Branchial Carbonic Anhydrase Activity Research Articles

Carrying eggs in a semi-terrestrial environment: Physiological responses to water deprivation of mothers and embryos of the tree-climbing crab Aratus pisonii

In semi-terrestrial crustaceans, mothers and the carried embryos form a unit. The ideal conditions for embryonic development place a sustained demand on the mothers' energy budget. A particular challenge for this unit is the transition between terrestrial and aquatic environments. Ecophysiological information on mothers and embryos contributes to the understanding of evolutionary pathways and constraints of terrestrialization in crabs. We evaluated the effects of 6, 12, and 18 h of water deprivation on embryonic development and maternal physiology of the tree-climbing crab Aratus pisonii (H. Milne Edwards, 1837). Specifically, we determined the effect of water deprivation on egg volume, osmolality, and larval survival, as well as on maternal body mass, hemolymph osmolality, and branchial carbonic anhydrase activity (CAA). We hypothesized that longer periods of water deprivation would negatively affect larvae viability, as well as disrupt the physiology of brooding females. Embryonic development, larval survival, and the mother's physiology were only altered after 18 h of water deprivation, possibly due to dehydration and/or the accumulation of metabolic wastes that could not be excreted in the absence of water. Osmolality was higher in females than in their eggs. CAA of the anterior gills was higher in ovigerous than in non-ovigerous females, possibly due to the increase in respiratory demands caused by carrying eggs. Our results confirmed that the gestation period represents a metabolically expensive period for mother crabs and that water deprivation of 18 h was critical for the embryos' survival.

Evaluation of impacted Brazilian estuaries using the native oyster Crassostrea rhizophorae: Branchial carbonic anhydrase as a biomarker

In this study, we investigated the use of branchial carbonic anhydrase activity in a sessile filter feeding species, the oyster Crassostrea rhizophorae, as a biomarker. The oysters were collected in three human impacted Brazilian estuaries, following a crescent latitudinal gradient: in Pernambuco state (Itamaracá), in Espírito Santo state (Piraquê), and in Paraná state (Paranaguá), in August/2003 (Winter in the southern hemisphere) and February/2004 (Summer). Three sites were chosen in each estuary for oyster sampling: Reference (R), Contaminated 1 (C1, close to industrial/harbor contamination), and Contaminated 2 (C2, near to sewage discharges). Comparing to values in oysters sampled in reference sites, there was apparent inhibition in carbonic anhydrase activity (CAA) in gills of oysters from C1 of Itamaracá and from C2 of Piraquê, both cases in Summer. On the other hand, increased CAA was noted in C2 oysters of Itamaracá in winter, and of Paranaguá, in both seasons. Branchial CAA in C. rhizophorae was thus very responsive to coastal contamination. Data are consistent with its usefulness as a supporting biomarker for inexpensive and rapid analysis in the assessment of estuaries using a sessile osmoconformer species, but preferably allied to other biomarkers and with knowledge on the suite of contaminants present.

Effects of the herbicide atrazine in neotropical catfish (Rhamdia quelen)

The exposure to a world-wide used herbicide atrazine (ATZ) (96h exposure to 2, 10, and 100μgL−1), was investigated on the freshwater fish Rhamdia quelen through a multi biomarker approach. Liver histopathology revealed leukocyte infiltration, hepatocyte vacuolization like steatosis and necrosis areas, leading to raised lesion index levels in all tested concentrations. The increase of free melanomacrophage numbers was observed. Gill filaments revealed considerable loss of the microridges on pavement cells at 10 and 100μgL−1 of ATZ, and a significantly increased of chloride cell (CC) number and density on apical surface area at 100μgL−1 of ATZ. CAT, GST, GPx, and GR activities were inhibited by all tested concentrations. GSH levels were reduced in individuals exposed to 100µgL−1. Osmoregulatory function was also disturbed. We observed an increase of plasma magnesium concentrations at 10µgL−1. Additionally the inhibition of branchial carbonic anhydrase activity was observed at 100µgL−1. In the kidney, carbonic anhydrase activity decreased only in the group exposed to 2µgL−1. These results suggest that ATZ, represents a potential ecotoxicological hazard and can be hepatotoxic and nephrotoxic even low concentrations. The current study was the first to show the nephrotoxic effect of ATZ in fish. Besides, in Brazil, the environmental protection agency (CONAMA) establishes that the maximum allowed level of dissolved ATZ in water is 2μgL−1, but the present results showed that this concentration may cause histopathological, biochemical and physiological changes in R. quelen.

