anti ent1 rabbit polyclonal antibody  (Alomone Labs)

Journal: Frontiers in Pharmacology

Article Title: Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters

doi: 10.3389/fphar.2022.837555

Figure Lengend Snippet: Effects of FPMINT analogues (listed in ) on [ 3 H]uridine uptake by ENT1 and ENT2 [ 3 H]uridine uptake (1 μM, 2 μCi/ml) in (A) PK15NTD/ENT1 and (B) PK15NTD/ENT2 cells was measured in the presence of various concentrations of FPMINT analogues listed in (10 nM–100 μM).

Article Snippet: The membranes were blocked with 5% (w/v) non-fat dry milk in PBS for 1 h at room temperature and then incubated with rabbit polyclonal anti-ENT1 or anti-ENT2 primary antibodies (Alomone Labs, Jerusalem, Israel) (1:200 [v/v] dilution in a blocking solution) at 4°C overnight.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters

doi: 10.3389/fphar.2022.837555

Figure Lengend Snippet: Effects of FPMINT analogues (listed in ) on [ 3 H]uridine uptake by ENT1 and ENT2 [ 3 H]uridine uptake (1 μM, 2 μCi/ml) in (A) PK15NTD/ENT1 and (B) PK15NTD/ENT2 cells was measured in the presence of various concentrations of FPMINT analogues listed in (10 nM–100 μM).

Article Snippet: The membranes were blocked with 5% (w/v) non-fat dry milk in PBS for 1 h at room temperature and then incubated with rabbit polyclonal anti-ENT1 or anti-ENT2 primary antibodies (Alomone Labs, Jerusalem, Israel) (1:200 [v/v] dilution in a blocking solution) at 4°C overnight.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters

doi: 10.3389/fphar.2022.837555

Figure Lengend Snippet: Competitive inhibition of ENT1 and ENT2 by compound 3c. Kinetic study of [ 3 H]uridine uptake (0.1 μM–1 mM) was measured in (A) PK15NTD/ENT1 and (D) PK15NTD/ENT2 cells in the presence of various concentrations of compound 3c (0, 0.01, 0.1, 1 and 10 µM) (B,E) 1/ V versus 1/[S] plots of each curve in (A) and (D) , respectively (C,F) The plot of slopes of each line in (B) and (E) , respectively, versus the concentration of compound 3c . Data are presented as mean ± SE of three experiments.

Article Snippet: The membranes were blocked with 5% (w/v) non-fat dry milk in PBS for 1 h at room temperature and then incubated with rabbit polyclonal anti-ENT1 or anti-ENT2 primary antibodies (Alomone Labs, Jerusalem, Israel) (1:200 [v/v] dilution in a blocking solution) at 4°C overnight.

Techniques: Inhibition, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters

doi: 10.3389/fphar.2022.837555

Figure Lengend Snippet: Effects of compound 3c on cytotoxicity and protein expressions of ENT1 and ENT2. Cytotoxicity of compound 3c was measured after 24 h or 48 h treatment of PK15NTD/ENT1 and PK15NTD/ENT2 cells with various concentrations of compound 3c (0.5, 5 and 50 μM) or a vehicle (0.5% DMSO, as a control) (A) The cell viability and (B) cell membrane integrity were determined by the MTT assay and LDH release, respectively (C) PK15NTD/ENT1 and (D) PK15NTD/ENT2 cells were incubated with 50 μM of compound 3c or a vehicle (0.5% DMSO, as a control) for 24 or 48 h. Western blotting assay was performed to determine the protein expression levels of ENT1 and ENT2, with β-actin as an internal reference. Cell surface proteins of (E) PK15NTD/ENT1 and (F) PK15NTD/ENT2 cells were biotinylated and treated with 50 μM of compound 3c or a vehicle (0.5% DMSO, as a control) for 24 or 48 h. After cleavage of extracellular biotin, internalized biotinylated proteins were precipitated with immobilized streptavidin beads and detected with an anti-ENT1 and anti-ENT2 antibodies by western blotting. Total ENT1 and ENT2 from whole-cell lysates were also detected for a comparison. β-actin served as an internal reference. Representative blots are from three independent experiments. Data are presented as mean ± SE of three experiments.

Article Snippet: The membranes were blocked with 5% (w/v) non-fat dry milk in PBS for 1 h at room temperature and then incubated with rabbit polyclonal anti-ENT1 or anti-ENT2 primary antibodies (Alomone Labs, Jerusalem, Israel) (1:200 [v/v] dilution in a blocking solution) at 4°C overnight.

