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.
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.
Ikeda
M
M
Advanced research Institute for Science and Engineering, Waseda University, Tokyo, Japan. msikeda@aol.com
Sagara
M
M
Sekino
Y
Y
Shirao
T
T
Honda
K
K
Yoshioka
T
T
Allen
C N
CN
Inoué
S
S
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
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