An in vitro analysis of intestinal ammonia handling in fasted and fed freshwater rainbow trout (Oncorhynchus mykiss)

Ammonia transport and metabolism were investigated in the intestinal tract of freshwater rainbow trout which had been either fasted for 7 days, or fasted then fed a satiating meal of commercial trout pellets. In vivo, total ammonia concentrations (T _amm) in the chyme were approximately 1 mmol L^?1 across the entire intestine at 24 h after the meal. Highest chyme pH and P _NH3 values occurred in the posterior intestine. In vitro gut sac experiments examined ammonia handling with mucosal (Jm_amm) and serosal (Js_amm) fluxes under conditions of fasting and feeding, with either background (control ≤0.013 mmol L^?1) or high luminal ammonia concentrations (HLA = 1 mmol L^?1), the latter mimicking those seen in chyme in vivo. Feeding status (fasted or fed) appeared to influence ammonia handling by each individual section. The anterior intestine exhibited the greatest Jm_amm and Js_amm values under fasted control conditions, but these differences tended to disappear under typical post-feeding conditions when total endogenous ammonia production (Jt_amm = Js_amm ? Jm_amm, signs considered) was greatly elevated in all intestinal sections. Under fasted conditions, glutamate dehydrogenase (GDH) and glutaminase (GLN) activities were equal across all sections, but the ammonia-trapping enzyme glutamine synthetase (GS) exhibited highest activity in the posterior intestine, in contradiction to previous literature. Feeding clearly stimulated the total rate of endogenous ammonia production (Jt_amm), even in the absence of a high luminal ammonia load. This was accompanied by an increase in GDH activity of the anterior intestine, which was also the site of the largest Jt_amm. In all sections, during HLA exposure, either alone or in combination with feeding, there were much larger increases in endogenous Jt_amm, most of which was effluxed to the serosal solution. This is interpreted as a response to avoid potential cytotoxicity due to overburdened detoxification mechanisms in the face of elevated mucosal ammonia. Thus T _amm of the intestinal tissue remained relatively constant regardless of feeding status and exposure to HLA. Ammonia production by the gut may explain up to 18 % of whole-body ammonia excretion in vivo under fasting conditions, and 47 % after feeding, of which more than half originates from endogenous production rather than from absorption from the lumen.     

Effects of epinephrine on branchial and renal calcium handling in the rainbow trout

Acute exposure of rainbow trout (Salmo gairdneri) to low external calcium (25 microM) caused an immediate but transient increase in plasma epinephrine concentration that may have been related to a concomitant depression of blood pH. Intra-arterial infusion of epinephrine at normal ambient calcium levels (0.35 mM) for 4 h caused circulating levels of epinephrine to rise from 2.9 X 10(-9) to 8.0 X 10(-8) M but did not affect norepinephrine levels, or branchial unidirectional calcium fluxes. Active (ATP-dependent) calcium transport across basolateral plasma membranes prepared from gill epithelial cells was not affected by pretreatment of fish with epinephrine or by direct application of epinephrine or cAMP, in vitro. Epinephrine infusion elevated urine flow rate, decreased urine pH, and increased urine phosphate levels significantly. Net renal calcium efflux increased significantly as a result of the increased urine flow rate. It is concluded that epinephrine does not stimulate branchial calcium uptake or renal conservation of calcium in rainbow trout at normal external calcium levels and therefore we cautiously suggest that epinephrine is unlikely to be involved in calcium balance during periods of exposure to low external calcium. Instead, epinephrine may play a role in compensating the acid-base disturbances and the increased branchial water influx that are associated with exposure to low ambient calcium.     