Techniques: MTT Assay, Incubation, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters

doi: 10.3389/fphar.2022.837555

Figure Lengend Snippet: Reversibility of the inhibitory effects of compound 3c on ENT1 and ENT2 (A) PK15NTD/ENT1 and (B) PK15NTD/ENT2 cells were incubated with 50 μM of compound 3c or a vehicle (0.5% DMSO, as a control) for different time (0–60 min) The cells were then washed five times. After wash-out for every single time [ 3 H]uridine uptake (1 μM, 2 μCi/ml) was measured. Data are presented as mean ± SE of three experiments. * p < 0.05 compared with the fifth wash.

Article Snippet: The membranes were blocked with 5% (w/v) non-fat dry milk in PBS for 1 h at room temperature and then incubated with rabbit polyclonal anti-ENT1 or anti-ENT2 primary antibodies (Alomone Labs, Jerusalem, Israel) (1:200 [v/v] dilution in a blocking solution) at 4°C overnight.

Techniques: Incubation

Journal: Frontiers in Pharmacology

Article Title: Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters

doi: 10.3389/fphar.2022.837555

Figure Lengend Snippet: Molecular docking analysis of compound 3c against ENT1. 3D structural overview of ENT1 with (A,B) compound 3c and (D,E) draflazine docked complexes. The black box showed the location of compound 3c. A close-up of the docked protein displaying the amino acid residues of ENT1 involved in the binding with (C) compound 3c and (F) draflazine.

Article Snippet: The membranes were blocked with 5% (w/v) non-fat dry milk in PBS for 1 h at room temperature and then incubated with rabbit polyclonal anti-ENT1 or anti-ENT2 primary antibodies (Alomone Labs, Jerusalem, Israel) (1:200 [v/v] dilution in a blocking solution) at 4°C overnight.

Techniques: Binding Assay

Journal: British Journal of Pharmacology

Article Title: β 3 Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

doi: 10.1111/bph.14921

Figure Lengend Snippet: Primary and secondary antibodies used to stain human and rat detrusor strips

Article Snippet: Observations were performed and analysed with a laser‐scanning confocal microscope (Olympus FluoView, FV1000, Tokyo, Japan; RRID:SCR_016840) and analysed with the Fluoview FV10‐ASW software, RRID:SCR_014215. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antigen Code/identifier Host Dilution Supplier Primary antibodies Anti‐β 2 receptor AAR‐016/RRID:AB_2039718 Rabbit 1:50 Alomone Anti‐β 3 receptor AAR‐017/RRID:AB_2039720 Rabbit 1:50 Alomone Anti‐β 3 receptor MC‐4198/RRID:AB_590525 Rabbit 1:50 MBL International Anti‐EPAC1 ab21236/RRID:AB_2177464 Rabbit 1:75 Abcam Anti‐EPAC1 SC‐28366/RRID:AB_627521 Mouse 1:100 Santa Cruz Anti‐EPAC2 #4156/RRID:AB_1904112 Mouse 1:50 Cell Signaling Tech Anti‐ENT1 ANT‐051/RRID:AB_2341015 Rabbit 1:50 Alomone Secondary antibodies Alexa Fluor 488 anti‐rabbit A‐21206/RRID:AB_2535792 Donkey 1:1,000 Molecular Probes Alexa Fluor 633 anti‐mouse A‐21050/RRID:AB_2535718 Goat 1:1,000 Molecular Probes Open in a separate window Primary and secondary antibodies used to stain human and rat detrusor strips 2.6.

Techniques: Staining

Journal: British Journal of Pharmacology

Article Title: β 3 Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

doi: 10.1111/bph.14921

Figure Lengend Snippet: Mechanism of action of drugs used in this study

Article Snippet: Observations were performed and analysed with a laser‐scanning confocal microscope (Olympus FluoView, FV1000, Tokyo, Japan; RRID:SCR_016840) and analysed with the Fluoview FV10‐ASW software, RRID:SCR_014215. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antigen Code/identifier Host Dilution Supplier Primary antibodies Anti‐β 2 receptor AAR‐016/RRID:AB_2039718 Rabbit 1:50 Alomone Anti‐β 3 receptor AAR‐017/RRID:AB_2039720 Rabbit 1:50 Alomone Anti‐β 3 receptor MC‐4198/RRID:AB_590525 Rabbit 1:50 MBL International Anti‐EPAC1 ab21236/RRID:AB_2177464 Rabbit 1:75 Abcam Anti‐EPAC1 SC‐28366/RRID:AB_627521 Mouse 1:100 Santa Cruz Anti‐EPAC2 #4156/RRID:AB_1904112 Mouse 1:50 Cell Signaling Tech Anti‐ENT1 ANT‐051/RRID:AB_2341015 Rabbit 1:50 Alomone Secondary antibodies Alexa Fluor 488 anti‐rabbit A‐21206/RRID:AB_2535792 Donkey 1:1,000 Molecular Probes Alexa Fluor 633 anti‐mouse A‐21050/RRID:AB_2535718 Goat 1:1,000 Molecular Probes Open in a separate window Primary and secondary antibodies used to stain human and rat detrusor strips 2.6.

