The loop diuretic bumetanide (Bumex) is thought to have antiepileptic properties

The loop diuretic bumetanide (Bumex) is thought to have antiepileptic properties via modulate GABAA mediated signaling through their antagonism of cation-chloride cotransporters. showed that Intravenous injection of bumetanide (15.2 mg/kg) 30 min prior to the training session blocked inhibitory avoidance learning significantly. Subsequent control experiment’s results excluded the possible nonspecific effect of bumetanide on avoidance learning. We also found the phosphorylation of hippocampal MAPK was attenuated after bumetanide administration. These results suggested that hippocampal NKCC1 may via AMG-458 MAPK signaling cascade to possess its function. Introduction The cation-chloride cotransporters (CCCs) are intrinsic membrane proteins that transport chloride ions together with sodium and/or potassium ions across AMG-458 the plasma membrane [1]. The CCCs are divided into two main branches including the sodium-coupled CCC branch and the potassium-coupled CCC branch. The sodium-coupled CCC branch that comprises AMG-458 the Na-(K)-Cl-cotransporters NCC NKCC1 and NKCC2 load chloride ions into the cell to raise [Cl?]i above its electrochemical equilibrium. In adult neurons the level of intracellular chloride is low and the reversal potential for chloride currents is near the neuron’s resting membrane potential. Minor changes in the intracellular chloride concentration can significantly affect the strength Rabbit polyclonal to EREG. and even polarity of GABAergic neurotransmission [2] [3]. In addition to setting the direction of GABAA receptor-mediated currents the intracellular chloride concentration is also an important osmotic determinant of cell volume [4]. Neurons glia and most other cells in the brain alter their cellular chloride concentration to defend their cell volume against fluctuations of extracellular osmolality and/or intracellular solute content-perturbations that can imperil their structural integrity [5]-[8]. Bumetanide is a loop diuretic which is commonly used for the treatment of edema associated with congestive heart failure hepatic and renal disease for decades [9] [10]. Unlike another well-known loop diuretics furosemide bumetanide has an approximately 500-fold greater affinity for NKCC1 (inhibition constant [Ki] of approximately 0.1 μM) than for KCC2 (Ki of approximately 25-50 μM). Furosemide inhibits NKCC1 and KCC2 with equal potency (Ki of approximately 25-50 μM). Therefore at low dosages (2-10 μM) bumetanide can be a relatively particular inhibitor of NKCC1 in a few specific mobile assay [11] which managed to get an ideal device for learning the part of NKCC1 on hippocampus function. Latest studies also recommended some loop diuretics such as antiepileptic agent [1] [12] [13] [14]. Given that AMG-458 loop diuretics possibly act as antiepileptic agents that enhance GABAA inhibition we sought to investigate whether they also mediate the learning and memory function of hippocampus. Towards that end brain slice extracellular recording AMG-458 western blot and inhibitory avoidance paradigm were performed to evaluate the possible role of NKCC1 on hippocampal function. Materials and Methods Animals Adult male Wistar rats (obtained from the animal center of National Taiwan University) weighing between 250 and 350 g were used. Animals were housed in group cages of four rats each in a temperature (24°C) – controlled animal colony with continuous access to food and water. They were maintained on a 12∶12 light-dark cycle with lights on at 0700 hrs. All behavioral procedures took place during the animal light cycle. All procedures were conducted in accordance with the National institutes of Health Guide for Care and Use of Laboratory Animals and the guidelines set forth by the local institutional animal care and use committee (IACUC) at the National Taiwan Normal University. All efforts were made to minimize the animal numbers which are required to produce meaningful experimental data. Drugs administration For inhibitory avoidance bumetanide was dissolved in 0.5% saline with 0.5N NaOH [15] and injected intravenously 30 min prior to the training session (15.2 mg/kg equivalent AMG-458 to 400 μM). The chosen doses of bumetanide were based on our previous experiments [16] [17]. The test was carried out 24 hrs later. For extracellular recording bumetanide was first dissolved in 100% DMSO to make a 10 mM stock solution and diluted to 5 10 and 20 μM by ACSF [18]. Inhibitory avoidance task A conventional one-way inhibitory avoidance learning task was used to measure the retention performance in rats. It included the training phase and testing phase. Both of them were conducted between 9:00 a.m. and 4:00 p.m. Before experimentation.