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S Kokubun

Publications and source records attributed to S Kokubun.

At least 91 records · Page 5Linked to original sources
PubMed1991

Blockade of Cl channels by organic and inorganic blockers in vascular smooth muscle cells.

The effects of Cl channel blockers on large-conductance (LC-type) Cl channels of cultured vascular smooth muscle cells (VSMC) were studied in order to characterize the channel pharmacologically. Intracellular disulphonic stilbene derivatives, such as 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanostilbene-2,2'-disulphonic acid (SITS) inhibited Cl channel activity in a dose-dependent manner. An obvious inhibitory effect of DIDS in this condition was obtained at concentrations higher than 5 microM, and the complete inhibition was obtained at around 100 microM, which was almost 10 times less than the effective dose of SITS. The inhibitory effect of DIDS was reversible at a drug concentration of lower than 50 microM. Single-channel conductance decreased as the concentration of DIDS increased. This decrease in the conductance was a consequence of unresolved openings of the channel due to fast blocking and unblocking rates of the drug. The Cl channel was also obviously inhibited by extracellular DIDS at a concentration of 1 mM. In addition, in cell-attached patches, 500 microM DIDS applied extracellularly inhibited Cl channel activated by the application of polymyxin B. We also investigated the effect of Zn on Cl channels in VSMC. Intracellular Zn dose dependently and reversibly blocked the channel at the positive intracellular potential range, whereas at the negative intracellular potential range it did not block the channel activity. Results in this study suggest the diversity of Cl channels among various tissues.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

Protein kinase C may regulate resting anion conductance in vascular smooth muscle cells.

The blocker of protein kinase C(PKC) activated large-conductance channel(337.1 pS) in cell attached patch mode in cultured vascular smooth muscle cells. The channel showed time-dependent inactivation whose time course became faster as the amplitude of the command potential was increased. These characteristics of large-conductance channel activated by the application of the PKC blocker were very similar to those of voltage-dependent Cl channels in these cells, indicating the channel activated by the drug is Cl channel. Since voltage-dependent Cl channels were reported to be only activated by forming inside-out patch, these findings suggest Cl permeability of vascular smooth muscle cells is at least partially regulated by protein kinase C.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Single anion-selective channel and its ion selectivity in the vascular smooth muscle cell.

The properties of voltage-gated Cl channels of cultured smooth muscle cell prepared from embryonic rat aorta were studied. In the excised patch (inside-out configuration), we observed the activity of channels, opening and closing spontaneously, when the membrane potential was held at around 0 mV. The channels were active at a potential range between +10 and -10 mV. A step change of the membrane potential from the active potential range in either a positive or a negative direction closed the channel to an apparently inactivated state. The time course of this inactivation process became faster as the amplitude of the step change was increased. Returning the membrane potential to 0 mV allowed the channel to recover from the inactivated state. The channel had at least two open conductance states. Ca ions at the cytoplasmic face were not required for the activation of the channel. Adenosine nucleotides at the same side of the membrane had no effect on channel activity. The channels were selective to anions rather than cations, and they had a large single channel conductance of 340.5 +/- 20.4 pS in symmetrical 150 mM TEA-Cl. The reversal potential of the channel was shifted by -15.2 +/- 2.6 and 17.0 +/- 1.7 mV, when the Cl concentration at the intracellular side was changed to 75 mM or 300 mM, respectively. The permeability sequence of halides was I- greater than Br- greater than Cl- greater than F- (1.4:1.3:1.0:0.7), whereas the conductance sequence was Cl- greater than Br- greater than F- greater than I- (1.00:0.89:0.86:0.83).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Voltage-dependent cooperative interactions of calcium channel ligands in intact cardiac cells.

Voltage-dependent Ca channels were studied in living, tissue cultured rat heart cells using patch clamp analysis of single channels and radioligand binding studies at different membrane potentials. Ca channel activating and blocking dihydropyridines (DHP) show cooperative interaction and, therefore, bind to at least two different binding sites on the channel protein. Cooperative interactions between activating and blocking DHP, between DHP and verapamil and between DHP and diltiazem are all voltage dependent. The type of interaction in polarized cells and hence, the possible effects in vivo cannot be predicted from studies in cell homogenates.

Allosteric Site↗
PubMed1986

Properties and modulation of cardiac calcium channels.

Voltage-dependent calcium channels are widely distributed in excitable membranes and are involved in the regulation of many cellular functions. These channels can be modulated by neurotransmitters and drugs. There is one particular type of calcium channel in cardiac cells (L-type) whose gating is affected in different ways by beta-adrenoceptor and 1,4-dihydropyridine agonists. We have analysed single calcium channel currents (i) in myocytes from rat hearts in the absence and presence of isoproterenol or 8-bromo-cAMP. We have found that both compounds have similar effects on calcium channel properties. They increase the overall open state probability (po) of individual calcium channels while i remains unaffected. Analysis of the gating kinetics of calcium channels showed: a slight increase in the mean open times of calcium channels, a reduction in time intervals between bursts of channel openings, an increase in burst length and a prominent reduction in failures of calcium channels to open upon depolarization. These kinetic changes caused by isoproterenol and 8-bromo-cAMP can account for the increase in po. Since the macroscopic calcium current, ICa, can be described by ICa = N X po X i, the increase in po accounts for the well-known increase in ICa by beta-adrenergic catecholamines. Cyclic AMP-dependent phosphorylation of calcium channels is a likely metabolic step involved in this modulation. Another class of drug that modulates calcium channel gating is the 1,4-dihydropyridines which can either enhance or reduce ICa, either by prolonging the open state of the channels or by facilitating the inactivated state. Both effects depend strongly on membrane potential and are independent of cyclic AMP-dependent phosphorylation reactions.

