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...

We postulate the following: (1) DA modifies NHE-3 phosphorylation by activating PKA through DA1 and by other kinases/phosphatases via DA2. (2) DA1 is sufficient to inhibit NHE-3, while DA2 is insufficient but plays a synergistic role by altering NHE-3 phosphorylation.

Parathyroid hormone (PTH) is a potent inhibitor of mammalian renal proximal tubule sodium absorption via suppression of the apical membrane Na/H exchanger (NHE-3). We examined the mechanisms by which PTH inhibits NHE-3 activity by giving an acute intravenous PTH bolus to parathyroidectomized rats. Parathyroidectomy per se increased apical membrane NHE-3 activity and antigen. Acute infusion of PTH caused a time-dependent decrease in NHE-3 activity as early as 30 min. Decrease in NHE-3 activity at 30 and 60 min was accompanied by increased NHE-3 phosphorylation. In contrast to the rapid changes in NHE-3 activity and phosphorylation, decrease in apical membrane NHE-3 antigen was not detectable until 4 -12 h after the PTH bolus. The decrease in apical membrane NHE-3 occurred in the absence of changes in total renal cortical NHE-3 antigen. Pretreatment of the animals with the microtubuledisrupting agent colchicine blocked the PTH-induced decrease in apical NHE-3 antigen. We propose that PTH acutely cause a decrease in NHE-3 intrinsic transport activity possibly via a phosphorylation-dependent mechanism followed by a decrease in apical membrane NHE-3 antigen via changes in protein trafficking. PTH1 plays a paramount role in mammalian calcium homeostasis. The calcitropic actions of PTH include direct stimulation of bone turnover (1) and renal Ca absorption (2) and indirect enhancement of intestinal Ca absorption via its action on 1,25-vitamin D 3 (3). In the kidney, PTH exerts direct action on the proximal tubule, thick ascending limb, distal convoluted tubule, and connecting tubule (4, 5). In the proximal tubule, PTH is a potent inhibitor of NaHCO 3 absorption (6 -12). Because proximal tubule calcium and sodium absorption are tightly coupled (6, 13), the potent inhibitory action of PTH on proximal NaHCO 3 transport appears to be counterproductive for an anticalciuric hormone. Moreover, the acute inhibition of proximal tubule NaHCO 3 absorption seems to serve little purpose because the HCO 3 exiting the proximal tubule is largely reclaimed in the distal nephron as evident by the fact that acute PTH only cause modest reductions in plasma HCO 3 concentration (14 -16). However, in the distal convoluted tubule, luminal HCO 3 is an important stimulus for transcellular calcium absorption (17)(18)(19). Thus the shift of NaHCO 3 absorption from proximal to distal nephron results in minimal net change in acid-base balance but serves as a key enhancer of the anticalciuric effect of PTH.In the mammalian proximal tubule, two-thirds of the transcellular NaHCO 3 absorption is mediated by apical membrane Na/H exchange (20). Immunohistochemical (21-23), pharmacokinetic (24), and genetic (25) data all indicate that the NHE-3 isoform is predominantly responsible for proximal tubule apical membrane Na/H exchange. Direct inhibition of proximal tubule HCO 3 absorption by PTH (6 -12) is effected at least in part by inhibition of apical membrane Na/H exchange activity, which has been demonstrated in the suspended tubules (26), isolated perfu...