John C. McNamara

Characterization of (Na+, K+)-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii

To better understand the adaptive strategies that led to freshwater invasion by hyper-regulating Crustacea, we prepared a microsomal (Na+, K+)-ATPase by differential centrifugation of a gill homogenate from the freshwater shrimp Macrobrachium olfersii. Sucrose gradient centrifugation revealed a light fraction containing most of the (Na+, K+)-ATPase activity, contaminated with other ATPases, and a heavy fraction containing negligible (Na+, K+)-ATPase activity. Western blotting showed that M. olfersii gill contains a single alpha-subunit isoform of about 110 kDa. The (Na+, K+)-ATPase hydrolyzed ATP with Michaelis Menten kinetics with K5, = 165+/-5 microM and Vmax = 686.1+/-24.7 U mg(-1). Stimulation by potassium (K0.5 = 2.4+/-0.1 mM) and magnesium ions (K0.5 = 0.76+/-0.03 mM) also obeyed Michaelis-Menten kinetics, while that by sodium ions (K0.5 = 6.0+/-0.2 mM) exhibited site site interactions (n = 1.6). Ouabain (K0.5 = 61.6+/-2.8 microM) and vanadate (K0.5 = 3.2+/-0.1 microM) inhibited up to 70% of the total ATPase activity, while thapsigargin and ethacrynic acid did not affect activity. The remaining 30% activity was inhibited by oligomycin, sodium azide and bafilomycin A. These data suggest that the (Na+, K+)-ATPase corresponds to about 70% of the total ATPase activity; the remaining 30%, i.e. the ouabain-insensitive ATPase activity, apparently correspond to F0F1- and V-ATPases, but not Ca-stimulated and Na- or K-stimulated ATPases. The data confirm the recent invasion of the freshwater biotope by M. olfersii and suggest that (Na+, K+)-ATPase activity may be regulated by the Na+ concentration of the external medium.

Modulation by ammonium ions of gill microsomal (Na+,K+)-ATPase in the swimming crab Callinectes danae: a possible mechanism for regulation of ammonia excretion.

The modulation by Na(+), K(+), NH(4)(+) and ATP of the (Na(+),K(+))-ATPase in a microsomal fraction from Callinectes danae gills was analyzed. ATP was hydrolyzed at high-affinity binding sites at a maximal rate of V=35.4+/-2.1 Umg(-1) and K(0.5)=54.0+/-3.6 nM, obeying cooperative kinetics (n(H)=3.6). At low-affinity sites, the enzyme hydrolyzed ATP obeying Michaelis-Menten kinetics with K(M)=55.0+/-3.0 microM and V=271.5+/-17.2 Umg(-1). This is the first demonstration of a crustacean (Na(+),K(+))-ATPase with two ATP hydrolyzing sites. Stimulation by sodium (K(0.5)=5.80+/-0.30 mM), magnesium (K(0.5)=0.48+/-0.02 mM) and potassium ions (K(0.5)=1.61+/-0.06 mM) exhibited site-site interactions, while that by ammonium ions obeyed Michaelis-Menten kinetics (K(M)=4.61+/-0.27 mM). Ouabain (K(I)=147.2+/-7.microM) and orthovanadate (K(I)=11.2+/-0.6 microM) completely inhibited ATPase activity, indicating the absence of contaminating ATPase and/or neutral phosphatase activities. Ammonium and potassium ions synergistically stimulated the enzyme, increasing specific activities up to 90%, suggesting that these ions bind to different sites on the molecule. The presence of each ion modulates enzyme stimulation by the other. The modulation of (Na(+),K(+))-ATPase activity by ammonium ions, and the excretion of NH(4)(+) in benthic crabs are discussed.

Osmoregulation and respiratory metabolism in brazilian Macrobrachium (Decapoda, palaemonidae)

Abstract 1. 1. The effect of salinity (0, 7, 14, 21, 28 and 35‰) on osmoregulatory capability and metabolic rate in adult Macrobrachium acanthurus, M. heterochirus, M. olfersii and M. potiuna , was investigated at 20°C using a microcryoscope and Warburg respirometer. 2. 2. All species are strong hyperosomotic regulators in freshwater or low salinities (0–14‰) while at high salinities (21–35‰), with the exception of M. olfersii , they are hypoconformers; M. olfersii exhibits a fair degree of hypo-osmotic regulatory capability. 3. 3. Isosmotic points decrease in the sequence M. heterochirus > M. acanthurus > M. olfersii > M. potiuna . 4. 4. For M. acanthurus and M. olfersii , the metabolism-salinity curves assume a dome shape with a high peak at 21‰ S, close to the isosmotic point. For M. heterochirus and M. potiuna , metabolic rates tended to decline with salinity increase. 5. 5. These results are discussed in relation to the distribution patterns of the adult shrimps and to physiological modifications occurring during development.

