Makoto Arita

Mechanical stretch increases intracellular calcium concentration in cultured ventricular cells from neonatal rats

SummaryWe investigated the effects of mechanical stretch on intracellular calcium concentration ([Ca2+]i) of cultured neonatal rat ventricular cells using micro-fluorometry with fura-2. Myocytes were cultured on laminin-coated silicon rubber and stretched by pulling the rubber with a manipulator. Myocytes were either mildly stretched (to less than 115% of control length), moderately so (to 115%–125% of control length), or extensively (to over 125% of the control length). “Quick stretches” (accomplished within 10s) of moderate to extensive intensities produced a large transient increase of [Ca2+]i in the early phase of stretch (30s-2 min), followed by a small but sustained increase during the late phase of stretch (5–10 min). The initial transient increase in [Ca2+]i after the “quick stretch” was preserved in the presence of gallopamil (10−7M) or ryanodine (10−5 M), but was absent in Ca2+-free medium or in the presence of gadolinium (10−7M). The late or steady state [Ca2+]i increase was observed in the presence of gadolinium, gallopamil, or ryanodine but was abolished in Ca2+-free medium. A steady-state increase in [Ca2+]i was also evoked by “slow stretch” in which cells were slowly pulled to the final length within 1–2min. As the presence of external Ca2+ was indispensable, increased trans-sarcolemmal Ca2+ influx appears to be involved in both initial and steady-state increases in [Ca2+]i. The initial increase in [Ca2+]i after the “quick stretch” can be attributed to the activation of gadolinium-sensitive, stretch-activated channels.

Ionic mechanisms of action potential prolongation at low temperature in guinea‐pig ventricular myocytes.

1. We studied the effects of low temperature on the action potentials and membrane currents of guinea‐pig ventricular myocytes, using a tight‐seal whole‐cell clamp technique. 2. The action potential duration at 95% repolarization was prolonged from 146 +/‐ 33 ms (mean +/‐ S.D., n = 6) at 33‐34 degrees C (control temperature) to 314 +/‐ 83 ms at 24‐25 degrees C (low temperature). 3. In whole‐cell clamp experiments, low temperature decreased the calcium current (ICa), the delayed rectifier potassium current (IK), and the inwardly rectifying potassium current (IK1) with ‘apparent’ Q10 (temperature coefficient) values of 2.3 +/‐ 0.6 for ICa, 4.4 +/‐ 1.2 for IK tail current and 1.5 +/‐ 0.3 for IK1 (n = 7). 4. The effect of low temperature on IK was further studied in the presence of 0.6 microM nicardipine to block ICa. The decay phase of the IK tail consisted of two exponential components. The fast but not the slow component was highly sensitive to the temperature change with an apparent Q10 of 4.5. 5. We found that a component of time‐independent current is also sensitive to the temperature. The current had a linear I‐V relationship and remained almost unchanged after inhibition of Na(+) ‐K+ pump in K(+)‐free external solution. 6. Using our mathematical model of the ventricular action potential (a modification from the DiFrancesco‐Noble model), we simulated the action potential at low temperature by modifying some of the membrane currents, namely IK, IK1, ICa and a component of background current. It was shown that simultaneous changes in these currents could reproduce approximately 75% of the action prolongation induced by low temperature.

Pharmacological Evidence for the Persistent Activation of ATP-Sensitive K+ Channels in Early Phase of Reperfusion and Its Protective Role Against Myocardial Stunning

