[Giga-seal suction electrode and its application to single cardiac myocytes].
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Amplifiers, Electronic↗
Biomedical subjects
H Irisawa
Publications and source records attributed to H Irisawa.
Membrane currents in the rabbit atrioventricular node cell.
The rabbit A-V node was dissected into pieces (0.2 x 0.2 x 0.2 mm) smaller than its space constant of 692 +/- 96 micrometers (n = 5). These small specimens showed spontaneous action potentials whose configurations were similar to those of large specimens before dissection. The membrane time constant was 21.5 +/- 1.5 ms (n = 5). Voltage clamp experiments were performed on the above specimens using the two-microelectrode technique. On depolarization from the holding potential of -40 mV to various potential levels a transient inward current and delayed outward current were recorded. On repolarization an outward current tail was observed. The transient inward current was blocked by application of D 600 (2 x 10(-7) g/ml) but was insensitive to TTX (1 x 10(-7) g/ml). The inward current was decreased by superfusion with Na- or Ca-free Tyrode solution. Thus, this current was classified as the slow inward current (is). When the K concentration in the Tyrode solution was varied, the reversal potential of the outward current tail changed as expected for a K electrode, indicating that the outward current was carried by K ions. On hyperpolarization slow activation of inward current was recorded. The reversal potential of this current was between -20 and -30 mV, which was analogous to hyperpolarization activated current, ih, in the S-A node. A contribution of sodium current (iNa) to the action potential was obviously demonstrated from an inhibitory effect of TTX on the upstroke of the anodal break excitation. The ionic selectivity of each current system is compared with analogous current systems in other cardiac tissues and a possible mechanism for the slow conduction in the A-V node is discussed.
Action Potentials↗
Transient depolarization and spontaneous voltage fluctuations in isolated single cells from guinea pig ventricles. Calcium-mediated membrane potential fluctuations.
Under the influence of cardiotonic steroids, single ventricular cells exhibit transient depolarization after a train of driven action potentials or, in voltage clamp experiments, transient inward current after a depolarizing clamp pulse. Transient depolarization or transient inward current was abolished by an intracellular injection of ethyleneglycol-bis (beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) or by superfusion of 5 mM caffeine. Transient depolarization was elicited even in the control Tyrode's solution by an intracellular injection of CaCl2 or augmented by an injection of adenosine 3',5'-cyclic monophosphoric acid (cAMP). Along with transient depolarization or transient inward current, digitalis intoxication promoted spontaneous oscillatory fluctuations in membrane potential or in membrane current. Their power spectra showed peaks at frequencies ranging from 2 to 7 Hz, which coincided well with the frequency of repetitive transient depolarization or transient inward current. The fluctuations were eliminated by intracellular injections of EGTA and decreased in amplitude by 5 mM caffeine with a shift toward higher frequencies. Depolarization of the membrane caused a shift of the spectrum peak toward higher frequencies. These results suggest that an oscillatory release of Ca from intracellular storage sites is the common basis underlying both the transient events (depolarization or inward current) and the spontaneous miniature fluctuations in membrane potential or current.
Animals↗
Electrogenic sodium pump in rabbit atrio-ventricular node cell.
Electrogenicity of the Na pump was demonstrated in rabbit A-V node cells by analyzing the K-induced hyperpolarization occurring after a short period of K-free perfusion. The transient hyperpolarization was abolished completely by strophanthidin (10(-5)M). The membrane slope conductance remained unchanged during the transient hyperpolarization. On perfusion of 50 mM K and K-free incubation the transient hyperpolarization reached --69 mV which was more negative than the expected EK (about --28 mV). The order of potencies of monovalent cations to activate the K site of the Na pump was Tl greater than Rb equal to K greater than NH4 equal to Cs greater than Li, which was similar to the sequence reported in the literature. Michaelis-Menten type activation of the K site of the Na pump was suggested from the relationship between the decay rate constant of the K-induced outward current transient and [K]o. These findings obviously indicate that the Na pump in the A-V node cells shares common characteristics with those of other excitable tissues. Direct contribution of the pump activity to the membrane potential under physiological conditions was suggested by a significant depolarization occurring immediately after application of strophanthidin.
