PubMed Health⌕ Search

Biomedical subjects

T Mitsuiye

Publications and source records attributed to T Mitsuiye.

At least 19 recordsLinked to original sources
PubMed2000

Sustained inward current during pacemaker depolarization in mammalian sinoatrial node cells.

Several time- and voltage-dependent ionic currents have been identified in cardiac pacemaker cells, including Na(+) current, L- and T-type Ca(2+) currents, hyperpolarization-activated cation current, and various types of delayed rectifier K(+) currents. Mathematical models have demonstrated that spontaneous action potentials can be reconstructed by incorporating these currents, but relative contributions of individual currents vary widely between different models. In 1995, the presence of a novel inward current that was activated by depolarization to the potential range of the slow diastolic depolarization in rabbit sinoatrial (SA) node cells was reported. Because the current showed little inactivation during depolarizing pulses, it was called the sustained inward current (I(st)). A similar current is also found in SA node cells of the guinea pig and rat and in subsidiary pacemaker atrioventricular node cells. Recently, single-channel analysis has revealed a nicardipine-sensitive, 13-pS Na(+) current, which is activated by depolarization to the diastolic potential range in guinea pig SA node cells. This channel differs from rapid voltage-gated Na(+) or L-type Ca(2+) channels both in unitary conductance and gating kinetics. Because I(st) was observed only in spontaneously beating SA node cells, ie, it was absent in quiescent cells dissociated from the same SA or atrioventricular node, an important role of I(st) for generation of intrinsic cardiac automaticity was suggested.

Animals↗
PubMed1999

Nicardipine-sensitive Na+-mediated single channel currents in guinea-pig sinoatrial node pacemaker cells.

1. The Na+-dependent inward currents underlying slow diastolic depolarization of sinoatrial (SA) node cells were examined. Using a Na+-rich, Ca2+-free pipette solution a novel single channel current was recorded in addition to the conventional Na+ and L-type Ca2+ currents. The current (termed ist, as it reflects the whole-cell sustained inward current, Ist) does not show obvious inactivation during a 700 ms depolarization and is unique in having a smaller amplitude (1.1 +/- 0.18 pA at -60 mV, n = 12) than the Na+ current through conventional Na+ ( approximately 3.3 pA) and Ca2+ channels (9.6 +/- 0.32 pA at -60 mV, n = 8). The mean unitary conductance of ist channels was 13.3 pS. 2. The recording of ist was infrequent, was observed only in spontaneously beating SA node cells, and was facilitated by adding Bay-K 8644 to the pipette solution. Overlapping of ist events was observed and ist was abolished by bath application of nicardipine. 3. In the ensemble average, the activation of ist was evident by depolarization beyond -70 mV, and the dynamic voltage range of activation (-70 to -30 mV) encompassed the extent of the slow diastolic depolarization. The current density of ist was 0.33 pA pF-1 at -60 mV, as estimated from the number of channels per membrane patch, the open probability and the unitary amplitude. 4. Cumulative histograms for both open and closed times were fitted with a sum of two exponential components. The slow time constants decreased with depolarization, while the fast time constants and the fraction of the fast component were voltage independent. The number of bursts per sweep increased with depolarization. The time constant of the first latency histogram was about two orders of magnitude larger than those in cardiac L-type Ca2+ channels and decreased with depolarization. 5. It is suggested that the ist channels might be responsible for the whole-cell Ist.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1999

Sarcomere length during contraction of isolated guinea-pig ventricular myocytes.

An improved method was developed for measuring sarcomere length (SML) during twitch contractions of single cardiac ventricular myocytes, using a charge-coupled photodiode array self-scanning at a rate of 1.5 ms/element. The average resting SML of 111 cells was 1.88+/-0.04 microm (mean +/-SD). When contractions were triggered by action potentials under perforated-patch conditions, the time course of SML shortening closely followed changes in cell length. A large variation was observed in contraction time course between myocytes, some cells having a phasic component with a duration at 50% shortening (full-width at half-maximum; FWHM) of approximately 40 ms, while others shortened more slowly (FWHM of phasic component @100 ms). FWHM was highly correlated with relaxation half-time, but with neither action potential duration nor resting SML. The kinetics of slowly contracting cells could not be converted to the rapid type by using conditioning trains or applying isoprenaline. The steady-state SML/pCa relation in ventricular myocytes was measured by applying solutions of various pCa immediately after localized punctures of the surface membrane using a focal laser beam. The Hill coefficient, nH, was @4-5 and K1/2@400-500 nM, but there was no evidence of two populations of cells with different Ca2+ sensitivities.

