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

M. Ikeda; M. Sagara; Y. Sekino; T. Shirao; K. Honda; T. Yoshioka; C. N. Allen; S. Inoué

doi:10.1016/S0306-4522(01)00290-1

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

M. Ikeda, M. Sagara, Y. Sekino, T. Shirao, K. Honda, T. Yoshioka, C. N. Allen, S. Inoué

Oregon Institute of Occupational Health Sciences

Research output: Contribution to journal › Article › peer-review

12 Scopus citations

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 μmol/10 h) dose-dependently inhibited both non-rapid eye movement sleep and rapid eye movement sleep. A maximal dose of NEM (10 μmol/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 Ca²⁺ imaging analysis of acute hypothalamic slices further demonstrated that the A1R agonist N⁶-cyclopentyladenosine (CPA; 200 nM) inhibited spontaneous Ca²⁺ oscillations and high potassium (80 mM)-induced Ca²⁺ flux in the ventromedial preoptic nucleus, while NEM (100-300 μM) and an A1R antagonist 8-cyclopentyl-dipropylxanthine (300 nM) blocked the CPA actions and increased the high potassium-induced Ca²⁺ 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 Ca²⁺ concentrations in the ventromedial preoptic nucleus.

Original language	English (US)
Pages (from-to)	733-743
Number of pages	11
Journal	Neuroscience
Volume	106
Issue number	4
DOIs	https://doi.org/10.1016/S0306-4522(01)00290-1
State	Published - Oct 31 2001

Keywords

Ca imaging
Fura-2
GTP-binding protein
Immunostaining
Intracerebroventricular infusion
Voltage-sensitive Ca channels

ASJC Scopus subject areas

Neuroscience(all)

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10.1016/S0306-4522(01)00290-1

Cite this

Ikeda, M., Sagara, M., Sekino, Y., Shirao, T., Honda, K., Yoshioka, T., Allen, C. N., & Inoué, S. (2001). 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. Neuroscience, 106(4), 733-743. https://doi.org/10.1016/S0306-4522(01)00290-1

Ikeda, M, Sagara, M, Sekino, Y, Shirao, T, Honda, K, Yoshioka, T, Allen, CN & Inoué, S 2001, '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', Neuroscience, vol. 106, no. 4, pp. 733-743. https://doi.org/10.1016/S0306-4522(01)00290-1

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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 Gi/o protein-coupled receptor-mediated signaling pathways. The diurnal infusion of NEM (0.01-10 μmol/10 h) dose-dependently inhibited both non-rapid eye movement sleep and rapid eye movement sleep. A maximal dose of NEM (10 μmol/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 Gi/o protein-coupled receptor. Robust A1R-like immunoreactivity was found in the ventromedial preoptic nucleus and the supraoptic nucleus. Fura-2-based Ca2+ imaging analysis of acute hypothalamic slices further demonstrated that the A1R agonist N6-cyclopentyladenosine (CPA; 200 nM) inhibited spontaneous Ca2+ oscillations and high potassium (80 mM)-induced Ca2+ flux in the ventromedial preoptic nucleus, while NEM (100-300 μM) and an A1R antagonist 8-cyclopentyl-dipropylxanthine (300 nM) blocked the CPA actions and increased the high potassium-induced Ca2+ 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 Ca2+ concentrations in the ventromedial preoptic nucleus.",

keywords = "Ca imaging, Fura-2, GTP-binding protein, Immunostaining, Intracerebroventricular infusion, Voltage-sensitive Ca channels",

author = "M. Ikeda and M. Sagara and Y. Sekino and T. Shirao and K. Honda and T. Yoshioka and Allen, {C. N.} and S. Inou{\'e}",

note = "Funding Information: We thank Drs. S. Tanaka and Y. Ren (Gunma University) for their excellent technical assistance with the immunostaining assays. We also thank Dr. J. Noguchi (University of Occupational and Environmental Health) for his helpful comments on this manuscript. This work is supported in part by a grant from {\textquoteleft}Research for the Future (RFTF){\textquoteright} program 96L00310 from Japan Society for the Promotion of Science to T.Y., a Grant from the NIH (NS036607) to C.N.A., a grant from Ono pharmaceutical company to C.N.A., M.I., and T.Y., and a Special Coordination Fund for Promoting Science and Technology from the Science and Technology Agency, Prime Minister{\textquoteright}s Office to S.I.",

year = "2001",

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doi = "10.1016/S0306-4522(01)00290-1",

language = "English (US)",

volume = "106",

pages = "733--743",

journal = "Neuroscience",

issn = "0306-4522",

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TY - JOUR

T1 - 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

AU - Ikeda, M.

