Abstract P320: Cooperative Signaling and Membrane Recruitment of the Dopamine-1 (D 1 R) and Angiotensin Type 2 (AT 2 R) Receptors in Human Renal Proximal Tubule Cells (RPTC)

Abstract

The D 1 R and AT 2 R are interdependent natriuretic receptors that are critical for the regulation of renal sodium balance and are implicated in both hypertension and salt-sensitivity. D 1 R and AT 2 R stimulation with the D 1 R agonist fenoldopam (FEN) and Angiotensin III (AngIII), selectively increases cAMP and cGMP, respectively, leading to D 1 R and AT 2 R recruitment to the cell surface (measured by fluorescent extracellular receptor epitope specific antibodies). The interdependence of the D 1 R and AT 2 R recruitment was investigated following AT 2 R stimulation with AngIII (10 nmol/L 1 hour) which leads to D 1 R cell surface recruitment using a normally D 1 R/Gs coupled cell line (i22) (VEH 8209±863, AngIII 19485±3425 RFU, n=6, p<0.05) but not in a D 1 R uncoupled from Gs cell line (i19). These data were verified by increasing intracellular sodium with monensin (100 μM, 1 hr) and measuring cell surface binding with a fluorescent labelled D 1 R agonist SKF83566 (10 nmol/L, 1 hr, i22 1.67±0.7 fold, n=5, p<0.0001, i19, NS). The importance of dopamine in regulating these receptor actions was shown by stimulating the AT 2 R with AngIII and measuring D 1 R recruitment followed by blockade of amino acid decarboxylase using either carbidopa or benzeraside (100 μM each, 1 hour). Additionally, FEN stimulated D 1 R surface recruitment is blocked by the AT 2 R inhibitor PD123319 (1 μM, 1 hour) and the coupling defect is fully rescued by 8Br-cGMP (1 mmol/L, 1 hour), a cell permeable second messenger normally thought to signal specifically through the AT 2 R. We then show that FEN stimulation leads to increased production of AngIII only in the D 1 R/Gs coupled i22 cell line (5.82±0.43 fold, n=6, p<0.05) but not in i19 the D 1 R/Gs uncoupled cell line and this production was blocked with an aminopeptidase A inhibitor, EC-33 (10 μmol/L, 1 hour). Addition of 8Br-cAMP (1mmol/L 1 hour) not only leads to an increase in AngIII production, but also cGMP production (4.33±0.36 fold, n=6, p<0.05) that is blocked by PD123319 (potent, selective, non-peptide angiotensin AT 2 R antagonist, 1 μM, 1 hour). In summary we not only show that dopaminergic stimulation leads to increased AngIII production in a D 1 R coupled manner, but also that AngIII stimulation of AT 2 R leads to dopaminergic activation in a D 1 R coupled manner.