Action Potentials↗
PubMed1999

Changes in cell volume induced by ion channel flux in guinea-pig cardiac myocytes.

1. The cell width of guinea-pig ventricular myocytes was measured using an optic device during patch-clamp experiments and the relationship between the ion channel flux and changes in cell volume was examined. 2. On superfusing myocytes with 50, 70, 150 and 200% osmotic solutions, the relative cell width changed to 121.1 (n = 4), 110.8 (n = 27), 87.1 (n = 6) and 82.6% (n = 6) of control, respectively. Changes in cell length were less than 2% in these test solutions. 3. The application of 300 nmol/L isoprenaline to myocytes swollen in the 70% hypotonic solution induced a decrease in cell width from 111.2 to 106.2% (n = 13). The application of isoprenaline in the isotonic solution also induced a decrease in cell width to 96.5% in eight of 13 cells. A membrane depolarization of 2-4 mV accompanied the isoprenaline-induced decrease in volume. In the remaining five cells, neither an obvious isoprenaline-induced decrease in volume nor membrane depolarization was observed. Under ruptured whole-cell voltage clamp conditions, the activation of inward isoprenaline-induced Cl- current decreased cell width. 4. Cell width was seen to either decrease or increase when a large outward or inward K+ current, respectively, was induced by shifting the holding potential or by applying 200 mumol/L pinacidil. Under gramicidin-perforated whole-cell clamp conditions, the cell width did not change, even when a large inward K+ current was induced. 5. When the test solution was applied to half of an elongated myocyte by using a micropipette, the cell width increased or decreased in the part exposed to the hypotonic or hypertonic test solutions, respectively. In contrast, in the other half of the elongated myocyte, the cell width responded in the opposite direction. 6. It is concluded that a continuous ionic flux through ion channels is capable of inducing changes in cell volume by generating a localized osmotic gradient across the cardiac sarcolemma.

Animals↗
PubMed1999

Cold acclimation of guinea pig depressed contraction of cardiac papillary muscle.

Guinea pigs were exposed to 5 degrees C for 3 wk, and the contractions of myocardial papillary muscle were compared with preparations dissected from control animals kept at approximately 25 degrees C. Developed tension of the papillary muscle per cross-sectional area was significantly (t-test, P < 0.05) decreased after cold exposure (19,200 +/- 8,160 vs. 3,020 +/- 2,890 dyne/cm2; 1 Hz). Time to peak tension was significantly faster in cold-exposed guinea pigs (126.4 +/- 11.1 ms; 1 Hz) than in controls (162.7 +/- 8. 7 ms). The magnitude of the developed tension after application of ryanodine (2 mM) to muscles from cold-exposed animals was decreased to 37.5 +/- 8.3% of control at 1 Hz, whereas in muscles from control animals, tension was decreased to 82.4 +/- 7.7%. The ryanodine-sensitive component of contraction was not significantly changed in control guinea pigs at frequencies >0.5 Hz, whereas in muscles from cold-acclimated guinea pigs, there was a "positive staircase." These results suggested that reversal of the Na+/Ca2+ exchanger is predominantly involved in the positive staircase in control guinea pigs, whereas rate-dependent increases in the Ca2+ store in the sarcoplasmic reticulum may be involved in the staircase after cold acclimation.

Acclimatization↗
PubMed1998

Slow inactivation of cardiac L-type Ca2+ channel induced by cold acclimation of guinea pig.

Whole cell L-type Ca2+ current was recorded in ventricular myocytes dissociated from guinea pigs that were bred at ambient temperatures ranging between daily averages of 4 and 29 degrees C. The dynamic voltage range of inactivation, as measured using 400-ms conditioning pulses and a holding potential of -40 mV, extended from -50 to -20 mV in myocytes prepared in summer. In winter, the inactivation curve was shifted to more negative potentials than in summer. Double-pulse experiments revealed that the negative shift was due to slow-inactivation kinetics. The negative shift of inactivation could be induced in myocytes prepared from animals that had been kept at 5 degrees C for > 3 wk in the summer. The negative shift in Ca2+ current inactivation could be abolished by adding guanosine 5'-O-(2-thiodiphosphate) (5 mM) to the pipette solution, but not by adding staurosporine (2 microM) or 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (100 microM) to the bath. The cold acclimation may introduce the slow inactivation of the cardiac L-type Ca2+ channel through an unknown pertussis toxin-insensitive G protein.

