5-(N-Ethyl-N-isopropyl)amiloride sensitive Na+ currents in intact fetal distal lung epithelial cells

1993 ◽  
Vol 71 (1) ◽  
pp. 58-62 ◽  
Author(s):  
X. Wang ◽  
T. R. Kleyman ◽  
H. Tohda ◽  
Y. Marunaka ◽  
H. O'Brodovich
Keyword(s):  
Patch Clamp ◽  
Short Circuit ◽  
Primary Cultures ◽  
Alveolar Epithelium ◽  
Short Circuit Current ◽  
Lung Epithelial Cells ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Na Currents ◽  
Distal Lung

To determine whether primary cultures of rat fetal distal lung epithelium (FDLE) possessed L-type Na+ channels on their plasma membrane we performed experiments with 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and other amiloride analogs. Short-circuit current (Isc) was decreased by the apical application of amiloride and benzamil, but was unaffected by 10 μM dimethylamiloride (DMA). EIPA decreased Isc when added to either the apical or basal sides. Greatest effects were seen with bilateral EIPA, where half-maximal effects occurred in the micromolar range. Measurements of intracellular pH with the fluorescent dye BCECF demonstrated that DMA impaired (IC50 = 71 nM) the ability of FDLE to recover from intracellular acidification. Nystatin perforated patch clamp techniques snowed that FDLE had nonrectifying Na+ currents but no detectable Cl− currents. The whole-cell currents were reversibly decreased by 20 μM concentrations of EIPA, benzamil, and amiloride but were unaffected by 20 μM DMA. These studies indicate that there are EIPA-sensitive Na+ conductances in intact FDLE and suggest the presence of L-type Na+ conductances on their apical membrane and EIPA-sensitive K+ channels on the basolateral membrane.Key words: Na+ channels, 5-(N-ethyl-N-isopropyl)amiloride, alveolar epithelium, whole-cell patch clamp, K+ channels.

Inhibition of amiloride-sensitive sodium-channel activity in distal lung epithelial cells by nitric oxide

1998 ◽  
Vol 274 (3) ◽  
pp. L378-L387 ◽  
Author(s):  
Jin Wen Ding ◽  
John Dickie ◽  
Hugh O’Brodovich ◽  
Yutaka Shintani ◽  
Bijan Rafii ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Epithelial Cells ◽  
Cell Function ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Lung Epithelial Cells ◽  
Flufenamic Acid ◽  
Activated Macrophages ◽  
Lung Epithelial ◽  
Distal Lung

Distal lung epithelial cells (DLECs) play an active role in fluid clearance from the alveolus by virtue of their ability to actively transport Na+ from the alveolus to the interstitial space. The present study evaluated the ability of activated macrophages to modulate the bioelectric properties of DLECs. Low numbers of lipopolysaccharide (LPS)-treated macrophages were able to significantly reduce amiloride-sensitive short-circuit current ( I sc) without affecting total I sc or monolayer resistance. This was associated with a rise in the flufenamic acid-sensitive component of the I sc. The effect was reversed by the addition of N-monomethyl-l-arginine to the medium, implying a role for nitric oxide. We hypothesized that macrophages exerted their effect by expressing inducible nitric oxide synthase (iNOS) in DLECs. The products of LPS-treated macrophages increased the levels of iNOS protein and mRNA transcripts in DLECs as well as causing a rise in iNOS activity. Immunofluorescence microscopy of LPS-stimulated macrophage-DLEC cocultures with anti-nitrotyrosine antibodies provided evidence for the generation of peroxynitrite in macrophages but not in DLECs. These data indicate that activated macrophages in the lung may contribute to impaired resolution of acute respiratory distress syndrome and suggest a novel mechanism whereby nitric oxide might alter cell function by altering its ion-transporting phenotype.

O2-induced ENaC expression is associated with NF-κB activation and blocked by superoxide scavenger

1998 ◽  
Vol 275 (4) ◽  
pp. L764-L770 ◽  
Author(s):  
Bijan Rafii ◽  
A. Keith Tanswell ◽  
Gail Otulakowski ◽  
Olli Pitkänen ◽  
Rose Belcastro-Taylor ◽  
...  
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Nuclear Factor Κb ◽  
Lung Epithelial Cells ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Lung Epithelial ◽  
Distal Lung ◽  
Superoxide Scavenger ◽  
Transcription Factor Nuclear Factor