Risks of waterborne copper exposure to a cultivated freshwater Neotropical catfish (Rhamdia quelen)

As it is the case in all animal food production systems, it is often necessary to treat farmed fish for diseases and parasites. Quite frequently, fish farmers still rely on the aggressive use of copper to control bacterial infections and infestations by ecto-parasites, and to manage the spread of diseases. The susceptibility of the neotropical fish Rhamdia quelen to copper was here evaluated at different waterborne copper concentrations (2, 7 or 11μgCuL−1) for 96h, through a multi biomarkers approach. Liver histopathological findings revealed leukocyte infiltration, hepatocyte vacuolization and areas of necrosis, causing raised levels of lesions upon exposure to 7 and 11μgCuL−1. Decreased occurrence of free melano-macrophages and increased densities of melano-macrophage centers were noted upon exposure to 11μgCuL−1. Gills showed damages on their secondary lamellae already at 2μgCuL−1; hypertrophy and loss of the microridges of pavement cells at 7 and 11μgL−1, and increased in chloride cell (CC) apical surface area (4.9-fold) and in CC density (1.5-fold) at 11μgCuL−1. In the liver, catalase (CAT), glutathione peroxidase activities (GPx) and glutathione concentration (GSH) remained unchanged, compared to the control group. However, there was inhibition of 7-ethoxyresorufin-O-deethylase (EROD) at all copper concentrations tested. Glutathione reductase activity (GR) was reduced and levels of lipid peroxidation (LPO) were increased at 11μgCuL−1. Glutathione S-transferase activity (GST) at 7μgCuL−1 and superoxide dismutase activity (SOD) at both 7 and 11μgCuL−1 were reduced. However, copper exposure did not alter brain and muscle acetylcholinesterase (AChE) activity. Osmoregulatory function was also disturbed, in agreement with the above-mentioned changes noted in the gills, as detected by plasma osmolality reduction in the group exposed to 11μgCuL−1, and plasma chloride reduction at 2μgCuL−1. These concentrations also, coherently, lead to inhibition of branchial carbonic anhydrase activity. In the kidney, increased carbonic anhydrase activity was measured in the groups exposed to 2 and 7μgCuL−1. When these effects are compared to data available in the literature for other freshwater fish, also for 96h of exposure, R. quelen appears as a relatively sensitive species. In addition, the concentrations employed here were quite low in comparison to levels used for disease control in real culture practices (ranging from 4μgCuL−1 used against bacteria to 6000μgCuL−1 against fungal infections). We can conclude that the concentrations frequently employed in aquaculture are in fact not safe enough for this species. Such data are essential for the questioning and establishment of new policies to the sector.

Waterborne copper exposure inhibits ammonia excretion and branchial carbonic anhydrase activity in euryhaline guppies acclimated to both fresh water and sea water

