Bumetanide, an inhibitor of cation-chloride cotransporter isoform 1, inhibits ?-aminobutyric acidergic excitatory actions and enhances sedative actions of midazolam in neonatal rats

Background: It has been shown that ?-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 ?-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 Ca2+ 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. Conclusion: These results suggest that ?-aminobutyric acid A receptor-mediated excitation plays an important role in attenuated sedative effects of midazolam in immature rats.