Cyclic GMP kinase II (cGKII) inhibits NHE3 by altering its trafficking and phosphorylating NHE3 at three required sites: identification of a multifunctional phosphorylation site.

The epithelial brush-border Na(+)/H(+) exchanger NHE3 is acutely inhibited by cGKII/cGMP, but how cGKII inhibits NHE3 is unknown. This study tested the hypothesis that cGMP inhibits NHE3 by phosphorylating it and altering its membrane trafficking. Studies were carried out in PS120/NHERF2 and in Caco-2/Bbe cells overexpressing HA-NHE3 and cGKII, and in mouse ileum. NHE3 activity was measured with 2',7'-bis(carboxyethyl)-S-(and 6)carboxyfluorescein acetoxy methylester/fluorometry. Surface NHE3 was determined by cell surface biotinylation. Identification of NHE3 phosphorylation sites was by iTRAQ/LC-MS/MS with TiO2 enrichment and immunoblotting with specific anti-phospho-NHE3 antibodies. cGMP/cGKII rapidly inhibited NHE3, which was associated with reduced surface NHE3. cGMP/cGKII increased NHE3 phosphorylation at three sites (rabbit Ser(554), Ser(607), and Ser(663), equivalent to mouse Ser(552), Ser(605), and Ser(659)), all of which had to be present at the same time for cGMP to inhibit NHE3. NHE3-Ser(663) phosphorylation was not necessary for cAMP inhibition of NHE3. Dexamethasone (4 h) stimulated wild type NHE3 activity and increased surface expression but failed to stimulate NHE3 activity or increase surface expression when NHE3 was mutated to either S663A or S663D. We conclude that 1) cGMP inhibition of NHE3 is associated with phosphorylation of NHE3 at Ser(554), Ser(607), and Ser(663), all of which are necessary for cGMP/cGKII to inhibit NHE3. 2) Dexamethasone stimulates NHE3 by phosphorylation of a single site, Ser(663). The requirement for three phosphorylation sites in NHE3 for cGKII inhibition, and for phosphorylation of one of these sites for dexamethasone stimulation of NHE3, is a unique example of regulation by phosphorylation.

A phase I open-label study to investigate the potential drug-drug interaction between single-dose dacomitinib and steady-state paroxetine in healthy volunteers.

Dacomitinib is currently in development for the treatment of non-small cell lung cancer. Formation of the major circulating metabolite (PF-05199265) is mediated by cytochrome P450 (CYP) 2D6 and CYP2C9. This phase I, single fixed-sequence, two-period study evaluated the effect of paroxetine, a CYP2D6 inactivator, on dacomitinib pharmacokinetics in healthy volunteers who were extensive CYP2D6 metabolizers. Subjects received a single 45-mg dacomitinib dose alone and in combination with paroxetine (30 mg/day for 10 consecutive days, with dacomitinib administered on day 4) at steady-state levels. Blood samples were collected through 240 hours post-dacomitinib dosing. Dacomitinib exposure (area under the concentration-time curve from 0 to infinity; AUCinf) increased 37%; however a reduction in PF-05199265 AUCinf of approximately 90% was observed during the paroxetine treatment period. The maximum concentration of dacomitinib changed minimally. Adverse events reported with single-dose dacomitinib administered alone or in the presence of steady-state levels of paroxetine were mostly mild, and no serious adverse events were reported. While paroxetine significantly inhibited CYP2D6-mediated metabolism of a single dose of dacomitinib, the modest effect on dacomitinib exposure is unlikely to be clinically relevant when dacomitinib is given daily. Dose adjustment of dacomitinib may therefore not be required upon coadministration with a CYP2D6 inhibitor.

Ezrin is required for the functional regulation of the epithelial sodium proton exchanger, NHE3.

The sodium hydrogen exchanger isoform 3 (NHE3) mediates absorption of sodium, bicarbonate and water from renal and intestinal lumina. This activity is fundamental to the maintenance of a physiological plasma pH and blood pressure. To perform this function NHE3 must be present in the apical membrane of renal tubular and intestinal epithelia. The molecular determinants of this localization have not been conclusively determined, although linkage to the apical actin cytoskeleton through ezrin has been proposed. We set out to evaluate this hypothesis. Functional studies of NHE3 activity were performed on ezrin knockdown mice (Vil2(kd/kd)) and NHE3 activity similar to wild-type animals detected. Interpretation of this finding was difficult as other ERM (ezrin/radixin/moesin) proteins were present. We therefore generated an epithelial cell culture model where ezrin was the only detectable ERM. After knockdown of ezrin expression with siRNA, radixin and moesin expression remained undetectable. Consistent with the animal ultrastructural data, cells lacking ezrin retained an epithelial phenotype but had shortened and thicker microvilli. NHE3 localization was identical to cells transfected with non-targeting siRNA. The attachment of NHE3 to the apical cytoskeleton was unaltered as assessed by fluorescent recovery after photobleaching (FRAP) and the solubility of NHE3 in Triton X-100. Baseline NHE3 activity was unaltered, however, cAMP-dependent inhibition of NHE3 was largely lost even though NHE3 was phosphorylated at serines 552 and 605. Thus, ezrin is not necessary for the apical localization, attachment to the cytoskeleton, baseline activity or cAMP induced phosphrylation of NHE3, but instead is required for cAMP mediated inhibition.

