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We have studied intracellular pH (pH(i)) recovery in isolated trout gill mitochondrion-rich (MR) cells following acidification by the NH(4)Cl pre-pulse technique. Within a mixed MR cell population, one cell type displayed Na(+)-independent pH(i) recovery while the other cell type lacked a Na(+)-independent pH(i) recovery. Cells displaying Na(+) independent recovery exhibited a significantly higher buffering capacity compared to cells lacking Na(+)-independent pH(i) recovery. Cells displaying Na(+) independent recovery were identified as PNA(+) (peanut lectin agluttinin binding) MR cells while those unable to recover were identified as PNA(-) (non-peanut lectin agluttinin binding) MR cells. Therefore, recovery from acidification in the absence of Na(+) provides a direct functional marker for PNA(+) and PNA(-) MR cells. Re-addition of Na(+) to acidified cells resulted in a transient pH(i) recovery in both cell types. This event was abolished by amiloride (500 microM) but it was insensitive to phenamil (50 microM). The phorbol ester PMA (1 microM) potentiated the Na(+) induced pH(i) recovery suggesting that activation by PKC is required for continuous Na(+)/H(+) exchanger activity in trout gill MR cells. This study is the first functional description of pH(i) recovery in lectin-identified trout gill MR cells and provides insight into a putative cellular signaling mechanism that may control pH(i) regulation in the gill epithelium., (2009 Elsevier Inc. All rights reserved.)

The lack of a suitable flat epithelial preparation isolated directly from the freshwater fish gill has led, in recent years, to the development of cultured gill epithelia on semipermeable supports. To date, their minimal capacity to actively transport ions has limited their utility as ionoregulatory models. The current study describes a new method of culturing gill epithelia consisting either of an enriched population of pavement (PV) cells or a mixed population of PV cells and mitochondria-rich (MR) cells from the gills of adult rainbow trout. Although the cell culture approach is similar to the double-seeded insert (DSI) technique described previously, it makes use of Percoll density centrifugation to first separate populations of PV and MR cells, which are then seeded on cell culture supports in varying proportions on successive days so as to produce preparations enriched in one or the other cell types. Based on rhodamine staining, the MR cell-rich epithelia exhibited a threefold higher enrichment of MR cells compared to traditional DSI preparations. In general, MR cell-rich epithelia developed extremely high transepithelial resistances (TER; >30 kOmega cm(2)) and positive transepithelial potentials (TEP) under symmetrical conditions (i.e., L15 medium on both apical and basolateral sides). Apical exposure of cell cultures to freshwater reduced TER and produced a negative TEP in all the epithelial preparations, although MR cell-rich epithelia maintained relatively high TER and negative TEP for over 2 d under these asymmetrical conditions. Measurement of unidirectional Na(+) fluxes and application of the Ussing flux ratio criterion demonstrated active Na(+) uptake in PV cell-rich and MR cell-rich epithelia under both symmetrical and asymmetrical conditions. In comparison, Ca(2+) uptake and Na(+)/K(+)-ATPase activity were significantly elevated in MR cell-rich preparations relative to the traditional DSI or PV cell-rich cultures under symmetrical conditions. This new methodology enhances our ability to tailor cultured gill epithelia on semipermeable supports with different proportions of PV cells and MR cells, thereby illuminating the ionoregulatory functions of the two cell types.

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.

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.

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]

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: We have studied intracellular pH (pHi) recovery in isolated trout gill mitochondrion-rich (MR) cells following acidification by the NH4Cl pre-pulse technique. Within a mixed MR cell population, one cell type displayed Na+-independent pHi recovery while the other cell type lacked a Na+-independent pHi recovery. Cells displaying Na+ independent recovery exhibited a significantly higher buffering capacity compared to cells lacking Na+-independent pHi recovery. Cells displaying Na+ independent recovery were identified as PNA+ (peanut lectin agluttinin binding) MR cells while those unable to recover were identified as PNA− (non-peanut lectin agluttinin binding) MR cells. Therefore, recovery from acidification in the absence of Na+ provides a direct functional marker for PNA+ and PNA− MR cells. Re-addition of Na+ to acidified cells resulted in a transient pHi recovery in both cell types. This event was abolished by amiloride (500µM) but it was insensitive to phenamil (50µM). The phorbol ester PMA (1µM) potentiated the Na+ induced pHi recovery suggesting that activation by PKC is required for continuous Na+/H+ exchanger activity in trout gill MR cells. This study is the first functional description of pHi recovery in lectin-identified trout gill MR cells and provides insight into a putative cellular signaling mechanism that may control pHi regulation in the gill epithelium. [Copyright &y& Elsevier]