Raymond P. Henry

The mechanism of acute silver nitrate toxicity in freshwater rainbow trout (Oncorhynchus mykiss) is inhibition of gill Na+ and Cl−1 transport

Rainbow trout (Oncorhynchus mykiss) were exposed to 2 and 10 μg l−1 silver (as AgNO3) for up to 75 h in moderately hard freshwater. At 10 μg l−1 total Ag, branchial Na+ and Cl− influxes were inhibited by over 50% immediately and by almost 100% at 8 h, and showed no signs of recovery over the duration of the experiment. Na+ and Cl− effluxes were much less affected. These changes in unidirectional fluxes resulted in a large net loss of both Na+ and Cl− across the gills and a significant decrease in plasma [Na+] and [Cl−1]. The effects of exposure to 2 μg l−1 Ag on Na+ and Cl−1 transport were generally similar to those at 10 μg l−1, but were of a lesser magnitude. Unidirectional Na+ fluxes recovered immediately following removal of silver, after 48 h exposure to 2 μg l−1. Michaelis-Menten kinetic analysis demonstrated that the maximal rate of Na+ influx (Jmax) was significantly reduced after 48 h exposure to 2 μg l−1 Ag, whereas the affinity of the transport sites for Na+ (1/Km) was unaffected, indicating that the inhibition of Na+ influx by silver was of a non-competitive nature. Fish exposed to 10 μg l−1 Ag for 48 h also had significantly lower activities of the branchial enzymes Na+K+ ATPase (85% inhibition) and carbonic anhydrase (28% inhibition). The results of this study suggest that a disturbance of branchial ionoregulation, as a result of inhibition of branchial enzymes involved in ion transport, is the principal mechanism of the physiological toxicity of silver nitrate to freshwater fish.

Multiple functions of the crustacean gill: osmotic/ionic regulation, acid-base balance, ammonia excretion, and bioaccumulation of toxic metals

The crustacean gill is a multi-functional organ, and it is the site of a number of physiological processes, including ion transport, which is the basis for hemolymph osmoregulation; acid-base balance; and ammonia excretion. The gill is also the site by which many toxic metals are taken up by aquatic crustaceans, and thus it plays an important role in the toxicology of these species. This review provides a comprehensive overview of the ecology, physiology, biochemistry, and molecular biology of the mechanisms of osmotic and ionic regulation performed by the gill. The current concepts of the mechanisms of ion transport, the structural, biochemical, and molecular bases of systemic physiology, and the history of their development are discussed. The relationship between branchial ion transport and hemolymph acid-base regulation is also treated. In addition, the mechanisms of ammonia transport and excretion across the gill are discussed. And finally, the toxicology of heavy metal accumulation via the gill is reviewed in detail.

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 enzymes 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.

Characterization of the Carcinus maenas neuropeptidome by mass spectrometry and functional genomics.

Carcinus maenas, commonly known as the European green crab, is one of the best-known and most successful marine invasive species. While a variety of natural and anthropogenic mechanisms are responsible for the geographic spread of this crab, its ability to adapt physiologically to a broad range of salinities, temperatures and other environmental factors has enabled its successful establishment in new habitats. To extend our understanding of hormonal control in C. maenas, including factors that allow for its extreme adaptability, we have undertaken a mass spectral/functional genomics investigation of the neuropeptides used by this organism. Via a strategy combining MALDI-based high resolution mass profiling, biochemical derivatization, and nanoscale separation coupled to tandem mass spectrometric sequencing, 122 peptide paracrines/hormones were identified from the C. maenas central nervous system and neuroendocrine organs. These peptides include 31 previously described Carcinus neuropeptides (e.g. NSELINSILGLPKVMNDAamide [beta-pigment dispersing hormone] and PFCNAFTGCamide [crustacean cardioactive peptide]), 49 peptides only described in species other than the green crab (e.g. pQTFQYSRGWTNamide [Arg(7)-corazonin]), and 42 new peptides de novo sequenced here for the first time (e.g. the pyrokinins TSFAFSPRLamide and DTGFAFSPRLamide). Of particular note are large collections of FMRFamide-like peptides (25, including nine new isoforms sequenced de novo) and A-type allatostatin peptides (25, including 10 new sequences reported here for the first time) in this study. Also of interest is the identification of two SIFamide isoforms, GYRKPPFNGSIFamide and VYRKPPFNGSIFamide, the latter peptide known previously only from members of the astacidean genus Homarus. Using transcriptome analyses, 15 additional peptides were characterized, including an isoform of bursicon beta and a neuroparsin-like peptide. Collectively, the data presented in this study not only greatly expand the number of identified C. maenas neuropeptides, but also provide a framework for future investigations of the physiological roles played by these molecules in this highly adaptable species.

