Characterization of ion transport in the isolated epipodite of the lobster Homarus americanus.

Unfolded epipodite isolated from American lobsters (Homarus americanus) acclimated to dilute seawater was mounted in an Ussing-type chamber for ion transport studies. The split epipodite is an electrically polarized, one-cell-layer epithelium supported with cuticle. Under open-circuit conditions, the transepithelial potential was -4.2+/-1.0 mV (N=38). In the short-circuited epithelium, the current averaged over all of the preparations was -185.4+/-20.2 A cm(-2) (N=38) with a high conductance of 55.2+/-11.4 mS cm(-2) (N=38), typical for a leaky epithelium. The Na:Cl absorptive flux ratio was 1:1.6; ion substitution experiments indicated that the transport of Na+ and Cl- is coupled. Basolateral application of the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino) benzoate (NPPB) and niflumic acid (NFA) dose-dependently inhibited short-circuit current (ISC). Secretory K+ (Rb+) fluxes exceeded influxes and were inhibited by the Na+/K+-ATPase inhibitor ouabain and the K+ channel blocker cesium. Western blot analysis showed that Na+/K+-ATPase alpha-subunit protein was more highly expressed in the epipodite of lobsters acclimated to 20 p.p.t. compared with animals acclimated to seawater (34 p.p.t.). 3-Isobutyl-1-methyl-xanthine (IBMX) stimulated a negative ISC and enhanced apical secretory K+ flux. Basolateral application of NPPB inhibited JRbB-->A fluxes, suggesting the interaction of K+ channels with NPPB-sensitive Cl- channels. The results are summarized in a transport model, suggesting apical Na+/K+/2Cl- co-transport, a dominant apical K+-secreting channel and basolaterally located Cl- and K+ channels. This study represents the first comprehensive characterization of ion transport processes across the lobster epipodite epithelium and indeed in any tissue within the branchial cavity of the American lobster.

Identification of a calcitonin-like diuretic hormone that functions as an intrinsic modulator of the American lobster, Homarus americanus, cardiac neuromuscular system.

In insects, a family of peptides with sequence homology to the vertebrate calcitonins has been implicated in the control of diuresis, a process that includes mixing of the hemolymph. Here, we show that a member of the insect calcitonin-like diuretic hormone (CLDH) family is present in the American lobster, Homarus americanus, serving, at least in part, as a powerful modulator of cardiac output. Specifically, during an ongoing EST project, a transcript encoding a putative H. americanus CLDH precursor was identified; a full-length cDNA was subsequently cloned. In silico analyses of the deduced prepro-hormone predicted the mature structure of the encoded CLDH to be GLDLGLGRGFSGSQAAKHLMGLAAANFAGGPamide (Homam-CLDH), which is identical to a known Tribolium castaneum peptide. RT-PCR tissue profiling suggests that Homam-CLDH is broadly distributed within the lobster nervous system, including the cardiac ganglion (CG), which controls the movement of the neurogenic heart. RT-PCR analysis conducted on pacemaker neuron- and motor neuron-specific cDNAs suggests that the motor neurons are the source of the CLDH message in the CG. Perfusion of Homam-CLDH through the isolated lobster heart produced dose-dependent increases in both contraction frequency and amplitude and a dose-dependent decrease in contraction duration, with threshold concentrations for all parameters in the range 10(-11) to 10(-10) mol l(-1) or less, among the lowest for any peptide on this system. This report is the first documentation of a decapod CLDH, the first demonstration of CLDH bioactivity outside the Insecta, and the first detection of an intrinsic neuropeptide transcript in the crustacean CG.

The peptide hormone pQDLDHVFLRFamide (crustacean myosuppressin) modulates the Homarus americanus cardiac neuromuscular system at multiple sites.

