A maxi Cl(−) channel in cultured pavement cells from the gills of the freshwater rainbow trout Oncorhynchus mykiss

Primary cultures of pavement cells from the gills of a freshwater fish, the rainbow trout Oncorhynchus mykiss, have been studied for the first time using the patch-clamp technique. Gigaohm seals were obtained in approximately 95 % of cells studied, and channel activity was evident in a high proportion (>90 %). A large-conductance Cl(−) channel was evident in 6 % of cell-attached and in 31 % of inside-out patches. Single-channel conductance in inside-out patches was 372 pS, and current/voltage relationships were linear over the range −60 to +60 mV. The channel was activated by patch excision, and activation was often associated with polarization of the patch. The mean number of channels per patch was 1.9, and there were several subconductance states. The relationship between channel activity (NP(o)) and voltage was in the form of an inverted U, and channel activity was highest between 0 and +20 mV. Large-conductance Cl(−) channels showed a progressive time-dependent reduction in current in response to sustained polarization to voltages outside the range −20 mV to +20 mV. Permeability ratios (P) of Cl(−) to other anions were P(HCO3)/P(Cl)=0.81, P(SO4)/P(Cl)=0.31 and P(isethionate)/P(Cl)=0.53. The channel was blocked by Zn(2+), SITS, DIDS and diphenylamine carboxylate. This is the first description of a large-conductance Cl(−) channel in gill cells from freshwater or marine species. Possible functions of the channel are discussed.

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Chloride channels in apical membrane of primary cultures of rabbit distal bright convoluted tubule

AJP Renal Physiology ◽

10.1152/ajprenal.1994.266.4.f543 ◽

1994 ◽

Vol 266 (4) ◽

pp. F543-F553 ◽

Cited By ~ 2

Author(s):

V. Poncet ◽

M. Tauc ◽

M. Bidet ◽

P. Poujeol

Keyword(s):

Chloride Channels ◽

Apical Membrane ◽

Primary Cultures ◽

Transmembrane Conductance Regulator ◽

Patch Clamp Technique ◽

Cl Channel ◽

Current Voltage ◽

Cl Channels ◽

Inside Out ◽

Small Conductance

Using the patch clamp technique on the apical membrane of primary cultures of rabbit distal bright convoluted tubule cells (DCTb), two types of Cl- channel were identified. A small channel of 9 pS was observed in 9% of the patches. Cells pretreated with 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) or 5 microM forskolin increased the expression of Cl- channels by 26 and 37%, respectively. In cell-attached and excised inside-out patches, the current-voltage (I-V) relationships of the 9-pS channel were linear. In only 1 out of 47 active patches was the small-conductance Cl- channel still active 1 h after membrane excision. The addition of 0.1 microM of the catalytic subunit protein kinase A with 2 mM ATP to the cytoplasmic side restored channel activity in 8 out of 15 excised membrane patches. In 5 out of 467 patches of stimulated or nonstimulated cells, a larger Cl- conductance of 30 pS was also recorded. In excised inside-out patches this channel outwardly rectified and was activated by strong depolarization. In cultured DCTb cells, the small-conductance, cAMP-activated Cl- channel shares many properties with the cystic fibrosis transmembrane conductance regulator. Our results suggest that at least the small-conductance channel may participate in Cl- secretion across the apical membrane of DCTb in primary culture. This secretion may increase the rate of the apical Cl-/HCO3- exchange indirectly by enhancing the inwardly-directed Cl- gradient.

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

Canadian Journal of Fisheries and Aquatic Sciences ◽

10.1139/f91-241 ◽

1991 ◽

Vol 48 (10) ◽

pp. 2028-2033 ◽

Cited By ~ 36

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.

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Development of a monoclonal antibody for ELISA of CYP1A in primary cultures of rainbow trout Oncorhynchus mykiss hepatocytes

Biomarkers ◽

10.1080/135475097231517 ◽

1997 ◽

Vol 2 (5) ◽

pp. 287-294 ◽

Cited By ~ 23

Author(s):

S. Scholz ◽

I. Behn ◽

H. Honeck ◽

C. Hauck ◽

T. Braunbeck ◽

...

Keyword(s):

Monoclonal Antibody ◽

Rainbow Trout ◽

Oncorhynchus Mykiss ◽

Primary Cultures ◽

Rainbow Trout Oncorhynchus Mykiss

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Induction of CYP1A in Primary Cultures of Rainbow Trout (Oncorhynchus mykiss) Liver Cells: Concentration–Response Relationships of Four Model Substances

Ecotoxicology and Environmental Safety ◽

10.1006/eesa.1999.1786 ◽

1999 ◽

Vol 43 (3) ◽

pp. 252-260 ◽

Cited By ~ 31

Author(s):

S. Scholz ◽

H. Segner

Keyword(s):

Rainbow Trout ◽

Oncorhynchus Mykiss ◽

Primary Cultures ◽

Liver Cells ◽

Rainbow Trout Oncorhynchus Mykiss ◽

Model Substances

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An in vitro examination of selenium–cadmium antagonism using primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes

Metallomics ◽

10.1039/c5mt00232j ◽

2016 ◽

Vol 8 (2) ◽

pp. 218-227 ◽

Cited By ~ 14

Author(s):

Ankur Jamwal ◽

Mohammad Naderi ◽

Som Niyogi

Keyword(s):

Rainbow Trout ◽

Oncorhynchus Mykiss ◽

Primary Cultures ◽

Ros Generation ◽

Antagonistic Effects ◽

Low Concentration ◽

Rainbow Trout Oncorhynchus Mykiss ◽

Dose Dependent ◽

In Vitro Examination

Se has antagonistic effects on Cd-induced cytotoxicityviaboth enzymatic and non-enzymatic antioxidative mechanisms and the effects are strictly dose dependent. Confocal fluorescent images of isolated rainbow trout hepatocytes exposed to 100 µM Cd, alone or in combination with low (25 µM) or high (250 µM) concentration of Se, show reduced ROS generation with low concentration of Se.