Branchial and renal excretion of urea and urea analogues in the plainfin midshipman,Porichthys notatus

This study investigated whether urea transport mechanisms were present in the gills of the ammoniotelic plainfin midshipman (Porichthys notatus), similar to those recently documented in its ureotelic relative (family Batrachoididae), the gulf toadfish (Opsanus beta). Midshipmen were fitted with internal urinary and caudal artery catheters for repetitive sampling of urine and blood in experiments and radiolabeled urea analogues ([(14)C]-thiourea and [(14)C]-acetamide) were used to evaluate the handling of these substances. Isosmotically balanced infusions of urea were used to raise plasma and urine urea concentrations to levels surpassing physiological levels by 8.5-fold and 6.4-fold, respectively. Despite these high urea levels, there was no observable transport maximum in either renal or branchial urea excretion rate, a result mirrored by the total uptake of fish exposed to a range of environmental urea concentrations. Permeability to urea appeared to be symmetrical in the two directions. At comparable plasma concentrations the branchial clearance rate of acetamide was 74% that of urea while branchial clearance rate of thiourea was 55% that of urea. For influx, the comparable values were 60% and 36%, indicating the same pattern. In contrast, the secretion clearance rate of acetamide by the kidney was 56% that of urea while the rate of thiourea secretion clearance was 137% greater than that of urea, with both urea and thiourea being more concentrated in the urine than in the plasma. In addition, the secretion clearance rates of thiourea and urea were significantly greater than those of water and Cl(-), whereas acetamide, water and Cl(-) were found equally in the plasma and urine, appearing to passively equilibrate between the two fluids. Based on our findings, there appear to be two distinct transport mechanisms involved in urea excretion in the plainfin midshipmen, one in the gill (a facilitated diffusion type transporter) and one in the kidney (an active transport mechanism), each of which does not saturate even at plasma urea concentrations that greatly exceed physiological levels. These transporters appear to be similar to those in the midshipman's ureotelic relative, the gulf toadfish.     

Peptidylarginine deiminase gene is differentially expressed in freshwater and brackish water rainbow trout

Peptidylarginine deiminase (PADI)-like cDNA sequence was isolated from rainbow trout (Oncorhynchus mykiss). It consists of a 111-bp 5′-untranslated region, a 731-bp 3′-UTR, and a 2,010-bp open reading frame encoding a protein of 669 amino acids. In the presence of calcium ions, PADI enzymes catalyze the post-translational modification reaction generating citrulline residues. Mammalian PADI enzymes are involved in a number of regulatory processes during cell differentiation and development such as skin keratinization, myelin maturation, and histone deimination. Though five PADI isotypes have been isolated from mammals, in bony fish only one PADI enzyme is present, which contains conserved amino acid residues responsible for catalysis and calcium ion-binding. Sequence identity of piscine PADI protein sequences available at gene databases exceeds 67%. Phylogenetic analyses revealed that not only piscine, but also amphibian and avian PADI-like proteins share most identical amino acid residues with mammalian PADI2. mRNA level of trout PADI-like gene is high in skin, fin, gills, brain, and spleen of rainbow trout. Quantitative Real-Time RT-PCR revealed that PADI gene is differentially expressed in liver, trunk kidney, and spleen of two trout strains, the freshwater-cultured STEELHEAD trout and the brackish water strain BORN.     

A comparative study of antioxidant enzyme activities in freshwater and seawater-adapted rainbow trout.

Comparative studies were performed on the antioxidant enzyme activities and thiobarbituric acid reactive substance (TBARS) concentration in liver and red cells of two groups of rainbow trout (Oncorhynchus mykiss). The fish of the first group were cultured in freshwater and the others were adapted to sea-water by by being transferred from freshwater at 5-6 months of age. Catalase (CAT), glutathione peroxidase (GPX), and glutathione S-transferase (GST) activities were significantly higher in hepatic and extrahepatic tissues in both of the fish groups. Superoxide dismutase (SOD) activities were found lower in the seawater-adapted trout than in the freshwater-cultured trout. In both tissues, TBARS were found significantly higher in the seawater-adapted trout than in the freshwater trout. It was also observed that the red cells of the seawater-adapted trout were much more resistant to oxidative stress than the red cells of the freshwater-cultured trout. The results implicate that antioxidant capacities in the seawater-adapted trout and freshwater trout may be related to physical and chemical characteristics of the environment.     

