Abstract P429: Human Renal Proximal Tubule Cell Paracrine Production Of Dopamine Is Increased In High Sodium Stress And Reduced In Dopamine D ₁ Receptor Uncoupled Cells

Angiotensin II type-2 receptor (AT 2 R) stimulation with Ang III leads to the dopamine D1 Receptor (D 1 R) plasma membrane recruitment in normally cAMP coupled human renal proximal tubule cells (nRPTC). While extending these studies to include D 1 R cAMP uncoupled (uRPTC), we determined that Ang III stimulation did not lead to D 1 R plasma membrane recruitment. We then hypothesized that local production and secretion of dopamine may be stimulated by sodium stress as well as in this AT 2 R to D 1 R recruitment pathway. A stable clone of Dlight 1.3 HEK293 cell line expressing the GFP dopamine biosensor functioned properly through multiple titration experiments using fenoldopam (D 1 R/D 5 R agonist) direct binding, showing dose dependent increase in the Dlight biosensor fluorescence, as well as a dose dependent increase and decrease of intracellular dopamine production in high or low sodium conditions respectively. Dopamine secretion was examined in a co-culture experiment using a stable clone of D 5 R and ICUE3 cAMP reporter HEK293 cell line. Local extracellular dopamine produces a change in the FRET biosensor. Both nRPTCs and uRPTCs make and secrete dopamine and this is increased further with incubation with L-Dopa. Interestingly 8Br-cGMP lead to an increase in dopamine secretion only in nRPTCs by 18.28±3.2 fold FRET nRPTC vs uRPTC (p<0.001, N=9 per group). Further experiments examined autocrine D 1 R signaling second messenger cAMP levels after Ang III stimulation at differing sodium levels. Only nRPTCs produced more cAMP (8.53±0.73 fold increase vs NS VEH control, p<0.01, N=8 per group) when stimulated with Ang III in high sodium conditions, this being from local dopamine production, secretion and D 1 R stimulation. These studies provide new insights into the autocrine dopamine production by RPTCs and D 1 R/AT 2 R trans-regulation that play a role in maintaining sodium and blood pressure homeostasis.