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First published online November 30, 2007
Journal of Experimental Biology 210, 4286-4297 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.009969

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Endothelin and endothelin converting enzyme-1 in the fish gill: evolutionary and physiological perspectives

Kelly A. Hyndman^* and David H. Evans

Department of Zoology, University of Florida, 221 Bartram Hall, Gainesville, FL 32608, USA and Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672, USA

View larger version (16K): [in this window] [in a new window]   Fig. 1. Maximum likelihood analyses of the vertebrate preproendothelin amino acid sequences. Following the WAG model of amino acid substitutions (Whelan and Goldman, 2001) and a gamma=1.023, there are three distinct groups of preproendothelins, preproendothelin-1 (abbreviated as only EDN1 to save space), preproendothelin-2 (EDN2) and preproendothelin-3 (EDN3). Numbers at nodes represent the percent bootstrap (BS=500 replications). GenBank accession or Ensembl numbers: Chicken EDN1, XP_418943; chicken EDN2, XP_417707; chicken EDN3, XP_001231488; frog EDN1, AAS13535.1; frog EDN3, AAS13536.1; human EDN1, NP_001946; human EDN2, NP_001947; human EDN3, NP_000105; killifish EDN1A, EU009474; killifish EDN1B, EU009475; medaka EDN1, ENSORLP00000011633; medaka EDN2, ENSORLP00000010557; medaka EDN3, ENSORLP00000011814; mouse EDN1, NP_034234; mouse EDN2, P22389; mouse EDN3, NP_031929; rat EDN1, NP_036680; rat EDN2, NP_036681; rat EDN3, NP_001071118; salmon EDN1, BAF30875.1; Takifugu EDN2, NEWSINFRUP00000182956; Takifugu EDN3, NEWSINFRUP00000181774; Tetraodon EDN3, GSTENT00028275001; Tetraodon EDN2, GSTENT00026224001; Zebrafish EDN1, NP_571594; Zebrafish EDN2, NP_001038650. Scale bar represents the number of replacements per site.

View larger version (52K): [in this window] [in a new window]   Fig. 2. An alignment of vertebrate preproendothelin-1 protein sequences. (A) Similar amino acid residues [based on the BLOSUM 62 score table (Eddy, 2004)] are highlighted in gray, as compared to human preproendothelin-1. The cleavage sites for furin (inverted triangles) and ECE1 () are indicated. Asterisks mark every 20th amino acid. (B) Predicted active EDN1 structure from the killifish, modeled after Webb (Webb, 1997). Amino acids different from human EDN1 are highlighted in gray. Two disulfide bonds are indicated at Cys1–Cys15 and Cys3–Cys10. GenBank and Ensembl accession numbers are listed in Fig. 1, except for platypus EDN1, ENSOANP00000011454 and opossum EDN1, XP_001377153.1.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Maximum likelihood analyses of the ECE family of proteins following the WAG model of amino acid substitutions (Whelan and Goldman, 2001), and a gamma distribution of 1.03. There are three distinct groups of ECE: ECE1, ECE2 and non-vertebrate ECE. Accession numbers: Archaea ECE, NP_616924; Bacteria ECE, NP_812722; chicken ECE1, NP_990048; chicken ECE2, ENSGALP00000010123; frog ECE1, AAH46653; frog ECE2, ENSXETP00000037627; fungus ECE, XP_754379; human ECE1, NP_001388; human ECE2, NP_055508; hydra ECE, AAD46624; killifish ECE1, EU009476; lancelet ECE, 86342 scaffold_150000101; locust ECE, AAN73018; medaka ECE2, ENSORLP00000025753; medaka ECE1, ENSORLP00000021332; mouse ECE1, NP_955011; mouse ECE2, NP_647454; opossum ECE1, ENSMODP00000019967; opossum ECE2, ENSMODP00000002887; platypus ECE1, ENSOANP00000023244; platypus ECE2, ENSOANP00000003016; rat ECE1, NP_446048; rat ECE2, NP_001002815; sea squirt ECE, ENSCSAVP00000016300; sea urchin ECE, XP_798822; stickleback ECE1, ENSGACP00000006069; stickleback ECE2, ENSGACP00000005922; Takifugu ECE1, NEWSINFRUP00000136873; Takifugu ECE2, NEWSINFRUP00000151424; Tetraodon ECE1, GSTENP00006535001; Tetraodon ECE2, CAG02177; zebrafish ECE1, XP_694687. Scale bar represents the number of replacements per site.

