Na+, K+-ATPase activity in gill microsomes from the blue crab, Callinectes danae, acclimated to low salinity: Novel perspectives on ammonia excretion

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Abstract

This investigation provides an extensive characterization of the modulation by ATP, Mg2+, Na+, K+ and NH4+ of a gill microsomal (Na+,K+)-ATPase from Callinectes danae acclimated to 15‰ salinity. Novel findings are the lack of high-affinity ATP-binding sites and a 10-fold increase in enzyme affinity for K+ modulated by NH4+, discussed regarding NH4+ excretion in benthic marine crabs. The (Na+,K+)-ATPase hydrolyzed ATP at a maximum rate of 298.7 ± 16.7 nmol Pi min 1 mg 1 and K0.5 = 174.2 ± 9.8 mmol L 1, obeying cooperative kinetics (nH = 1.2). Stimulation by sodium (V = 308.9 ± 15.7 nmol Pi min 1 mg 1, K0.5 = 7.8 ± 0.4 mmol L 1), magnesium (299.2 ± 14.1 nmol Pi min 1 mg 1, K0.5 = 767.3 ± 36.1 mmol L 1), potassium (300.6 ± 15.3 nmol Pi min 1 mg 1, K0.5 = 1.6 ± 0.08 mmol L 1) and ammonium (V = 345.1 ± 19.0 nmol Pi min 1 mg 1, K0.5 = 6.0 ± 0.3 mmol L 1) ions showed site–site interactions. Ouabain inhibited (Na+,K+)-ATPase activity with KI = 45.1 ± 2.5 μmol L 1, although affinity for the inhibitor increased (KI = 22.7 ± 1.1 μmol L 1) in 50 mmol L 1 NH4+. Inhibition assays using ouabain plus oligomycin or ethacrynic acid suggest mitochondrial F0F1- and K+-ATPase activities, respectively. Ammonium and potassium ions synergistically stimulated specific activity up to 72%, inferring that these ions bind to different sites on the enzyme molecule, each modulating stimulation by the other.

Introduction

The (Na+, K+)-ATPase (E.C.3.6.1.37), or sodium pump, is a member of the P2C ATPase family. This ion-transporting enzyme couples ATP hydrolysis to the uphill transfer of two K+ ions into, and three Na+ ions out of cells, generating an electrochemical gradient that establishes their resting membrane potential, determines their excitable properties and underlies their osmotic equilibrium. Such ion transport across the plasma membrane involves repeated cycling between at least two phosphorylated or dephosphorylated enzyme conformations: E1, which exhibits high affinity for intracellular Na+, and E2 in which extracellular K+ ions bind with high affinity (for reviews see Kaplan, 2002, Horisberger, 2004, Morth et al., 2007, Pedersen, 2007).

Most marine crustaceans are osmoconformers, essentially isosmotic with their surrounding medium. Usually stenohaline, they show little tolerance of external salinity changes, and exposure to dilute or fluctuating media may be lethal (Péqueux, 1995, Lucu and Towle, 2003, Kirschner, 2004, Tsoi et al., 2005). In contrast, euryhaline crustaceans inhabit media of variable salinity and hyper-regulate their hemolymph osmolality when in dilute media, employing ion uptake mechanisms that involve considerable energy expenditure (Péqueux, 1995, Guerin and Stickle, 1997, Henry et al., 2002, Genovese et al., 2004). In such species, coordinated ion transport across the gills maintains hemolymph osmotic and ionic equilibria and compensates for diffusive and urinary ion losses (Péqueux, 1995, Onken and Riestenpatt, 1998, Onken and Riestenpatt, 2002, Lucu and Towle, 2003, Kirschner, 2004).

Crustacean gills are multifunctional organs, performing respiratory gas exchange, hemolymph acid–base and osmo-ionic regulation, as well as the excretion of nitrogenous metabolites (for reviews see Lucu and Towle, 2003, Weihrauch et al., 2004, Tresguerres et al., 2008, Freire et al., 2008). Covered by a thin cuticle, the gill epithelium constitutes a selective interface between the animal's extracellular space and the environment, and across which Na+ and Cl are actively absorbed from dilute media. Both weak and strong hyperosmoregulating crustaceans possess a common set of ion transporters, including the (Na+, K+)-ATPase and K+ and Cl channels located in the basolateral epithelial cell membranes. However, while Na+ channels, and a V-type proton pump and Cl/HCO3 antiporter furnished with H+ and HCO3 via the cytoplasmic carbonic anhydrase, characterize the apical membranes of strong hyperosmoregulators, weak hyperosmoregulators possess an apical Na+/K+/2Cl symporter, Na+/H+ and Cl/HCO3 antiporters and K+ channels (see reviews by Kirschner, 2004, Freire et al., 2008). The basolateral (Na+, K+)-ATPase constitutes a key enzyme in ion uptake in both cases (Péqueux, 1995, Furriel et al., 2000, Lucu and Towle, 2003, Kirschner, 2004, Lovett et al., 2006, Jayasundara et al., 2007) and there is an apparent relationship between enzyme activity, hyperosmoregulation and ammonia excretion in many species (Péqueux, 1995, Lucu and Towle, 2003, Kirschner, 2004, Weihrauch et al., 2004, Leone et al., 2005b, Masui et al., 2005, Freire et al., 2008). However, while ammonia excretion rates correlate with Na+ absorption in C. sapidus (Pressley et al., 1981), C. maenas (Lucu et al., 1989) and E. sinensis (Péqueux, 1995), a direct relationship between active ion uptake and ammonia excretion remains tenuous.

