Norio Inomata

Protective effects of polysaccharide fucoidin on myocardial ischemia-reperfusion injury in rats.

We tested whether polysaccharide fucoidin, which inhibits leukocyte rolling in the mesenteric venule, has protective effects in the rat myocardial 30-min ischemia and 6-h reperfusion injury model. Intravenous infusion of fucoidin (27 microg/kg/min from 10 min before to 6 h after reperfusion) significantly attenuated myocardial infarct size 6 h after reperfusion. In this ischemia and reperfusion heart model, expression of P-selectin (determined immunohistochemically) was observed on the venular endothelial cells in the heart 30 min after reperfusion and also was sustained after 6 h. Neutrophil infiltration as estimated by myeloperoxidase activity significantly increased 2 h after reperfusion and kept increasing with time until 6 h after reperfusion. Four-hour infusion of fucoidin after reperfusion significantly reduced neutrophil infiltration, whereas the 2-h infusion of fucoidin did not. These results indicate that neutrophil infiltration and myocardial injury are attributed to expression of P-selectin after reperfusion, and that one of the inhibitory mechanisms of fucoidin seems to be blockade of P-selectin-mediated neutrophil rolling on the vessel wall.

Effect of human plasma-type platelet-activating factor acetylhydrolase in two anaphylactic shock models.

The effect of human recombinant plasma-type platelet-activating factor (PAF) acetylhydrolase was examined in two murine models, PAF-induced death and active anaphylactic models. In the PAF-induced death model where mice were injected intravenously with 40 microg/kg of PAF, the administration of PAF acetylhydrolase reduced mortality in a dose-dependent manner, showing complete prevention of mortality at 1.0 mg/kg. Myeloperoxidase activity, the marker for neutrophils, was increased in the lung by PAF injection, and the PAF acetylhydrolase treatment significantly reversed the increase in myeloperoxidase activity. As in the PAF-induced model, PAF acetylhydrolase also decreased mortality in the active anaphylactic shock model where bovine serum albumin was injected intravenously to mice previously immunized with bovine serum albumin. The protective effect of PAF acetylhydrolase on mortality in this model was significant at 1.0 mg/kg. These results suggest that PAF is an important mediator in the lethality of systemic anaphylaxis, and that PAF acetylhydrolase may be beneficial for treatment of anaphylactic shock.

Antiarrhythmic agents act differently on the activation phase of the ACh‐response in guinea‐pig atrial myocytes

1 Anti‐acetylcholine effects of pilsicainide, flecainide, disopyramide and propafenone on the acetylcholine (ACh)‐induced K+ current (IK.ACh) were examined in dissociated guinea‐pig atrial myocytes under whole‐cell voltage clamp by the use of the ‘concentration‐clamp’ technique. 2 The IK.ACh was activated with a latency of about 100 ms after 1 μm ACh application and desensitized to a steady‐state level. The latent period and the time to peak response were shortened with increasing ACh concentration. 3 The values of half‐maximal inhibition (IC50) on the peak and steady state responses were 25 and 25 μm for pilsicainide, 1.7 and 2.0 μm for disopyramide, 19 and 2.0 μm for flecainide and 0.7 and 0.2 μm for propafenone, respectively. 4 Pilsicainide and disopyramide increased the latent period and the time to peak of IK.ACh in a concentration‐dependent manner. Flecainide and propafenone did not change the latent period, but shortened the time to peak and hastened the decay of IK.ACh in a voltage‐independent manner. 5 The results suggest that the mechanisms underlying the anti‐acetylcholine effect of antiarrhythmic drugs are different among these drugs: i.e., pilsicainide and disopyramide mainly block the muscarinic ACh receptors while flecainide and propafenone inhibit the K+ channel itself as open channel blockers.

