No abstract available.
Calcium* ; Cyclic GMP* ; Muscle Cells*

In single atrial and ventricular cells isolated from the guinea-pig and the rabbit heart, action potentials and membrane currents were recorded by using the whole cell voltage clamp technique. In rabbit atrial cells the repolarization showed two distinctive phases, referred as the early and late phases(early and late plateau phase), but in guinea-pig atrial cells there was a maintained plateau and less distinctive two phases of repolartization. Increasing intracellular sodium or reducing external sodium by replacement with lithium suppressed the late phase of the action potential in rabbit atrial cells and shortened the plateau of action potential in rabbit ventricle and guinea-pig atrial cells. Reducing external sodium decreased Ca-current and late inward current in voltage clamp. Ouabain in the concentration of 10(-5)M shortened the duration of action potential and shifted the holding current level to outward direction, decreased Ca-current and moved late inward current to outward direction. Ryanodine 10(-6)M which is known to be an inhibitor of Ca-release in the intracellular store, suppressed the late phase of action potential in rabbit atrial cells and shortened the plateau of action potential in rabbit ventricular cells. Ryanodine also decreased Ca-current and shifted late inward current to outward direction. It is concluded that an inward current activated by intracellular calcium contributes to the late Phase of the action potential in rabbit atrial cells and to the late plateau in rabbit ventricular cells and in guinea-pig atrial cells. It may be carried by the Na-Ca exchange precess and/or by calcium-activated non-specific channels but preferably Na-Ca exchange machanism.
Action Potentials* ; Calcium ; Heart* ; Lithium ; Membranes* ; Myocytes, Cardiac ; Ouabain ; Ryanodine ; Sodium

No abstract available.
B-Lymphocytes*

The types of K+ channel which determine the pattern of spontaneous action potential (SAP) were investigated using whole-cell variation of patch clamp techniques under current- and voltage-clamp recording conditions in rat clonal pituitary GH3 cells. Heterogeneous pattern of SAP activities was changed into more regular mode with elongation of activity duration and afterhyperpolarization by treatment of TEA (10 mM). Under this condition, exposure of the class III antiarrhythmic agent E-4031 (5 micrometer) to GH3 cells hardly affected SAP activities. On the other hand, the main GH3 stimulator thyrotropin-releasing hormone (TRH) still produced its dual effects (transient hyperpolarization and later increase in SAP frequency) in the presence of TEA. However, addition of BaCl2 (2 mM) in the presence of TEA completely blocked SAP repolarization process and produced membrane depolarization in all tested cells. This effect was observed even in TEA-untreated cells and was not mimicked by higher concentration of TEA (30 mM). Also this barium-induced membrane depolarization effect was still observed after L-type Ca2+ channel was blocked by nicardipine (10 micrometer). These results suggest that barium-sensitive current is important in SAP repolarization process and barium itself may have some depolarizing effect in GH3 cells.
Action Potentials* ; Animals ; Barium ; Cell Line* ; Hand ; Membranes ; Nicardipine ; Patch-Clamp Techniques ; Rats* ; Tea ; Thyrotropin-Releasing Hormone

