Masaaki Kageyama

Three susceptible loci associated with primary open-angle glaucoma identified by genome-wide association study in a Japanese population

Primary open-angle glaucoma (POAG) is the major type of glaucoma. To discover genetic markers associated with POAG, we examined a total of 1,575 Japanese subjects in a genome-wide association study (stage 1) and a subsequent study (stage 2). Both studies were carried out at a single institution. In the stage 1 association study, we compared SNPs between 418 POAG patients and 300 control subjects. First, low-quality data were eliminated by a stringent filter, and 331,838 autosomal SNPs were selected for analysis. Poorly clustered SNPs were eliminated by a visual assessment, leaving 255 that showed a significant deviation (P < 0.001) in the allele frequency comparison. In the stage 2 analysis, we tested these 255 SNPs for association in DNA samples from a separate group of 409 POAG and 448 control subjects. High-quality genotype data were selected and used to calculate the combined P values of stages 1 and 2 by the Mantel–Haenszel test. These analyses yielded 6 SNPs with P < 0.0001. All 6 SNPs showed a significant association (P < 0.05) in stage 2, demonstrating a confirmed association with POAG. Although we could not link the SNPs to the annotated gene(s), it turned out that we have identified 3 genetic loci probably associated with POAG. These findings would provide the foundation for future studies to build on, such as for the metaanalysis, to reveal the molecular mechanism of the POAG pathogenesis.

Common variants in CDKN2B-AS1 associated with optic-nerve vulnerability of glaucoma identified by genome-wide association studies in Japanese.

Background To date, only a small portion of the genetic variation for primary open-angle glaucoma (POAG), the major type of glaucoma, has been elucidated. Methods and Principal Findings We examined our two data sets of the genome-wide association studies (GWAS) derived from a total of 2,219 Japanese subjects. First, we performed a GWAS by analyzing 653,519 autosomal common single-nucleotide polymorphisms (SNPs) in 833 POAG patients and 686 controls. As a result, five variants that passed the Bonferroni correction were identified in CDKN2B-AS1 on chromosome 9p21.3, which was already reported to be a significant locus in the Caucasian population. Moreover, we combined the data set with our previous GWAS data set derived from 411 POAG patients and 289 controls by the Mantel-Haenszel test, and all of the combined variants showed stronger association with POAG (P<5.8×10−10). We then subdivided the case groups into two subtypes based on the value of intraocular pressure (IOP)—POAG with high IOP (high pressure glaucoma, HPG) and that with normal IOP (normal pressure glaucoma, NPG)—and performed the GWAS using the two data sets, as the prevalence of NPG in Japanese is much higher than in Caucasians. The results suggested that the variants from the same CDKN2B-AS1 locus were likely to be significant for NPG patients. Conclusions and Significance In this study, we successfully identified POAG-associated variants in the CDKN2B-AS1 locus using a Japanese population, i.e., variants originally reported as being associated with the Caucasian population. Although we cannot rule out that the significance could be due to the differences in sample size between HPG and NPG, the variants could be associated specifically with the vulnerability of the optic nerve to IOP, which is useful for investigating the etiology of glaucoma.

Selective prostaglandin D2 receptor stimulation elicits ocular hypotensive effects in rabbits and cats

The effects of the selective prostaglandin D2 (DP) receptor agonists, 572C85 ((+/-)-5-(3-carboxypropylthio)-1-(2-cyclohexyl-2-hydroxyethyl- amino)hexahydrocyclopenta(d)imidazol-2(1H)-one) and 192C86 ((+/-)-5-(3-carboxypropylthio)-1-(2-cyclohexyl-2-hydroxyethylidene - amino)-3-ethylhexahydrocyclopenta(d)imidazol-2(1H)-one), were determined on intraocular pressure regulation in rabbits and cats. 572C85 (50 micrograms) in rabbits maximally lowered intraocular pressure by 4.3 mm Hg, and significantly for 4 h compared to control. In cats 572C85 had a similar effect. 192C86 (50 micrograms) reduced intraocular pressure by 2.8 mm Hg for 2 h in rabbits. Following exposure to the specific DP receptor antagonist, BW A868C ((+/-)-3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2-hydroxyethylamin o)- hydantoin; 50 micrograms), which had no effect on intraocular pressure by itself, 572C85 (50 micrograms) did not reduce intraocular pressure in rabbits and cats. The intraocular pressure lowering effect of the mixed DP and EP receptor agonist, BW245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)-hydantoin; 50 micrograms), in cats was suppressed by only 64% by BW A868C (50 micrograms). These results clearly show that the DP receptors in rabbit and cat eyes are involved in intraocular pressure regulation. However, under baseline conditions DP receptor activity does not contribute to this regulation.

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.

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.

Involvement of both L- and N-type voltage-dependent Ca2+ channels in KCl- and veratridine-evoked transmitter release from non-adrenergic, non-cholinergic nerves in the rabbit iris sphincter muscle

