Transport properties of cultured branchial epithelia from freshwater rainbow trout: a novel preparation with mitochondria-rich cells

2000 ◽  
Vol 203 (10) ◽  
pp. 1523-1537 ◽  
Author(s):  
M. Fletcher ◽  
S.P. Kelly ◽  
P. Part ◽  
M.J. O'Donnell ◽  
C.M. Wood
Keyword(s):  
Rainbow Trout ◽  
Flux Ratio ◽  
Culture Conditions ◽  
Chloride Cells ◽  
Pavement Cells ◽  
Ratio Criterion ◽  
Peg 4000 ◽  
Tight Epithelia ◽  
Gill Filaments

A new double-seeded insert (DSI) technique is described for culture of branchial epithelial preparations from freshwater rainbow trout on filter supports. DSI epithelia contain both pavement cells and mitochondria-rich (MR) cells (15.7+/−2.5 % of total cell numbers). MR cells occur singly or in clusters, are voluminous, open apically to the ‘external environment’ and exhibit ultrastructural characteristics similar to those found in the ‘chloride cells’ of freshwater fish gills. After 6–9 days in culture with Leibovitz's L-15 medium on both surfaces (symmetrical conditions), transepithelial resistance (TER) stabilized at values as high as 34 k capomega cm(2), indicative of electrically ‘tight’ epithelia. The density of MR cells, the surface area of their clusters and transepithelial potential (TEP; up to +8 mV basolateral positive, mean +1.9+/−0.2 mV) were all positively correlated with TER. In contrast, preparations cultured using an earlier single-seeded insert (SSI) technique contained only pavement cells and exhibited a negligible TEP under symmetrical conditions. Na(+)/K(+)-ATPase activities of DSI preparations were comparable with those in gill filaments, but did not differ from those of SSI epithelia. Replacement of the apical medium with fresh water to mimic the in vivo situation (asymmetrical conditions) induced a negative TEP (−6 to −15 mV) and increased permeability to the paracellular marker PEG-4000. Under symmetrical conditions, unidirectional Na(+) and Cl(−) fluxes were in balance, and there was no active transport by the Ussing flux ratio criterion. Under asymmetrical conditions, there were large effluxes, small influxes and evidence for active Cl(−) uptake and Na(+) extrusion. Unidirectional Ca(2+) fluxes were only 0.5-1.0 % of Na(+) and Cl(−) fluxes; active net Ca(2+) uptake occurred under symmetrical conditions and active net extrusion under asymmetrical conditions. Thus, DSI epithelia exhibit some of the features of the intact gill, but improvements in culture conditions are needed before the MR cells will function as true freshwater ‘chloride cells’.

scholarly journals Calcium transport by isolated skin of rainbow trout

1992 ◽  
Vol 166 (1) ◽  
pp. 297-316 ◽  
Author(s):  
W. S. Marshall ◽  
S. E. Bryson ◽  
C. M. Wood
Keyword(s):  
Rainbow Trout ◽  
Cell Density ◽  
Flux Ratio ◽  
Adenosine Monophosphate ◽  
Mucosal Surface ◽  
Chloride Cells ◽  
Serosal Surface ◽  
Ratio Criterion

The skin overlying the cleithrum bone of freshwater-acclimated rainbow trout contains numerous mitochondria-rich (MR) cells, as detected by DASPEI fluorescence. This tissue was mounted in vitro in an Ussing-style chamber with fresh water on the mucosal surface and saline supplemented with bovine serum albumin on the serosal surface. The preparation developed a high transepithelial resistance and a small transepithelial potential (Vt), positive on the serosal side. Radioisotopic flux measurements indicated that the preparation actively transported Ca2+ from the mucosal to the serosal surface, as assessed by the Ussing flux ratio criterion. Ca2+ transport was positively correlated with MR cell density. Cortisol pretreatment in vivo reduced MR cell density and increased Vt but did not significantly alter Ca2+ fluxes. Ca2+ transport was unaffected by adrenergic agonists (10(−5) mol l-1 adrenaline, clonidine, isoprenaline) or cyclic AMP stimulants (10(−3) mol l-1 dibutyryl cyclic adenosine monophosphate, db-cAMP, plus 10(−4) mol l-1 isobutylmethylxanthine, IBMX) applied to the serosal surface. The Ca2+ ionophore ionomycin (1 × 10(−6)-3.2 × 10(−6) mol l-1 on the mucosal surface) increased both unidirectional Ca2+ fluxes and caused Ca2+ to accumulate within the epithelium. Lanthanum (10(−4) mol l-1) did not inhibit unidirectional Ca2+ fluxes, but apparently displaced Ca2+ from binding sites on the mucosal surface. Unlike Ca2+, movements of Na+ and Cl- across the epithelium were passive, as assessed by the flux ratio criterion, and neither adrenaline nor db-cAMP plus IBMX had any effect on Na+ or Cl- fluxes or electrical properties. These results indicate that ion transport across the skin mediated by MR cells (‘chloride cells’) contributes to Ca2+ but not to NaCl balance in freshwater trout.

