Acute Regulation of Na+/H+ Exchanger NHE3 by Parathyroid Hormone via NHE3 Phosphorylation and Dynamin-dependent Endocytosis

Abstract

Parathyroid hormone (PTH) is a potent inhibitor of mammalian renal proximal tubule Na؉ transport via its action on the apical membrane Na ؉ /H ؉ exchanger NHE3. In the opossum kidney cell line, inhibition of NHE3 activity was detected from 5 to 45 min after PTH addition. Increase in NHE3 phosphorylation on multiple serines was evident after 5 min of PTH, but decrease in surface NHE3 antigen was not detectable until after 30 min of PTH. The decrease in surface NHE3 antigen was due to increased NHE3 endocytosis. When endocytic trafficking was arrested with a dominant negative dynamin mutant (K44A), the early inhibition (5 min) of NHE3 activity by PTH was not affected, whereas the late inhibition (30 min) and decreased surface NHE3 antigen induced by PTH were abrogated. We conclude that PTH acutely inhibits NHE3 activity in a biphasic fashion by NHE3 phosphorylation followed by dynamin-dependent endocytosis.The calcitropic effect of PTH 1 is effected by altering calcium flux between the plasma and the skeleton, intestine, and kidney in a concerted fashion. In the mammalian kidney, PTH is the principal anti-calciuric hormone exerting its action on the proximal tubule, thick ascending limb, and the distal convoluted tubule (1-4). In the renal proximal tubule, PTH is a potent inhibitor of NaHCO 3 absorption (5-11) that results in increased delivery to the distal nephron where NaHCO 3 acts as an important stimulus for active transcellular Ca 2ϩ absorption (12-14). In the mammalian proximal tubule, two-thirds of the transcellular NaHCO 3 absorption is mediated by apical membrane Na ϩ /H ϩ exchange (15) Acute inhibition of NHE3 can potentially be mediated by multiple mechanisms including NHE3 phosphorylation, binding to regulatory factors, and alteration of surface NHE3 protein abundance (37, 38). In cultured cells, acute regulation of NHE3 has been shown to be accompanied by NHE3 phosphorylation (39 -45), and the functional role of phosphorylation has been demonstrated for PKA and PKC (40,42,44). The signal transduction cascade mediating the effect of PTH on NHE3 is complex, likely involving multiple pathways, but at least part of the effect of PTH on NHE3 is 35,36). In the intact animal, acute infusion of PTH increases NHE3 phosphorylation as assayed by mobility retardation (26) which lends additional support for the importance of NHE3 phosphorylation in its regulation. This paper further characterizes the time course and pattern of NHE3 phosphorylation in OK cells in response to PTH.It is presently unclear how phosphorylation of NHE3 alters its transport activity. One mechanism that has been shown to alter acutely NHE3 transport activity is the change in cell surface protein abundance. This has been demonstrated in cultured cells (46 -48) as well as in intact kidney (26, 49 -53). Two studies have shown that acute infusion of PTH leads to a decrease in proximal tubule apical membrane NHE3 using immunohistochemistry (53) and membrane vesicles (26). It is unclear whether this acute decrement in apical membrane NHE3 is d...

Regulation of sodium-proton exchanger isoform 3 (NHE3) by PKA and exchange protein directly activated by cAMP (EPAC)

“…As reported in ref. 4, a reduction in surface expression was observed after a 4-h incubation in the presence of PTH (Fig. 2D), indicating that the biotinylation assay is sensitive enough to detect changes in surface-expressed NHE3.…”

“…The ratiometric emission of 490͞440 was converted to intracellular pH after calibration (37) by using the high K ϩ ͞nigericin technique (38,39). In brief, after a 20 mM NH 4 Cl prepulse, cells were washed with a Na-free solution (Hepes buffer with NaCl replaced by of N-methyl-D-glucamine). NHE3 activity was calculated from the initial slope of intracellular alkalinization upon readdition of Na.…”

“…About 50 g was subjected to SDS͞PAGE. Nitrocellulose membranes (Schleicher & Schüll) were incubated overnight at 4°C with a polyclonal antibody against NHE3 (1:1,000; kindly provided by O. W. Moe) (4). Upon incubation with HRP-linked secondary antibodies, immunoreactive signals were detected by chemiluminiscence (ECL, Amersham Pharmacia Biotech).…”

“…This transporter is down-regulated by parathyroid hormone (PTH) and dopamine. Acute treatment of rats with PTH (2, 3) or short exposure of opossum kidney (OK) cells to PTH or dopamine (4,5) inhibits NHE3 activity without reducing its expression in the BBM. The renal type IIa Na ϩ -coupled inorganic phosphate cotransporter (NaPi-IIa; SLC34A1) is also expressed in the BBM of PT where it mediates the bulk of Pi reabsorption from primary urine (6).…”

Disorders of Renal Tubular Phosphate Transport

“…Other brush border membrane proteins, e.g., the Na/sulfate cotransporter, are not internalized, whereas others that are internalized are recycled back to the brush border membrane (e.g., NHE-3 [2,44]). Specificity of retrieval can be achieved at the level of the transporter and/or at the level of the cellular machinery involved in recognition and internalization of the transporter (see below).…”