pQDLDHVFLRFamide is a highly conserved crustacean neuropeptide with a structure that places it within the myosuppressin subfamily of the FMRFamide-like peptides. Despite its apparent ubiquitous conservation in decapod crustaceans, the paracrine and/or endocrine roles played by pQDLDHVFLRFamide remain largely unknown. We have examined the actions of this peptide on the cardiac neuromuscular system of the American lobster Homarus americanus using four preparations: the intact animal, the heart in vitro, the isolated cardiac ganglion (CG), and a stimulated heart muscle preparation. In the intact animal, injection of myosuppressin caused a decrease in heartbeat frequency. Perfusion of the in vitro heart with pQDLDHVFLRFamide elicited a decrease in the frequency and an increase in the amplitude of heart contractions. In the isolated CG, myosuppressin induced a hyperpolarization of the resting membrane potential of cardiac motor neurons and a decrease in the cycle frequency of their bursting. In the stimulated heart muscle preparation, pQDLDHVFLRFamide increased the amplitude of the induced contractions, suggesting that myosuppressin modulates not only the CG, but also peripheral sites. For at least the in vitro heart and the isolated CG, the effects of myosuppressin were dose-dependent (10(-9) to 10(-6) mol l(-1) tested), with threshold concentrations (10(-8)-10(-7) mol l(-1)) consistent with the peptide serving as a circulating hormone. Although cycle frequency, a parameter directly determined by the CG, consistently decreased when pQDLDHVFLRFamide was applied to all preparation types, the magnitudes of this decrease differed, suggesting the possibility that, because myosuppressin modulates the CG and the periphery, it also alters peripheral feedback to the CG.

Anti-lipopolysaccharide factors in the American lobster Homarus americanus: molecular characterization and transcriptional response to Vibrio fluvialis challenge.

Two partial mRNA sequences predicted to encode anti-lipopolysaccharide factors (ALFs) were identified among expressed sequence tags generated from the American lobster Homarus americanus and complete cDNA sequences were obtained from library clones. Comparison of the translated amino acid sequences to those publicly available confirmed similarity to arthropod anti-lipopolysaccharide factors. Both protein sequences, designated ALFHa-1 and ALFHa-2, contained an N-terminal signal peptide and two half-cysteines participating in a disulfide bridge, features conserved in other ALFs. Predicted secondary structures were similar to that described for the ALF from the horseshoe crab Limulus polyphemus. As part of an exploratory study of immunity in H. americanus, lobsters were injected with the bacterium Vibrio fluvialis and gill, hematopoietic, and hepatopancreas tissues were sampled for analysis of gene expression of ALFHa-1 and ALFHa-2 by quantitative PCR. The relative abundance of ALFHa-2 mRNA was not significantly affected by Vibrio injection in any of the three tissues tested. In contrast, ALFHa-1 mRNA levels in gills were increased by the treatment some 17-fold. Our results support a molecularly specific regulation of antimicrobial proteins in response to bacterial infection in H. americanus.

Expression of Na(+)/K(+)-ATPase alpha-subunit mRNA during embryonic development of the crayfish Astacus leptodactylus.

Astacus leptodactylus is a decapod crustacean fully adapted to freshwater where it spends its entire life cycle after hatching under huge osmoconcentration differences between the hemolymph and surrounding freshwater. We investigated the expression of mRNA encoding one ion transport-related protein, Na(+)/K(+)-ATPase alpha-subunit, and one putative housekeeping gene, beta-actin, during crayfish ontogenesis using quantitative real-time PCR. A 216-amino acid part of the open reading frame region of the cDNA coding for the Na(+)/K(+)-ATPase alpha-subunit was sequenced from total embryo, juvenile and adult gill tissues. The predicted amino acid sequence showed a high percentage similarity to those of other invertebrates (up to 95%) and vertebrates (up to 69%). beta-actin expression exhibited modest changes through embryonic development and early post-embryonic stage. The Na(+)/K(+)-ATPase alpha-subunit gene was expressed in all studied stages from metanauplius to juvenile. Two peaks of expression were observed: one in young embryos at 25% of embryonic development (EI=100 mum), and one in embryos just before hatching (at EI=420 mum), continuing in the freshly hatched juveniles. The Na(+)/K(+)-ATPase expression profile during embryonic development is time-correlated with the occurrence of other features, including ontogenesis of excretory antennal glands and differentiation of gill ionocytes linked to hyperosmoregulation processes and therefore involved in freshwater adaptation.

Effect of salinity on osmoregulatory patch epithelia in gills of the blue crab Callinectes sapidus.

