The inositol Inpp5k 5-phosphatase affects osmoregulation through the vasopressin-aquaporin 2 pathway in the collecting system.

Inositol Inpp5k (or Pps, SKIP) is a member of the inositol polyphosphate 5-phosphatases family with a poorly characterized function in vivo. In this study, we explored the function of this inositol 5-phosphatase in mice and cells overexpressing the 42-kDa mouse Inpp5k protein. Inpp5k transgenic mice present defects in water metabolism characterized by a reduced plasma osmolality at baseline, a delayed urinary water excretion following a water load, and an increased acute response to vasopressin. These defects are associated with the expression of the Inpp5k transgene in renal collecting ducts and with alterations in the arginine vasopressin/aquaporin-2 signalling pathway in this tubular segment. Analysis in a mouse collecting duct mCCD cell line revealed that Inpp5k overexpression leads to increased expression of the arginine vasopressin receptor type 2 and increased cAMP response to arginine vasopressin, providing a basis for increased aquaporin-2 expression and plasma membrane localization with increased osmotically induced water transport. Altogether, our results indicate that Inpp5k 5-phosphatase is important for the control of the arginine vasopressin/aquaporin-2 signalling pathway and water transport in kidney collecting ducts.

The renal connecting tubule: Resolved and unresolved issues in Ca(2+) transport.

The renal connecting tubule (CNT) localizes to the distal part of the nephron between the distal convoluted tubule and the collecting duct, and consists of two different cell types: segment-specific and intercalated cells. The former reabsorb water (H(2)O), sodium (Na(+)) and calcium (Ca(2+)) ions to the blood compartment, while secreting potassium ions (K(+)) into the pro-urine. The latter cells contribute to the renal control of the acid-base balance. Several factors and hormones tightly regulate these transport processes. Although the CNT reabsorbs only ∼15% of filtered Ca(2+) load, this segment is finally decisive for the amount of Ca(2+) that appears in the urine. Impaired Ca(2+) transport across CNT can provoke severe urinary Ca(2+) excretion, called hypercalciuria. This review mainly focuses on the activity, abundance and expression of the epithelial Ca(2+) channel named Transient Receptor Potential Vanilloid 5 (TRPV5) that is the gatekeeper of active Ca(2+) reabsorption in the CNT.

5-Stabilized phosphatidylinositol 3,4,5-trisphosphate analogues bind Grp1 PH, inhibit phosphoinositide phosphatases, and block neutrophil migration.

Metabolically stabilized analogues of PtdIns(3,4,5)P3 have shown long-lived agonist activity for cellular events and selective inhibition of lipid phosphatase activity. We describe an efficient asymmetric synthesis of two 5-phosphatase-resistant analogues of PtdIns(3,4,5)P3, the 5-methylene phosphonate (MP) and 5-phosphorothioate (PT). Furthermore, we illustrate the biochemical and biological activities of five stabilized PtdIns(3,4,5)P3 analogues in four contexts. First, the relative binding affinities of the 3-MP, 3-PT, 5-MP, 5-PT, and 3,4,5-PT3 analogues to the Grp1 PH domain are shown, as determined by NMR spectroscopy. Second, the enzymology of the five analogues is explored, showing the relative efficiency of inhibition of SHIP1, SHIP2, and phosphatase and tensin homologue deleted on chromosome 10 (PTEN), as well as the greatly reduced ability of these phosphatases to process these analogues as substrates as compared to PtdIns(3,4,5)P3. Third, exogenously delivered analogues severely impair complement factor C5a-mediated polarization and migration of murine neutrophils. Finally, the new analogues show long-lived agonist activity in mimicking insulin action in sodium transport in A6 cells.

Synthesis and biological activity of PTEN-resistant analogues of phosphatidylinositol 3,4,5-trisphosphate.

The activation of phosphatidylinositol 3-kinase (PI 3-K) and subsequent production of PtdIns(3,4,5)P3 launches a signal transduction cascade that impinges on a plethora of downstream effects on cell physiology. Control of PI 3-K and PtdIns(3,4,5)P3 levels is an important therapeutic target in treatments for allergy, inflammation, cardiovascular, and malignant human diseases. We designed metabolically stabilized, that is, phosphatase resistant, analogues of PtdIns(3,4,5)P3 as probes for long-lived potential agonists or potential antagonists for cellular events mediated by PtdIns(3,4,5)P3. In particular, two types of analogues were prepared containing phosphomimetics that would be selectively resistant to the lipid 3-phosphatase PTEN. The total asymmetric synthesis of the 3-phosphorothioate-PtdIns(3,4,5)P3 and 3-methylenephosphonate-PtdIns(3,4,5)P3 analogues is described. These two analogues showed differential binding to PtdIns(3,4,5)P3 binding modules, and both were potential long-lived activators that mimicked insulin action in sodium transport in A6 cells.

