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Biomedical subjects

H Irisawa

Publications and source records attributed to H Irisawa.

At least 37 records · Page 2Linked to original sources
PubMed1987

Cell-to-cell diffusion of fluorescent dyes in paired ventricular cells.

The intracellular and cell-to-cell diffusion of fluorescent dyes of various molecular sizes were studied in enzymatically isolated paired ventricular cells of the guinea pig heart. Fluorescein sodium (mol wt 332), 6-carboxyfluorescein (mol wt 376), Lucifer yellow CH (mol wt 457), lissamine rhodamine B-200 (mol wt 559), and tetraglycine-conjugated lissamine rhodamine B-200 (mol wt 859) were all diffused into the single ventricular cell through the patch-clamp pipette. All these dyes were able to diffuse through the gap junction of the paired cells. The diffusion coefficient of 6-carboxyfluorescein in the cytoplasm was 5.8 X 10(-6) cm2/s, Lucifer yellow CH was 3.0 X 10(-6) cm2/s, and lissamine rhodamine B-200 was 8.6 X 10(-7) cm2/s. Permeability of the gap-junctional membrane was 6.8 X 10(-4) cm/s for 6-carboxyfluorescein, 2.8 X 10(-4) cm/s for Lucifer yellow CH and 7.4 X 10(-5) cm/s for lissamine rhodamine B-200. Thus lissamine rhodamine B-200 diffused in the cytoplasm and through the gap junction approximately 10 times slower than 6-carboxyfluorescein. Tetraglycine-conjugated lissamine rhodamine B-200 (mol wt 859) passed through the gap junction, suggesting that the critical pore size of the ventricular gap junction may be somewhat larger than that of the conjugated tetraglycine rhodamine.

Animals↗
PubMed1987

Rectification of muscarinic K+ current by magnesium ion in guinea pig atrial cells.

Rectifying properties of the acetylcholine (ACh)-sensitive K+ channels were studied using a patch-clamp method in single atrial cells prepared enzymatically from adult guinea pig hearts. In the presence of micromolar concentration of nonhydrolyzable guanosine 5'-triphosphate (GTP) analogue 5'-guanylylimidodiphosphate (GppNHp) and the absence of Mg2+ at the inner surface of patch membrane [( Mg2+]i), the channel activity recovered in inside-out patch condition. The single channel conductance became ohmic between -80 and +80 mV (symmetrical 150 mM K+ solutions). The rapid relaxation of outward single channel currents was disclosed on a depolarization. [Mg2+]i blocked the outward current through the channel dose- and voltage-dependently and also induced a dose-dependent increase in the channel activation. The apparent paradoxical role of [Mg2+]i is important for the cholinergic control in the heart; voltage-dependent Mg block ensures a low K+ conductance of cell membrane at the plateau of action potentials during the exposure to ACh, thereby slowing the heart rate without unfavorable shortening of the action potentials.

Acetylcholine↗
PubMed1987

Intracellular Ca2+ and protein kinase C modulate K+ current in guinea pig heart cells.

Effects of protein kinase C (PKC) and intracellular calcium ion (Cai2+) on the delayed rectifier K+ current (IK) were investigated in the single ventricular cells of guinea pig by use of an internal-dialysis method and a whole cell voltage-clamp technique. 12-O-tetradecanoylphorbol-13-acetate (TPA, 10(-9) M), an activator of PKC, increased the amplitude of IK in the presence of Cai2+ higher than 10(-10) M. This effect of TPA was mimicked by a synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), 50 micrograms/ml, 125 microM, and was blocked by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (10 microM). The above findings suggest that IK channels were phosphorylated by PKC and thereby the amplitude of IK was increased. IK was also increased by elevating the concentration of Cai2+ in the absence of TPA. It is thus indicated that IK channels are modulated by Cai2+ not only through activation of PKC but also directly. Our observation may provide a possible mechanism by which Cai2+ mediates the link between the Ca2+ transients during contraction and the action potential duration.

Animals↗
PubMed1986

Simulation analysis of excitation conduction in the heart: propagation of excitation in different tissues.

