James B. Claiborne

Gene expression after freshwater transfer in gills and opercular epithelia of killifish: insight into divergent mechanisms of ion transport

SUMMARY We have explored the molecular basis for differences in physiological function between the gills and opercular epithelium of the euryhaline killifish Fundulus heteroclitus. These tissues are functionally similar in seawater, but in freshwater the gills actively absorb Na+ but not Cl–, whereas the opercular epithelium actively absorbs Cl– but not Na+. These differences in freshwater physiology are likely due to differences in absolute levels of gene expression (measured using real-time PCR), as several proteins important for Na+ transport, namely Na+,H+-exchanger 2 (NHE2), carbonic anhydrase 2 (CA2), Na+,HCO3–cotransporter 1, and V-type H+-ATPase, were expressed at 3- to over 30-fold higher absolute levels in the gills. In gills, transfer from 10% seawater to freshwater increased the activity of Na+,K+-ATPase by twofold (from 12 h to 7 days), increased the expression of NHE2 (at 12 h) and CA2 (from 12 h to 7 days), and decreased the expression of NHE3 (from 12 h to 3 days). In opercular epithelium, NHE2 was not expressed; furthermore, Na+,K+-ATPase activity was unchanged after transfer to freshwater, CA2 mRNA levels decreased, and NHE3 levels increased. Consistent with their functional similarities in seawater, killifish gills and opercular epithelium expressed Na+,K+-ATPase α1a, Na+,K+,2Cl–cotransporter 1 (NKCC1), cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel and the signalling protein 14-3-3a at similar absolute levels. Furthermore, NKCC1 and CFTR were suppressed equally in each tissue after freshwater transfer, and 14-3-3a mRNA increased in both. These results provide insight into the mechanisms of ion transport by killifish gills and opercular epithelia, and demonstrate a potential molecular basis for the differences in physiological function between these two organs.

Expression of Na(+) / H(+) exchanger mRNA in the gills of the Atlantic hagfish (Myxine glutinosa) in response to metabolic acidosis.

Sodium/proton exchangers (NHE) are transmembrane proteins that facilitate the exchange of a Na(+) ion for a H(+) ion across cellular membranes. The NHE are present in the gills of fishes and are believed to function in acid-base regulation by driving the extrusion of protons across the branchial epithelium in exchange for Na(+) in the water. In this study, we have used reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the presence of a branchial NHE in the gills of the Atlantic hagfish, Myxine glutinosa. The subsequent partial cDNA sequence shares homology with other vertebrate and invertebrate NHE isoforms. In addition, using semi-quantitative, multiplex RT-PCR we demonstrate that mRNA expression of hagfish gill NHE is upregulated following an induced metabolic acidosis. Expression was increased to 4.4 times basal levels at 2-h post-infusion and had decreased to 1.6 times basal by 6 h. Expression had returned to basal levels by 24-h post-infusion. The inference from this study is that a gill NHE which is potentially important in acid-base regulation has been present in the vertebrate lineage since before the divergence of the hagfishes from the main vertebrate line.

Immunological detection of Na+/H+ exchangers in the gills of a hagfish, Myxine glutinosa, an elasmobranch, Raja erinacea, and a teleost, Fundulus heteroclitus

Na(+)/H(+) exchangers (NHE) are a family of ion exchangers with diverse functions that are well defined in mammals. NHE-1 is expressed in the plasma membrane of most mammalian cells where it regulates intracellular pH, and usually in the basolateral membrane of epithelial cells. It has also been detected in teleost gills where it may participate in systemic pH regulation. NHE-3 is usually expressed in the apical membrane of mammalian epithelial cells where it helps reabsorb Na(+) and HCO(3)(-); it has also been detected in teleost gills. We used Western blotting and heterologous antibodies to screen for expression of NHE-1 and NHE-3 in gills of an agnathan (Myxine glutinosa) and an elasmobranch (Raja erinacea), and NHE-3 in gills of a teleost (Fundulus heteroclitus). Positive NHE-1 bands were detected in gills from the agnathan and elasmobranch. Using the NHE-3 antibody, bands were detected in the gills of the elasmobranch and teleost. These data are some of the first direct evidence of NHEs in the gills of an agnathan and elasmobranch, and confirm the presence of NHEs in the gills of teleosts.

