Nobuaki Miyawaki

Protective effects of timolol against the neuronal damage induced by glutamate and ischemia in the rat retina

The purpose of this study was to determine whether timolol, an ocular hypotensive drug, has retinal neuroprotective effects in experimental in vitro and in vivo models. For in vitro studies, we used retinal neuron cultures from rat embryos and purified retinal ganglion cells (RGCs) from newborn rats. In the former, neurotoxicity was induced using 1 mM glutamate and cell viability was assessed. In RGCs, neurotoxicity was induced using 25 microM glutamate for 3 days and cell viability was assessed. For the in vivo study, we used a rat model of retinal ischemic injury induced by elevating intraocular pressure (IOP) by raising the hydrostatic pressure. The retinal damage was evaluated by counting the number of cells in the ganglion cell layer (GCL) and by examining the a- and b-waves in the electroretinogram (ERG). For the intraocular distribution study, 0.5% [3H]timolol was topically applied to rat eyes, and these were enucleated after various intervals and divided into parts. Each part was combusted and the radioactivity measured. Timolol (0.1 and 1 microM) markedly reduced the glutamate-induced neuronal cells in retinal neuron cultures and in RGCs. After ischemic-reperfusion, both the number of cells in the GCL and a- and b-waves in the ERG decreased; however, topically applied 0.5% timolol reduced these effects. Topically applied 0.5% timolol was detected at a concentration of approximately 1 microg/g wet tissue in retina-choroid at 30 min after its application. In conclusion, timolol was effective against retinal neuron damage both in vitro and in vivo. Furthermore, topically applied timolol reached the retina-choroid. These findings suggest that timolol has a direct neuroprotective effect against retinal damage.

Electrophysiological properties of SD-3211, a novel putative Ca2+ antagonist, in isolated guinea pig and rabbit hearts

Summary: The cardiac effects of SD-3211, a novel nondihydropyridine type of Ca2+ antagonist, were examined in isolated guinea pig and rabbit hearts using an electrophysiological technique. SD-3211 (10 -6-10-5 M) shortened the action potential duration of guinea pig papillary muscles in a concentration-dependent manner without affecting the resting potential or the maximum upstroke velocity (&OV0622; max). The &OV0622; max of slow responses induced by high extracellular K+ and isoproterenol was inhibited by SD-3211 at concentrations of >10-6 M. Elevation of extracellular Ca2+ by 2 m M reversed this inhibited response. The inhibitory effect of SD-3211 on the slow response was enhanced as the stimulation frequency was increased. In Langendorff-perfused rabbit hearts electrically driven at 2.0 Hz, SD-3211 (10 -8-10-6 M) produced a concentration-dependent prolongation of the atrium- His bundle conduction time (A-H interval) as well as a reduction in the developed tension of ventricular muscle, whereas SD-3211 did not affect the His bundle-ventricular conduction time (A-H interval) significantly. The potency of SD-3211 in A-H prolongation was greater than those of diltiazem and bepridil, but weaker than those of nicardipine, nifedipine, and verapamil. The effect of SD-3211 on the A-H interval was more pronounced at higher stimulation frequencies. SD-3211 was intermediate between nicardipine, nifedipine, and verapamil in its intensity of frequency- dependent effects on the A-H interval. These results suggest that SD-3211 has a preferential and frequency- dependent inhibitory action on cardiac slow Ca2 + channels

SD-3211, a novel benzothiazine calcium antagonist, alone and in combination with a beta-adrenoceptor antagonist, produces antihypertensive effects without affecting heart rate and atrioventricular conduction in conscious renal hypertensive dogs.