Cultured rat fetal distal lung epithelial cells (FDLEs), when switched from fetal (3%) to postnatal (21%) O2 concentrations, have increased epithelial Na+ channel (ENaC) mRNA levels and amiloride-sensitive Na+transport [O. Pitkänen, A. K. Tanswell, G. Downey, and H. O’Brodovich. Am. J. Physiol. 270 ( Lung Cell. Mol. Physiol. 14): L1060–L1066, 1996]. The mechanisms by which O2 mediates these effects are unknown. After isolation, FDLEs were kept at 3% O2 overnight, then switched to 21% O2 (3–21% O2 group) or maintained at 3% O2 (3–3% O2 group) for 48 h. The amiloride-sensitive short-circuit current ( I sc) in the 3–21% O2 group was double that in the 3–3% O2 group. Amiloride-sensitive I sc could not be induced by medium conditioned by 21% O2-exposed FDLEs but was reversed by returning the cells to 3% O2. Neither the cyclooxygenase inhibitor ibuprofen, liposome-encapsulated catalase, nor hydroperoxide scavengers (U-74389G or Trolox) blocked the O2-induced amiloride-sensitive I sc. In contrast, the cell-permeable superoxide scavenger tetramethylpiperidine- N-oxyl (TEMPO) eliminated the O2-induced increases in amiloride-sensitive I sc and ENaC mRNA levels. The switch from 3 to 21% O2 induced the transcription factor nuclear factor-κB, which could also be blocked by TEMPO. We conclude that 1) the O2-induced increase in amiloride-sensitive I sc is reversible and 2) the O2-induced increase in amiloride-sensitive I sc and ENaC mRNA levels is associated with activation of nuclear factor-κB and may be mediated, at least in part, by superoxide.

Astrocytic inwardly rectifying potassium currents are dependent on external sodium ions

1996 ◽  
Vol 76 (1) ◽  
pp. 626-630 ◽  
Author(s):  
C. B. Ransom ◽  
H. Sontheimer ◽  
D. Janigro
Keyword(s):  
Primary Cultures ◽  
Potassium Currents ◽  
Input Resistance ◽  
Current Amplitude ◽  
Sodium Ions ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Na Currents ◽  
Cell Current ◽  
Inwardly Rectifying

1. Two subtypes of astrocytes that expressed distinctly different ion channel complements were identified in primary cultures from rat spinal cord and hippocampus using whole cell patch-clamp techniques. One population of cells expressed voltage-activated Na+ currents and displayed outwardly rectifying I-V relationships; the other group of cells had no detectable Na+ currents and pronounced inwardly rectifying I-V curves. 2. Astrocytes expressing Na+ currents were hyperpolarized (by approximately 7 mV) upon removal of external sodium, suggesting a resting Na+ conductance in these cells. In contrast, cells expressing primarily inwardly rectifying K+ currents, Kir, depolarized (by approximately 4-6 mV) in low-sodium solutions. 3. Removal of external Na+ ions increased the input resistance (189% of control) and reduced the whole cell current amplitude (60% of control at -120 mV) of cells with Kir. The reduction in current amplitude was dose-dependent and became apparent after a 10% reduction of [Na+]0 in 7/7 cells tested. At -120 mV, the effect was near maximal in response to a 50% reduction of [Na+]0. 4. The outward potassium currents of cells expressing Na(+)-currents were unaffected by removal of bath Na+. 5. We conclude that the conductance of glial inwardly rectifying K+ channels is dependent on external sodium ions via a mechanism that does not involve sodium ion permeation or blockade of these channels.

Novobiocin forms cation-permeable ion channels in rat fetal distal lung epithelium

1993 ◽  
Vol 264 (6) ◽  
pp. C1532-C1537 ◽  
Author(s):  
H. O'Brodovich ◽  
X. Wang ◽  
C. Li ◽  
B. Rafii ◽  
J. Correa ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Lipid Membranes ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Monovalent Cation ◽  
Lung Epithelium ◽  
Ussing Chambers ◽  
Distal Lung ◽  
Perforated Patch Clamp

The antibiotic novobiocin has been previously reported to increase Na+ transport in frog skin, presumably by attenuation of Na+ self-inhibition of Na+ channels. To determine whether novobiocin had similar effects and utilized a similar mechanism in mammalian Na(+)-transporting tissues, we studied its effect on ion transport by primary cultures of fetal distal lung epithelium (FDLE) cultured from 20-day gestationally aged rats (term = 22 days). Novobiocin (10 mM) increased short-circuit current and markedly decreased the resistance in FDLE monolayers mounted in Ussing chambers. Fura-2 single-cell studies showed that novobiocin increased intracellular Ca2+ concentration and that this resulted from extracellular sources. Nystatin-perforated patch-clamp techniques demonstrated that novobiocin increased nonrectifying cation whole cell currents without inducing detectable anion currents. Novobiocin created nonrectifying monovalent cation-selective channels in lipid bilayers. These studies demonstrated that novobiocin affects the bioelectric properties of Na+ transporting lung epithelium and that this likely occurs by the formation of ion-permeant channels in their lipid membranes.

