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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 PN[A.sup.+] MR cell (PNA is peanut lectin agglutinin), was found to accumulate over threefold more [sup.109]Cd than did PN[A.sup.-] MR cells, pavement cells (PV cells), and mucous cells during a 1-h in vivo exposure at 2.4 [micro]g/1 [sup.109]Cd. In vitro [sup.109]Cd exposures, performed in standard PBS and [Cl.sup.-]- free PBS, at concentrations from 1 to 16 [micro]g/1 [sup.l09]Cd, were also carried out to further characterize [Cd.sup.2+] uptake kinetics. As observed during in vivo experiments, PN[A.sup.+] MR cells accumulated significantly more [sup.109]Cd than did other cell types when exposures were performed by an in vitro procedure in PBS. Under such conditions, [Cd.sup.2+] accumulation kinetics in all cell types could be described with Michaelis-Menten relationships, with [K.sub.m] values of ~3.0 [micro]g/l Cd (27 nM) for both MR cell subtypes and 8.6 [micro]g/1 Cd (77 nM) for PV cells. In similar experiments performed in [Cl.sup.-]-free conditions, a significant reduction in [sup.109]Cd accumulation in PN[A.sup.+] MR cells was seen but not in PN[A.sup.+] MR or in PV cells. In vitro [sup.45]Ca fluxes were also performed to determine the cellular localization of [Ca.sup.2+] transport in these functionally distinct populations of gill cells. [sup.45]Ca uptake was most pronounced in PN[A.sup.+] MR cells, with levels over threefold higher than those found in either PN[A.sup.-] MR or in PV cells. Results from the present study suggest that the PN[A.sup.+] MR cell type is a high-affinity and high-capacity site for apical entry of [Cd.sup.2+] and [Ca.sup.2+] in the gill epithelium of rainbow trout. freshwater fish gill epithelium; peanut lectin agglutinin; metal binding; MINEQL+

A magnetic cell separation technique (MACS) was developed for isolating and characterizing peanut lectin agglutinin positive ([PNA.sup.+]) 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.sup.+] MR cells were enriched to >95% purity. Transmission electron microscopy analysis of both the [PNA.sup.+] and PNA negative ([PNA.sup.-]) fraction showed that PNA binds to MR chloride cells while the [PNA.sup.-] cell fraction is comprised of MR cells with features characteristic of pavement cells. Western blotting demonstrated that both [PNA.sup.+] and [PNA.sup.-] fractions had high levels of [Na.sup.+]-[K.sup.+]-ATPase and Sco1 expression; however, relative expression of [H.sup.+]-ATPase in [PNA.sup.+] and [PNA.sup.-] cells demonstrated that untreated fish had twofold higher [H.sup.+]-ATPase levels in [PNA.sup.-] cells relative to the [PNA.sup.+] cells. Furthermore, hypercapnic acidosis significantly increased the relative [H.sup.+]-ATPase expression on [PNA.sup.-] cells only, whereas metabolic alkalosis had no significant effect. peanut lectin; pavement cell; fish; hypercapnia; hydrogen-adenosine-triphosphatase

Peanut lectin binds to a subpopulation of mitochondria-rich cells in the rainbow trout gill epithelium. Am J Physiol Regulatory Integrative Comp Physiol 281: R1718-R1725, 2001.--Fluorescently labeled peanut lectin agglutinin (PNA-FITC) was used to identify a subtype of mitochondria-rich (MR) cells in the gills of freshwater rainbow trout. In situ binding of PNA-FITC was visualized by inverted fluorescence microscopy and found to bind to cells on the trailing edge of the filament epithelium as demonstrated by differential interference contrast optics. The amount of PNA-FITC binding on the filament epithelium increased with cortisol pretreatment concomitant with an increased chloride cell fractional area as demonstrated by scanning electron microscopy. Dispersed gill cells were isolated by trypsinization and separated using a discontinuous Percoll density gradient. Cells migrating to the 1.06-1.09 g/ml interface were found to be MR as demonstrated by staining with the vital mitochondrial dye 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide and transmission electron microscopy (TEM). However, only ~40% of the MR cells were found to bind PNA-FITC. Cortisol pretreatment increased the relative numbers of MR cells isolated from the dispersed gill cell population, but the relative proportions of PNA binding cells remained unchanged. Ultrastructural analysis of isolated cells in the TEM demonstrated that the MR cell fraction was comprised of a mixed population of chloride cells and pavement cells. chloride cells; pavement cells; peanut lectin; peanut lectin agglutinin; scanning electron microscopy; transmission electron microscopy; fish