 5), which is statistically equal to 1 (two-tailed P = 0.5). At higher concentrations of NaHS, the 5HT-induced contractions of the treated demibranchs were larger than those of the controls; that is, the contraction ratios were greater than 1. The threshold for the response was at 10–8 M NaHS. The external contraction ratios at 10–7 and 10–6 M NaHS are statistically greater than 1 (one-tailed P = 0.043; n = 7 and one-tailed P = 0.01, n = 5, respectively; Fig. 6).



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  		Figure 5. Top: contraction of an outer demibranch in response to 5HT. Bottom: contraction of an outer demibranch from the same clam, again in response to 5HT but after pretreatment with NaHS. The resulting external contraction ratio was 1.7.

 


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  		Figure 6. The effect of an H2S donor (NaHS) on 5HT-induced contraction. Mean external contraction ratios were constructed as follows: the treatment demibranchs (numerator) were exposed to NaHS for 5 min, then to 2 x 10–5 M 5HT; the control demibranchs (denominator) were exposed only to 2 x 10–5 M 5HT. The open circle represents data collected in August, when endogenous potentiation is absent. Error bars are ± 1 se; n = 5–7 at each concentration of NaHS.

 
The experiments described above were performed during February and March, when the gill muscles are potentiated endogenously. In August, when the gill muscles are not potentiated (Gainey and Greenberg, 2003), we repeated the experiment at 10–6 M NaHS. The external contraction ratio is statistically equal to 1 (Fig. 6, open symbol; two-tailed P = 0.533; n = 7), indicating that there was no potentiation by H2S.

Mode of action of H2S
To test whether the effects of H2S on the gill muscle might be due to stimulation of sGC, we exposed one inner and one outer demibranch to various concentrations of the sGC inhibitor ODQ for 55 min, then to 10–6 M NaHS for 5 min. Then all the demibranchs were exposed to 2 x 10–5 M 5HT, and external contraction ratios were constructed. ODQ inhibited the effect of H2S in a dose-dependent manner: the threshold for an effect was at 10–5 M, and the effect of H2S was abolished at 5 x 10–5 M ODQ; that is, the external contraction ratio at 5 x 10–5 M ODQ is equal to 1 (two-tailed P = 0.26; Fig. 7).



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  		Figure 7. The effect of the sGC inhibitor ODQ on H2S-induced potentiation. Mean external contraction ratios were constructed as follows: the treated demibranchs (numerator) were exposed to varying concentrations of ODQ for 55 min and then to 10–6 M NaHS for an additional 5 min. The control demibranchs (denominator) and the treated demibranchs were then exposed to 2 x 10–5 M 5HT. Error bars are ± 1 se; n = 4 at each concentration of ODQ.

 
To confirm our results with ODQ, we tested the effect of the protein kinase G (PK-G) inhibitor Rp-8-CPT-cGMPS. One demibranch was exposed to 10–5 M Rp-8-CPT- cGMPS for 10 min—a concentration and time sufficient to abolish endogenous potentiation (Gainey and Greenberg, 2003). This demibranch and one not exposed to RP-8 were then exposed to 10–6 M NaHS for 5 min. Finally, these two demibranchs, along with an untreated control, were exposed to 2 x 10–5 5HT. Again, external contraction ratios, with the response of the untreated demibranch as the denominator, were calculated. The mean external contraction ratio for the demibranchs exposed only to NaHS and 5HT was 2.45 (se = 0.76; n = 8), while that of the demibranchs exposed to NaHS, Rp-8-CPT-cGMPS, and 5HT was 0.32 (se = 0.13; n = 8); the latter mean is significantly less than the former (one-tailed P < 0.001).

H2S synthesis and endogenous potentiation
Although 5HT stimulates the rate of H2S synthesis, we wondered whether the gas is produced rapidly enough to contribute, along with NO, to endogenous potentiation. Unfortunately, neither of the inhibitors of H2S synthesis that we tested had an effect, but DTT stimulated H2S synthesis. Therefore, we used DTT to test the hypothesis that increased endogenous H2S production would increase the magnitude of endogenous potentiation. We used internal contraction ratios (Fig. 1b) on the same demibranch in response to two exposures of 5HT. One each of the inner and outer demibranchs served as untreated controls, while the others were exposed to 2.2 mM DTT for 30 min between the first and second exposures to 5HT. Data were analyzed with a paired t test between each pair of inner and outer demibranchs. The mean internal contraction ratio of the demibranchs treated with DTT was 3.78 (se = 0.97; n = 22), while that of the untreated controls was 1.49 (se = 0.19; n = 22); the mean of the treated gills is significantly greater than that of the controls (one-tailed P = 0.007).