Characterization of branchial transepithelial calcium fluxes in freshwater trout, Salmo gairdneri

1988 ◽  
Vol 254 (3) ◽  
pp. R491-R498 ◽  
Author(s):  
S. F. Perry ◽  
G. Flik
Keyword(s):  
Active Transport ◽  
Calcium Uptake ◽  
Chloride Cells ◽  
Salmo Gairdneri ◽  
Apical Surface ◽  
Electrochemical Gradient ◽  
Plasma Membrane Vesicles ◽  
Pavement Cells ◽  
Calcium Fluxes

Experiments were performed to determine whether gill transepithelial calcium fluxes in the freshwater trout (Salmo gairdneri) are passive or require active transport and to characterize the mechanisms involved. A comparison of the in vivo unidirectional flux ratios with the flux ratios calculated according to the transepithelial electrochemical gradients revealed that calcium uptake from the water requires active transport of Ca2+. The inhibition of calcium uptake by external lanthanum, the specific deposition of lanthanum on the apical surface of chloride cells, and the favorable electrochemical gradient for calcium across the apical membrane suggest that the initial step in branchial calcium uptake is the passive entry of calcium into the cytosol of chloride cells through apical channels that are permeable to calcium. The study of gill basolateral plasma membrane vesicles demonstrated the existence of a high-affinity calmodulin-dependent calcium-transporting system [half-maximal Ca2+ concentration (K0.5) = 160 nM, Vmax = 1.86 nmol.min-1.mg protein-1]. This system actively transports calcium from the cytosol of chloride cells into the plasma against a sizeable electrochemical gradient, thereby completing the transepithelial uptake of calcium. Calcium efflux occurs passively through paracellular pathways between chloride cells and adjacent pavement cells or between neighboring pavement cells.

Effect of cortisol on the physiology of cultured pavement cell epithelia from freshwater trout gills

2001 ◽  
Vol 281 (3) ◽  
pp. R811-R820 ◽  
Author(s):  
Scott P. Kelly ◽  
Chris M. Wood
Keyword(s):  
Direct Action ◽  
Flux Ratio ◽  
Culture Conditions ◽  
Total Protein Content ◽  
Pavement Cell ◽  
Ratio Criterion ◽  
Basolateral Side ◽  
Active Ion Transport ◽  
Flux Measurements ◽  
Dose Dependent

Cortisol had dose-dependent effects on the electrophysiological, permeability, and ion-transporting properties of cultured pavement cell epithelia derived from freshwater rainbow trout gills and grown on cell culture filter supports. Under both symmetrical (L15 media apical/L15 media basolateral) and asymmetrical (freshwater apical/L15 media basolateral) culture conditions, cortisol treatment elevated transepithelial resistance, whereas permeability of epithelia to a paracellular permeability marker (polyethylene glycol-4000) decreased. Cortisol did not alter the Na+-K+-ATPase activity or the total protein content of the cultured preparations. During 24-h exposure to asymmetrical conditions, the net loss rates of both Na+ and Cl− to the water decreased with increasing cortisol dose, an important adaptation to dilute media. Unidirectional Na+ and Cl− flux measurements and the application of the Ussing flux-ratio criterion revealed cortisol-induced active uptake of both Na+ and Cl− under symmetrical culture conditions together with an increase in transepithelial potential (positive on the basolateral side). Under asymmetrical conditions, cortisol did not promote active ion transport across the epithelium. These experiments provide evidence for the direct action of cortisol on cultured pavement cell epithelia and, in particular, emphasize the importance of cortisol for limiting epithelial permeability.

scholarly journals Mechanisms of zinc uptake in gills of freshwater rainbow trout: interplay with calcium transport

1996 ◽  
Vol 270 (5) ◽  
pp. R1141-R1147 ◽  
Author(s):  
C. Hogstrand ◽  
P. M. Verbost ◽  
S. E. Bonga ◽  
C. M. Wood
Keyword(s):  
Rainbow Trout ◽  
Control Level ◽  
Inhibitory Effect ◽  
Chloride Cells ◽  
Water Addition ◽  
Uptake Mechanism ◽  
Basolateral Membranes ◽  
Control Value