Abstract To determine which types of voltage-dependent Ca2+ channels mediate tachykinin release in the isolated rabbit iris sphincter muscle, we examined the effects of several Ca2+ channel modulators on contractions induced by either an elevation of the extracellular KCl concentration or application of the Na+ channel activator veratridine. Contractions caused by either 45.9mMKCl or veratridine (10μM) were inhibited by spantide (10μM), a tachykinin receptor antagonist, and capsaicin (10μM), a tachykinin-depleting agent, but were not changed by atropine. Nicardipine, an L-type Ca2+ channel blocker, inhibited contractions induced by KCl and veratridine in a concentration-dependent manner. ω-Conotoxin GVIA (1μM), an N-type Ca2+ channel blocker, inhibited only contractions induced by lower concentrations of KCl, both when applied alone and when combined with nicardipine. Bay K8644, an L-type Ca2+ channel activator, caused a spantide- and nicardipine-sensitive contraction in muscles partially depolarized with 15.9mMKCl, and enhanced contractions induced by 15.9mMKCl and veratridine (2μM). ω-Agatoxin IVA (0.3μM), a P-type voltage-dependent Ca2+ channel blocker, did not affect contractions induced by either KCl or veratridine. Contractions induced by exogenous substance P were not modified by any of the Ca2+ channel blockers or by Bay K8644. These results suggest that, in the rabbit iris sphincter muscle, L- and N-type voltage-dependent Ca2+ channels are involved in neurotransmitter release from tachykininergic nerves elicited by high KCl and by veratridine.

Novel common variants and susceptible haplotype for exfoliation glaucoma specific to Asian population

The common variants in lysyl oxidase-like 1 gene (LOXL1) are associated with exfoliation glaucoma (XFG) patients developed through exfoliation syndrome (XFS). However, the risk allele of a variant in LOXL1 has been found to be inverted between Asian and Caucasian populations. Therefore, we newly performed a genome-wide association study using 201 XFS/XFG and 697 controls in Japanese, and identified 34 genome-wide significant single-nucleotide polymorphisms (SNPs) distributing in not only LOXL1 but also TBC1D21 and PML at the 15q24.1 locus. These SNPs were confirmed by an independent population consisted of 121 XFS/XFG and 263 controls in Japanese. Moreover, further analyses revealed a unique haplotype structure only from the combination of TBC1D21 and LOXL1 variants showing a high XFS/XFG susceptibility specific for the Asian population. Although there still should be other gene(s) in the other region(s) contributing to the disease process, these results suggested that the combination of newly discovered variants in these genes might be useful for precise XFG risk assessment, as well as for elucidating the molecular mechanism of XFG pathogenesis through XFS.

Effects of the immunosuppressant cyclosporin A on neurotransmitter release from peripheral non-adrenergic, non-cholinergic nerves

Abstract This study was undertaken to determine the effect of the immunosuppressant cyclosporin A on neurotransmitter release from non-adrenergic, non-cholinergic nerves (tachykininergic nerves) in the rabbit iris sphincter muscle. Cumulative application of cyclosporin A (0.1 to 10 μM) caused a slow onset of contraction in a concentration-dependent manner. Both FK888 (1 μM) and capsaicin (10 μM), a substance P receptor antagonist and a substance P-depleting agent, respectively, inhibited the contractile effect of cyclosporin A, whereas atropine (1 μM) had no effect. Both cyclosporin A and capsaicin (10 μM) stimulated the release of substance P-like immunoreactivity in the iris. Neither the sodium channel blocker tetrodotoxin (1 μM), the N-type voltage-dependent Ca2+ channel blocker ω-conotoxin GVIA (1 μM) nor the P-type channel blocker ω-agatoxin IVA (0.2 μM) affected cyclosporin A (1 μM)-induced contraction. In contrast, the L-type Ca2+ channel blocker nicardipine (10 μM) inhibited this contractile effect. These results suggest that cyclosporin A stimulates substance P-like tachykinin release by activating L-type voltage-dependent Ca2+ channels, resulting in contraction of the rabbit iris sphincter muscle.

Inhibitory effect of nisoldipine on angiotensin-II-induced renal actions in anesthetized dogs.

Renal effects of nisoldipine, a potent calcium channel blocker, were examined in anesthetized dogs. In-trarenal arterial infusion of nisoldipine (2, 10, and 50 ng/kg per min) had no effect on mean systemic blood pressure and heart rate and there was no significant change in renal hemodynamics during infusion of various doses of the drug. Urine flow and urinary excretions of sodium, chloride, and potassium were increased by nisoldipine in a dose-related manner. Fractional excretions of sodium and chloride were markedly elevated with the highest dose given thereby indicating that tubular reab-sorptions of sodium and chloride were inhibited by nisoldipine. Nisoldipine (50 ng/kg per min) abolished the decreasing effects of angiotensin-II on glomerular filtration rate, urine flow, and urinary excretion of electrolytes but not the decrease in renal blood flow by the peptide. Angiotensin-II-induced reduction of fractional excretion of electrolytes was completely blocked by nisoldipine. Renal responses to norepinephrine were unaffected by nisoldipine. Thus, nisoldipine administered intrarenally to anesthetized dogs exerts a diuretic action by way of tubular effects, as is the case with other dihydropyridine calcium channel blockers. Nisoldipine seems to effectively antagonize the renal response to angiotensin-II. Thus, the preferential inhibition of angiotensin-II-in-duced antidiuresis may mean that nisoldipine interferes with stimulatory effects of angiotensin-II on the renal tubular reabsorption of electrolytes and water.

The different effects of exogenous and neuronally released norepinephrine on renin release in rat kidney cortical slices

The effects of veratrine on renin release from rat kidney cortical slices were compared with those of norepinephrine (NE). Veratrine (10-100 micrograms/ml) produced a concentration-dependent increase in renin release. This action was markedly attenuated by propranolol but not influenced by prazosin. On the other hand, relatively higher concentrations of NE produced an inhibition of renin release and the inhibitory action was reversed by prazosin. These results suggest that exogenous NE inhibits renin release by preferential activation of alpha 1-adrenoceptors while neuronally released NE stimulates renin release by exclusive activation of beta-adrenoceptors.