Yuichi Tsumamoto

In situ localization of nitric oxide synthase and direct evidence of NO production in rat retinal ganglion cells

The expression of isoforms of nitric oxide synthase (NOS), enzymes responsible for NO production, and the synthesis of nitric oxide (NO) in rat retinal ganglion cells (RGCs) during synaptogenesis for various phases of the pre- and postnatal developmental periods were investigated. The retinas from prenatal, lactating, young, and adult rats were fixed in paraformaldehyde. The cryosections or paraformaldehyde-fixed ganglion cells purified from rat pups were immunostained for constitutive isoforms of NOS (n and eNOS) and observed with a confocal laser scanning microscope. Synthesis of NO in the RGCs was achieved by in vitro stimulation with glutamate. The intracellular NO levels were measured in real time using diaminofluorescein-2 diacetate, a fluorescence indicator of NO. Immunohistochemical analysis revealed nNOS and eNOS expressed in retinal ganglion cells during the first 2 postnatal weeks. Cultured RGCs also expressed nNOS and eNOS in vitro. Intracellular NO levels in cultured RGCs showed spontaneous fluctuation during a 20-min observation. The presence of both a non-specific NOS inhibitor, L-NAME, and a specific nNOS inhibitor, 7-NI, significantly inhibited (P<0.001) the increase of intracellular NO 6 and 8 min after the introduction of L-arginine and glutamate to the medium. This study revealed that all constitutive NOS isoforms are expressed in RGCs and demonstrated that NO is produced by nNOS mainly through stimulation by glutamate in cultured RGCs.

Differences in nitric oxide production: a comparison of retinal ganglion cells and retinal glial cells cultured under hypoxic conditions

The aim of this study was to compare the effects of hypoxia on nitric oxide synthase (NOS) expression and the production of NO between isolated retinal ganglion cells (RGCs) and retinal glial cells. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to examine the presence of neuronal NOS mRNA, inducible NOS mRNA, and endothelial NOS mRNAs in the two cell types. RGCs and retinal glial cells were separately cultured under hypoxic (10% O(2)) or control (20% O(2)) conditions. Changes in NOS-mRNA expression were quantified by real-time PCR, and nitrite in the medium was measured up to 96 h of culture. The effects of non-NOS- and iNOS-selective inhibitors on hypoxia-induced release of nitrite in the culture medium were evaluated. RT-PCR revealed the presence of three types of NOSs in the two types of cultured cells. Hypoxic culture conditions significantly changed the expression of all NOS mRNAs in retinal glial cells but not in RGCs. NO production showed significant changes corresponding to those of NOS mRNAs in retinal glial cells but not in RGCs, and both NOS inhibitors significantly reduced hypoxia-induced nitrite release in retinal glial cells. Retinal glial cells but not RGCs may be the major source of NO under hypoxic conditions.

Additive effect of bunazosin on intraocular pressure when topically added to treatment with latanoprost in patients with glaucoma

PURPOSE To investigate whether an alpha-1 blocker, bunazosin, has an additive effect on lowering intraocular pressure (IOP) when topically added to latanoprost treatment in patients with glaucoma. METHODS Bunazosin twice a day was added topically to the treatment for 12 patients with glaucoma who had been instilling latanoprost once a day for more than 1 month. IOP was measured and adverse events were checked 2, 4 and 8 weeks after the addition of bunazosin to their treatment. RESULTS One of the 12 patients dropped out in the course of the study. Therefore, 11 patients were included for the analysis of IOP, and 12 for the analysis of adverse events. IOPs were decreased significantly (P=.008, Wilcoxon signed rank test) from 18.2+/-3.4 mm Hg to 16.6+/-3.5 mm Hg 8 weeks after the addition of bunazosin. Adverse events were seen in 5 of the 12 patients. CONCLUSION Bunazosin has an additive effect on lowering IOP when topically added to latanoprost treatment in glaucoma patients.