[Effect of KB-R7943 on Na(+)-Ca2+ exchange current in guinea pig ventricular myocytes].
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To study whether KB-R7943 has selective inhibitory effect on the inward and outward Na(+)-Ca2+ exchange current (INa-Ca) in guinea pig ventricular myocytes.Through setting up the model of intracellular Na(+)-overload during myocardial ischemia and reperfusion, the current-voltage relationship of INa-Ca was recorded using whole-cell patch clamp technique under bi-directional ionic conditions.Currents were elicited by a declining ramp pulse depolarized immediately from holding potential of -40 mV to +60 mV, then repolarized to -100 mV at a speed of 80 mV.s-1 and returned to the holding potential under bi-directional ionic conditions, while the [Na+] was 25 mmol.L-1 in the pipette solution. The currents increased time-dependently and voltage-dependently which reached from (2.51 +/- 0.15) pA.pF-1 to (5.94 +/- 0.13) pA.pF-1 at mV and from (-1.92 +/- 0.13) pA.pF-1 to (-3.17 +/- 0.16) pA.pF-1 at -80 mV (n = 12) after 3 min and there is no significant run-down of the current. KB-R7943 10(-6) mol.L-1 was found to decrease the current to (4.62 +/- 0.05) pA.pF-1 by 29.4% at mV and to (-2.30 +/- 0.18) pA.pF-1 by 22.1% at -80 mV (n = 5) after 5 min. While 10(-5) mol.L-1 KB-R7943 was shown to decrease the current to (3.13 +/- 0.03) pA.pF-1 by 61.7% at mV and to (-1.62 +/- 0.03) pA.pF-1 by 56.9% at -80 mV (n = 7).KB-R7943 can block INa-Ca in guinea pig ventricular myocytes. But, it did not show selective inhibition effect on inward and outward currents.Keywords:
Sodium-calcium exchanger
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1. Stretch-activated channels (SACs) were studied in isolated rat atrial myocytes using the whole-cell and single-channel patch clamp techniques. Longitudinal stretch was applied by using two patch electrodes. 2. In current clamp configuration, mechanical stretch of 20 % of resting cell length depolarised the resting membrane potential (RMP) from -63.6 +/- 0.58 mV (n = 19) to -54.6 +/- 2.4 mV (n = 13) and prolonged the action potential duration (APD) by 32.2 +/- 8.8 ms (n = 7). Depolarisation, if strong enough, triggered spontaneous APs. In the voltage clamp configuration, stretch increased membrane conductance in a progressive manner. The current-voltage (I-V ) relationship of the stretch-activated current (ISAC) was linear and reversed at -6.1 +/- 3.7 mV (n = 7). 3. The inward component of ISAC was abolished by the replacement of Na+ with NMDG+, but ISAC was hardly altered by the Cl- channel blocker DIDS or removal of external Cl-. The permeability ratio for various cations (PCs:PNa:PLi = 1.05:1:0.98) indicated that the SAC current was a non-selective cation current (ISAC,NC). The background current was also found to be non-selective to cations (INSC,b); the permeability ratio (PCs:PNa:PLi = 1.49:1:0.70) was different from that of ISAC,NC. 4. Gadolinium (Gd3+) acted on INSC,b and ISAC,NC differently. Gd3+ inhibited INSC,b in a concentration-dependent manner with an IC50 value of 46.2 +/- 0.8 microM (n = 5). Consistent with this effect, Gd3+ hyperpolarised the resting membrane potential (-71.1 +/- 0.26 mV, n = 9). In the presence of Gd3+ (0.1 mM), stretch still induced ISAC,NC and diastolic depolarisation. 5. Single-channel activities were recorded in isotonic Na+ and Cs+ solutions using the inside-out configuration. In NMDG+ solution, outward currents were abolished. Gd3+ (100 microM) strongly inhibited channel opening both from the inside and outside. In the presence of Gd3+ (100 microM) in the pipette solution, an increase in pipette pressure induced an increase in channel opening (21.27 +/- 0.24 pS; n = 7), which was distinct from background activity. 6. We concluded from the above results that longitudinal stret in rat atrial myocytes induces the activation of non-selective cation channels that can be distinguished from background channels by their different electrophysiology and pharmacology.
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When the patch-clamp technique was used, a slowly activating, time-dependent outward current was identified in both cell-attached and excised membrane patches obtained from guinea pig ventricular myocytes. This macroscopic patch current was present in approximately 50% of patches studied and could be observed both in the presence and absence of unitary single channel activity (i.e., ATP-sensitive K+ channels). The time course of activation of the patch current resembled that of the whole cell delayed-rectifier K+ current (IK) recorded under similar ionic conditions, and the patch current and IK were activated over a similar membrane potential range. The time-dependent patch current could be eliminated when the Nernst potential for K+ equaled that of the pulse voltage. The patch current was inhibited by external addition of the tertiary ammonium compound LY 97241 (50 microM) and was augmented after internal application of the catalytic subunit of adenosine 3',5'-cyclic monophosphate-dependent protein kinase (500 nM). Deactivating tail currents with kinetics similar to those of IK could be recorded to cell-attached and excised patches. Unitary single channel events underlying the time-dependent patch current could not be resolved despite various attempts to increase single channel conductance. Thus our results suggest that a major component of delayed rectification in guinea pig ventricular cells is due to the activity of a high-density, extremely low conductance K+ channel.
