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Natural organic matter (NOM) is an important constituent of aquatic environments; however, its influence on aquatic biota remains poorly studied. In the current study, NOM was isolated from nine different sites in southern Ontario, Canada, by the on-site treatment of water by reverse osmosis, followed by cation exchange. NOM from each site was reconstituted to 10 mg of C/L and pH 7.0 and exposed to either adult rainbow trout implanted with indwelling catheters or to in vitro primary cultures of the gill epithelium grown on semipermeable membranes. In both the in vivo and in vitro preparations, NOM was found to hyperpolarize transepithelial potential (TEP), with the magnitude of this change correlating extremely well to the absorptivity of the NOM at 340 nm, which is an index of its aromaticity. Gill hyperpolarization appeared to be independent of Ca2+ complexation by the NOM in all but two samples tested. We argue that NOM has direct actions on the ionic transport and/or permeability properties of fish gills. While NOM effects on the bioavailability of contaminants are well-known, NOM actions on such fundamental physiological properties of the gills have previously been overlooked. These may be of comparable or greater magnitude than commonly reported for other water-quality variables (e.g., hardness, pH, salinity) and therefore of critical importance in ecological understanding and risk assessment. [ABSTRACT FROM AUTHOR]

Na+ and Ca2+ regulation were compared in two euryhaline species, killifish (normally estuarine-resident) and rainbow trout (normally freshwater-resident) during an incremental salinity increase. Whole-body unidirectional fluxes of Na+ and Ca2+, whole body Na+ and Ca2+, and plasma concentrations (trout only), were measured over 1-h periods throughout a total 6-h protocol of increasing salinity meant to simulate a natural tidal flow. Killifish exhibited significant increases in both Na+ influx and efflux rates, with efflux slightly lagging behind efflux up to 60% SW, but net Na+ balance was restored by the time killifish reached 100% SW. Whole body Na+ did not change, in agreement with the capacity of this species to tolerate daily salinity fluctuations in its natural habitat. In contrast, rainbow trout experienced a dramatic increase in Na+ influx (50-fold relative to FW values), but not Na+ efflux between 40 and 60% SW, resulting in a large net loading of Na+ at higher salinities (60–100% SW), and increases in plasma Na+ and whole body Na+ at 100% SW. Killifish were in negative Ca2+ balance at all salinities, whereas trout were in positive Ca2+ balance throughout. Ca2+ influx rate increased two- to threefold in killifish at 80 and 100% SW, but there were no concomitant changes in Ca2+ efflux. Ca2+ flux rates were affected to a larger degree in trout, with twofold increases in Ca2+ influx at 40% SW and sevenfold increases at 100% SW. Again, there was no change in Ca2+ efflux with salinity, so plasma Ca2+ concentration increased in 100% SW. As the killifish is regularly submitted to increased salinity in its natural environment, it is able to rapidly activate changes in unidirectional fluxes in order to ensure ionic homeostasis, in contrast to the trout. [ABSTRACT FROM AUTHOR]

A novel cell isolation technique was used to characterize cadmium and calcium uptake in distinct populations of gill cells from the adult rainbow trout (Oncorhynchus mykiss). A specific population of mitochondria-rich (MR) cell, termed the PNA+ MR cell (PNA is peanut lectin agglutinin), was found to accumulate over threefold more 109Cd than did PNA- MR cells, pavement cells (PV cells), and mucous cells during a 1-h in vivo exposure at 2.4 μg/l 109Cd. In vitro 109Cd exposures, performed in standard PBS and Cl--free PBS, at concentrations from 1 to 16 μg/l 109Cd, were also carried out to further characterize Cd2+ uptake kinetics. As observed during in vivo experiments, PNA+ MR cells accumulated significantly more 109Cd than did other cell types when exposures were performed by an in vitro procedure in PBS. Under such conditions, Cd2+ accumulation kinetics in all cell types could be described with Michaelis-Menten relationships, with Km values of ∼3.0 μg/l Cd (27 nM) for both MR cell subtypes and 8.6 μg/l Cd (77 nM) for PV cells. In similar experiments performed in Cl--free conditions, a significant reduction in 109Cd accumulation in PNA+ MR cells was seen but not in PNA- MR or in PV cells. In vitro 45Ca fluxes were also performed to determine the cellular localization of Ca2+ transport in these functionally distinct populations of gill cells. 45Ca uptake was most pronounced in PNA+ MR cells, with levels over threefold higher than those found in either PNA- MR or in PV cells. Results from the present study suggest that the PNA+ MR cell type is a high-affinity and high-capacity site for apical entry of Cd2+ and Ca2+ in the gill epithelium of rainbow trout. [ABSTRACT FROM AUTHOR]