The distribution and physiological significance of carbonic anhydrase in vertebrate gas exchange organs.

The enzyme carbonic anhydrase (CA) catalyzes the reversible hydration/dehydration of CO(2) and water, maintaining a near-instantaneous equilibrium among all chemical species involved in the reaction. CA is found in association with all tissue and organ systems involved in the transport and excretion of CO(2), from the site of CO(2) production, metabolically active tissue such as muscle, to circulating red blood cells in the vasculature, to the various organs of gas exchange, the lungs and gills. The presence of the enzyme in every fluid compartment along the pathway of CO(2) transport appears necessary in order to allow the dehydration of HCO(3)(-) to keep pace with the rapid diffusion of CO(2) across biological membranes. Within the actual organ of gas exchange, CA is compartmentalized in multiple subcellular fractions, with the specific subcellular localization determining the enzymes physiological function.

The distribution of carbonic anhydrase type I and II isozymes in lamprey and trout : possible co-evolution with erythrocyte chloride/bicarbonate exchange

The subcellular distribution and kinetic properties of carbonic anhydrase were examined in red blood cells and gills of the lamprey, Petromyzon marinus, a primitive agnathan, and rainbow trout, Oncorhynchus mykiss, a modern teleost, in relation to the evolution of rapid Cl−/HCO3−exchange in the membrane of red blood cells. In the lamprey, which either lacks or has minimal red cell Cl−/HCO3−exchange, there has been no compensatory incorporation of carbonic anhydrase into the membrane fraction of either the red cell or the gill. Carbonic anhydrase activity in red cells is exclusively cytoplasmic, and the single isozyme displays kinetic properties typical of the type I, slow turnover, isozyme. In the red blood cells of the trout, however, which possess high amounts of the band-3 Cl−/HCO3−exchange protein, the single carbonic anhydrase isozyme appears to be kinetically similar to the type II, fast turnover, isozyme. It thus appears that the type I isozyme present in the red blood cells of primitive aquatic vertebrates was replaced in modern teleosts by the kinetically more efficient type II isozyme only after the incorporation and expression of a significant amount of the band-3 exchange protein in the membrane of the red cell.

Techniques for Measuring Carbonic Anhydrase Activity in Vitro

The isozymes of carbonic anhydrase (CA) (EC 4.2.1.1.) catalyze the reversible hydration—dehydration of carbon dioxide and water, with H+ ions being transferred between the active site of the enzyme and a surrounding buffer.3,16,17 This results in a change in pH as the reaction proceeds toward equilibrium. With the advent of rapid-responding pH and reference electrodes coupled to sensitive pH meters, these changes in H+ ion concentration could be accurately measured. That measurement, which can be performed either directly or indirectly, forms the underlying principle upon which the electrometric methods for the assay of CA activity are built.

