Abstract 463: Transregulation and Plasma Membrane Recruitment of the Dopamine-1 (D1R) and Angiotensin Type 2 (AT2R) Receptors in Human Renal Proximal Tubule Cells (RPTC)

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Both the D1R and AT2R are key natriuretic receptors in that stimulation by dopamine agonists or AT2R agonist angiotensin III (AngIII) increases cAMP and cGMP, respectively, causing inhibition of sodium reabsorption. The interdependence of D1R and AT2R stimulation on receptor translocation has not been studied. We utilized normally coupled RPTC (nRPTC) and RPTC with defective D1R coupling to adenylyl cyclase (uRPTC). A 1 hr addition of the cell permeable cyclic nucleotide analogs 8Br-cGMP (1 mM) or 8Br-cAMP (1 mM) caused a similar recruitment of both D1R and AT2R, as measured by cell surface biotinylation and western blotting (see table below).Flow cytometry confirmed these results using fluorescently labeled extracellular epitope-specific D1R and AT2R antibodies. When 8Br-cAMP and 8Br-cGMP were added together, no added effects were seen. AngIII-alone increased plasma membrane recruitment of D1R (1.23±0.04 fold, n=6, p<0.05, VEH=0.63) and AT2R (1.65±0.10 fold, n=6, p<0.05, VEH=0.69) in nRPTC but not in uRPTC. The nRPTC response was completely blocked by the AT2R specific antagonist PD123319 or the D1R antagonist LE300. Inhibition of either PKA with adenosine-3’, 5’-cyclic monophosphorothioate Rp-Isomer (100 μM) or PKG with 8-(4-chlorophenylthio) guanosine-3’, 5’ -cyclic monophosphorothioate Rp-isomer (10 μM) completely inhibited the cell surface recruitment of both D1R and AT2R when stimulated with 8Br-cAMP or 8Br-cGMP, respectively.In conclusion, we found a novel AngIII-interdependent AT2R and D1R cell surface recruitment in human nRPTC cells. Furthermore, defects in this pathway in uRPTC can be bypassed by incubating the cells with either 8Br-cAMP or 8Br-cGMP.

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