Accessory cells in the gill epithelium of the freshwater rainbow trout Salmo gairdneri

Two types of mitochondria-rich cells were identified in the gill epithelium of the freshwater-adapted rainbow trout, Salmo gairdneri, after selective impregnation of their tubular system with reduced osmium. A first type consisted of large cells with a poorly developed and loosely anastomosed tubular system; thus, that resembled the chloride cells commonly encountered in the gill epithelium of freshwater-adapted euryhaline fishes. A second type comprised smaller cells with an extensively developed and tightly anastomosed tubular system. These never reached the basal lamina of the gill epithelium and were adjacent to chloride cells, to which they were linked by shallow apical junctions (100-200 nm); thus, they resembled accessory cells, which are currently found in the gill epithelium of seawater-adapted fishes but are usually lacking in freshwater living fishes. Transfer of the freshwater-adapted trout into seawater induced the proliferation of the tubular system in the chloride cells and the formation of lateral plasma membrane interdigitations between accessory cells and the apical portion of the chloride cells. The length of the apical junction sealing off this extended intercellular space was reduced to 20-50 nm. The tubular system of the accessory cells was not modified. The extension of the tubular system in the chloride cells of the seawater-adapted fishes indicated that, as in most euryhaline fishes, these cells have a role in the adaptation of the rainbow trout to seawater. In contrast, the function of the presumptive accessory cells in freshwater trout remains to be established.     

Calcitonin induces hypercalcemia in grey mullet and immature freshwater and sea-water adapted rainbow trout

1. Changes in plasma calcium levels, in response to salmon calcitonin injections, were studied in freshwater and sea-water adapted trout (Salmo gairdnerii) and in grey mullet (Chelon labrosus). 2. Low doses (0.1 ng sCT/100 g body weight) elicited hypercalcemia in the two species studied. 3. High doses (0.5 microgram) provoked hypocalcemia only in freshwater and sea-water adapted trout. 4. An hypercalcemic response appears as the primordial effects of CT injections, higher doses of CT leading to hypocalcemic effects.     

Effect of feeding time on postprandial nitrogen excretion and energy expenditure in rainbow trout

The effect of feeding time (dawn or midnight) on nitrogen excretion and energy expenditure was studied in immature rainbow trout using measurements of respiratory gas exchange. Fish (mean individual weight 70 g) were maintained indoors under natural photoperiod and fed by hand (commercial food pellets) at a rate of 1% weight/day⁻¹. Rates of ammonia and CO₂ excretion and O₂ uptake were measured every hour. Ammonia excretion increased immediately after feeding in fish fed at midnight, and 2h after feeding in fish fed at dawn. Ammonia excretion and energy supply from protein catabolism, were higher in trout fed at midnight than in those fed at dawn, while total energy expenditure was the same in both groups. The results suggested that trout fed in phase with their natural feeding rhythm use dietary protein more efficiently for growth than do trout fed out of phase with the natural rhythm.     

An in vitro investigation of gastrointestinal Na+ uptake mechanisms in freshwater rainbow trout

In vitro gut-sac preparations of all four sections (stomach, anterior, mid, and posterior intestine) of the gastrointestinal tract (GIT) of freshwater rainbow trout, together with radiotracer (²²Na) techniques, were used to study unidirectional Na⁺ uptake rates (UR, mucosal → blood space) and net absorptive fluid transport rates (FTR) under isosmotic conditions (mucosal = serosal osmolality). On an area-specific basis, unidirectional Na⁺ UR was highest in the mid-intestine, but when total gut area was taken into account, the three intestinal sections contributed equally, with very low rates in the stomach. The theoretical capacity for Na⁺ uptake across the whole GIT is sufficient to supply all of the animal’s nutritive requirements for Na⁺. Transport occurs by low affinity systems with apparent K _m values 2–3 orders of magnitude higher than those in the gills, in accord with comparably higher Na⁺ concentrations in chyme versus fresh water. Fluid transport appeared to be Na⁺-dependent, such that treatments which altered unidirectional Na⁺ UR generally altered FTR in a comparable fashion. Pharmacological trials (amiloride, EIPA, phenamil, bafilomycin, furosemide, hydrochlorothiazide) conducted at a mucosal Na⁺ concentration of 50 mmol L^?1 indicated that GIT Na⁺ uptake occurs by a variety of apical mechanisms (NHE, Na⁺ channel/H⁺ ATPase, NCC, NKCC) with relative contributions varying among sections. However, at a mucosal Na⁺ concentration of 10 mmol L^?1, EIPA, phenamil, bafilomycin, and hydrochlorothiazide were no longer effective in inhibiting unidirectional Na⁺ UR or FTR, suggesting the contribution of unidentified mechanisms under low Na⁺ conditions. A preliminary model is presented.     

