Ionic currents in rat pulmonary and mesenteric arterial myocytes in primary culture and subculture

1993 ◽  
Vol 264 (2) ◽  
pp. L107-L115 ◽  
Author(s):  
X. J. Yuan ◽  
W. F. Goldman ◽  
M. L. Tod ◽  
L. J. Rubin ◽  
M. P. Blaustein
Keyword(s):  
Cultured Cells ◽  
Primary Cultures ◽  
Outward Current ◽  
Ionic Currents ◽  
Patch Clamp Technique ◽  
Electrophysiological Properties ◽  
Bath Solution ◽  
Transient Component ◽  
Whole Cell Patch Clamp ◽  
Three Components

The electrophysiological properties of cultured single vascular smooth muscle (VSM) cells from rat pulmonary (PA) and mesenteric (MA) arteries were studied using the whole cell patch-clamp technique. Cells were studied at 3–7 days as primary cultures, or were replated after 10–20 days and subcultured for 2–5 days. In the standard physiological bath solution (containing 1.8 mM Ca2+), and with 125 mM K+ + 10 mM ethylene glycol-bis(beta-aminoethyl ether)- N,N,N',N'-tetraacetic acid (EGTA)-filled pipettes, both PA and MA primary cultured cells had high input resistances (mean = 2-3 G omega) and resting membrane potentials of about -40 mV. The cells were clamped at a holding potential of -70 mV. Depolarization to -20 mV or more evoked a transient inward current (Iin) that was eliminated in Ca(2+)-free bath solution; this indicates that Iin was carried by Ca2+. Iin was substantially smaller in subcultured cells from both PA and MA. Depolarization also activated three components of outward current (Iout) in primary cultured PA and MA cells: a rapidly inactivating transient component (Irt), a slowly inactivating transient component (Ist), and a steady-state (noninactivating) component (Iss). All three components of Iout were inhibited to varying degrees by 5 mM 4-aminopyridine and were eliminated by replacing intracellular K+ with Cs+, but were only minimally affected by removal of extracellular Ca2+. These results suggest that this Iout was carried by K+ and was voltage gated. Little external Ca(2+)-dependent Iout was observed under these conditions, but a substantial Ca(2+)-dependent component was seen when the EGTA concentration in the pipettes was reduced to 0.1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloride currents in primary cultures of rabbit proximal and distal convoluted tubules

1998 ◽  
Vol 275 (5) ◽  
pp. F651-F663 ◽  
Author(s):  
Isabelle Rubera ◽  
Michel Tauc ◽  
Michel Bidet ◽  
Chantal Poujeol ◽  
Béatrice Cuiller ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Physiological Role ◽  
Cell Types ◽  
Patch Clamp Technique ◽  
Chloride Currents ◽  
Hypotonic Stress ◽  
Quantitative Measurements ◽  
Whole Cell Patch Clamp

Cl− conductances were studied in cultured rabbit proximal convoluted tubule (PCT) epithelial cells and compared with those measured in cultured distal bright convoluted tubule (DCTb) epithelial cells. Using the whole cell patch-clamp technique, three types of Cl− conductances were identified in DCTb cultured cells. These consisted of volume-sensitive, Ca2+-activated, and forskolin-activated Cl−currents. In PCT cultured cells, only volume-sensitive and Ca2+-activated Cl− currents were recorded. The characteristics of Ca2+-activated currents in PCT cells closely resembled those in DCTb cells. Volume-sensitive Cl− currents could be elicited both in PCT and in DCTb cells by hypotonic stress. The pharmacological profile of this conductance was established for both cell types. Forskolin activated a linear Cl− current in DCTb cells but not in PCT cells. This conductance was insensitive to DIDS and corresponds to cystic fibrosis transmembrane conductance regulator (CFTR)-like channels. Quantitative measurements of SPQ fluorescence showed that only the apical membrane of DCTb cells possessed a Cl− pathway that was sensitive to forskolin. RT-PCR experiments showed the presence of CFTR mRNA in both cultures, whereas immunostaining experiments revealed the expression of CFTR in DCTb cells only. The physiological role of the different types of channels is discussed.