Techniques:

Journal: British Journal of Pharmacology

Article Title: β 3 Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

doi: 10.1111/bph.14921

Figure Lengend Snippet: Effects of mirabegron (0.1 μM, ai–iv), isoprenaline (Isop, 1 μM, bi–iv), CL316,243 (1 μM, ci–ii), fenoterol (1 μM, d), 8‐CPT‐2Me cAMP (20 μM, ei), and 8‐pCPT‐2‐O‐Me‐cAMP‐AM (20 μM, eii) on adenosine outflow from urothelium‐denuned human (a) and rat (b–e) detrusor strips, respectively, in the absence and in the presence of β3‐adrenoceptors antagonists (L‐748,337, 30 nM; SR592230A, 100 nM) and of inhibitors of ENT1 (dipyridamole, 0.5 μM; NBTI, 30 μM) and EPAC (ESI‐09, 10 μM). Mirabegron, isoprenaline, CL316,243, fenoterol, 8‐CPT‐2Me cAMP, and 8‐pCPT‐2‐O‐Me‐cAMP‐AM contacted with the preparations for 15 min before sample collection. Antagonists/inhibitors were applied 15 min before mirabegron, isoprenaline or CL316,243 and were maintained throughout the assay. The ordinates represent the amount of adenosine (ADO, white bars) and inosine (INO, black bars) in pmol·mg−1 of wet weight of the preparations detected by HPLC with diode array in samples collected from the incubation media at 15 min intervals (for details, see Section 2). Data are means ± SD of five to eight individuals; duplicates were performed for each individual experiment. * P < .05 (two‐way ANOVA followed by the Sidak's multiple comparison test) represent significant differences when compared to the control situation (basal/inhibitor alone)

Article Snippet: Observations were performed and analysed with a laser‐scanning confocal microscope (Olympus FluoView, FV1000, Tokyo, Japan; RRID:SCR_016840) and analysed with the Fluoview FV10‐ASW software, RRID:SCR_014215. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antigen Code/identifier Host Dilution Supplier Primary antibodies Anti‐β 2 receptor AAR‐016/RRID:AB_2039718 Rabbit 1:50 Alomone Anti‐β 3 receptor AAR‐017/RRID:AB_2039720 Rabbit 1:50 Alomone Anti‐β 3 receptor MC‐4198/RRID:AB_590525 Rabbit 1:50 MBL International Anti‐EPAC1 ab21236/RRID:AB_2177464 Rabbit 1:75 Abcam Anti‐EPAC1 SC‐28366/RRID:AB_627521 Mouse 1:100 Santa Cruz Anti‐EPAC2 #4156/RRID:AB_1904112 Mouse 1:50 Cell Signaling Tech Anti‐ENT1 ANT‐051/RRID:AB_2341015 Rabbit 1:50 Alomone Secondary antibodies Alexa Fluor 488 anti‐rabbit A‐21206/RRID:AB_2535792 Donkey 1:1,000 Molecular Probes Alexa Fluor 633 anti‐mouse A‐21050/RRID:AB_2535718 Goat 1:1,000 Molecular Probes Open in a separate window Primary and secondary antibodies used to stain human and rat detrusor strips 2.6.