The normal excitation and conduction in the heart are maintained by the coordination between the dynamics of ionic conductance of each cell and the electrical coupling between cells. To examine functional roles of these two factors, we proposed a spatially-discrete model of conduction of excitation in which the individual cells were assumed isopotential. This approximation was reasoned by comparing the apparent space constant with the measured junctional resistance between myocardial cells. We used the four reconstruction models previously reported for five kinds of myocardial cells. Coupling coefficients between adjacent cells were determined quantitatively from the apparent space constants. We first investigated to what extent the pacemaker activity of the sinoatrial node depends on the number and the coupling coefficient of its cells, by using a one-dimensional model system composed of the sinoatrial node cells and the atrial cells. Extensive computer simulation revealed the following two conditions for the pacemaker activity of the sinoatrial node. The number of the sinoatrial node cells and their coupling coefficients must be large enough to provide the atrium with the sufficient electric current flow. The number of the sinoatrial node cells must be large so that the period of the compound system is close to the intrinsic period of the sinoatrial node cell. In this simulation the same sinoatrial node cells produced action potentials of different shapes depending on where they were located in the sinoatrial node. Therefore it seems premature to classify the myocardial cells only from their waveforms obtained by electrical recordings in the compound tissue. Second, we investigated the very slow conduction in the atrioventricular node compared to, for example, the ventricle. This was assumed to be due to the inherent property of the membrane dynamics of the atrioventricular node cell, or to the small value of the coupling coefficient (weak intercellular coupling), or to the electrical load imposed on the atrioventricular node by the Purkinje fibers, because the relatively small atrioventricular node must provide the Purkinje fibers with sufficient electric current flow. Relative contributions of these three factors to the slow conduction were evaluated using the model system composed of only the atrioventricular cells or that composed of the atrioventricular and Purkinje cells. We found that the weak coupling has the strongest effect. In the model system composed of the atrioventricular cells, the propagation failure was not observed even for very small values of the coupling coefficient.(ABSTRACT TRUNCATED AT 400 WORDS)

Atrioventricular Node↗
PubMed1986

Intra- and extracellular actions of proton on the calcium current of isolated guinea pig ventricular cells.

External or internal application of proton on the calcium current was investigated using the whole-cell patch clamp technique in single ventricular cells of the guinea pig. The effect of acid pHo on the calcium current depended on the strength of the buffer in the internal solution. In the presence of 5 mM HEPES in the pipette, external acidification depressed calcium current with the half maximum inhibition of pHo 5.5. When the intracellular pH was buffered strongly to pH 7.2 by 50 mM HEPES within the pipette solution, half maximum inhibition of the calcium current was shifted to the acidic side markedly. When acid was applied in the patch electrode, calcium current was hardly affected by the pH values down to 6.0 in the pipette solution but was depressed by approximately half on further acidification to pH 4.5. When the Na+-H+ exchange system was blocked by superfusing the cell with either amiloride or Na+-deficient solutions (Tris substituted), calcium current was decreased by half at a pipette pH of around 6.5 and was completely and irreversibly blocked at pHpip 6.0. From the above results, we concluded that the calcium current of the ventricular cell is much more sensitive to intracellular protons than extracellular ones.

Animals↗
PubMed1986

Observations on the fine structure of nodal, Purkinje and working myocardial cells isolated from rabbit hearts.

Sinoatrial and atrioventricular nodal cells as well as Purkinje and working myocardial cells isolated enzymatically from rabbit hearts were examined by transmission electron microscopy. The cardiac cell fine structure remained well organized in the storage solution after isolation. In normal Tyrode solution, the nodal cells changed from spindle shaped to spherical, whereas the shapes of Purkinje and working myocardial cells remained unchanged. The nodal cell fine structure became disorganized with respect to its sarcomeric arrangement in normal Tyrode solution. This was assumed to result from a combination of several factors seen in the nodal cells: the loss of the normal anchoring of the myofibrils at the previous intercalated discs, breakdown of the Z bands and the alteration of the integrity of the intermediate filaments. Also, extensive restoration of plasma membrane damaged by of the intermediate filaments. Also, extensive restoration of plasma membrane damaged by the isolation procedure was observed in this solution. This might correspond to the retention of the normal physiological properties of the plasma membrane of the nodal cells despite gross morphological changes undergone in normal Tyrode solution.

Animals↗
PubMed1985

Membrane currents and their modification by acetylcholine in isolated single atrial cells of the guinea-pig.