Animals↗
Electrogenic Na pump evidenced by injecting various Na salts into the isolated A-V node cells of rabbit heart.
Electrogenicity of the Na pump was confirmed by injecting Na salts into a small cluster of A-V node cells. Injection of Na glutamate or Na acetate induced marked hyperpolarization, accompanying with cessation of spontaneous activity. The hyperpolarization exceeded EK in 27 mM K Tyrode solution and was inhibited by 10(-5) M strophanthidin. Injection of NaCl or NaI depolarized the membrane. These data showed that inward-going current carried by the injected anion antagonized the outward-going pump current and thus determined the net effect of the injection.
Action Potentials↗
Effect of procaine amide on the membrane currents of the sino-atrial node cells of rabbits.
Using small rabbit sino-atrial node preparations, the effects of procaine amide in concentrations from 0.01 to 2 mg/ml on the membrane potentials currents were studied by both current-clamp and voltage-clamp experiments. Procaine amide in concentrations over 0.1 mg/ml reduced the peak of the action potential, maximum diastolic potential and the maximum rate of depolarization. The action potential duration was prolonged, the resting potential was decreased and the heart rate was reduced. In the voltage-clamp experiments, procaine amide (0.1 mg/ml) reduced the slow inward current (is), the outward current (iK) and the inward current activated by hyperpolarization (ih). The major effect, however, was the reduction of the outward current. Sine the degree of the steady-state activation of iK and its time constant were unchanged, the observed reduction of iK could have been caused by a reduction of iK.
Action Potentials↗
Kinetics and rectification of the slow inward current in the rabbit sinoatrial node cell.
The slow inward current (is) in the rabbit sinoatrial node cell was studied by the conventional two-microelectrode voltage clamp technique. When is was measured as the difference between two records obtained before and after blocking is with D 600, the fully activated current (is)-voltage relation was non-linear; the conductance decreased in the negative potential range resulting in an almost constant amplitude of is negative to -10 mV. The degree of steady-state activation was about 1 at -5mV and 0 at -65mV. The recovery time course of is during repolarization was measured by varying the interval between two sequential depolorizing pulses with various holding potentials. The time constant of the exponential recovery time course was about 120 msec at -40 mV and decreased to about 40 msec at -70 mV. The steady-state conductance of is, calculated from the activation and inactivation curves, produced a large hump in the steady-state current voltage relation between -60 and -20 mV, which was not observed in the experiment. When the above kinetics were incorporated, the S-A node model failed to discharge the spontaneous activity. The activation and inactivation curves which can simulate the experimental I-V curve and the action potential were proposed.
Action Potentials↗
Spontaneously active cells isolated from the sino-atrial and atrio-ventricular nodes of the rabbit heart.
Single cells or cell clusters composed of 3-10 cells were isolated from the S-A and A-V nodes of the rabbit heart by coronary perfusion of collagenase dissolved in Ca-free Tyrode solution (0.04%, for 1 hr). For comparison, atrial and ventricular cells were also isolated from the same heart. Shapes of the isolated nodal cell were either rod or round and nodal cells were slightly smaller than ventricular cells. Spontaneous activity was observed in both rod and round nodal cells. The action potentials had the configurations similar to those recorded from larger conventional preparations. The membrane current recorded from the small nodal cell clusters (5-10 cells) by the two-microelectrode voltage clamp technique showed a time course similar to that of previous recordings from conventional preparations, but the amplitude of the currents was 5-10 times smaller. The isolated cells showed normal sensitivities to both acetylcholine and epinephrine. Findings given in this study indicate that the isolated cells maintain the typical membrane characteristics of the nodal cells and that they are suitable for electrophysiological studies of the cardiac pacemaker cell.
Acetylcholine↗
Slow current systems in the A-V node of the rabbit heart.
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Action Potentials↗
Slow inward current and its role mediating the chronotropic effect of epinephrine in the rabbit sinoatrial node.