Stimulus-secretion coupling of hypotonicity-induced insulin release in BRIN-BD11 cells.

The stimulus-secretion coupling for hypotonicity-induced insulin release was investigated in BRIN-BD11 cells. A 50 mM decrease in extracellular NaCl caused a twofold increase in insulin release. The release of insulin evoked by hypotonicity progressively decreased in an exponential manner. The response to extracellular hypotonicity displayed a threshold value close to 20 mOsmol/L and a maximal response at about 70 mOsmol/ L. Hypotonicity also caused a rapid increase in cell volume followed by a regulatory volume decrease (RVD), cell membrane depolarization with induction of spike activity, and a rise in cytosolic Ca2+ concentration. 5-Nitro-2-(3-phenylpropylamino)benzoate inhibited the secretory response to hypoosmolarity, failed to affect the early increase in cell volume but prevented the RVD, and suppressed the hypotonicity-induced plasma membrane depolarization. Insulin release provoked by hypotonicity was inhibited by verapamil, absence of Ca2+, thapsigargin, furosemide, tributyltin, and diazoxide. On the contrary, tolbutamide augmented modestly insulin release recorded in the hypoosmolar medium. Last, a rise in extracellular K+ concentration, while augmenting basal insulin output, failed to affect insulin release in the hypoosmolar medium. Thus, the insulin secretory response to hypotonicity apparently represents a Ca2+-dependent process triggered by the gating of volume-sensitive anion channels with subsequent depolarization and gating of voltage-sensitive Ca2+ channels.

Hydrogen peroxide and epidermal growth factor activate phosphatidylinositol 3-kinase and increase sodium transport in A6 cell monolayers.

Activation of phosphatidylinositol 3-kinase (PI 3-kinase) is required for insulin stimulation of sodium transport in A6 cell monolayers. In this study, we investigate whether stimulation of the PI 3-kinase by other agents also provoked an increase in sodium transport. Both epidermal growth factor (EGF) and H2O2 provoked a rise in sodium transport that was inhibited by LY-294002, an inhibitor of PI 3-kinase activity. PI 3-kinase activity was estimated in extracts from A6 cell monolayers directly by performance of a PI 3-kinase assay. We also estimated the relative importance of the PI 3-kinase pathway by two different methods: 1) coprecipitation of the p85 regulatory subunit with anti-phosphotyrosine antibodies and 2) phosphorylation of PKB on both Ser 473 and Thr 308 residues observed by Western blotting. Since the mitogen-activated protein kinase (MAPK) pathway has also been implicated in the regulation of sodium transport, we also investigated whether this pathway is turned on by insulin, H2O2, or EGF. Phosphorylation of ERK1/2 was increased only transiently by insulin and H2O2 but quite sustainedly by EGF. Inhibitors of this pathway (U-0126 and PD-98059) failed to affect the insulin and H2O2 stimulation of sodium transport but increased substantially the stimulation induced by EGF. The latter effect was associated with an increase in PKB phosphorylation, thus suggesting that the stimulation of the MAPK pathway prevents, in part, the stimulation of the PI 3-kinase pathway in the transport of sodium stimulated by EGF.

Phosphatidylinositol 3,4,5-trisphosphate: an early mediator of insulin-stimulated sodium transport in A6 cells.

Insulin stimulates sodium transport across A6 epithelial cell monolayers. Activation of phosphatidylinositol 3-kinase (PI 3-kinase) was suggested as an early step in the insulin-stimulated sodium reabsorption (Ref. 35). To establish that the stimulation of the PI 3-kinase signaling cascade is causing stimulation of apical epithelial Na channel, we added permeant forms of phosphatidylinositol (PI) phosphate (P) derivatives complexed with a histone carrier to A6 epithelium. Only PIP(3) and PI(3,4)P(2) but not PI(4,5)P(2) stimulated sodium transport, although each of them penetrated into A6 cell monolayers as assessed using fluorescent permeant phosphoinositides derivatives. By Western blot analysis of A6 cell extracts, the inositol 3-phosphatase PTEN and the protein kinase B PKB were both detected. To further establish that the stimulation of sodium transport induced by insulin is related to PIP(3) levels, we transfected A6 cells with human PTEN cDNA and observed a 30% decrease in the natriferic effect of insulin. Similarly, the increase in sodium transport observed by addition of permeant PIP(3) was also reduced by 30% in PTEN-overexpressing cells. PKB, a main downstream effector of PI 3-kinase, was phosphorylated at both Thr 308 and Ser 473 residues upon insulin stimulation of the A6 cell monolayer. PKB phosphorylation in response to insulin stimulation was reduced in PTEN-overexpressing cells. Permeant PIP(3) also increased PKB phosphorylation. Taken together, the present results establish that the d-3-phosphorylated phosphoinositides PIP(3) and PI(3,4)P(2) mediate the effect of insulin on sodium transport across A6 cell monolayers.