Techniques: Incubation

Journal: British Journal of Pharmacology

Article Title: β 3 Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

doi: 10.1111/bph.14921

Figure Lengend Snippet: Immunolocalization of (a) β3‐ and (b) β2‐adrenoceptors, (c) EPAC1 and (d) EPAC2 isoforms, and (e) ENT1 in transverse sections of the human (left hand‐side panels) and rat (right hand‐side panels) detrusor by confocal microscopy. β3‐Adrenoceptor and ENT1 immunoreactivities follow a plasma membrane staining pattern, whereas EPAC1 is compatible with a diffuse cytoplasmic distribution. Two distinct β3 (AAR‐017 and MC‐4198 raised in rabbits) and EPAC (ab21236 and SC‐28366 raised in rabbits and mice, respectively) antibodies were used as indicated. (f) Micrographs show that β3‐adrenoceptors (green) and EPAC (red) co‐localize in human detrusor, as identified by the yellow staining in the merge image shown in the right hand‐side image. Images are representative of four different individuals. Differential interference contrast (DIC) images are shown for comparison. Scale bar = 50 μm. Bottom panels are representative immunoblots of β2‐ and β3‐adrenoceptors (g) and EPAC1 and EPAC2 isoforms (h) in urothelium‐denuded detrusor homogenates of one male human subject (H) and one rat (R) run in parallel using the same antibodies as for the immunohistochemical staining. The highly conserved GAPDH enzyme (EC 1.2.1.12, MW~37 kDa) and β‐actin (MW~43 kDa) were used as reference proteins for β−adrenoceptors and EPAC respectively. Images are representative of three different individuals. Gels were loaded with 150 μg of protein. Host species for antibody production were rabbit (rb) and mouse (ms)

Article Snippet: Observations were performed and analysed with a laser‐scanning confocal microscope (Olympus FluoView, FV1000, Tokyo, Japan; RRID:SCR_016840) and analysed with the Fluoview FV10‐ASW software, RRID:SCR_014215. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antigen Code/identifier Host Dilution Supplier Primary antibodies Anti‐β 2 receptor AAR‐016/RRID:AB_2039718 Rabbit 1:50 Alomone Anti‐β 3 receptor AAR‐017/RRID:AB_2039720 Rabbit 1:50 Alomone Anti‐β 3 receptor MC‐4198/RRID:AB_590525 Rabbit 1:50 MBL International Anti‐EPAC1 ab21236/RRID:AB_2177464 Rabbit 1:75 Abcam Anti‐EPAC1 SC‐28366/RRID:AB_627521 Mouse 1:100 Santa Cruz Anti‐EPAC2 #4156/RRID:AB_1904112 Mouse 1:50 Cell Signaling Tech Anti‐ENT1 ANT‐051/RRID:AB_2341015 Rabbit 1:50 Alomone Secondary antibodies Alexa Fluor 488 anti‐rabbit A‐21206/RRID:AB_2535792 Donkey 1:1,000 Molecular Probes Alexa Fluor 633 anti‐mouse A‐21050/RRID:AB_2535718 Goat 1:1,000 Molecular Probes Open in a separate window Primary and secondary antibodies used to stain human and rat detrusor strips 2.6.

Techniques: Confocal Microscopy, Staining, Western Blot, Immunohistochemical staining

Journal: British Journal of Pharmacology

Article Title: β 3 Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

doi: 10.1111/bph.14921

Figure Lengend Snippet: (a) Inhibitory effect of mirabegron on electrically evoked [3H]ACh release from urothelium‐denuded human detrusor strips. Ordinates represent tritium outflow expressed in scintillations per min (cpm). Abscissa indicates the times at which samples were collected. [3H]ACh release was elicited by electrical field stimulation (10 Hz, 200 pulses of 0.2‐ms duration) twice, starting at 4th (S1) and 13th (S2) minutes after the end of washout (zero time). Mirabegron (0.1 μM) was added to the incubation media 6 min before S2 (black horizontal bar). Panels b, c and d show the inhibitory effects of mirabegron, isoprenaline and forskolin on evoked [3H]ACh release human (b) and rat (c and d) detrusor strips respectively. Mirabegron (0.1 μM), isoprenaline (1 μM), and forskolin (3 μM) were applied 6 min before S2 either in the absence or in the presence of selective inhibitors of PKA (H‐89, 10 μM), EPAC (ESI‐09, 10 μM), and ENT1 (Dipy, 0.5 μM), as well as of the adenosine A1 receptor antagonist, DPCPX (0.1 μM). All inhibitors and A1 receptor antagonist were present throughout the assay, including S1 and S2. The ordinates are changes in S2/S1 ratios compared to the S2/S1 ratio obtained without addition of any drug (dotted horizontal line). The data are means ± SD of an n number of individuals (black dots). #,* P < .001 (one‐way ANOVA followed by the Dunnett's multicomparison test with a single pooled variance) represent significant differences when compared to the control situation and to the inhibitory effects of mirabegron (b), isoprenaline (c) and forskolin (d) applied alone respectively