Isolation and characterization of mitochondria-rich cell types from the gill of freshwater rainbow trout

A magnetic cell separation technique (MACS) was developed for isolating and characterizing peanut lectin agglutinin positive (PNA(+)) cells from rainbow trout gills. Percoll density separated mitochondria-rich (MR) cells were serially labeled with PNA-FITC and an anti-FITC antibody covalently coupled to a 50-nm iron particle and then applied to a magnetic column. PNA(+) MR cells were enriched to >95% purity. Transmission electron microscopy analysis of both the PNA(+) and PNA negative (PNA(-)) fraction showed that PNA binds to MR chloride cells while the PNA(-) cell fraction is comprised of MR cells with features characteristic of pavement cells. Western blotting demonstrated that both PNA(+) and PNA(-) fractions had high levels of Na(+)-K(+)-ATPase and Sco1 expression; however, relative expression of H(+)-ATPase in PNA(+) and PNA(-) cells demonstrated that untreated fish had twofold higher H(+)-ATPase levels in PNA(-) cells relative to the PNA(+) cells. Furthermore, hypercapnic acidosis significantly increased the relative H(+)-ATPase expression on PNA(-) cells only, whereas metabolic alkalosis had no significant effect.     

Physiological and molecular analysis of the interactive effects of feeding and high environmental ammonia on branchial ammonia excretion and Na+ uptake in freshwater rainbow trout

Recently, a “Na⁺/NH₄ ⁺ exchange complex” model has been proposed for ammonia excretion in freshwater fish. The model suggests that ammonia transport occurs via Rhesus (Rh) glycoproteins and is facilitated by gill boundary layer acidification attributable to the hydration of CO₂ and H⁺ efflux by Na⁺/H⁺ exchanger (NHE-2) and H⁺-ATPase. The latter two mechanisms of boundary layer acidification would occur in conjunction with Na⁺ influx (through a Na⁺ channel energized by H⁺-ATPase and directly via NHE-2). Here, we show that natural ammonia loading via feeding increases branchial mRNA expression of Rh genes, NHE-2, and H⁺-ATPase, as well as H⁺-ATPase activity in juvenile trout, similar to previous findings with ammonium salt infusions and high environmental ammonia (HEA) exposure. The associated increase in ammonia excretion occurs in conjunction with a fourfold increase in Na⁺ influx after a meal. When exposed to HEA (1.5 mmol/l NH₄HCO₃ at pH 8.0), both unfed and fed trout showed differential increases in mRNA expression of Rhcg2, NHE-2, and H⁺-ATPase, but H⁺-ATPase activity remained at control levels. Unfed fish exposed to HEA displayed a characteristic reversal of ammonia excretion, initially uptaking ammonia, whereas fed fish (4 h after the meal) did not show this reversal, being able to immediately excrete ammonia against the gradient imposed by HEA. Exposure to HEA also led to a depression of Na⁺ influx, demonstrating that ammonia excretion can be uncoupled from Na⁺ influx. We suggest that the efflux of H⁺, rather than Na⁺ influx itself, is critical to the facilitation of ammonia excretion.     

Renal function in the freshwater rainbow trout after dietary cadmium acclimation and waterborne cadmium challenge

