title: Bumetanide, an inhibitor of cation-chloride cotransporter isoform 1, inhibits γ-aminobutyric acidergic excitatory actions and enhances sedative actions of midazolam in neonatal rats.
abstract: BACKGROUND
  It has been shown that γ-aminobutyric acid exerts excitatory actions on the immature brain due to the increased expression of Na(+)-K(+)-2Cl(-) cotransporter isoform 1. The authors sought to clarify whether midazolam, a γ-aminobutyric acid-mimetic hypnotic agent, causes neuronal excitation that can be blocked by bumetanide, a selective inhibitor of Na(+)-K(+)-2Cl(-) cotransporter isoform 1. Furthermore, the authors examined whether bumetanide potentiates the sedative effects of midazolam in neonatal rats.
  
  
  METHODS
  The authors measured the effects of midazolam with or without bumetanide on the cytosolic Ca(2+) concentration ([Ca](2+)(i)) in hippocampal slices (n=3 in each condition) from rats at postnatal days 4, 7, and 28 (P4, P7, and P28) using fura-2 microfluorometry. Neuronal activity in the hippocampus and thalamus after intraperitoneal administration of midazolam with or without bumetanide was estimated by immunostaining of phosphorylated cyclic adenosine monophosphate-response element-binding protein (n=12 in each condition). Furthermore, the authors assessed effects of bumetanide on the sedative effect of midazolam by measuring righting reflex latency (n=6 in each condition).
  
  
  RESULTS
  Midazolam significantly increased [Ca](2+)(i) in the CA3 area at P4 and P7 but not at P28. Bumetanide inhibited midazolam-induced increase in [Ca](2+)(i). Midazolam significantly up-regulated phosphorylated cyclic adenosine monophosphate-response element-binding protein expression in a bumetanide-sensitive manner in the hippocampus at P7 but not P28. Bumetanide enhanced the sedative effects of midazolam in P4 and P7 but not P28 rats.
  
  
  CONCLUSIONS
  These results suggest that γ-aminobutyric acid A receptor-mediated excitation plays an important role in attenuated sedative effects of midazolam in immature rats.
authors.affiliation: * Graduate Course Student, # Assistant Professor, ** Professor and Chair, Department of Anesthesiology and Critical Care Medicine, ‡ Assistant Professor, Department of Neuroanatomy, § Professor, Department of Biostatistics and Epidemiology, ‖ Assistant Professor, Departments of Anesthesiology and Critical Care Medicine and Physiology, Yokohama City University Graduate School of Medicine, Yokohama, Japan. † Visiting Professor, Department of Anesthesiology and Critical Care Medicine, Yokohama City University Graduate School of Medicine, and Professor, Department of Anesthesiology, Mizonokuchi Hospital, Teikyo University School of Medicine, Kawasaki, Japan.
authors.forename: Yukihide
authors.initials: Y
authors.lastname: Koyama
authors.forename: Tomio
authors.initials: T
authors.lastname: Andoh
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kamiya
authors.forename: Satoshi
authors.initials: S
authors.lastname: Morita
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Miyazaki
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Uchimoto
authors.forename: Takahiro
authors.initials: T
authors.lastname: Mihara
authors.forename: Takahisa
authors.initials: T
authors.lastname: Goto
mesh_terms: D000375:Aging / Q000502:physiology; D000818:Animals; D000831:Animals, Newborn; D002034:Bumetanide / Q000494:pharmacology*; D056654:CA3 Region, Hippocampal / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004232:Diuretics / Q000494:pharmacology*; D004357:Drug Synergism; D018756:GABA Antagonists; D006993:Hypnotics and Sedatives / Q000494:pharmacology*; D008874:Midazolam / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012018:Reflex / Q000187:drug effects; D064506:Solute Carrier Family 12, Member 2 / Q000187:drug effects*; D015854:Up-Regulation / Q000187:drug effects; D005680:gamma-Aminobutyric Acid / Q000502:physiology*
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D004232:Diuretics; D018756:GABA Antagonists; D006993:Hypnotics and Sedatives; C578879:Slc12a2 protein, rat; D064506:Solute Carrier Family 12, Member 2; D002034:Bumetanide; D005680:gamma-Aminobutyric Acid; D008874:Midazolam; D002118:Calcium