Article Snippet: Observations were performed and analysed with a laser‐scanning confocal microscope (Olympus FluoView, FV1000, Tokyo, Japan; RRID:SCR_016840) and analysed with the Fluoview FV10‐ASW software, RRID:SCR_014215. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antigen Code/identifier Host Dilution Supplier Primary antibodies Anti‐β 2 receptor AAR‐016/RRID:AB_2039718 Rabbit 1:50 Alomone Anti‐β 3 receptor AAR‐017/RRID:AB_2039720 Rabbit 1:50 Alomone Anti‐β 3 receptor MC‐4198/RRID:AB_590525 Rabbit 1:50 MBL International Anti‐EPAC1 ab21236/RRID:AB_2177464 Rabbit 1:75 Abcam Anti‐EPAC1 SC‐28366/RRID:AB_627521 Mouse 1:100 Santa Cruz Anti‐EPAC2 #4156/RRID:AB_1904112 Mouse 1:50 Cell Signaling Tech Anti‐ENT1 ANT‐051/RRID:AB_2341015 Rabbit 1:50 Alomone Secondary antibodies Alexa Fluor 488 anti‐rabbit A‐21206/RRID:AB_2535792 Donkey 1:1,000 Molecular Probes Alexa Fluor 633 anti‐mouse A‐21050/RRID:AB_2535718 Goat 1:1,000 Molecular Probes Open in a separate window Primary and secondary antibodies used to stain human and rat detrusor strips 2.6.

Techniques: Incubation

Journal: British Journal of Pharmacology

Article Title: β 3 Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

doi: 10.1111/bph.14921

Figure Lengend Snippet: Activation of EPAC (with 8‐CPT‐2Me cAMP, a) and PKC (with phorbol 12‐myristate 13‐acetate, PMA, b) decrease electrical evoked (10 Hz, 200 pulses of 0.2‐ms duration) [3H]ACh release from urothelium‐denuded human and rat detrusor strips. In rat detrusor strips, 8‐CPT‐2Me cAMP (20 μM) and PMA (10 μM) were applied 6 min before S2 either in the absence or in the presence of dipyridamole (Dipy, 0.5 μM) and ABT 702 (0.1 μM) to inhibit ENT1 and adenosine kinase, respectively; ESI‐09 (10 μM) and chelerythrine (CHL, 5 μM) were also used to show that the inhibitory effects of CPT‐2Me cAMP (20 μM) and PMA (10 μM) were due to EPAC and PKC activation respectively. All inhibitors were present throughout the assay, including S1 and S2. The ordinates are changes in S2/S1 ratios compared to the S2/S1 ratio obtained without addition of any drug (dotted horizontal line). The data are means ± SD of an n number of individuals (black dots). #,* P < .05 (one‐way ANOVA followed by the Dunnett's multicomparison test with a single pooled variance) represent significant differences when compared to the control situation and to the inhibitory effects of 8‐CPT‐2Me cAMP (a) and PMA (b) applied alone to rat detrusor strips respectively

Article Snippet: Observations were performed and analysed with a laser‐scanning confocal microscope (Olympus FluoView, FV1000, Tokyo, Japan; RRID:SCR_016840) and analysed with the Fluoview FV10‐ASW software, RRID:SCR_014215. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antigen Code/identifier Host Dilution Supplier Primary antibodies Anti‐β 2 receptor AAR‐016/RRID:AB_2039718 Rabbit 1:50 Alomone Anti‐β 3 receptor AAR‐017/RRID:AB_2039720 Rabbit 1:50 Alomone Anti‐β 3 receptor MC‐4198/RRID:AB_590525 Rabbit 1:50 MBL International Anti‐EPAC1 ab21236/RRID:AB_2177464 Rabbit 1:75 Abcam Anti‐EPAC1 SC‐28366/RRID:AB_627521 Mouse 1:100 Santa Cruz Anti‐EPAC2 #4156/RRID:AB_1904112 Mouse 1:50 Cell Signaling Tech Anti‐ENT1 ANT‐051/RRID:AB_2341015 Rabbit 1:50 Alomone Secondary antibodies Alexa Fluor 488 anti‐rabbit A‐21206/RRID:AB_2535792 Donkey 1:1,000 Molecular Probes Alexa Fluor 633 anti‐mouse A‐21050/RRID:AB_2535718 Goat 1:1,000 Molecular Probes Open in a separate window Primary and secondary antibodies used to stain human and rat detrusor strips 2.6.

