Journal: Advanced Science

Article Title: Oxytocin Improves Autistic Behaviors by Positively Shifting GABA Reversal Potential via NKCC1 in Early‐Postnatal‐Stage

doi: 10.1002/advs.202415432

Figure Lengend Snippet: Oxytocin regulates GABA A reversal potential by modulating the protein expression of NKCC1. (A) The mRNA expression levels of NKCC1 and KCC2 at P7 (the control group) were examined via RT‒qPCR ( n = 9; unpaired t ‐test; * p < 0.01). (B) The GABA A reversal potential of P7 mice in the different treatment groups. The VPA + Oxt group received DCZ injections from P1 to P7. The GABA A reversal potential of the VPA + Oxt + bumetanide group was recorded following bath application of bumetanide (10 µ m , an NKCC1 antagonist). The Con (N = 7, n = 11), VPA (N = 7, n = 11), VPA + Oxt (N = 5, n = 10), and VPA + Oxt + bumetanide (N = 4, n = 10) groups were analyzed, with N mice and n recorded neurons. The data are expressed as the means ± SEMs, and the statistical significance is indicated as *** p < 0.001 (one‐way ANOVA + Tukey post hoc test). (C) The mRNA expression level of NKCC1 in different groups (Con and VPA) was examined via RT‒qPCR (NKCC1: Con N = 13, VPA N = 12, unpaired t ‐test, ** p < 0.01). (D) The mRNA expression level of KCC2 in different groups (Con and VPA) was examined via RT‒qPCR (Con, N = 6; VPA, N = 5; unpaired t ‐test; ns, p = 0.8632). (E) The protein level of KCC2 in the control and VPA groups at P7 (Con n = 7, VPA n = 7, ns, p = 0.7136). (F) The protein level of NKCC1 in the Con or VPA group at P7 ( n = 5, *** p < 0.001). (G) The protein level of NKCC1 in the saline‐injected or DCZ‐injected group of P7 OXT‐hM4Di mice ( n = 7, * p < 0.05). (H) The protein level of NKCC1 in the saline‐injected or DCZ‐injected group of P7 OXT‐hM3Dq mice. ( n = 4, * p < 0.05). The data are expressed as the means ± SEMs, and statistical significance is indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001. Significance was determined via an unpaired t ‐test.

Article Snippet: Bumetanide (10 μ m , Tocris Bioscience, cat. number: No. 3108), a selective antagonist of NKCC1, was administered through a brief bath application (10 min) to examine the contribution of NKCC1 to the reversal potential of the GABA A receptor.

Techniques: Expressing, Control, Saline, Injection

Journal: Nature immunology

Article Title: WNK1 kinase balances T cell adhesion versus migration in vivo

doi: 10.1038/ni.3495

Figure Lengend Snippet: OXSR1, STK39 and SLC12A2 positively regulate chemokine-induced migration. ( a ) CXCL12-induced migration of Jurkat T cells transfected with the indicated siRNAs normalized to migration of cells transfected with NT siRNAs. Data for NT and WNK1 siRNA is the same as that shown in and is repeated here for clarity. ( b ) CCL21-induced migration of mouse CD4+ T cells of the indicated genotypes. ( c ) CCL21-induced migration of mouse CD4+ T cells of the indicated genotypes. ( d ) Speed and displacement of mouse CD4 + T cells measured by time-lapse microscopy in response to CCL21. Flower plots show overlay of migration tracks of individual T cells. ( e ) CCL21-induced migration of mouse CD4+ T cells of the indicated genotypes in the presence or absence of bumetanide. * P < 0.01, ** P < 0.001, *** P < 0.0001 (Mann-Whitney). Sample sizes: a (5), b (8), c (10, control cells; 11, mutant cells), d (630 control cells no CCL21; 1773 control cells with CCL21; 643 mutant cells no CCL21; 1105, mutant cells with CCL21), e (6). Data are representative of 2 ( d ) independent experiments or were pooled from 2 ( b,e ), 4 ( c ), or 5 ( a ) independent experiments. Graphs show mean±SEM.

