Human kidney-2 cells harbor functional dopamine D1 receptors that require Giαfor Gq/11αsignaling

Abstract

A recent study demonstrated that the dopamine D1 receptor (D1R) is nonfunctional in human kidney cells, HK2 cells, in terms of their inability to couple to G s protein in response to the D1R agonist fenoldopam. Since D1R also couples to G q protein, we tested whether D1R is functional in HK2 cells in terms of their ability to couple to G q and produce downstream signaling. For comparison, we also studied another receptor, angiotensin II type 1 receptor (AT1R) known to couple to G q. Protein kinase C (PKC) and 86rubidium transport activities were determined as surrogate downstream signaling markers. Fenoldopam and angiotensin II increased PKC activity, which decreased in the presence of respective receptor antagonists (SCH23390 for D1R; candesartan for AT1R), PKC (chelerythrine chloride) and G i protein (pertussis toxin) inhibitors and G q/11α siRNA. Furthermore, fenoldopam and angiotensin II increased 35S-GTPγS binding, an index of receptor-G protein coupling, which decreased with pertussis toxin and in G q/11α-depleted cells. Also, fenoldopam-mediated inhibition of 86rubidium transport (an index of Na-K-ATPase activity) was attenuated with SCH23390, chelerythrine chloride, pertussis toxin, and G q/11α siRNA. Moreover, fenoldopam caused a decrease in cytosolic and increase in membranous abundance of G q/11α. The immunoprecipitated levels of G q/11α in the membranes were greater in fenoldopam-treated cells, and G iα coimmunoprecipitated with G q/11α. Our results suggest that both D1R and AT1R are functional in HK2 cells, enabling G q-mediated downstream signaling in a G i dependent manner.

Grape Powder Improves Age‐Related Decline in Mitochondrial and Kidney Functions in Fischer 344 Rats

“…Western immunoblotting in cytosolic and nuclear fractions and tissue homogenates were performed using standard methods as described previously [ 29 ]. Tissue homogenates were made in lysis buffer (mM: 20 Tris-HCl (pH 7.5), 150 NaCl, 1 Na 2 EDTA, 1 EGTA, 1% Triton, 2.5 sodium pyrophosphate, 1 beta-glycerophosphate, 1 Na 3 VO 4 , 1 μ g/mL leupeptin, and protease inhibitor cocktail) as described [ 29 ]. Protein concentrations in the cytosolic and nuclear fractions and in homogenates were determined by BCA protein assay kit (Thermo Scientific, Rockford, IL).…”

Age-related hypertension and salt sensitivity are associated with unique cortico-medullary distribution of D1R, AT1R, and NADPH-oxidase in FBN rats

“…Western immunoblotting on tissue homogenates and membranes was performed by standard methods as described previously (31). Briefly, tissue homogenates were made in lysis buffer (20mM Tris-HCl (pH 7.5),150mM NaCl, 1mM Na 2 EDTA, 1mM EGTA, 1% Triton, 2.5mM sodium pyrophosphate, 1mM beta-glycerophosphate, 1mM Na 3 VO 4 , 1 μg/ml leupeptin, protease inhibitor cocktail) as described.…”

“…Briefly, tissue homogenates were made in lysis buffer (20mM Tris-HCl (pH 7.5),150mM NaCl, 1mM Na 2 EDTA, 1mM EGTA, 1% Triton, 2.5mM sodium pyrophosphate, 1mM beta-glycerophosphate, 1mM Na 3 VO 4 , 1 μg/ml leupeptin, protease inhibitor cocktail) as described. Crude plasma membranes from tissues were also made in lysis buffer using our published method (31). Protein concentrations in the homogenate and membrane samples as well as in bladder urine were determined by the BCA protein assay kit (Pierce BCA Protein Assay Kit, Thermo Scientific, Rockford, IL).…”

Ligand-dependent serum response factor activation by the human CC chemokine receptors CCR2a and CCR2b is mediated by G proteins of the Gq family

“…Interestingly, regulation of receptor function, including chemokine receptors, by concerted activity of G q and G i proteins has been described. Pokkunuri et al [50] reported a G q -mediated signaling of D1R and angiotensin receptor 1 in a G i -dependent manner in human kidney HK2 cells. They showed that signaling of the Ga q -coupled D1R receptor [51] was ablated in the presence of Ga q/11 small interfering RNA and by pretreatment with PTX.…”