Increased Po2 alters the bioelectric properties of fetal distal lung epithelium

1996 ◽  
Vol 270 (6) ◽  
pp. L1060-L1066 ◽  
Author(s):  
O. Pitkanen ◽  
A. K. Transwell ◽  
G. Downey ◽  
H. O'Brodovich
Keyword(s):  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Na Channel ◽  
Lung Epithelium ◽  
Ambient Oxygen ◽  
Ion Substitution ◽  
Distal Lung ◽  
Dose Dependent ◽  
Oxygen Concentrations

At birth the lung must efficiently clear the liquid from its air spaces and permanently convert from a fluid-secreting to a fluid-absorbing organ. When primary cultures of rat fetal distal lung epithelium (FDLE) grown on permeable supports were switched from a fetal (3%) to a postnatal (21%) oxygen environment, there was an increase in epithelial permeability as reflected by a dose-dependent decline in transepithelial resistance (Rt) 4 h later (3% = 239 +/- 19 omega.cm2; 21% = 170 +/- 28 omega.cm2; 50% = 98 +/- 20 omega.cm2; P < 0.05). The effect was transient, since monolayers initially maintained at 3% and switched to these higher oxygen concentrations subsequently had Rt values comparable to the 3% group at 48 h (3% = 153 +/- 17 omega.cm2; 21% = 181 +/- 19 omega.cm2; 50% = 192 +/- 21 omega.cm2; P = NS). Changes in Rt were associated with expected changes in the histological appearance of the interepithelial tight junctions, but intracellular actin content and distribution remained constant. Amiloride-sensitive equivalent short-circuit current increased within 18 h, with further increases after 48 h of exposure to postnatal oxygen concentrations. Ion substitution experiments suggested diminished FDLE Cl transport and increased Na transport. The amount of FDLE-alpha, -beta, and -gamma rat epithelial Na channel mRNA increased within 48 h of increasing the ambient oxygen concentration. These results suggest that the physiological increase in alveolar Po2 at birth is, at least in part, responsible for distal lung's permanent switch from Cl secretion to Na absorption at birth.

β-Adrenoceptor-mediated control of apical membrane conductive properties in fetal distal lung epithelia

2002 ◽  
Vol 282 (4) ◽  
pp. L621-L630 ◽  
Author(s):  
A. Collett ◽  
S. J. Ramminger ◽  
R. E. Olver ◽  
S. M. Wilson
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Anion Channel ◽  
Short Circuit Current ◽  
Lung Epithelial Cells ◽  
Disulfonic Acid ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Distal Lung ◽  
Stimulation Of

Distal lung epithelial cells isolated from fetal rats were cultured (48 h) on permeable supports so that transepithelial ion transport could be quantified electrometrically. Unstimulated cells generated a short-circuit current ( I sc) that was inhibited (∼80%) by apical amiloride. The current is thus due, predominantly, to the absorption of Na+ from the apical solution. Isoprenaline increased the amiloride-sensitive I sc about twofold. Experiments in which apical membrane Na+ currents were monitored in basolaterally permeabilized cells showed that this was accompanied by a rise in apical Na+ conductance ( G Na+ ). Isoprenaline also increased apical Cl− conductance ( G Cl− ) by activating an anion channel species sensitive to glibenclamide but unaffected by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). The isoprenaline-evoked changes in G Na+ and G Cl− could account for the changes in I sc observed in intact cells. Glibenclamide had no effect upon the isoprenaline-evoked stimulation of I sc or G Na+ demonstrating that the rise in G Cl− is not essential to the stimulation of Na+ transport.

scholarly journals Inhibition of cardiac Na+ currents by isoproterenol

1990 ◽  
Vol 258 (4) ◽  
pp. H977-H982 ◽  
Author(s):  
B. Schubert ◽  
A. M. Vandongen ◽  
G. E. Kirsch ◽  
A. M. Brown
Keyword(s):  
Patch Clamp ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Na Channel ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Na Currents ◽  
Voltage Dependent