Microarray-detected changes in gene expression in gills of green crabs (Carcinus maenas) upon dilution of environmental salinity

The interaction between environmental salinity and gene expression was studied in gills of the euryhaline green shore crab Carcinus maenas. A 4462-feature oligonucleotide microarray was used to analyze changes in transcript abundance in posterior ion-transporting gills at 8 time periods following transfer of animals from 32 to 10 or 15 ppt salinity. Transcripts encoding Na(+)/K(+)-ATPase α-subunit and cytoplasmic carbonic anhydrase were upregulated with significant changes between 6 and 24h post-transfer. Other transport proteins showing similar transcriptional upregulation were an organic cation transporter, a sodium/glucose cotransporter, an endomembrane protein associated with regulating plasma membrane protein composition, and a voltage-gated calcium channel. Transport proteins showing little transcriptional response included Na(+)/H(+) exchanger, Na(+)/K(+)/2Cl(-) cotransporter, and V-type H(+)-ATPase B subunit, all of which have been implicated in osmoregulatory ion transport across crustacean gill. Interestingly, there was little affect of salinity dilution on transcriptional expression of stress proteins, suggesting that salinity acclimation is part of normal physiology for C. maenas. Expression of transcripts encoding a variety of mitochondrial proteins was significantly upregulated between 4 days and 7 days post-transfer, consistent with the proliferation of mitochondria-rich cells previously observed at this time.

Extracellular Carbonic Anhydrase Activity and Carbonic Anhydrase Inhibitors in the Circulatory System of Fish

Carbonic anhydrase activity in the extracellular fluid of lower vertebrates is considered to be minimal, either because of the absence of carbonic anhydrase or because of the presence of naturally occurring inhibitors. The presence of carbonic anhydrase activity and circulating inhibitors was measured in plasma and subcellular fractions of gill tissue in elasmobranchs and teleosts. Plasma carbonic anhydrase activity was confirmed in the former but in extremely low amounts, especially compared with activity in red cells. The activity was correlated with plasma iron concentration and red cell hemolysis, which suggests that it is a byproduct of endogenous hemolysis during red cell turnover. A subcellular fraction of dogfish gills rich in microsomes contained significantly higher carbonic anhydrase activity than previously found in teleosts, making elasmobranchs the only aquatic lower vertebrates to possess putative basolateral membrane‐associated carbonic anhydrase in the gill vasculature. It is suggested that branchial membrane‐associated carbonic anhydrase is correlated more with a pH and/or CO2‐sensitive ventilatory drive than with the maintenance of resting CO2 excretion. The occurrence and effectiveness of plasma carbonic anhydrase inhibitors were highly species‐specific, with the salmonids having the most potent inhibitor. Cross‐reactivity of inhibitor to red cell carbonic anhydrase appeared to be related to phylogenetic proximity. Selection for the presence of carbonic anhydrase inhibitors in fish plasma appears to be the result of multiple physiological pressures, including preservation of red cell intracellular pH, ventilatory control, and red cell fragility.

Inhibition of carbonic anhydrase in the gills of two euryhaline crabs, Callinectes sapidus and Carcinus maenas, by heavy metals

Two subcellular fractions of gill tissue, cytoplasm and basolateral membranes, from two species of euryhaline decapod crustaceans, Callinectes sapidus and Carcinus maenas, acclimated to low salinity, were isolated via differential centrifugation. Carbonic anhydrase activity from both fractions was titrated against a variety of heavy metals in vitro. The metals Ag(+), Cd(2+), Cu(2+) and Zn(+) showed inhibitory action against the enzyme. Ki values for these metals against cytoplasmic CA from C. sapidus were in the range of 0.05-0.5 microM (for Ag(+), Cd(2+) and Cu(2+)) and 2-6 microM for Zn(+), some of the highest sensitivities reported for CA from an aquatic organism. The Ki values for these same metals were approximately 2-3 orders of magnitude higher for cytoplasmic CA from C. maenas, indicating that there are significant differences in heavy metal sensitivity in branchial CA from the two species, and that C. maenas possesses a metal-resistant CA isoform. It required concentrations of metals in the millimolar range, however, to inhibit CA activity from the membrane fraction of the gill of both species. There were no effects on either mortality or on hemolymph osmotic and ionic concentrations in C. maenas that were exposed to 10 microM Cd or Zn(+) at 32 per thousand salinity and subsequently transferred to 10 per thousand. The presence of a metal-resistant CA isoform in the gills of C. maenas suggests that this species would not be restricted from its normal estuarine environment by heavy metal pollution.