title: The sulphydryl reagent, N-ethylmaleimide, disrupts sleep and blocks A1 adenosine receptor-mediated inhibition of intracellular calcium signaling in the in vitro ventromedial preoptic nucleus.
abstract: To explore the neuronal signaling mechanisms underlying sleep regulation in the rat, the present study examined continuous intra-third ventricle infusion of N-ethylmaleimide (NEM), a sulphydryl reagent that inhibits G(i/o) protein-coupled receptor-mediated signaling pathways. The diurnal infusion of NEM (0.01-10 micromol/10 h) dose-dependently inhibited both non-rapid eye movement sleep and rapid eye movement sleep. A maximal dose of NEM (10 micromol/10 h) dramatically inhibited day-time sleep (-57% for non-rapid eye movement sleep and -89% for rapid eye movement sleep) with a compensatory increase of sleep during the subsequent night-time (+33% for non-rapid eye movement sleep and +259% for rapid eye movement sleep). The day-time brain temperature was also increased by NEM, demonstrating effects of NEM on both sleep and body temperature levels. Immunostaining of the rat hypothalamus with a monoclonal antibody against the A1 adenosine receptor (A1R) was used to explore the distribution of a sleep-related G(i/o) protein-coupled receptor. Robust A1R-like immunoreactivity was found in the ventromedial preoptic nucleus and the supraoptic nucleus. Fura-2-based Ca(2+) imaging analysis of acute hypothalamic slices further demonstrated that the A1R agonist N(6)-cyclopentyladenosine (CPA; 200 nM) inhibited spontaneous Ca(2+) oscillations and high potassium (80 mM)-induced Ca(2+) flux in the ventromedial preoptic nucleus, while NEM (100-300 microM) and an A1R antagonist 8-cyclopentyl-dipropylxanthine (300 nM) blocked the CPA actions and increased the high potassium-induced Ca(2+) flux. From these results we suggest that NEM-sensitive G protein-coupled receptor(s) may play an important role in the regulation of sleep and body temperature in the rat and one possible mechanism is an A1R-mediated regulation of intracellular Ca(2+) concentrations in the ventromedial preoptic nucleus.
authors.affiliation: Advanced research Institute for Science and Engineering, Waseda University, Tokyo, Japan. msikeda@aol.com
authors.forename: M
authors.initials: M
authors.lastname: Ikeda
authors.forename: M
authors.initials: M
authors.lastname: Sagara
authors.forename: Y
authors.initials: Y
authors.lastname: Sekino
authors.forename: T
authors.initials: T
authors.lastname: Shirao
authors.forename: K
authors.initials: K
authors.lastname: Honda
authors.forename: T
authors.initials: T
authors.lastname: Yoshioka
authors.forename: C N
authors.initials: CN
authors.lastname: Allen
authors.forename: S
authors.initials: S
authors.lastname: Inoué
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001831:Body Temperature / Q000187:drug effects / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002940:Circadian Rhythm / Q000187:drug effects / Q000502:physiology; D005033:Ethylmaleimide / Q000494:pharmacology*; D016257:Fura-2; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D018698:Glutamic Acid / Q000494:pharmacology; D007031:Hypothalamus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007150:Immunohistochemistry; D008297:Male; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D011301:Preoptic Area / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018047:Receptors, Purinergic P1 / Q000187:drug effects / Q000378:metabolism*; D015290:Second Messenger Systems / Q000187:drug effects / Q000502:physiology*; D012890:Sleep / Q000187:drug effects / Q000502:physiology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D013779:Tetrodotoxin / Q000494:pharmacology; D014970:Xanthines / Q000494:pharmacology
chemical_list: D015220:Calcium Channels; D018047:Receptors, Purinergic P1; D013439:Sulfhydryl Reagents; D014970:Xanthines; D018698:Glutamic Acid; C048599:N(6)-cyclopentyladenosine; D013779:Tetrodotoxin; C051360:1,3-dipropyl-8-cyclopentylxanthine; D019204:GTP-Binding Proteins; D000241:Adenosine; D005033:Ethylmaleimide; D011188:Potassium; D002118:Calcium; D016257:Fura-2