Whole-cell Cl− currents in a human peripheral airway epithelial cell line

1993 ◽  
Vol 71 (9) ◽  
pp. 662-670 ◽  
Author(s):  
Xiaodong Wang ◽  
Ludwik Fedorko ◽  
Yoshinori Marunaka ◽  
Hugh O'Brodovich
Keyword(s):  
Cyclic Amp ◽  
Patch Clamp ◽  
Cell Line ◽  
Pertussis Toxin ◽  
Outward Current ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Peripheral Airway

We have used the whole-cell patch-clamp technique to identify and characterize Cl− currents in a cell line derived from human peripheral airway epithelium (NCI-H-441-4). The permeability sequence and relative selectivity for different anions was Br− (1.4) ~ I− (1.3) > Cl− (1.0) > F− (0.6) > gluconate (0.4) > glutamate (0.2). The current–voltage relationship displayed rectification in the outward direction. Diphenylamine-2-carboxylate (10−4 M) applied intracellularly blocked the outward-rectified current, while extracellularly applied diphenylamine-2-carboxylate had no effect on Cl− current. This current was also blocked by extracellularly applied 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), with an estimated IC50 of 15.2 μM. Dibutyryl-cyclic AMP (10−4 M) increased outward current, whereas pretreatment with 100 ng/mL pertussis toxin almost completely abolished the Cl− current. Pertussis toxin inhibition of this current could be partially reversed by dialysis of the cell interior with the activated αi–2 subunit of Gi protein. This cell line provides an opportunity to study directly the regulation of Cl− channels in cells derived from the peripheral human lung airways.Key words: chloride secretion, whole-cell patch clamp, GTP binding protein, cyclic AMP, pertussis toxins, 5-nitro-2-(3-phenylpropylamino)benzoate, diphenylamine-2-carboxylate, cell line H441.

scholarly journals Baro-excited neurons in the caudal ventrolateral medulla (CVLM) recorded using the whole-cell patch-clamp technique

2011 ◽  
Vol 35 (5) ◽  
pp. 500-506 ◽  
Author(s):  
Naoki Oshima ◽  
Hiroo Kumagai ◽  
Kamon Iigaya ◽  
Hiroshi Onimaru ◽  
Akira Kawai ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Ventrolateral Medulla ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Caudal Ventrolateral Medulla ◽  
Clamp Technique ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp

scholarly journals Neutrophil Secretion Induced by an Intracellular Ca2+Rise and Followed by Whole-Cell Patch-Clamp Recordings Occurs Without any Selective Mobilization of Different Granule Populations

2006 ◽  
Vol 2006 ◽  
pp. 1-7 ◽  
Author(s):  
Daniel Granfeldt ◽  
Olle Harbecke ◽  
Åse Björstad ◽  
Anna Karlsson ◽  
Claes Dahlgren
Keyword(s):  
Patch Clamp ◽  
Human Neutrophils ◽  
Lag Phase ◽  
Measuring Time ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
High Concentrations ◽  
Kinetics Of

We have investigated calcium-induced secretion in human neutrophils, using a whole-cell patch-clamp technique. Mobilization of subcellular granules to the cell membrane was followed as the change in membrane capacitance (△Cm). Both the magnitude and the kinetics of the response differed between low and high concentrations of Ca2+. A sustained secretion following a short lag phase was induced by high concentrations of Ca2+(100μM and higher). A stable plateau was reached after 5–7 minutes at△Cmvalues corresponding to values expected after all specific as well as azurophil granules have been mobilized. Capacitance values of the same magnitude could be obtained also at lower Ca2+concentrations, but typically no stable plateau was reached within the measuring time. In contrast to previous studies, we were unable to detect any pattern of secretion corresponding to a distinct submaximal response or selective mobilization of granule subsets specified by their Ca2+-sensitivity.

GABA-Mimetic Actions of Withania somnifera on Substantia Gelatinosa Neurons of the Trigeminal Subnucleus Caudalis in Mice

2013 ◽  
Vol 41 (05) ◽  
pp. 1043-1051 ◽  
Author(s):  
Hua Yin ◽  
Dong Hyu Cho ◽  
Soo Joung Park ◽  
Seong Kyu Han
Keyword(s):  
Patch Clamp ◽  
Receptor Antagonist ◽  
Withania Somnifera ◽  
Substantia Gelatinosa ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Inward Currents