scholarly journals Effect of Auxin (IAA) on the Fast Vacuolar (FV) Channels in Red Beet (Beta vulgaris L.) Taproot Vacuoles

2020 ◽  
Vol 21 (14) ◽  
pp. 4876
Author(s):  
Zbigniew Burdach ◽  
Agnieszka Siemieniuk ◽  
Waldemar Karcz
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
K Channel ◽  
Single Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Inward Current ◽  
Bath Solution ◽  
Outward Currents ◽  
Inward Currents

In contrast to the well-studied effect of auxin on the plasma membrane K+ channel activity, little is known about the role of this hormone in regulating the vacuolar K+ channels. Here, the patch-clamp technique was used to investigate the effect of auxin (IAA) on the fast-activating vacuolar (FV) channels. It was found that the macroscopic currents displayed instantaneous currents, which at the positive potentials were about three-fold greater compared to the one at the negative potentials. When auxin was added to the bath solution at a final concentration of 1 µM, it increased the outward currents by about 60%, but did not change the inward currents. The imposition of a ten-fold vacuole-to-cytosol KCl gradient stimulated the efflux of K+ from the vacuole into the cytosol and reduced the K+ current in the opposite direction. The addition of IAA to the bath solution with the 10/100 KCl gradient decreased the outward current and increased the inward current. Luminal auxin reduced both the outward and inward current by approximately 25% compared to the control. The single channel recordings demonstrated that cytosolic auxin changed the open probability of the FV channels at the positive voltages to a moderate extent, while it significantly increased the amplitudes of the single channel outward currents and the number of open channels. At the positive voltages, auxin did not change the unitary conductance of the single channels. We suggest that auxin regulates the activity of the fast-activating vacuolar (FV) channels, thereby causing changes of the K+ fluxes across the vacuolar membrane. This mechanism might serve to tightly adjust the volume of the vacuole during plant cell expansion.

Effects of thyroid hormone on action potential and repolarizing currents in rat ventricular myocytes

2000 ◽  
Vol 278 (2) ◽  
pp. E302-E307 ◽  
Author(s):  
Zhuo-Qian Sun ◽  
Kaie Ojamaa ◽  
William A. Coetzee ◽  
Michael Artman ◽  
Irwin Klein
Keyword(s):  
Action Potential ◽  
Cardiac Electrophysiology ◽  
Ventricular Myocytes ◽  
Outward Current ◽  
Mechanisms Of Action ◽  
Transient Outward Current ◽  
Prolonged Action ◽  
Electrophysiological Properties ◽  
Whole Cell Patch Clamp ◽  
Prolonged Action Potential Duration

Thyroid hormones play an important role in cardiac electrophysiology through both genomic and nongenomic mechanisms of action. The effects of triiodothyronine (T3) on the electrophysiological properties of ventricular myocytes isolated from euthyroid and hypothyroid rats were studied using whole cell patch clamp techniques. Hypothyroid ventricular myocytes showed significantly prolonged action potential duration (APD90) compared with euthyroid myocytes, APD90 of 151 ± 5 vs. 51 ± 8 ms, respectively. Treatment of hypothyroid ventricular myocytes with T3 (0.1 μM) for 5 min significantly shortened APD by 24% to 115 ± 10 ms. T3 similarly shortened APD in euthyroid ventricular myocytes, but only in the presence of 4-aminopyridine (4-AP), an inhibitor of the transient outward current ( I to), which prolonged the APD by threefold. Transient outward current ( I to) was not affected by the acute application of T3 to either euthyroid or hypothyroid myocytes; however, I to density was significantly reduced in hypothyroid compared with euthyroid ventricular myocytes.