Techniques: Activation Assay

Journal: British Journal of Pharmacology

Article Title: β 3 Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

doi: 10.1111/bph.14921

Figure Lengend Snippet: Schematic representation of the mechanisms involved on β3‐adrenoceptors inhibition of cholinergic neurotransmission in human and rat urinary bladders. β3‐adrenoceptors (β3‐AR) predominantly located in detrusor smooth fibres are positively coupled to AC leading to increases in intracellular cAMP accumulation. The enzymatic breakdown of cAMP by intracellular PDEs and 5′‐nucleotidase (5′NTase) results in the formation of high levels of adenosine (ADO) forcing its translocation to the extracellular milieu, via dipyridamole‐sensitive ENT1 transporters. Besides acting as a source of ADO, cAMP triggers an intracellular signalling cascade leading to preferential EPAC1 over PKA activation on detrusor smooth muscle fibres. Data suggest that EPAC‐induced activation of calcium‐dependent conventional PKC isoforms may be necessary to stimulate ENT1‐mediated ADO outflow. The way PKC facilitates ADO transport to the extracellular compartment is still unknown, but it may result from PKC phosphorylation‐induced increases in the nucleoside transporters capacity and/or by favouring their translocation to the plasma membrane. Once in the extracellular milieu, ADO is free to activate inhibitory A1 receptors (A1R) on cholinergic nerve terminals causing the inhibition of nerve‐evoked ACh release

Article Snippet: Observations were performed and analysed with a laser‐scanning confocal microscope (Olympus FluoView, FV1000, Tokyo, Japan; RRID:SCR_016840) and analysed with the Fluoview FV10‐ASW software, RRID:SCR_014215. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antigen Code/identifier Host Dilution Supplier Primary antibodies Anti‐β 2 receptor AAR‐016/RRID:AB_2039718 Rabbit 1:50 Alomone Anti‐β 3 receptor AAR‐017/RRID:AB_2039720 Rabbit 1:50 Alomone Anti‐β 3 receptor MC‐4198/RRID:AB_590525 Rabbit 1:50 MBL International Anti‐EPAC1 ab21236/RRID:AB_2177464 Rabbit 1:75 Abcam Anti‐EPAC1 SC‐28366/RRID:AB_627521 Mouse 1:100 Santa Cruz Anti‐EPAC2 #4156/RRID:AB_1904112 Mouse 1:50 Cell Signaling Tech Anti‐ENT1 ANT‐051/RRID:AB_2341015 Rabbit 1:50 Alomone Secondary antibodies Alexa Fluor 488 anti‐rabbit A‐21206/RRID:AB_2535792 Donkey 1:1,000 Molecular Probes Alexa Fluor 633 anti‐mouse A‐21050/RRID:AB_2535718 Goat 1:1,000 Molecular Probes Open in a separate window Primary and secondary antibodies used to stain human and rat detrusor strips 2.6.

Techniques: Inhibition, Translocation Assay, Activation Assay

Journal: British Journal of Pharmacology

Article Title: β 3 Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

doi: 10.1111/bph.14921

Figure Lengend Snippet: Primary and secondary antibodies used to stain human and rat detrusor strips

Article Snippet: Observations were performed and analysed with a laser‐scanning confocal microscope (Olympus FluoView, FV1000, Tokyo, Japan; RRID:SCR_016840) and analysed with the Fluoview FV10‐ASW software, RRID:SCR_014215. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Antigen Code/identifier Host Dilution Supplier Primary antibodies Anti‐β 2 receptor AAR‐016/RRID:AB_2039718 Rabbit 1:50 Alomone Anti‐β 3 receptor AAR‐017/RRID:AB_2039720 Rabbit 1:50 Alomone Anti‐β 3 receptor MC‐4198/RRID:AB_590525 Rabbit 1:50 MBL International Anti‐EPAC1 ab21236/RRID:AB_2177464 Rabbit 1:75 Abcam Anti‐EPAC1 SC‐28366/RRID:AB_627521 Mouse 1:100 Santa Cruz Anti‐EPAC2 #4156/RRID:AB_1904112 Mouse 1:50 Cell Signaling Tech Anti‐ENT1 ANT‐051/RRID:AB_2341015 Rabbit 1:50 Alomone Secondary antibodies Alexa Fluor 488 anti‐rabbit A‐21206/RRID:AB_2535792 Donkey 1:1,000 Molecular Probes Alexa Fluor 633 anti‐mouse A‐21050/RRID:AB_2535718 Goat 1:1,000 Molecular Probes Open in a separate window Primary and secondary antibodies used to stain human and rat detrusor strips

Techniques: Staining

Journal: British Journal of Pharmacology

Article Title: β 3 Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

doi: 10.1111/bph.14921

Figure Lengend Snippet: Primary and secondary antibodies used to stain human and rat detrusor strips

Article Snippet: Anti‐ENT1 , ANT‐051/RRID:AB_2341015 , Rabbit , 1:50 , Alomone.