The plant Withania somnifera (WS), also known as Ashwagandha, has been used widely in traditional medicine systems in India and Nepal (Ayurveda), and has been accepted to cure various ailments. In this study, the whole-cell patch clamp technique was performed to examine the mechanism of action of WS on the SG neurons of the Vc from mouse brainstem slices. In whole-cell patch clamp mode, methanol extract of Withania somnifera (mWS) induced short-lived and repeatable inward currents in all SG neurons tested (31.3±8.51 pA, n = 7) using a high chloride pipette solution. The mWS-induced inward currents were concentration dependent and maintained in the presence of tetrodotoxin (TTX), a voltage gated Na + channel blocker, CNQX, a non-NMDA glutamate receptor antagonist, AP5, an NMDA receptor antagonist and strychnine, a glycine receptor antagonist. The mWS induced currents were blocked by picrotoxin, a GABAA receptor antagonist. These results show that mWS has an inhibitory effects on SG neurons of the Vc through GABAA receptor-mediated activation of chloride ion channels, indicating that mWS contains compounds with sedative effects on the central nervous system. These results also suggest that mWS may be a potential target for modulating orofacial pain processing.

scholarly journals Expression and function of KV2-containing channels in human urinary bladder smooth muscle

2012 ◽  
Vol 302 (11) ◽  
pp. C1599-C1608 ◽  
Author(s):  
Kiril L. Hristov ◽  
Muyan Chen ◽  
Serge A. Y. Afeli ◽  
Qiuping Cheng ◽  
Eric S. Rovner ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Electrical Field Stimulation ◽  
Bladder Smooth Muscle ◽  
Rt Pcr ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp

The functional role of the voltage-gated K+ (KV) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of KV2.1, KV2.2, and the electrically silent KV9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of KV2.1, KV2.2, and KV4.2 homotetrameric channels and of KV2.1/9.3 heterotetrameric channels, was used to examine the role of these channels in human DSM function. Human DSM tissues were obtained during open bladder surgeries from patients without a history of overactive bladder. Freshly isolated human DSM cells were studied using RT-PCR, immunocytochemistry, live-cell Ca2+ imaging, and the perforated whole cell patch-clamp technique. Isometric DSM tension recordings of human DSM isolated strips were conducted using tissue baths. RT-PCR experiments showed mRNA expression of KV2.1, KV2.2, and KV9.3 (but not KV4.2) channel subunits in human isolated DSM cells. KV2.1 and KV2.2 protein expression was confirmed by Western blot analysis and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the voltage step-induced KV current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca2+ level in DSM cells. In human DSM isolated strips, ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude and muscle force, and enhanced the amplitude of the electrical field stimulation-induced contractions within the range of 3.5–30 Hz stimulation frequencies. These findings reveal that ScTx1-sensitive KV2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.

scholarly journals Are there functional gap junctions or junctional hemichannels in macrophages?

Blood ◽  
1996 ◽  
Vol 88 (1) ◽  
pp. 328-334 ◽  
Author(s):  
LA Alves ◽  
R Coutinho-Silva ◽  
PM Persechini ◽  
DC Spray ◽  
W Savino ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Gap Junctions ◽  
Cell Line ◽  
Connexin 43 ◽  
Lucifer Yellow ◽  
Dye Coupling ◽  
Dye Uptake ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp

Abstract The existence of functional gap junctions in migratory cells of the immune system is a controversial issue. In this report, we have focused on one particular cell type, namely the macrophages, because connexin- 43, a protein that forms gap junctions, has been described in peritoneal macrophages and a macrophage cell line (J774), by Northern and Western blot analysis. To test whether these cell types expressed functional gap junctions, we assayed dye coupling by intracellular injection of Lucifer Yellow. We observed that nonstimulated macrophages are not coupled among themselves and did not form functional gap junctions with an epithelial cell line, which expresses functional gap junctions formed by connexin-43. Dye coupling was also not detected between macrophages previously activated by lipopolysaccharide or interferon-gamma. We further examined the presence of functional coupling using the more sensitive technique of dual whole cell patch- clamp, and again, did not find electrical coupling between macrophages, consistent with the dye microinjection data. We also examined the possible presence of hemigap junction channels activated by extracellular adenosine triphosphate (ATP) using a dye uptake assay and the whole cell patch-clamp technique. Conditions expected to close gap junction hemichannels (exposure to octanol and low intracellular pH) did not decrease ATP-induced Lucifer Yellow uptake, whereas conditions expected to increase hemichannel opening either did not affect ATP permeabilization (dibutyryl adenosine monophosphate) or decreased it (zero extracellular CA+2). Finally, in experiments using resident macrophages derived from conexin-43 knockout mice, we observed ATP induced dye uptake. Our experimental data thus indicate that macrophages in vitro do not form functional gap junctions and that the permeability pathway activated by extracellular ATP is not formed by a hemigap junction channel.

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