We compared the effects of SD-3211, a novel calcuim antagonist, on blood pressure, heart rate, and atrioventricular conduction with those of diltiazen usinng conscius renal hypertensice dogs (one-kidney, one-clip type). We also examined the combined effects of these calcium antagonists with a beta-adrenceptor antagonist. propranolo, on these variabes. Oral administration of SD-3211 (1.25. 2.5 and 5 mg/kg) resulted ion a dosedependent decrease in blood pressure without affecting heart rate. SD-3211 at all three doses significantly decreased systolic blood pressure. At 2.5 and 5 mg/kg the compound elicited significant decreases in mean blood presure and diastolic blood pressure. Hypotension obtained with the highest dose of SD-3211 lasted for at least 9 h. No significant alteration in PR interval was observed in electrocardiograms after administration of SD-3211. Diltiazem, given orally at doses of 2.5 and 5 mg/kg but not 1.25 mg/kg, produced significant hypotension with little change in heart rate. The duration of hypotension induced by the highest dose of dilitiazem was only 3 h. Diltiazem prolonged PR interval in a dose-dependent manner, causing second-degree atrioventricular block in some dogs. Combined adminstration of SD-3211 or diltiazem (2.5 mg/kg) with propranolol (30 mg/kg) resulted in enhanced hypotension with no alteration in heart rate. SD-3211 plus prolpranolol had little effect on the PR interval, whereas diltiazem plus propranlol caused a markedly enhanced prolongation. These results indicate that SD-3211 is an antihypertensive agent with long-lasting action and little effect on heart rate and atrioventricular conduction with propranolol. may be useful in the treatment of hypertension.

Cardiovascular characterization of SD-3211, a novel benzothiazine calcium channel blocker, in isolated rabbit hearts

The cardiovascular effects of SD-3211, a novel benzothiazine Ca++ channel blocker, were compared with those of diltiazem and nicardipine in Langendorff-perfused rabbit hearts. SD-3211 was more potent in increasing coronary artery flow than in depressing cardiac function (i.e., contractile force, heart rate and conduction time). The relative specificity of SD-3211 for coronary vasodilation to cardiodepression was clearly greater than that of diltiazem, but less than that of nicardipine. Thus, the present study demonstrates that SD-3211, despite a non-dihydropyridine type of Ca++ channel blocker, can be characterized as a potent coronary vasodilator with a little effect on cardiac function.

Dual effects of interleukin-1β on N-methyl-d-aspartate-induced retinal neuronal death in rat eyes

In this study we determine if interleukin-1beta (IL-1beta) modulates N-methyl-D-aspartate (NMDA)-induced retinal damage. Sprague-Dawley rats were anesthetized with inhalation of halothane, after which a single injection of 5 microl of IL-1beta (0.1 to 10 ng/eye) (and/or IL-1 receptor antagonist (IL-1ra)) for experimental eyes was administered. Two days later (or simultaneously), NMDA (20 nmol) was injected into the vitreous space. One week later, each eye was enucleated and transverse sections were subjected to morphometric analysis. Enzyme-linked immunosorbent assay (ELISA) was conducted for the determination of IL-1beta levels in retina. Immunohistochemical and immunoblot studies were also performed. In eyes that received an intravitreal injection of IL-1beta (0.1 to 10 ng/eye), significant thinning of the inner plexiform layer (IPL) was observed (P<0.05). Immunohistochemical and ELISA studies demonstrated upregulated expression of IL-1beta in retinas that had undergone NMDA injection. Treatment with 10 ng of IL-1ra induced a protective effect against NMDA-induced retinal damage. Pretreatment with IL-1beta induced a significant protective effect on NMDA-induced retinal damage. Our studies suggest that IL-1beta induces neuronal cell death directly, as shown by the protective effects of IL-1ra, but has a protective effect on NMDA-induced retinal damage indirectly after an incubation time of at least 2 days.

Synthesis of azido derivatives of semotiadil, a novel 1,4-benzothiazine calcium antagonist, for photoaffinity probes of calcium channels

Abstract Aliphatic and aromatic azido derivatives of semotiadil ( 1 ), a novel calcium antagonist with a 1,4-benzothiazine skeleton, were synthesized for developing photoaffinity probes of L type calcium channels. The azidophenoxy derivative 12 proved to be a potent calcium antagonist and its [ 3 H]-labeled compound 16 would be a useful tool to clarify the binding sites of 1 to the calcium channels.

Antihypertensive effect of a novel calcium antagonist, SD-3211, in experimental hypertensive rats.

