Abstract 480: Dopamine d1 Receptor Requires Both gQ/11? and gI for Downstream Signaling in Human Kidney 2 Cells

Abstract

Renal dopamine D1 receptor (D1R) plays pivotal role in maintaining sodium homeostasis and blood pressure (BP). It does so by either coupling to G s or G q proteins and engaging downstream signaling molecules. Renal proximal tubular cells from human kidney, HK-2 cells, are important ex vivo tool to study mechanistic aspect of D1R signaling cascade involved in BP regulation. Recently, however, it is reported that D1R is uncoupled from G s protein suggesting attenuation of the receptor’s downstream signaling in HK-2 cells. In this study we wanted to determine whether D1R is coupled to Gq proteins in HK-2 cells . To test this, we measured the activity of a downstream surrogate marker of G q stimulation, protein kinase C (PKC), by undertaking following experimental manipulations and comparing them with a known activator of G q , angiotensin II (Ang II). Fenoldopam (FD, 1μM, 10 min), a D1R agonist, increased PKC activity to the levels of a direct activator (PMA, a phorbol ester, 1μM, 15 min) of PKC activity (in ng/μl: Control vs FD vs PMA: 0.31 + 0.04 vs 1.08 + 0.05 vs 1.02 + 0.07). FD-mediated PKC stimulation was attenuated in the presence of D1R antagonist ( SCH23390 , 10μM), PKC inhibitor (chelerythrin chloride, 1μM), G q depletion (siRNA, 200 nM) and pertussis toxin (PTX, an inhibitor of G i protein, 100 ng/ml). Similarly, Ang II (1μM, 10 min) increased PKC activity (in ng/μl: Control vs Ang II: 0.31 + 0.04 vs 1.05 + 0.15) which was attenuated in the presence of Ang II AT1 receptor blocker (candesartan, 1μM), chelerythrin chloride, G q siRNA and PTX. Furthermore, FD increased 35 S-GTPγS binding, an index of D1R function (cpm/μg protein: Control vs FD: 414 + 104 vs 1188 + 43), and abundance of G q (Density units: Control vs FD: 30 + 8 vs 47 + 12) in the cellular membranes. Our results suggest that (i) D1R is functional; (ii) not only D1R but also AT1R downstream signaling is intact, which requires (iii) both G q and G i G-proteins in HK-2 cells.