Abstract 480: Dopamine d1 Receptor Requires Both gQ/11? and gI for Downstream Signaling in Human Kidney 2 Cells
Renal dopamine D1 receptor (D1R) plays pivotal role in maintaining sodium homeostasis and blood pressure (BP). It does so by either coupling to Gs or Gq 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 Gs 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 Gq stimulation, protein kinase C (PKC), by undertaking following experimental manipulations and comparing them with a known activator of Gq, 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), Gq depletion (siRNA, 200 nM) and pertussis toxin (PTX, an inhibitor of Gi 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, Gq siRNA and PTX. Furthermore, FD increased 35S-GTPγS binding, an index of D1R function (cpm/μg protein: Control vs FD: 414 + 104 vs 1188 + 43), and abundance of Gq (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 Gq and Gi G-proteins in HK-2 cells.
- © 2012 by American Heart Association, Inc.