Journal: Kidney International

Article Title: β 3 adrenergic receptor in the kidney may be a new player in sympathetic regulation of renal function

doi: 10.1016/j.kint.2016.03.020

Figure Lengend Snippet: Ex vivo β 3 -AR activation in a kidney tubule: intracellular cAMP measurements, AQP2 subcellular localization, and NKCC2 phosphorylation. ( a ) 1-Deamino-8- d -arginine-vasopressin (dDAVP)– and BRL37344 (BRL)-induced cAMP production in mouse kidney tubule suspensions. Freshly isolated tubule suspensions from wild-type mice (12-week-old males, 3 per individual experiment) were pooled and equally distributed into 24-well plates. Samples were treated with dDAVP (10 −7 M) or with the indicated concentrations of BRL for 60 minutes at 37 °C. Total cAMP-generated in each well was normalized to the protein content. Three independent experiments were carried out. Data are expressed as the percentage of the cAMP content measured in resting cells ± SEM. Significant differences between means were tested by 1-way analysis of variance with the Newman-Keuls posttest. Significance was accepted for P values < 0.05. *** P < 0.0001, ** P < 0.001 compared with resting tubules. ## P < 0.001 compared with 1 μM BRL. ( b ) Freshly isolated kidney slices (250 μm) were rapidly cut after sacrifice, maintained in CO 2 -equilibrated culture medium at 37 °C, left untreated (resting), or incubated with dDAVP (10 −7 M) or with BRL (10 μM BRL). BRL was also incubated after preincubation with either the β 3 -AR–antagonist (L748,337, 10 −7 M) or the protein kinase A inhibitor (H89) (10 −5 M). Slices were treated as described and fixed, and ultrathin sections were stained for aquaporin 2 (AQP2) and β 3 -adrenergic receptor (β 3 -AR) and subjected to confocal microscopy. BRL was as effective as dDAVP in promoting AQP2 expression at the apical plasma membrane of cortical and outer medullary collecting duct cells (white arrows) compared with the intracellular localization of AQP2 observed in untreated samples (resting) or samples incubated with BRL after preincubation with L748,337 or H89 (white arrowheads) (bar = 15 μm). ( c ) Kidney slices were treated as described, then lysed, and total protein extracts underwent Western blotting analysis using the anti–phosphorylated Na-K-Cl cotransporter (pNKCC2) and the anti–total NKCC2 antibodies. ( d ) Densitometric analysis showed a five-fold increase in pNKCC2 (normalized to total NKCC2) in samples treated with BRL or dDAVP, and the effect of BRL was significantly prevented by L748,337 and H89. Data are provided as mean ± SEM and expressed as a percentage of the resting condition. Significant differences between means were tested by 1-way analysis of variance with the Newman-Keuls posttest. *** P < 0.001. Comparable results were obtained in 3 different mice. NKCC2, Na-K-Cl cotransporter; NS, not significant.

Article Snippet: Polyclonal antibodies against β 3 -AR (cat. nos. sc-50436 and sc-1473) were obtained from Santa Cruz Biotechnology (Dallas, TX) and were previously validated for Western blotting and immunofluorescence analysis., Antibodies against AQP1 (cat. no. sc-20810), and CD-31 (cat. no. sc-1506), BRL37344 (cat. no. sc-200154), L748,337 (cat. no. sc-204044) were from Santa Cruz Biotechnology.

Techniques: Ex Vivo, Activation Assay, Phospho-proteomics, Isolation, Generated, Incubation, Staining, Confocal Microscopy, Expressing, Clinical Proteomics, Membrane, Western Blot

Journal: Kidney International

Article Title: β 3 adrenergic receptor in the kidney may be a new player in sympathetic regulation of renal function