Na + /H + antiporter, V-H + -ATPase and Na + /K + -ATPase immunolocalization in a marine teleost (Myoxocephalus octodecemspinosus)

SUMMARY Long-term pH compensation in a marine teleost requires the transepithelial excretion of H+ across the gill epithelium. H+ efflux in the longhorn sculpin (Myoxocephalus octodecemspinosus) is dependent on external sodium ion concentration and is inhibited by known inhibitors of Na+/H+ exchangers. Our model for proton transport suggests acid-excreting cells in the gill with an apical Na+/H+ antiporter and basolateral Na+/K+-ATPase. This model is similar to mammalian kidney and elasmobranch gill epithelium in which a basolateral electrogenic-vacuolar proton pump (V-H+-ATPase) localizes to base-excreting cells. The objective of this study was to detect the presence and location of membrane transporters in marine fish gills using immunohistochemical staining. Our data indicate the presence of an apical and subapical Na+/H+-exchanger 2 (NHE2) in the sculpin gill. NHE2 is present in large, ovoid chloride cells and often colocalizes in the same cells as Na+/K+-ATPase. We also detected V-H+-ATPase immunoreactivity, predominantly in cells at the base of the lamellae, with staining patterns indicative of a basolateral location. The 85 kDa protein detected on immunoblots with anti-NHE2 antibodies was found in both control and acid-infused animals and did not change following a large acute acidosis over 8 h.

Immunolocalization of Na+/K+-ATPase in mitochondrion-rich cells of the atlantic hagfish (Myxine glutinosa) gill

Using a monoclonal antibody for the alpha-subunit of the Na+/K(+)-ATPase, DASPEI (a vital mitochondria dye), and confocal laser scanning microscopy, the presence of Na+/K(+)-ATPase in mitochondrion-rich cells of the hagfish gill was confirmed. In addition, the level of Na+/K(+)-ATPase expression in the hagfish gill was compared to that of fishes with different osmoregulatory strategies (little skate, Raja erinacea and mummichog, Fundulus heteroclitus). Immunocytochemistry detected a high density of columnar cells expressing Na+/K(+)-ATPase in the afferent filamental epithelium. Positive cells were also found in the lamellar epithelium but at a much lower density. The distribution of DASPEI staining was similar to that of the Na+/K(+)-ATPase antibody, indicating that the enzyme is expressed in mitochondrion-rich cells. Immunoblot analysis confirmed the specificity of the antibody for the 97 kDa alpha-subunit of the enzyme. The immunoreactive band intensity for the Atlantic hagfish was similar to that of the little skate, but less than half that of the full-strength seawater mummichog. These results are discussed in relation to gill function in early craniates.

MECHANISMS OF ACID-BASE EXCRETION ACROSS THE GILLS OF A MARINE FISH

Na+/H+ and Cl–/HCO3–exchanges in the branchial epithelium are thought to be primarily responsible for acid-base transfers in fish. Several different cellular mechanisms have been proposed to drive these exchanges in fresh water and marine species. We measured the acid-base balance and net H+ transfers (ΔH+) in the marine long-horned sculpin (Myoxocephalus octodecimspinosus) following acidosis. ΔH+ was determined in different groups of acid loaded (2–3 meq kg–1) animals which were: 1) adapted to seawater (SW); 2) adapted to 20% SW; 3) exposed to water with artificially low [Na+] or [Cl–]; 4) exposed to water containing 1 × 10–4 M amiloride, 5-(N,N-hexamethylene)-amiloride (HMA), or 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). Both seawater and 20% SW adapted fish were able to completely compensate for the infused load and over 24 hours typically over-excreted more than 2× the amount infused. A 30% decrease in plasma PCO2 following the metabolic acidosis in sculpin adpated to 20% SW (presumably secondary to respiratory alterations) contributed to the rapid recovery of blood pH. Low ambient [Na+] reversed normal acid excretion to an uptake (HCO3– loss; even after acid infusion). 20–30 mM Na+ in the water was necessary to induce a positive ΔH+. A reversible inhibition of ΔH+ was also observed in sculpin exposed to either amiloride or HMA during the acidosis. In contrast, low [Cl–] or DIDS enhanced ΔH+ excretion. We conclude that net H+ excretion measured following acidosis in these seawater or brackish water adapted animals is the sum of parallel (and counter acting) apical gill Na+/H+ and Cl–/HCO3– exchanges. The Na+/H+ transfers are most likely via an antiporter of the NHE family and occur on the background of continued “band-3” Cl–/HCO3– exchange. J. Exp. Zool. 279:509–520, 1997.© 1997 Wiley-Liss, Inc.

Molecular identification of Na(+)-H(+) exchanger isoforms (NHE2) in the gills of the euryhaline teleost Fundulus heteroclitus.

In the current study, reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR were used to clone full-length putative Na(+)-H(+) exchanger isoforms (NHE2a) cDNA from the gills of Fundulus heteroclitus. The 2480 bp cDNA includes a coding region for a protein that shows a 57% amino acid homology to rabbit NHE2. These sequences allowed data mining of available fish genome data, which revealed at least three NHE2 subtypes in some teleost species.