Abstract
Dopamine inhibited phosphate transport in isolated renal brush border membrane vesicles and in cultured renal proximal tubule cells from wild-type but not from NHERF-1 null mice. Co-immunoprecipitation experiments established that NHERF-1 associated with D1-like receptors. In wild-type mice, dopamine stimulated cAMP accumulation and protein kinase C (PKC) activity in renal proximal tubule cells, an effect that was abolished by SCH-23390, a D1-like receptor antagonist. In NHERF-1 null kidney tissue; however, dopamine failed to stimulate either cAMP accumulation or PKC activity. Infection of proximal tubule cells from NHERF-1 null mice with adenovirus-green fluorescent protein-NHERF-1 restored the ability of dopamine to stimulate cAMP and PKC. Finally, in (32)P-labeled wild-type proximal tubule cells and in opossum kidney cells, dopamine increased NHERF-1 phosphorylation at serine 77 of the PDZ I domain of NHERF-1, a site previously shown to attenuate binding of cellular targets including the Npt2a (sodium-dependent phosphate transporter 2a). Together, these studies establish that NHERF-1 plays a key role in dopamine signaling and is also a downstream target of D1-like receptors in the mouse kidney. These studies suggest a novel role for the PDZ adapter protein NHERF-1 in coordinating dopamine signals that inhibit renal phosphate transport.
Highlights
Dopamine inhibited phosphate transport in isolated renal brush border membrane vesicles and in cultured renal proximal tubule cells from wild-type but not from NHERF-1 null mice
Phosphate transport was inhibited 31.8 Ϯ 2.1% (p Ͻ 0.05) by dopamine in adenovirus GFP-NHERF-1-infected cells compared with 1.9 Ϯ 4.1% (p ϭ NS) in cells infected with control adenovirus GFP (n ϭ 4) (Fig. 1)
The signaling pathways activated by dopamine in mouse kidney involves activation of adenylyl cyclase, generation of cAMP, and subsequent activation of protein kinase A (PKA) but not EPAC (2, 16 –19)
Summary
Measurement of Sodium-dependent Phosphate Uptake in BBM Vesicles and Primary Cultures of Proximal Tubule Cells—The uptake of phosphate (100 M) in BBM vesicles harvested from control and dopamine-treated kidney slices (10Ϫ4 M for 45 min) was measured at 30 s (initial rate) and 90 min (steady state) by the rapid filtration technique using a transport medium containing 137 mM NaCl. 5.4 mM KCl, 2.8 mM CaCl2, 1.2 mM MgSO4, 0.1 mM KH2PO4, and [32P]orthophosphate [26]. In vivo phosphorylation of full-length NHERF-1 was assayed in wild-type cultured proximal tubule cells incubated for 3 h in phosphate-free Dulbecco’s modified Eagle’s medium containing [32P]orthophosphate. The cells were incubated in phosphate-free Dulbecco’s modified Eagle’s medium containing [32P]orthophosphate and studied under control conditions or after treatment with dopamine (10Ϫ4 M for 45 min). Sodium-dependent phosphate uptake (nmol/mg of protein) was determined in BBM vesicles harvested from freshly prepared kidney slices from control (ϪD) and dopamine-treated (ϩD) tissues from wild-type and NHERF-1 null mice. Statistical analyses were performed using Peritz analysis of variance. p values less than 0.05 were considered statistically significant
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