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Localization and Characterization of Nitric Oxide Synthase in the Rat Suprachiasmatic Nucleus: Evidence for a Nitrergic Plexus in the Biological Clock
Author(s) -
Chen Dong,
Hurst William J.,
Ding Jian M.,
Faiman Lia E.,
Mayer Bernd,
Gillette Martha U.
Publication year - 1997
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1997.68020855.x
Subject(s) - suprachiasmatic nucleus , nitric oxide synthase , citrulline , immunocytochemistry , nitric oxide , medicine , endocrinology , biology , chemistry , gene isoform , arginine , biochemistry , central nervous system , amino acid , gene
Behavioral and electrophysiological evidence indicates that the biological clock in the hypothalamic suprachiasmatic nuclei (SCN) can be reset at night through release of glutamate from the retinohypothalamic tract and subsequent activation of nitric oxide synthase (NOS). However, previous studies using NADPH‐diaphorase staining or immunocytochemistry to localize NOS found either no or only a few positive cells in the SCN. By monitoring conversion of l ‐[ 3 H]arginine to l ‐[ 3 H]citrulline, this study demonstrates that extracts of SCN tissue exhibit NOS specific activity comparable to that of rat cerebellum. The enzymatic reaction requires the presence of NADPH and is Ca 2+ /calmodulin‐dependent. To distinguish the neuronal isoform (nNOS; type I) from the endothelial isoform (type III), the enzyme activity was assayed over a range of pH values. The optimal pH for the reaction was 6.7, a characteristic value for nNOS. No difference in nNOS levels was seen between SCN collected in day versus night, either by western blot or by enzyme activity measurement. Confocal microscopy revealed for the first time a dense plexus of cell processes stained for nNOS. These data demonstrate that neuronal fibers within the rat SCN express abundant nNOS and that the level of the enzyme does not vary temporally. The distribution and quantity of nNOS support a prominent regulatory role for this nitrergic component in the SCN.