The mechanism by which the beta-adrenergic agonist isoproterenol (ISO) modulates voltage-dependent cardiac Na+ currents (INa) was studied in single ventricular myocytes of neonatal rat using the gigaseal patch-clamp technique. ISO inhibited INa reversibly, making the effect readily distinguishable from the monotonic decrease of INa caused by the shift in gating that customarily occurs during whole cell patch-clamp experiments (E. Fenwick, A. Marty, and E. Neher, J. Physiol. Lond. 331: 599-635, 1982; and J. M. Fernandez, A. P. Fox, and S. Krasne, J. Physiol. Lond. 356: 565-585, 1984). The inhibition was biphasic, having fast and slow components, and was voltage-dependent, being more pronounced at depolarized potentials. In whole cell experiments the membrane-permeable adenosine 3',5'-cyclic monophosphate (cAMP) congener 8-bromo-cAMP reduced INa. In cell-free inside-out patches with ISO present in the pipette, guanosine 5'-triphosphate (GTP) applied to the inner side of the membrane patch inhibited single Na+ channel activity. This inhibition could be partly reversed by hyperpolarizing prepulses. The nonhydrolyzable GTP analogue guanosine-5'-O-(3-thiotriphosphate) greatly reduced the probability of single Na+ channel currents in a Mg2(+)-dependent manner. We propose that ISO inhibits cardiac Na+ channels via the guanine nucleotide binding, signal-transducing G protein that acts through both direct (membrane delimited) and indirect (cytoplasmic) pathways.

Effect of K channel blockers on basal and β-agonist stimulated ion transport by fetal distal lung epithelium

1993 ◽  
Vol 71 (1) ◽  
pp. 54-57 ◽  
Author(s):  
Hugh O'Brodovich ◽  
Bijan Rafii
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Alveolar Epithelium ◽  
Short Circuit Current ◽  
K Channel ◽  
Channel Blockers ◽  
Lung Epithelium ◽  
Na Transport ◽  
Ussing Chambers ◽  
Distal Lung

To determine whether basolateral K channels play an important role in the basal and β-agonist stimulated ion transport by fetal distal lung epithelium we harvested these cells from fetal rats (20 days gestation, term = 22 days) and studied them in Ussing chambers. Short-circuit current (Isc) fell with basal 3 mM BaCl2 (3.0 ± 0.2 (±SEM) to 2.0 ± 0.2 μA∙cm−2, n = 18, p < 0.01) without affecting monolayer resistance (R = 693 ± 57 Ω∙cm2). Basal quinine sulfate (1 mM) also decreased Isc (3.7 ± 0.15 to 3.0 ± 0.10 μA∙cm−2; n = 4, p < 0.01). None of apical BaCl2 (3 mM), apical quinine (1 mM), nor bilaterally applied tetraethylammonium (10 mM), lidocaine (1 mM), or 4-aminopyridine (2 mM) decreased Isc. Cell monolayers treated with basal BaCl2 (3 mM) demonstrated an impaired ability to increase their Isc in response to the β2-agonist terbutaline (1 mM). Basal 3 mM BaCl2 also decreased Isc in amiloride (0.1 mM) and furosemide (1 mM) treated monolayers, indicating that barium also affected the previously described amiloride-insensitive Na transport by these cells (n = 8, p < 0.01). Together these experiments suggest that normal basolateral K channel function is required for normal and β2-stimulated Na transport in fetal distal lung epithelium.Key words: type II alveolar epithelium, potassium channels, β-agonist, sodium transport, Na channels.

scholarly journals Functional Expression of TRPV1 Ion Channel in the Canine Peripheral Blood Mononuclear Cells

2021 ◽  
Vol 22 (6) ◽  
pp. 3177
Author(s):  
Joanna K. Bujak ◽  
Daria Kosmala ◽  
Kinga Majchrzak-Kuligowska ◽  
Piotr Bednarczyk
Keyword(s):  
Patch Clamp ◽  
Ion Channel ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Functional Expression ◽  
Peripheral Blood Mononuclear ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Blood Mononuclear Cells

TRPV1, known as a capsaicin receptor, is the best-described transient receptor potential (TRP) ion channel. Recently, it was shown to be expressed by non-excitable cells such as lymphocytes. However, the data regarding the functional expression of the TRPV1 channel in the immune cells are often contradictory. In the present study, we performed a phylogenetical analysis of the canine TRP ion channels, we assessed the expression of TRPV1 in the canine peripheral blood mononuclear cells (PBMC) by qPCR and Western blot, and we determined the functionality of TRPV1 by whole-cell patch-clamp recordings and calcium assay. We found high expression of TRPV2, -M2, and -M7 in the canine PBMCs, while expression of TRPV1, -V4 and, -M5 was relatively low. We confirmed that TRPV1 is expressed on the protein level in the PBMC and it localizes in the plasma membrane. The whole-cell patch-clamp recording revealed that capsaicin application caused a significant increase in the current density. Similarly, the results from the calcium assay show a dose-dependent increase in intracellular calcium level in the presence of capsaicin that was partially abolished by capsazepine. Our study confirms the expression of TRPV1 ion channel on both mRNA and protein levels in the canine PBMC and indicates that the ion channel is functional.

Sign in / Sign up

Export Citation Format

Share Document