Dopamine D1‐like receptors (D1R and D5R) differently regulate phospho‐PKC in HEK‐293 cells heterologously expressing human D1R or D5R (1097.6)

Abstract

Dopamine cellular signaling via the D1 receptor (D1R) involves a cross talk between protein kinase A (PKA) and protein kinase C (PKC) in HEK‐293 cells heterologously expressing human D1R (hD1R). We tested the hypothesis that dopamine D1‐like receptors (D1R and D5R) differentially regulate phospho‐PKC isoforms in HEK‐293 cells heterologously expressing hD1R or hD5R. In hD1R but not hD5R cells, short‐term (15, 30, and 60min) stimulation with the D1‐like receptor agonist, fenoldopam, (1μM) increased phosphorylated (corrected by total PKC, relative to vehicle treatment) PKCθS676 (1.79±0.21, 15 min; 1.54±0.10, 30min; and 1.40±0.10, 60min, n=6), PKCµS744 (1.65±0.25, 15 min), and PKCµS916 (1.60±0.09, 15min) (n=5‐6). The effect on PKCµS916 and PKCµS744 decreased with the time; no other phospho‐PKC isoforms were affected. In hD5R but not hD1R cells, long‐term (8 and 24hr) stimulation with fenoldopam (1µM) caused an increase in PKCβ2S660 and PKCηS674; there were no other phospho‐PKC proteins affected. The long‐term fenoldopam‐mediated increase in PKCβ2S660 and PKCηS674 proteins was blocked by the D5R (in the absence of D1R) antagonist Sch23990 (5µM) (PKCβ2S660: fenoldopam=1.66±0.14 vs. control=1.06±0.04, Sch 23390=0.89±0.25 and Sch+fenoldopam=1.11±0.143; PKCηS674: Fen=1.45±0.05 vs. Con=1.04±0.04, Sch=0.71±0.20 and S+F=1.02±0.06) (P<0.05, n=4, Newman‐Keuls test). The D5R‐mediated stimulation (1μM/24 hr) of PKCβ2S660 and PKCηS674 proteins was also blocked by a PKC inhibitor bisindolylmaleimide II (1µM) and two PKA inhibitors, H89 (10µM) and Rp‐cAMP (50µM). The D5R‐mediated (1μM/24 hr) increase in PKCβ2S660 and PKCηS674 was also blocked by phosphoinositide 3‐kinase inhibitor LY294002 (1µM). We conclude that short‐term stimulation of D1R increases phospho‐PKCηS676 and PKCµS proteins while long‐term stimulation of D5R increases PKCβ2S660, and PKCηS674 proteins via PKA and PI3K signaling pathways.Grant Funding Source: HL023081, HL074940, DK039308