Techniques: Staining

Journal: British Journal of Pharmacology

Article Title: β 3 Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

doi: 10.1111/bph.14921

Figure Lengend Snippet: Mechanism of action of drugs used in this study

Article Snippet: Anti‐ENT1 , ANT‐051/RRID:AB_2341015 , Rabbit , 1:50 , Alomone.

Techniques:

Journal: British Journal of Pharmacology

Article Title: β 3 Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

doi: 10.1111/bph.14921

Figure Lengend Snippet: Effects of mirabegron (0.1 μM, ai–iv), isoprenaline (Isop, 1 μM, bi–iv), CL316,243 (1 μM, ci–ii), fenoterol (1 μM, d), 8‐CPT‐2Me cAMP (20 μM, ei), and 8‐pCPT‐2‐O‐Me‐cAMP‐AM (20 μM, eii) on adenosine outflow from urothelium‐denuned human (a) and rat (b–e) detrusor strips, respectively, in the absence and in the presence of β3‐adrenoceptors antagonists (L‐748,337, 30 nM; SR592230A, 100 nM) and of inhibitors of ENT1 (dipyridamole, 0.5 μM; NBTI, 30 μM) and EPAC (ESI‐09, 10 μM). Mirabegron, isoprenaline, CL316,243, fenoterol, 8‐CPT‐2Me cAMP, and 8‐pCPT‐2‐O‐Me‐cAMP‐AM contacted with the preparations for 15 min before sample collection. Antagonists/inhibitors were applied 15 min before mirabegron, isoprenaline or CL316,243 and were maintained throughout the assay. The ordinates represent the amount of adenosine (ADO, white bars) and inosine (INO, black bars) in pmol·mg−1 of wet weight of the preparations detected by HPLC with diode array in samples collected from the incubation media at 15 min intervals (for details, see Section 2). Data are means ± SD of five to eight individuals; duplicates were performed for each individual experiment. * P < .05 (two‐way ANOVA followed by the Sidak's multiple comparison test) represent significant differences when compared to the control situation (basal/inhibitor alone)

Article Snippet: Anti‐ENT1 , ANT‐051/RRID:AB_2341015 , Rabbit , 1:50 , Alomone.

Techniques: Incubation

Journal: British Journal of Pharmacology

Article Title: β 3 Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

doi: 10.1111/bph.14921

Figure Lengend Snippet: Immunolocalization of (a) β3‐ and (b) β2‐adrenoceptors, (c) EPAC1 and (d) EPAC2 isoforms, and (e) ENT1 in transverse sections of the human (left hand‐side panels) and rat (right hand‐side panels) detrusor by confocal microscopy. β3‐Adrenoceptor and ENT1 immunoreactivities follow a plasma membrane staining pattern, whereas EPAC1 is compatible with a diffuse cytoplasmic distribution. Two distinct β3 (AAR‐017 and MC‐4198 raised in rabbits) and EPAC (ab21236 and SC‐28366 raised in rabbits and mice, respectively) antibodies were used as indicated. (f) Micrographs show that β3‐adrenoceptors (green) and EPAC (red) co‐localize in human detrusor, as identified by the yellow staining in the merge image shown in the right hand‐side image. Images are representative of four different individuals. Differential interference contrast (DIC) images are shown for comparison. Scale bar = 50 μm. Bottom panels are representative immunoblots of β2‐ and β3‐adrenoceptors (g) and EPAC1 and EPAC2 isoforms (h) in urothelium‐denuded detrusor homogenates of one male human subject (H) and one rat (R) run in parallel using the same antibodies as for the immunohistochemical staining. The highly conserved GAPDH enzyme (EC 1.2.1.12, MW~37 kDa) and β‐actin (MW~43 kDa) were used as reference proteins for β−adrenoceptors and EPAC respectively. Images are representative of three different individuals. Gels were loaded with 150 μg of protein. Host species for antibody production were rabbit (rb) and mouse (ms)

Article Snippet: Anti‐ENT1 , ANT‐051/RRID:AB_2341015 , Rabbit , 1:50 , Alomone.

