Schoner W. may be due, not only to inhibition of Na+/K+-ATPase activity, but also its ability to interfere with MR-dependent expression of the Na/K/H exchanger in the late distal nephron. essentially as previously explained [20]. Briefly, 1 g of the MR expression plasmid was transfected into Cos-1 cells using Lipofectamine and 24 h thereafter media was aspirated from wells and replaced with DMEM made up of 5% sFBS and 1.0 nM [3H]aldosterone (39.0 Ci/mmol) (Perkin Elmer Life Sciences, Shelton, CT) in the presence of either vehicle, up to 200 nM aldosterone or increasing concentrations (from 10?8 to 10?6 M) of unlabeled MBG. After incubation for 2 hours at 37C media was aspirated, cells were washed 3 times with ice-cold PBS and incubated in 100% ethanol for 10 minutes at room temperature to extract bound steroid. The amount of MR-bound [3H]aldosterone in the ethanol extract was quantified with a Beckman LS 6500 scintillation counter (Beckman Instrument, Fullerton, CA) and Biodegradable Counting Scintillant (Amersham). Coimmunoprecipitation Cos-1 cells were plated at a density of 1 1 106 cells/100 mm Petri dish in phenol-red free DMEM made up of 10% sFBS, and transfected with 500 ng each of pCMV-Flag-SRC-3 and pRShMR using Lipofectamine. After 24 h, cells were treated with either 0.1% ethanolic vehicle, 10?8 M aldosterone or 10?8 M aldosterone with 10?6 MBG for 30 min and then harvested and incubated in lysis buffer [20 mM Tris (pH 7.5) containing 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 5% Rabbit Polyclonal to CATZ (Cleaved-Leu62) glycerol, 1mM Na3VO4 and 1mM NaF) supplemented with a complete Mini-Tablets protease inhibitor tablet (Roche Diagnostics) at 4C with rotation for 30-60 min. The cell lysate was centrifuged for 5 min at 20,000 g, and 1.5 mg cell lysate protein was pre-cleared with 25 l of a 50% slurry of prewashed protein G agarose beads (Santa Cruz) in a total volume of 1 ml lysis buffer. The producing lysate was incubated with rotation with 20 l EZview? Red ANTI-FLAG? M2 Affinity Gel (Sigma) or 3 g normal mouse IgG (Santa Cruz) for 2 hours at 4C, prior to the addition of 25 l of a 50% slurry of prewashed protein G agarose beads and an additional 2 h incubation with rotation. The immunocomplex was washed 4 occasions with lyses buffer at 4C and subsequently heated to 95C for 10 min in 25 l of 1x Laemmli buffer BET-IN-1 and resolved by a NuPAGE Novex 3-8% Tris-Acetate gel (Invitrogen). The Western blot was probed with anti-AIB1 (BD Biosciences, San Jose, CA), anti-MR or anti-actin antibodies in PBS-T made up of 5% skim milk powder, followed by anti-mouse or anti-goat antibodies conjugated to horseradish peroxidase. Protein bands were detected using ECL Plus reagents as explained above. RESULTS In order to ascertain whether MBG altered the transcriptional activity of the mineralocorticoid receptor, expression plasmids for this receptor along with the PRE-E1b-Luc reporter gene were transfected into Cos-1 kidney cells. The PRE-E1b-Luc synthetic target gene possesses two copies of a DNA sequence, termed the progesterone response element, which is identical to a response element for the MR [21], linked upstream to TATA box and luciferase reporter gene. As expected, treatment of Cos-1 cells with aldosterone led to a strong induction of luciferase gene expression (Fig. 1A). Although exposure of these cells to MBG alone did not impact the very low basal activity of the MR, it was observed that treatment of cells with 10?6 M MBG reduced MR transcriptional activity induced by 10?9 M aldosterone by 65% and activity induced by 10?8 M aldosterone was inhibited by 50%. Western blot analysis (Fig. 1B) revealed the anticipated reduction in MR expression in cells treated with aldosterone [22]. MBG alone did not impact MR expression, and MR levels in cells treated with aldosterone and MBG were much like those treated with aldosterone alone indicating that MBG did not interfere with MR transcriptional activity via alterations in MR expression. BET-IN-1 Open in a separate window Physique 1 Inhibition of MR transcriptional activity by MBG. A) Cos-1 cells were transfected with 100 ng pRShMR and 1 g of PRE-E1b-Luc and treated with vehicle (0.1% ethanol) or the indicated concentrations of aldosterone (Ald) or marinobufagenin (MBG). B) Cos-1 cells were transfected with 500 ng pRShMR and 1 g.