Divalent cations modulate the transient outward current in rat ventricular myocytes

1991 ◽  
Vol 261 (2) ◽  
pp. C310-C318 ◽  
Author(s):  
Z. S. Agus ◽  
I. D. Dukes ◽  
M. Morad
Keyword(s):  
Hippocampal Neurons ◽  
Voltage Dependence ◽  
Ventricular Myocytes ◽  
Divalent Cations ◽  
Outward Current ◽  
Transient Outward Current ◽  
Patch Clamp Technique ◽  
Rat Ventricular Myocytes ◽  
Specific Effects ◽  
Whole Cell Patch Clamp

The modulation of the transient outward K+ current (Ito) by divalent cations was studied in enzymatically isolated rat ventricular myocytes with the whole cell patch-clamp technique. At holding potentials negative to -70 mV, 1 mM Cd2+ suppressed Ito, whereas, at potentials positive to -50 mV, the current was augmented. These effects were caused by shifts in the voltage dependence of both activation and inactivation of Ito toward more positive potentials. Cd2+ also slowed the activation kinetics of Ito by shifting the voltage dependence of its rate of activation, but the rate of inactivation was unaffected. Other divalent cations produced similar shifts but at markedly different concentrations. Thus, in the millimolar range, a rightward shift of approximately 20 mV was produced by 3 Co2+, 5 Ni2+, and 10 Ca2+, whereas 10 microM concentrations of Cu2+ and Zn2+ produced equivalent shifts. Similar effects were seen in hippocampal neurons with micromolar concentrations of Zn2+. Thus divalent cations have marked and specific effects on the kinetics and voltage dependence of Ito and may serve as a regulatory mechanism in its activation, particularly in cells with resting potentials positive to -60 mV.

Ionic currents of cultured horizontal cells isolated from white perch retina

1986 ◽  
Vol 55 (3) ◽  
pp. 499-513 ◽  
Author(s):  
E. M. Lasater
Keyword(s):  
White Perch ◽  
Slow Component ◽  
Outward Current ◽  
Ionic Currents ◽  
Cell Types ◽  
Horizontal Cells ◽  
Inward Current ◽  
Whole Cell Patch Clamp ◽  
Anomalous Rectifier ◽  
K Concentration

Horizontal cells from the retinas of white perch were isolated and maintained in cell culture for 3 days to 3 wk. Four morphologically distinct types of horizontal cells could be identified in culture and were labeled types H1, H2, H3, and H4. Whole-cell patch-clamp techniques were used to study the ionic currents present in the four cell types. In all cells, depolarizing commands above threshold elicited a fast-inward current followed by an outward current. The fast-inward current was abolished by tetrodotoxin (TTX) or 0 Na+ Ringer's, indicating the current was carried by Na+. In H1, H2, and H3 cells, the outward current, carried by K+, consisted of two components: a transient current (IA), blockable with 4-aminopyridine (4-AP), tetraethylammonium (TEA), or intracellular cesium and a sustained current that could be blocked with TEA. The H4 cell had only the sustained current. An inward rectifying K+ current (anomalous rectifier) was observed in the four cell types. The current was sensitive to the extracellular K+ concentration. Its activation showed two components: an instantaneous component and a slower component. The slow component becomes faster with greater hyperpolarizations. The four cell types possessed a small, sustained Ca2+ current that, under normal conditions, was masked by the inward Na+ current and outward K+ currents.

Calcium-activated chloride currents in primary cultures of rabbit distal convoluted tubule

1996 ◽  
Vol 271 (4) ◽  
pp. F940-F950 ◽  
Author(s):  
M. Bidet ◽  
M. Tauc ◽  
I. Rubera ◽  
G. de Renzis ◽  
C. Poujeol ◽  
...  
Keyword(s):  
Ion Selectivity ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Distal Convoluted Tubule ◽  
Disulfonic Acid ◽  
Resting Membrane Potential ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Steady State Current ◽  
Cl Permeability