BACKGROUND The activation of cardiac ATP-sensitive potassium channels is reported to protect myocardium during ischemia. However, the behavior and role of this channel during reperfusion remain uncertain. METHODS AND RESULTS Guinea pig right ventricular walls were studied by use of microelectrodes and a force transducer. Each preparation was perfused via the coronary artery at a constant flow rate and was stimulated at 3 Hz. In the first protocol, the preparation was subjected to 10 minutes of no-flow ischemia, which was followed by 60 minutes of reperfusion. Introduction of ischemia shortened the action potential duration (APD) to 58.7 +/- 3.1% of the preischemic values, in association with a decrease in the resting membrane potential (by 12 +/- 0.8 mV) and action potential amplitude (by 34.6 +/- 1.8 mV). On reperfusion, although the APD was restored, it remained shortened for up to approximately 30 minutes of reperfusion. In the presence of glibenclamide (10 mumol/L), the shortening of the APD during ischemia was significantly attenuated and the restoration of APD after reperfusion was significantly facilitated. When glibenclamide was applied from the onset of reperfusion, the persistent APD shortening was significantly suppressed. The developed tension decreased during ischemia and recovered after 60 minutes of reperfusion (up to 92.0 +/- 6.4% of preischemic values) in the untreated preparations. The application of glibenclamide that was started before ischemia or from the onset of reperfusion significantly suppressed the recovery of contractility (P < .05 versus untreated preparations). In the second series of experiments, 20 minutes of no-flow ischemia and 60 minutes of reperfusion were applied. This protocol produced a sustained contractile dysfunction after reperfusion (to 34.0 +/- 3.2% of preischemic values). In the presence of cromakalim (2 mumol/L), the APD shortening was enhanced during both ischemia and the early reperfusion period. Cromakalim significantly improved the contractile recovery (to 79.3 +/- 4.1% of preischemic values, P < .05 versus untreated preparations). The application of cromakalim that was started from the onset of reperfusion also improved the contractile recovery during this phase and this effect was associated with enhanced APD shortening. However, the cromakalim-treated preparations demonstrated a higher incidence of ventricular fibrillation during reperfusion. CONCLUSIONS Cardiac ATP-sensitive potassium channels are activated by ischemia, and a fraction of these channels remains activated during the early reperfusion phase. The resulting shortening of the APD prevents the heart from developing myocardial stunning.

Bepridil differentially inhibits two delayed rectifier K+ currents, IKr and IKs, in guinea-pig ventricular myocytes

We investigated the effects of bepridil on the two components of the delayed rectifier K+ current, i.e., the rapidly activating (IKr) and the slowly activating (IKs) currents using tight‐seal whole‐cell patch‐clamp techniques in guinea‐pig ventricular myocytes, under blockade of L‐type Ca2+ current with nitrendipine (5 μM) or D600 (1 μM). Bepridil decreased IKs under blockade of IKr with E4031 (5 μM), in a concentration‐dependent manner. The concentration‐dependent inhibition of IKs by bepridil was fitted by a curve, assuming one‐to‐one interactions between the channel and the drug molecule. The concentration of half‐maximal inhibition (IC50) was found to be 6.2 μM. The effect of bepridil on IKr was assessed using an envelope‐of‐tails test. In the control condition, a ratio of the tail current to the time‐dependent current measured during depolarization was large (>1) at shorter pulses (<200 ms), and it decreased to a steady state value of ∼0.4 with increases in the pulse duration. Bepridil at a concentration of 2 μM did not decrease this ratio at shorter pulses. In a short‐pulse (duration=50 ms) experiment that largely activates IKr, the drug was found to block IKr in a cooperative manner (Hill coefficient=3.03) and the IC50 was 13.2 μM. These results suggest that bepridil at a clinical therapeutic concentration (∼2 μM) selectively blocks IKs but does not inhibit IKr. This may relate to the characteristic frequency‐dependent effects of bepridil on the action potential duration (APD), e.g., the non‐reverse use‐dependent prolongation of APD.

Effects of lysophosphatidylcholine on resting potassium conductance of isolated guinea pig ventricular cells

AbstractWe studied the effects of lysophosphatidylcholine (LPC), a toxic metabolite of ischemia, on the inward rectifier potassium channel current in isolated guinea pig ventricular cells.1)LPC (10–50 μM) added to the external solution decreased the resting membrane potential and occasionally induced repetitive action potential discharges, with or without loss of repolarization.2)In voltage clamp studies, LPC (20 μM) decreased the conductance at the levels of resting potentials (≃ −80 mV) from 26±8 nS to 16±3 nS (mean and SD,n=4) within 10 min. Prolonged application of LPC (>12 min) produced transient inward currents after depolarizing clamp pulses, thereby suggesting that the LPC elevated intracellular Ca2+ concentrations.3)The effect of LPC on the single inward rectifier K channel current was examined using the patch clamp technique in a cell-attached mode. LPC decreased the single channel conductance, depending on the concentration (5–100 μM). The slope conductance in the presence of 150 mM K+ in the pipette decreased from 45±7 pS (control) to 32±17, 20±19, and 14±10 pS for 5, 20 and 100 μM LPC, respectively. LPC induced little change with regard to probability of the channel opening. These results suggest that LPC depolarizes membrane by decreasing single channel conductance of the inward rectifier K channel. This reduction partially contributes to the alleged LPC-induced abnormal automaticities and conduction disturbances in the heart.