View larger version (9K): [in this window] [in a new window]   Fig. 4. Tissue distribution of killifish EDN1A, EDN1B and ECE1 determined by duplexing semi-quantitative PCR with 18S as an internal control.

View larger version (116K): [in this window] [in a new window]   Fig. 5. Representative pictures of in situ hybridization of EDN1A and EDN1B mRNA in lamellar cross-sections of the seawater killifish gill. (A) The EDN1A antisense probe was localized to epithelial cells in the interlamellar region of the gill. Little staining was seen in the gill when the EDN1A sense probe was used (B). (C) The NKA antisense probe was localized to mitochondrion-rich cells. (D) EDN1B antisense probes bound to pillar cells and epithelial cells adjacent to the environment. Little staining was seen with the sense probe (E). (F) A magnification of the pillar cell EDN1B staining. Scale bars, 50 µm.

View larger version (108K): [in this window] [in a new window]   Fig. 6. Representative pictures of killifish lamellar cross-sections, labeled with anti-proendothelin-1 (brown) and anti-NKA (blue). (A,B) Gill sections from a chronic (>30 day) FW acclimated killifish and (C,D) a chronic SW acclimated killifish. (B,D) Sections were incubated in normal goat serum as a negative control for proendothelin immunoreactivity, and doubled labeled with anti-NKA (blue) to illustrate the position of the mitochondrion-rich cells (MRC) in the gill. Proendothelin immunoreactivity was found in a cell adjacent to the NKA immunoreactivity (MRC) and on gill pillar cells in both the FW and SW killifish. The immunoreactivity presented here matches the in situ hybridization of mRNA probes for EDN1s and NKA in Fig. 5. Scale bars, 50 µm.

View larger version (18K): [in this window] [in a new window]   Fig. 7. Acute changes in killifish gill EDN1A, EDN1B and ECE1 mRNA levels as determined by quantitative RT-PCR. FWSW transfers are indicated by solid lines and solid circles and FWFW sham controls are represented by open circles and dotted lines (A,C,E). SWFW transfers are represented by solid lines and solid squares and SWSW sham controls by dotted lines and open squares (B,D,F). N=5 or 6 killifish per time and treatment and values are means ± s.e.m. Note the y axis is logarithmic. All values are normalized to L8 and standardized to chronic SW mRNA levels (see Fig. 9). (A,B) EDN1A mRNA levels, (C,D) EDN1B mRNA levels and (E,F) ECE1 mRNA levels. The asterisks indicate statistical significance (P<0.05) compared to sham value.

View larger version (6K): [in this window] [in a new window]   Fig. 8. Chronic changes in gill EDN1A, EDN1B and ECE1 mRNA levels as measured by quantitative RT-PCR. Killifish (N=6) were transferred from FWSW (SW treatment, black bars) or SWFW (FW treatment, gray bars) and sacrificed 30 days later. Values are means ± s.e.m. Note the y axis is logarithmic. No significant changes in mRNA level were found between the SW and FW killifish for EDN1A, EDN1B or ECE1.

View larger version (21K): [in this window] [in a new window]   Fig. 9. A model of paracrine and autocrine EDN1 signaling in the fish gill. The diagram shows a lamellar cross-section (see Figs 5, 6, 7) with pillar cells (PCs) in gray, pavement cells (PVCs) and a lamellar arteriole (LA) adjacent to the interlamellar region of the gill containing mitochondrion rich-cells (MRCs), a neuroendocrine cell (NEC), and the gill vasculature. Cyclo-oxygenase-2 (COX-2) and neuronal nitric oxide were previously immunolocalized in the killifish gill (Choe et al., 2006; Hyndman et al., 2006). NKA and the cystic fibrosis transmembrane conductance regulator (CFTR) are used as markers for the MRC (Katoh et al., 2001). See text for details.