Inhibition of ammonia excretion (Jamm) is a common response to Cu exposure in freshwater (FW) and seawater (SW) organisms. To determine the mechanism of this response, a euryhaline species of guppy (Poecilia vivipara) was exposed to 20μg Cu/l in FW (0ppt) and SW (25ppt) for 96h. In both salinities, Cu transiently inhibited ammonia excretion (Jamm) followed by a full recovery by the end of the 96h exposure. The activities of Na+/K+-ATPase, H+-ATPase, and carbonic anhydrase (CA) were examined in the gills at 12 and 96h of Cu exposure. In both salinity acclimations, CA activity was significantly inhibited following 12h of Cu exposure in P. vivipara, marking the first in vivo evidence of Cu-induced inhibition of CA in fish. Moreover, the inhibition and recovery of this enzyme were correlated with the inhibition and recovery of Jamm in both salinity acclimations. The blockade of CA potentially acts as a common mechanism of Jamm inhibition in FW and SW. There were no significant effects on Na+/K+-ATPase or H+-ATPase activity at either time point or salinity. However, H+-ATPase activity was upregulated at 96h relative to the 12h time point, potentially involving this enzyme in re-establishing Jamm.

Monitoring water quality in reservoirs for human supply through multi-biomarker evaluation in tropical fish

Paraíba do Sul River is located at a very densely inhabited region of Brazil crossing the three most industrialized states of the country (São Paulo, Minas Gerais and Rio de Janeiro states). As a result, industrial and farming residues as well as urban sewage are frequently disposed without appropriate treatment. The current study aimed at investigating the water quality in three reservoirs along the Paraíba do Sul River (Ilha dos Pombos, Santa Cecília and Santa Branca), through physiological, morphological, biochemical, and genetic biomarkers. The bioindicator chosen was the catfish Pimelodus maculatus, sampled during the dry (June 2008) and rainy (February 2009) seasons. Also, some water physicochemical parameters were analyzed from the sampling sites, but displayed no alterations according to the Brazilian Agency for Water Quality Legislation. Branchial carbonic anhydrase activity was inhibited in the dry season, while renal carbonic anhydrase activity was inhibited in the rainy season in the Santa Branca reservoir, indicating disturbance of osmoregulatory and acid-base regulation processes. Histopathological alterations were observed in the gills (neoplasic and tissue hyperplasia processes) and liver (necrosis), indicating serious damage to the functional integrity of these organs. A high incidence of melanomacrophage centers was observed in the liver, suggesting an intense immune response in all reservoirs. Acetylcholinesterase and catalase activity showed also differences corroborating some morphological results. Likewise, the induction of the micronucleus and DNA damage indicate genotoxicity, but mainly in the Santa Branca reservoir. Thus, the health status of P. maculatus warrants caution in the use of the water from the 3 reservoirs for direct human consumption, particularly after the accidental spill of endosulfan in November 2008, three months before the rainy season sampling.

Down-regulation of activity and expression of three transport-related proteins in the gills of the euryhaline green crab, Carcinus maenas, in response to high salinity acclimation

The euryhaline green crab, Carcinus maenas, undergoes an annual cycle of salinity exposure, having to adapt to low salinity during its annual spring migration into estuaries, and then having to re-adapt to high salinity when it moves off-shore at the end of summer. Most studies have focused on low salinity acclimation, the activation of osmoregulatory mechanisms, and the induction of transport protein and transport-related enzyme activity and gene expression. In this study we followed the changes in hemolymph osmolality, carbonic anhydrase activity, and mRNA expression of three proteins through a complete cycle of low (15 ppt) and high (32 ppt) salinity acclimation. One week of low salinity acclimation resulted in hemolymph osmoregulation and a four-fold induction of branchial carbonic anhydrase activity. Relative mRNA expression increased for two CA isoforms (CAc 100-fold, and CAg 7-fold) and the α-subunit of the Na/K-ATPase (8-fold). Upon re-exposure to high salinity, hemolymph osmolality increased to 32 ppt acclimated levels by 6 h, and mRNA levels returned to high salinity, baseline levels within 1 week. However, CA activity remained unchanged in response to high salinity exposure for the first week and then gradually declined to baseline levels over 4 weeks. The relative timing of these changes suggests that while whole-organism physiological adaptations and regulation at the gene level can be very rapid, changes at the level of protein expression and turnover are much slower. It is possible that the high metabolic cost of protein synthesis and/or processing could be the underlying reason for long biological life spans of physiologically important proteins.