NHERF2 is necessary for basal activity, second messenger inhibition, and LPA stimulation of NHE3 in mouse distal ileum.

To test the hypothesis that Na(+)/H(+) exchanger (NHE) regulatory factor 2 (NHERF2) is necessary for multiple aspects of acute regulation of NHE3 in intact mouse small intestine, distal ileal NHE3 activity was determined using two-photon microscopy/SNARF-4F in a NHERF2-null mouse model. The NHERF2-null mouse ileum had shorter villi, deeper crypts, and decreased epithelial cell number. Basal rates of NHE3 activity were reduced in NHERF2-null mice, which was associated with a reduced percentage of NHE3 in the apical domain and an increase in intracellular NHE3 amount but no change in total level of NHE3 protein. cAMP, cGMP, and elevated Ca(2+) due to apical exposure to UTP all inhibited NHE3 activity in wild-type mouse ileum but not in NHERF2-null mice, while inhibition by hyperosmolarity occurred normally. The cAMP-increased phosphorylation of NHE3 at aa 552; levels of PKAIIα and cGMP-dependent protein kinase II (cGKII); and elevation of Ca(2+) were similar in wild-type and NHERF2-null mouse ileum. Luminal lysophosphatidic acid (LPA) stimulated NHE3 in wild-type but not in NHERF2-null ileum. In conclusion, 1) there are subtle structural abnormalities in the small intestine of NHERF2-null mouse which include fewer villus epithelial cells; 2) the decreased basal NHE3 activity and reduced brush border NHE3 amount in NHERF2-null mice show that NHERF2 is necessary for normal basal trafficking or retention of NHE3 in the apical domain; 3) hyperosmolar inhibition of NHE3 occurs similarly in wild-type and NHERF2-null ileum, demonstrating that some inhibitory mechanisms of NHE3 are not NHERF2 dependent; 4) cAMP inhibition of NHE3 is NHERF2 dependent at a step downstream of cAMP/PKAII phosphorylation of NHE3 at aa 552; 5) cGMP- and UTP-induced inhibition of NHE3 are NHERF2 dependent at steps beyond cGKII and the UTP-induced increase of intracellular Ca(2+); and 6) LPA stimulation of NHE3 is also NHERF2 dependent.

NHE3 function and phosphorylation are regulated by a calyculin A-sensitive phosphatase.

Na+/H+ exchanger 3 (NHE3) is phosphorylated and regulated by multiple kinases, including PKA, SGK1, and CK2; however, the role of phosphatases in the dephosphorylation and regulation of NHE3 remains unknown. The purpose of this study was to determine whether serine/threonine phosphatases alter NHE3 activity and phosphorylation and, if so, at which sites. To this end, we first examined the effects of calyculin A [a combined protein phosphatase 1 (PP1) and PP2A inhibitor] and okadaic acid (a PP2A inhibitor) on general and site-specific NHE3 phosphorylation. Calyculin A induced a phosphorylation-dependent NHE3 gel mobility shift and increased NHE3 phosphorylation at serines 552 and 605. No change in NHE3 phosphorylation was detected after okadaic acid treatment. An NHE3 gel mobility shift was also evident in calyculin A-treated COS-7 cells transfected with either wild-type or mutant (S552A, S605G, S661A, S716A) rat NHE3. Since the NHE3 gel mobility shift occurred despite mutation of known phosphorylation sites, novel sites of phosphorylation must also exist. Next, we assayed NHE3 activity in response to calyculin A and okadaic acid and found that calyculin A induced a 24% inhibition of NHE3 activity, whereas okadaic acid had no effect. When all known NHE3 phosphorylation sites were mutated, calyculin A induced a stimulation of NHE3 activity, demonstrating a functional significance for the novel phosphorylation sites. Finally, we established that the PP1 catalytic subunit can directly dephosphorylate immunopurified NHE3 in vitro. In conclusion, our data demonstrate that a calyculin A-sensitive phosphatase, most likely PP1, is involved in the regulation and dephosphorylation of NHE3 at known and novel sites.