Effects of hydroxyl radicals on KATP channels in guinea-pig ventricular myocytes.

Abstract We studied the effects of oxygen free radicals on the ATP-sensitive potassium channel (KATP channel) of guinea-pig ventricular myocytes. Single KATP channel currents were recorded from inside-out patches in the presence of symmetrical K+ concentrations (140 mM in both bath and pipette solutions). Reaction of xanthine oxidase (0.1 U/ml) on hypoxanthine (0.5 mM) produced superoxide anions (·O2-) and hydrogen peroxide (H2O2). Exposure of the patch membrane to ·O2- and H2O2 increased the opening of KATP channels, but this activation was prevented by adding 1 µM glibenclamide to the bath solution. In the presence of ferric iron (Fe3+: 0.1 mM), the same procedure produced hydroxyl radicals (·OH) via the iron-catalysed Haber-Weiss reaction. ·OH also activated KATP channels; however, this activation could not be prevented by, even very high concentrations of glibenclamide (10 µM). These different effects of glibenclamide suggest that the mode of action of these oxygen free radicals on KATP channels is different and that ·OH is more potent than ·O2-/H2O2 in activating KATP channels in the heart.

Wide bandgap engineering of (AlGa)2O3 films

Bandgap tunable (AlGa)2O3 films were deposited on sapphire substrates by pulsed laser deposition (PLD). The deposited films are of high transmittance as measured by spectrophotometer. The Al content in films is almost the same as that in targets. The measurement of bandgap energies by examining the onset of inelastic energy loss in core-level atomic spectra using X-ray photoelectron spectroscopy is proved to be valid for determining the bandgap of (AlGa)2O3 films as it is in good agreement with the bandgap values from transmittance spectra. The measured bandgap of (AlGa)2O3 films increases continuously with the Al content covering the whole Al content range from about 5 to 7 eV, indicating PLD is a promising growth technology for growing bandgap tunable (AlGa)2O3 films.

Modification of cardiac sodium current by intracellular application of cAMP

We examined the effects of intracellular perfusion of cyclic adenosine monophosphate (cAMP) on the sodium current (INa) of guinea-pig ventricular myocytes, using the whole-cell clamp technique. INa was elicited by depolarizing voltage steps (−20 mV) from a variety of holding potentials (−120 to −50 mV), under conditions of 60 mM extracellular Na+ concentration ([Na+]0) and at the temperature of 24–26°C.Intracellular perfusion of cAMP decreased the INa elicited from the holding potentials less negative than −90 mV. In the presence of 1 mM cAMP, for example, the peak INa elicited from −80 mV decreased from 6.0±2.0 nA to 4.0±2.2 nA (mean±SD, P<0.02, n=7) within 3–6 min. In the presence of extracellular 3-isobutyl-1-methylxanthine (IBMX, 20 μM), much lower concentrations of cAMP (0.2 mM) yielded a comparable effect. On the other hand, intracellular perfusion of cAMP increased the INa elicited from very negative holding potentials (<−100 mV). For instance, the application of cAMP (1 mM) increased the INa elicited by step depolarizations from −120 mV (to −20 mV), from 9.9±2.1 nA to 11.0±3.1 nA (P<0.05, n=5).The former effect was attributed to a marked shift of the steady-state inactivation curve of INa to the negative direction; the voltage of half-inactivation shifted from −77.9±1.0 to −83.5±1.4 mV, or by −5.6 mV. The latter effect may be explained by increases in maximum available conductance of INa. Extracellular application of isoproterenol (1 μM) also decreased the INa evoked from a holding potential of −80 mV, whereas it increased the INa elicited from more negative potentials of −120 mV. These effects of isoproterenol were reversible and markedly attenuated in the presence of a specific inhibitor of cAMP-dependent protein kinase, H-89 {N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide}, an isoquinolinesulphonamide derivative, in the extracellular medium (2–10 μM) and a protein kinase inhibitor (Walsh inhibitor) in the pipette solution (40 μM). H-89 (10 and 30 μM) affected neither the adenylate cyclase activity prepared from rabbit ventricular muscles, nor the isoproterenol-mediated increases in the cAMP content in guinea-pig ventricular muscles. Our observations suggest that the increase in intracellular cAMP modulates the function of cardiac Na channels, preferentially by stimulating cAMP-dependent protein kinase, with subsequent phosphorylation of the channel protein.