doi: 10.1016/j.kint.2016.03.020

Figure Lengend Snippet: BRL37344 failed to induce AQP2 apical expression and NKCC2 phosphorylation in the kidney of β 3 -AR–null mice. ( a ) Freshly isolated kidney slices (250 μm) were obtained from β 3 −/− AR (β 3 -adrenergic receptor) mice, maintained in CO 2 -equilibrated culture medium at 37 °C, and left untreated (resting) or incubated with desmopressin (dDAVP, 10 −7 M) or BRL37344 (BRL, 10 μM). Slices were fixed, and ultrathin sections (5 μm) were stained for AQP2 (aquaporin 2) and subjected to confocal laser-scanning microscopy. In β 3 −/− AR mice, BRL was unable to promote AQP2 expression at the apical plasma membrane of cortical and outer medullary collecting duct cells. dDAVP was used as an internal control to promote AQP2 apical expression (bar = 10 μm). Arrows indicate apical plasma membrane staining. Arrowheads indicate intracellular staining. ( b ) Slices were also lysed and protein extracts subjected to Western blotting analysis with antiphosphorylated Na-K-Cl cotransporter (pNKCC2) and total Na-K-Cl cotransporter (NKCC2) antibodies. ( c ) Densitometric analysis of pNKCC2, normalized to total NKCC2, showed that in β 3 −/− AR mice, BRL was unable to increase NKCC2 phosphorylation compared with dDAVP. Data are provided as mean ± SEM and expressed as the percentage of the resting condition. Significant differences between means were tested by 1-way analysis of variance with the Newman-Keuls posttest. *** P < 0.0001. Comparable results were obtained in 3 different mice.

Article Snippet: Polyclonal antibodies against β 3 -AR (cat. nos. sc-50436 and sc-1473) were obtained from Santa Cruz Biotechnology (Dallas, TX) and were previously validated for Western blotting and immunofluorescence analysis., Antibodies against AQP1 (cat. no. sc-20810), and CD-31 (cat. no. sc-1506), BRL37344 (cat. no. sc-200154), L748,337 (cat. no. sc-204044) were from Santa Cruz Biotechnology.

Techniques: Expressing, Phospho-proteomics, Isolation, Incubation, Staining, Confocal Laser Scanning Microscopy, Clinical Proteomics, Membrane, Control, Western Blot

Journal: Kidney International

Article Title: β 3 adrenergic receptor in the kidney may be a new player in sympathetic regulation of renal function

doi: 10.1016/j.kint.2016.03.020

Figure Lengend Snippet: Effect of β 3 -AR stimulation on urine concentrating ability in β 3 -AR +/+ mice. β 3 -adrenergic receptor (β 3 -AR)–null (β 3 -AR −/− ) mice and their age-matched controls (β 3 -AR +/+ ) (10 of each genotype) were individually acclimatized in metabolic cages for 48 hours; 5 received a single i.p. injection of BRL37344 (BRL) (0.6 mg/kg), whereas 5 control animals received phosphate-buffered saline alone (vehicle). Urine samples were collected for 4 hours after injection. Urine output ( a ) and urine osmolality ( b ) were measured in β 3 -AR +/+ and β 3 -AR −/− mice and expressed as a percentage of control values measured in vehicle-injected animals ± SEM. Urine output of β 3 -AR +/+ mice decreased ∼70% and urine osmolality increased ∼40% after BRL37344 (BRL) injection. No significant effect was seen in β 3 -AR −/− mice. Significant differences between means were tested by 1-way analysis of variance with the Newman-Keuls posttest. * P < 0.05, *** P < 0.0001. ( c , d , e ) Urine excretion of Na + , K + , and Cl – , normalized for the urine volume, measured in β 3 -AR +/+ mice. Data are reported as a percentage of the values measured in vehicle-injected mice ± SEM. Significant differences between means were tested by the Mann-Whitney U test. * P < 0.05. ( f ) Glomerular filtration rate (GFR) of β 3 -AR +/+ conscious mice was measured at 1, 2, 3, and 4 hours after injection of BRL or vehicle alone. No significant difference was found at each time point between BRL- and vehicle-injected mice. Significant differences between means were tested by 2-way analysis of variance with a Bonferroni posttest.

Article Snippet: Polyclonal antibodies against β 3 -AR (cat. nos. sc-50436 and sc-1473) were obtained from Santa Cruz Biotechnology (Dallas, TX) and were previously validated for Western blotting and immunofluorescence analysis., Antibodies against AQP1 (cat. no. sc-20810), and CD-31 (cat. no. sc-1506), BRL37344 (cat. no. sc-200154), L748,337 (cat. no. sc-204044) were from Santa Cruz Biotechnology.