Aquatic crustaceans are ammoniotelic, excreting their nitrogenous metabolites across the gill epithelium largely as ammonia (NH3 + NH4+), which at physiological pH, is mostly in the ionic form (Weihrauch et al., 1998, Weihrauch et al., 1999, Weihrauch et al., 2004). The demonstration that NH4+ can substitute for K+ in the gill (Na+,K+)-ATPase initially suggested a role in active NH4+ transport into the gill cell cytoplasm from the hemolymph (Weihrauch et al., 2004, Weiner and Hamm, 2007). However, NH4+ and K+ synergistically stimulate the Callinectes danae gill (Na+, K+)-ATPase in a manner regulated by Mg2+, suggesting additional NH4+-binding sites on the enzyme, which may guarantee the outwardly directed, active transport of ammonia even at usual hemolymph K+ concentrations (Masui et al., 2002, Masui et al., 2005). These findings corroborate current models of ammonia transport across the crustacean gill epithelium (reviewed by Weihrauch et al., 2004, Freire et al., 2008) and open new perspectives on ammonia excretion.

Callinectes danae Smith 1869, is a euryhaline, benthic brachyuran crab widely distributed in the Western Atlantic from Florida to southern Brazil (Melo, 1996, Chacur and Negreiros-Fransozo, 2001). In Ubatuba Bay, Brazil, C. danae is often found in seawater affected by freshwater runoff (Mantelatto and Fransozo, 1999, Mantelatto and Fransozo, 2000) and is exposed to variable salinities; efficient mechanisms of hyperosmoregulation are vital for survival. Since C. danae characteristically buries itself in bottom sediments, local ammonia concentration may also increase, reducing passive efflux and even leading to ammonia influx across the gill epithelium (Weihrauch et al., 1999). Since both osmoregulation and ammonia excretion in aquatic brachyurans occur mainly across the gill epithelium (Péqueux, 1995, Weihrauch et al., 1999), we have employed C. danae as a model to characterize the crab gill (Na+,K+)-ATPase (Masui et al., 2002). In fresh-caught, unacclimated C. danae, total gill microsomal ATPase activity is predominantly (Na+,K+)-ATPase activity. The enzyme exhibits two distinct sites for ATP hydrolysis and is synergistically stimulated by K+ and NH4+ (Masui et al., 2002).

The present study provides a kinetic characterization of the (Na+, K+)-ATPase expressed in the gill tissue of C. danae acclimated to dilute seawater of 15‰ salinity. Our data confirm the synergistic stimulation of the enzyme by K+ and NH4+ but, in contrast to fresh-caught, unacclimated crabs (Masui et al., 2002), modulation of enzyme activity by NH4+ increases K+ affinity 10-fold. These findings provide a better understanding of the biochemical mechanisms underlying ammonia excretion in crustaceans.

Section snippets

Material

All solutions were prepared using Millipore MilliQ ultrapure, apyrogenic water and all reagents were of the highest purity commercially available. Imidazole, pyruvate kinase (PK), phosphoenolpyruvate (PEP), NAD+, NADH, N-(2-hydroxyethyl) piperazine-N′-ethanesulfonic acid (Hepes), lactate dehydrogenase (LDH), ouabain, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate (G3P), alamethicin, 3-phosphoglyceraldehyde diethyl acetal, ATP ditris

Results

A maximum ATPase activity of V = 298.8 ± 16.7 nmol Pi min 1 mg 1 was obtained for gill microsomes from C. danae acclimated to 15‰ salinity. The residual ouabain-insensitive ATPase activity (V = 29.9 ± 1.7 nmol Pi min 1 mg 1), corresponding to about 10% of total ATPase activity, suggests the presence of hydrolyzing ATPases other than the (Na+, K+)-ATPase. The continuous sucrose gradient centrifugation analysis of the microsomal fraction showed a single protein peak coincident with the ATPase activities (

Discussion

In this investigation, we provide an extensive kinetic characterization of the gill (Na+, K+)-ATPase from C. danae acclimated to a dilute medium of 15‰ salinity. Novel findings are the lack of high-affinity ATP binding sites on the (Na+, K+)-ATPase, and a 10-fold increase in enzyme apparent affinity for K+, in the presence of NH4+. Comprehending just how NH4+ modulates the apparent affinity of the enzyme for K+ may aid our understanding of the relationship between hyperosmoregulation and

Acknowledgements

This work constitutes part of a Ph.D. thesis by D.C.M. and was supported by research grants from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). F.A.L., J.C.M. and F.L.M.M. received research scholarships from CNPq. D.C.M. thanks FAPESP for a doctoral scholarship. All experiments conducted in this study comply with currently applicable state and federal laws. We thank Nilton Rosa Alves for technical

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