AU - Sagara, M.

AU - Sekino, Y.

AU - Shirao, T.

AU - Honda, K.

AU - Yoshioka, T.

AU - Allen, C. N.

AU - Inoué, S.

N1 - Funding Information: We thank Drs. S. Tanaka and Y. Ren (Gunma University) for their excellent technical assistance with the immunostaining assays. We also thank Dr. J. Noguchi (University of Occupational and Environmental Health) for his helpful comments on this manuscript. This work is supported in part by a grant from ‘Research for the Future (RFTF)’ program 96L00310 from Japan Society for the Promotion of Science to T.Y., a Grant from the NIH (NS036607) to C.N.A., a grant from Ono pharmaceutical company to C.N.A., M.I., and T.Y., and a Special Coordination Fund for Promoting Science and Technology from the Science and Technology Agency, Prime Minister’s Office to S.I.

PY - 2001/10/31

Y1 - 2001/10/31

N2 - 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 Gi/o protein-coupled receptor-mediated signaling pathways. The diurnal infusion of NEM (0.01-10 μmol/10 h) dose-dependently inhibited both non-rapid eye movement sleep and rapid eye movement sleep. A maximal dose of NEM (10 μmol/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 Gi/o protein-coupled receptor. Robust A1R-like immunoreactivity was found in the ventromedial preoptic nucleus and the supraoptic nucleus. Fura-2-based Ca2+ imaging analysis of acute hypothalamic slices further demonstrated that the A1R agonist N6-cyclopentyladenosine (CPA; 200 nM) inhibited spontaneous Ca2+ oscillations and high potassium (80 mM)-induced Ca2+ flux in the ventromedial preoptic nucleus, while NEM (100-300 μM) and an A1R antagonist 8-cyclopentyl-dipropylxanthine (300 nM) blocked the CPA actions and increased the high potassium-induced Ca2+ 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 Ca2+ concentrations in the ventromedial preoptic nucleus.

AB - 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 Gi/o protein-coupled receptor-mediated signaling pathways. The diurnal infusion of NEM (0.01-10 μmol/10 h) dose-dependently inhibited both non-rapid eye movement sleep and rapid eye movement sleep. A maximal dose of NEM (10 μmol/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 Gi/o protein-coupled receptor. Robust A1R-like immunoreactivity was found in the ventromedial preoptic nucleus and the supraoptic nucleus. Fura-2-based Ca2+ imaging analysis of acute hypothalamic slices further demonstrated that the A1R agonist N6-cyclopentyladenosine (CPA; 200 nM) inhibited spontaneous Ca2+ oscillations and high potassium (80 mM)-induced Ca2+ flux in the ventromedial preoptic nucleus, while NEM (100-300 μM) and an A1R antagonist 8-cyclopentyl-dipropylxanthine (300 nM) blocked the CPA actions and increased the high potassium-induced Ca2+ 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 Ca2+ concentrations in the ventromedial preoptic nucleus.

KW - Ca imaging

KW - Fura-2

KW - GTP-binding protein

KW - Immunostaining

KW - Intracerebroventricular infusion

KW - Voltage-sensitive Ca channels

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U2 - 10.1016/S0306-4522(01)00290-1

DO - 10.1016/S0306-4522(01)00290-1

M3 - Article

C2 - 11682159

AN - SCOPUS:0035980493

SN - 0306-4522

VL - 106

SP - 733

EP - 743

JO - Neuroscience

JF - Neuroscience

IS - 4

ER -

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

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