The ionic mechanism underlying the chronotropic effect of epinephrine on the rabbit sinoatrial (S-A) node has been studied. Epinephrine (5.5 X 10(-6) M) increased the spontaneous rate from 206 +/- 25 min-1 to 242 +/- 39 min-1. The effect of epinephrine was reproducible on repetitive applications. Voltage clamp experiments using the two microelectrode technique revealed the following changes in the membrane current: epinephrine (5.5 X 10(-7) M) increased the limiting conductance for the slow inward current (is) by approximately 30% and the potassium current (ik) by about 10%, keeping the kinetics of is and ik constant. From the holding potential of -70 mV the activation of is was observed on step depolarization positive to -60 or -55 mV in both control and epinephrine solution. The hyperpolarization-activated current (ih) was also increased by about 20% at -70 mV, and its time course was slightly accelerated. Participation of is for the chronotropic effect of epinephrine was strongly suggested by the findings that is was partially available positive to -60 mV and that epinephrine could not increase the slope of diastolic depolarization when is was blocked by D 600.
Action Potentials↗
Inward current activated during hyperpolarization in the rabbit sinoatrial node cell.
Inward current activated by hyperpolarization (ih) was dissected from the K-current by the difference in its activation voltage range and the selective blocking effect of Ba2+ on the K-current. The ih shows little specificity to any particular ion, and its reversal potential was -25 mV. The current system can be expressed well by Hodgkin-Huxley type kinetics. The time constant of ih ranged from 2-4 s at about -70 mV, but it became shorter at about -10 mV. The ih began to activate at -50 mV and fully saturated at about -100 mV. The fully activated current-voltage relation shows no rectifying property. Activation and deactivation time courses were fitted by a single exponential with the same time constant at a given membrane potential. Although ih plays only a small role during the normal action potential in the isolated preparation, it plays a significant role in keeping the pacemaker cell at a low membrane potential
Animals↗
Potassium current during the pacemaker depolarization in rabbit sinoatrial node cell.
Voltage clamp experiments were carried out on the rabbit sinoatrial (S-A) node. The delayed outward current in the voltage range between --60 mV and --22 mV almost disappeared in the presence of 5 mM Ba2+. The slow inward current and the hyperpolarization-activated current remained unaffected. In the absence of the time-dependent potassium current the S-A node cell generated spontaneous action potentials, provided that the membrane was hyperpolarized by constant outward current. Therefore it seems unlikely that the potassium current plays an essential role in generating the pacemaker potential in the S-A node. The time course of the potassium current (iK) during the cardiac cycle was calculated using equations simulating the kinetics of iK. According to this computation, the change of iK in the S-A node is small during pacemaker depolarization. It is proposed that the gradual decay of potassium conductance is less important for the development of the pacemaker potential than the contribution of the slow inward current.
Action Potentials↗
The spontaneous action potential of rabbit atrioventricular node cells.
The rabbit atrioventricular (A-V) node, with dimensions of approximately 5 X 3 mm was dissected into 15 small specimens (0.5 X 0.5 mm). A majority of the specimens continued to discharge spontaneous action potentials, the action potential configuration being almost identical in different specimens. The amplitude (98 mV) and the maximum rate of rise of the action potential (11 V/sec) were similar to those recorded from the intact A-V node (amplitude 98 mV, maximum rate of rise of action potential 12 V/sec). In these small specimens, the "resting membrane potential (-44 mV)" was approximately 20 mV less negative than that in the intact A-V node preparation (-62 mV) before the dissecting procedure. The spontaneous discharge in these small A-V node specimens was attributed to the low resting membrane potential. The small specimen became inexcitable under the effects of blockers of is, though TTX had no significant effect on the action potential. The after-hyperpolarization was observed after cessation of the depolarizing current pulse. It is concluded that the slow inward current and the slow kinetics of the outward current contribute to the generation of the spontaneous A-V node action potentials as in the sinoatrial node.
Action Potentials↗
Reconstruction of sino-atrial node pacemaker potential based on the voltage clamp experiments.