Acclimatization↗
PubMed1997

The sustained inward current in sino-atrial node cells of guinea-pig heart.

Single myocytes were dissociated from the sino-atrial (SA) node of guinea-pig hearts. Only a quite small fraction of the cell population showed spontaneous action potentials and these cells were characterized by the presence of the hyperpolarization-activated cation current If , the delayed rectifier K+ current IK and the L-type Ca2+ current ICa,L as well as by the absence of both the transient outward current Ito and the inward rectifier K+ current IK, 1. After blocking If and IK, depolarizing pulses from -80 mV revealed a large nicardipine-sensitive late current (NSLC). The NSLC was scarcely affected by decreasing extracellular [Ca2+] ([Ca2+]o) from 1.8 to 0.1 mM, while it was decreased significantly by depleting [Na+]o, differently from ICa,L. NSLC was blocked by nicardipine and was increased by Bay K 8644. NSLC was increased by isoprenaline and the additional application of acetylcholine reversed the increase of this current. We conclude that NSLC is largely composed of Ist described in the rabbit SA node pacemaker cells, and that Ist is unique for the pacemaker cells in mammalian SA node cells. Most of the quiescent cells showed neither If nor Ist.

Animals↗
PubMed1997

Regulatory volume decrease of cardiac myocytes induced by beta-adrenergic activation of the Cl- channel in guinea pig.

A new method was developed to automatically measure the thickness of a single ventricular myocyte of guinea-pig heart. A fine marker was attached on the cell's upper surface and changes in its vertical position were measured by focusing it under the microscope. When the osmolarity of the bath solution was varied, the cell thickness reached a new steady level without any obvious regulatory volume change within the period of observation up to 15 min. The cell thickness was 7.8 +/- 0.2 microns (n = 94) in the control Tyrode solution and was varied to 130.4 +/- 3.1% (n = 10), 119.1 +/- 1.1% (n = 50), 87.2 +/- 1.9% (n = 9), and 75.6 +/- 3.2% (n = 5) of control at 50, 70, 130, and 200% osmolarity, respectively. The application of a Cl- channel blocker, 500 microM anthracene-9-carboxylic acid (9AC) did not modify these osmotic volume changes. We discovered that the application of isoprenaline induced a regulatory volume decrease (RVD) in cells inflated by hypotonic solutions. This isoprenaline-induced RVD was inhibited by antagonizing beta-adrenergic stimulation with acetylcholine. The isoprenaline-induced RVD was mimicked by the external application of 8-bromoadenosine 3':5'-cyclic monophosphate. The RVD was inhibited by blocking the cAMP-dependent Cl- channel (ICl, rAMP) with 9AC but was insensitive to 4,4'-diisothiocyanostilbene-2,2'-dissulphonate (DIDS). Taken together these data suggest an involvement of ICl, cAMP activation in the RVD. Whole cell voltage clamp experiments revealed activation of ICl, cAMP by isoprenaline under the comparable conditions. The cardiac cell volume may be regulated by the autonomic nervous activity.

Adrenergic beta-Agonists↗
PubMed1997

Effects of tilisolol, a nonselective beta-adrenergic blocker, on the membrane currents of isolated guinea pig ventricular myocytes.

The effects of tilisolol, a nonselective beta-adrenoceptor blocker, on transmembrane ionic currents were studied in single guinea pig ventricular myocytes by using the whole-cell voltage clamp technique. In the absence of beta-adrenergic stimulation, 10 microM tilisolol, a concentration higher than that used in the clinical therapeutic regimen, did not affect the L-type Ca2+ current (ICa), the inwardly rectifying K+ current (IK1), or the delayed rectifying K+ current (IK). In addition, it did not induce currents through the adenosine triphosphate (ATP)-sensitive K+ channels. However, under the nonselective beta-adrenergic stimulation with 1 microM isoproterenol, 1 microM tilisolol almost completely reversed the agonist-induced increase of IK. The increase of ICa by isoproterenol was blocked only by approximately 30% with tilisolol. We concluded that, at therapeutic concentrations (0.01-0.15 microM), tilisolol is a pure beta-adrenoceptor antagonist that has no direct effects on the transmembrane ionic currents of mammalian ventricular myocytes, such as ICa, IK1, or IK. Comparison of the dose-dependent effects of tilisolol on ICa and IK suggested that tilisolol may selectively inhibit catecholamine-induced increase of IK at the therapeutic concentrations. The virtually selective inhibition of IK, leaving ICa intact, may be favorable to prevent the catecholamine-induced arrhythmia without inhibiting contraction.