The uptake mechanism of Zn2+ through the gill epithelium of freshwater rainbow trout was investigated both in intact animals and in isolated basolateral membranes. Involvement of the apical Ca2+ uptake sites in Zn2+ uptake was examined in vivo by pharmacological manipulation of the apical Ca2+ permeability. The apical entries of Ca2+ and Zn2+, but not Na2+ and Cl-, were inhibited by addition of La to the water. Addition of 1.0 microM La reduced the influxes of Ca2+ and Zn2+ to 22 +/- 3 and 53 +/- 7% (mean +/- SE) of the control value, respectively. Injection of CaCl2 also reduced the branchial influxes of Ca2+ and Zn2+. This treatment decreased the influx of Ca2- to 45 +/- 4% of the control level and the Zn2+ influx to 68 +/- 5%. These results strongly imply that Zn2+ passes across the apical membrane of the chloride cells of the gills via the same pathway as Ca2+. The presence of an active basolateral transporter for Zn2+ was investigated in vitro on isolated basolateral membranes. There was no ATP-dependent or Na2+(-)gradient driven transport of Zn2+ at physiological Zn2+ activities. The same system was used to study potential effects of Zn2+ on the basolateral Ca2+(-)adenosinetri-phosphatase. Zn2+ was found to be a potent blocker of this transporter, causing a mixed inhibitory effect on the ATP driven Ca2+ transport at a free Zn2+ activity of 100 pM.

Shortening of Branchial Tight Junction Acid-Exposed Rainbow Trout (Oncorhynchus mykiss)

1991 ◽  
Vol 48 (10) ◽  
pp. 2028-2033 ◽  
Author(s):  
J. Freda ◽  
D. A. Sanchez ◽  
H. L. Bergman
Keyword(s):  
Rainbow Trout ◽  
Oncorhynchus Mykiss ◽  
Tight Junctions ◽  
Relative Magnitude ◽  
Chloride Cells ◽  
Pavement Cells ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Plasma Electrolytes ◽  
Gill Structure ◽  
Electrolyte Loss

The objective of this study was to investigate possible sites for Na+ loss in fish exposed to low environmental pH. In rainbow trout (Oncorhynchus mykiss) exposed to pH 4.0 for 1 h, a net loss of Na+ was stimulated, and changes in gill structure occurred. In addition to epithelial lifting and necrosis in the gills of acid-exposed fish, tight junctions between pavement epithelial cells and chloride cells decreased in length by 25% whereas tight junctions between adjacent pavement cells did not significantly change. In a second experiment where fish were moved from pH 4.0 or 3.5 water to pH 6.5 water, we observed that Na+ loss declined immediately and approached control levels. The reversible nature of the stimulation of Na+ loss indicates that the site of Na+ loss in the fish gill can be reversibly opened and closed, which is consistent with the known properties of tight junctions. We hypothesize that the opening of tight junctions contributes to the loss of plasma electrolytes at low environmental pH. However, the relative magnitude of electrolyte loss through the tight junctions remains unknown.

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

2006 ◽  
Vol 291 (1) ◽  
pp. R170-R176 ◽  
Author(s):  
Fernando Galvez ◽  
Denise Wong ◽  
Chris M. Wood
Keyword(s):  
Rainbow Trout ◽  
Cellular Localization ◽  
Calcium Uptake ◽  
High Capacity ◽  
Cell Types ◽  
Isolation Technique ◽  
Pavement Cells ◽  
Gill Cells

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.

The effect of highly alkaline water (pH 9.5) on the morphology and morphometry of chloride cells and pavement cells in the gills of the freshwater rainbow trout: relationship to ionic transport and ammonia excretion

2000 ◽  
Vol 78 (2) ◽  
pp. 307-319 ◽  
Author(s):  
Pierre Laurent ◽  
Michael P Wilkie ◽  
Claudine Chevalier ◽  
Chris M Wood
Keyword(s):  
Rainbow Trout ◽  
Surface Area ◽  
Ammonia Excretion ◽  
Chloride Cells ◽  
Apical Surface ◽  
Influx Rate ◽  
Pavement Cells ◽  
Alkaline Water ◽  
Partial Restoration ◽  
Water Ph

Exposure of rainbow trout (Oncorhynchus mykiss) to alkaline water (pH 9.5) impairs ammonia excretion (JAmm) and gill-mediated ion-exchange processes, as characterized by decreased Cl- (JC1in) and Na+ influx (JNain) across the gill. Scanning electron microscopy suggested that the depression of JC1in was concomitant with an early decrease in the population of the most active chloride cells (CCs), partly compensated for by an increasing number of immature CCs. However, within 72 h after the onset of exposure to alkaline water, there was a 2-fold increase in the fractional apical surface area of CCs that paralleled complete recovery of the maximal Cl- influx rate (JC1max). These results suggest that recovery of JC1max was associated with greater CC surface area, resulting in more transport sites on the gill epithelium. Morphometric analysis of the outermost layer of pavement cells on the lamellar epithelium showed a greater density of microvilli during exposure to alkaline water, which may have contributed to partial restoration of the number of Na+ transport sites (JNamax). Finally, the blood-to-water gill-diffusion distance decreased by 27% after 72 h at pH 9.5, and likely contributed to progressive restoration of ammonia excretion in alkaline water.

scholarly journals Occluding junctions and cell behavior in primary cultures of normal and neoplastic mammary gland cells.