BACKGROUND: We have previously reported that not only cGMP but also 8-Br-cGMP or 8-pCPT-cGMP, specific and potent stimulators of cGMP-dependent protein kinase (cGMP-PK), increased basal L-type calcium current (ICa) in rabbit ventricular myocytes. Our findings in rabbit ventricular myocytes were entirely different from the earlier findings in different species, suggesting that the activation of cGMP-PK is involved in the facilitation of ICa by cGMP. However, there is no direct evidence that cGMP-PK can stimulate ICa in rabbit ventricular myocytes. In this report, we focused on the direct effect of cGMP-PK an ICa in rabbit ventricular myocytes. METHODS AND RESULTS: We isolated single ventricular myocytes of rabbit hearts by using enzymatic dissociation. Regulation of ICa by cGMP-PK was investigated in rabbit ventricular myocytes using whole-cell voltage clamp method. ICa was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Extracellular 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP), potent stimulator of cGMP-dependent protein kinase (cGMP-PK), increased basal ICa. cGMP-PK also increased basal ICa. The stimulation of basal ICa by cGMP-PK required both 8-Br-cGMP in low concentration and intracellular ATP to be present. The stimulation of basal ICa by cGMP-PK was blocked by heat inactivation of the cGMP-PK and by bath application of 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-pCPT-cGMP), a phosphodiesterase-resistant cGMP-PK inhibitor. When ICa was increased by internal application of cGMP-PK, IBMX resulted in an additional stimulation of ICa. In the presence of cGMP-PK, already increased ICa was potentiated by bath application of isoprenaline or forskolin or intracellular application of cAMP. CONCLUSIONS: We present evidence that cGMP-PK stimulated basal ICa by a direct phosphorylation of L-type calcium channel or associated regulatory protein in rabbit ventricular myocytes.
1-Methyl-3-isobutylxanthine ; Adenosine Triphosphate ; Baths ; Calcium Channels, L-Type ; Calcium* ; Colforsin ; Heart ; Hot Temperature ; Isoproterenol ; Muscle Cells* ; Phosphorylation ; Protein Kinases*

The intermediate conductance Ca2+-activated K+ channels (SK4, IKCa1) are present in lymphocytes, and their membrane expression is upregulated by various immunological stimuli. In this study, the activity of SK4 was compared between Bal-17 and WEHI-231 cell lines which represent mature and immature stages of murine B lymphocytes, respectively. The whole-cell patch clamp with high-Ca2+ (0.8microM) KCl pipette solution revealed a voltage-independent K+ current that was blocked by clotrimazole (1 mM), an SK4 blocker. The expression of mRNAs for SK4 was confirmed in both Bal-17 and WEHI-231 cells. The density of clotrimazole-sensitive SK4 current was significantly larger in Bal-17 than WEHI-231 cells (-11.4+/-3.1 Vs. -5.7+/-1.15 pA/pF). Also, the chronic stimulation of B cell receptors (BCR) by BCR-ligation (anti-IgM Ab, 3microgram/ml, 8~12 h) significantly upregulated the amplitude of clotrimazole-sensitive current from -11.4+/-3.1 to -53.1+/-8.6 pA/pF in Bal-17 cells. In WEHI-231 cells, the effect of BCR-ligation was significantly small (-5.7+/-1.15 to -9.0+/-1.00 pA/pF). The differential expression and regulation by BCR-ligation might reflect functional changes in the maturation of B lymphocytes.
B-Lymphocytes* ; Cell Line ; Clotrimazole ; Lymphocytes ; Membranes ; Potassium Channels ; Potassium Channels, Calcium-Activated ; RNA, Messenger

We developed a cardiac cell model to explain the phenomenon of mechano-electric feedback (MEF), based on the experimental data with rat atrial myocytes. It incorporated the activity of ion channels, pumps, exchangers, and changes of intracellular ion concentration. Changes in membrane excitability and Ca2+ transients could then be calculated. In the model, the major ion channels responsible for the stretch-induced changes in electrical activity were the stretch-activated channels (SACs). The relationship between the extent of stretch and activation of SACs was formulated based on the experimental findings. Then, the effects of mechanical stretch on the electrical activity were reproduced. The shape of the action potential (AP) was significantly changed by stretch in the model simulation. The duration was decreased at initial fast phase of repolarization (AP duration at 20% repolarization level from 3.7 to 2.5 ms) and increased at late slow phase of repolarization (AP duration at 90% repolarization level from 62 to 178 ms). The resting potential was depolarized from -75 to -61 mV. This mathematical model of SACs may quantitatively predict changes in cardiomyocytes by mechanical stretch.
Action Potentials ; Animals ; Ion Channels ; Membrane Potentials ; Membranes ; Models, Theoretical ; Muscle Cells* ; Myocytes, Cardiac ; Rats*