Adrenergic beta-Antagonists↗
PubMed1997

Temperature dependence of the inward rectifier K+ channel gating in guinea-pig ventricular cells.

Whole-cell and single-channel currents of the inward rectifier K+ channels from guinea-pig ventricular myocytes were recorded over the range between 5 and 37 degrees C. The conductance for inward currents was decreased by lowering the temperature with a Q10 of 1.28 (whole cell), or 1.41 (single channel) between 20 and 30 degrees C. The open probability of the channel at -100 mV remained high (> 0.9). The distribution of open times was single exponential at all temperatures, confirming a single open state. The entropy change (delta S) for the closing rate of the channel obtained from open-time distribution was -14.0 e.u. (cal/mol/K), and enthalpy change (delta H) was 11.9 kcal/mol. The configuration of closed-time distribution varied markedly by altering the temperature, and three exponentials were necessary to fit the histogram. The slowest component showed higher temperature dependency (delta S = 13.6 e.u. and delta H = 19.0 kcal/mol) than the other two faster components. By assuming a reduced model of C-O at 37 degrees C, the difference in Gibb's free energy (GOC) between the open and closed states was approximately 2 kcal/mol, and the height of the energy barrier for the C-O transition was estimated to be approximately 15 kcal/mol.

Animals↗
PubMed1996

Cell distension-induced increase of the delayed rectifier K+ current in guinea pig ventricular myocytes.

Single ventricular myocytes of guinea pig heart were distended by applying a positive pressure of 5 to 20 mm Hg in the pipette during the whole-cell voltage clamp. The amplitude of delayed rectifier K+ current (I(K)) was increased by approximately 1.5 times, whereas the inward rectifier K+ current was scarcely affected. The increase of I(K) was reversible by applying a negative pressure of -10 to -30 mm Hg accompanied by shrinkage of the inflated cell. This response of I(K) was largely attributed to the E-4031-insensitive component of I(K). The fully activated current amplitude, measured using long-lasting depolarizing pulses (> 30 seconds) to +60 mV, was increased by the cell distension. The activation time course of I(K) during the long pulse consisted of more than three exponential components, and the slowest time constant was decreased by the distension from control 20.2 +/- 7.7 seconds (n=4) to 7.6 +/- 1.6 seconds (n=5). We failed to detect an involvement of microtubules or microfilaments, protein kinase C, and Ca2+ in the inflation-mediated increase of I(K).

Action Potentials↗
PubMed1995

Inactivation of the cardiac Na+ channels in guinea-pig ventricular cells through the open state.

1. The inactivation kinetics of the Na+ current were investigated using the improved oil-gap voltage clamp method in single ventricular cells of guinea-pig hearts. 2. Activation of the Na+ current was observed on depolarization more positive than -50 mV from a holding potential of -100 mV, and inactivation was complete during these depolarizations. The time course of current decay was fitted by a double exponential at potentials between -40 and -15 mV, and virtually by a single exponential at more positive potentials. The decay time courses examined either by the double-pulse protocol or the single-pulse protocol were similar. 3. The double-pulse protocol clearly revealed a sigmoidal onset of inactivation on depolarization. The initial delay of inactivation decreased with more positive potentials. The time course of double-pulse inactivation was reconstructed by integrating the Na+ current recorded by a continuous depolarization. 4. These findings are consistent with the hypothesis that the cardiac Na+ channel inactivates exclusively through the open state.

Animals↗
PubMed1994

Increase of the delayed rectifier K+ and Na(+)-K+ pump currents by hypotonic solutions in guinea pig cardiac myocytes.

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 activation of Cl- current. The hypertonic superfusion did not cause any consistent change in the membrane Cl- conductance. Since the response of IK was observed in all experiments, its mechanism was studied. We failed to observe marked changes in the kinetic and conductance properties of IK in the hypotonic solution. The involvements of either the protein kinases or Ca2+ were also ruled out as major mechanisms underlying the IK response.

Animals↗
PubMed1993

Quantification of exponential Na+ current activation in N-bromoacetamide-treated cardiac myocytes of guinea-pig.