In euryhaline crabs, ion-transporting cells are clustered into osmoregulatory patches on the lamellae of the posterior gills. To examine changes in the branchial osmoregulatory patch in the blue crab Callinectes sapidus in response to change in salinity and to correlate these changes with other osmoregulatory responses, crabs were acclimated to a range of salinities between 10 and 35 ppt. When crabs that had been acclimated to 35 ppt were subsequently transferred to 10 ppt, both the size of the osmoregulatory patch on individual gill lamellae and the specific activity of Na+, K+-ATPase in whole-gill homogenates increased only after the first 24 h of exposure to dilute seawater. Enzyme activity and size of patch area increased gradually and reached their maxima (increasing by 200% and 60%, respectively) 6 days following transfer to 10 ppt seawater and then remained at these levels. Patch size at acclimation varied inversely with the salinity for seawater dilutions below 26 ppt (the isosmotic point of the crab), although it did not vary in salinities at or above 26 ppt. Thus, the size of the patch clearly is modulated with acclimation salinity, but it increases only in those salinities in which the crab hyperosmoregulates. An increase in the total RNA/DNA ratio in gill homogenates, the lack of mitotic figures in the lamellae, and the lack of incorporation of bromodeoxyuridine into nuclei of lamellar epithelial cells during acclimation to dilute seawater were interpreted as evidence that no cell proliferation had occurred and that increases in the size of the osmoregulatory patch occurred through differentiation of existing gas exchange cells or of undifferentiated epithelial cells into ion-transporting cells.

Expression of Ca2+-ATPase and Na+/Ca2+-exchanger is upregulated during epithelial Ca2+ transport in hypodermal cells of the isopod Porcellio scaber.

It is thought that a plasma membrane Ca(2+)-transport ATPase (PMCA) and a Na(+)/Ca(2+)-exchange (NCE) mechanism are involved in epithelial Ca(2+) transport (ECT) in a variety of crustacean epithelia. The sternal epithelium of the terrestrial isopod Porcellio scaber was used as a model for the analysis of Ca(2+)-extrusion mechanisms in the hypodermal epithelium. Using RT-PCR, we amplified a cDNA fragment of 1173 bp that encodes a protein sequence possessing 72% identity to the PMCA from Drosophila melanogaster and a cDNA fragment of 791 bp encoding a protein sequence with 50% identity to the NCE from Loligo opalescens. Semiquantitative RT-PCR revealed that the expression of both mRNAs increases from the non-Ca(2+)-transporting condition to the stages of CaCO(3) deposit formation and degradation. During Ca(2+)-transporting stages, the expression of PMCA and NCE was larger in the anterior sternal epithelium (ASE) than in the posterior sternal epithelium (PSE). The results demonstrate for the first time the expression of a PMCA and a NCE in the hypodermal epithelium of a crustacean and indicate a contribution of these transport mechanisms in ECT.

Molecular characterization of V-type H(+)-ATPase (B-subunit) in gills of euryhaline crabs and its physiological role in osmoregulatory ion uptake.

The vacuolar-type H(+)-ATPase (V-ATPase) has been implicated in osmoregulatory ion uptake across external epithelia of a growing variety of species adapted to life in fresh water. In the present study, we investigated whether the V-ATPase may also function in a euryhaline species that tolerates brackish water (8 salinity) but not fresh water, the shore crab Carcinus maenas. cDNA coding for the regulatory B-subunit of the V-ATPase was amplified and sequenced from C. maenas gills and partially sequenced from four other crab species. Two isoforms differing in the 3'-untranslated region were found in C. maenas. In this species, the abundance of B-subunit mRNA was greater in the respiratory anterior gills than the ion-transporting posterior gills and was not increased by acclimation to dilute salinity. Immunocytochemical analysis showed that the B-subunit protein is not targeted to the apical membrane but is distributed throughout the cytoplasmic compartment. Physiological studies of isolated perfused gills indicated that the V-ATPase inhibitor bafilomycin had no effect on transepithelial potential difference. Thus, in contrast to the freshwater-tolerant Chinese crab Eriocheir sinensis, in which the V-ATPase appears to play an important osmoregulatory role, the V-ATPase in C. maenas probably functions in acidification of intracellular organelles but not in transbranchial NaCl uptake.

Na(+)+K(+)-ATPase in gills of the blue crab Callinectes sapidus: cDNA sequencing and salinity-related expression of alpha-subunit mRNA and protein.