Article Snippet: For SLC12A2 inhibitor experiments cells were pre-incubated with either DMSO (1:1000) or with 20 μM Bumetanide (Santa Cruz) in DMSO for 20 min and all subsequent steps were performed in the presence of DMSO or the inhibitor.

Techniques: Migration, Transfection, Time-lapse Microscopy, MANN-WHITNEY, Control, Mutagenesis

Journal: Biochemical pharmacology

Article Title: Endoplasmic reticulum transporter OAT2 regulates drug metabolism and interaction.

doi: 10.1016/j.bcp.2024.116322

Figure Lengend Snippet: Fig. 3. Role of Oat2 in drug metabolism in slc22a7 knockout mice. (A) Genotyping of wild-type and slc22a7 knockout mice. (B, C) Hepatocytes were treated with bumetanide (1 μM) for 30 min at 37 ◦C. Production of bumetanide β-D-glucuronide (B) and accumulation of bumetanide into the hepatocytes (C) were measured. (D) The metabolic efficiency of bumetanide calculated by dividing production of bumetanide β-glucuronide by accumulation of bumetanide into the cells. Data are presented as the mean ± S.D. (n = 3, biological replicate wells). Statistical significance was determined using the Student’s t-test; *P < 0.05. (E) Glucuronidation activity of bumetanide in wild-type and slc22a7-/- mice directly estimated using hepatic microsomes isolated from these mice. Data are presented as the means ± S.D. (n = 3, biological replicate wells). Statistical significance was determined using the Student’s t-test; *P < 0.05.

Article Snippet: Ketoprofen, bumetanide, bumetanide β-D-glucuronide, zidovudine β-D-glucuronide, ER-Tracker Red, and DAPI-Fluoromount-G were purchased from Cayman Chemical (Ann Arbor, MI, USA), Enzo Life Science (Farmingdale, NY, USA), Toronto Research Chemicals (North York, Canada), Biosynth (Berkshire, UK), Thermo Fisher Scientific (Waltham, MA, USA), and Southern Biotech (Alabama, USA), respectively.

Techniques: Knock-Out, Activity Assay, Isolation

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Exacerbated brain edema in a rat streptozotocin model of hyperglycemic ischemic stroke: Evidence for involvement of blood–brain barrier Na–K–Cl cotransport and Na/H exchange

doi: 10.1177/0271678X18770844

Figure Lengend Snippet: Intravenous bumetanide and HOE-642 attenuate hyperglycemia-elevated brain Na uptake during MCAO. (a) Rats made hyperglycemic for 48 h and normoglycemic rats were subjected to MCAO and then assessed for brain Na uptake by localized 23Na chemical shift imaging (CSI) magnetic resonance spectroscopy (MRS) as described in Materials and Methods. Values shown are ratios of left (ipsilateral) to right (contralateral) [Na] for cortical ROI 2. Data are presented as Tukey box and whisker plots as described in Figure 1. For all plots, n values are 6 and 16 for normoglycemic and hyperglycemic rats, respectively. (b,c) Rats made hyperglycemic for 48 h were given vehicle, bumetanide (30 mg/kg) or HOE-642 (15 mg/kg) intravenously immediately before induction of MCAO and then brain [Na] ratios assessed by Na CSI as in (a). Values shown are ratios of left (ipsilateral) to right (contralateral) [Na] for cortical ROIs 1 (b) and 2 (c). Data are presented as Tukey box and whisker plots as described in Figure 1. For all plots, n values are 16, 10 and 10 for vehicle, bumetanide and HOE642, respectively. For statistical analysis (a–c), brain Na ratios were log-transformed and evaluated in Stata Version 14 using mixed-effects regression models as described in Figure 4. For Figure 5(a), holding time since the start of MCAO constant, the hyperglycemia vs. normoglycemia adjusted geometric mean ratio (GMR) of L/R brain Na ratios is 1.18 (95% CI: 1.08, 1.28), indicating that hyperglycemia could plausibly increase the typical Na ratio anywhere from 8 to 28% in the population. Geometric mean ratios (GMR) for brain Na data in B and C were: L1/R1 bumetanide vs. vehicle GMR (95% CI): 0.90 (0.82, 0.98); L1/R1 HOE642 vs. vehicle GMR (95% CI): 0.80 (0.74, 0.87); L2/R2 bumetanide vs. vehicle GMR (95% CI): 0.96 (0.89, 1.04); L2/R2 HOE642 vs. vehicle GMR (95% CI): 0.85 (0.77, 0.93).