The handling of salt by the neotropical cultured freshwater catfish Rhamdia quelen

The jundiá ( Rhamdia quelen) is a Siluriform native to South America, intensely cultivated in Southern Brazil, and which has been increasingly considered as a potential substitute for the culture of exotic species. Due to this interest, there is a constant need of methods to reduce parasite infestations common to the species. The use of sodium chloride (NaCl) is one of such practices. However, not much is known about the effects of NaCl on the osmotic homeostasis of this and other commercially relevant tropical freshwater species. This study thus aimed at evaluating the effect of adding NaCl (5, 15, or 25 g/L) to the water for 1 h, on the osmoregulation, and capacity for muscle tissue water regulation of the jundiá. In addition, muscle slices were subjected in vitro to isosmotic (control, animal in fresh water) and hyper-osmotic (120, 150, and 180% of control) salines, for evaluation of their capacity to control their water content. Some changes have been observed, when fishes submitted to 25 g/L were compared to freshwater controls: plasma ions and osmolality increased, the last from 260 ± 5 to 419 ± 12 mOsm/kg H 2O; plasma glucose increased from 47.8 to 92.8 mg/dL, and muscle water content decreased from 80 to 76.7%; plasma cortisol levels remained unchanged (~ 150 ng/mL). Branchial carbonic anhydrase activity also increased in 25 g/L, probably as a response to the metabolic acidosis that develops upon severe saline challenges. In vitro experiments indicated a high capacity for water content regulation, even when muscle slices were exposed to an 80% increase in osmolality. Exposure of jundiás for 1 h to 25 g/L was indeed harmful to their osmotic homeostasis, also affecting sensory and motor functions (observed as loss of barbel function, and swim impairment). However, the high degree of euryhalinity shown both in vivo and in vitro by this freshwater siluriform is coherent with its occasional presence in dilute estuarine waters and to the positioning of its family within a group of seawater-related Siluriforms. Thus, a reasonable amount of salt (15 g/L) for an intermediate time period (1 h) could be employed against parasites of the jundiá, while still being harmless to both the jundiás and humans that consume its flesh.

Branchial carbonic anhydrase activity and ninhydrin positive substances in the Pacific white shrimp, Litopenaeus vannamei, acclimated to low and high salinities

The Pacific white shrimp, Litopenaeus vannamei, acclimated to 30 ppt salinity, was transferred to either low (15 and 5 ppt), or high (45 ppt) salinity for 7 days. Hemolymph osmolality, branchial carbonic anhydrase activity, and total ninhydrin-positive substances (TNPS) in abdominal muscle were then measured for each condition. Hemolymph osmotic concentration was regulated slightly below ambient water osmolality in shrimp acclimated to 30 ppt. At 15 and 5 ppt, shrimp were strong hyper-osmotic regulators, maintaining hemolymph osmolality between 200 and 400 mOsm above ambient. Shrimp acclimated to 30 ppt and transferred to 45 ppt salinity were strong hypo-osmotic and hypo-ionic regulators, maintaining hemolymph osmolality over 400 mOsm below ambient. Branchial carbonic anhydrase (CA) activity was low (approximately 100 micromol CO(2) mg protein(-1) min(-1)) and uniform across all 8 gills in shrimp acclimated to 30 ppt, but CA activity increased in all gills after exposure to both low and high salinities. Anterior gills had the largest increases in CA activity, and levels of increase were approximately the same for low and high salinity exposure. Branchial CA induction appears to be functionally important in both hyper- and hypo-osmotic regulations of hemolymph osmotic concentrations. Abdominal muscle TNPS made up between 19 and 38% of the total intracellular osmotic concentration in shrimp acclimated to 5, 15, and 30 ppt. TNPS levels did not change across this salinity range, over which hemolymph osmotic concentrations were tightly regulated. At 45 ppt, hemolymph osmolality increased, and muscle TNPS also increased, presumably to counteract intracellular water loss and restore cell volume. L. vannamei appears to employ mechanisms of both extracellular osmoregulation and intracellular volume regulation as the basis of its euryhalinity.