The ionic currents in isolated single atrial cells of the guinea-pig heart were analysed by the patch-clamp technique applied to whole-cell recordings and the effects of acetylcholine (ACh) on the membrane potential and currents were studied. Resting and action potentials of single isolated cells were normal. Upstroke velocity was sensitive to tetrodotoxin. Action potential duration was slightly shorter than in multicellular preparations. Voltage-clamp experiments demonstrated the presence of a Ca2+ current (iCa), and inward rectifying and outward rectifying K+ currents. The Ca2+ current was abolished by 2 mM-Co2+ or 10(-6) M-D600, and the K+ currents were greatly reduced by intracellular application of Cs+ using the patch electrode and simultaneously superfusing the cell with 5 mM-Cs+ Tyrode solution. Acetylcholine shortened the action potential duration in a dose-dependent manner. The threshold dose of ACh was about 10(-9) M and the maximal effect was obtained with 10(-6) M. The resting membrane potential was hyperpolarized by 1-3 mV. ACh (10(-8)-10(-6) M) increased the K+ currents both on depolarization and on hyperpolarization in a dose-dependent manner in the presence of 10(-6) M-D600. The ACh-induced K+ outward current revealed a progressive deactivation ('relaxation'), with a time constant of 111 +/- 16 ms at around 0 mV. When the K+ outward currents were minimized with Cs+, the reduction of iCa by 10(-8) M-ACh was insignificant, and became 17 +/- 2.5, 26 +/- 3.5 and 26 +/- 5% of the control value in 10(-7), 10(-6) and 10(-5) M-ACh, respectively. The inactivation time course of iCa recorded from the Cs+-loaded cells was not affected by 10(-7) M-ACh. These results suggest that ACh activates predominantly a K+ conductance in the guinea-pig atrium. The superimposition of a relaxing K+ outward current on iCa may lead to over-estimation of the decrease in iCa.

Acetylcholine↗
PubMed1985

Transient outward current carried by potassium and sodium in quiescent atrioventricular node cells of rabbits.

Single atrioventricular node cells were dispersed by treating the rabbit heart with collagenase. In Tyrode's solution, the cells became rounded, and about 20% of them showed spontaneous activity, whereas the rest remained quiescent. When those quiescent cells were whole-cell clamped, depolarizing clamp pulses from the holding potential of -83 mV induced an outward current which decayed quickly, with a time course similar to that of the transient outward current in the Purkinje fiber. The amplitude of the current became larger when progressively more positive clamp pulses were given from a very negative holding potential. The inactivation time course of this current consisted of two exponential components. Single-channel current recordings from those cells revealed a class of channels that activated more frequently during the initial part of depolarizing pulses. Summation of those unitary currents reproduced activation and inactivation time courses of the macroscopic current well, suggesting that this channel corresponds to the transient outward current. The current-voltage relationship of the channel was linear with the slope conductance of 19.9 +/- 1.8 pS (n = 7), and the reversal potential was near the resting potential of the atrioventricular node cell with 5.4 mM potassium chloride and 134.6 mM sodium chloride in the pipette. The channel was passing mainly potassium ions, but sodium ions also seemed to carry a fraction of the current. The possible role of the transient outward current in the quiescent node cell is discussed.

Animals↗
PubMed1985

Effects of intracellular acidification on membrane currents in ventricular cells of the guinea pig.

The membrane currents of single ventricular cells were measured under whole cell voltage clamp using a giga-sealed patch electrode, and the effects of intracellular acidification were examined by perfusing the electrode pipette with different pH solutions. The plateau of the action potential was shortened when the pH of the pipette solution was lowered from the control of 7.2 to 6, and finally to 5. The pH 6 pipette solution evoked a time-independent outward current at positive potentials and increased the slope conductance near the resting potential. These changes were suppressed by removal of both intra- and extracellular potassium ion, indicating that these currents were carried by potassium ions, but not by protons. Increasing the calcium concentration in the pipette from pCa 8 to pCa 6 induced a time-dependent outward current which had a reversal potential of about -13 mV. This result clearly differed from the changes induced by the acidic pipette solution, suggesting that the calcium-mediated conductance was not involved in the genesis of the acidic effects. The calcium current was not significantly affected by perfusion at pH 6, but was decreased by the more acidic (pH 5) solution. When the calcium current was recorded in sodium- and potassium-free external solution but with a cesium-rich internal solution, however, the calcium current was suppressed even with a weak acidic (pH 6.8) pipette solution. This effect was attributed not to an increased sensitivity of the calcium channel to protons, but to a more extensive intracellular acidification, which might have been caused by a depressed extrusion of proton via a sodium-hydrogen exchange mechanism on the surface membrane.

Action Potentials↗
PubMed1984

Action potential and membrane currents of single pacemaker cells of the rabbit heart.