1975 ◽  
Vol 66 (2) ◽  
pp. 316-332 ◽  
Author(s):  
P B Pickett ◽  
D R Pitelka ◽  
S T Hamamoto ◽  
D S Misfeldt
Keyword(s):  
Primary Cultures ◽  
Freeze Fracture ◽  
Culture Conditions ◽  
Pavement Cells ◽  
Thin Sections ◽  
Occluding Junctions ◽  
Bulk Medium ◽  
Mammary Gland Cells ◽  
Junction Structure

Cells dissociated from normal prelactating mouse mammary glands or from spontaneous mammary adenocarcinomas, when grown at high density on an impermeable substrate, form nonproliferating, confluent, epithelial pavements in which turgid, blister-like domes appear as a result of fluid accumulation beneath the cell layer. To compare the structure of the fluid-segregating cell associations in normal and tumor cell cultures with that of lactating gland in vivo, we have examined such cultures alive and in thick and thin sections and freeze-fracture replicas. Pavement cells in all cases are polarized toward the bulk medium as a lumen equivalent, with microvilli and continuous, well-developed occluding junctions at this surface. Between the pavement and the substrate are other cells, of parenchymal or stromal origin, scattered or in loose piles; these sequestered cells are relatively unpolarized and never possess occluding junctions. Small gap junctions have been found in the pavement layer, and desmosomes may link epithelial cells in any location. Under the culture conditions used, development of the epithelial secretory apparatus is not demonstrable; normal and neoplastic cells do not differ consistently in any property examined. A dome's roof is merely a raised part of the epithelial pavement and does not differ from the latter in either cell or junction structure. We suggest that dome formation demonstrates the persistence of some transport functions and of the capacity to form effective occluding junctions. These basic epithelial properties can survive both neoplastic transformation and transition to culture.

Permeability properties of the teleost gill epithelium under ion-poor conditions

2012 ◽  
Vol 302 (6) ◽  
pp. R727-R739 ◽  
Author(s):  
Helen Chasiotis ◽  
Dennis Kolosov ◽  
Scott P. Kelly
Keyword(s):  
Gill Tissue ◽  
Transcript Abundance ◽  
Mrna Abundance ◽  
Gill Epithelium ◽  
Pavement Cells ◽  
Peg 4000 ◽  
Cell Fractions ◽  
Interfering Rna

Permeability properties of the goldfish gill epithelium were examined in vivo and in vitro following exposure to ion-poor water (IPW) conditions. In gill tissue of IPW-acclimated goldfish, transcript abundance of tight junction (TJ) proteins occludin, claudin-b, -d, -e, -h, -7, and -8d increased, whereas ZO-1 and claudin 12 mRNA decreased and claudin-c was unaltered. In association with these changes, TJ depth increased among gill pavement cells (PVCs) and gill PVCs and mitochondria-rich cells (MRCs). PVC and MRC gill cell fractions were isolated using Percoll. Transcripts encoding for occludin, claudin-b, -c, -d, -e, -h, -7, -8d, -12, and ZO-1 were present in both fractions. After IPW acclimation, occludin, claudin-b and -e, and ZO-1 mRNA abundance increased in both fractions. In contrast, claudin-8d mRNA abundance increased in PVCs only while claudin-h decreased in MRCs. Gill permeability was examined using primary cultured goldfish PVC epithelia supplemented with serum derived from IPW-acclimated goldfish. IPW serum supplementation increased transepithelial resistance, reduced [3H]PEG-4000 permeability, and enhanced epithelial integrity during in vitro IPW exposure. IPW serum increased mRNA abundance of occludin, claudin-8d and -e in vitro. Using small interfering RNA, we found that occludin abundance was decreased in cultured gill epithelia, resulting in an increase in [3H]PEG-4000 flux. As occludin increased in the gills of IPW-acclimated fish as well as cultured gill epithelia exposed to IPW serum, results suggest that occludin is a barrier-forming TJ protein in fish gill epithelia. These studies support the idea that TJ proteins play an important role in regulating gill permeability in IPW.

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

2002 ◽  
Vol 282 (3) ◽  
pp. R658-R668 ◽  
Author(s):  
Fernando Galvez ◽  
Scott D. Reid ◽  
Guy Hawkings ◽  
Greg G. Goss
Keyword(s):  
Rainbow Trout ◽  
Iron Particle ◽  
Cell Types ◽  
Chloride Cells ◽  
Transmission Electron Microscopy Analysis ◽  
Pavement Cells ◽  
Isolation And Characterization ◽  
Transmission Electron ◽  
Electron Microscopy Analysis ◽  
Magnetic Cell Separation

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.

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