Short‐ and long‐term A₃ adenosine receptor activation inhibits the Na⁺/H⁺ exchanger NHE3 activity and expression in opossum kidney cells

Abstract

The renal function of the A(3) adenosine receptor (A3AR) is poorly characterized. In this study, we report that the A3AR-selective agonist, 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purine-9-yl]-1-deoxy-N-methyl-b-D-ribofuranuronamide (2-Cl-IBMECA) regulates the Na+/H+ exchanger-3 (NHE3) in a dose- and time-dependent fashion. In opossum kidney (OK) cells, 2-Cl-IBMECA at high (10(-6) M) and low (10(-8) M) dose inhibits NHE3 by a multiphasic time course with an acute phase of NHE3 inhibition from 15 min to 1 h, followed by a chronic phase of NHE3 inhibition from 24 to 48 h. Pre-incubation with either the selective A3AR-antagonist MRS1523 (10(-7) M) or the protein kinase C inhibitor, Calphostin C (10(-8) M) completely blocked 10(-6) M 2-Cl-IBMECA-induced acute (15 min) and chronic (24 h) phases of NHE3 inhibition. In contrast, the acute inhibitory phase (15 min) of 10(-8) M 2-Cl-IBMECA was completely prevented only when Calphostin C (10(-8) M) was added in conjunction with the protein kinase A inhibitor, H89 (10(-7) M). Acute (15 or 30 min depending on the A3AR-agonist concentration) A3AR-dependent inhibition of NHE3 activity was accompanied by decrease in cell surface NHE3 protein with no change in total NHE3 antigen. Chronic (24 h) A3AR-mediated down-regulation of NHE3 was associated with reduction of surface NHE3, decreased total NHE3 protein (70%) and a paradoxical rise of NHE3 RNA (40%). In summary, these results indicate that A3AR directly regulates NHE3 at multiple levels in a complex pattern. A3AR-dependent short- and long-term inhibition of NHE3 may be a fundamental mechanism of net sodium and fluid balance.

Chronic regulation of the renal Na+/H+exchanger NHE3 by dopamine: translational and posttranslational mechanisms

“…Immunocytochemistry was performed where OKP cells were fixed (4% paraformaldehyde in PBS ϫ 20 min) and permeabilized (0.2% saponin, 3% gelatin ϫ 20 min). The cells were then incubated with primary antibody (monoclonal anti-NHE3 3H3) (23,26,32,33,41,68), followed by incubation with FITC-conjugated donkey anti-mouse polyclonal antibodies (Jackson ImmunoResearch Laboratories). Confocal fluorescent images were visualized through a Zeiss ϫ100 objective lens using a Zeiss LSM-410 laser-scanning confocal microscope.…”

The Calcineurin Homologous Protein-1 Increases Na+/H+-Exchanger 3 Trafficking via Ezrin Phosphorylation

“…The enhanced chemoluminescence system was from Amersham Biosciences (Piscataway, NJ). The following primary antibodies were used: monoclonal mouse anti-opossum NHE3 antibody (#3H3) to detect NHE3, 78 anti-CHP rabbit polyclonal antibody (kindly provided by Dr. Diane L. Barber, University of California, San Francisco, CA) to detect CHP1, 48 anti-ezrin rabbit polyclonal antibodies (Abcam, Cambridge, MA) to detect total and phospho-threonine 567 ezrin, anti-myc rabbit polyclonal antibody (Abcam, Cambridge, MA) to detect myc-tagged proteins, anti-GFP rabbit polyclonal antibody (Invitrogen, Carlsbad, CA) to detect eGFP-tagged proteins, and ␤-actin mouse monoclonal antibody (Sigma, St. Louis, MO) to detect ␤-actin.…”

“…Cultures were incubated in a humidified 95% air/5% CO 2 atmosphere at 37°C and subcultured weekly by trypsinization using 0.1% trypsin/0.5 mM EGTA in PBS. 48,78 Cells generally reached confluence within 3 to 4 d, and confluent cells were serum-deprived for 2 d before assay. Studies on OK cells were performed between passages 23 and 57.…”

“…48,78 OK cells were grown to confluence on 100-mm culture dishes. Cells were rinsed in Ca/Mg/PBS (containing 150 mM NaCl, 10 mM Na 2 HPO 4 , pH 7.4, 0.1 mM CaCl 2 , 1 mM MgCl 2 ) and incubated with the arginine-and lysine-reactive NHS-SS-biotin (2 mg/ml) in buffer (containing 150 mM NaCl, 10 mM triethanolamine, pH 7.4, 2 mM CaCl 2 ) for 1 h. After being quenched in Ca/Mg/PBS supplemented with 100 mM glycine, cells were lysed in radio-immunoprecipitation assay (RIPA) buffer (containing 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 1% (vol/vol) Triton X-100, 0.5% (w/v) deoxycholate, 0.1% (w/v) SDS), and centrifuged at 100,000 ϫ g for 25 min, at 4°C.…”

The reduction of Na/H exchanger-3 protein and transcript expression in acute ischemia–reperfusion injury is mediated by extractable tissue factor(s)

“…63,64,69,70 RNA isolation was carried out using an RNeasy Mini Kit (QIAGEN, Valencia, CA) and reverse transcription was carried out using the ThermoScript RT-PCR system (Invitrogen, Carlsbad, CA). In the summary of data of total NHE3 antigen, the antigen signal was normalized to b-actin mouse monoclonal antibody (Sigma).…”