Ion-motive ATPases and active, transbranchial NaCl uptake in the red freshwater crab, Dilocarcinus pagei (Decapoda, Trichodactylidae)

SUMMARY The present investigation examined the microanatomy and mRNA expression and activity of ion-motive ATPases, in anterior and posterior gills of a South American, true freshwater crab, Dilocarcinus pagei. Like diadromous crabs, the anterior gills of this hololimnetic trichodactylid exhibit a highly attenuated (2–5 μm), symmetrical epithelium on both lamellar surfaces. In sharp contrast, the posterior gill lamellar epithelia are markedly asymmetrical. Their proximal side consists of thick (18–20μ m) cells, displaying features typical of a transporting epithelium, while the distal epithelium is thin (3–10 μm) and formed entirely by apical pillar cell flanges. Both anterior and posterior gills express Na+/K+- and V-ATPases. Phylogenetic analysis of partial cDNA sequences for the Na+/K+-ATPase α-subunit and V-ATPase B-subunit among various crab species confirmed the previous classification and grouping of D. pagei based on morphological criteria. Semi-quantitative RT-PCR clearly showed that mRNA for both ion pump subunits is more intensely expressed in posterior gills. Na+/K+-ATPase activity in the posterior gills was nearly fourfold that of anterior gills, while V-ATPase and F-ATPase activities did not differ. A negative short-circuit current (Isc) was measured using the distal side of split, posterior gill lamellae, mounted in a modified Ussing chamber and perfused symmetrically with identical hemolymph-like salines. Although hemolymph-side ouabain did not affect this current, concanamycin significantly reduced Isc without altering preparation conductance, suggesting V-ATPase-driven Cl– absorption on the distal side of the posterior gill lamellae, as known to occur in diadromous crabs adapted to freshwater. These findings suggest that active Na+ uptake predominates across the thick proximal epithelium, and Cl– uptake across the thin, distal epithelium of the posterior gill lamellae.

Regulation of hemolymph osmolytes and gill Na+/K+-ATPase activities during acclimation to saline media in the freshwater shrimp Macrobrachium olfersii (Wiegmann, 1836) (Decapoda, Palaemonidae)

To evaluate the mechanisms of long-term osmotic adaptation to saline media, total hemolymph osmolytes, hemolymph sodium and chloride concentrations, and gill Na+/K+-ATPase activities were measured in the freshwater shrimp Macrobrachium olfersii (Wiegman) after acclimation for 10 or 20 days to media of <0.5, 21 and 28‰ salinity. Total hemolymph osmolytes are maintained strongly hyperosmotic to the external medium in low and moderate salinities, becoming slightly hyperosmotic at high salinity. In contrast, however, hemolymph [Na+] and [Cl−] are distinctly hyporegulated at the higher salinities. This difference in response pattern to acclimation apparently results from the presence of free amino acids in the hemolymph as a consequence of the synthesis of intracellular organic osmolytes. The resulting hyperosmotic regulation both avoids water loss from the animal in saline media and provides an osmotic gradient, allowing the uptake of water from the medium for excretion of the salt load. Gill Na+/K+-ATPase activities decrease by ≈35% after acclimation to saline media, suggesting that the rates of the cellular mechanisms responsible for salt uptake in freshwater become reduced, avoiding excessive salt loading. A Na+-ATPase activity is salinity independent. These data are examined with regard to the intrinsic coupling between the physiological alterations taking place in hemolymph osmolytes during acclimation to saline media and the concomitant ultrastructural rearrangements in the salt transporting tissues of the gills, and their possible neurosecretory control mechanisms.

THE ONTOGENY OF ISOSMOTIC INTRACELLULAR REGULATION IN THE DIADROMOUS, FRESHWATER PALAEMONID SHRIMPS, MACROBRACHIUM AMAZONICUM AND M. OLFERSI (DECAPODA)