Mitochondria-rich cells (MR cells) of the gills of rainbow trout undergo changes in relative distribution and biochemical function during acclimation to partialstrength (10‰) and full-strength (30‰) seawater. In isolated total gill cells, Na+/K+-ATPase activity increased fivefoid and H+-ATPase activity decreased fourfold when trout were acclimated to either 10&permi;; or 30‰ seawater. When total MR gill cells were separated based on differential binding to peanut lectin agglutinin (PNA), the PNA subtypes underwent a change in relative distribution in seawater-acclimated fish. In freshwater, the ratio of PNA-:PNA+ was 65:35 while in seawater the distribution changed to 20:80 PNA-:PNA+. Additionally, differential changes in Na+/K+-ATPase and H+-ATPase activity in each of the independent cell types occurred during seawater acclimation; Na+/K+-ATPase activity in the PNA- cells increased by 197% while in PNA+ cells Na+/K+ATPase decreased by 57%. However, H+-ATPase activity was decreased in both PNA- (84%) and PNA+ (72%) subtypes during acclimation to seawater. [ABSTRACT FROM AUTHOR]

Presents a study that developed a magnetic cell separation (MACS) technique for isolating and characterizing peanut lectin agglutin positive cells from rainbow trout gills. Experimental procedure; Gill digestion protocol; Isolation of mitochondria-rich (MR) chloride and MR pavement cells by MACS.

Juvenile rainbow trout (Oncorhynchus mykiss) were subjected to a 2-day radioactive pulse of 110mAg at 11.9 μg/l (as AgNO3), followed by a 19-day post-tracer exposure to non-radioactive Ag(I) (3.8 μg/l). The distribution of 110mAg in the gills, liver, intestine, kidney, brain and remaining carcass was investigated over a 19-day post-tracer period. Initially, the intestine contained the highest proportion of the 110mAg burden (34%), however, by day 8, less than 5% of the total radioactivity remained in this tissue. The majority of the 110mAg eliminated from the intestine appeared to distribute to the liver. Eventually, the 110mAg content in the liver accounted for as much as 65% of the total radioactivity in the fish. Apart from the liver and intestine, only the gills and carcass contained any appreciable amount (>5%) of the total body 110mAg content. Liver and gill samples were fractionated using differential centrifugation techniques to discern the subcellular distribution of 110mAg in these tissues. In the liver, the 110mAg levels in the cytosolic fraction increased from 35% to 72% of the total cellular burden between days 8 and 19, respectively. The radioactive pulse in the gills was predominantly found in a membrane compartment termed the nuclear fraction (∼60% of the total). Little change was observed over time (day 8 to day 19) to the subcellular distribution of Ag in the gills. Using size-exclusion chromatography, most (∼70%) of the 110mAg content in the liver cytosol eluted at a molecular weight characteristic of metallothionein. The cytosolic distribution of 110mAg in gills was quite diffuse, occurring primarily in the heavy molecular weight fractions. [Copyright &y& Elsevier]

Studies a subtype of mitochondria-rich (MR) cells in gills of freshwater rainbow trout using fluorescently labeled peanut lectin agglutinin (PNA-FITC). Change in PNA-FITC binding on filament epithelium with cortisol pretreatment contamination; Evidence for presence of mixed population of chloride and pavement cells in MR cells.

Abstract: The rate of acid-stimulated and phenamil-sensitive sodium (Na+) uptake was measured in three different cell lineages: pavement cells (PVC), total mitochondrion-rich (MR) cell populations, and peanut lectin agglutinin-negative mitochondrion-rich cells (PNA− MR) isolated from the rainbow trout gill epithelium. Despite the presence of basal levels of Na+ uptake in PVC, this transport was not enhanced by acidification, nor was it inhibited by independent treatment with bafilomycin (i.e., a V-type H+-ATPase inhibitor), phenamil (i.e., a specific inhibitor of ENaC), or Ag (a specific inhibitor of active Na+ transport in fish). In contrast, Na+ uptake in PNA− MR cells was increased by ~220% above basal levels following acidification of near 0.4 pH units in the presence of 1.0mM external Na+. Acid-stimulated Na+ transport was entirely inhibited by both phenamil and bafilomycin. Silver (Ag) and copper (Cu), which are known to interfere with active Na+ transport in fish, were also responsible for inhibiting acid stimulated Na+ uptake in PNA− MR cells, but by themselves had no effect on basal Na+ transport. Thus, we demonstrate that Ag specifically prevented acid-stimulated Na+ uptake in PNA− MR cells in a dose-dependent manner. We also demonstrate rapid (<1min) and significant inhibition of carbonic anhydrase (CA) by Ag in PNA− MR cells, but not in PVC. These data lend further support to the idea of a PNA− MR cell type as the primary site for Na+ uptake in the freshwater (FW) gill phenotype of rainbow trout. Moreover, these findings provide support for the importance of intracellular protons in regulating the movement of Na+ across the apical surface of the fish gill. [Copyright &y& Elsevier]