Quantitative changes in branchial carbonic anhydrase activity and expression in the euryhaline green crab,Carcinus maenas, in response to low salinity exposure

Hemolymph osmolality, and changes in gill carbonic anhydrase (CA) activity, relative mRNA expression, and CA protein concentration were measured in the green crab Carcinus maenas acclimated to 32 ppt salinity and transferred to 10 ppt. Hemolymph osomolality stabilized at new, acclimated values, by 24 hr after transfer. There was a large increase in CA mRNA concentrations, as measured by quantitative PCR, in the posterior gills by 24 hr post-transfer that remained elevated through 4 days. By 7 days, however, CA mRNA levels began to decline. CA activity, on the other hand, did not begin to increase until 48 hr after transfer to 10 ppt, but it continued to increase through 7 days. CA protein concentration increased by 5-fold in posterior gills in crabs acclimated to 10 ppt. CA activity, mRNA expression, and CA protein concentrations did not change in anterior gills. These results indicate that low salinity-stimulated CA induction is under transcriptional regulation, and that the increase in CA activity is a result of the increase in gene expression and synthesis of new enzyme. Changes in mRNA appear to be transient, but once synthesized, the CA protein appears to persist in the gill for an extended time. In a separate set of experiments, green crabs acclimated to 32 ppt were transferred directly to salinities of 25, 20, 15, and 10 ppt. CA activity and mRNA concentrations increased with decreasing salinity, peaking at 15 ppt but decreasing between 15 and 10 ppt. The decrease may represent a breakdown in the mechanism of transport-related protein induction near the lower salinity limit of this species.

Mechanisms behind Pb-induced disruption of Na+and Cl−balance in rainbow trout (Oncorhynchus mykiss)

The mechanism of Pb-induced disruption of Na(+) and Cl(-) balance was investigated in the freshwater rainbow trout (Oncorhynchus mykiss). Na(+) and Cl(-) influx rates were reduced immediately in the presence of 2.40 +/- 0.24 and 1.25 +/- 0.14 muM Pb, with a small increase in efflux rates occurring after 24-h exposure. Waterborne Pb caused a significant decrease in the maximal rate of Na(+) influx without a change in transporter affinity, suggesting a noncompetitive disruption of Na(+) uptake by Pb. Phenamil and bafilomycin markedly reduced Na(+) influx rate but did not affect Pb accumulation at the gill. Time-course analysis in rainbow trout exposed to 0, 0.48, 2.4, and 4.8 microM Pb revealed time- and concentration-dependent branchial Pb accumulation. Na(+)-K(+)-ATPase activity was significantly reduced, with 4.8 microM exposure resulting in immediate enzyme inhibition and 0.48 and 2.4 microM exposures inhibiting activity by 24 h. Reduced activity was weakly correlated with gill Pb accumulation after 3- and 8-h exposures; this relationship strengthened by 24 h. Reduced Na(+) uptake was correlated with gill Pb burden after exposures of 3, 8, and 24 h. Immediate inhibition of branchial carbonic anhydrase activity occurred after 3-h exposure to 0.82 +/- 0.05 or 4.30 +/- 0.05 microM Pb and continued for up to 24 h. We conclude that Pb-induced disruption of Na(+) and Cl(-) homeostasis is in part a result of rapid inhibition of carbonic anhydrase activity and of binding of Pb with Na(+)-K(+)-ATPase, causing noncompetitive inhibition of Na(+) and Cl(-) influx.

