Abstract 469: The Role of PP2A in Angiotensin AT2R and Dopamine-1 Receptor Membrane Recruitment in Human Renal Proximal Tubule Cells

Abstract

The renal dopamine-1 receptor (D1R) and angiotensin type 2 receptor (AT2R) are two important receptors that regulate renal sodium reabsorption through heterologous and autologous plasma membrane recruitment following stimulation of protein kinase A (PKA) via D1R and protein kinase G (PKG) via AT2R. Using the cell permeable analog of PKA (8Br-cAMP) along with the PKG analog 8Br-cGMP, we did not find additive effects, which led to our hypothesis that downstream protein phosphatase 2A (PP2A) is the common link in the D1R/AT2R interaction. To test our hypothesis, we used two human renal proximal tubule cell lines (RPTC): one with normal D1R coupling (nRPTC) to adenylyl cyclase (AC) and a 2nd cell line with a defect in D1R coupling to AC (uncoupled or uRPTC). A dose response curve for 8Br-cAMP and 8Br-cGMP using cell surface Dylight-649-AngIII binding for 1 hr in the presence of losartan (a measure of surface AT2R) showed maximal recruitment at 1 mM for both 8Br-cAMP and 8Br-cGMP. EC50 of 8Br-cAMP was 129.9±60.58 μM (log EC50 was -3.9). EC50 of 8Br-cGMP was 99.17±48.13 μM (log EC50 was -4.0) A 1 hr addition of 1 mM 8Br-cAMP or 8Br-cGMP resulted in increased PP2A activity. Specifically, PP2A activity increased by 1.59±0.24 fold (N=3, P<0.05 vs vehicle (VEH, baseline value of 1216 pmoles of phosphate at 650 nm) when 8Br-cAMP was added. The addition of 8Br-cGMP induced a 1.48±0.33 fold increase in PP2A (N=3, P< 0.05 vs VEH at 1216 pmoles of phosphate at 650 nm). Using flow cytometry for cell surface D1R and AT2R, recruitment of either receptor (AT2R or D1R) was completely blocked by okadaic acid (OA, a specific PP2A phosphatase inhibitor at 10 nM), whether stimulating D1R with fenoldopam (FEN), AT2R with AngIII, or using cell permeable downstream second messenger agonists 8Br-cAMP or 8Br-cGMP. Similarly, addition of the AC inhibitor SQ 22536 (10 μM) or the guanylyl cyclase (GC) inhibitor ODQ (10 μM) completely blocked FEN, AngIII, 8Br-cAMP and 8Br-cGMP-induced D1R and AT2R cell surface recruitment. In summary, we determined that PP2A is the common mediator of D1R and AT2R cell surface recruitment and may be an important target for regulating renal sodium homeostasis.