Single, viable pacemaker cells were isolated from sinoatrial (S-A) and atrioventricular (A-V) nodes by treating with collagenase. In normal Tyrode solution containing 1.8 mM Ca2+, these pacemaker cells had a round configuration and contracted rhythmically at a frequency of about 150-260/min. The amplitude, duration, and maximum rate of rise of the spontaneous action potentials recorded using patch clamp electrodes were similar to those obtained from multicellular preparations. Amplitudes of the recorded membrane current were normalized with reference to the surface area of the cell by assuming the cell shape as a plane oblate spheroid. The membrane resistance of the isolated nodal cells was 14.9 +/- 4.0 k omega . cm2 (n = 12) at about -35 mV and the membrane capacitance was 1.30 +/- 0.24 microF/cm2 (n = 18). The inactivation time course of the slow inward current, isi, was fitted with a sum of two exponentials with time constants of 6.7 +/- 0.6 ms and 46.6 +/- 15.3 ms (n = 4) at +10 mV. The amplitude of isi peaked at 0 approximately +10 mV in the current-voltage relationship and was 18.2 +/- 8.4 microA/cm2. The potassium current, iK, was activated in the voltage range positive to -50 mV and was saturated at about +20 mV. The amplitude of the fully-activated iK at -40 mV was 3.3 +/- 1.4 microA/cm2 (n = 10) and showed an inward-going rectification. The activation of the hyperpolarization-activated current was observed at potentials negative to -70 mV in seven of 14 experiments. The current density and membrane capacitance calculated could be overestimated and the membrane resistance underestimated, because of the presence of caveolae on the cell surface. However, these data give the nearest possible estimates of the electrical constants in the nodal cells, which cannot be measured accurately in the conventional multicellular preparations.

Animals↗
PubMed1984

Resting K conductances in pacemaker and non-pacemaker heart cells of the rabbit.

Currents through the inward rectifier K channel (iK X rec) and the ACh-operated K channel (iK X ACh) were recorded in isolated heart cells of rabbit using the patch clamp technique with electrodes having 0.5-1 micron tip inner diameter. The maximum number of overlaps of open iK X rec channels per patch was measured over 347 experiments. An average of 2.3 was found in ventricular cells and 0.03-0.06 in sinoatrial (S-A) and atrioventricular (A-V) node cells. The estimated total number of the iK X rec channels for each ventricular cell was great enough to supply the resting K conductance of the cell. The iK X ACh channel was present in S-A and A-V node cells, but was never observed in the ventricular cells. The resting conductance of the nodal cells measured with whole cell clamp recordings was about 16 times smaller than that of the ventricular cells, and was hardly decreased at all by the removal of K+ from the bath solution. Thus, the lower membrane potential of the nodal cells compared with that of the ventricular cells was attributed to the smaller K conductance of the resting membrane, which is due to the very low density of the iK X rec channel. On the other hand, the iK X ACh channel, when activated by neural regulation, may play a major role in generating the resting K conductance of the nodal cells.

Animals↗
PubMed1984

Electrophysiology of single cardiac cells.

Whatever techniques of isolation one may use, it is certainly true that the isolated single heart cells are very useful in various physiological experiments. For electrophysiology, the application of the single cell serves to test findings previously obtained in the multicellular preparation. Therefore, summary and comparison of the data will become necessary in the near future. Furthermore, the application of the single cell opens new areas for electrophysiology of the heart, because when using only the single cell preparation, one can dialyse intracellular millieu and can also measure single channel analyses.

Action Potentials↗
PubMed1984

Effects of various intracellular Ca ion concentrations on the calcium current of guinea-pig single ventricular cells.

The effects of changing the intracellular Ca concentration ([Ca]i) on the calcium current (iCa) were studied in isolated single ventricular cells of the guinea-pig. [Ca]i was varied by an intracellular perfusion technique using a suction pipette. iCa measured from internally perfused cells at a pCa lower than 9.0 was dependent on the extracellular Ca concentration ([Ca]o). Increasing [Ca]o from 1.8 to 5.4 mM increased the amplitude of iCa, and reduction of [Ca]o from 1.8 to 0.01 mM decreased the amplitude. The inactivation time course of iCa became faster as [Ca]o was increased and slower as [Ca]o was reduced. By decreasing the pCa of the internal perfusate from 9.0 to 6.8, the amplitude of iCa was decreased markedly, but no significant change was observed in its time course. Analysis of the I-V curve led to the conclusion that a change in the driving force was not a major factor in the reduction of iCa. The "steady state inactivation" of iCa was measured by a double-pulse method. The amplitude of iCa elicited by the test pulse was smaller at pCa 7.4 than at pCa 9.0 at potentials of between -27 and +33 mV. By normalizing the iCa amplitude, however, the "steady state inactivation" curve in the control and at high [Ca]i overlapped. Similar results were obtained in Sr-Tyrode solution. The degree of "steady state inactivation" of iCa at the potentials positive to +10 mV was larger in Ca-Tyrode than in Sr-Tyrode solution. It is proposed that the reduction in amplitude of iCa at higher [Ca]i is caused by a reduction of the maximum conductance of iCa (gCa) and that Ca ions passing through iCa channels have a remarkable effect on its inactivation.