The antihypertensive effect of SD-3211. a structurally novel type of nondihydropyridine calcium antagonist, was assessed using several types of experimental hypertensive rats. Oral administration of SD-3211 (10. 20, and 30 mg/kg) to conscious spontaneously hypertensive rats (SHR). deoxycorticosterone acetate-salt hypertensive rats (DHR) and 2-kidney, 1-clip renal hypertensive rats (RHR) resulted in a dose-dependent decrease in systolic blood pressure (SBP). The hypotensive effect of SD-3211 in these hypertensive rats was more pronounced than in normotensive rats (NR). The potencies of SD-3211 for the hypotensive effect in the hypertensive rats and NR were 5–7 times greater than that of diltiazem but 2–3 times less than that of nicardipine. Furthermore, SD-3211 showed longer-lasting hypotensive action than diltiazem and nicardipine, at the respective equihypotensive dose. During the course of hypotension. SD-3211 did not exert any influence on heart rate (HR) in any type of hypertensive rats or NR, in contrast to the appearance of tachycardia with nicardipine in SHR, DHR, and NR and of bradycardia with diltiazem in DHR. At doses of 10 and 30 mg/kg, the hypotensive doses; SD-3211 elicited a dose dependent natriuresis but no kaliuresisin SHR. In the chronic study using SHR. SD-3211 at 10 mg/kg/day showed an antihypertensive effect during anadministration period of 12 consecutive weeks. These results allow us to conclude that SD-3211 has a potent and long-lasting hypotensive action with little cardiac effect.

Brain-Derived Neurotrophic Factor Inhibits Changes in Soma-Size of Retinal Ganglion Cells Following Optic Nerve Axotomy in Rats

To determine if optic nerve axotomy affects the cell soma size of retinal ganglion cells and to establish whether such quantitative analysis is useful as a new way of evaluating retinal ganglion cell damage, we measured the changes in both the number and soma size of retinal ganglion cells after optic nerve axotomy in rats. Retinal ganglion cells were retrogradely labeled by fluoro-gold injection into the superior colliculus, and the soma size was measured using image-analysis software. We detected a decrease in the proportion of large-sized retinal ganglion cells that was significant at 3, 5 and 7 days after the axotomy, and an increased proportion of small-sized ones that was significant at 5 and 7 days after the axotomy, indicating that retinal ganglion cells shrank following axotomy, that there was a shift away from the largest category of retinal ganglion cells towards the smallest category. On days 3 and 5 post-axotomy, there was no significant change in the proportion of medium-sized retinal ganglion cells. Intravitreal injection of brain-derived neurotrophic factor one hour before the axotomy significant inhibited the increase in the proportion of small-sized retinal ganglion cells otherwise seen at 3 days after the axotomy. These results may suggest that larger retinal ganglion cells are more sensitive to optic nerve axotomy than small- and medium-sized ones, and that a quantitative analysis of soma size is a useful way of detecting retinal ganglion cell damage in the early phase after axotomy.

Electrophysiologic effects of a novel Ca antagonist, SA2572, in isolated rabbit and guinea pig hearts

Cardiac effects of SA2572, a newly synthesized Ca antagonist, were evaluated in guinea pig and rabbit hearts with electrophysiologic technique. SA2572(10−6–10−5 M) decreased the upstroke velocity (&OV0312;max) and the duration of the action potential (APD30) in guinea pig papillary muscles in a concentration- and frequency-dependent manner without affecting the resting potential. The slow responses (high K+ + isoproterenol) were suppressed by SA2572 at 10−6 M. In rabbit sinus node, SA2572(10−7–5 × 10−6 M) caused a concentration-dependent decrease in the amplitude and &OV0312;max of the action potential and tended to prolong the spontaneous cycle length. In Langendorff-perfused rabbit hearts electrically driven at 2 Hz, SA2572(5 × 10−8–10−6 M) produced concentration-dependent prolongations of the atrio-His bundle conduction time (A-H interval) and the His bundle-ventricular conduction time (H-V interval) concomitantly with a reduction of the developed tension of the ventricular muscle. These effects of SA2572 on the A-H and H-V intervals were more pronounced at higher stimulation frequency. Enantiospecificity was observed in these actions of SA2572, (–)-isomer of SA2572 having more potent inhibitory effects on slow channel-dependent than on fast channel-dependent phenomena. These results indicate that SA2572 has characteristics of both slow and fast channel blockers, and that these inhibitory effects are frequency dependent.

ANTI‐ISCHAEMIC AND VASOSPASMOLYTIC EFFECTS OF A NOVEL Ca2+ CHANNEL BLOCKER, SD‐3211, IN VITRO

1. The present study was undertaken to determine the vasospasmolytic activity of a novel non‐dihydropyridine type of Ca2+ channel blocker, SD‐3211, in isolated canine coronary arteries and its ability to reduce myocardial ischaemic damage in isolated perfused rabbit hearts.