Adrenergic beta-Agonists↗
PubMed1986

Studies on Ca channels in intact cardiac cells: voltage-dependent effects and cooperative interactions of dihydropyridine enantiomers.

We have investigated the effects of two oppositely acting enantiomers of the 1,4-dihydropyridine derivative 202-791 on voltage-dependent Ca channels by combining electrophysiological techniques and binding studies. The (S)-enantiomer of 202-791 promoting prolonged openings of single Ca channels, and thereby increasing transmembrane Ba currents, was classified as channel activator. The (R)-enantiomer favoring a closed state of the channel, and thereby reducing Ba currents, was classified as a channel blocker. Both compounds shifted the steady state current inactivation curve toward more negative potentials. At holding potentials positive to -20 mV, the Ca channel-activating effect of the (S)-enantiomer turned over into a blocking effect. In cells with normal resting potential the combination of the two enantiomers revealed a possible positive cooperative effect resulting in an enhancement of the open state probability of the channels. At depolarized holding potentials the activator enhanced the inhibitory effect of the blocker. Binding studies in intact cells were performed by using the radiolabeled channel-blocking dihydropyridine 3H-(+)-PN 200-110. The results showed a strong increase in binding affinity but no change in binding capacity when the cells were depolarized. Analysis of the interactions of (S)- and (R)-202-791 with this radioligand indicated stimulation of 3H-(+)-PN 200-110 binding by the (S)-enantiomer in polarized cells (membrane potential -38 +/- 4 mV). This effect could be attributed to an increase in binding affinity. The (R)-enantiomer had no such positive cooperative effect, but acted as a purely competitive ligand. Depolarization to 0 mV increased the apparent affinity of both enantiomers by factors of 38 (blocker) and 12 (activator), but abolished the cooperative effect of (S)-202-791 on the binding of the radioligand. Ca ions had little effect on the binding of 3H-(+)-PN 200-110 in polarized cells. However, in the presence of the activating (S)-enantiomer, Ca transformed the usual hyperbolic binding isotherm of the radioligand into a strongly sigmoid curve. Sigmoidicity was minimal with 3-5 microM Ca and maximal with 0.5 mM Ca. Together these data demonstrate homotropic and heterotropic cooperative interactions between channel activator and channel blocker. They indicate that at least two high affinity binding sites for dihydropyridines are associated with voltage-dependent Ca channels. Voltage dependence of both--binding affinity and cooperativity--suggests that these binding sites are located close to a structural component of the channel which is involved in the potential-sensitive gating process.

Aging↗
PubMed1986

The voltage-dependent effect of 1,4-dihydropyridine enantiomers on Ca channels in cardiac cells.

We studied voltage-dependent binding and action of 1,4-dihydropyridine enantiomers (Sandoz (+)-(S) & (-)-(R)-202-791) in intact cardiac cells. Drug action was studied by patch clamp method. (+)-enantiomer primarily prolonged open time of Ca channel, thus enhancing Ca currents, while (-)-enantiomer predominantly favored closed state of the channel, reducing the currents. Electrophysiological study also revealed that steady-state inactivation was greatly enhanced by both enantiomers. Therefore, it seems that both enantiomers have a capability of activating and blocking effects. Strongly voltage-dependent binding affinity of these enantiomers was revealed by displacement of 3H-(+)-PN200-110 with each enantiomers. Binding affinity of both enantiomers in depolarized cells was much higher than in polarized cells. These results indicate that both enantiomers have higher affinity when Ca channels are inactivated.

Animals↗
PubMed1984

Dihydropyridine derivatives prolong the open state of Ca channels in cultured cardiac cells.

The dihydropyridine derivatives CGP 28392 and BAY K 8644 exert a strong positive inotropic effect in mammalian cardiac muscle, presumably by increasing Ca influx during the action potential. Analysis of the drug effects at the level of single Ca channels by means of the patch-clamp method revealed complicated changes in the kinetics of channel gating. The prevailing effect is a prolongation of the mean open time of Ca channels. In addition, the intervals between channel openings can be slightly prolonged. Ensemble averages of single-channel traces showed a concentration-dependent increase in the mean current amplitude by the drugs. In contrast to beta-adrenoceptor agonists, the increase in Ca current by the dihydropyridine derivatives is not associated with an increase in the intracellular cyclic AMP level.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
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

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

Sodium channels in cultured cardiac cells.

Primary cardiac cell cultures were prepared from the hearts of neonatal rats. The patch-clamp method (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) was applied for studying whole-cell Na+ currents and single-channel Na+ currents, respectively. Whole-cell recordings yielded voltage- and time-dependent Na+ currents which could be blocked by tetrodotoxin. Single-channel Na+ currents were directly compared in cell-attached patches and in inside-out patches. In cell-attached patches the elementary current was about -1 pA at -10 mV and the slope conductance over a 50 mV voltage range was 15.1 +/- 1.6 pS (mean +/- S.D.). Inactivation during depolarization and after conditioning clamp steps, in the steady state, resulted from a reduced opening probability of Na+ channels. In inside-out patches, with identical solutions at both membrane surfaces, there was a large (40-50 mV) shift of channel opening and inactivation kinetics towards more negative potentials. However, for levels of comparable opening probabilities, mean open times of Na+ channels were similar in cell-attached and inside-out patches. Tetrodotoxin (10-20 microM) had no effect on Na+ channels when applied from the inside, but blocked them completely after application to the outside membrane surface.

Action Potentials↗
PubMed1982

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↗
PubMed1981

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↗
PubMed1981

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↗