Salinity-mediated carbonic anhydrase induction in the gills of the euryhaline green crab, Carcinus maenas

The euryhaline green crab, Carcinus maenas, is a relatively strong osmotic and ionic regulator, being able to maintain its hemolymph osmolality as much as 300 mOsm higher than that in the medium when the crab is acclimated to low salinity. It makes the transition from osmoconformity to osmoregulation at a critical salinity of 26 ppt, and new acclimated concentrations of hemolymph osmotic and ionic constituents are reached within 12 h after transfer to low salinity. One of the central features of this transition is an 8-fold induction of the enzyme carbonic anhydrase (CA) in the gills. This induction occurs primarily in the cytoplasmic pool of CA in the posterior, ion-transporting gills, although the membrane-associated fraction of CA also shows some induction in response to low salinity. Inhibition of branchial CA activity with acetazolamide (Az) has no effect in crabs acclimated to 32 ppt but causes a depression in hemolymph osmotic and ionic concentrations in crabs acclimated to 10 ppt. The salinity-sensitive nature of the cytoplasmic CA pool and the sensitivity of hemolymph osmotic/ionic regulation to Az confirm the enzyme's role in ion transport and regulation in this species. CA induction is a result of gene activation, as evidenced by an increase in CA mRNA at 24 h after transfer to low salinity and an increase in protein-specific CA activity immediately following at 48 h post-transfer. CA gene expression appears to be under inhibitory control by an as-yet unidentified repressor substance found in the major endocrine complex of the crab, the eyestalk.

Calcium metabolism related markers during the growth of Haliotis tuberculata

Summary In order to search for a physiological role of calcitonin gene-related peptide (CGRP) during the growth of abalone, the presence of CGRP-like molecules in haemolymph and mantle extracts was investigated. In addition, the activities of carbonic anhydrase and alkaline phosphatase, two enzymes implicated in calcium metabolism of vertebrates, were quantified. The CGRP circulating concentration increased progressively from the 3- to 7-cm-long abalone, whereas the mantle ir-CGRP strongly decreased in 5-cm-long abalone. Moreover, branchial carbonic anhydrase activity, a specific osteoclastic marker in vertebrates, increased during abalone development from 11.8 to 39 WA Units per mg of gill membrane proteins in 4- and 9-cm-long animals, respectively. Mantle alkaline phosphatase activity, a vertebrate specific osteoblastic marker, showed a twofold increase in 6-cm-long animals (8.11 Units per mg of proteins). These studies suggest that CGRP may be involved in molluscan growth, and that gill carbonic anhydrase and the mantle alkaline phosphatase undergo important variations, in relation to the sexual maturation of abalone.

Effect of cadmium on carbonic anhydrase and Na +-K +-ATPase in eel, Anguilla anguilla, intestine and gills

The effect of the heavy metal cadmium on in vitro activities of Na +-K +-ATPase and carbonic anhydrase (CA) in intestine and gills of the European eel, Anguilla anguilla, was analyzed. Na +-K +-ATPase enzymatic activities, measured in intestinal and branchial homogenates, were inhibited by increasing cadmium concentrations (0.5–50 μM, after 1 h of incubation) in a dose-dependent manner; the maximal inhibition (about 80%) was observed at 5 μM and 50 μM CdCl 2 for the branchial and intestinal Na +-K +-ATPase respectively. Carbonic anhydrase activities, measured in gill homogenate and in cytosolic and brush border membrane fractions isolated from intestinal mucosa, were significantly inhibited by pre-incubation (1 h) with CdCl 2. Maximal inhibition (about 80%) of branchial CA was noted at approximately 60 μM; higher concentrations evoked no further significant inhibition. Intestinal CA isoforms, cytosolic and membrane-bound, exhibited lower sensitivity to the heavy metal with respect to the branchial CA activity, since the highest concentration of CdCl 2 tested (600 μM) produced an inhibition of about 30% and 50% respectively. The results of the present study suggest that cadmium, by inhibiting the activity of CA and Na +-K +-ATPase enzymes in intestine and gills, could alter both acid-base balance and osmoregulation in teleostean fish.

Quaternary ammonium sulfanilamide: a membrane-impermeant carbonic anhydrase inhibitor.

