Acute regulation of renal Na⁺/H⁺ exchanger NHE3 by dopamine: role of protein phosphatase 2A

Abstract

mine is a potent natriuretic paracrine/autocrine hormone that is central for mammalian sodium homeostasis. In the renal proximal tubule, dopamine induces natriuresis partly via inhibition of the sodium/ proton exchanger NHE3. The signal transduction pathways and mechanisms by which dopamine inhibits NHE3 are complex and incompletely understood. This manuscript describes the role of the serine/ threonine protein phosphatase 2A (PP2A) in the regulation of NHE3 by dopamine. The PP2A regulatory subunit B56␦ (coded by the Ppp2r5d gene) directly associates with more than one region of the carboxy-terminal hydrophilic putative cytoplasmic domain of NHE3 (NHE3-cyto), as demonstrated by yeast-two-hybrid, coimmunoprecipitation, blot overlay, and in vitro pull-down assays. Phosphorylated NHE3-cyto is a substrate for purified PP2A in an in vitro dephosphorylation reaction. In cultured renal cells, inhibition of PP2A by either okadaic acid or by overexpression of the simian virus 40 (SV40) small T antigen blocks the ability of dopamine to inhibit NHE3 activity and to reduce surface NHE3 protein. Dopamineinduced NHE3 redistribution is also blocked by okadaic acid ex vivo in rat kidney cortical slices. These studies demonstrate that PP2A is an integral and critical participant in the signal transduction pathway between dopamine receptor activation and NHE3 inhibition. natriuresis; sodium transport; signal transduction THE INTRARENAL AUTOCRINE/PARACRINE dopamine natriuretic system is critical for mammalian sodium homeostasis (5,8,28,29,37,39,47,49,53). The dopamine precursor 3,4-dihydroxy-Lphenylalanine (L-DOPA) is taken up from the glomerular filtrate and plasma into the proximal tubule (48, 50), decarboxylated to dopamine by the action of the cytoplasmic aromatic amino acid decarboxylase (61), and dopamine is extruded into the urinary lumen as well as the interstitial space (43,51,58). Dopamine locally controls multiple aspects of renal function including renal blood flow, glomerular filtration rate, the setting of tubuloglomerular feedback, renin release, and Na ϩ absorption by the renal tubules (5, 37). Quantitatively, the most important mechanism by which dopamine induces natriuresis is direct suppression of Na ϩ absorption by the renal tubular epithelium (9,20,27,40). Inhibition of proximal Na ϩ absorption translates into effective whole kidney natriuresis because downstream of the proximal tubule, dopamine also inhibits Na ϩ absorption in the thick ascending loop of Henle, tubuloglomerular feedback, and Na ϩ transporters in the distal nephron (37). In the proximal tubule, dopamine inhibits the two principal Na ϩ transporters: the apical membrane Na ϩ /H ϩ exchanger NHE3 (7,23,26,33,59,60) and the basolateral Na ϩ -K ϩ -ATPase (4, 12, 13). Inhibition of NHE3 by dopamine involves reduced transport activity and increased endocytosis (7,33,59) and is associated with NHE3 phosphorylation (33, 64). Two PKA phosphorylation sites (serines 552 and 605 in rat NHE3) are necessary but not sufficient for PKA-mediated NHE3 regulation ...

Chronic regulation of the renal Na+/H+exchanger NHE3 by dopamine: translational and posttranslational mechanisms

“…Just like acute regulation by DA, chronic action of DA also involves increased endocytosis of NHE3 as evidenced by the lower amount and half-life of cell surface NHE3, but it is in contradistinction to the acute effect of DA which internalizes NHE3 but not degrade it (12,26,59). The chronic effect of DA stimulates NHE3 internalization and directs it to destruction.…”

“…Immunocytochemistry was performed where OKP cells were fixed (4% paraformaldehyde in PBS ϫ 20 min) and permeabilized (0.2% saponin, 3% gelatin ϫ 20 min). The cells were then incubated with primary antibody (monoclonal anti-NHE3 3H3) (23,26,32,33,41,68), followed by incubation with FITC-conjugated donkey anti-mouse polyclonal antibodies (Jackson ImmunoResearch Laboratories). Confocal fluorescent images were visualized through a Zeiss ϫ100 objective lens using a Zeiss LSM-410 laser-scanning confocal microscope.…”

Sodium and Depression: Hypothetical Associations

“…Dopamine modulates various renal functions including renal blood flow, glomerular filtration rate, tubuloglomerular feedback, renin release and renal reabsorption of sodium (23). A positive correlation has been reported between sodium intake and renal dopamine production.…”

Functional Roles of a Putative B' Delta Regulatory Subunit and a Catalytic Subunit of Protein Phosphatase 2A in the Cereal Pathogen Fusarium graminearum

“…Although the B' regulatory subunit is the most diverse class among the three families (B, B', and B'') of the B-regulatory subunits, its functional roles have been intensively determined only in mammals (Bobulescu et al, 2010;Flegg et al, 2010;Hahn et al, 2010;Louis et al, 2011;Muneer et al, 2002). Specifically expressed in both embryonic cells and brain, it is involved in the dephosphorylation of proteins in various signaling pathways (McCright et al, 1996).…”