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Figures
- Fig. 1.
Schematic drawing of the position (A,B) and structure (C) of the ventral photophores of the hatchetfish Argyropelecus hemigymnus. A common chamber for the photocytes (p) is surrounded by a reflector (r) (B,C). There are several exits for light from the photocyte chamber. The exits contain pigmented filter (f) cells and, more peripherally, unpigmented transparent tissue called the gelatinous body (gb; C) (Bassot, 1966; Denton et al., 1985). Arrows indicate the main entrance points for nerve fibres through the reflector to the photocyte chamber found in the present study (C).
- Table 1.
Antibodies and peptides used for immunohistochemistry
Antibody/peptide Host Working dilution Antigen (peptide sequence) Source Primary eNOS N30030 Rabbit 1:500 Human eNOS c-terminal (1030-1209) Transduction Laboratories iNOS N32030 Rabbit 1:1000 Mouse iNOS c-terminal (961-1144) Transduction Laboratories nNOS N31030 Rabbit 1:100 Human nNOS c-terminal (1095-1289) Transduction Laboratories nNOS SC1025 Rabbit 1:150 Human/mouse nNOS n-terminal Santa Cruz Biotechnology uNOS PA1-039 Rabbit 1:100 Murine i and nNOS c-terminal (1113-1122) Affinity Bioreagents Tubulin acetylated T-6793 Mouse 1:1000 Sea urchin acetylated tubulin Sigma Secondary DaM-biotin 715-065-151 Donkey 1:100 Jackson ImmunoResearch Laboratories DaM-FITC 715-095-150 Donkey 1:100 Jackson ImmunoResearch Laboratories DaR-biotin 711-065-152 Donkey 1:400 Jackson ImmunoResearch Laboratories DaR-Cy3 711-165-152 Donkey 1:800 Jackson ImmunoResearch Laboratories DaR-FITC 711-095-152 Donkey 1:100 Jackson ImmunoResearch Laboratories Block peptide NOS1 (K-20) P sc-1025P 1:30 Santa Cruz Biotechnology -
All antibodies against NOS are polyclonal. The antibody against acetylated tubulin is monoclonal.
DaM, donkey anti-mouse; DaR, donkey anti-rabbit; eNOS, enothelial nitric oxide synthase; iNOS, inducible NOS; nNOS, neuronal NOS; uNOS, universal NOS, i.e. antibody reacting to all NOS isoforms.
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- Fig. 2.
Control experiments on sections of a ventral photophore from the hatchetfish Argyropelecus hemigymnus, showing (A) lack of unspecific staining after omission of primary antibody, and specific staining among the photocytes and in the filter cells, with (B) nNOS 31030 and (C) nNOS sc1025. f, filter cell area; p, photocytes. Bars, 50 μm.
- Fig. 3.
Control experiments on sections of a ventral photophore from the hatchetfish, Argyropelecus hemigymnus. Preabsorption using antigen (block peptide, Table 1) and the antibody nNOS sc1025 (A and B) resulted in quenching of the immunoreaction both in the filter cells (compare A and C) and among the photocytes (compare B and D). f, filter cell area; p, photocytes; r, reflector. Bars, 50 μm.
- Fig. 4.
Sections of ventral photophores from the hatchetfish, Argyropelecus hemigymnus showing acetylated tubulin-like (A–C; AcT-LI IR, green fluorescence) and nitric oxide synthase-like immunoreactivity (D–H; NOS-LI IR, brownish precipitate). Blue fluorescence in A–C is autofluorescence from the photocytes. (A) AcT-LI IR nerve fibres in the filter area, and among the photocytes. (B) Nerve bundle (arrow) entering the photocyte chamber through the reflector. (C) Nerves branching among the photocytes from a common point (arrow) at the median narrowing of the photophore. (D) NOS-LI IR (nNOS sc1025) in the cytoplasm of the outer filter cells (arrows). (E) NOS-LI IR (nNOS sc1025) in nerve bundle (arrows) leading to photophore. (F,G) Varicose nerve fibres (arrows) among photocytes showing NOS-LI IR (F, nNOS sc1025; G, nNOS 31030). (H) Intra- or extra-cellular structures with NOS-LI IR (arrows; nNOS 31030) at the cell membrane of a photocyte. f, filter cell area; p, photocytes; r, reflector. Bars, 50μ m.
- Fig. 5.
SNP (circles) and SNAP (triangles) at 10–3 mol l–1 in 22 cases out of 34 decreased the adrenaline-induced luminescence of photophores. Insert, blow-up showing comparison of the relationship ΔLmax–Lmax for lower Lmax values. (The 12 instances where SNP or SNAP increased the adrenaline-induced luminescence are reported in the text.) The ordinate shows ΔLmax (differences between pairs of treated and control photophores in Mq s–1); the abscissa, Lmax (Mq s–1) of the control photophores.
- Table 2.
Mean values of adrenergic light response parameters after treatments with NO donors as compared to control (Δ)
SNP (N=21) SNAP (N=13) ΔTLmax (min) 5.47±3.55 3.07±7.29 ΔTL1/2 (min) -0.97±7.30 -19.23±4.60*** -
SNAP, S-nitroso-N-acetylpenicillamine; SNP, sodium nitroprusside; TLmax, time to maximal light emission; TL1/2, time from maximal to half extinction of light emission.
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↵*** P<0.001.
Adrenaline controls: TLmax=36.40±3.9 min; TL1/2=43.78±6.27 min (N=21).
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- Fig. 6.
(A) Comparison of the maximal amplitude of adrenergic response of 8 pairs of control and hydroxylamine (10–3 mol l–1) treated photophores. (B) Extinction time course for luminescence from adrenaline stimulated photophores. On addition of hydroxylamine (H, arrow), treated photophores show an acceleration of light extinction as compared with control photophores.
- Fig. 7.
(A) Lmax differences (ΔLmax) between pairs of l-thiocitrulline treated and control photophores plotted in function of Lmax (Mq s–1) of control photophores. (B) Relationship between rates of light production (TLmax) and light extinction (TL1/2, min) in l-thiocitrulline treated photophores.
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