Chloride (Cl-) conductances were studied in primary cultures of rabbit distal convoluted tubule (very early distal “bright” convoluted tubule, DCTb) by the whole cell patch-clamp technique. We identified a Cl- current activated by 2 microM extracellular ionomycin. The kinetics of the macroscopic current were time dependent for depolarizing potentials with a slow developing component. The steady state current presented outward rectification, and the ion selectivity sequence was I- > Br- > > Cl > glutamate. The current was inhibited by 0.1 mM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and 1 mM diphenylamine-2-carboxylate. To identify the location of the Cl- conductance, 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence experiments were carried out in confluent cultures developed on collagen-coated permeable filters. Cl- removal from the apical solution induced a Cl- efflux that was stimulated by 10 microM forskolin. Forskolin had no effect on the basolateral Cl- permeability Cl- substitution in the basolateral solution induced an efflux stimulated by 2 microM ionomycin or 50 microM extracellular ATP Ionomycin had no effect on the apical Cl- fluxes. Thus cultured DCTb cells exhibit Ca(2+)-activated Cl- channels located in the basolateral membrane. This Cl- permeability was active at a resting membrane potential and could participate in the Cl- reabsorption across the DCTb in control conditions.

Properties of potassium currents in Purkinje cells of failing human hearts

2002 ◽  
Vol 283 (6) ◽  
pp. H2495-H2503 ◽  
Author(s):  
Wei Han ◽  
Liming Zhang ◽  
Gernot Schram ◽  
Stanley Nattel
Keyword(s):  
Purkinje Cells ◽  
Ventricular Myocytes ◽  
Outward Current ◽  
Ionic Currents ◽  
Time Dependent ◽  
Transient Outward Current ◽  
Whole Cell Patch Clamp ◽  
Cardiac Purkinje Fibers ◽  
Cardiac Purkinje Cells ◽  
Hyperpolarization Activated Current

Cardiac Purkinje fibers play an important role in cardiac arrhythmias, but no information is available about ionic currents in human cardiac Purkinje cells (PCs). PCs and midmyocardial ventricular myocytes (VMs) were isolated from explanted human hearts. K+ currents were evaluated at 37°C with whole cell patch clamp. PCs had clear inward rectifier K+current ( I K1), with a density not significantly different from VMs between −110 and −20 mV. A Cs+-sensitive, time-dependent hyperpolarization-activated current was measurable negative to −60 mV. Transient outward current ( I to) density was smaller, but end pulse sustained current ( I sus) was larger, in PCs vs. VMs. I to recovery was substantially slower in PCs, leading to strong frequency dependence. Unlike VM I to, which was unaffected by 10 mM tetraethylammonium, Purkinje I to was strongly inhibited by tetraethylammonium, and Purkinje I to was 10-fold more sensitive to 4-aminopyridine than VM. PC I sus was also reduced strongly by 10 mM tetraethylammonium. In conclusion, human PCs demonstrate a prominent I K1, a time-dependent hyperpolarization-activated current, and an I towith pharmacological sensitivity and recovery kinetics different from those in the atrium or ventricle and compatible with a different molecular basis.

scholarly journals LOCUST MEDIAL NEUROSECRETORY CELLS IN VITRO: MORPHOLOGY, ELECTROPHYSIOLOGICAL PROPERTIES AND EFFECTS OF TEMPERATURE

1993 ◽  
Vol 183 (1) ◽  
pp. 323-339
Author(s):  
W. Rossler ◽  
U. Bickmeyer
Keyword(s):  
Culture Medium ◽  
Locusta Migratoria ◽  
Ionic Currents ◽  
Neurosecretory Cells ◽  
Patch Clamp Technique ◽  
Pars Intercerebralis ◽  
Serum Free ◽  
Electrophysiological Properties ◽  
Effects Of Temperature

The medial neurosecretory cells of the pars intercerebralis in the protocerebrum of larval and adult locusts (Locusta migratoria) were cultured in a chemically defined serum-free culture medium. The morphology of the cells was investigated by light microscopy and the electrophysiological properties were studied using the patch-clamp technique in the whole-cell configuration. The dissociated neurosecretory cells grew new processes under these conditions and were maintained in culture for up to 2 months. The percentage of cells showing outgrowth was significantly higher in third-instar larvae than in instars 4 and 5 and adults. A primary axonal stump promoted a unipolar cell morphology; in other cases, most neurosecretory cells became multipolar. The presence of glial cells in undissociated groups of neurosecretory cells improved outgrowth and the formation of neurite bundles. A considerable number of the recorded cells showed spiking activity in response to depolarization. The influences of temperature on spike frequency, duration and amplitude as well as on membrane potential and ionic currents were investigated. The results suggest that temperature may directly affect the function of neurosecretory cells.