Renal function was examined in adult rainbow trout (Oncorhynchus mykiss) after chronic exposure to a sublethal level of dietary Cd (500 mg/kg diet) for 52 d and during a subsequent challenge to waterborne Cd (10 microg/L) for 72 h. Dietary Cd had no major effects on UFR (urine flow rate) and GFR (glomerular filtration rate) but caused increased renal excretion of glucose, protein, and major ions (Mg(2+), Zn(2+), K(+), Na(+), Cl(-) but Ca(2+)). However, dietary Cd did not affect any plasma ions except Na(+) which was significantly elevated in the Cd-acclimated trout. Plasma glucose and ammonia levels fell by 25% and 36% respectively, but neither plasma nor urine urea was affected in Cd-acclimated fish. Dietary Cd exposure resulted in a remarkable increase of Cd load in the plasma (48-fold, approximately 22 ng/mL) and urine (60-fold, 8.9 ng/mL), but Cd excretion via the kidney was negligible on a mass-balance basis. Clearance ratio analysis indicates that all ions, Cd, and metabolites were reabsorbed strongly (58-100%) in both na?ve and dietary Cd exposed fish, except ammonia which was secreted in both groups. Mg(2+), Na(+), Cl(-) and K(+) reabsorption decreased significantly (3-15%) in the Cd-exposed fish relative to the control. Following waterborne Cd challenge, GFR and UFR were affected transiently, and only Mg(2+) and protein excretion remained elevated with no recovery with time in Cd-acclimated trout. Urinary Ca(2+) and Zn(2+) excretion rates dropped with an indication of renal compensation towards plasma declines of both ions. Cadmium challenge did not cause any notable effects on urinary excretion rates of metabolites. However, a significant decrease in Mg(2+) reabsorption but an increase in total ammonia secretion was observed in the Cd-acclimated fish. The study suggests that dietary Cd acclimation involves physiological costs in terms of renal dysfunction and elevated urinary losses.     

Physical characterization of high-affinity gastrointestinal Cu transport in vitro in freshwater rainbow trout Oncorhynchus mykiss

This study investigated the transport of copper (Cu) in the gut of trout. Examination of the spatial distribution of Cu along the digestive tract and a physical characterization of the uptake process was carried out using an in vitro gut sac technique and ⁶⁴Cu as a tracer. Unidirectional Cu uptake was highest in the anterior intestine followed in decreasing order by the posterior intestine, mid intestine and the stomach. Cu uptake was resistant to hypoxia and appeared to be fueled equally well by Cu(II) or Cu (I) at Cu concentrations typically found in the fluid phase of the chyme in vivo in the trout intestine. Transport demonstrated saturation kinetics (e.g. K _m = 31.6 μM, J _max = 17 pmol cm⁻² h⁻¹, in mid intestine) at low Cu levels representative of those measured in the chyme in vivo, with a diffusive component at higher Cu concentrations. Q ₁₀ analysis indicated Cu uptake is via diffusion across the apical membrane and biologically mediated across the basolateral membranes of enterocytes. The presence of l-histidine but not d-histidine stimulated both Cu and Na uptake suggesting a common pathway for the transport of Cu/Na with l-histidine.     

Differential conservation and divergence of fertility genes boule and dazl in the rainbow trout

Background

The genes boule and dazl are members of the DAZ (Deleted in Azoospermia) family encoding RNA binding proteins essential for germ cell development. Although dazl exhibits bisexual expression in mitotic and meiotic germ cells in diverse animals, boule shows unisexual meiotic expression in invertebrates and mammals but a bisexual mitotic and meiotic expression in medaka. How boule and dazl have evolved different expression patterns in diverse organisms has remained unknown.

Methodology and Principal Findings

Here we chose the fish rainbow trout (Oncorhynchus mykiss) as a second lower vertebrate model to investigate the expression of boule and dazl. By molecular cloning and sequence comparison, we identified cDNAs encoding the trout Boule and Dazl proteins, which have a conserved RNA-recognition motif and a maximal similarity to their homologs. By RT-PCR analysis, adult RNA expression of trout boule and dazl is restricted to the gonads of both sexes. By chromogenic and two-color fluorescence in situ hybridization, we revealed bisexual and germline-specific expression of boule and dazl. We found that dazl displays conserved expression throughout gametogenesis and concentrates in the Balbinani''s body of early oocytes and the chromatoid body of sperm. Surprisingly, boule exhibits mitotic and meiotic expression in the male but meiosis-specific expression in the female.

Conclusions

Our data underscores differential conservation and divergence of DAZ family genes during vertebrate evolution. We propose a model in which the diversity of boule expression in sex and stage specificity might have resulted from selective loss or gain of its expression in one sex and mitotic germ cells.     