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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.[
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Abstract Transient currents are activated by spontaneous Ca 2+ oscillations in rabbit ventricular myocytes. We investigated the ionic basis for these transient currents under conditions in which K + currents would be expected to be blocked. Holding cells under voltage clamp at positive potentials leads to a rise in intracellular Ca 2+ via reversal of the Na + -Ca 2+ exchanger and subsequently to the initiation of spontaneous Ca 2+ transients, presumably from a Ca 2+ -overloaded sarcoplasmic reticulum. The current transients associated with these Ca 2+ transients reversed at about +10 to +15 mV under conditions of approximately symmetrical Cl − . In the absence of Cl − , this current was inward at all potentials examined over the range from −88 to +72 mV, consistent with a Na + -Ca 2+ exchanger current. In the absence of Na + , the repetitive spontaneous Ca 2+ transients could be initiated by a brief train of depolarizations to activate the inward Ca 2+ current. Under such conditions, the current was found to reverse at −3 mV when the equilibrium potential of Cl − (E Cl ) was −2 mV, and the reversal potential shifted to −32 mV when internal Cl − was lowered, to make E Cl −33 mV. Thus, in the absence of Na + , it appears that the current is exclusively a Ca 2+ -activated Cl − current. There is no evidence to indicate the presence of a Ca 2+ -activated cationic conductance. Further, our results demonstrate that the Ca 2+ -activated Cl − conductance can carry inward current at potentials more negative to E Cl in rabbit ventricular myocytes and is therefore likely to contribute to the arrhythmogenic delayed afterdepolarizations that occur in Ca 2+ -overloaded cells.
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Objective: This study examines the relative ability of sodium current (INa)-stimulated reverse mode Na/Ca exchange and the L-type calcium current (ICa) to trigger calcium-induced calcium release (CICR) in guinea-pig ventricular myocytes. Methods: Cytosolic Ca2+ transients were recorded from enzymatically dissociated guinea-pig ventricular mycocytes using Indo-1. Macroscopic membrane currents were simultaneously recorded using the whole-cell patch-clamp technique. Results: At room temperature (22–25°C) Ca2+ transients were associated with the activation of INa, ICa or INa plus ICa in combination. However, after ICa was blocked by verapamil (10 μM), no Ca2+ transient could be evoked by the activation of INa alone at either −40 or +5 mV. Similar results were obtained with 5 and 8 mM intracellular sodium, and when the temperature of the bathing solution was raised to 35°C and cAMP (10 μM) added to the pipette solution. Conclusions: From consideration of the relative magnitudes of the Ca2+ influx via ICa and Na/Ca exchange and thermodynamic considerations, we suggest that ICa is the major source of 'trigger' calcium for CICR (and cardiac contraction) under normal conditions. Although the Na/Ca exchanger was incapable of triggering CICR under the conditions of these experiments, we suggest that it may become more important when cytosolic Ca2+ is elevated, a condition which will also lead to decrease the amplitude of ICa.
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The Na(+)-HCO(3)(-) cotransporter (NBC) is an important sarcolemmal acid extruder in cardiac muscle. The characteristics of NBC expressed functionally in heart are controversial, with reports suggesting electroneutral (NBCn; 1HCO(3)(-) : 1Na(+); coupling coefficient N= 1) or electrogenic forms of the transporter (NBCe; equivalent to 2HCO(3)(-) : 1Na(+); N= 2). We have used voltage-clamp and epifluorescence techniques to compare NBC activity in isolated ventricular myocytes from rabbit, rat and guinea pig. Depolarization (by voltage clamp or hyperkalaemia) reversibly increased steady-state pH(i) while hyperpolarization decreased it, effects seen only in CO(2)/HCO(3)(-)-buffered solutions, and blocked by S0859 (cardiac NBC inhibitor). Species differences in amplitude of these pH(i) changes were rat > guinea pig approximately rabbit. Tonic depolarization (-140 mV to -0 mV) accelerated NBC-mediated pH(i) recovery from an intracellular acid load. At 0 mV, NBC-mediated outward current at resting pH(i) was +0.52 +/- 0.05 pA pF(-1) (rat, n= 5), +0.26 +/- 0.05 pA pF(-1) (guinea pig, n= 5) and +0.10 +/- 0.03 pA pF(-1) (rabbit, n= 9), with reversal potentials near -100 mV, consistent with N= 2. The above results indicate a functionally active voltage-sensitive NBCe in these species. Voltage-clamp hyperpolarization negative to the reversal potential for NBCe failed, however, to terminate or reverse NBC-mediated pH(i)-recovery from an acid load although it was slowed significantly, suggesting electroneutral NBC may also be operational. NBC-mediated pH(i) recovery was associated with a rise of [Na(+)](i) at a rate approximately 25% of that mediated via NHE, and consistent with an apparent NBC stoichiometry between N= 1 and N= 2. In conclusion, NBCe in the ventricular myocyte displays considerable functional variation among the three species tested (greatest in rat, least in rabbit) and may coexist with some NBCn activity.
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To investigate the membrane current changes induced by membrane stretching, single guinea pig ventricular myocytes were superfused with solutions of various osmolarities, and the whole-cell current was recorded by the patch-clamp technique. The application of 70% and 130% osmolar bath solutions increased and decreased the amplitude of delayed rectifier K+ current (IK), respectively, whereas no obvious change was observed in the L-type Ca2+ current or the inward rectifier K+ current. When the Na(+)-K+ pump current (Ipump) was recorded by the use of high-Na+ (> 35 mmol/L) pipette solutions, Ipump was also increased and decreased by the superfusion of hypotonic and hypertonic solutions, respectively, in approximately half of the cells. An increase of the Ipump was also observed in the absence of external Na+, excluding a possibility that the enhancement of Ipump was secondary to an elevation of cytosolic Na+. In most cells that did not show the increase of Ipump, the hypotonic superfusion induced a gradual a...
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