Article Snippet: Bumetanide (30 mg/kg, ICN Biomedicals, Costa Mesa, CA) or/and HOE-642 (15 mg/kg; gift from Sanofi-Aventis Pharmaceuticals, Bridgewater, NJ) were administered intravenously via the femoral vein cannula immediately before occlusion of the MCA.

Techniques: Imaging, Spectroscopy, Whisker Assay, Transformation Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Exacerbated brain edema in a rat streptozotocin model of hyperglycemic ischemic stroke: Evidence for involvement of blood–brain barrier Na–K–Cl cotransport and Na/H exchange

doi: 10.1177/0271678X18770844

Figure Lengend Snippet: Hyperglycemia-exacerbated edema formation during MCAO is attenuated by bumetanide and HOE-642. Rats made hyperglycemic for 48 h were given vehicle, bumetanide (30 mg/kg) or HOE-642 (15 mg/kg) intravenously immediately before induction of MCAO. ADC ratios are shown for cortical ROIs 1–3 (a–c) and striatal ROI 4 (d). Data are presented as Tukey box and whisker plots as described in Figure 1. For all plots, n values are 14, 10 and 6 for vehicle, bumetanide and HOE642, respectively. For statistical analysis, ADC ratios were log-transformed and evaluated in Stata Version 14 using mixed-effects regression models that expressed the mean of the log-transformed ratios (equivalently, the log of the geometric mean ratio) as a function of time and group. Pairwise contrasts between groups were used for hypothesis testing, with contrasts back-transformed and thus expressed as adjusted between-group geometric mean (GM) within-animal (left:right hemisphere) Na ratios with robust 95% CI. One is excluded from the 95% CI when and only when p < 0.05. The best fitting model by the Akaike information criteria is a parallel slopes model (in the log-transformed outcome), with random intercepts for animals to capture within-animal correlations among longitudinal measurements. For ADC ratios, geometric mean ratios (GMR) were: L1/R1 bumetanide vs. vehicle GMR (95% CI): 1.18 (1.04, 1.33); L1/R1 HOE642 vs. vehicle GMR (95% CI): 1.36 (1.13, 1.64); L2/R2 bumetanide vs. vehicle GMR (95% CI): 0.99 (0.89, 1.10);L2/R2 HOE642 vs. vehicle GMR (95% CI): 1.15 (0.98,1.35); L3/R3 bumetanide vs. vehicle GMR (95% CI): 1.12 (0.99, 1.28); L3/R3 HOE642 vs. vehicle GMR (95% CI): 1.41 (1.20, 1.65); L4/R4 bumetanide vs. vehicle GMR (95% CI): 1.10 (1.01, 1.19); and L4/R4 HOE642 vs. vehicle GMR (95% CI): 1.21 (1.05, 1.4).

Article Snippet: Bumetanide (30 mg/kg, ICN Biomedicals, Costa Mesa, CA) or/and HOE-642 (15 mg/kg; gift from Sanofi-Aventis Pharmaceuticals, Bridgewater, NJ) were administered intravenously via the femoral vein cannula immediately before occlusion of the MCA.