Electrophysiological actions of N-(2,6-dimethylphenyl)-8-pyrrolizidine-acetamide hydrochloride hemihydrate (SUN 1165), a new antiarrhythmic agent

Summary: The electrophysiological actions of SUN 1165 on isolated guinea pig atrial and papillary muscles, canine Purkinje fibers, and rabbit sinoatrial node were studied using standard microelectrode techniques. SUN 1165 in low (10-7 g/ml) concentration had little effect on any of the action potential parameters measured. Intermediate (10-6 g/ml) concentration of the compound shortened the duration of action potential of canine Purkinje fibers and increased ratio of the effective refractory period to the duration of action potential at 90% repolarization in guinea pig atrial muscles. At high (10-5 g/ml) concentration, the compound reduced the maximum rate of rise of phase 0 in guinea pig atrial, papillary muscles, and canine Purkinje fibers, though the change in the latter was not statistically significant, and also decreased the action potential amplitude in guinea pig atrial muscles and canine Purkinje fibers. At all concentrations (10-7-10-5 g/ml) tested, the compound exerted little effect both on spontaneous action potentials in rabbit sinoatrial node cells and on Ca2+ -mediated slow responses in partially depolarized guinea pig papillary muscles. These results indicate that SUN 1165 may selectively inhibit cardiac sodium channels and is likely to be of value in correcting not only ventricular but also supraventricular tachyarrhythmias.

Effects of diuretics on GABA‐gated chloride current in frog isolated sensory neurones

1 Effects of three diuretics (furosemide, amiloride and α‐human atrial natriuretic polypeptide (α‐hANP)) on GABA‐activated chloride current (ICl) were investigated in frog isolated sensory neurones, following suppression of Na+, K+ and Ca2+ currents, by use of a ‘concentration‐clamp’ technique. 2 Furosemide inhibited the GABA‐activated ICl in a non‐competitive manner and facilitated the inactivation phase, while amiloride inhibited the GABA response in a competitive manner, both inhibitions being concentration‐dependent. α‐hANP had no effects on the GABA‐induced ICl. 3 The reversal potential of GABA‐activated ICl (EGABA) was not shifted in the presence of amiloride or furosemide. 4 The results suggest that amiloride may act at the GABA binding site while furosemide may act on the GABA‐gated chloride channel.

Sun 1165: A new antiarrhythmic na current blocker in ventricular myocytes of guinea-pig

1. We compared the effect of a new antiarrhythmic compound, SUN 1165, on Na and Ca channels in papillary muscles and enzymatically dispersed single ventricular cells of guinea-pig. Action potential and contractile force in papillary muscle were measured by the conventional microelectrode technique and a strain gauge. The membrane currents were measured in internally perfused and voltage clamped cells by a single suction pipette technique. 2. In papillary muscles, SUN 1165 depressed the maximum rate of rise of action potential (Vmax) in a concentration dependent manner (IC30 = 1.7 X 10(-5) M) more markedly (about six times) than the contractile force. 3. In single ventricular cells, the Na current (INa) was reduced by the drug in a concentration dependent manner (IC30 = 9.1 X 10(-6) M). 4. It showed frequency-dependent block and the steady-state inactivation curve was shifted to more negative potentials. 5. The recovery of INa from inactivation was prolonged by SUN 1165. 6. The Ca current (ICa) was also blocked by the drug in a concentration dependent manner but much less than INa (IC30 = 5.5 X 10(-5) M). 7. These results suggested that SUN 1165 causes a selective inhibition of Na channels in guinea-pig ventricular cells at the antiarrhythmic concentrations.

The anion selectivity of the γ-aminobutyric acid controlled chloride channel in the perfused spinal ganglion cell of frog

The selectivity of the GABA-controlled Cl- channel in the membrane of the dorsal root ganglion cell of the frog has been measured in internally perfused cells by means of current and voltage clamp. When Cl- was replaced by various anions, the 10(-5) or 10(-4) M GABA-induced reversal potentials (EGABA) for Br-, I-, NO3-, ClO4-, SCN-, BF4- and ClO3- were more negative than that for Cl-, despite the fact that, in solution, the test anions are either larger than or similar in size to Cl-.

The role of C-terminal part of ghrelin in pharmacokinetic profile and biological activity in rats.