The pacemaker activity of the S-A node cell was explained by reconstructing the pacemaker potential using a Hodgkin-Huxley type mathematical model which was based on the reported voltage clamp data. In this model four dynamic currents, the sodium current iNa, the slow inward current, is, the potassium current, iK, and the hyperpolarization-activated current, ih, in addition to a time-dependent leak current, i1 were included. The model simulated the spontaneous action potential the current voltage relationship, and the voltage clamp experiment in normal Tyrode solution of the rabbit S-A node. Furthermore, the changes of activity induced by the potassium current blocker Ba2+, by applying constant current, acetylcholine, and epinephrine were also reconstructed. It was strongly suggested that the pacemaker depolarization in the S-A node cell is mainly due to a gradual increase of iS during diastole, and that the contribution of iK is much less compared to the potassium current iK2 in the Purkinje fiber pacemaker depolarization. The rising phase of the action potential was due to iS and the plateau phase is determined by both the inactivation of iS and activation of iK.
Acetylcholine↗
Comparative physiology of the cardiac pacemaker mechanism.
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Action Potentials↗
Inward current of the rabbit sinoatrial node cell.
Inward currents of the rabbit sinoatrial node cell were examined in voltage-clamp experiments using the two-microelectrode technique. A fast and slow inward current could be separated from each other. The slow inward current was blocked by Mn and D 600, but it was insensitive to TTX. On the contrary, the fast inward current was blocked by TTX, but not by Mn and D 600. Both the fast and slow inward currents disappeared on Na removal. The fast inward current system was fully inactivated by holding the membrane potential positive to -40 to -50 mV, while the slow inward current system was recorded with the holding potential up to -20 mV. The voltage dependence of the inactivation of the 2 inward current systems and their dependence on [Na]0 suggests that the rising phase of the spontaneous action potential in the S-A node cell is produced mainly by Na current through the slow inward current system.
Animals↗
Effect of hemolysin produced by Vibrio parahaemolyticus on membrane conductance and mechanical tension of rabbit myocardium.
The mechanisms of the action of hemolysin extracted from Vibrio parahaemolyticus in the S-A node and right atrium cells of rabbit were studied by means of the single sucrose gap and isometric tension recording methods. Hemolysin caused the membrane to depolarize reversibly without affecting the action potential generating mechanism. Lowering of [Na+]o inhibited membrane depolarization in the presence of hemolysin while the readmission of normal Tyrode solution induced depolarization. Tetrodotoxin (TTX) barely antagonized the depolarizing action of hemolysin but slowed the rate of development of depolarization. Therefore, this depolarization is considered to be primarily due to the increase in conductance to Na which TTX may not block. The dose-response relationship was obtained by measuring a change in membrane resistance. The concentration necessary to yield one-half of the maximum reduction of the membrane was determined to by 7.5 micrograms/ml. Accumulation of Na within the cell may be responsible for an increase of twitch tension observed during the action of a low concentration of hemolysin. On the other hand, a higher concentration of hemolysin seemed to promote exchange of intracellar Na with extracellular Ca, especially when the Na concentration of the perfusing solution was reduced, and led to stronger contracture.
Animals↗
A time- and voltage-dependent potassium current in the rabbit sinoatrial node cell.
Voltage clamp experiments were conducted in the rabbit sinoatrial node (S-A node) using the double microelectrode technique. When the membrane was repolarized to the resting potential after depolarizing test pulses, the outward current slowly decayed to the steady level (outward current tail). The magnitude of the outward current tail was a sigmoid function of the amplitude of the preceding depolarization. The degree of activation of this current varied from 0 at about -50 mV to 1 at about +20 mV. The time course of the current change was a simple exponential and was independent of the preceding depolarization. The reciprocal time constant appeared to be a U-shaped function of the membrane potential with a minimum value of about 3s-1 at -40 mV. The instantaneous current voltage relation was an inward-going rectifier, but showed no detectable negative slope. The reversal potential, obtained between 10 and 50 mM [K]o, decreased with a slope of 58 mV for a 10-fold increase in [K]o. These findings indicate that the outward current tail in the S-A node cell is attributable to a single component of K current (pacemaker current component). The pacemaker current component is mainly responsible for the slow diastolic depolarization.
Membrane Potentials↗