Abstract To elucidate the osmoregulatory mechanisms underpinning the invasion of fresh water by the palaemonid Crustacea, we investigate the contribution of free amino acids (FAA) to intracellular isosmotic regulation in selected ontogenetic stages of two diadromous, neotropical shrimps, Macrobrachium amazonicum and M. olfersi, exposed to fresh water or to saline media. We also evaluate anisosmotic/ionic extracellular regulatory capability in adult M. amazonicum alone; all data for adult M. olfersi are from McNamara et al. (2004). Adult shrimps show similar osmotic and ionic regulatory capabilities, including elevated hemolymph osmolality in fresh water, moderate isosmotic points, hyper-regulatory capability up to 20‰, and good tolerance of saline media. However, the two species rely on brackish water to different degrees to complete their life cycles: while M. olfersi zoeae 1 and 2 survive well in fresh water, those of M. amazonicum die within two hours. Total FAA titers increase significantly over the ontogenetic sequence in both species, independently of salinity exposure, concentrations increasing sharply in M. amazonicum zoeae 1 alone, but steadily from embryos to adult M. olfersi. While total FAA titers increase significantly on transfer of zoeae 1 (+ 43%) and adult (muscle + 72%, gill + 62%) M. amazonicum to elevated salinity (25‰), their effective contribution to hemolymph and intracellular osmolality is unaltered (≈16% in zoea 1, 6-8% in zoea 2 and adult tissues). Total FAA titers in M. olfersi increase in embryos (+ 95%), zoeae 1 (+ 23%) and post larvae (+ 28%), and in adult tissues (muscle + 69%, gill + 110%, nerve + 187%) after salinity exposure. However, effective contribution to intracellular osmolality increases only in embryos (5 to 6%) and adult nervous tissue (6 to 13%). In both species, total FAA increase is due to the most abundant non-essential FAA, glycine, alanine and proline, and arginine. Our analysis shows that diadromous species like M. amazonicum and M. olfersi exhibit lower total FAA titers compared to marine species. Such findings allow a better understanding of the physiological mechanisms underlying the invasion of fresh water by these recent colonizers.

Ultracytochemical location of Na+/K+-atpase activity and effect of high salinity acclimation in gill and renal epithelia of the freshwater shrimp Macrobrachium olfersii (Crustacea, Decapoda)

Accumulation sites of lead phosphate reaction product consequent to Na(+)/K(+)-ATPase activity in gill and renal epithelia of the freshwater shrimp Macrobrachium olfersii were located ultracytochemically by para-nitrophenyl-phosphate hydrolysis and lead precipitation, and quantified per unit membrane area and cytoplasmic volume. In shrimps in freshwater (<0.5 per thousand S, 20 mOsm/kg H(2)O, 0.7 mEq Na(+)/liter), numerous sites of electron-dense, Na(+)/K(+)-ATPase reaction product accumulation were demonstrated in the membrane invaginations of the mitochondria-rich, intralamellar septal cells (12.5 +/- 1.7 sites/microm(2) membrane, 179 +/- 22 sites/microm(3) cytoplasm, mean+/- SEM, N </= 7) and in the basal region of the medial renal tubules (19.8 +/- 1.8 sites/microm(2) membrane, 437 +/- 53 sites/microm(3) cytoplasm), but not in the pillar cells whose apical flanges form the primary interface with the external medium. A putative, ouabain-insensitive Na(+)- or H(+)-ATPase was found in the apical microvilli of the medial renal tubules (17.4 +/- 1.7 sites/microm(2) membrane, 629 +/- 101 sites/microm(3) cytoplasm). This restricted location of Na(+)/K(+)-ATPase activity within the gill epithelium suggests that during uptake, Na(+) moves across the apical pillar cell membrane, passes through specialized, basolateral coupling junctions into the septal cell cytoplasm and is pumped into the hemolymph via the Na(+)/K(+)-ATPase in the invagination membranes. In shrimps acclimated to a high-salinity medium (21 per thousand S, 630 mOsm/kg H(2)O, 280 mEq Na(+)/liter) for 2 and 5 days, the mean number of sites of para-nitrophenylphosphatase activity/microm(2) membrane and /microm(3) cytoplasm for both epithelia increases markedly by 83 and 163%, respectively. However, after 10 days acclimation, the number of sites declines dramatically, attaining values far below those for shrimps in freshwater. These acclimation-induced alterations in numerical density/microm(3) cytoplasm cannot be accounted for by corresponding changes in membrane surface density (microm(2) membrane/microm(3) cytoplasm) and reflect a real alteration in the number of Na(+)/K(+)-ATPase reaction product sites/unit membrane area. These data suggest that neither the gill nor the renal Na(+)/K(+)-ATPase systems function at maximal activity in shrimps in freshwater, possibly due to the low Na(+) concentration, and are initially stimulated by the increase in external ionic concentration. However, these powerful Na(+) transport systems respond to salt loading by a notable reduction in the number of hydrolysis sites, possibly through the incorporation of the Na(+)/K(+)-ATPase into isolated membrane vesicles in the basal invaginations of the medial renal tubules, together with ultrastructural alterations like the spatial isolation of the mitochondria by multiple membrane stacks in the intralamellar septal cells. J. Exp. Zool. 284:617-628, 1999.