Many studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enhanced Na(+)+K(+)-ATPase activity in the posterior gills, but it remains unclear whether the response is due to regulation of pre-existing enzyme or to increased gene transcription and mRNA translation. To address this question, the complete open reading frame and 3' and 5' untranslated regions of the mRNA coding for the alpha-subunit of Na(+)+K(+)-ATPase from the blue crab Callinectes sapidus were amplified by reverse transcriptase/polymerase chain reaction (RT-PCR) and sequenced. The resulting 3828-nucleotide cDNA encodes a putative 1039-amino-acid protein with a predicted molecular mass of 115.6 kDa. Hydrophobicity analysis of the amino acid sequence indicated eight membrane-spanning regions, in agreement with previously suggested topologies. The alpha-subunit amino acid sequence is highly conserved among species, with the blue crab sequence showing 81-83 % identity to those of other arthropods and 74-77 % identity to those of vertebrate species. Quantitative RT-PCR analysis showed high levels of alpha-subunit mRNA in posterior gills 6-8 compared with anterior gills 3-5. Western blots of gill plasma membranes revealed a single Na(+)+K(+)-ATPase alpha-subunit protein band of the expected size. The posterior gills contained a much higher level of alpha-subunit protein than the anterior gills, in agreement with previous measurements of enzyme activity. Immunocytochemical analysis showed that the Na(+)+K(+)-ATPase alpha-subunit protein detected by alpha5 antibody is localized to the basolateral membrane region of gill epithelial cells. Transfer of blue crabs from 35 to 5 per thousand salinity was not accompanied by notable differences in the relative proportions of alpha-subunit mRNA and protein in the posterior gills, suggesting that the enhanced Na(+)+K(+)-ATPase enzyme activity that accompanies the hyperosmoregulatory response may result from post-translational regulatory processes.

Expression of arginine kinase enzymatic activity and mRNA in gills of the euryhaline crabs Carcinus maenas and Callinectes sapidus.

Phosphagen kinases catalyze the reversible dephosphorylation of guanidino phosphagens such as phosphocreatine and phosphoarginine, contributing to the restoration of adenosine triphosphate concentrations in cells experiencing high and variable demands on their reserves of high-energy phosphates. The major invertebrate phosphagen kinase, arginine kinase, is expressed in the gills of two species of euryhaline crabs, the blue crab Callinectes sapidus and the shore crab Carcinus maenas, in which energy-requiring functions include monovalent ion transport, acid-base balance, nitrogen excretion and gas exchange. The enzymatic activity of arginine kinase approximately doubles in the ion-transporting gills of C. sapidus, a strong osmoregulator, when the crabs are transferred from high to low salinity, but does not change in C. maenas, a more modest osmoregulator. Amplification and sequencing of arginine kinase cDNA from both species, accomplished by reverse transcription of gill mRNA and the polymerase chain reaction, revealed an open reading frame coding for a 357-amino-acid protein. The predicted amino acid sequences showed a minimum of 75 % identity with arginine kinase sequences of other arthropods. Ten of the 11 amino acid residues believed to participate in arginine binding are completely conserved among the arthropod sequences analyzed. An estimation of arginine kinase mRNA abundance indicated that acclimation salinity has no effect on arginine kinase gene transcription. Thus, the observed enhancement of enzyme activity in C. sapidus probably results from altered translation rates or direct activation of pre-existing enzyme protein.

Sodium/proton antiporter in the euryhaline crab Carcinus maenas: molecular cloning, expression and tissue distribution.

Gill epithelial cells of euryhaline crustaceans demonstrate net inward transport of sodium ions, possibly via apical Na+/H+ antiporters, Na+/K+/2Cl- cotransporters or Na+ channels working in series with the basolateral Na(+) + K(+)-ATPase. We have identified and sequenced the cDNA coding for a crustacean Na+/H+ antiporter, starting with mRNA isolated from gills of the euryhaline green shore crab Carcinus maenas. The complete 2595-base-pair cDNA includes an open reading frame coding for a 673-amino-acid protein. A search of GenBank revealed more than 20 high-scoring matches, all Na+/H+ antiporter sequences from mammalian, amphibian, teleost and nematode species. Injection of Xenopus laevis oocytes with cRNA transcribed from the cloned crab sequence substantially enhanced Na(+)-dependent H+ efflux from the oocytes. Analysis of crab tissue antiporter mRNA levels by semi-quantitative reverse transcription-polymerase chain reaction revealed that posterior and anterior gills of Carcinus maenas expressed this antiporter the most strongly, followed in decreasing order by skeletal muscle, hepatopancreas, hypodermis and heart. Hydropathy and transmembrane alpha-helix analysis suggested a 10-helix membrane-spanning topology of the antiporter protein. It is clear from this study that Carcinus maenas gills vigorously transcribe a gene coding for a Na+/H+ antiporter. Whether these gills also express a gene coding for an epithelial Na+ channel or Na+/K+/2Cl- cotransporter remains to be demonstrated.

