Figure 1.
Histology of the fugu swimbladder.
A, A picture of the ventral wall of the swimbladder of fugu (Takifugu rubripes). The gas gland and rete mirabile exist in the area surrounded by the dotted line. Hematoxylin/eosin-stained images (B–D) are cross-section views cut along a vertical dotted line. B, Paraffin-embedded sections of the ventral wall of the fugu swimbladder were stained with hematoxylin and eosin. C, D, Higher magnification images of the gas gland (C) and rete mirabile (D). RBC, red blood cell.
Figure 2.
Transmission electron microscopy of fugu gas gland and rete mirabile cells.
A, Transmission electron micrograph of a gas gland cell. B, Higher magnification image of the basolateral side of the gas gland cell. C, Transmission electron microscopy of the rete mirabile. D, E, Higher magnification images of the endothelial cells of arterial (D) and venous (E) capillaries in the rete mirabile. RBC, red blood cell; A, arterial capillary; V, venous capillary; EC, endothelial cell; Cav, caveolae-like structure.
Figure 3.
Phylogenetic analyses of fugu MCT and SMCT families.
Phylogenetic trees of SMCT (A) and MCT (B) families were constructed using the maximum likelihood method with ClustalW and MEGA4. Numbers indicate bootstrap values and the scale bar represents a genetic distance of amino acid substitutions per site. Trub, fugu; Tnig, Tetraodon; Gacu, stickleback; Olat, medaka; Drer, zebrafish; Xtro, X. tropicalis; Ggal, chicken; Acar, anole lizard; Mmus, mouse; and Hsap, human.
Figure 4.
Synteny analysis of MCT and SMCT gene families.
Synteny of neighboring genes of MCTs and SMCTs in the genome databases of human (Hsap), chicken (Ggal), X. tropicalis (Xtro), zebrafish (Drer), medaka (Olat), stickleback (Gacu), Tetraodon (Tnig), and fugu (Trub) are shown. chr, chromosome; gr, group; sc, scaffold; and uctg, ultracontig.
Table 1.
Numbers of MCT and SMCT genes in vertebrate genome databases.
Table 2.
Chromosomal localization of MCT and SMCT genes in vertebrate genome databases.
Figure 5.
Expression analyses of fugu MCT and SMCT families.
Tissue-specific expression analyses of fugu MCT and SMCT families determined by semiquantitative RT-PCR.
Figure 6.
In situ hybridization analysis of the distribution of MCT1b and MCT4b mRNAs in the fugu swimbladder.
Paraffin-embedded sections from a fugu swimbladder (specifically, the gas gland and rete mirabile) were stained with digoxin-labeled antisense or sense (control) cRNA probes for MCT1b (A) and MCT4b (B). Higher magnification images are shown to the right.
Figure 7.
Immunohistochemistry of the fugu swimbladder.
Swimbladder sections were stained with anti-MCT1b (A), anti-GAPDH (B), and anti-MCT4b (C) antisera. (D) Control samples stained with nonimmune rabbit serum. The right panels show sections double- or triple-stained with Alexa Fluor 594-labeled concanavalin A (ConA) and Hoechst 33342.
Figure 8.
Subcellular localization of MCT1b and MCT4b in the fugu swimbladder.
A, High-magnification images of immunohistochemical sections of the rete mirabile stained with anti-MCT1b antiserum. The right panels show sections triple-stained with Hoechst 33342 and/or Alexa Fluor 594-labeled ConA and/or fluorescently labeled phalloidin. A, arterial capillary; V, venous capillary. B, Arterial or venous small blood vessels near the rete mirabile were stained with anti-MCT1b antiserum, Hoechst 33342, and fluorescently labeled phalloidin. A, artery; V, vein. C, High-magnification images of immunohistochemical sections of the gas gland stained with anti-MCT4b antiserum. The right panels show sections triple-stained with Alexa Fluor 594-labeled ConA and Hoechst 33342. E, endothelial cell; G, gas gland cell.
Figure 9.
Electroneutral H+/lactate cotransport mediated by fugu MCT1b and MCT4b.
A, Representative traces of intracellular pH (pHi) and membrane potential (Vm) of oocytes injected with MCT1b (red), MCT4b (blue), or water (gray) are shown. The H+/lactate cotransport activities were monitored via pHi changes when 20 mM lactate was added. Gray boxes indicate results during a solution change from 70 mM Cl− ND96 (70-Cl ND96) to 70-Cl ND96 containing 20 mM lactate. Numbers above pHi traces are the initial rates of change in ΔpHi/s. B, Michaelis–Menten curves fitted to lactate-elicited ΔpHi in oocytes expressing MCT1b and MCT4b. ΔpHi was measured by addition of 0, 1, 3.3, 10, or 33 mM lactate in the presence of 70 mM Cl−. Maximum ΔpHi (ΔpHi-max) and the Michaelis–Menten constant (Km) are shown. Values are expressed as means ± SE, n = 3–12. C, Inhibition of MCT1b and MCT4b by cinnamate (α-cyano-4-hydroxycinnamic acid). In the continuous presence of 0, 1, 3.3, and 10 mM cinnamate, the initial rates of change in ΔpHi/s were measured when 10 mM lactate was added. Values are expressed as means ± SE, n = 4–12.
Figure 10.
Roles of MCT1b and MCT4b in the fugu swimbladder.
A, A model of lactic acid transfer mediated by MCT1b and MCT4b. In this model, the presence of MCT1b in tightly sealed arterial capillaries of the rete mirabile allows back-diffusion of lactate from the venous side to the arterial side (box ii), despite the presence of tight junctions that are essential for the efficient delivery of glucose to gas gland cells (box i), by preventing its shunt diffusion directly to the venous side (box iv). The expression of MCT1b and MCT4b in arterial capillaries and gas gland cells was demonstrated in this study. A loose (leaky) association between venous capillary endothelial cells and a tight (impermeable) adhesion between arterial capillary endothelial cells were demonstrated by Wagner [25], [26]. Gas gland cells' glucose requirement and lactic acid secretion have been demonstrated by others [14]. B, A model of the rete mirabile assuming free permeability between arterial and venous capillaries. In the absence of restricted permeability (tight junctions), delivery of glucose to gas gland cells becomes inefficient (box iii).