Cl- current in IMCD cells activated by hypotonicity: time course, ATP dependence, and inhibitors

1996 ◽  
Vol 271 (3) ◽  
pp. F552-F559 ◽  
Author(s):  
K. A. Volk ◽  
C. Zhang ◽  
R. F. Husted ◽  
J. B. Stokes
Keyword(s):  
Time Course ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Renal Medulla ◽  
Cell Types ◽  
Disulfonic Acid ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent

The hypertonic environment of the renal medulla can change rapidly according to the state of hydration of the animal. We used primary cultures of rat inner medullary collecting duct (IMCD) cells to investigate the characteristics of Cl- currents activated by an acute reduction in osmolarity (ICl(osm)). Using the whole cell patch-clamp technique, we identified an outwardly rectifying current that decayed slowly at strongly depolarizing voltages. The onset of ICl(osm) began 6.7 min after the fall in bath osmolarity, a delay longer than reported in other cell types. Hypotonicity did not induce an increase in intracellular Ca2+ concentration, and activation of ICl(osm) did not require the presence of Ca2+. Intracellular ATP was needed to evoke ICl(osm) when the hypotonic stimulus was modest (50 mosmol/l or less) but was not necessary when the stimulus was stronger (100 mosmol/ l). ICl(osm) was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid but not by tamoxifen or glibenclamide. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid produced a voltage-dependent block. Acute reduction in osmolarity using cells grown on filters did not induce a Cl- secretory current. The ICl(osm) of IMCD cells appears to be on the basolateral membrane and displays some unique features.

Comparison of a Ca(2+)-gated conductance and a second-messenger-gated conductance in rat olfactory neurons

2000 ◽  
Vol 203 (3) ◽  
pp. 567-573
Author(s):  
Y. Okada ◽  
R. Fujiyama ◽  
T. Miyamoto ◽  
T. Sato
Keyword(s):  
Outward Current ◽  
Patch Clamp Technique ◽  
External Application ◽  
Olfactory Neurons ◽  
Internal Solution ◽  
Inward Current ◽  
Whole Cell Patch Clamp ◽  
Cation Conductance ◽  
Intracellular Ca ◽  
Sustained Outward Current

The effect of a rise in intracellular Ca(2+) concentration was analyzed in isolated rat olfactory neurons using a whole-cell patch-clamp technique. Intracellular dialysis of 1 mmol l(−)(1) Ca(2+) in a standard-K(+), low-Cl(−) internal solution (E(Cl)=−69 mV) from the patch pipette into the olfactory neurons induced a sustained outward current of 49+/−5 pA (N=13) at −50 mV in all the cells examined. The outward currents were inhibited by external application of 100 micromol l(−)(1) 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB). External application of a Ca(2+) ionophore, 3 micromol l(−)(1) ionomycin, induced an inward current in three of eight cells whose voltages were clamped using the gramicidin-perforated technique, but ionomycin elicited an outward current in the other five cells, suggesting that natural intracellular Cl(−) concentration in the olfactory neurons was heterogeneous. While intracellular dialysis of 50 micromol l(−)(1) inositol 1,4,5-trisphosphate (1,4,5-InsP(3)) in the standard-K(+), low-Cl(−) internal solution induced the NPPB-sensitive outward current in 31 % of cells, and 500 micromol l(−)(1) cAMP induced it in 21 % of cells, a large proportion of the cells displayed an inward current in response to 1,4,5-InsP(3) and cAMP. The results suggest that 1,4,5-InsP(3) and cAMP can elicit Ca(2+)-dependent Cl(−) conductance and Ca(2+)-independent cation conductance in rat olfactory neurons.

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