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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
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-
-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
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