Techniques: Confocal Microscopy, Staining, Western Blot, Immunohistochemical staining

Journal: British Journal of Pharmacology

Article Title: β 3 Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

doi: 10.1111/bph.14921

Figure Lengend Snippet: (a) Inhibitory effect of mirabegron on electrically evoked [3H]ACh release from urothelium‐denuded human detrusor strips. Ordinates represent tritium outflow expressed in scintillations per min (cpm). Abscissa indicates the times at which samples were collected. [3H]ACh release was elicited by electrical field stimulation (10 Hz, 200 pulses of 0.2‐ms duration) twice, starting at 4th (S1) and 13th (S2) minutes after the end of washout (zero time). Mirabegron (0.1 μM) was added to the incubation media 6 min before S2 (black horizontal bar). Panels b, c and d show the inhibitory effects of mirabegron, isoprenaline and forskolin on evoked [3H]ACh release human (b) and rat (c and d) detrusor strips respectively. Mirabegron (0.1 μM), isoprenaline (1 μM), and forskolin (3 μM) were applied 6 min before S2 either in the absence or in the presence of selective inhibitors of PKA (H‐89, 10 μM), EPAC (ESI‐09, 10 μM), and ENT1 (Dipy, 0.5 μM), as well as of the adenosine A1 receptor antagonist, DPCPX (0.1 μM). All inhibitors and A1 receptor antagonist were present throughout the assay, including S1 and S2. The ordinates are changes in S2/S1 ratios compared to the S2/S1 ratio obtained without addition of any drug (dotted horizontal line). The data are means ± SD of an n number of individuals (black dots). #,* P < .001 (one‐way ANOVA followed by the Dunnett's multicomparison test with a single pooled variance) represent significant differences when compared to the control situation and to the inhibitory effects of mirabegron (b), isoprenaline (c) and forskolin (d) applied alone respectively

Article Snippet: Anti‐ENT1 , ANT‐051/RRID:AB_2341015 , Rabbit , 1:50 , Alomone.

Techniques: Incubation

Journal: British Journal of Pharmacology

Article Title: β 3 Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

doi: 10.1111/bph.14921

Figure Lengend Snippet: Activation of EPAC (with 8‐CPT‐2Me cAMP, a) and PKC (with phorbol 12‐myristate 13‐acetate, PMA, b) decrease electrical evoked (10 Hz, 200 pulses of 0.2‐ms duration) [3H]ACh release from urothelium‐denuded human and rat detrusor strips. In rat detrusor strips, 8‐CPT‐2Me cAMP (20 μM) and PMA (10 μM) were applied 6 min before S2 either in the absence or in the presence of dipyridamole (Dipy, 0.5 μM) and ABT 702 (0.1 μM) to inhibit ENT1 and adenosine kinase, respectively; ESI‐09 (10 μM) and chelerythrine (CHL, 5 μM) were also used to show that the inhibitory effects of CPT‐2Me cAMP (20 μM) and PMA (10 μM) were due to EPAC and PKC activation respectively. All inhibitors were present throughout the assay, including S1 and S2. The ordinates are changes in S2/S1 ratios compared to the S2/S1 ratio obtained without addition of any drug (dotted horizontal line). The data are means ± SD of an n number of individuals (black dots). #,* P < .05 (one‐way ANOVA followed by the Dunnett's multicomparison test with a single pooled variance) represent significant differences when compared to the control situation and to the inhibitory effects of 8‐CPT‐2Me cAMP (a) and PMA (b) applied alone to rat detrusor strips respectively

Article Snippet: Anti‐ENT1 , ANT‐051/RRID:AB_2341015 , Rabbit , 1:50 , Alomone.

Techniques: Activation Assay

Journal: British Journal of Pharmacology

Article Title: β 3 Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

doi: 10.1111/bph.14921

Figure Lengend Snippet: Schematic representation of the mechanisms involved on β3‐adrenoceptors inhibition of cholinergic neurotransmission in human and rat urinary bladders. β3‐adrenoceptors (β3‐AR) predominantly located in detrusor smooth fibres are positively coupled to AC leading to increases in intracellular cAMP accumulation. The enzymatic breakdown of cAMP by intracellular PDEs and 5′‐nucleotidase (5′NTase) results in the formation of high levels of adenosine (ADO) forcing its translocation to the extracellular milieu, via dipyridamole‐sensitive ENT1 transporters. Besides acting as a source of ADO, cAMP triggers an intracellular signalling cascade leading to preferential EPAC1 over PKA activation on detrusor smooth muscle fibres. Data suggest that EPAC‐induced activation of calcium‐dependent conventional PKC isoforms may be necessary to stimulate ENT1‐mediated ADO outflow. The way PKC facilitates ADO transport to the extracellular compartment is still unknown, but it may result from PKC phosphorylation‐induced increases in the nucleoside transporters capacity and/or by favouring their translocation to the plasma membrane. Once in the extracellular milieu, ADO is free to activate inhibitory A1 receptors (A1R) on cholinergic nerve terminals causing the inhibition of nerve‐evoked ACh release

Article Snippet: Anti‐ENT1 , ANT‐051/RRID:AB_2341015 , Rabbit , 1:50 , Alomone.

Techniques: Inhibition, Translocation Assay, Activation Assay