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.
Tsumamoto
Yuichi
Y
Department of Ophthalmology, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
Yamashita
Keisuke
K
Takumida
Masaya
M
Okada
Koji
K
Mukai
Satoshi
S
Shinya
Makoto
M
Yamashita
Hidetoshi
H
Mishima
Hiromu K
HK
D000375:Aging / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D000918:Antibody Specificity; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005333:Fetus; D019793:Fluorescein / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D007150:Immunohistochemistry; D007202:Indicators and Reagents / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D016900:Neurofilament Proteins / Q000378:metabolism; D026602:Nitrergic Neurons / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011247:Pregnancy; D051381:Rats; D017208:Rats, Wistar; D012160:Retina / Q000196:embryology / Q000201:enzymology* / Q000254:growth & development; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D013569:Synapses / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure
C414689:4,5-diaminofluorescein diacetate; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D007527:Isoenzymes; D016900:Neurofilament Proteins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D019793:Fluorescein