1. The activation kinetics of the Na+ current was investigated in single ventricular cells of the guinea-pig heart using an improved oil-gap voltage clamp method. The inactivation of the current was removed by an intracellular application of N-bromoacetamide (NBA) for less than 1 min. Although the NBA treatment slightly decreased the peak amplitudes (81.7 +/- 13.4% of control, n = 15), the Na+ current remained stable after the removal of inactivation. 2. On depolarization, the activation of Na+ current took an exponential time course after the capacitive current decreased to 5% of its peak amplitude (40-100 microseconds after the pulse onset). The time course of deactivation, recorded on repolarization from 1.2 ms depolarization, was also a single exponential. 3. The time constants of activation and deactivation were almost identical when compared at a given test potential within a range of -50 to -30 mV. These findings indicate that the cardiac Na+ current activation is determined by m1 kinetics, or one rate-limiting step. 4. At potentials negative to -60 mV, the deactivation was complete, and its time constant decreased e-fold per 20.3 +/- 1.8 mV hyperpolarization (n = 7). 5. The degree of steady-state activation (m(infinity)) was fitted to a Boltzmann equation with a slope factor of 7.4 +/- 0.3 mV and a half-maximum potential of -33.3 +/- 0.8 mV (n = 8). 6. Rate constants for the rate-limiting activation step between a closed state and an open state (alpha m, beta m), were determined from m(infinity) and tau m over a potential range between -100 and +50 mV. On a logarithmic scale, beta m-1 was a linear function of the membrane potential over the range -100 and -30 mV. 7. Fitting the newly determined activation kinetics to the rising phase of the action potential indicated that the activation kinetics in the present study is relevant to the physiological action potential. The density of the Na+ channels thus obtained was 1075 +/- 186 pF-1 (n = 6). 8. The measurements in the NBA-treated Na+ current were compared with those obtained without treatment.

Acetamides↗
PubMed1992

Exponential activation of the cardiac Na+ current in single guinea-pig ventricular cells.

1. The cardiac Na+ current of guinea-pig was recorded using an improved oil-gap voltage clamp method. When a single ventricular cell was stretched between the internal and external solution compartments across an oil gap of about 40 microns in width, the sealing resistance in the oil gap was higher than 1 G omega and the time constant of the capacitive current was between 10 and 40 microseconds. Effective series resistance (Rs) was less than 50 k omega after Rs compensation. 2. The activation time course (I'Na) was separated from inactivation by dividing the digitized record of Na+ current with the inactivation variable h(t), which was obtained by fitting exponential functions to the decaying phase of current. I'Na started as a single exponential activation at time 0, which was defined by the decay of the capacitive current to 5% of its peak. 3. The Na+ tail current was recorded on repolarization after a short (1.2 ms) depolarizing pulse to -10 mV. Its single exponential decay at potentials negative to -50 mV, or its major exponential component of decay between -50 and -30 mV, was attributed to deactivation. The time constants of deactivation were similar to those of activation which were measured from I'Na on depolarization to comparable potentials. The m1 kinetics gave a better fit for Na+ activation than the m3 kinetics. 4. The time constant of deactivation was a linear function of the membrane potential on a semilogarithmic scale with an e-fold increase per 21.6 +/- 1.3 mV (n = 8) depolarization. The steady-state activation value (m(infinity)) was obtained from the amplitude of I'Na. Fitting a Boltzmann equation indicated a half-activation potential of -21.9 +/- 1.7 mV and a slope factor of 7.9 +/- 0.4 mV (n = 9). 5. m1 kinetics are more pertinent to a description of the cardiac Na+ current. Limitations in analysing the activation kinetics of Na+ current are discussed for the improved oil-gap voltage clamp method.

Animals↗
PubMed1992

Effects of mechanical stretch on membrane currents of single ventricular myocytes of guinea-pig heart.

A technique to record whole cell membrane current during stretching single cardiac myocytes was developed. Ventricular myocytes were dissociated by treating guinea-pig hearts with collagenase. One end of the cell was fixed either to a microglasstool tip or to a glass plate, while the other end was attached either to a microglasstool tip or to a suction pipette, which was mounted on a micromanipulator. A time-independent current showing a reversal potential of -15 +/- 4 mV (n = 7) was activated when the myocytes were stretched more than 20% of the length between two fixed point. The current gradually relaxed during the maintained stretch, and disappeared on releasing the stretch. We failed to detect any consistent change in either the L-type Ca2+ current or the inward rectifier K+ current. For comparison, current changes induced by inflating the cell using a hypo-osmotic solution were recorded. The inflation was not accompanied by any change in the time-independent current. Instead, the delayed rectifier K+ current was increased to 170 +/- 48% control by the 70% hypo-osmotic solution. Thus, the effect of mechanical stretch on the time-independent current is different from those of hypo-osmotic cell inflation. The stretch-induced time-independent current is compared with reported current changes induced by the intracellular microinjection of Ca2+.

Animals↗