The effect of eyestalk ablation on haemolymph osmotic and ionic concentrations during acute salinity exposure in the freshwater shrimp Macrobrachium olfersii (Wiegmann)(Crustacea, Decapoda)

The effect of bilateral eyestalk ablation on haemolymph osmotic and Na+, Cl−, K+, Mg2+ and Ca2+ concentrations was investigated in the freshwater shrimp Macrobrachium olfersii during acute exposure (1, 3, 6, 12 and 24 h) to sea-water of 21% S. Analysis of variance treatment indicated that haemolymph osmotic and Na+, Cl−, K+, Mg2+ and Ca+ concentrations were affected by exposure time (p ≤ 0.01). However, only [Na+] was affected by eyestalk ablation (p < 0.01), destalked shrimps exhibiting haemolymph Na+ concentrations approximately 15 to 30% lower than control shrimps. These results are interpreted to indicate that less Na+ enters the haemolymph of eyestalkless shrimps on exposure to seawater of 21% S, presumably resulting from the absence of an eyestalk-located neurofactor. A factor causing the influx of Na+ into the haemolymph is thus proposed to be present in the eyestalk ganglionic system of intact Macrobrachium olfersii in freshwater. Reduction in secretion of the factor may act to reduce Na+ entry in high salt environments.

Differential adjustment in gill Na+/K+- and V-ATPase activities and transporter mRNA expression during osmoregulatory acclimation in the cinnamon shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae)

SUMMARY We evaluate osmotic and chloride (Cl–) regulatory capability in the diadromous shrimp Macrobrachium amazonicum, and the accompanying alterations in hemolymph osmolality and [Cl–], gill Na+/K+-ATPase activity, and expression of gill Na+/K+-ATPase α-subunit and V-ATPase B subunit mRNA during salinity (S) acclimation. We also characterize V-ATPase kinetics and the organization of transport-related membrane systems in the gill epithelium. Macrobrachium amazonicum strongly hyper-regulates hemolymph osmolality and [Cl–] in freshwater and in salinities up to 25‰ S. During a 10-day acclimation period to 25‰ S, hemolymph became isosmotic and hypo-chloremic after 5 days, [Cl–] alone remaining hyporegulated thereafter. Gill Na+/K+-ATPase α-subunit mRNA expression increased 6.5 times initial values after 1 h, then decreased to 3 to 4 times initial values by 24 h and to 1.5 times initial values after 10 days at 25‰ S. This increased expression was accompanied by a sharp decrease at 5 h then recovery of initial Na+/K+-ATPase activity within 24 h, declining again after 5 days, which suggests transient Cl– secretion. V-ATPase B-subunit mRNA expression increased 1.5-fold within 1 h, then reduced sharply to 0.3 times initial values by 5 h, and remained unchanged for the remainder of the 10-day period. V-ATPase activity dropped sharply and was negligible after a 10-day acclimation period to 21‰ S, revealing a marked downregulation of ion uptake mechanisms. The gill epithelium consists of thick, apical pillar cell flanges, the perikarya of which are coupled to an intralamellar septum. These two cell types respectively exhibit extensive apical evaginations and deep membrane invaginations, both of which are associated with numerous mitochondria, characterizing an ion transporting epithelium. These changes in Na+/K+- and V-ATPase activities and in mRNA expression during salinity acclimation appear to underpin ion uptake and Cl– secretion by the palaemonid shrimp gill.

Na,K-ATPase activity and epithelial interfaces in gills of the freshwater shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae)

Diadromous freshwater shrimps are exposed to brackish water both as an obligatory part of their larval life cycle and during adult reproductive migration; their well-developed osmoregulatory ability is crucial to survival in such habitats. This study examines gill microsomal Na,K-ATPase (K-phosphatase activity) kinetics and protein profiles in the freshwater shrimp Macrobrachium amazonicum when in fresh water and after 10-days of acclimation to brackish water (21 per thousand salinity), as well as potential routes of Na+ uptake across the gill epithelium in fresh water. On acclimation, K-phosphatase activity decreases 2.5-fold, Na,K-ATPase alpha-subunit expression declines, total protein expression pattern is markedly altered, and enzyme activity becomes redistributed into different density membrane fractions, possibly reflecting altered vesicle trafficking between the plasma membrane and intracellular compartments. Ultrastructural analysis reveals an intimately coupled pillar cell-septal cell architecture and shows that the cell membrane interfaces between the external medium and the hemolymph are greatly augmented by apical pillar cell evaginations and septal cell invaginations, respectively. These findings are discussed regarding the putative movement of Na+ across the pillar cell interfaces and into the hemolymph via the septal cells, powered by the Na,K-ATPase located in their invaginations.