Characterization of an endogenous Na+/H+ antiporter in Xenopus laevis oocytes.

The amiloride-sensitive Na+/H+ antiporter in defolliculated oocytes of Xenopus laevis was characterized by measurements of 22Na+ influx and apparent H+ efflux. Uptake of 22Na+ was linear over a 90-min incubation period and was inhibited approximately 80% with 5 x 10(-4) mol l-1 amiloride. Amiloride-sensitive sodium uptake was reduced following collagenase treatment or oocyte aging. K0.5 for amiloride inhibition was 4.13 x 10(-6) +/- 1.33 x 10(-6)mol l-1 and the Km for Na+ was 4.25 x 10(-3) mol l-1. Hill analysis of the kinetic data for Na+ revealed an nH value of 1.14, indicating an absence of interacting binding sites for Na+. Parallel measurements of amiloride-sensitive Na+ uptake and H+ efflux indicated a Na+/H+ exchange ratio of 0.88:1. Our conclusion is that the Na+/H+ antiporter of Xenopus oocytes exhibits a nominal 1:1 Na+/H+ exchange stoichiometry and is similar in its properties to the antiporter of other vertebrate cells.

Electrogenic sodium-proton exchange in membrane vesicles from crab (Carcinus maenas) gill.

Plasma membrane vesicles from Na+-absorbing posterior gills of the green crab Carcinus maenas were shown to contain a Na+-H+ antiporter sensitive to amiloride (Ki = 280 mumol/l), using a fluorometric assay based on acridine orange. Lithium was found to support Li+-H+ exchange, giving Michaelis-Menten kinetics (K0.5 = 20 mmol/l) and a Hill coefficient of 1.32 +/- 0.06, indicating a stoichiometry of 1 Li+:1 H+. The stoichiometry of Na+-H+ exchange, however, appeared to be 2 Na+:1 H+. The dependence of exchange rate on Na+ concentration followed a sigmoid relationship (K0.5 = 34 mmol/l), producing a Hill coefficient of 1.99 +/- 0.18. Measurements made with the potential-sensitive dye 3,3'-dipropylthiadicarboxocyanine iodide directly demonstrated electrogenicity of Na+-H+ exchange. Na+-loaded vesicles showed greater potential change upon addition to Na+-free medium than upon addition to Na+-containing medium, and the developed potential was reversed by the addition of external Na+. Amiloride reduced the rate of membrane polarization and abolished the reversal by external Na+. The crustacean Na+-H+ antiporter therefore bears similarities to the vertebrate antiporter but is uniquely electrogenic.

Effects of tributyltin compounds on ionic regulation and gill ATPase activity in estuarine fish.

1. Striped bass (Morone saxatilis) were exposed to 0, 0.10, 0.34, or 1.09 micrograms/l tributyltin (TBT) for 14 days. Gill Mg2+ ATPase and serum Na+, K+, Ca2+ and Mg2+ were not altered. Na+K+ATPase was significantly (P less than 0.05) increased (+48%) at 0.10 microgram/l. 2. In striped bass gill homogenates exposed to TBT in vitro, there was significant inhibition of Na+K+ATPase at 106.0 micrograms/l (17.2%) and Mg2+ATPase at 53.0 and 106.0 micrograms/l (16.0% and 24.3%, respectively). 3. In mummichog (Fundulus heteroclitus) gill homogenates, there was significant inhibition of Na+K+ATPase at 25.3 and 50.5 micrograms/l (10.9% and 16.1%) and Mg2+ATPase at 5.1, 25.3, and 50.5 micrograms/l (26.7%, 32.2%, and 36.2%).

Ammonium ion substitutes for K+ in ATP-dependent Na+ transport by basolateral membrane vesicles.