Animals↗
PubMed1983

Does the "pacemaker current" generate the diastolic depolarization in the rabbit SA node cells?

Small preparations of spontaneously beating rabbit sino-atrial node (SA node) were voltage clamped with the two-microelectrode technique. The effects of 0.25-5 mM Cs+ on the spontaneous pacing rate and the time-dependent inward "pacemaker" current, ih, were studied. In the presence of 2 mM Cs+, the spontaneous pacing rate decreased only slightly even though ih was strongly depressed at potentials negative to -60 mV Cs+ had little or no effect on other time-dependent currents observed with clamp pulses less negative than -50 mV. Since no voltage-dependence to the Cs+ effect on ih could be measured (between -90 mV and -20 mV), it was considered unlikely that the lack of Cs+ effect on the rate of diastolic depolarization results from a voltage-dependent effect of Cs+ on the ih channel. Adrenaline produced a marked positive chronotropic effect in Cs+-treated SA node cells. This effect was accompanied by marked enhancement of the slow inward current (isi) with no change in the Cs+-blocked ih current. These results are consistent with the idea that ih plays a minor role in generation of pacemaker depolarization, and suggest a more prominent role of isi in the generation of diastolic depolarization in SA nodal cells.

Animals↗
PubMed1983

Modulation by intracellular ATP and cyclic AMP of the slow inward current in isolated single ventricular cells of the guinea-pig.

Effects of ATP and of cyclic AMP on membrane current systems were investigated in isolated single ventricular cells from guinea-pig hearts by applying the suction electrode method. The intracellular milieu was dialysed with various solutions which were perfused continuously through the suction pipette. The presence of ATP, cyclic AMP and EGTA in the perfusion solution kept the plateau phase of the action potential almost intact for as long as 30 min. With depolarizing voltage-clamp pulses from holding potentials between -30 and -40 mV, the slow inward current (isi) was activated at potentials positive to -20 mV. The inactivation time course of isi was fitted by two exponential components in the potential range between -10 mV and +30 mV. By increasing ATP from 2 to 9.5 mM in the solution, the amplitude of isi was increased and the slow component of inactivation was accelerated. The steady-state current-voltage relationship (I-V curve), exhibited a negative slope that became steeper after increasing the ATP concentration. The current was shifted towards the outward direction between -40 mV and -10 mV and became more inward between -10 mV and +40 mV. Increase of the cyclic AMP concentration from 30 to 60 microM also enhanced the amplitude of isi, but the negative slope in the steady-state I-V curve was unaffected. Assuming that the concentration of free Ca2+ in the cell was well buffered at a low level by the EGTA-Ca buffer solution in the pipette, it was concluded that [ATP]i and [cyclic AMP]i exert a direct influence on membrane current systems of the ventricular cell.

Action Potentials↗
PubMed1983

Modification of the cardiac action potential by intracellular injection of adenosine triphosphate and related substances in guinea pig single ventricular cells.

Effects of varying the intracellular adenosine triphosphate level on both the action potential and the membrane current were studied in single ventricular cells isolated from the guinea pig heart, using collagenase. Intracellular injection of adenosine triphosphate elevated the plateau potential level and prolonged the action potential duration. Similar results were obtained by injecting adenosine diphosphate, adenosine monophosphate, or creatine phosphate, i.e., substances considered to increase the intracellular concentration of adenosine triphosphate. In contrast, the action potential was depressed by procedures which could reduce the intracellular adenosine triphosphate level, such as an injection of creatine, superfusion of glucose-free Tyrode's solution containing 5.4 mM cyanide ion, or an injection of adenosine monophosphate into the cyanide-superfused cell. When the membrane current was recorded under the voltage clamp, it was found that the injection of adenosine triphosphate increased the amplitude of the slow inward current, whereas the superfusion of cyanide ion did not significantly decrease the slow inward current, although the action potential became considerably shorter. It was also found that the adenosine monophosphate injection decreased the amplitude of the net outward membrane current at the plateau level and increased it at around -40 mV, and thus intensified the N-shape of the isochronal 0.3-second current-voltage curve. The cyanide ion superfusion produced the opposite effect; in response to depolarizing clamp pulses more positive to the plateau level, the membrane current increased significantly with cyanide ion, but increased only slightly with adenosine triphosphate. These results suggest that intracellular adenosine triphosphate modifies the membrane currents at the plateau potential range, thus altering the action potential duration.

Action Potentials↗