A novel carbonic anhydrase (CA) inhibitor, quaternary ammonium sulfanilamide (QAS), was tested for potency as a CA inhibitor and for its ability to be excluded from permeating biological membranes. Inhibitor titration plots of QAS vs. pure bovine CA II and CA from the gills of the blue crab, Callinectes sapidus, yielded Ki values of approximately 15 microM; thus QAS is a relatively weak but effective CA inhibitor. Permeability of the QAS was directly tested by two independent methods. The inhibitor was excluded from human erythrocytes incubated in 5 mM QAS for 24 h as determined using an 18O-labeled mass spectrometer CA assay for intact cells. Also QAS injected into the hemolymph of C. sapidus (1 or 10 mM) did not cross the basal membrane of the gill. The compound was cleared from the hemolymph by 96 h after injection, and at no time during that period could the QAS be detected in homogenates of gill tissue. Total branchial CA activity was only slightly reduced following the QAS injection. These data indicate that QAS is a CA inhibitor to which biological membranes are impermeable and that can be used in vivo or in vitro in the study of membrane-associated CA.

The Role of Carbonic Anhydrase in Blood Ion and Acid-Base Regulation

The role of carbonic anhydrase (CA) in ion transport processes of aquatic and terrestrial arthropod species is reviewed. In both insects and crustaceans CA is found in a variety of ion transporting tissues. The bulk of CA activity in crustaceans is concentrated in the posterior gills, which are morphologically and biochemically adapted for ion transport. The enzyme can be specifically localized to gill lamellae which contain large populations of salt transporting chloride cells. Enzyme activity in the posterior gills of species having the ability to regulate blood ion concentrations increases when these organisms are acclimated to environmental salinities in which they ion regulate. In stenohaline, ion conforming species branchial CA activity is uniformly low, being only 5–10% that in regulating species. Studies on the blue crab, Callinectes sapidus , using the specific CA inhibitor acetazolamide have shown that the enzyme is indeed important in blood ion regulation. Blood Na$ and Cl− concentrations are both severely lowered in drug-treated animals acclimated to low salinity, while they remain virtually unaffected in animals acclimated to high salinity, in which the animal is an ion conformer. High salinity acclimated crabs treated with acetazolamide do not survive transfer to low salinity, and mortality is related to a breakdown in the ion regulatory mechanism. Branchial CA most likely functions in the hydration of respiratory CO2 to H$ and HCO3−, which serve as counterions for the active uptake of Na$ and Cl−, respectively. In terrestrial species the role of CA is unclear and merits further investigation.

Carbonic anhydrase (acetazolamide-sensitive esterase) activity in the blood, gill and kidney of the thermally acclimated rainbow trout, Salmo gairdneri.

1. Gill, kidney and blood levels of acetazolamide-sensitive esterase (carbonic anhydrase) activity were estimated at acclimation temperature and at a common temperature (25 degrees C) in rainbow trout acclimated to 2, 10 and 18 degrees C. Plasma levels of sodium, potassium and chloride were also examined for possible acclimatory variations. 2. Plasma sodium and chloride levels, and the sodium:chloride ratio were unaffected by thermal acclimation; potassium concentrations were significantly elevated at 18 degrees C. 3. Significant, but modest changes in renal and branchial carbonic anhydrase activity were observed under physiologically realistic incubation temperature conditions. Blood carbonic anhydrase activity was sharply elevated at higher acclimation temperatures. 4. The data are discussed in relation to the hypothesis that carbonic anhydrase in this relatively stenothermal freshwater salmonid, through its intimate association with the coupled HCO-3/CL- and H+ +NH+4/Na+ exchange systems may provide for relatively thermostable basal rates of sodium and chloride uptake from the medium and recovery from urine. The renal, and more notably the branchial (Na+/K+)-stimulated ATPase systems, and erythrocytic carbonic anhydrase may then serve primarily as high-temperature amplifiers of sodium and chloride recruitment respectively.