[PubMed] [Google Scholar]. reduced interaction between the SRC-3 coactivator and the MR. Thus, the ability of MBG to cause a natriuresis may be due, not only to inhibition of Na+/K+-ATPase activity, but also its ability to interfere with MR-dependent expression of the Na/K/H exchanger in the late distal nephron. essentially as previously explained [20]. Briefly, 1 g of the MR expression plasmid was transfected into Cos-1 cells using Lipofectamine and 24 h thereafter media was aspirated from wells and replaced with DMEM made up of 5% sFBS and 1.0 nM [3H]aldosterone (39.0 Ci/mmol) (Perkin Elmer Life Sciences, Shelton, CT) in the presence of either vehicle, up to 200 nM aldosterone or increasing concentrations (from 10?8 to 10?6 M) of unlabeled MBG. After incubation for 2 hours at 37C media was aspirated, cells were washed 3 times with ice-cold PBS and incubated in 100% ethanol for 10 minutes at room temperature to extract bound steroid. The amount of MR-bound [3H]aldosterone in the ethanol extract was quantified with a Beckman LS 6500 scintillation counter (Beckman Instrument, Fullerton, CA) and Biodegradable Counting Scintillant (Amersham). Coimmunoprecipitation Cos-1 cells were plated at a density of 1 1 106 cells/100 mm Petri dish in phenol-red free DMEM made up of 10% sFBS, and transfected with 500 ng each of pCMV-Flag-SRC-3 and pRShMR using Lipofectamine. After 24 h, cells were treated with either 0.1% ethanolic vehicle, 10?8 M aldosterone or 10?8 M aldosterone with 10?6 MBG for 30 min and then harvested and incubated in lysis buffer [20 mM Tris (pH 7.5) containing 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 5% glycerol, 1mM Na3VO4 and 1mM NaF) supplemented with a complete Mini-Tablets protease inhibitor tablet (Roche Diagnostics) at 4C with rotation for 30-60 min. The cell lysate was centrifuged for 5 min at 20,000 g, and 1.5 mg cell lysate protein was pre-cleared with 25 l of a 50% slurry of prewashed protein G agarose beads (Santa Cruz) in a total volume of 1 ml lysis buffer. The producing lysate was incubated with rotation with 20 l EZview? Red ANTI-FLAG? M2 Affinity Gel (Sigma) or 3 g normal mouse IgG (Santa Cruz) for 2 hours at 4C, prior to the addition of 25 l of a 50% slurry of prewashed protein G agarose beads and an additional 2 h incubation with rotation. The immunocomplex was washed 4 occasions with lyses buffer at 4C and subsequently heated to 95C for 10 min in 25 l of 1x Laemmli buffer and resolved by a NuPAGE Novex 3-8% Tris-Acetate gel (Invitrogen). The Western blot was probed with anti-AIB1 (BD Biosciences, San Jose, CA), anti-MR or anti-actin antibodies in PBS-T made up of 5% skim milk powder, followed by anti-mouse or anti-goat antibodies conjugated to horseradish peroxidase. Protein bands were detected using ECL Plus BET-IN-1 reagents as explained above. RESULTS In order to ascertain whether MBG altered the transcriptional activity of the mineralocorticoid receptor, expression plasmids for this receptor along with the PRE-E1b-Luc reporter gene were transfected into Cos-1 kidney cells. The PRE-E1b-Luc synthetic target gene possesses two copies of a DNA sequence, termed the progesterone response element, which is identical to a response element for the MR [21], linked upstream to TATA box and luciferase reporter gene. As expected, treatment of Cos-1 cells with aldosterone led to BET-IN-1 a strong induction of luciferase gene expression (Fig. 1A). Although exposure of these cells to MBG alone did not impact the very low basal activity of the MR, it was observed that treatment of cells with 10?6 M MBG reduced MR transcriptional activity induced by 10?9 M aldosterone by 65% and activity induced by 10?8 M aldosterone was inhibited by 50%. Western blot analysis (Fig. 1B) revealed the anticipated reduction in MR expression in cells treated with aldosterone [22]. MBG alone did not impact MR expression, and MR levels in cells treated with aldosterone and MBG were much like those treated with aldosterone alone indicating that MBG did not interfere with MR transcriptional activity.