Nitric oxide (NO) modulates epithelial ion transport pathways in mammals, but this remains largely unexamined in fish. We explored the involvement of NO in controlling NaCl secretion by the opercular epithelium of seawater killifish using an Ussing chamber approach. Pharmacological agents were used to explore the mechanism(s) triggering NO action. A modified Biotin-switch technique was used to investigate S-nitrosation of proteins. Stimulation of endogenous NO production via the nitric oxide synthase (NOS) substrate l-arginine (2.0 mmol l−1), and addition of exogenous NO via the NO donor SNAP (10−6 to 10−4 mol l−1), decreased the epithelial short-circuit current (Isc). Inhibition of endogenous NO production by the NOS inhibitor l-NAME (10−4 mol l−1) increased Isc and revealed a tonic control of ion transport by NO in unstimulated opercular epithelia. The NO scavenger PTIO (10−5 mol l−1) supressed the NO-mediated decrease in Isc, and confirmed that the effect observed was elicited by release of NO. The effect of SNAP on Isc was abolished by inhibitors of the soluble guanylyl cyclase (sGC), ODQ (10−6 mol l−1) and Methylene Blue (10−4 mol l−1), revealing NO signalling via the sGC/cGMP pathway. Incubation of opercular epithelium and gill tissues with SNAP (10−4 mol l−1) led to S-nitrosation of proteins, including Na+/K+-ATPase. Blocking of NOS with l-NAME (10−6 mol l−1) or scavenging of NO with PTIO during hypotonic shock suggested an involvement of NO in the hypotonic-mediated decrease in Isc. Yohimbine (10−4 mol l−1), an inhibitor of α2-adrenoceptors, did not block NO effects, suggesting that NO is not involved in the α-adrenergic control of NaCl secretion.
The authors declare no competing or financial interests.
F.B.J. and S.S.M. conceived the study; L.G., F.B.J., S.S.M. and W.S.M. designed the experiments; L.G. and W.S.M. performed the experiments; L.G. analysed the data and drafted the manuscript; F.B.J., S.S.M. and W.S.M. revised the manuscript.
This work was supported by the Det Frie Forskningsråd | Natur og Univers (F.B.J. and S.S.M.) and a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (W.S.M.).
- Received June 21, 2016.
- Accepted August 22, 2016.
- © 2016. Published by The Company of Biologists Ltd