Identification and expression of natriuretic peptide receptor type-A and -B mRNA in freshwater and seawater rainbow trout

Natriuretic peptide receptors mediate the physiological response of natriuretic peptide hormones. One of the natriuretic peptide receptor types is the particulate guanylyl cyclase receptors, of which there are two identified: NPR-A and NPR-B. In fishes, these have been sequenced and characterized in eels, medaka, and dogfish shark (NPR-B only). The euryhaline rainbow trout provides an opportunity to further pursue examination of the system in teleosts. In this study, partial rainbow trout NPR-A-like and NPR-B-like mRNA sequences were identified via PCR and cloning. The sequence information was used in real-time PCR to examine mRNA expression in a variety of tissues of freshwater rainbow trout and rainbow trout acclimated to 35 parts per thousand seawater for a period of 10 days. In the excretory kidney and posterior intestine, real-time PCR analysis showed greater expression of NPR-B in freshwater fish than in those adapted to seawater; otherwise, there was no difference in the expression of the individual receptors in fresh water or seawater. In general, the expression of the NPR-A and NPR-B type receptors was quite low. These findings indicate that NPR-A and NPR-B mRNA expression is minimally altered under the experimental regime used in this study.     

Genetic comparison between sympatric anadromous steelhead and freshwater resident rainbow trout in British Columbia, Canada

Sympatrically occurring steelhead and rainbowtrout (n = 100) were collected from fiveriver systems in British Columbia, Canada, and1300 bp from the mitochondrial genome and 270bp of the nuclear growth hormone 2 gene weresequenced to determine if the two forms aregenetically differentiated. ND3 and D-loopsequence differences produced nine haplotypeswhile two length variants of the GH2 intron Dwere present. In one river system, steelheadand rainbow were genetically divergent based onmtDNA data but overall, no consistent geneticdifference between life history types wasfound. Rather, genetic differences wereassociated with geography, suggesting thatsteelhead and rainbow trout are polyphyleticand the result of parallel evolution ratherthan members of two distinct lineages.     

Ammonia excretion and urea handling by fish gills: present understanding and future research challenges

In fresh water fishes, ammonia is excreted across the branchial epithelium via passive NH(3) diffusion. This NH(3) is subsequently trapped as NH(4)(+) in an acidic unstirred boundary layer lying next to the gill, which maintains the blood-to-gill water NH(3) partial pressure gradient. Whole animal, in situ, ultrastructural and molecular approaches suggest that boundary layer acidification results from the hydration of CO(2) in the expired gill water, and to a lesser extent H(+) excretion mediated by apical H(+)-ATPases. Boundary layer acidification is insignificant in highly buffered sea water, where ammonia excretion proceeds via NH(3) diffusion, as well as passive NH(4)(+) diffusion due to the greater ionic permeability of marine fish gills. Although Na(+)/H(+) exchangers (NHE) have been isolated in marine fish gills, possible Na(+)/NH(4)(+) exchange via these proteins awaits evaluation using modern electrophysiological and molecular techniques. Although urea excretion (J(Urea)) was thought to be via passive diffusion, it is now clear that branchial urea handling requires specialized urea transporters. Four urea transporters have been cloned in fishes, including the shark kidney urea transporter (shUT), which is a facilitated urea transporter similar to the mammalian renal UT-A2 transporter. Another urea transporter, characterized but not yet cloned, is the basolateral, Na(+) dependent urea antiporter of the dogfish gill, which is essential for urea retention in ureosmotic elasmobranchs. In ureotelic teleosts such as the Lake Magadi tilapia and the gulf toadfish, the cloned mtUT and tUT are facilitated urea transporters involved in J(Urea). A basolateral urea transporter recently cloned from the gill of the Japanese eel (eUT) may actually be important for urea retention during salt water acclimation. A multi-faceted approach, incorporating whole animal, histological, biochemical, pharmacological, and molecular techniques is required to learn more about the location, mechanism of action, and functional significance of urea transporters in fishes.     

Cadmium and calcium uptake in isolated mitochondria-rich cell populations from the gills of the freshwater rainbow trout

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 microg/l 109Cd. In vitro 109Cd exposures, performed in standard PBS and Cl- -free PBS, at concentrations from 1 to 16 microg/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 approximately 3.0 microg/l Cd (27 nM) for both MR cell subtypes and 8.6 microg/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.