   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Literature Cited
 
The results reported here show that the gills of Mercenaria mercenaria synthesize H2S at a low basal rate, which is markedly increased by 5HT and DTT, and that the effects of 5HT have a seasonal profile. Moreover, H2S—like NO—potentiates 5HT-induced branchial muscle contraction; this action of the gas is seasonal, and this effect is mediated by a sGC/cGMP/protein kinase-G signaling cascade. Finally, we have established that H2S not only acts very similarly to NO but also participates with NO in the endogenous potentiation of 5HT-induced branchial muscle contraction; that is, NO and H2S seem to be closely coordinated in their effects, mechanism of action, and seasonality.

H2S synthesis
Our finding that isolated demibranchs synthesize H2S at a basal rate significantly greater than zero is central to the notion that the gas is an endogenous modulator of muscle contraction in Mercenaria. This finding is also consistent with the prior work of Julian et al. (2002), which showed that tissue homogenates of the venerid clam Tapes philppinarum synthesize H2S, and that, of the six tissues examined, the gills had the highest rate of synthesis—a rate comparable to those measured in some mammalian tissues (e.g., rat brain, guinea pig smooth muscle).

Our observation of H2S synthesis by the gills—an organ that filters particles from seawater—immediately raises the possibility that the synthesis is due to bacteria. We therefore treated the gills with the antibiotic chloramphenicol at a concentration that was sufficient to suppress bacterial H2S production in an experimental system containing the clam Macoma baltica (de Zwaan et al., 2001). This treatment did not affect the rate of H2S production, suggesting that bacteria were not involved in the synthesis. The result is also consistent with the finding that chloramphenicol does inhibit H2S uptake in Solemya velum, a species that maintains symbiotic bacteria in its gills (Joyner et al., 2003). Thus, we conclude that, in Mercenaria, H2S is most likely synthesized by gill cells.

Our attempt to identify the enzymes that catalyze the synthesis of H2S from L-cysteine in Mercenaria gill was based on the biochemical analyses of Julian et al. (2002), who studied tissue homogenates from Tapes philippinarum, another venerid clam. Julian et al. found that PAG—an inhibitor of cystathionine-{gamma}-lyase (CSE)—had no effect on H2S synthesis, whereas AOAA—an inhibitor of cystathionine-ß-synthase (CBS)—completely inhibited synthesis. In addition, they found that the addition of either 2-mercaptoethanol or DTT stimulated H2S synthesis, and they concluded than an activated L-serine sulfhydrase pathway is also present in Tapes. Based upon the close phylogenetic relationship between Tapes and Mercenaria, our finding that PAG did not block H2S synthesis in Mercenaria gills was to have been expected. AOAA was also ineffective as an inhibitor, but the permeability of cell membranes to AOAA is in question (Hosoki et al., 1997), and the Mercenaria experiments were carried out on whole tissues rather than homogenates. But, like Julian et al., we found that DTT significantly increased the rate of H2S synthesis, implying the existence of an activated L-serine sulfhydrase pathway in Mercenaria.

Seasonality
Synthesis of H2S in response to 5 x 10–3 M 5HT is seasonal; that is, the rates were significantly higher from July through November. These data are perplexing for two reasons. First, endogenous potentiation, though seasonal, is absent during most of this same period. Second, when gills are pretreated, when potentiation is absent, with NaHS (this report), the NO generator DEANO (Gainey and Greenberg, 2003), or the cGMP analog dibutyryl cGMP (Gainey, pers. obs.), branchial muscle contraction is not potentiated. However, our earlier studies showed that, although immunoreactive NOS and sGC are absent from the branchial muscles when potentiation is absent, they are still present in the gill, but confined to the filaments (Gainey and Greenberg, 2003). Recently, moreover, one of us (L. F. G.) has found that both H2S and NO stimulate quiescent lateral cilia to beat, and that the time needed to start the cilia beating is dose dependent. Thus, the higher levels of H2S synthesis from July through November may reflect this activity.

