title: Structural and biochemical correlates of Na+,K+-ATPase driven ion uptake across the posterior gill epithelium of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae).
abstract: To better comprehend the structural and biochemical underpinnings of ion uptake across the gills of true freshwater crabs, we performed an ultrastructural, ultracytochemical and morphometric investigation, and kinetically characterized the Na(+),K(+)-ATPase, in posterior gill lamellae of Dilocarcinus pagei. Ultrastructurally, the lamellar epithelia are markedly asymmetrical: the thick, mushroom-shaped, proximal ionocytes contain elongate mitochondria (41% cell volume) associated with numerous (≈14 µm² membrane per µm³cytoplasm), deep invaginations that house the Na(+),K(+)-ATPase, revealed ultracytochemically. Their apical surface is amplified (7.5 µm² µm⁻²)) by stubby evaginations whose bases adjoin mitochondria below the subcuticular space. The apical membrane of the thin, distal ionocytes shows few evaginations (1.6 µm² µm⁻²), each surrounding a mitochondrion, abundant in the cytoplasm below the subcuticular space; basolateral invaginations and mitochondria are few. Fine basal cytoplasmic bridges project across the hemolymph space, penetrating into the thick ionocytes, suggesting ion movement between the epithelia. Microsomal Na(+),K(+)-ATPase specific activity resembles marine crabs but is ≈5-fold less than in species from fluctuating salinities, and freshwater shrimps, suggesting ion loss compensation by strategies other than Na(+) uptake. Enzyme apparent K(+) affinity attains 14-fold that of marine crabs, emphasizing the relevance of elevated K(+) affinity to the conquest of fresh water. Western blotting and biphasic ouabain inhibition disclose two α-subunit isoforms comprising distinct functional isoenzymes. While enzyme activity is not synergistically stimulated by NH(4) (+) and K(+), each increases affinity for the other, possibly assuring appropriate intracellular K(+) concentrations. These findings reveal specific structural and biochemical adaptations that may have allowed the establishment of the Brachyura in fresh water.
authors.affiliation: Departamento de Química Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brasil. rosapmfi@ffclrp.usp.br
authors.forename: Rosa Prazeres Melo
authors.initials: RP
authors.lastname: Furriel
authors.forename: Kelly Cristina Silva
authors.initials: KC
authors.lastname: Firmino
authors.forename: Douglas Chodi
authors.initials: DC
authors.lastname: Masui
authors.forename: Rogério Oliveira
authors.initials: RO
authors.lastname: Faleiros
authors.forename: Antonio Hernandes
authors.initials: AH
authors.lastname: Torres
authors.forename: John Campbell
authors.initials: JC
authors.lastname: McNamara
mesh_terms: D000818:Animals; D015153:Blotting, Western; D003386:Brachyura / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004848:Epithelium / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D005618:Fresh Water; D005880:Gills / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D017136:Ion Transport; D007527:Isoenzymes; D007700:Kinetics; D046529:Microscopy, Electron, Transmission; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D010042:Ouabain / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase