The volume-sensitive chloride channel in mouse cardiac myocytes
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Objective:To characterize the volume_sensitive chloride channel (I Cl,Vol ) in isolated cardiac myocytes of mouse. Methods:The cardiac myocytes were isolated from mouse heart and the whole_cell patch clamp was employed.Results:Affer exposure of the cardiac myocytes to a hypotonic solution,a volume_sensitive chloride current was activated. Time_dependent inactivation was observed at large positive potentials. The current_voltage relationship showed that the reversal potential of the hypotonic_activated current (_34.5±0.8?mV) was close to the calculated equilibrium potential for Cl _(E Cl =_38.6?mV). When the extracellular Cl _ concentration changed, the reversal potential (E rev ) of the current also shifted. The E rev shifted per 10_fold of o was 43.9?mV . The activation of the current depended on intracellular ATP. The anion permeability order of volume_sensitive Cl _ current was I _Br _ Cl _. A relative permeability of I _ and Br _ to Cl _ was 2.32∶1.28∶1.Conclusions:In mouse cardiac myocytes , the volume_sensitive Cl _ current that is dependent on intracellular ATP has an outward rectification and time_dependent inactivation at positive potential.[Keywords:
Reversal potential
Chloride channel
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1. Hyperpolarization‐activated Cl‐ currents (ICl,hyp) were investigated in the T84 human adenocarcinoma cell line, using the patch‐clamp whole‐cell configuration. 2. During whole‐cell recording with high‐chloride and ATP‐containing internal solutions, hyperpolarizing jumps from a holding potential of 0 mV elicited slow inward current relaxations, carried by Cl‐ and detected at membrane potentials more negative than ‐40 mV. Analysis of the relative permeabilities to monovalent anions gave the following sequence: Cl‐ > Br‐ > I‐ > glutamate. 3. ICl,hyp was partially inhibited by 1 mM diphenylamine‐2‐carboxylic acid or 0.1 mM 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate, and was completely blocked by Cd2+ (> 300 microM). It was insensitive to 1 mM external 4,4'‐diisothiocyanatostilbene‐2,2'‐disulphonic acid or 1 mM Ba2+. 4. ICl,hyp was inhibited by external application of 500 microM cptcAMP (8‐(4‐chlorophenylthio)‐adenosine 3':5'‐cyclic monophosphate) or 500 nM of the protein kinase C activator, phorbol 12‐myristate, 13‐acetate. 5. (i) Omission of ATP from the pipette solution, (ii) ATP replacement by the non‐hydrolysable ATP analogue 5'‐adenylylimidodiphosphate, and (iii) inhibition of protein kinase C by staurosporine or calphostin C accelerated the activation kinetics of the current and increased its amplitude, but did not alter its pharmacological properties. 6. We conclude that hyperpolarization‐activated Cl‐ channels similar to those of ClC‐2 channels (mammalian homologue of Torpedo chloride channel ClC‐0) are present in T84 cells, and that their gating properties are modulated by phosphorylation.
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Calphostin C
Chloride channel
Staurosporine
Phorbol
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Abstract Oxygen-derived free radicals (O-Rs) cause alterations in cardiac electrical activity, including sustained depolarization, which may contribute to arrhythmic activity in reperfusion after ischemia. The ionic current(s) and cellular mechanism(s) underlying the sustained depolarization are not well defined. We used the whole-cell variant of the patch-clamp technique to study sustained depolarization in guinea pig ventricular myocytes during the extracellular application of O-Rs (generating system: dihydroxyfumaric acid, 3 to 6 mmol/L; FeCl 3 /ADP, 0.05:0.5 mmol/L). Myocytes superfused with O-Rs (pipette EGTA, 0.1 mmol/L) showed (1) sustained depolarization to between −40 and −10 mV, (2) oscillations in membrane potential, and (3) triggered activity. The depolarization resulted from an increase in quasi–steady state difference current reversing at ≈−18 mV, and the oscillations were due to transient inward current. The latter were inhibited with ryanodine (10 μmol/L) or high pipette EGTA (5 mmol/L), but the steady state current was unaffected. Nonselective cation current (I NSC ) (recorded with Cs + , Li + , and Mg 2+ replacing K + , Na + , and Ca 2+ , respectively; 20 mmol/L tetraethylammonium chloride [TEA] and 5 mmol/L BAPTA in the pipette solution and 10 mmol/L TEA, 10 μmol/L tetrodotoxin, and 10 μmol/L nicardipine in the bath solution) was activated by O-Rs; the increase in current was unaffected by preventing changes in [Ca 2+ ] i but was inhibited with dithiothreitol. Oxidizing agents (diamide and thimerosal) or caffeine (pipette EGTA, 0.1 mmol/L) produced a similar increase in membrane conductance. I NSC activated with O-Rs, oxidizing agents, or caffeine was sensitive to SK&F 96365. O-R treatment was without effect when I NSC was already activated with caffeine. The data suggest that (1) extracellular O-Rs activate a Ca 2+ -sensitive I NSC in the absence of changes in [Ca 2+ ] i , (2) oxidative modification of extracellular sulfhydryl groups may be involved, and (3) this mechanism is different from the Ca 2+ -dependent activation of I NSC by intracellular O-Rs, indicating that O-Rs may alter ion channel activity by differential mechanisms, depending on the compartment, extracellular or intracellular, in which they are present.
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The fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to measure pHi in the spontaneously hypertensive rat (SHR) and in normal rat cardiac myocytes under nominally HCO3-free (20 mmol/L HEPES-buffered) conditions. When only the Na-H exchanger was blocked, the intrinsic buffering power (beta i) in SHR myocytes was significantly higher than when both the Na-H exchanger and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)-sensitive pHi regulators (the Na-HCO3 cotransporter and the Cl-HCO3 exchanger) were blocked. Similar low values for beta i were also found for normal rat myocytes in Na(+)-free conditions. In Cl(-)-free solution under nominally HCO3-free conditions, in both normal and SHR myocytes, the pHi slowly alkalinized (by 0.16 +/- 0.02 and 0.11 +/- 0.02 pH units, respectively); this alkalinization was also DIDS sensitive. The reacidification during NH4+ perfusion was inhibited 30.2 +/- 7.4% by DIDS. In addition, in the nominal absence of HCO3-, 100 mumol/L ATP ac...
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HEPES
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Bicarbonate
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When swollen in hypotonic media, HL-60 cells exhibit a regulatory volume decrease (RVD) response as a result of net losses of K+ and Cl-. This is primarily caused by a dramatic increase in Cl- permeability, which may reflect the opening of volume-sensitive channels (11). To test this hypothesis, we measured volume-activated Cl- currents in HL-60 cells using the patch-clamp technique. The whole cell Cl- conductance (in nS/pF at 100 mV) increased from 0.09 +/- 0.06 to 1.15 +/- 0.19 to 1.64 +/- 0.40 as the tonicity (in mosmol/kgH2O) of the external medium was decreased from 334 to 263 to 164, respectively. Cl- currents showed no significant inactivation during 800-ms pulses. Current-voltage curves exhibited outward rectification and were identical at holding potentials of 0 or -50 mV, suggesting that the gating of the channels is voltage independent. The selectivity sequence, based on permeability ratios (PX/PCl) calculated from the shifts of the reversal potentials, was SCN- > I- approximately NO3- > Br- > Cl- >> gluconate. 4-Acetamido-4'- isothiocyanostilbene-2,2'-disulfonic acid (SITS; 0.5 mM) inhibits HL-60 Cl- channels in a voltage-dependent manner, with approximately 10-fold increased affinity at potentials greater than +40 mV. Voltage-dependent blockade by SITS indicates that the binding site is located near the outside, where it senses 20% of the membrane potential. These Cl- channels were also inhibited in a voltage-independent manner by the oxonol dye bis-(1,3-dibutylbarbituric acid)pentamethine oxonol [diBA-(5)-C4] with a concentration that gives half inhibition (IC50) of 1.8 microM at room temperature. A similar apparent IC50 value (1.2 microM) was observed for net 36Cl- efflux into a Cl(-)-free hypotonic medium at 21 degrees C. It seems likely, therefore, that the volume-activated Cl- channels are responsible for the net Cl- efflux during RVD. These Cl- channels have properties similar to the “mini-Cl-” channels described in lymphocytes and neutrophils and are strongly inhibited by low concentrations of diBA-(5)-C4.