Inhibitory effects of palmitoylcarnitine and lysophosphatidylcholine on the sodium current of cardiac ventricular cells

We investigated the effects of ischemia-related amphipathic compounds, palmitoylcarnitine (PamCar, 0.5–50 μM) and lysophosphatidylcholine (lysoPtdCho, 5–50 μM) on sodium current (INa) of guinea-pig ventricular myocytes. The cells were perfused with low-Na+ (60 mM) Tyrodes solution, and Ca2+ and K+ currents were blocked by external Co2+ (3 mM) and internal Cs+ (140 mM), respectively. INa was elicited by depolarizing voltage steps from a holding potential of −100mV at a temperature of 33 °C. PamCar (5 μM) decreased the peak INa (attained at −20mV or −30mV) from 6.1±2.1 nA to 3.9±1.4 nA (n=11), or by 36.1% within 2 min, and shifted the curve of steady-state INa inactivation by 5.4 mV in the positive direction (from −76.3±4.6 mV, control to −70.9±4.0 mV, in PamCar, n=4). Partial restoration of the amplitude and the shift of the steady-state inactivation curve of INa was attained after washout of PamCar. In contrast, lysoPtdCho at concentrations over 10 μM irreversibly depressed the INa within 0.5–3 min and the reduction of IinNa was followed by cell contracture or cell death (n=9). The survival time, defined as a period from the start of lysoPtdCho application to the time of the last successful recording of the INa (before evolution of sudden changes in the holding current), depended on the concentrations of lysoPtdCho. Both PamCar and lysoPtdCho retarded the time course of activation and inactivation of INa. These findings are compatible with the idea that PamCar and lysoPtdCho decrease the maximum Na+ conductance and alter the surface negative charge of the membrane, perhaps via amphiphilic intervention in the phospholipid bilayers. However, PamCar had an additional effect that indicates more direct but reversible incorporation of this agent with the Na+ channels or integral membrane proteins.

Isoproterenol inhibits residual fast channel via stimulation of β-adrenoceptors in guinea-pig ventricular muscle

When perfused with high K+ (8.1 to 14.9 mM)-Tyrodes solution, the upstroke of action potentials in the isolated guinea-pig ventricular muscle is composed of two components and there are two separable peaks in the first derivative, i.e., Vmax, fast and Vmax, slow. The Vmax, fast was a measure of activation of the residual fast channel, while the Vmax, slow was that of the slow channel. Isoproterenol depressed Vmax, fast with increase in Vmax, slow, in a concentration-dependent manner (10(-8) to 10(-6) M). This depression of Vmax, fast was greater at more depolarized levels of membrane potential. Therefore, the isoproterenol-induced depression of Vmax, fast may be due to a negative shift of the curve relating Vmax, fast to the take-off potential (Em) (Vmax--Em relationship), along the voltage axis. The negative shift of Vmax--Em relationship by isoproterenol was also recognized in small preparations the size of which is well within the space constant. The negative shift was inhibited in the presence of beta-blockers (pindolol 1 microgram/ml or atenolol 10 micrograms/ml) but not by a calcium antagonist, 1-verapamil (1 microgram/ml). These results suggest that isoproterenol blocks sodium channels in the depolarized ventricular muscle via stimulation of the beta-adrenoceptors and that the depression of Vmax, fast is not mediated by the well-known effects of isoproterenol on Vmax, slow, i.e., increased influx of Ca2+ ions.