Ghrelin is an endogenous ligand for growth hormone secretagogue receptor 1a (GHS-R1a), and consists of 28 amino acid residues with octanoyl modification at Ser(3). The previous studies have revealed that N-terminal part of ghrelin including modified Ser(3) is the active core for the activation of GHS-R1a. On the other hand, the role of C-terminal (8-28) region in ghrelin has not been clarified yet. In the present study, we prepared human ghrelin, C-terminal truncated ghrelin derivatives and anamorelin, a small molecular GHS compound which supposedly mimics the N-terminal active core, and examined GHS-R1a agonist activity in vitro, pharmacokinetic (PK) profile and growth hormone (GH) releasing activity in rats. All compounds demonstrated potent GHS-R1a agonist activities in vitro. Although the lack of C-terminal two amino acids did not modify PK profile and GH releasing activity, the deletion of C-terminal 8 and 20 amino acids affected them, and ghrelin(1-7)-Lys-NH(2) exhibited very short plasma half-life and low GH releasing activity in vivo. In rat plasma, ghrelin(1-7)-Lys-NH(2) was degraded more rapidly than ghrelin, suggesting that C-terminal part of ghrelin protected octanoylation of Ser(3) from plasma esterases. Subdiaphragmatic vagotomy significantly attenuated GH response to ghrelin but not to anamorelin. These results suggest that the C-terminal part of ghrelin has an important role in the biological activity in vivo. We also found that ghrelin stimulated GH release mainly via a vagal nerve pathway but anamorelin augmented GH release possibly by directly acting on brain in rats.

Different time courses of the blockade of sodium current by lignocaine and SUN 1165 in single myocytes isolated from guinea-pig atrium

1 The time course of the blockade of sodium currents (INa) by the antiarrhythmic agents, lignocaine and SUN 1165, was studied in single myocytes isolated enzymatically from guinea‐pig atrium, by a new concentration‐jump termed as a ‘concentration‐clamp’ technique. This technique combines an intracellular perfusion and a rapid exchange of external solution surrounding the voltage‐clamped myocyte within 2 ms. 2 Lignocaine (3.7 × 10−5 m to 3.7 × 10−4 M) inhibited the peak amplitude of INa in a concentration‐dependent fashion. It took 2 to 5 s to reach apparent steady‐state inhibition at the concentrations used. Complete recovery from the inhibition also took 2 to 5 s after washing out the agent. In contrast, the inhibitory effect of SUN 1165 (1 × 10−5 M) on the peak INa gradually progressed and reached a steady‐state level about 2 min after the start of drug‐application. The recovery required more than 10 min after washing out of the agent. 3 In cardiomyocytes treated with scorpion toxin (5 μg ml−1), the inactivation of INa was greatly inhibited, resulting in the relatively persistent Na inward current (persistent INa) during the depolarizing pulse. Lignocaine (1.1 × 10−4 M) applied during the depolarizing pulse, reduced in a single‐exponential fashion the amplitude of the persistent INa in milliseconds. On the other hand, lignocaine applied several tens of milliseconds before the depolarizing pulse induced only a small reduction of the peak amplitude of the first persistent INa. When SUN 1165 (1 × 10−5 M) was applied during the depolarizing pulse, there was only a small instantaneous reduction of the amplitude of the persistent INa, although it did inhibit time‐dependently, the peak INa. Both agents accelerated the decay phase of the persistent INa in a time‐dependent manner. 4 These results suggest that lignocaine and SUN 1165 may preferentially interact with the open‐state of the sodium channel rather than with the rested one, although SUN 1165 does so much more slowly.

Mechanism of inhibition by SUN 1165, a new Na channel blocking antiarrhythmic agent, of cardiac glycoside-induced triggered activity

The mechanism of the antiarrhythmic action of SUN 1165, a selective Na channel blocker, in digitalis-induced arrhythmias was investigated by means of conventional microelectrode and suction pipette methods with isolated canine Purkinje fibers and guinea-pig single ventricular cells, respectively. SUN 1165 decreased acetylstrophanthidin-induced delayed afterdepolarization and completely blocked the initiation of triggered activity by acetylstrophanthidin in Purkinje fibers. Delayed afterdepolarization, which was completely abolished either by intracellular dialysis with EGTA or by extracellular superfusion with caffeine, was decreased by SUN 1165 in a concentration-dependent manner in single ventricular cells. These results suggest that an increase in intracellular Ca2+ concentration is a requirement for delayed afterdepolarization. Furthermore, the antiarrhythmic action of SUN 1165 in cardiac glycoside-induced arrhythmias in dogs could be mediated not only by an inhibition of sodium channels and subsequent reduction in the intracellular sodium activity, but also by a reduction of intracellular calcium activity due to the Na+-Ca2+ exchange mechanism.