The mechanism of action of H2S
In the experiments described here, the sGC inhibitor ODQ and the protein kinase-G inhibitor Rp-8-CPT-cGMPS blocked the potentiating effect of H2S on the branchial muscles; these data are similar to those found previously for the inhibition of endogenous potentiation (Gainey and Greenberg, 2003), and we tentatively conclude that the effect of H2S on Mercenaria gills is also mediated by the stimulation of sGC. Elsewhere, studies of vertebrate muscle continue to demonstrate that H2S is a gaseous transmitter, but the mechanism of action is unclear, and the data are often contradictory. For example, the response of trout vascular smooth muscle to H2S is triphasic: an initial relaxation (phase 1) is followed by contraction (phase 2), and then by a second relaxation (phase 3) (Dombkowski et al., 2004). These workers found that the sGC inhibitor ODQ stimulated phases 1 and 2, but inhibited phase 3; however, they measured a decrease in cGMP after the muscle was exposed to H2S.

H2S synthesis and endogenous potentiation
We have shown that 5HT and DTT stimulate the rate of H2S synthesis in isolated gills. In addition, our data show that DTT enhances endogenous potentiation of muscle contraction. Taken together, these experiments imply that H2S is synthesized in sufficient concentrations, and quickly enough, to contribute—along with NO—to endogenous potentiation of branchial muscle.

Gaseous signaling, the environment, and seasonality: a speculation
Among terrestrial vertebrates, gaseous signaling occurs internally; that is, NO, H2S, and CO are generated enzymatically and produce their effects near their points of origin (recent reviews include Wang, 2002, Wang, 2003; Boehning and Snyder, 2003; Moore et al., 2003). Environmental exposure of air-breathing species to these gases is atypical, and when exposure does occur, it is usually unplanned, physiologically detrimental, and often lethal. In contrast, clams and other water-breathing organisms are regularly and naturally exposed to NO, H2S, and CO, for these gases occur normally in seawater. But the concentrations and residence times of the three gases are variable because they depend upon temperature, salinity, season, water clarity, and oxygen concentration. In general, the concentrations found in free (i.e., not interstitial) seawater are in the following order: NO ({approx}2 x 10–12 M) < CO ({approx} 80 nM to 22 µM) < H2S ({approx} 80 µM) (NO, McFarland et al., 1979; CO, Conrad et al., 1982; Butler et al., 1987; H2S, Jonas, 1997). In fact, seawater is supersaturated with CO (Schmidt, 1979), and there is a net transfer of CO from the oceans to the atmosphere (Erickson, 1989). These data suggest that CO would be unsuitable as a gaseous signaling molecule in marine animals. Because the environment and its resident water breathing fauna are supersaturated with CO, internal concentrations of CO cannot be increased by enzymatic synthesis. Moreover, sensitivity to CO, typically through the action of sGC, would result in a continuous signal. Not surprisingly, CO has no effect, either direct or modulatory, upon the branchial muscles of Mercenaria, even in the presence of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1; Gainey, pers. obs.), which enhances the effects of CO in mammals (McLaughlin et al., 2000). Finally, there should be a seasonal component to the functioning of NO and H2S as signaling molecules because the concentrations of H2S, and probably NO, are highest during the summer, when oxygen concentrations are lowest (Jonas, 1997). The disappearance of endogenous potentiation during the summer, and the pharmacological inactivity of both NO and H2S at that time, may be an adaptation to seasonal changes in environmental levels of O2, NO, and H2S. Although we don’t know how widespread the phenomenon of muscle potentiation is, we do know that it is present in the gills of two other heterodont bivalves, Mya arenaria and Spisula solidissima. Moreover, potentiation seems to be mediated by NO—at least, in these two species—and it also follows the same seasonal pattern as that in Mercenaria (Gainey and Greenberg, 2004).


   Acknowledgments
 
We thank David Chicoine, John Concannon, Melanie Gainey, and James Walton for their assistance in various phases of this project. Lynn Milstead, at The Whitney Lab, assisted with Figures 1 and 4. Funding was provided to LFG by the University of Southern Maine.


   Footnotes
 
Received 20 April 2005; accepted 21 June 2005.


   Literature Cited
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

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