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Chloride channel
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Effect of agmatine on intracellular free calcium concentration in isolated rat ventricular myocytes.
The present study was to investigate the effects of agmatine (Agm) on free intracellular calcium concentration ([Ca(2+)]( i )) of isolated rat ventricular myocytes. [Ca(2+)]( i ) was measured by confocal microscopy in single rat ventricular myocytes which were dissociated by enzymatic dissociation method and loaded with Fluo 3-AM. The changes in [Ca(2+)]( i ) were represented by fluorescence intensity (FI) or relative fluorescence intensity (F/F(0)%). The results showed that the control level of FI value of single rat ventricular myocytes was 128.8+/-13.8 and 119.6+/-13.6 in the presence of normal Tyrode's solution containing Ca(2+) 1.0 mmol/L and Ca(2+)-free Tyrode's solution, respectively. There was no difference between these two groups (P>0.05). Agm 0.1, 1, and 10 mmol/L significantly reduced the [Ca(2+)]( i ) in both extracellular solutions in a concentration-dependent manner. The similar effect of Agm on [Ca(2+)]( i ) was also observed in the presence of EGTA 3 mmol/L. KCl 60 mmol/L, PE 30 micromol/L, and Bay-K-8644 10 micromol/L, all these substances induced [Ca(2+)]( i ) elevations in ventricular myocytes. Agm (0.1, 1, and 10 mmol/L) markedly inhibited the increase in [Ca(2+)]( i ) induced by KCl, phenylephrine (PE), and Bay-K-8644. When Ca(2+) waves were produced by increasing extracellular Ca(2+) concentration from 1 to 10 mmol/L, 1 mmol/L Agm could block the propagating waves of elevated [Ca(2+)]( i ), and reduce the velocity and duration of propagating waves. These results suggest that Agm possesses an inhibitory effects on [Ca(2+)]( i ) via blocking voltage-dependent Ca(2+) channel, and possibly by alleviating calcium release from SR in single isolated rat ventricular myocytes.
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Phenylephrine
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Cardiac ventricular myocytes from several species, including the guinea pig, possess a cAMP-dependent protein kinase A (PKA)-activated Cl- channel. In the present study, the properties of a protein kinase C (PKC)-activated Cl- current were studied in isolated guinea pig ventricular myocytes using the whole-cell arrangement of the patch-clamp technique. Intracellular dialysis of ventricular cells with PKC resulted in the activation of a large background current that displayed time-independent kinetics. In the presence of 146 mmol/L external Cl- and 71 mmol/L internal Cl-, the reversal potential (Erev) of the background current (-17 +/- 1 mV) was close to that of the Cl- equilibrium potential (-18 mV), and the current versus voltage relation for the current was outward rectifying in shape. When [Cl-]i or [Cl-]o was reduced by substitution of Cl- with aspartic acid, Erev for the background current shifted in a manner expected for a Cl(-)-selective channel. Based on Erev measurements, the permeability sequence for this PKC-activated Cl- channel was determined to be SCN- > I- > Br- congruent to Cl-. The PKC-activated Cl- current was not inhibited by the Cl- channel blocker 4,4'-dinitrostilbene-2,2'-disulfonic acid (100 mumol/L) but could be blocked by anthracene-9-carboxylic acid (1 mmol/L). Activation of the current was abolished in the presence of the PKC inhibitor staurosporine (2.5 mumol/L). Under conditions designed to cause a maximal activation of the Cl- channels by PKC, the addition of forskolin (1 mumol/L) to stimulate PKA caused only a slight further increase in the amplitude of the Cl- current. Thus, PKC activates a Cl- channel in guinea pig ventricular cells with properties similar but not identical to the PKA-activated channel.
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Staurosporine
Chloride channel
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