Techniques: Injection, Control, Saline, MANN-WHITNEY, Filtration

Journal: Kidney International

Article Title: β 3 adrenergic receptor in the kidney may be a new player in sympathetic regulation of renal function

doi: 10.1016/j.kint.2016.03.020

Figure Lengend Snippet: β 3 -Adrenergic receptor stimulation promotes antidiuresis in mice lacking functional expression of the arginine vasopressin receptor type 2. Ten V2R fl/y Esr1-Cre mice were acclimatized in mouse metabolic cages for 48 hours; 5 received a single i.p. injection of BRL37344 (0.6 mg/kg), whereas 5 control mice received phosphate-buffered saline alone (vehicle). Urine samples were collected every hour for 3 hours from both groups, and urine output ( a ) and urine osmolality ( b ) at each time point were reported. One hour after the injection, the urine output of BRL37344-treated mice was reduced to zero compared with vehicle-injected animals (vehicle) (diuresis, 1 hour). Two hours after injection, urine output of treated mice was still dramatically reduced compared with control animals (diuresis, 2 hours). Urine osmolality increased in BRL37344-injected animals (urine osmolality, 2 hours). At 3 hours postinjection, the effect of BRL on the urine output still persisted (urine output, 3 hours), whereas the effect on urine osmolality partially reversed (urine osmolality, 3 hours). The analysis reports the mean ± SEM values. Significant differences between measurements were tested by 2-way analysis of variance with a Bonferroni posttest for diuresis and by 1-way analysis of variance with a Bonferroni posttest. * P < 0.05; *** P < 0.001.

Article Snippet: Polyclonal antibodies against β 3 -AR (cat. nos. sc-50436 and sc-1473) were obtained from Santa Cruz Biotechnology (Dallas, TX) and were previously validated for Western blotting and immunofluorescence analysis., Antibodies against AQP1 (cat. no. sc-20810), and CD-31 (cat. no. sc-1506), BRL37344 (cat. no. sc-200154), L748,337 (cat. no. sc-204044) were from Santa Cruz Biotechnology.

Techniques: Functional Assay, Expressing, Injection, Control, Saline

Journal: Frontiers in Pharmacology

Article Title: Salmeterol, a β2 Adrenergic Agonist, Promotes Adult Hippocampal Neurogenesis in a Region-Specific Manner

doi: 10.3389/fphar.2019.01000

Figure Lengend Snippet: β 2 -AR agonists exert proneurogenic effects on ahNPCs. (A) Representative confocal images of MAP-2 (red) and nestin (green) immunolabeling in ahNPCs after 24 h treatment with vehicle (left panel) and 10 nM salmeterol (right panel). Nuclei were stained with DAPI (blue). MAP-2 + /nestin − cells are indicated (arrowheads). Magnification 400X, scale bar = 20 µm. (B-C) Effects of β 2 -AR agonists salmeterol (0.1–10 nM) (B) and formoterol (0.01–10 nM) (C) on neuronal differentiation of ahNPCs. (D-E) Effects of 24-h treatment with salmeterol (SAL) and formoterol (FORM) on GFAP + (D) and NG-2 + (E) cells. (F) Effect of 3 nM SAL in presence of vehicle, ICI118551, SR59230A, or CGP20712 antagonists. (G) Effects of 1 µM NE, 10 nM SAL, 10 nM FORM, and 100 nM BRL37344 (BRL) on neuronal differentiation of ahNPCs derived from β 2 -AR knockout mice. (H) Effects of salmeterol (0.0001–1 µM) on ahNPC proliferation. Fold change compared to vehicle-treated (−) ahNPCs. All experiments (n = 3) were run in triplicates. Data are expressed as mean ± S.D. ** p < 0.01, *** p < 0.001 vs. vehicle-treated cells; ### p < 0.001 vs. 3-nM SAL-treated cells (one-way ANOVA, Tukey’s post hoc analysis).

Article Snippet: The following drugs were utilized: L-(-)-norepinephrine-(+)-bitartrate salt monohydrate (NE) purchased from Sigma–Aldrich (Milan, Italy), salmeterol xinafoate, formoterol hemifumarate, ICI118,551 hydrochloride, CGP20712 dihydrochloride, SR59230A hydrochloride, and BRL37344 sodium salt purchased from Tocris (Bioscience, Bristol, UK).

Techniques: Immunolabeling, Staining, Derivative Assay, Knock-Out