Ion-transporting cells from posterior gills of blue crabs (Callinectes sapidus) acclimated to low salinity were used as starting material for the preparation of microsomal membrane vesicles by density gradient centrifugation. The Na+-K+-adenosinetriphosphatase (ATPase)-enriched basolateral vesicles were loaded with KCl- or NH+4-containing medium by dilution and centrifugation, and initial rates of 22Na+ uptake into the vesicles were measured by a rapid filtration procedure. Varying the extravesicular sucrose concentration altered equilibrium uptake of 22Na+, indicating the existence of osmotically sensitive vesicles. Monensin, a sodium-specific ionophore, enhanced passive uptake of 22Na+ across the vesicle membrane in the absence of ATP. With 100 mM KCl in the intravesicular medium, addition of ATP to the extravesicular medium increased initial rates of 22Na+ uptake 10- to 20-fold over levels measured without ATP. A nonhydrolyzable ATP analog failed to stimulate 22Na+ uptake. Intravesicular K+ could be replaced by NH+4 but not by choline. With NH+4 as counterion, Na+ transport was inhibited by digitoxin, but valinomycin had no effect. A study of the kinetic effects of intravesicular K+ and NH+4 on initial rates of 22Na+ uptake indicated the existence of two classes of binding sites, one responding to counterion concentrations in the millimolar range and a second class responding to counterion concentrations over 50 mM. Our results indicate that ATP-dependent 22Na+ uptake by membrane vesicles from Callinectes sapidus gill, mediated by Na+-K+-ATPase, can utilize either K+ or NH+4 as counterion.

Evaluation of the rapid penicillinase paper strip test for detection of beta-lactamase.

The penicillin-starch paper strip method was compared with the acidometric and iodometric methods for assaying beta-lactamase production, using fresh isolates of clinically important bacteria. Results obtained by the three methods were compared for rapidity, accuracy, and stability of reagents. Of the 210 isolates tested by the paper strip method, 301 isolates tested by the acidometric method, and 117 isolates tested by the iodometric method, all were in perfect agreement with the disk diffusion susceptibility test except one strain each of Haemophilus influenzae, Staphylococcus aureus, and Staphylococcus epidermidis. The H. influenzae isolate was penicillin resistant and failed to give a positive test for beta-lactamase in all three tests. The staphylococci (intermediate and resistant in susceptibility, respectively) failed to give a positive test for beta-lactamase with the iodometric method. The results of the paper strip method, in which 3,241 strains representing nine species of bacteria were used, correlated completely with disk susceptibility tests except for 2 and 69 strains, respectively, of penicillin-resistant, beta-lactamase-negative H. influenzae and H. parainfluenzae. The results of this study indicate that the paper strip method is accurate, simple to perform, extremely economical, and uses materials that are stable when stored frozen. It is eminently suitable for routine laboratory use.

Na++K+-ATPase in the osmoregulating clam Rangia cuneata.

Of six tissues sampled from the osmoregulating clam Rangia cuneata, mantle contained the highest concentration of Na++K+-ATPase activity and ouabain binding sites. Acclimation to low salinities was accompanied by adaptive increases in Na++K+-ATPase activity in mantle but not in gill. Since the number of ouabain binding sites did not show parallel increases, the mechanism of acclimation to reduced salinity in Rangia appears to involve activation of pre-existing pump sites in the mantle epithelium.

Rapid modulation of gill Na+ + K+-dependent ATPase activity during acclimation of the killifish Fundulus heteroclitus to salinity change.

The enzymatic properties of membrane-bound Na+ + K+-ATPase from gills of killifish acclimated to fresh water, to 16% sea water, or to 30% sea water appear to be identical, indicating that the same enzyme may function to absorb Na+ in low salinities and excrete Na+ at the gills in high salinities. Ammonium ion is an effective substitute for K+: in the ATPase reaction itself, in blocking phosphorylation of the ATPase protein, and in inhibiting the binding of ouabain to the enzyme. The specific activities of the Na+ + K+-ATPase in the three different salinities are consistent with the expected Na+ pumping rates: higher in fresh water and 30% sea water than in 16% sea water. Within one-half hour after transfer of killifish from one salinity to another, gill Na+ + K+-ATPase activities reach equilibrium levels. The rapid increase in Na+ + K+-ATPase activity in gill microsomes of fish acclimating from fresh water to 30% sea water is accompanied by a slow decrease in the number of binding sites for ouabain, supporting the idea that acclimation to short-term salinity changes may involve modifications in the catalytic rate rather than the number of Na+ + K+-ATPase molecules.