Human kidney-2 cells harbor functional dopamine D1 receptors that require Giα for Gq/11α signaling

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.

Potassium and Rubidium Uptake in Freshwater Bivalves

Potassium transport characteristics were investigated in three species of freshwater bivalves: a corbiculid, Corbiculafluminea, and two unionids, Carunculina texasensis and Ligumia subrostrata. Using 42K, all three were found to take up potassium from dilute artificial pondwater ([K+] about 0.05 mmoll+1). The influx (Ji) was 0.72μequiv g−1dry tissue h−1 in the corbiculid, significantly higher than the value of about 0.40μequiv g−1dry tissuer g−1 in the unionids. The K+ uptake displayed saturation kinetics in the range 0.05-0.36 mmoll−1: in Co. fluminea, there was a Jmax of 3.56μequiv g−1 dry tissue h−1 and the affinity coefficient (Km) was 0.27mmoll−1; in Ca. texasensis, Jmax had a value of 1.8 μequiv g−1dry tissue h−1 and Km was 0.16mmoll−1. Using K+-free artificial pondwater containing 0.03-0.04 mmoll−1 Rb+, the Rb+ influx was 0.41μequiv g−1 dry tissue h−1 in the corbiculid and 0.28 μequiv g−1 dry tissue h−1 in Ca. texasensis. All animals lost K+ during the rubidium flux studies, and since they contained no Rb+, the Rb+ efflux was zero and the net flux was equal to the influx. The Jmax values for Rb+ were lower than the corresponding values for potassium: in Co. fluminea, Jmax was 1.4μequiv g−1 dry tissueh+1, significantly higher than in Ca. texasensis, which had Jmax of 0.84μequiv g−1 dry tissue h−1. The rubidium Km (approx. 0.05 mmoll−1) values were significantly lower than corresponding values for potassium. Salt depletion increased the rubidium transport rate fourfold for both Co. fluminea and Ca. texasensis. High rates of net K+ uptake may account for the bivalves' inability to tolerate elevated environmental potassium.