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First evidence of the pore-forming properties of a keratin from skin mucus of rainbow trout (Oncorhynchus mykiss, formerly Salmo gairdneri)

Biochemical Journal ◽

10.1042/bj20070801 ◽

2008 ◽

Vol 411 (1) ◽

pp. 33-40 ◽

Cited By ~ 24

Author(s):

Virginie Molle ◽

Sylvie Campagna ◽

Yannick Bessin ◽

Nathalie Ebran ◽

Nathalie Saint ◽

...

Keyword(s):

Rainbow Trout ◽

Oncorhynchus Mykiss ◽

Single Channel ◽

Antibacterial Properties ◽

Enzymatic Digestion ◽

Salmo Gairdneri ◽

Human Beings ◽

Skin Mucus ◽

Rainbow Trout Oncorhynchus Mykiss ◽

Bacteria And Fungi

The epidermis of fish is covered with a layer of mucus, which contributes to the defence of the species against parasites, bacteria and fungi. We have previously extracted glycoproteins from various mucus samples from fish and have shown that they present pore-forming activities well correlated with strong antibacterial properties [Ebran, Julien, Orange, Saglio, Lemaitre and Molle (2000) Biochim. Biophys. Acta 1467, 271–280]. The present study focuses on the 65 kDa glycoprotein, Tr65, from the rainbow trout (Oncorhynchus mykiss, formerly Salmo gairdneri). Enzymatic digestion of Tr65 yielded a fragment pattern with strong homology with that of trout type II cytokeratin. Sequence analysis of the cDNA clone obtained by PCR confirmed this homology. We thus constructed a plasmid to overproduce the recombinant Tr65. We extracted and purified this recombinant Tr65, using it for multichannel and single-channel experiments in azolectin bilayers. Our results with recombinant Tr65 confirmed the pore-forming properties already shown with native antibacterial Tr65. These findings offer new insights into the function of keratin proteins present in various mucosal surfaces of animals and human beings.

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Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes.

The Journal of General Physiology ◽

10.1085/jgp.104.2.357 ◽

1994 ◽

Vol 104 (2) ◽

pp. 357-373 ◽

Cited By ~ 34

Author(s):

S Koumi ◽

R Sato ◽

T Aramaki

Keyword(s):

Guinea Pig ◽

Single Channel ◽

Hill Coefficient ◽

Disulfonic Acid ◽

Dependent Manner ◽

Channel Activity ◽

Patch Clamp Technique ◽

Whole Cell ◽

Cl Channels ◽

Inside Out

Macroscopic and unitary currents through Ca(2+)-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 1 microM (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+]i and was inactivated in Ca(2+)-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9-anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = 11). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The relationship between [Ca2+]i and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 microM. These results suggest that guinea-pig hepatocytes possess Ca(2+)-activated Cl- channels.

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Ca(2+)-dependent Cl- channels in undifferentiated human colonic cells (HT-29). I. Single-channel properties

AJP Cell Physiology ◽

10.1152/ajpcell.1993.264.4.c968 ◽

1993 ◽

Vol 264 (4) ◽

pp. C968-C976 ◽

Cited By ~ 29

Author(s):

A. P. Morris ◽

R. A. Frizzell

Keyword(s):

Single Channel ◽

Voltage Sensitivity ◽

Channel Activity ◽

Patch Clamp Technique ◽

Channel Activation ◽

Whole Cell ◽

Multiple Channel ◽

Cl Channel ◽

Cl Channels ◽

Ht 29

The patch-clamp technique was combined with camera-based intracellular Ca2+ concentration ([Ca2+]i) imaging to identify the single-channel basis of the Ca(2+)-dependent Cl- conductance in human colonic adenocarcinoma cells (HT-29). Cl- channels were activated when membrane patches were excised into solutions containing high (1 microM) Ca2+ concentrations. Their single-channel conductance, measured by amplitude histogram analysis, averaged 13 pS at -90 mV and 16 pS at +90 mV membrane potential (MP). In multiple channel patches, Cl- currents showed properties similar to Ca(2+)-activated whole cell currents: outward rectification and time-dependent activation at depolarizing MP. Channel activity disappeared shortly after patch excision from the cell. In cell-attached patches, Cl- channel opening was infrequent at resting [Ca2+]i values (96 +/- 18 nM), but when [Ca2+]i was increased by the Ca2+ ionophore ionomycin (1 microM), Cl- channels were activated with a time course that paralleled the [Ca2+]i rise. Repetitive ionophore exposure produced equivalent rises in [Ca2+]i, but the corresponding Cl- channel activity became progressively reduced. The Ca(2+)-mediated agonist neurotensin (50 nM) elicited a transient Cl- channel activation that preceded the generalized cellular [Ca2+]i rise. Channel activation with neurotensin occurred in the absence of pipette Ca2+ but was abolished by preloading cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Thus, in response to the Ca(2+)-mediated agonist neurotensin, Cl- channel activation results from Ca2+ mobilization from intracellular pools localized within the vicinity of the plasma membrane. The Ca2+ dependency, voltage sensitivity, and kinetics of this 15-pS Cl- channel indicate that it is the basis of the whole cell Ca(2+)-activated Cl- current.

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