Techniques: Whisker Assay, Transformation Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Exacerbated brain edema in a rat streptozotocin model of hyperglycemic ischemic stroke: Evidence for involvement of blood–brain barrier Na–K–Cl cotransport and Na/H exchange

doi: 10.1177/0271678X18770844

Figure Lengend Snippet: Bumetanide and HOE-642 reduce brain acute ischemic injury in hyperglycemic rats. (a) Representative DWI images from hyperglycemic rats treated with vehicle, bumetanide or HOE-642. Images shown are from B990 scans acquired 170 min after the start of MCAO. (b) Quantitation of acute ischemia area. Data are presented as Tukey box and whisker plots with n values of 11, 10 and 5 for vehicle, bumetanide and HOE642, respectively. The DWI B990-defined ischemic injury in hyperglycemic rats treated with bumetanide or HOE-642 is significantly different than hyperglycemic rats treated with Vehicle by ANOVA with Bonferroni’s multiple comparisons test, *p < .05.

Article Snippet: Bumetanide (30 mg/kg, ICN Biomedicals, Costa Mesa, CA) or/and HOE-642 (15 mg/kg; gift from Sanofi-Aventis Pharmaceuticals, Bridgewater, NJ) were administered intravenously via the femoral vein cannula immediately before occlusion of the MCA.

Techniques: Quantitation Assay, Whisker Assay

Journal: Neuroscience letters

Article Title: Subsequent maternal separation exacerbates neurobehavioral abnormalities in rats neonatally exposed to sevoflurane anesthesia

doi: 10.1016/j.neulet.2017.09.063

Figure Lengend Snippet: Treatment groups.

Article Snippet: To study the role of GABA A R-mediated depolarization, subgroups of P6 rats received a single injection of the NKCC1 inhibitor, bumetanide (1.82 mg kg −1 , intraperitoneally, IP, Ben Venue Laboratories, Inc., Bedford, OH), 15 min prior to initiation of anesthesia with sevoflurane (Bumetanide plus Sevoflurane plus MS10 group).

Techniques: Control

Journal: Neuroscience letters

Article Title: Subsequent maternal separation exacerbates neurobehavioral abnormalities in rats neonatally exposed to sevoflurane anesthesia

doi: 10.1016/j.neulet.2017.09.063

Figure Lengend Snippet: The brain hypothalamus tissue samples were collected 4 days after the onset of anesthesia with sevoflurane for qRT-PCR analyses. (A) Illustration of the experimental protocol. Shown are the respective levels of NKCC1 mRNA (B), KCC2 mRNA (C), the resulting NKCC1/KCC2 mRNA ratios (D) and CRH mRNA (E). Data normalized against Control are means ± SD from 5–6 rats per treatment group. *P = 0.02 vs. Control; #P = 0.025 vs. Bumetanide plus Sevoflurane plus maternal separation.

Article Snippet: To study the role of GABA A R-mediated depolarization, subgroups of P6 rats received a single injection of the NKCC1 inhibitor, bumetanide (1.82 mg kg −1 , intraperitoneally, IP, Ben Venue Laboratories, Inc., Bedford, OH), 15 min prior to initiation of anesthesia with sevoflurane (Bumetanide plus Sevoflurane plus MS10 group).

Techniques: Quantitative RT-PCR, Control

Journal: Neuroscience letters

Article Title: Subsequent maternal separation exacerbates neurobehavioral abnormalities in rats neonatally exposed to sevoflurane anesthesia

doi: 10.1016/j.neulet.2017.09.063

Figure Lengend Snippet: (A) Illustration of the experimental protocol. (B) Plots showing the values of escape latencies during the 6-day training period from P54 to P59. (C) Representative swimming tracks made by rats during the MWM test at P60. Location of the escape platform is illustrated by small circle in the left-top quadrant. (D) Histograms showing time spent in the target quadrant during the MWM test. N = 12 for each treatment group. MS10 means maternal separation at P10, See Methods for details. *P = 0.012 vs. the Control group; #P = 0.01 vs. the Bumetanide plus sevoflurane plus MS10 group. Color coding in B and D is the same as in C.

Article Snippet: To study the role of GABA A R-mediated depolarization, subgroups of P6 rats received a single injection of the NKCC1 inhibitor, bumetanide (1.82 mg kg −1 , intraperitoneally, IP, Ben Venue Laboratories, Inc., Bedford, OH), 15 min prior to initiation of anesthesia with sevoflurane (Bumetanide plus Sevoflurane plus MS10 group).

Techniques: Control