Ion channel inhibitors are widely used for pharmacological discrimination between the different channel types as well as for determination of their functional role. In the present study, we tested the hypothesis that 4-aminopyridine (4-AP) could affect the large conductance Ca2+ -activated K+ channel (BKCa) currents using perforated-patch or cell-attached configuration of patch-clamp technique in the rabbit pulmonary arterial smooth muscle. Application of 4-AP reversibly inhibited the spontaneous transient outward currents (STOCs). The reversal potential and the sensitivity to charybdotoxin indicated that the STOCs were due to the activation of BKCa. The BKCa currents were recorded in single channel resolution under the cell-attached mode of patch-clamp technique for minimal perturbation of intracellular environment. Application of 4-AP also inhibited the single BKCa currents reversibly and dose-dependently. The membrane potential of rabbit pulmonary arterial smooth muscle cells showed spontaneous transient hyperpolarizations (STHPs), presumably due to the STOC activities, which was also inhibited by 4-AP. These results suggest that 4-AP can inhibit BKCa currents in the intact rabbit vascular smooth muscle. The use of 4-AP as a selective voltage-dependent K+ (KV) channel blocker in vascular smooth muscle, therefore, must be reevaluated.
4-Aminopyridine* ; Charybdotoxin ; Discrimination (Psychology) ; Ion Channels ; Membrane Potentials ; Muscle, Smooth* ; Muscle, Smooth, Vascular ; Myocytes, Smooth Muscle* ; Patch-Clamp Techniques ; Pulmonary Artery

A regulating mechanism of the ATP-sensitive potassium channels (KATP channels) is yet to fully explained. This study was carried out to investigate the effects of intracellular application of monocarboxylates (acetate, formate, lactate, and pyruvate) on KATP channels in isolated rabbit ventricular myocytes. Single channel currents of KATP channels were recorded using the excised inside-out or permeabilized attached (open-cell) patch-clamp technique at room temperature. Intracellular application of acetate, formate and pyruvate led to an inhibition of channel activity, whereas intracellular application of lactate increased channel activity. These effects were reversible upon washout. Analysis of single channel kinetics showed that monocarboxylates did not affect open-time constant and close-time constant. These results suggest that monocarboxylates participate in modulating KATP channels activity in cardiac cells and that modulation of KATP channels activity may resolve the discrepancy between the low Ki in excised membrane patches and high levels of intracellular ATP concentration during myocardial ischemia or hypoxia.
Adenosine Triphosphate ; Anoxia ; KATP Channels* ; Kinetics ; Lactic Acid ; Membranes ; Muscle Cells* ; Myocardial Ischemia ; Patch-Clamp Techniques ; Pyruvic Acid

In the present study, we have investigated the effect of metabolic inhibition on the inward rectifer K current (IK1). Using whole cell patch clamp technique we applied voltage ramp from +80 mV to -140 mV at a holding potential of -30 mV and recorded the whole cell current in single ventricular myocytes isolated from the rabbit heart. The current-voltage relationship showed N-shape (a large inward current and little outward current with a negative slope) which is a characteristic of IK1. Application of 0.2 mM dinitrophenol (DNP, an uncoupler of oxidative phosphorylation as a tool for chemical hypoxia) to the bathing solution with the pipette solution containing 5 mM ATP, produced a gradual increase of outward current followed by a gradual decrease of inward current with little change in the reversal potential (-80 mV). The increase of outward current was reversed by glibenclamide (10 muM), suggesting that it is caused by the activation of KATP. When DNP and glibenclamide were applied at the same time or glibenclamide was pretreated, DNP produced same degree of reduction in the magnitude of the inward current. These results show that metabolic inhibition induces not only the increase of KATP channel but also the decrease of IK1. Perfusing the cell with ATP-free pipette solution induced the changes very similar to those observed using DNP. Long exposure of DNP (30 min) or ATP-free pipette solution produced a marked decrease of both inward and outward current with a significant change in the reversal potential. Above results suggest that the decrease of IK1 may contribute to the depolarization of membrane potential during metabolic inhibition.
Adenosine Triphosphate ; Architectural Accessibility ; Baths ; Glyburide ; Heart ; Membrane Potentials ; Muscle Cells* ; Oxidative Phosphorylation