NHE3 phosphorylation at serines 552 and 605 does not directly affect NHE3 activity.

Direct phosphorylation of sodium hydrogen exchanger type 3 (NHE3) is a well-established physiological phenomenon; however, the exact role of NHE3 phosphorylation in its regulation remains unclear. The objective of this study was to evaluate whether NHE3 phosphorylation at serines 552 and 605 is physiologically regulated in vivo and, if so, whether changes in phosphorylation at these sites are tightly coupled to changes in transport activity. To this end, we directly compared PKA-induced NHE3 inhibition with site-specific changes in NHE3 phosphorylation in vivo and in vitro. In vivo, PKA was activated using an intravenous infusion of parathyroid hormone in Sprague-Dawley rats. In vitro, PKA was activated directly in opossum kidney (OKP) cells using forskolin and IBMX. NHE3 activity was assayed in microvillar membrane vesicles in the rat model and by (22)Na uptake in the OKP cell model. In both cases, NHE3 phosphorylation at serines 552 and 605 was determined using previously characterized monoclonal phosphospecific antibodies directed to these sites. In vivo, we found dramatic changes in NHE3 phosphorylation at serines 552 and 605 with PKA activation but no corresponding alteration in NHE3 activity. This dissociation between NHE3 phosphorylation and activity was further verified in OKP cells in which phosphorylation clearly preceded transport inhibition. We conclude that although phosphorylation of NHE3 at serines 552 and 605 is regulated by PKA both in vivo and in vitro, phosphorylation of these sites does not directly alter Na(+)/H(+) exchange activity.

Membrane curvature alters the activation kinetics of the epithelial Na+/H+ exchanger, NHE3.

The epithelial Na(+)/H(+) exchanger, NHE3, was found to activate slowly following an acute cytosolic acidification. The sigmoidal course of activation could not be explained by the conventional two-state model, which postulates that activation results from protonation of an allosteric modifier site. Instead, mathematical modeling predicted the existence of three distinct states of the exchanger: two different inactive states plus an active form. The interconversion of the inactive states is rapid and dependent on pH, whereas the conversion between the second inactive state and the active conformation is slow and pH-independent but subject to regulation by other stimuli. Accordingly, exposure of epithelial cells to hypoosmolar solutions activated NHE3 by accelerating this latter transition. The number of surface-exposed exchangers and their association with the cytoskeleton were not affected by hypoosmolarity. Instead, NHE3 is activated by the membrane deformation, a result of cell swelling. This was suggested by the stimulatory effects of amphiphiles that induce a comparable positive (convex) deformation of the membrane. We conclude that NHE3 exists in multiple states and that different physiological parameters control the transitions between them.

Use of phospho-specific antibodies to determine the phosphorylation of endogenous Na+/H+ exchanger NHE3 at PKA consensus sites.

Transfection studies using mutant constructs have implicated one or both protein kinase A (PKA) consensus phosphorylation sites [serines 552 and 605 in rat Na(+)/H(+) exchanger type 3 (NHE3)] as critical for mediating inhibition of NHE3 in response to several stimuli including dopamine. However, whether one or both of these sites is actually phosphorylated in endogenous NHE3 in proximal tubule cells is unknown. The purpose of this study was to generate phosphospecific antibodies so that the state of phosphorylation of these serine residues in endogenous NHE3 could be assessed in vitro and in vivo. To this end, polyclonal and monoclonal phosphospecific peptide antibodies were generated against each PKA consensus site. Phosphospecificity was established by ELISA and Western blot assays. We then used these antibodies in vitro to evaluate the effect of dopamine on phosphorylation of the corresponding PKA sites (serines 560 and 613) in NHE3 endogenously expressed in opossum kidney cells. Baseline phosphorylation of both sites was detected that was significantly increased by dopamine. Next, we determined the baseline phosphorylation state of each serine in rat kidney NHE3 in vivo. We found that serine 552 of NHE3 is phosphorylated to a much greater extent than serine 605 at baseline in vivo. Moreover, we detected a distinct subcellular localization for NHE3 phosphorylated at serine 552 compared with total NHE3. Specifically, NHE3 phosphorylated at serine 552 localized to the coated pit region of the brush-border membrane, where NHE3 is inactive, while total NHE3 was found throughout the brush-border membrane. These findings strongly suggest that phosphorylation of NHE3 plays a role in its subcellular trafficking in vivo. In conclusion, we successfully generated phosphospecific antibodies that should be useful to assess the phosphorylation of endogenous NHE3 at its two PKA consensus sites under a variety of physiological conditions in vitro and in vivo.